Synthesis, Radioiodination and Biological distribution of 5-(5-(tributylstannyl) benzofuran-2-yl) pyridin-2-amine as an amyloid imaging agent

Pyridine derivatives of different heterocyclic nuclei have potent pharmacological properties including antifungal [1], antitubercular [2], antibacterial [3], antimicrobial [4-7], and insecticidal [8]. Heterocyclic Mannich bases are well known and important nitrogen compounds. Several methods have been applied for their synthesis. Most of the derivatives of heterocyclic Mannich bases have been found to possess considerable biological activities [9]. Alzheimer ’s disease (AD) is a leading cause of dementia with symptoms that include cognitive decline, irreversible memory loss, disorientation, language impairment, and an inability to carry out normal daily functions. Currently, it is dif icult for clinicians to differentiate between the cognitive decline associated with normal aging and that associated with AD [10]. A de initive diagnosis of AD can only be established by postmortem histopathological examination [11]. Postmortem brains of AD patients reveal neuropathological features including Senile Plaques (SPs) and Neuro-Fibrillary Tangles (NFTs), which contain β-Amyloid (Aβ) peptides and highly phosphorylated tau proteins, respectively [12]. Although several radioiodinated or 99mTc-labeled ligands for Aβ plaques have been reported, unfavorable pharmacokinetics including low uptake into the brain and slow washout have prevented clinical studies. [123I]-6-iodo-2-(4′-dimethylamino)phenyl-imidazo[1,2-a]pyridine (IMPY) is the irst Single Photon Emission Tomography (SPECT )imaging agent to be tested in humans [13]. Although [123I]-IMPY showed excellent properties as an Aβ imaging probe in preclinical studies, the preliminary clinical data displayed a low signal-to-noise ratio, making it dif icult to distinguish AD patients, possibly because of its high lipophilicity and low Abstract


Introduction
Pyridine derivatives of different heterocyclic nuclei have potent pharmacological properties including antifungal [1], antitubercular [2], antibacterial [3], antimicrobial [4][5][6][7], and insecticidal [8]. Heterocyclic Mannich bases are well known and important nitrogen compounds. Several methods have been applied for their synthesis. Most of the derivatives of heterocyclic Mannich bases have been found to possess considerable biological activities [9]. Alzheimer 's disease (AD) is a leading cause of dementia with symptoms that include cognitive decline, irreversible memory loss, disorientation, language impairment, and an inability to carry out normal daily functions. Currently, it is dif icult for clinicians to differentiate between the cognitive decline associated with normal aging and that associated with AD [10]. A de initive diagnosis of AD can only be established by postmortem histopathological examination [11]. Postmortem brains of AD patients reveal neuropathological features including Senile Plaques (SPs) and Neuro-Fibrillary Tangles (NFTs), which contain β-Amyloid (Aβ) peptides and highly phosphorylated tau proteins, respectively [12]. Although several radioiodinated or 99m Tc-labeled ligands for Aβ plaques have been reported, unfavorable pharmacokinetics including low uptake into the brain and slow washout have prevented clinical studies.
[ 123 I]-6-iodo-2-(4′-dimethylamino)phenyl-imidazo[1,2-a]pyridine (IMPY) is the irst Single Photon Emission Tomography (SPECT )imaging agent to be tested in humans [13]. Although [ 123 I]-IMPY showed excellent properties as an Aβ imaging probe in preclinical studies, the preliminary clinical data displayed a low signal-to-noise ratio, making it dif icult to distinguish AD patients, possibly because of its high lipophilicity and low

Abstract
In this work an effi cient method developed for the synthesis of 125 I-benzofuran-2-yl) pyridin-2-amine ( 125 IBPA), followed by radioiodination with 125 I by using Chloramine-T at pH 8. The reaction proceeds within 10 min at room temperature (20-25°C). The radiochemical yield determined by Thin-Layer Chromatography (TLC) using hexane:ethyl acetate (1:6 v/v) and the purity analyzed by high-performance liquid chromatography using a reversed-phase RP18column and acetonitrile:0.1 M ammonium bicarbonate (pH 7.5) (1:1) as the mobile phase at a fl ow rate of 1 ml×min −1 . The radiochemical yield using aH 2 O 2 oxidant found equal to 96.5% with a radiochemical purity of 125 I-BPA of over 96.5%. The biodistribution data in normal mice indicated a high initial uptake of 6.54±0.10 (% ID/g±SD) in the brain within 30 min post-injection. These results promote a further the use of 125 I-BPA as a novel agent for brain imaging. stability in vivo. Therefore, there is no SPECT imaging probe useful for the clinical diagnosis of AD [14]. In this work, a simple method for syntheses and radioiodination of 125 I-BPA was studied. A preliminary in vivo study of 125 I-BPA in normal mice was completed to elucidate the biological behavior of this labeled compound.

Experimental materials
The following instruments and materials were used. A gamma counter (Nucleous Model 2010, Oak Ridge, USA), connected with a well type NaI(Tl) crystal. The High-Performance Liquid Chromatography (HPLC) machine [Sykam Model] was equipped with a S-2100 solvent delivery system, a S-5111 injector valve bracket, a UV/VIS spectrophotometric detector S-3240 and a C-18 Lichrosorb analytical column (250 mm×4-6 mm, 5 mm inner diameter). All melting points were determined on an electrothermal melting point apparatus and are uncorrected. IR spectra were recorded (KBr discs) on a Shimadzu FT-IR-8201 PC spectrophotometer (Kyoto, Japan). 1 H-NMR and 13 C-NMR spectra were recorded in CDCl 3 and (CD 3 ) 2 SO solutions on a Varian Gemini 300 MHz (Varian Inc., Palo Alto, CA) and JNM-LA 400 FT-NMR system spectrometer (Japan Electronic Optics Laboratory Co. Ltd., Tokyo, Japan) and chemical shifts are expressed in δ units using TMS as an internal reference. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer (70 eV, Shimadzu, Kyoto, Japan). Elemental analyses were carried out at the Micro Analytical Center of the University of Cairo, Giza, Egypt. All other chemicals were purchased from Merck Co. Radioactive iodine-125 was purchased from Institute of Isotopes Co., Ltd. (Budapest, Hungary). Solution pH was adjusted to pH 7 before use. The ethanol used was 99 % ethanol. To prepare the AgCl-impregnated ilter, Whatman No. 3 ilter paper (0=5 mm and t=0.33 mm and basis weight 185 gm/m) was immersed into a 0.5 N aqueous solution of AgNO 3 . After drying in the dark, the ilter was immersed into 1 N HCl and then repeatedly washed with distilled water up to neutral reaction and kept in the darkness up to use.

Chromatographic analysis
The radiochemical yield and purity of the labeled product, 125 I-BPA, were determined by TLC and HPLC as follows. Silica gel G60 on alumina sheets was used for radiochemical yield determination. A 5-μL portion of the reaction mixture was placed at 2 cm from the base and developed using hexane: ethyl acetate (1:6 v/v) as the developing solvent. The strips were dried under N 2 gas, cut into 1-cm segments, and assayed for radioactivity. The radio-iodinated product, 125 I-BPA, has an Rf=0.7, whereas 125 I has an Rf=0.3. The radiochemical yield was calculated in a similar fashion. Typically, the radiochemical purity of the product was more than 96.5 % by TLC. A 10-μL portion of the reaction mixture was injected onto the HPLC column (RP18-Lichrosorb) and eluted with acetonitrile 0.1 M ammonium bicarbonate (pH 7.5) (1:1). The retention time (RT) of 125 I-BPA was 10 min, whereas the RT of 125 I was 2 min.

Animals
Swiss Albino mice weighing 30-35 gm, purchased from the Agriculture Research Center, Cairo, Egypt. The animals were exposed to constant environmental and nutritional conditions throughout the experimental period and were maintained at room temperature (22±2°C) with a 12 h on/off light schedule. Standard access to food and water was provided.

Bio-distribution in normal mice
Mice were injected directly into a tail vein with 100 μl of 10 % EtOH containing 125 I-BPA (37 kBq). The mice (male, n=5 for each time point) were sacri iced at 2, 10, 30, and 60 min post injection. The organs of interest were removed and weighed, and the radioactivity was measured with a gamma counter. The percentage dose per organ was calculated by comparing the tissue counts to suitably diluted aliquots of the injected material. The % dose/g of samples was calculated by comparing the sample counts with the count of the diluted initial dose.

Effect of oxidizing agent
The effect of the amount of the oxidizing agent CAT on the labeling yield of 125 I-BPA is demonstrated in igure 2. CAT is a mild oxidizing agent that can oxidize Iinto I + without forming I 2 . At low amounts of CAT (10 μg), the radiochemical yield of 125 I-BPA was low at 68.9%. A high radiochemical yield of 95.5% was achieved by increasing the amount of CAT to 250 μg. Increasing the CAT amount above 250 μg led to a decrease in the iodination yield where at 250 μg of CAT the decrease in yield may be attributed to the formation of undesirable oxidative byproducts like chlorination. Consequently, 250 μg of CAT is highly recommended to avoid the formation of any byproducts and obtain a high labeling yield igure 2.

Effect of substrate amounts
The dependence of radiochemical yield on the amount of 125 I-BPA is shown in igure 3. The reaction was performed at different BPA amounts (10-200 μg). The radiochemical yield of 125 I-BPA was only 80 % at 10 μg BPA. As the BPA concentration increased, the labeling yield increased to 96.5 % when 100 μg of BPA was used. This improvement in yield may be attributed to the fact that 100 μg of BPA is suf icient to capture the generated iodonium ion, and as a result, the maximum yield was achieved.

Effect of pH on the reaction
The effect of the reaction mixture pH on the labeling of BPA was studied by varying the pH from 2 to 10 as shown in igure 4. The experiment was performed using 100 μg of BPA. The CAT amount used was 250 μg, while the other factors were kept constant. The experiment was repeated using 200 μl of each buffer at different pH values. The labeling yield at each pH was measured and the 96.5% was obtained at pH 8. Increasing the pH of the medium above 8 led to a slight decrease in the iodination yield 95.5 % at pH 9 ( Figure 4).

Effect of reaction time
The labeling yield is strongly dependent on reaction time in the range from 2 to 30 min. It is evident from igure 5 that the yield was signi icantly increased as the reaction time increased. The reaction was very fast and after 10 min, the maximum radiochemical yield 96.5 % was obtained.

In vitro stability of 125 I BPA
The in vitro stability of 125 I-BPA was studied to determine the most suitable time for injection to avoid the formation of the undesired products that resulted from the radiolysis of the labeled compound. In vivo, these undesired radioactive products might be accumulated in non-target organs. As shown in Figure 6, no signi icant changes in the relative content of 125 I-BPA were observed for up to 24 h post labeling, indicating that the labeled compound is very stable ( Figure 6).
The biodistribution pattern of 125 I-BPA is shown in table 1. 125 I-BPA was injected in normal mice intravenously and was distributed throughout the body organs and luids. All radioactivity levels are expressed as the average percentage of injected dose per gram (% ID/g±SD). The biodistribution data showed substantial uptake of the imaging agent at 6.54±0.10 (% ID/g±SD) in the brain at 30 min post-injection. After this time, the point radioactivity dropped to 0.09±0.01 at 120 min post-injection. The high amounts of radioactivity in the intestine indicated that excretion of the 125 I-BPA  derivative occurred mainly through the hepatobiliary pathway. The low radioactivity located in the thyroid gland indicates that the iodocompounds are stable against in vivo deiodination. The high accumulation of 125 I-BPA in the lungs is likely because the lungs function as a reservoir for antidepressants with high af inity to the serotonin transporter. The biodistribution data showed substantial lung uptake of 4.18±0.54 (% ID/g± SD) at 15 min post injection. The maximum brain uptake of 125 I-BPA is analogous to that of the currently used radiopharmaceuticals for brain imaging, 99m Tc-ECD and 99m Tc-HMPAO, which have maximum brain uptake of 4.7 and 2.25 %, respectively. These preliminary results strongly suggest that BPA labeled with 125 I could be used as a novel agent for brain imaging.

Conclusion
BPA was successfully synthesized and radiolabeled with 125 I using CAT. 125 I-BPA showed good in vitro stability. In biodistribution experiments using normal mice, 125 I-BPA displayed good uptake into the brain followed by fast clearance. Radioactivity was also distributed to several other organs, such as the liver, kidneys, and intestines. In addition to accumulating in the brain, the lungs and stomach showed an initial high uptake resulting in moderate accumulation over time, while the liver and kidneys accumulated radioactivity over the irst 15 minutes followed by a rapid decrease. The radioactive probe accumulated in the intestines and did not wash out over the period monitored in this experiment.