RADIOISOTOPE 18F SUBSTITUTED THIAMINE, AND SYNTHESIS METHOD AND USE THEREOF

Abstract

The present invention discloses a radioisotope .sup.18F substituted thiamine, synthesis method, and use thereof in small animal PET/CT imaging. The radioisotope .sup.18F substituted thiamine has a structure of

##STR00001##

The synthesis method comprises radiochemical synthesis by using existing precursors. In the present invention, the hydroxyl in the hydroxyethyl on the thiazole cycle of thiamine is replaced by radioisotope .sup.18F, to prepare a PET tracer. The radioisotope .sup.18F substituted thiamine of the present invention successfully enters the brains of various strains of mice, and the uptake in the brains of thiamine-deficient mice is obviously greater than that in normal control. The present invention is useful in the preparation of a brain imaging tracer for clinical trials of Alzheimer's disease and tumors.

Claims

1. A radioisotope .sup.18F substituted thiamine or a salt thereof, which is 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-fluoroethyl)-4-methylthiazolium or a salt thereof, where the fluorine element in the fluoroethyl on the thiazole ring is radioisotope .sup.18F, having a chemical structure of Formula (I) below: ##STR00012##

2. The radioisotope .sup.18F substituted thiamine or a salt thereof according to claim 1, having a chemical structure of Formula (II) or (III) below: ##STR00013## ##STR00014##

3. The radioisotope .sup.18F substituted thiamine or a salt thereof according to claim 2, wherein the salt of the radioisotope .sup.18F substituted thiamine is specifically a radioisotope .sup.18F substituted thiamine hydrobromide having a chemical formula of C.sub.12H.sub.16.sup.18FN.sub.4SBr.HBr, and a chemical structure of Formula (IV) below: ##STR00015##

4. A method for synthesizing a radioisotope .sup.18F substituted thiamine hydrobromide according to claim 3, comprising: Step 1): generating H.sup.+ protons in a cyclotron, bombarding .sup.18O-heavy oxygen water with the H.sup.+ protons, and generating .sup.18F.sup. ions through the nuclear reaction .sup.18O(p, n).sup.18F; enriching .sup.18F.sup. ions by a QMA column, and eluting it into a reaction vial with an eluant, to obtain [K/Kryptofix].sup.+18F.sup., evaporating water to dryness; and reacting 2-(4-methylthiazol-5-yl)ethyl 4-methylbenzenesulfonate with [K/Kryptofix].sup.+18F.sup. to produce 5-(2-fluoroethyl)-4-methylthiazole through nucleophilic substitution in acetonitrile solvent, where the reaction equation for chemical synthesis is: ##STR00016## Step 2): reacting the product 5-(2-fluoroethyl)-4-methylthiazole obtained in Step 1) with 2-methyl-5-bromomethyl-6-aminopyrimidine hydrobromide in acetonitrile solvent to produce .sup.18F substituted thiamine hydrobromide, where the reaction equation for chemical synthesis is: ##STR00017##

5. The method for synthesizing radioisotope .sup.18F substituted thiamine hydrobromide according to claim 4, wherein the ratio of 2-(4-methylthiazol-5-yl)ethyl 4-methylbenzenesulfonate to the solvent in Step 1) is 5 mg:0.5-1 mL; the ratio of 2-(4-methylthiazol-5-yl)ethyl 4-methylbenzenesulfonate to .sup.18F.sup. ions is 5 mg:4-6 Ci; and the ratio of 2-(4-methylthiazol-5-yl)ethyl 4-methylbenzenesulfonate to the eluant is 5 mg:0.6-0.9 mL, where each 0.9 mL of the eluant contains 2.52 mg KHCO.sub.3, 9 mg Kryptofix 2.2.2, 0.72 mL acetonitrile, and 0.18 mL water.

6. The method for synthesizing radioisotope .sup.18F substituted thiamine hydrobromide according to claim 4, wherein the ratio of 2-methyl-5-bromomethyl-6-aminopyrimidine hydrobromide to the solvent in Step 2) is 5 mg:0.5-1 mL.

7. The method for synthesizing radioisotope .sup.18F substituted thiamine hydrobromide according to claim 4, wherein the reaction temperature in Steps 1) and 2) is 110 C.

8. The method for synthesizing radioisotope .sup.18F substituted thiamine hydrobromide according to claim 4, wherein the mass ratio of 2-(4-methylthiazol-5-yl)ethyl 4-methylbenzenesulfonate in Step 1) to 2-methyl-5-bromomethyl-6-aminopyrimidine hydrobromide in Step 2) is 1:1.

9. Use of the radioisotope .sup.18F substituted thiamine according to claim 1 in the preparation of a tracer for PET imaging of the brain in rodents.

10. Use of the radioisotope .sup.18F substituted thiamine according to claim 1 in the preparation of a tracer for PET imaging of clinical trials for Alzheimer's disease.

11. Use of the radioisotope .sup.18F substituted thiamine according to claim 1 in the preparation of a tracer for PET imaging of clinical trials for tumors.

12. The method for synthesizing radioisotope .sup.18F substituted thiamine hydrobromide according to claim 5, wherein the reaction temperature in Steps 1) and 2) is 110 C.

13. The method for synthesizing radioisotope .sup.18F substituted thiamine hydrobromide according to claim 6, wherein the reaction temperature in Steps 1) and 2) is 110 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 shows a synthesis route in Step 1.1 in Example 1;

[0039] FIG. 2 shows the result of TLC analysis in Step 1.1.1 in Example 1;

[0040] FIG. 3 shows the result of TLC analysis in Step 1.1.2.3 in Example 1;

[0041] FIG. 4 shows the result of TLC analysis in Step 1.1.2.4 in Example 1;

[0042] FIG. 5 shows a HPLC chromatogram obtained in Step 2 of Example 1;

[0043] FIG. 6 shows time-activity curves (TACs) of .sup.18F substituted thiamine uptake in the brains of three ICR mice in a thiamine-deficient model group in Example 4;

[0044] FIG. 7 shows TACs of .sup.18F substituted thiamine uptake in the brains of three ICR mice in a control group in Example 4;

[0045] FIG. 8 shows a representative image of the uptake in the brains of ICR mice in the thiamine-deficient model group in Example 4;

[0046] FIG. 9 shows a representative image of the uptake in the brains of ICR mice in the control group in Example 4;

[0047] FIG. 10 shows TACs of .sup.18F substituted thiamine uptake in the brains of two C57BL/6 mice in a thiamine-deficient model group in Example 5;

[0048] FIG. 11 shows TACs of .sup.18F substituted thiamine uptake in the brains of three C57BL/6 mice in a control group in Example 5;

[0049] FIG. 12 shows a representative image of the uptake in the brains of C57BL/6 mice in the thiamine-deficient model group in Example 5; and

[0050] FIG. 13 shows a representative image of the uptake in the brains of C57BL/6 mice in the control group in Example 5.

DETAILED DESCRIPTION

[0051] To make the present invention more comprehensible and understandable, the present invention is described in further detail by way of preferred embodiments with reference to the accompanying drawings.

[0052] The eluant used in Examples 1-3 is formulated such that each 0.9 mL of the eluant contains 2.52 mg KHCO.sub.3, 9 mg Kryptofix 2.2.2, 0.72 mL acetonitrile, and 0.18 mL water.

Example 1

[0053] 1. Synthesis of Radioisotope .sup.18F Substituted Thiamine

[0054] 1.1. Synthesis of Precursors (Preparation Step, as Shown in FIG. 1)

[0055] 1.1.1 Synthesis of Precursor I: 2-(4-methylthiazol-5-yl)ethyl 4-methylbenzenesulfonate

[0056] Under nitrogen protection, 4-methyl-5-(beta-hydroxyethyl)thiazole (2.9 g, 20.0 mmol) and TsCl (19.1 g, 100 mmol) were added to a 250 mL one-neck flask, and then TEA (6.07 g, 60.0 mmol) was added, dissolved in anhydrous DCM (100 mL), and stirred for 16 hrs at normal temperature (RT). TLC analysis (n-hexane:ethyl acetate=3:1) was performed, as shown in FIG. 2. The resulting material was rotary evaporated, and purified by column chromatography (dichloromethane:methanol=100:1), to obtain a crude product (6.1 g), which was further purified by column chromatography (dichloromethane:methanol=80:1), to obtain the target product (3.2 g, yield 71%).

[0057] .sup.1H NMR (300 MHz, CDCl.sub.3) : 8.56 (s, 1H), 7.72 (d, 2H, J=6.6 Hz), 7.31 (d, 2H, J=8.1 Hz), 4.17 (t, 2H, J=6.6 Hz), 3.12 (t, 2H, J=6.6 Hz), 2.45 (s, 3H), 2.33 (s, 3H). MS (ESI): m/z 298 (M+1, 100).

[0058] 1.1.2. Synthesis of Precursor II: 2-methyl-5-bromomethyl-6-aminopyrimidine hydrobromide

[0059] 1.1.2.1. Synthesis of ethyl aldehyde group-3-ethoxypropionate sodium salt

[0060] To a 500 mL three-neck flask, sodium wires (12 g) soaked in anhydrous diethyl ether (200 mL) were added, and then a mixed solution containing ethyl 3-ethoxypropionate (73 g, 0.5 mol) and ethyl formate (40 g, 0.54 mol) was slowly added dropwise. The color of the reaction solution changed from colorless to yellow, and then to dark yellow. The reaction was completed within 8 hours, and stirred under RT to obtain a solid, which was directly used in the next step under nitrogen protection.

[0061] 1.1.2.2. Synthesis of 2-Methyl-5-ethoxymethyl-6-hydroxypyrimidine

[0062] Under nitrogen protection, sodium (12 g) was fed to anhydrous ethanol (200 mL), and reacted under reflux until sodium was completely reacted, to prepare a sodium ethoxide solution. Acetamidine hydrochloride (45 g, 0.48 mol) dissolved in anhydrous ethanol (100 mL) was slowly added dropwise to a mixture of sodium ethoxide and ethyl aldehyde group-3-ethoxypropionate sodium salt, and stirred and refluxed overnight at 80 C. after addition. The reaction was terminated, and adjusted to pH 6 with glacial acetic acid. The solvent was removed by rotary evaporation. Water (50 mL) was added, and extracted with DCM (2100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and purified by column chromatography (methanol:dichloromethane=1:100), to obtain the target product as a pale yellow solid (20.1 g, yield 24%).

[0063] .sup.1H NMR (300 MHz, CDCl.sub.3) : 8.02 (s, 1H), 4.40 (s, 2H), 3.62 (q, 2H, J=6.9 Hz), 2.48 (s, 3H), 1.26 (t, 3H, J=7.1 Hz). MS (ESI): m/z 169 (M+1, 100).

[0064] 1.1.2.3. Synthesis of 2-methyl-5-ethoxymethyl-6-chloropyrimidine

[0065] 2-methyl-5-ethoxymethyl-6-hydroxypyrimidine (2 g, 12.0 mmol) was fed to a 100 mL one-neck flask, then POCl.sub.3 (16 mL) was added and heated to 78 C., and then TEA (0.4 mL) was added dropwise. TLC analysis (methanol:dichloromethane=1:40) was performed 3 hrs later, as shown in FIG. 3. The reaction was terminated, and POCl.sub.3 was removed by rotary evaporation. Water (10 mL) was added, and then NaHCO.sub.3 was added to adjust the pH to neutral. The system was extracted with DCM (350 mL), and rotary evaporated to dryness, to obtain a crude product (2 g). The crude product was purified by column chromatography (dichloromethane:methanol=100:1), to obtain a yellow oil (1.7 g, yield 77%).

[0066] .sup.1H NMR (300 MHz, CDCl.sub.3) : 8.64 (s, 1H), 4.56 (s, 2H), 3.64 (q, 2H, J=7.1 Hz), 2.71 (s, 3H), 1.29 (t, 3H, J=6.9 Hz). MS (ESI): m/z 187 (M+1, 100).

[0067] 1.1.2.4. Synthesis of 2-methyl-5-ethoxymethyl-6-aminopyrimidine

[0068] 2-methyl-5-ethoxymethyl-6-chloropyrimidine (1.5 g, 8.1 mmol) was fed to an autoclave, and then a solution of ammonia in methanol (7.1 M, 39 mL) was added, heated to 140 C., and reacted for 4.5 hrs. TLC analysis (methanol:dichloromethane=1:50) was performed, as shown in FIG. 4. The reaction was terminated, rotary evaporated, and purified by chromatography on a dry-packed column (methanol:dichloromethane=1:100), to obtain the target product as a white solid (1.1 g, yield 82%).

[0069] .sup.1H NMR (300 MHz, CDCl.sub.3) : 7.99 (s, 1H), 5.47 (s, 2H), 4.43 (s, 2H), 3.49 (q, 2H, J=7.1 Hz), 2.50 (s, 3H), 1.23 (t, 3H, J=7.1 Hz). MS (ESI): m/z 168 (M+1, 100).

[0070] 1.1.2.5. Synthesis of 2-methyl-5-bromomethyl-6-aminopyrimidine hydrobromide (Precursor II)

[0071] 2-methyl-5-ethoxymethyl-6-aminopyrimidine (1.1 g, 6.6 mmol) was fed to a 200 mL one-neck flask, and then a HBr solution in acetic acid (10%, 73 mL) was added, heated to 100 C., and reacted for 2 hrs. The reaction was cooled to room temperature (where during the reaction, the solid was dissolved first, and then a solid was produced gradually). The reaction solution was suctioned, washed with diethyl ether, and recrystallized twice in methanol/anhydrous diethyl ether, to obtain a white solid (1.5 g, yield 81%).

[0072] .sup.1H NMR (300 MHz, DMSO-d.sub.6) : 9.31 (s, 1H), 8.62 (s, 1H), 8.50 (s, 1H), 4.67 (s, 2H), 2.49 (s, 3H). MS (ESI): m/z 202 (M+1, 20), 154 (M-Br+OCH.sub.3, 100).

[0073] 1.2. Radiochemical Synthesis Step:

[0074] .sup.18F substituted thiamine was synthesized through a two-step synthesis process using precursors I and II. The operation steps in radiochemical synthesis were all carried out in a closed hot cell (BBS2-V, COMECER Inc.), using RNplus synthesis module (RNplus, Synthra GmbH).

[0075] 1.2.1 (Step 1)

[0076] H.sup.+ protons were generated in a cyclotron (Cyclone18 Twin, IBA Inc.). .sup.18O-heavy oxygen water having an abundance of 98% or higher was bombarded with the accelerated protons, and .sup.18F.sup. ions (5 Ci) were generated through the nuclear reaction .sup.18O(p, n).sup.18F. The heavy oxygen water containing .sup.18F.sup. ions was passed through a QMA column (Sep-Pak Light QMA Carb, Waters), and then .sup.18F was eluted into the reaction vial 1 using 0.9 mL of an eluant (containing 2.52 mg KHCO.sub.3, 9 mg Kryptofix 2.2.2, 0.72 mL acetonitrile, and 0.18 mL water), where Kryptofix 2.2.2, K.sup.+ ions, and .sup.18F.sup. ions were complexed to form [K/Kryptofix].sup.+18F.sup.. Acetonitrile (1 mL) was added, and evaporated at 120 C. for 3 min. A nitrogen stream was blown into the reaction vial 1 at 120 C. for evaporation for further 3 min. After cooling to RT, additional acetonitrile (1 mL) was added, and evaporated to dryness at 120 C. for 3 min under a stream of nitrogen. After cooling to RT, precursor I (5 mg, dissolved in 0.5 mL acetonitrile) was added, and reacted for 20 min at 110 C. while the reaction vial 1 was sealed, to obtain an intermediate product 5-(2-fluoroethyl)-4-methylthiazole having a structure of:

##STR00009##

After cooling to RT, the reaction solution was stirred with a rotor for 20 s, and then transferred to a sample injector. A rinsing liquid (0.5 mL, water:acetonitrile=9:1) was further added to the reaction vial 1, stirred with a rotor for 20 s, and transferred to the sample injector. The solution (0.9 mL) was injected into a preparative column by the sample injector and separated by preparative chromatography. Pure 5-(2-fluoroethyl)-4-methylthiazole product was collected into an extraction flask (a 50 mL slender glass flask). Conditions for preparative chromatography: Waters Xbridge C.sub.18 preparative column (19100 mm, 5 um), mobile phase: 15% acetonitrile/85% water; flow rate: 14 mL/min. The desired liquid phase peak was collected based on the signal from a radio-detector, where the retention time of 5-(2-fluoroethyl)-4-methylthiazole was 10.5 min, and the collection was completed in 1 minute and 30 seconds.

[0077] 1.2.2 (Step 2)

[0078] The preparative peak of 5-(2-fluoroethyl)-4-methylthiazole was collected in an extraction flask containing diethyl ether (20 mL) and sodium chloride (6.6 g), during which the extraction flask (sodium chloride was used to saturate water in mobile phase) was stirred with a rotor. After collection, the stirring was continued for additional 1 min, then the solution in extraction flask was stood for 1 min, and the supernatant diethyl ether phase was transferred to an intermediate flask. The diethyl ether was transferred from the intermediate flask to the reaction vial 2 where the temperature was set to 55 C., and diethyl ether was evaporated to dryness over 12 min under a stream of nitrogen. After 12 min of evaporation, acetonitrile (1 mL) was added into the reaction vial 2 to dissolve the 5-(2-fluoroethyl)-4-methylthiazole, stirred for 10 sec and then passed through a magnesium sulfate column. Additional acetonitrile (1 mL) was added to rinse the reaction vial 2 and elute the 5-(2-fluoroethyl)-4-methylthiazole off from the magnesium sulfate column. The acetonitrile solution flowing through the magnesium sulfate column was transferred back to the reaction vial 2. Precursor II (5 mg, previously dissolved in 1 mL hot acetonitrile) was added, and reacted for 20 min at 110 C. while the reaction vial 2 was sealed, to obtain a .sup.18F substituted thiamine product. At the same time, the sample injector was washed with methanol repeatedly. In the heating process during the reaction, a nitrogen stream was blown to reduce the volume of the solvent acetonitrile. When the temperature reached 110 C., the nitrogen stream was shut off, and the reaction vial 2 was sealed. After reaction, the system was maintained at 110 C. for 3 min, during which a nitrogen stream was blown to contribute to evaporate the acetonitrile to dryness, and then the system was cooled to RT. After cooling to RT, water (0.5 mL) was added, stirred for 20 sec to dissolve .sup.18F substituted thiamine, and then transferred to the sample injector for separating and collecting a pure .sup.18F substituted thiamine product by preparative chromatography. Conditions for preparative chromatography: Waters Xbridge C18 preparative column (19100 mm, 5 um), mobile phase: 5% methanol/95% water (containing 50 mM ammonium acetate); flow rate: 14 mL/min. The desired liquid phase peak was collected based on the signal from a radio-detector, where the .sup.18F substituted thiamine retention time was 6.5 min, and the collection was completed in 30 sec.

[0079] The whole radiochemical synthesis process is completed in about 2 hours. The radiochemical output of .sup.18F substituted thiamine is >75 mCi, the radiochemical yield is >3% (decay-correction to end of bombardment), the radiochemical purity is >95% and the specific activity is >1.5 Ci/mol.

[0080] 2. the Purity of the Product was Detected by Analytical HPLC. The Result is Shown in FIG. 5.

[0081] 2.1. Conditions for Analytical Chromatography:

[0082] 2.2. Analytic chromatographic column: shim-pack VP-ODS 5 uM C.sub.18 (4.6150 mm) available from shimadzu.

[0083] 2.3. Mobile phases: Phase A: water (with 0.05% triethylamine and 50 mM ammonium acetate); and Phase B: methanol. 0-15 min: A:B=85%:15%; 15-25 min: Phase A gradient from 85% to 0%, and Phase B gradient from 15% to 100%; and 25-30 min: Phase B: 100%. Flow rate: 0.8 mL/min.

[0084] 2.4 Setting of radio-detection probe: .sup.18F. Setting of UV detection light source: deuterium lamp, and detection probe: 254 nm. The radio-detection probe is located behind the UV probe, and the regular time interval is 24 seconds at 0.8 ml/min flow rate.

[0085] 2.5. Other Remarks

[0086] In FIGS. 5A and B, the product collected in the preparation of .sup.18F substituted thiamine was mixed with the standard sample (.sup.19F substituted thiamine), and then the mixture was loaded into analytical HPLC. A shows the radio-signal, with the retention time being 7 minutes and 59 seconds; and B shows the UV-signal, with the retention time being 7 minutes and 35 seconds. The radio-probe is located behind the UV probe, and the time delay is 24 seconds. C shows the UV signal when the .sup.18F substituted thiamine is loaded alone.

Examples 2

[0087] 1. Synthesis of Radioisotope .sup.18F Substituted Thiamine

[0088] 1.1. Synthesis of Precursors (the Same as Step 1.1 in Example 1)

[0089] 1.2. Radiochemical Synthesis Step:

[0090] Hot-labeled 18F substituted thiamine was synthesized through a two-step synthesis process using precursors I and II. The operation steps in radiochemical synthesis were all carried out in a closed hot cell (BBS2-V, COMECER Inc.), using RNplus synthesis module (RNplus, Synthra GmbH).

[0091] 1.2.1 (Step 1)

[0092] H.sup.+ protons were generated in a cyclotron (Cyclone18 Twin, IBA Inc.). .sup.18O-heavy oxygen water having an abundance of 98% or higher was bombarded with the accelerated protons, and .sup.18F.sup. ions (4 Ci) were generated through the nuclear reaction .sup.18O(p, n).sup.18F. The heavy oxygen water was passed through a QMA column (Sep-Pak Light QMA Chloride type, Waters) that was previously activated with a NaHCO.sub.3 solution (10 mL, 0.5M) and water (10 mL), and then .sup.18F.sup. was eluted into a reaction vial 1 using 0.6 mL of an eluant, where Kryptofix 2.2.2, K.sup.+ ions, and .sup.18F.sup. ions were complexed to form [K/Kryptofix].sup.+18F.sup.. Acetonitrile (1 mL) was added, and evaporated at 120 C. for 3 min. A nitrogen stream was introduced into the reaction vial 1 at 120 C. for evaporation for further 3 min. After cooling to room temperature, additional acetonitrile (1 mL) was added, and evaporated to dryness at 120 C. over 3 min under a stream of nitrogen. After cooling to room temperature, precursor I (5 mg, dissolved in 0.6 mL acetonitrile) was added, and reacted for 20 min at 110 C. while the reaction flask was closed, to obtain an intermediate product 5-(2-fluoroethyl)-4-methylthiazole having a structure of:

##STR00010##

After cooling to room temperature, the reaction solution was stirred with a rotor for 20 s, and then transferred to a sample injector. A rinsing liquid (0.5 mL, water:acetonitrile=9:1) was further added to the reaction vial 1, stirred with a rotor for 20 s, and transferred to the sample injector. The solution (0.9 mL) was injected into a preparative column by the sample injector and separated by preparative chromatography. Pure 5-(2-fluoroethyl)-4-methylthiazole product was collected into an extraction flask (a 50 mL slender glass flask). Conditions for preparative chromatography: Waters Xbridge C.sub.18 preparative column (19100 mm, 5 um), mobile phase: 15% acetonitrile/85% water; flow rate: 14 mL/min. The desired liquid phase peak was collected based on the signal from a gamma detector, where the retention time of 5-(2-fluoroethyl)-4-methylthiazole was 10.5 min, and the collection was completed in 1 minute and 30 seconds.

[0093] 1.2.2 (Step 2)

[0094] The preparative peak of 5-(2-fluoroethyl)-4-methylthiazole was collected in an extraction flask containing diethyl ether (20 mL) and sodium chloride (6.6 g), during which the extraction flask (sodium chloride was used to saturate water in mobile phase) was stirred with a rotor. After collection, the stirring was continued for additional 1 min, then the extraction flask was stood for 1 min, and the supernatant diethyl ether phase was transferred to an intermediate flask. The diethyl ether was transferred from the intermediate flask to a reaction vial 2 where the temperature was set to 55 C., and diethyl ether was evaporated to dryness over 12 min under a stream of nitrogen. After 12 min of evaporation, acetonitrile (1 mL) was added to the reaction vial 2 to dissolve the 5-(2-fluoroethyl)-4-methylthiazole, stirred for 10 sec and then passed through a magnesium sulfate column. Additional acetonitrile (1 mL) was added to rinse the reaction vial 2 and elute the 5-(2-fluoroethyl)-4-methylthiazole off from the magnesium sulfate column. The acetonitrile solution flowing through the magnesium sulfate column was transferred back to the reaction vial 2. Precursor II (5 mg, previously dissolved in 0.5 mL hot acetonitrile) was added, and reacted for 20 min at 110 C. while the reaction flask was closed, to obtain a 18F substituted thiamine product. At the same time, the sample injector was washed with methanol repeatedly. In the heating process during the reaction, a nitrogen stream was introduced to reduce the volume of the solvent acetonitrile. When the temperature reached 110 C., the nitrogen stream was shut off, and the reaction vial 2 was closed. After reaction, the system was maintained at 110 C. for 3 min, during which a nitrogen stream was introduced to evaporate the acetonitrile to dryness, and then the system was cooled to room temperature. After cooling to room temperature, water (0.5 mL) was added, stirred for 20 sec to dissolve .sup.18F substituted thiamine, and then transferred to the sample injector for separating and collecting a pure .sup.18F substituted thiamine product by preparative chromatography. Conditions for preparative chromatography: Waters Xbridge C18 preparative column (19100 mm, 5 um), mobile phase, 5% methanol/95% water (containing 50 mM ammonium acetate); flow rate, 14 mL/min. The desired liquid phase peak was collected based on the signal from a gamma detector, where the .sup.18F substituted thiamine retention time was 6.5 min, and the collection was completed in 30 sec.

[0095] The whole radiochemical synthesis step is completed in about 2 hours. The radiochemical output of .sup.18F substituted thiamine is >50 mCi, the radiochemical yield is >2% (with attenuation correction), the radiochemical purity is >95% and the specific activity is >1 Ci/mol.

[0096] 2. The purity of the product was detected by analytical HPLC (as described in Step 2 of Example 1).

Examples 3

[0097] 1. Synthesis of Radioisotope .sup.18F Substituted Thiamine.

[0098] 1.1. Synthesis of Precursors (the Same as Step 1.1 in Example 1)

[0099] 1.2. Radiochemical Synthesis Step:

[0100] Hot-labeled .sup.18F substituted thiamine was synthesized through a two-step synthesis process using Precursors I and II. The operation steps in radiochemical synthesis were all carried out in a closed hot cell (BBS2-V, COMECER Inc.), using RNplus synthesis module (RNplus, Synthra GmbH).

[0101] 1.2.1 (Step 1)

[0102] H.sup.+ protons were generated in a cyclotron (Cyclone18 Twin, IBA Inc.). .sup.18O-heavy oxygen water having an abundance of 98% or higher was bombarded with the accelerated protons, and .sup.18F.sup. ions (6 Ci) were generated through the nuclear reaction .sup.18O(p, n).sup.18F. The heavy oxygen water was passed through a QMA column (Sep-Pak Light QMA Carb, Waters), and then .sup.18F.sup. was eluted into a reaction vial 1 using 0.8 mL of an eluant, where Kryptofix 2.2.2, K.sup.+ ions, and .sup.18F.sup. ions were complexed to form [K/Kryptofix].sup.+18F.sup.. Acetonitrile (1 mL) was added, and evaporated at 120 C. for 3 min. A nitrogen stream was introduced into the reaction vial 1 at 120 C. for evaporation for further 3 min. After cooling to room temperature, additional acetonitrile (1 mL) was added, and evaporated to dryness at 120 C. over 3 min under a stream of nitrogen. After cooling to room temperature, precursor I (5 mg, dissolved in 1 mL acetonitrile) was added, and reacted for 20 min at 110 C. while the reaction flask was closed, to obtain an intermediate product 5-(2-fluoroethyl)-4-methylthiazole having a structure of:

##STR00011##

After cooling to room temperature, the reaction solution was stirred with a rotor for 20 s, and then transferred to a sample injector. A rinsing liquid (0.5 mL, water:acetonitrile=9:1) was further added to the reaction vial 1, stirred with a rotor for 20 s, and transferred to the sample injector. The solution (0.9 mL) was injected into a preparative column by the sample injector and separated by preparative chromatography. Pure 5-(2-fluoroethyl)-4-methylthiazole product was collected into an extraction flask (a 50 mL slender glass flask). Conditions for preparative chromatography: Waters Xbridge Cis preparative column (19100 mm, 5 um), mobile phase: 15% acetonitrile/85% water; flow rate: 14 mL/min. The desired liquid phase peak was collected based on the signal from a gamma detector, where the retention time of 5-(2-fluoroethyl)-4-methylthiazole was 10.5 min, and the collection was completed in 1 minute and 30 seconds.

[0103] 1.2.2 (Step 2)

[0104] The preparative peak of 5-(2-fluoroethyl)-4-methylthiazole was collected in an extraction flask containing diethyl ether (15 mL) and sodium chloride (6.6 g), during which the extraction flask (sodium chloride was used to saturate water in mobile phase) was stirred with a rotor. After collection, the stirring was continued for additional 1 min, then the extraction flask was stood for 1 min, and the supernatant diethyl ether phase was transferred to an intermediate flask. The diethyl ether was transferred from the intermediate flask to a reaction vial 2 where the temperature was set to 55 C., and diethyl ether was evaporated to dryness over 15 min under a stream of nitrogen. After 15 min of evaporation, acetonitrile (1 mL) was added to the reaction vial 2 to dissolve the 5-(2-fluoroethyl)-4-methylthiazole, stirred for 10 sec and then passed through a magnesium sulfate column. Additional acetonitrile (1 mL) was added to rinse the reaction vial 2 and elute the 5-(2-fluoroethyl)-4-methylthiazole off from the magnesium sulfate column. The acetonitrile solution flowing through the magnesium sulfate column was transferred back to the reaction vial 2. Precursor II (5 mg, previously dissolved in 0.8 mL hot acetonitrile) was added, and reacted for 30 min at 100 C. while the reaction flask was closed, to obtain a 18F substituted thiamine product. At the same time, the sample injector was washed with methanol repeatedly. In the heating process during the reaction, a nitrogen stream was introduced to reduce the volume of the solvent acetonitrile. When the temperature reached 100 C., the nitrogen stream was shut off, and the reaction vial 2 was closed. After reaction, the system was maintained at 110 C. for 3 min, during which a nitrogen stream was introduced to evaporate the acetonitrile to dryness, and then the system was cooled to room temperature. After cooling to room temperature, water (0.5 mL) was added, stirred for 20 sec to dissolve .sup.18F substituted thiamine, and then transferred to the sample injector for separating and collecting a pure .sup.18F substituted thiamine product by preparative chromatography. Conditions for preparative chromatography: Waters Xbridge C18 preparative column (19100 mm, 5 um), mobile phase: 5% methanol/95% water (containing 50 mM ammonium acetate); flow rate, 14 mL/min. The desired liquid phase peak was collected based on the signal from a gamma detector, where the .sup.18F substituted thiamine retention time was 6.5 min, and the collection was completed in 30 sec.

[0105] The whole radiochemical synthesis step is completed in about 2 hours. The radiochemical output of .sup.18F substituted thiamine is >50 mCi, the radiochemical yield is >2% (with attenuation correction), the radiochemical purity is >95% and the specific activity is >1 Ci/mol.

[0106] 2. The purity of the product was detected by analytical HPLC (as described in Step 2 of Example 1).

Examples 4

[0107] 1. Experiments on Scanning of ICR Mice by Using .sup.18F Substituted Thiamine as a PET/CT Tracer.

[0108] 1.1. Experimental Drug: .sup.18F Substituted Thiamine (Synthesized and Identified as in Example 1).

[0109] 1.2. Animals and treatments: 8-week-old ICR mice, SPF grade, purchased from Shanghai SLAC Laboratory Animal Co., Ltd, were housed in a barrier environment and assigned to a treatment group and a control group randomly. The animals in the treatment group were fed with thiamine deficient diet (n=3), and the animals in the control group were fed with normal diet (n=3). After 28 days, PET/CT scan of the mice was performed.

[0110] 1.3. PET/CT scan of the mice: small animal PET/CT scanner (Inveon, Siemens) thickness of PET layer: 0.78 mm, 128*128 matrix, energy window: 350-650 kev, CT tube current: 500 uA, tube voltage: 80 kv; scan mode: dynamic scan; scan time: 90 min.

[0111] 1.4. Processing of PET/CT image: After the acquisition, the images were reconstructed with two iterations of 3D OSEM. The whole brain was delimited as the region of interest (ROI) by the image processing software coming with the Inveon small animal PET/CT scanner. The Standard uptake value (SUV) and TAC within 90 min were calculated.

[0112] 1.5. Experimental Results

[0113] In the thiamine-deficient group, the peaks of .sup.18F substituted thiamine absorption in the brains of two mice occurred at 22.5 and 35 minutes, respectively, at which the SUV values were 0.98 and 0.97, respectively, followed by a slow decline. The peak of .sup.18F substituted thiamine absorption in the brain of the third mice occurred at 65 minutes, at which the SUV value was 0.61, followed by a slow decline (as shown in FIG. 6). In the control group, the absorption peaks in the brains of two mice occurred at about 20 min, at which the SUV values were 0.18 and 0.36, respectively. The SUV value in the third mice was stable within 20 min and was between 0.26 and 0.27, followed by a slow decline (as shown in FIG. 7). It can be seen from FIGS. 8 and 9 that the .sup.18F substituted thiamine uptake in the brains of thiamine-deficient group was obviously increased.

Examples 5

[0114] 1. Experiments on Scanning of C57BL/6 Mice by Using .sup.18F Substituted Thiamine as a PET/CT Tracer.

[0115] 1.1. Experimental Drug: .sup.18F Substituted Thiamine (Synthesized and Identified as in Example 1).

[0116] 1.2. Animals and treatments: 8-week-old C57BL/6 mice, SPF grade, purchased from Shanghai SLAC Laboratory Animal Co., Ltd, were housed in a barrier environment and assigned to a treatment group and a control group, randomly. The animals in the treatment group were fed with thiamine deficient diet (n=2), and the animals in the control group were fed with normal diet (n=3). After 28 days, PET/CT scan of the mice was performed.

[0117] 1.3. PET/CT scan of the mice: Small animal PET/CT scanner (Inveon, Siemens), thickness of PET layer: 0.78 mm, 128*128 matrix, energy window: 350-650 kev, CT tube current: 500 uA, tube voltage: 80 kv; scan mode: dynamic scan; scan time: 90 min.

[0118] 1.4. Processing of PET/CT image: After the acquisition, the images were reconstructed with two iterations of 3D OSEM. The whole brain was delimited as the region of interest (ROI) by the image processing software coming with the Inveon small animal PET/CT scanner. The SUV and TAC within 90 min were calculated.

[0119] 1.5. Experimental results: In the thiamine-deficient group, the accumulation of .sup.18F substituted thiamine in the brains of two C57BL/6 mice rose continuously over 90 min, and gradually reached a peak at 90 min, at which the SUV values are 0.48 and 0.55, respectively (as shown in FIG. 10). In the control group, the absorption peaks in the brains of three mice occurred from 35 to 55 min, at which the SUV values were 0.17, 0.25, and 0.3, respectively (as shown in FIG. 11). It can be seen from FIGS. 12 and 13 that the .sup.18F substituted thiamine uptake in the brains of thiamine-deficient C57BL/6 mice was obviously increased.