Patent Application
20080063599
A 1-3 mCi solution of Na125I is introduced in a 10 mL balloon and is evaporated to dryness at 100° C. under a nitrogen current. The corresponding 7-iodoquinoline is added (100 mg) dissolved in 2 mL of a suitable solvent. The balloon is joined to a reflux condenser and the bath temperature is raised. The reaction mixture is stirred under nitrogen atmosphere for a certain time and is let to cool. Water is added and the product that is collected by filtration is rinsed well with water. It is recrystallised and the purity is determined by means of thin layer radiochromatography.
Hexamethylditin (1.31 mmol) is added to an 0.88 mmol mixture of iodine derivative (5-chloro-8-hydroxy-7-iodoquinoline) and tetrakis-triphenylphosphine palladium(0) (0.05 g) in 12 mL of 1,4-dioxane and the mixture is heated under reflux under nitrogen atmosphere for 6.5 hours. After cooling, the crude reaction product is filtered and the insoluble material is washed with ethyl acetate. The solvent is eliminated under reduced pressure, obtaining a yellowish oil which is subject to silica gel column chromatography to produce a solid with a 65% yield.
Synthesis of 5-chloro-8-hydroxy-7-[123I]iodoquinoline from the Organotin Derivative Using Chloramine T
[123I]sodium iodide (2-20 mCi) is added to a solution of the trimethyltin derivative described in Example 2 (200-300 ug) in 300 uL of ethanol, followed by chloramine T (100 ug) in 1N HCl (100 uL) . After stirring for 5 minutes the reaction is stopped with an aqueous solution of metabisulphite (50 mg/mL, 100 uL) and is injected in the RP-HPLC. The radioiodinated derivative fraction obtained is collected with a 98% radiochemical purity.
The radioiodinated derivative is obtained from the tributyltin derivative prepared in an analogous manner to the organotin derivative described in example 2 by suspension in methanol and treatment with Na131I and hydrogen peroxide at room temperature, as described below. The reaction takes place by adding 50 uL of H2O2 (3% w/v) to a mixture formed by 50 uL of the tributyltin derivative (100 ug/50 uL EtOH), 1.5 mCi of [125I]sodium iodide (specific activity 2200 Ci/mmol) and 100 uL of 1N HCl in a sealed vial. The reaction is -stirred at room temperature for 10 minutes and is finalised after adding 100 uL of a saturated solution of anhydrous Na2SO3 and the solvent is evaporated by means of a nitrogen current. It is neutralized with sodium bicarbonate and extracted with ethyl acetate. The extract is dried by passing the solution over a column of anhydrous Na2SO4 and the solvent is evaporated by means of nitrogen current. The crude reaction product is purified by HPLC obtaining a radiochemical purity over 85%. The fractions are dried and the residue is extracted again with EtOAc. The different EtOAc extracts are concentrated under nitrogen atmosphere until dry. The residue is dissolved in EtOH. The radiochemical purity of the final product exceeds 98% (by HPLC).
1.0 mL of an 0.02 M KH2PO4 buffer solution (pH 4.8) is added to a vial containing 5-chloro-8-hydroxyquinoline. The mixture is heated to 40° C. and is stirred to obtain a transparent solution which is cooled to room temperature. 10 mL of an 0.1 N NaOH solution containing 10.0 mCi of [131I]sodium iodide is added and the vial is closed with a Teflon stopper. A 7.5 ug chloramine T (N-chloro-p-toluenesulphonamide sodium salt) solution in 30 uL of the 0.02 M KH2PO4 buffer solution is added. It is mechanically stirred at room temperature for 5 minutes and then manually for some seconds. After continuing stirring for 5 more minutes an aqueous solution of NaHSO3 (1.4 mg/mL) is added and the reaction mixture is analysed by thin layer chromatography. The solution is passed over an anionic exchange column to eliminate free radioiodine.
A 30 nmol solution of 5-chloro-8-hydroxyquinoline in 30 uL of ethanol is added to 10 mCi of [125I]sodium iodide (4.5 ug, 330 Ci/mmol, 30 nmol) in 0.001 N NaOH (30 uL) . A solution of chloramine T (13 ug, 50 nmol) in water (10 uL) is added. The mixture is heated to 60° C. during 45 minutes. 100 uL of 0.1 N NH4Cl are added and it is extracted with ether. The reaction result is analysed by thin layer chromatography (Merck F254 Silica gel) in the appropriate eluent and then treated as in the previous example.
100 uL of a Iodogen solution (1,3,4,6-tetrachloro-3-alpha, 6-alpha-diphenylglycoluril) (1.0 mg/mL in CH2Cl2) are added to a 5 mL vial. Dichloromethane evaporates from the vial due to nitrogen current. A 5-chloro-8-hydroxyquinoline solution (0.5 mg/0.5 mL) in 0.02M KH2PO4, pH 4.8 is added to the vial, which is then closed with a Teflon stopper and a solution containing approximately 10 mCi of [131I]sodium iodide is introduced by means of a syringe. The mixture is stirred at room temperature for 30 minutes. A radiochemical purity exceeding 99% is observed by thin layer chromatography in silica gel using suitable eluents.
The Iodo-beads are previously washed with a 0.1 M sodium phosphate buffer solution at pH=6.5. The necessary amount of Iodo-beads is added to an Na125I solution in buffer solution at a concentration of 1 mCi for each 100 ug of product to be iodinated. Between 5 and 500 ug of the phenol derivative dissolved in the same buffer solution are then added thereto. It is left to react between 2 and 15 minutes at room temperature. The reaction is stopped by separating the Iodo-beads from the solution by decantation. The Iodo-beads are washed with the buffer solution in order to recover all the radioiodinated derivative.
0.19 umol of 5-chloro-8-hydroxyquinoline precursor dissolved in 50 uL of ethanol are mixed in a vial with a known volume of a buffered solution at pH 2 which is twice the volume of the basic commercial [123I]iodine solution. This solution is introduced in the vial containing the radioactive iodine, followed by 50 uL of a 3.2% solution of peracetic acid (obtained from a stock solution at 32% w/v) . The sealed vial is heated to 65° C. for 12 minutes. It is left to cool and an aqueous solution of NaHSO3 is added. The radiochemical yield is purified and determined by chromatographic methods such as those described in previous examples.
Direct radiofluorination of 100 umol of the phenol derivative 5-chloro-8-hydroxyquinoline is performed by dissolving the compound in a mixture of trifluoroacetic acid and glacial acetic acid (1:1) through which freshly prepared acetyl hypofluorite ([18F]CH3—COOF) is bubbled. The solvent is evaporated and the residue is purified by means of HPLC.
The radioactive fluoride [18F] is transferred to a 5 mL borosilicate reaction vial and is azeotropically dried with acetonitrile in the presence of 4.0 mg of K2CO3 and 14.6 mg of Kryptofix 2.2.2®. The precursor fluoride derivative dissolved in 0.5 mL of DMSO is added to the vial containing the dry radioactive fluoride, the K2CO3 and the Kryptofix 2.2.2® and the isotope exchange reaction is performed heating to 110, 130 and 160° C. for 0-30 minutes to optimise the reaction. Purification of the radiofluorinated derivative is performed by reverse phase HPLC. The corresponding fractions are collected and concentrated under reduced pressure.
The starting material dissolved in 600 uL of ethanol is added to 100 uL of a solution A (containing 200 umol of SnSO4, 2 mmol of gentisic acid, 2 mmol of monohydrate citric acid, 10 ul of glacial acetic acid dissolved in 10% acetic acid), 25 uL of a solution B (containing 400 umol of CuSO4.5H2O dissolved in 10 mL of water) and 65 uL of glacial acetic acid. The vial is placed in an ultrasound bath for 15 minutes, and 10 uL of Na123I (about 1 mCi) are added. N2 is passed through the mixture and it is heated to 60° C. for 1 hour. It is left to cool at room temperature and the crude reaction product is purified by means of HPLC.
The corresponding halo- or nitro- derivative is made to react with the fluorine-18 ion by aromatic nucleophilic substitution using the [18F]FK-K222 complex in DMSO and
heating to 150-180° C. in the conventional manner for 10 minutes or activating the reaction mixture with a 100 watt microwave during 1-2.5 minutes. The characterisation and isolation of the radiolabelled product is performed as in Example 11.
The electrophilic radiofluorination agent [18F]CH3COOF, prepared from 100 umol [18F]F2, or [18F]OF2 (100 umol), is made to bubble for 10 minutes at room temperature in a solution (101 umol) of the trimethyltin derivative 5-chloro-8-hydroxy-7-trimethylstannyl-quinoline or 5-chloro-8-hydroxy-7-tributylstannyl-quinoline in 10 mL of Freon-11 (CFCl3). The solvent is evaporated at 50° C. with a nitrogen current and the residue is dissolved in 10 mL of methylene chloride and is transferred to a chromatographic column packed with Na2S2O3 (2.5 cm) and silica gel (9.5 cm) which has previously been equilibrated with ether. It is eluted with ether. The solvent is evaporated and the solution is purified by means of semi-preparative HPLC.
The electrophilic radiofluorination agent [18F]CH3COOF, prepared from 100 umol [18F]F2, or [18F]OF2 (100 umol), is made to bubble for 10 minutes at room temperature in a solution (101 umol) of the trimethyltin derivative 5-chloro-8-t-butoxycarbonyloxy-7-trimethylstannyl-quinoline or 5-chloro-8-t-butoxycarbonyloxy-7-tributylstannyl-quinoline in 10 mL of Freon-11 (CFCl3). The solvent is evaporated at 50° C. with a nitrogen current and the residue is dissolved in 10 mL of methylene chloride and is transferred to a chromatographic column packed with Na2S2O3 (2.5 cm) and silica gel (9.5 cm) which has previously been equilibrated with ether. It is eluted with ether. The solvent is evaporated and the derivative is hydrolysed in the presence of a 48% hydrobromic acid solution at 130° C. for 10 min. After cooling, the reaction mixture is partially neutralised with a 3 N NaOH solution and purified by means of HPLC.
260 mg (1.5 equiv.) of HNMe2.HCl and 430 mg (1.5 equiv.) of K2CO3 are added to a 2.1 mmol solution of the precursor fluorine derivative in a mixture of 30 mL of DMSO and 10 mL of water at 10° C. After stirring at 10° C. for 10 minutes, the solution is heated under reflux for 24 hours and left to cool, diluted with water (50 mL) and the product is extracted with ether. After processing the reaction, the residue is purified by silica gel chromatography. In a second reaction step the following process is followed: 1.45 equiv. of methyl trifluoromethanesulphonate are added to a solution of 0.65 mmol of dimethyl amino derivative in 2 mL of toluene. The solution is stirred for 1 hour at room temperature under nitrogen atmosphere and is then diluted with 100 mL of water. The product is extracted with dichloromethane, the solvent is evaporated and the remaining product is ground with diethyl ether. In a final stage, about 2.0-3.5 mg (6.5-11.3 umol) of the methyl trifluoromethanesulphonate derivative are dissolved in 600 uL of freshly distilled DMSO and added directly to the tube containing the K[18F]F-K222 anhydrous complex. The tube is heated on a heating block at 145-150° C. without stirring for 2 minutes or is placed in a 100 w microwave oven for 1 minute. The reaction mixture is left to cool and is diluted with 3 mL of water and filtered through a C18 Sep Pack cartridge. The cartridge is washed with 3.0 mL of water and partially dried for 0.5 minutes applying nitrogen current. The radiofluorinated derivative is eluted from the cartridge with DCM (3 mL) followed by two more washings each of 1 mL.
A mixture of 5-chloro-8-hydroxy-7-iodoquinoline (50 mg, 0.164 mmol), methyl 1-bromo-1-deoxy-2,3,4-tri-O-acetyl-D-glucopyranoside uronate (65 mg, 0.164 mmol), CaSO4.H2O (35 mg) in pyridine (1.5 mL) is stirred at room temperature for a few minutes. Ag2CO3 (35 mg) is added to the reaction and the suspension is stirred at room temperature for 20 hours, protected from light. Once the reaction has finished and after isolating the product, the deprotection of the hydroxyl protecting groups is performed using a 1 N NaOH solution. The reaction is diluted with dichloromethane, filtered and the solvent is removed at reduced pressure. The glycoconjugate is purified by means of flash column chromatography. (TLC: CH2Cl2/MeOH 99/1, eluent: CH2Cl2/MeOH 99.5/0.5). NMR (400 MHz, CDCl3) 2.04 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.13 (s, 3H, Ac), 3.68 (s, 3H, Me), 3.99 (d, 1H, 5′H), 5.40-5.52 (m, 3H, 2′, −3′, −4′-H), 6.29 (d, 1H, 1′-H), 7.56 (m, 1H, 3H), 7.99 (s, 1H, 6-H), 8.52 (d, 1H, 4-H), 8.93 (s, 1H, 2-H).
10 mg of Iodogen™ are dissolved in 2 mL of dichloromethane in a tube containing small crystals in order to increase contact of the solution with the Iodogen™ (1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluril). The solvent is evaporated and a white film is observed inside the tube and on the small crystals. 1 mL of an aqueous solution of the corresponding glucuronide (4 mg/mL) is added and then 7.4×107 Bq (2 mCi) of Na131I is added. The solution is kept at room temperature while stirring for 10 minutes. The reaction is followed by TLC and the Rf of the radiochromatography-labelled product is determined. It can be observed that a single radioiodinated product has been obtained with a radioactive yield between 90 and 95%.
A 4 mg/mL solution of chloramine T in 50 mM of disodium phosphate at pH 7 is prepared. 25 uL of a standard solution of the glycoconjugate in 50 uM of sodium phosphate at pH 7 is added to 2 uL of a 0.5 mCi/uL Na125I solution. The vial is closed and 25 uL of the chloramine T solution are added, this mixture is stirred for 30 seconds. Once the reaction has finished 100 uL of 12.6 mM sodium metabisulphite are added. It is stirred for 10 seconds. The reaction product is purified by filtration on gel using a Sephadex G-25 or G-50 column.
Radioiodination of the Clioguinol Glucuronide with Iodo-beads: Synthesis of the 5-chloro-8-hydroxy-7-[125I]iodoquinoline Glucuronide
25 uL of a solution of the glycoconjugate in 50 uM of sodium phosphate at pH 7 are added to 2 uL of a 0.5 mCi/uL Na125I solution. The vial is closed and some Iodo-beads are added and it is stirred for 30 seconds. Once the reaction is finished the polymer is separated from the supernatant. 100 uL of 12.6 mM sodium metabisulphite are added to this solution. It is stirred for 10 seconds. The reaction product is purified by filtration on gel using a Sephadex G-25 or G-50 column.
The trihydrate [111In]indium chloride (111InCl3.3H2O, 1.37 g, 5.0 mmol) and the 5,7-dichloro-8-hydroxy-quinoline (2.14 g, 10 mmol) are dissolved in ethanol (200 mL) and the solution is concentrated to dryness at reduced pressure. The yellowish crude product obtained is recrystallised from ethanol after adding water to give the 5,7-dichloro-8-hydroxyquinoline [111In]indium complex.
The [67Ga]gallium chelator is prepared by adding [67Ga]gallium trichloride (67GaCl3) dissolved in an 0.05 M HCl solution to a 7 mM aqueous solution of 8-hydroxyquinoline at pH 3.5. After about 25 minutes of stirring the pH increases to 6. The extraction of the product with chloroform leads to the chelator, which is obtained with a 90% yield.
The 8-hydroxyquinoline (0.51 mmol) is dissolved in 3 mL of a 0.1 N NaOH solution and the solution pH is adjusted to pH=3.5 with 1 N HCl. 0.3 mL of an SnCl2 solution (20 mg, 0.11 mmol in 10 mL of 1 N HCl) are added and the pH is again adjusted with 0.1 N NaOH. It is stirred for 5 minutes and 80 uCi of technetium-99m are added in the form of sodium pertechnetate. The chelator is obtained by extracting with chloroform with a yield exceeding 90%.
A [64Cu] copper (II) chloride (64CuCl2) solution is diluted 10 times with a 0.1 M ammonium acetate buffer solution of pH 5.5. The [64Cu] copper acetate is added to 8-hydroxyquinoline and the final volume is adjusted to 1.0-1.5 mL with the buffer solution. After incubating at room temperature for 45 minutes, the extraction of the 64Cu derivative product with chloroform leads to the chelator, which is obtained with a 90% yield.
An aliquot of the starting solution (67Cu2+/HCl) is evaporated to dryness under nitrogen current and reconstituted with an 0.25 N acetate buffered solution (pH =5.5). The ligand (0.2 mg) is dissolved in DMSO (1 mg/10 uL) and is added to 50 uL of the [67Cu] copper acetate solution (50 uL) . After adding 50 uL of ethanol, the [67Cu] copper derivative is filtered through a polytetrafluoroethylene (PFTE) membrane. The radiochemical purity is determined by thin layer chromatography in silica gel plaques eluting with ethanol.
Method A: Chelation of 5-chloro-8-hydroxy-7-[125I]iodoquinoline
A copper (II) chloride solution is diluted 10 times with 0.1 M ammonium acetate of pH 5.5. This solution is added to 5-chloro-8-hydroxy-7-[125I]iodoquinoline and the final volume is adjusted to 1.0-1.5 mL with a 0.1 M ammonium acetate buffer solution at pH =5.5. After incubating at room temperature for 45 minutes, the extraction of the solution with chloroform leads to the chelator, which is obtained with a 90% yield.
Method B: Radioiodination of the 5-chloro-8-hydroxyquinoline Copper Chelator
A 30 nmol solution of 5-chloro-8-hydroxyquinoline copper chelator in 30 uL of ethanol is added to 10 mCi of [125I]sodium iodide (4.5 ug, 330 Ci/mmol, 30 nmol) in 0.001 N NaOH (30 uL). A chloramine T solution (13 ug, 50 nmol) in water (10 uL) is added. The mixture is heated to 60° C. for 45 minutes. 100 uL of 0.1 N NH4Cl is added and extracted with ether. The product is analysed by thin layer chromatography (Silica gel, Merck F254) in the appropriate eluent.
Method A: Chelation of 5-chloro-B-hydroxy-7-[125I]iodoquinoline.
Zn(OAc)2.2H2O (60 mmol) is added to a suspension of 5-chloro-8-hydroxy-7-[125I]iodoquinoline derivative (60 mmol) in methanol and the mixture is stirred for 19 hours at room temperature. The product is filtered and washed with methanol followed by petroleum ether and it is left to dry in the dryer.
Method B: Radioiodination of the 5-chloro-8-hydroxyquinoline Zinc Chelator
A solution of 5-chloro-8-hydroxyquinoline zinc chelator in 30 uL of ethanol is added to 10 mCi of [125I]sodium iodide (4.5 ug, 330 Ci/mmol, 30 nmol) in 0.001 N NaOH (30 uL). A solution of chloramine T (13 ug, 50 nmol) in water (10 uL) is added. The mixture is heated to 60° C. for 45 minutes. 100 uL of 0.1 N NH4Cl are added and extracted with ether. The reaction product is analysed by thin layer chromatography (Silica gel, Merck F254) in the appropriate eluent.
A solution of 5-chloro-8-hydroxyquinoline manganese (II) chelator in 30 uL of ethanol is added to 10 mCi of [125I]sodium iodide (4.5 ug, 330 Ci/mmol, 30 nmol) in 0.001 N NaOH (30 uL). A solution of chloramine T (13 ug, 50 nmol) in water (10 uL) is then added. The mixture is heated to 60° C. for 45 minutes. After adding 100 uL of 0.1 N NH4Cl it is extracted with ether. The obtained product is analysed by thin layer chromatography (Silica gel, Merck F254) in the appropriate eluent.
A solution of 5-chloro-8-hydroxyquinoline iron (II) chelator in 30 uL of ethanol is added to 10 mCi of [125I]sodium iodide (4.5 ug, 330 Ci/mmol, 30 nmol) in 0.001 N NaOH (30 uL). After adding a solution of chloramine T (13 ug, 50 nmol). in water (10 uL), the mixture is heated to 60° C. for 45 minutes. The reaction is processed by adding 100 uL of 0.1 N NH4Cl and extracting with ether. The reaction monitoring is performed by thin layer chromatography (Silica gel, Merck F254) in the appropriate eluent.
The 5-chloro-8-hydroxy-7-[123I]iodoquinoline radioactive derivative is injected in transgenic mice (Tg2576) and control mice via the jugular vein under anaesthesia using a 30 g syringe. A series of controls and transgenic mice are examined. After 2, 4, 6 hours of the intravenous injection, the brains-of each mouse are removed, they are quickly cooled with dry ice and dissected using a Mokron HM 505E cryostat apparatus, placing them on glass sheets and drying them at room temperature. For the film autoradiography the sheets are placed in a MICROM optical densitometer obtaining greyscale images of the distribution of the radiopharmaceutical in the histological sections. Measured optical density is expressed as a percentage (%) of the maximum. Images were obtained with an optimal detection of amyloid plaques of the mice brains.
All the animal experiments were performed according to the established animal protocol. The transgenic mice (Tg2576) were anaesthetised via intravenous injection with a 40 uL solution of ketamine and xylazine (4:1) before the injection of the tracer radiolabelled with 18F: 5-[18F]-fluoro-8-hydroxy-7-iodoquinoline. The mice were placed in supine position and scanned using microPET. A dynamic microPET study was performed using 15 planes and 15 to 16 frames (dynamic sequence: 6×30 s, 4×1 min, 2×5 min, 2×10 min, and 1-2×20 min) for a total acquisition time of 55-77 min. Images were reconstructed using the appropriate algorithms (Filter-back projection algorithm) and a cut-off frequency of 0.5. Images were reconstructed by means of a filtered iteration resulting in an image resolution of 1.8 mm and a volumetric resolution of about 6 mm3. Transverse planes were reoriented to obtain the coronal and axial sections of the mice brains. Regional uptake is expressed in activity per gram of tissue. The images showed an adequate detection of the amyloid plaques in mice brains.
The study was performed using transgenic mice (Tg2576) in groups of 5 animals for each time value. The tracers (with SPECT-detectable radionuclides) (1-5 uCi) dissolved in 100 uL of PBS or in the appropriate solvent were injected via the tail vein.
SPECT images were obtained after 4.5 hours from the administration of the 5-chloro-8-hydroxy-7-[123I]iodoquinoline tracer by means of a double-head camera (MULTISPECT II, Siemens Medical Systems) equipped with parallel low energy high resolution collimators, using an energy window centred on the photopeak energy of the corresponding radionuclide (159 KeV for 123I).
57Co sources were used which were placed on the head of the animal in order to aid reconstruction by means of anatomical references. 120 projections were obtained in a 360 degree turn, acquired in a 64×64 matrix. Two simultaneous acquisitions are performed, one using a window of 20% of the energy centred at 159 KeV for 123I and another of 4% of the energy centred at 122 KeV for the 57Co labels. Total exploration time was about 40 minutes. The SPECT images were reconstructed using a filter-back projection algorithm. The attenuation correction was performed using the Chang algorithm.
The semiquantitative analysis of the uptake was performed comparing the average/pixel counts in the region of interest with the average/pixel counts in control regions. The results were compared with those obtained in the macroautoradiographies. The images showed the distribution of the amyloid plaques in the mice brains.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US02/00537 | 1/14/2002 | WO | 00 | 5/31/2005 |
