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研究生(外文):Ming Li Lee
論文名稱(外文):Synthesis and Feasibility Study of Iodo-GTS-21 as a Model to Develop New Tracer for Imaging Central Nicotinic Receptors
指導教授(外文):S. P. Wey
外文關鍵詞:GTS-21alpha7 nAChRAlzheimer's disease
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首先我們先合成出含天然碘的GTS-21 (碘化GTS-21),因此在本研究中利用競爭性結合分析 (competition binding assay) 與若干已知化合物作為平行試驗,以便探討碘化GTS-21對尼古丁a7亞型受體的結合性。
由競爭性結合分析結果,顯示新的化合物 (碘化GTS-21) 並未提高原本GTS-21對尼古丁a7亞型受體的親和力 (對a7亞型受體的抑制常數 [Ki] >1000 nM)。雖然實驗結果不符合本研究的目的, 然而我們卻意外的發現:雖然碘化GTS-21並無加強GTS-21對尼古丁a7亞型受體的親和力,卻比GTS-21更具有受體選擇性 (subtype selectivity);碘化GTS-21對於 a7與a4β2兩亞型受體的選擇性大過於GTS-21與上述兩受體的選擇性。
GTS-21 has been demonstrated to show moderate binding affinity to 7 subtype of nicotinic acetylcholine receptor (nAChR). Change of the 7 concentration has been proven to be correlated with several neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease and schizophrenia. Previous studies showed the number of 7 nAChR declined significantly in patients suffering from Alzheimer’s disease. Although GTS-21 has capability to bind to 7 nAChR, however, it binds to 4β2 subtype as well. Several studies have attempted to improve the binding affinity and subtype selectivity of GTS-21 by modifying its functional group. The purpose of this study was attempting to enhance the binding affinity of a novel compound, which was based on GTS-21, to 7 nAChR.
First of all, chemical synthesis was carried out for the novel compound, iodo-GTS-21. In vitro competition binding assay experiments were performed to estimate the binding affinity of iodo-GTS-21 using rat brain homogenates. Meanwhile, there were several known cold ligands to serve as parallel comparison and methodology confirmation.
The results from competition binding experiments indicated that adding an iodo group to the parent molecule did not increase the binding affinities, as compared to the parent compound GTS-21, for either 7 nAChR or 4β2 subtype. However, iodo-GTS-21 possessed significant subtype selectivity than that of GTS-21, with Ki values >1,000 nM and 13 nM for 7 nAChR and 4β2 subtype, respectively.
It is to our disappointment that the results were contradictory with the goal we set beforehand, that is iodo-GTS-21 won’t be capable of improving the binding affinity and selectivity to 7 nAChR. However, the results led to another finding, that was even iodo-GTS-21 did not show better affinity than GTS-21, but iodo-GTS-21 did have a feature of subtype selectivity for 4β2 subtype that was better than GTS-21. This kind of characteristic may become an issue that will be worthwhile for further investigation in the future.
Chapter I Introduction
1.1 Acetylcholine Receptors
The cholinergic neurons, with acetylcholine as the neurotransmitter, are widely distributed in the human brain and play important roles in attention, memory and cognition [1-5]. There are two classes of acetylcholine receptors (AChR). Muscarinic acetylcholine receptors belong to a class of metabotropic receptors which use G proteins as their signaling mechanism. Four subtypes of muscarinic receptors (named M1-M4) have been determined [6]. Another main class of acetylcholine receptors is nicotinic acetylcholine receptors (nAChR), which have also been implicated in memory, cognitive and learning processes [5, 7-10]. nAChR consist of eight alpha (2 to 9) and three beta (2 to 4) subunits [11], of which the major subtypes concentrated in hippocampus and cerebral cortex in the human brain are the 42 and 7 subtypes [12, 13]. The 42 subtype is most abundant in human brain and occupies merely 90% in the cerebrum; the α7 subtype is the second abundance, which contains almost 10% of nicotinic receptors in human brain [14]. nAChR function as ion-gated channels and, particularly α7 subtype, modulate influx of calcium into cells [14] and subsequently offer neuroprotective ability to neurons [15], of which capability has influence on cognition and memory function. The correlation between modulation of calcium influx of α7 nAChR and neuroprotection has evoked huge interest [16-18]. However, reductions in nAChR have been reported in various neurodegenerative disorders, including Alzheimer’s disease (AD) [19-21], dementia with Lewy bodies (DLB) [22] and Parkinson’s disease (PD) [23]. Accumulated evidences suggested that those neurodegenerative disorders were correlated with α7 subtype of nAChR [24-26].

1.2 Radiotracers for nAChR Imaging
Since many studies have demonstrated that the function and the amount of nAChR was related to neurodegenerative disorder, efforts to evaluate both the function and the amount of nAChR have raised huge interests by applying radioactive ligands to image nAChR. Imaging nAChR provides several advantages on understanding pharmacokinetics and biochemical functions of nAChR.
A lot of radiotracers for imaging nAChR, particularly 42 subtype, have been developed and well studied. Doll et al [27] identified the characteristic of A-85380, which only binds to 42 subtype but not 7 nAChR. Ryu et al [28] reviewed several radioligands, such as 11C-MP4A, 11C-nicotine and 18F-A-85380, about their recent progress and development of AD imaging agents in PET. Sabri et al [29] reported the use of 18F-A-85380 PET to clarify the change in 42 subtype in the early stage of AD patients and indicated a decline in 42 nAChR in AD patients. 123I-labeled A-85380 was used for demonstrating the relation between DLB and visual hallucinations [22]. Even though radiolabeled A-85380 displays promising performance on detecting 42 nAChR, however, the slow equilibrium of A-85380 may limit its application. Brust et al [30] proposed 18F labeled norchloro-fluoro-homoepibatidine which possessed faster equilibrium than A-85380 and showed comparable function as A-85380.
Compare to large amount of studies with radiotracers targeting 42 nAChR, progress on 7 subtype was relatively slow. Kulak et al [31] examined the binding of [125I]iodoMLA to nAChR, which was competed with -bungarotoxin (BTX), a very potent 7 nAChR agonist [32]. Kulak et al concluded [125I]iodoMLA might be a potential tracer for selectively imaging 7 nAChR.

1.3 Anabaseine Derivative
Anabaseine has been proved as a potent agonist on α7 nAChR [33]. Many derivatives of anabaseine have been developed and evaluated for their potentials to improve treatment of AD. Among them, a novel compound, GTS-21 [3E-(2,4-dimethoxybenzylidene)anabaseine; DMXBA], has recently been exploited and well characterized as a selective agonist for α7 nAChR. Previous studies proved that GTS-21 significantly enhanced the memory and learning on experimental animals and healthy human [3, 34, 35]. For pharmacokinetic studies of GTS-21, Kim et al [33] reported the synthesis of 11C-labeled GTS-21and positron emission tomography (PET) imaging in small animals. In attempts to developing radiotracer for imaging, radioactive iodine would be one of the best choices. 123I decays with the emission of 159 keV photons which are ideal for single photon emission computed tomography (SPECT) imaging; 124I is a positron decay radionuclide which can be used for PET studies. The physical half-lives of 123I and 124I are 13.2 hr and 4.18 days, respectively. Comparing to 11C, the longer half-live of 123I and 124I and rather simple iodination chemistry should be the strengths of radioactive iodine in the development of new radiotracers for clinical imaging practice.

1.4 Objective of This Study
The aim of this study was to evaluate if a newly synthesized compound, iodo-GTS-21, can be a candidate model for further development of radioiodine labeled tracers for imaging nAChR. In vitro competition binding experiments were conducted to evaluate the binding affinities of iodo-GTS-21 for 42 and 7 nAChR, and compared with other known inhibitors, such as α-bungarotoxin and (-)-cytisine. The rat brain was the tissue source for binding targets and the homogenates were prepared to determine the binding affinity.
Chapter II Materials and Methods
2.1 General
Solvents and reagents were reagent grade and used without further purification. Piperidone was purchased from Arcos. Trimethylsil chloride was commercial available from Fluka. Lithium diisopropylamide (LDA), tetraethylamine (TEA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and bovine serum albumin (BSA) were obtained from Sigma-Aldrich. Ethyl nicotinate, 2,4-dimethoxybenzaldehyde and iodine monochloride (ICl) were purchased from Alfa Aesar. Ammonium acetate, sodium chloride, potassium chloride, calcium chloride, potassium chloride, (-)-cytisine, tris(hydroxymethyl)aminomethane (Tris), polyethylenimine (PEI) and silica gel 60 F254 thin-layer chromatography plates were obtained from Merck. (-)-Nicotine ditartrate and α-bungarotoxin (BTX) were purchased from CALBIOCHEM. [125I]Tyr54-α-bungarotoxin ([125I]BTX), [3H]cytosine and [125I]sodium iodide (without reductant) were commercial available from Perkin Elmer. GF-B filter paper was obtained from Whatman. Ready Safe™ cocktail for liquid scintillation counting was purchased from Beckman Coulter.

2.2 Instruments
The instruments employed in this study included a Wallac Wizard 1470 automatic gamma counter, a Packard Tri-Carb 2900TR liquid scintillation counter, a Heraeus Biofuge pico and an Eppendorf Centrifuge 5424 desktop centrifuges, a Sartoriu pH meter, an AND HR200 analytical balance, a Branson 5510 ultrasonic cleaner, a Biodex Medical Systems AUTOMAB 100 isotope calibrator, a Fargo HMS-520 heating plate, a Scientific Industries Vortex-2 gene vortex, a BRANSTEAD EASYpure UF compact bioresearch water system, micropipettes (BRAND and Gilson, with volume ranges of 0.5 to 10 µl, 5 to 50 µl, 50 to 200 µl, and 200 to 1000 µl), a BRANDEL M-24T cell harvester, a Digisystem water bath with shaker, a FIRSTEK OV-80 oven. High performance liquid chromatography (HPLC) analyses were performed on a Waters 600 HPLC system equipped with a Waters 2996 photodiode array (PDA) detector and Waters Empower 2 software for analysis. Proton nuclear magnetic resonance (NMR) spectra were performed at the Institute of Nuclear Energy Research (INER) on a Varian Mercury Plus-300 MHz spectrometer using tetramethylsilane as an internal standard.

2.3 Chemical Synthesis
The synthesis of GTS-21 was followed the method described by Kem et al [36] with some modification. Figure 1 illustrates the synthesis scheme of GTS-21 and iodo-GTS-21.

2.3.1 Synthesis of 2,4-dimethoxy-5-iodobenzaldehyde (I)
A mixture of 2,4-dimethoxybenzaldehyde (A; 2 g, 12 mmol) and iodochloride (2.35 g) in absolute ethanol (50 ml) was refluxed for half a day. After removal of solvent by rotary evaporation, the residue was redissolved in a small portion of dichloromethane and was subjected to further purification on silica gel column chromatography eluted with pure dichloromethane. Upon drying in a rotary evaporator, 1.83 g (52% yield) of product was obtained as white powers. Proton NMR at 300 MHz in CD3OD:  3.94 (s, 3 H, -OCH3); 3.96 (s, 3 H, -OCH3); 6.38 (s, 1 H, 3-aromatic H); 8.18 (s, 1 H, 6-aromatic H) (Figure 2).

2.3.2 Synthesis of lithium 3-nicotinoyl-2-piperidone enolate (II)
A 250-ml round bottom flask was filled with nitrogen gas to evacuate air and then was corked with a plug. A balloon inflated with nitrogen gas was dug into the plug. The nitrogen gas filled round bottom flask was placed into a liquid nitrogen bath and was injected with 25 ml of tetrahydrofuran (THF). A 20-ml (30 mmol) portion of lithium diisopropylamide (LDA) and 3 g of piperidone (B) dissolved in 10 ml of THF were injected into the round bottom flask subsequently. The reaction mixture was stirred for 15 min. A portion of 3.75 ml (30 mmol) trimethylsil chloride was added to the mixture and stirred for another 15 min. The liquid nitrogen bath was removed and the mixture was stirred at room temperature for additional 2 hr.
The reaction mixture in the round bottom flask was put back to the liquid nitrogen bath. An aliquot of 20-ml (30 mmol) LDA was added dropwise with stirring for 15 min. Following injection of ethyl nicotinate (2.75 ml, 20 mmol) and being stirred for 15 min, the liquid nitrogen bath was removed again and the reaction mixture was stirred overnight at room temperature.
After adding approximately 1 ml of purified water and being stirred for 20 min, the reaction mixture was filtered and the pale yellow solid was collected. The solid was then washed with a small amount of purified water. Upon drying in vacuum, 3.9 g of yellow solid was yielded. Proton NMR at 300 MHz in D2O:  3.13 (m, 3 H, 3-H and 6-H); 7.24 (dd, J = 8.1 and 5.2 Hz, 1 H, 5’-aromatic H); 7.28 (dt, J = 8.1 and 1.6 Hz, 1 H, 4’-aromatic H); 8.15 (d, J = 1.6 Hz, 1 H, 2’-aromatic H); 8.23 (dd, J = 5.2 and 1.6 Hz, 1 H, 6’-aromatic H) (Figure 3).

2.3.3 Synthesis of anabaseine dihydrochloride (III)
A 250-ml round bottom flask in an ice bath was added 10 ml of HCl. One gram of solid lithium 3-nicotinoyl-2-piperidone enolate (II) was slowly added into the flask. After being refluxed overnight, the reaction mixture was cooled down to room temperature and was then slowly added with cold isopropanol. The solution was stored in a 4C refrigerator overnight to speed up the crystallization. The silk needle-like crystals with pale grey color were collected by filtration and were washed by cold isopropanol. Upon drying in vacuum, 0.7 g of product was yielded. Proton NMR at 300 MHz in CD3OD:  1.69 (m, 4 H, 4-H and 5-H); 2.85 (t, J = 5.6 Hz, 2 H, 3-H); 3.98 (t, J = 5.7 Hz, 2 H, 6-H); 8.20 (dd, J = 8.0 and 5.1 Hz, 1 H, 5’H-aromatic-H); 8.76 (d, J = 5.1 Hz, 1 H, 6’-aromatic H); 8.93 (m, 1 H, 4’-aromatic H); 9.40 (s, 1 H, 2’-aromatic H) (Figure 4).

2.3.4 Synthesis of 3E-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) (IV)
To a 250-ml round bottom flask was added 1 g (3.8 mmol) of anabaseine dihydrochloride (III), 1.45 g (8.73 mmol) of 2,4-dimethoxybenzaldehyde (A), a small amount of HCl and 45 ml of ethanol. After being refluxed overnight, the solution was cooled down to room temperature. The solution volume was reduced by rotary evaporation to approximately 10 ml. Following addition of a large amount of diethyl ether with vigorous stirring to remove impurities, 0.88 g of solid with bright yellow color was obtained upon drying in vacuum. Proton NMR at 300 MHz in CD3OD:  2.11 (qn, J = 5.6 Hz, 2 H, 4-H); 3.30 (t, J = 5.6 Hz, 2 H, 5-H); 3.79 (s, 3 H, OCH3); 3.90 (s, 3 H, OCH3); 3.94 (t, J = 5.7 Hz, 2 H, 6-H); 6.61 (d, J = 2.4 Hz, 2 H, 7-H); 6.71 (dd, J = 8.8 and 2.4 Hz, 1 H, 12-aromatic H); 7.58 (s, 1 H, 10-aromatic H); 7.70 (d, J = 8.8 Hz, 1 H, 13- aromatic H); 8.26 (dd, J = 8.0 and 5.2 Hz, 1 H, 5’-aromatic H); 8.79 (d, J = 7.9 Hz, 1 H, 4’-aromatic H); 9.18 (d, J = 5.1 Hz, 1 H, 6’-aromatic H); 9.27 (s, 1 H, 2’-aromatic H) (Figure 5).

2.3.5 Synthesis of 3E-(2,4-dimethoxy-5-iodobenzylidene)anabaseine (Iodo-GTS-21) (V)
A mixture of 2,4-dimethoxy-5-iodobenzaldehyde (I; 0.4 g, 1.6 mmol) and anabaseine dihydrochloride (III, 1.4 g, 3 mmol) was dissolved in 40 ml of ethanol in a 250-ml round bottom flask. A small amount of HCl, was added to the solution before reflux overnight. After cooling to room temperature, the solution was poured gently into a large amount of diethyl ether in a 500-ml round bottom flask with vigorous stirring. The yellow precipitates were collected by filtration and dried in vacuum. The solids were dissolved in a small amount of ethanol, and then added with approximately 5 ml of THF. After neutralization with 0.2 ml of tetraethylamine (TEA), 0.9 g of orange color oil was obtained upon drying in vacuum. Proton NMR at 300 MHz in CD3OD:  2.18 (qn, J = 5.6 Hz, 2 H, 4-H); 3.08 (t, J = 5.8 Hz, 5-H); 3.85 (s, 3 H, OCH3); 3.92 (t, J = 5.8 Hz, 2 H, 6-H); 3.99 (s, 3 H, OCH3); 6.68 (s, 2 H, 7-H), 7.47 (s, 1 H, 10-aromatic H); 7.88 (dd, J = 8.0 and 5.4 Hz, 1 H, 5’-aromatic H); 8.05 (s, 1 H, 13-aromatic H); 8.33 (d, J = 8.0 Hz, 1 H, 4’-aromatic H); 8.95 (d, J = 1.5 Hz, 1 H, 2’-aromatic H); 8.98 (d, J = 5.4 Hz, 1 H, 6’-aromatic H), (Figure 6).

2.4 HPLC Analysis
The purity of the synthesized GTS and iodo-GTS were analyzed using reverse-phase HPLC. A Waters 600 HPLC system with a reverse-phase Phenomenex Gemini 5 C18 110A (4.6 × 250 mm) column was equilibrated with a mobile phase containing 20% 5 mM ammonium acetate (pH 7.4) and 80% acetonitrile. After sample injection, an isocratic elution of the mobile phase at a flow rate of 0.5 ml/min was initiated. The detection of absorbance was carried out at 260 nm. All the peaks detected 0-20 min were integrated.

2.5 Binding Assay
Rat brain homogenates were the source of nAChR for binding assay. Two radioligands, [125I]Tyr54-α-bungarotoxin ([125I]BTX) and [3H]cytisine, were used in the experiments. The specific activity of [3H]cytisine and [125I]BTX were 40 Ci/mmol and 147 Ci/mmol, respectively. Inhibitors employed in the study included (-)-nicotine ditartrate, (-)-cytisine, α-bungarotoxin (BTX), GTS-21 and iodo-GTS-2.

2.5.1 Preparation of HEPES Buffer and Binding Buffer
HEPES buffer was prepared by dissolving 3.5 g of sodium chloride (120 mM in final concentration), 0.186 g of potassium chloride (5 mM in final concentration), 0.11 g of calcium chloride (2 mM in final concentration), 0.1 g of magnesium chloride (1 mM in final concentration) and 2.383 g of HEPES (20 mM in final concentration) in 500 ml of de-ionized water, pH value of the buffer was adjusted to 7.4.
Binding buffer was the abovementioned HEPES buffer containing 0.1% bovine serum albumin (BSA).

2.5.2 Preparation of Rat Brain Homogenates
Male Sprague-Dawley rats weighting 250-300 g were used for the experiments. After arrival, four of them were sacrificed by cervical decapitation. The brains were quickly removed. The whole brain minus cerebellums and pons were homogenized in 20 volumes of ice-cold HEPES buffer using a glass homogenizer. Following centrifugation of the homogenates at 16,000 rpm for 22 min at 4°C, the supernatant was removed. The pellet was added with approximately 20 ml ice-cold de-ionized water. The homogenates were vortexed at intervals and stored at ice temperature for 1 hour. The mixture was subjected to centrifugation again. After removal of the supernatant, a portion of ice-cold HEPES buffer was added to the pellet. The homogenates were vortexed to reach homogeneity, dispensed into 1 ml-portions, and then stored at a -70 °C freezer.
At the day of binding assay, the homogenates were removed from the freezer and kept at ice temperature. Before dilution to appropriate concentration with ice-cold binding buffer, the homogenates were vortexed again to assure the homogeneity.
Protein quantification was followed by standard protein quantification procedure and the measured concentration of rat brain homogenates was 390 g/l.

2.5.3 Preparation of Cell Harvester and Filter Papers
Right before binding assay, a BRANDEL M-24T cell harvester (Figure 8) was washed with 25 mM Tris buffer (pH 7.4) three times. Whatman GF-B filter papers were pre-soaked in 0.5% PEI solution.

2.5.4 Inhibitor Competition of [125I]BTX Binding
[125I]BTX was a radioligand specifically targeting α7 nAChR (Kd = 1.7 nM) [37]. The inhibitors used to compete [125I]BTX binding were BTX, (-)-cytisine, (-)-nicotine ditartrate, GTS-21 and iodo-GTS-21. Several solutions were prepared as follows:
Serial dilution of inhibitor solution: The stock solution of each inhibitor was prepared in a concentration of 1 mg/ml in de-ionized water (for BTX, (-)-cytisine and (-)-nicotine ditartrate) or in ethanol (for GTS-21 and iodo-GTS-21). The stock solutions were further diluted in series with the binding buffer. The No. 4 solutions of (-)-cytisine, (-)-nicotine ditartrate, GTS-21, iodo-GTS-21 and BTX were prepared to a concentration of 1/10, 1/10, 1/100, 1/100 and 1/1,000, respectively, of their corresponding stock solutions. The No. 3, No. 2 and No. 1 solutions were obtained by 10, 100 and 1000 folds dilution, respectively, of their corresponding No.4 solutions.
BTX solution for non-specific binding: The BTX stock solution was prepared by reconstitution of BTX (1 mg) with 1 ml of de-ionized water. The concentration of BTX used to define non-specific binding was 1/50 of the stock solution (0.5 mM).
[125I]BTX solution: The specific activity of [125I]BTX was 147 Ci/mmol. Right before study, appropriate quantity of [125I]BTX was diluted with the binding buffer to a concentration of 120,000 cpm in 100 μl, a volume required to add to each assay tube. Since 1 Ci equals 2.2 × 106 dpm, and the counting efficiency for 125I was 70%, therefore, the specific activity of [125I]BTX would be 147 × 2.2 × 106 × 0.7 cpm/nmol. The radioactivity of appropriate input [125I]BTX (120,000 cpm) in the reaction volume of 0.25 ml corresponded to a concentration of 2.12 nM in final 250-μl reaction mixture.
To initiate the competition study, an aliquot of 50-μl binding buffer was added to each of two assay tubes as the control; whereas 50 μl of the binding buffer containing 1 M BTX was added to each of two assay tubes as the non-specific binding. An aliquot of 50 μl of various inhibitors with different concentrations was added in next order. Cross contamination of any assay tube with different inhibitor concentration should avoid. Subsequently, 120,000 cpm of [125I]BTX in 100 μl solution was added to each assay tube. Rat brain homogenates (100 μl) were added the last. The control, the inhibitors and the non-specific binding were duplicated when binding assay was performed. The arrangement of the assay tubes was illustrated in Figure 7. The binding assay was repeated at least 3 times to meet statistical significance.
After all materials (the binding buffer, the inhibitors, the radioligand and the brain homogenates) were put in order, the assay tubes were vortexed to reach homogeneity, and were incubated in a 37C water bath for 2 hr with shaking at a speed of 90-100 rpm.
The cell harvester was used to separate the receptor-bound from the unbound ligand. After harvesting, the [125I]BTX bound to α7 nAChR was retained on the filter paper, and the radioactivity was measured with a Wallac Wizard 1470 automatic gamma counter.

2.5.5 Inhibitor Competition of [3H]Cytisine Binding
[3H]Cytisine was a potent radioligand specifically targeting α4β2 nAChR (Kd = 0.19 nM) [37]. The inhibitors used to compete [3H]cytisine binding in this study were (-)-cytisine, (-)-nicotine ditartrate, GTS-21 and iodo-GTS-21. Several solutions were prepared as follows:
Serial dilution of inhibitor solution: Serial dilution method as mentioned above was adopted. The only exception was the concentrations of (-)-cytisine and (-)-nicotine ditartrate in No.4 solutions were 1,000 folds dilution from their stock solutions.
Nicotine solution for non-specific binding: (-)-Nicotine ditartrate (1.6224 g) was reconstituted with 1 ml of de-ionized water to prepare 10 mM stock solution. The non-specific binding solution was prepared by 100 folds dilution of the stock solution.
[3H]Cytisine solution: The specific activity of [3H]cytisine was 40 Ci/mmol. Prior to experiment, appropriate amount of [3H]cytisine was diluted with the binding buffer to a concentration of 60,000 cpm in 100 μl, a volume required to add to each assay tube. Because 1 Ci is equivalent to 2.2 × 106 dpm, and the counting efficiency for 3H was 30%, therefore, the specific activity of [3H]cytisine would be 40 × 2.2 × 106 × 0.3 cpm/nmol. The radioactivity of appropriate input [3H]cytisine (60,000 cpm) in the reaction volume of 0.25 ml corresponded to a concentration of 9.09 nM in final 250-μl reaction mixture.
To trigger the competition study, an aliquot of 50-μl binding buffer was added to each of two assay tubes as the control; whereas 50 μl of the binding buffer containing 100 μM (-)-nicotine ditartrate was added to each of two assay tubes as the non-specific binding. An aliquot of 50 μl of each inhibitor with various concentrations were added in next order. Cross contamination of among assay tubes with different inhibitor concentration should avoid. Each assay tube was then added with 60,000 cpm (100 μl) of [3H]cytisine, and followed by addition of 100 μl abovementioned dilution of rat brain homogenates. Assay tubes were duplicated for the control, with the inhibitors and the non-specific binding, and repeated at least 3 times to meet statistical significance. After being vortexed for homogeneity, all assay tubes were stood still at room temperature for 20 min. Upon harvesting, the [3H]cytisine bound to α42 nAChR was not be filtered and remained on the filter paper.
For measuring the radioactivity of 3H, 4 ml of Ready SafeTM cocktail was added to each assay tube with the filter. The beta radioactivity was measured on a liquid scintillation counter (Packard Tri-Carb 2900TR) with a detection efficiency of 30%. The acquisition time was set as 5 min.

2.5.6 Determination of IC50 for Inhibitors
IC50 stands for a measure of the effectiveness of a cold compound in inhibiting 50% of the radioligand binding to the receptor. That is, the concentration of an inhibitor to displace 50% of a radioligand binding. IC50 is an approximate estimate so that it cannot be regarded as accurate as the Kd values obtained with the radioligand.
The percentage specific binding for each concentration of an inhibitor was calculated by Equation 1:
Counts is the total binding in cpm of various concentrations of inhibitors; NS is the radioactivity in cpm of the non-specific binding; Control is the total binding in cpm of the control.
The percentage of specific binding was plotted as a function of the inhibitor concentration, and then the IC50 can be determined with the corresponding concentration that the percentage specific binding reaches 50%

2.5.7 Calculation of Ki for Inhibitors
Ki, the inhibition constant, is used to estimate the binding affinity of a cold compound. The experimental Ki was calculated by the Cheng and Prusoff equation (Equation 2) as follows:
where, the dissociation constant, Kd, of the radioligan, was obtained from the literature (Kd of [125I]BTX and [3H]cytisine were reported as 1.7 nM [37] and 0.19 nM [38], respectively). IC50 was calculated from competition binding experiment. The term [radioligand] stands for the concentration of radioligand (nM), and were 2.12 nM and 9.09 nM for [125I]BTX and [3H]cytosine, respectively. Therefore, the formula to calculate Ki of an inhibitor which competed the binding of [125I]BTX was simplified as Equation 3.
Similarly, the Ki of an inhibitor which competed the binding of [3H]cytisine can be computed by Equation (4).

Chapter III Results
3.1 Chemical Syntheses and HPLC Analyses of GTS-21 and Iodo-GTS-21
Both GTS-21 and iodo-GTS-21 were synthesized successfully. The chemical purity of both synthesized compounds was determined by reverse-phase HPLC analysis. The UV 260 nm absorbance chromatograms of synthesized GTS-21 and iodo-GTS-21 were shown in Figures 9 and 10, respectively. The retention time of GTS-21 in the HPLC system was 8.07 min with a chemical purity of 92.48%.; whereas the retention time of iodo-GTS-21 was 9.77 min with a chemical purity of 98.26%.

3.2 Inhibitor Competition of [125I]BTX Binding
The results of competition studies of BTX, (-)-cytisine, (-)-nicotine ditartrate, GTS-21 and iodo-GTS-21 against the [125I]BTX binding to rat brain homogenates were shown in Figures 11-15.
For iodo-GTS-21, there was no curve extended and crossed 50% indicating that iodo-GTS-21 was not able to compete [125I]BTX binding, therefore, the IC50 was much larger than 1 M by rough estimation. The Ki of iodo-GTS-21 would be greater than 1 M.
The IC50 and the Ki of inhibitors were summarized in the Table 1.

Table 1. Summary of IC50 of inhibitors against [125I]BTX binding and calculated Ki of inhibitors
Inhibitor IC50 (nM) Ki (nM)
BTX 5.2± 0.8 2.3 ± 0.4
(-)-Cytisine 3,310 ± 31 1,478 ± 137
(-)-Nicotine ditartrate 15,000 ± 5,420 6,696 ± 2438
GTS-21 1,540 ± 36 688 ± 165
Iodo-GTS-21 >>1,000 >1,000

3.3 Inhibitor Competition of [3H]Cytisine Binding
The results of competition studies of (-)-cytisine, (-)-nicotine ditartrate, GTS-21 and iodo-GTS-21 against the [3H]cytisine binding to rat brain homogenates were shown in Figures 16-19.
Since the calculated data of experimental percentage specific binding of [3H]cytisine far excessed 100%, normalization was performed in cases of competition with (-)-cytisine, (-)-nicotine ditartrate and iodo-GTS-21. Normalization was performed on the data set in which the maximal percentage specific binding was exceeded 120% by normalizing the maximum to 100%. Normalized plots are shown in Figure 16, 17 and 19.
The IC50 and the Ki of inhibitors were summarized in the Table 2.

Table 2. Summary of IC50 of inhibitors against [3H]cytisine binding and calculated Ki of inhibitors
Inhibitor IC50 (nM) Ki (nM)
(-)-Cytisine 9.1 ± 2.0 0.18 ± 0.04
(-)-Nicotine ditartrate 84 ± 2 1.7 ± 0.5
GTS-21 646 ± 30 13 ± 6
Iodo-GTS-21 619 ± 49 13 ± 10

Chapter IV Discussion
4.1 Chemical Synthesis
In the beginning of iodo-GTS-21 synthesis, a straightforward iodination of GTS-21 with ICl had been proposed. However, the direct iodination was failed because the addition of iodine requires high electron density at the bounding site, and unfortunately the methoxy group is not a strong electron donator. After then, we modified the synthesis method by adding iodine to 2,4-dimethoxybenzaldehyde to prepare 2,4-dimethoxy-5-iodobenzaldehyde (I) first. After coupling of I with anabaseine (III), the iodo-GTS-21 was successfully synthesized (Figure 1).
There was 3-nicotinoyl-2-piperidone appeared with the product lithium 3-nicotinoyl-2-piperidone enolate (II) and it could not be separated from II due to their structural similarity. Since the presence of 3-nicotinoyl-2-piperidone would not affect the subsequent synthesis, therefore, we left it alone and continued further synthesis.
In attempts to prepare [125I]iodo-GTS-21 for further characterization study, we tried to synthesize tri-n-butyltin-GTS-21 as the precursor of 125I-iodo-GTS-21. The tri-n-butyltin function group would serve as an excellent leaving group and provide an electron-rich active site in GTS-21 molecule for electrophilic iodination. We proposed the coupling reaction of GTS-21 (IV) with bis(tributyltin) in THF in the presence of tetrakis(triphenylphosphine) palladium, however, the synthesis was failed. Another possibility to prepare [125I]iodo-GTS-21 by direct iodination of IV with [125I]sodium iodide in the presence of peracetic acid was also proved in vain.

4.2 Binding Assay
The purpose of competition binding assay experiments was to estimate the binding potential of the newly synthesized compound, iodo-GTS-21. Several known compounds, such as (-)-nicotine ditartrate, (-)-cytisine, BTX, and even synthesized GTS-21 were performed in parallel to help estimate IC50 of I-GTS-21. These compounds were served as references for method confirmation in this study.
The calculated inhibition constants (Ki) to α7 nAChR were 1,478 nM, 6,696 nM, 688 nM and 2.3 nM, respectively, for (-)-cytisine, (-)-nicotine ditartrate, GTS-21, and BTX. These data were comparable to the literature reports in which the Ki of (-)-cytisine ranged from 1400 nM to 3883 nM [39-42]; the Ki of (-)-nicotine ditartrate was estimated from 400 nM to 8,900 nM [39, 40, 42-45]; the Ki of GTS-21 was reported as 212-652 nM [40, 42]; and the Ki of BTX ranged from 0.35 nM to 3.5 nM [39, 41, 42, 44, 46]. The Ki values obtained from this study almost fell within the ranges of previous reports confirmed the appropriateness of this study. The Ki of iodo-GTS-21 calculated in this study was much greater than 1,000 nM indicating this novel iodinated compound showed much lower binding affinity to α7 nAChR.
As to the competition experiment to α4β2 nAChR, the calculated Ki of (-)-cytisine, (-)-nicotine ditartrate and GTS-21 in this study were 0.18, 1.7 and 13 nM, respectively. The literature reported Ki of these known compounds to α4β2 nAChR were 0.14-2.7 nM [40, 47, 48], 1-11 nM [39, 43, 47, 49], and 85 nM [40] for (-)-cytisine, (-)-nicotine ditartrate and GTS-21, respectively. The calculated Ki of BTX to α4β2 nAChR was larger than 1,000 nM consistent with the face that BTX is a specific ligand with good affinity to α7 nAChR but not to α4β2 subtype. The calculated Ki of iodo-GTS-21 to α4β2 nAChR in this study was determined to be 13 nM, suggesting subtype binding selectivity toward α4β2 nAChR.
Comparing the binding affinity of iodo-GTS-21 to α7 and α4β2 nAChR, we found that iodo-GTS-21 showed higher binding affinity to α4β2 nAChR instead of to α7 nAChR. The Ki of iodo-GTS-21 to α7 nAChR was much more than 1,000 nM. On the other hand, it bound to α4β2 subtype showing a Ki value of 13 nM. Those two values demonstrated the selectivity of iodo-GTS-21 for α4β2 subtypes. Even though the original purpose of this study was aiming to develop a novel compound which would show better affinity to α7 nAChR than GTS-21. However, the results shown in the study did not reach the goal for binding affinity. Instead iodo-GTS-21 showed unexpected subtype selective different from the parent compound GTS-21. This interesting profile of iodo-GTS-21 may be worthwhile for further investigation.

Chapter V Conclusions
A novel derivative of GTS-21, iodo-GTS-21, was synthesized with high chemical purity. The attachment of a large element, such as iodine, might reduce the affinity of GTS-21 binding to α7 nAChR. Moreover, the amount of α7 nAChR was much lower than the dominant α4β2 subtype, which might contribute to the difficulty of binding assay with α7 nAChR.
Although iodo-GTS-21 did not show its binding affinity to α7 nAChR in competition binding experiment in this study, nevertheless, it demonstrates significant subtype selectivity between α4β2 and α7 two different subtypes of nicotinic receptors. Iodo-GTS-21 showed the more specific subtype selectivity than GTS-21 to α4β2 than to α7 nAChR.
Even though iodo-GTS-21 did not display its binding affinity for targeting α7 nAChR, GTS-21 still holds its potential and access to α7 nAChR for Alzheimer’s disease treatment. Further modification of GTS-21 to improve binding affinity are still deserved to be encouraged.
Table of figures
Figure 1. Synthesis scheme of Iodo-GTS-21 (V) and GTS-21 (IV)………………………………………………….32
Figure 2. Proton NMR spectrum of 2,4-dimethoxy-5-iodobenzaldehyde (I)……………..33
Figure 3. Proton NMR spectrum of lithium 3-nicotinoyl-2-piperidone enolate (II)………………34
Figure 4. Proton NMR spectrum of anabaseine dihydrochloride (III)…………………………………………………..35
Figure 5. Proton NMR spectrum of 3E-(2,4-dimethoxybenzylidene) anabaseine (GTS-21) (IV)…………………………………………………..36
Figure 6. Proton NMR spectrum of 3E-3-(2,4-dimethoxy-5-iodo- benzylidene)anabaseine (V)…………………………37
Figure 7. Arrangement of the assay tubes in inhibitors competition study……………………………………38
Figure 8. The BRANDEL M-24T cell harvester used in this study………………………………………………....39
Figure 9. The reverse-phase HPLC chromatogram of synthesized GTS-21. The peak signal corresponds to the absorbance of UV 260 nm…………………………..40
Figure 10. The reverse-phase HPLC chromatogram of synthesized iodo-GTS-21. The peak signal corresponds to the absorbance of UV 260 nm……………………41
Figure 11. Plot of percentage specific binding of [125I]BTX to rat brain homogenates as a function of BTX concentration (nM) (n = 3)………………………….42
Figure 12. Plot of percentage specific binding of [125I]BTX to rat brain homogenates as a function of (-)-cytisine concentration (nM) (n = 3)………………………….43
Figure 13. Plot of percentage specific binding of [125I]BTX to rat brain homogenates as a function of (-)-nicotine ditartrate concentration (nM) (n = 3)………………..44
Figure 14. Plot of percentage specific binding of [125I]BTX to rat brain homogenates as a function of GTS-21 concentration (nM) (n = 3)…………………………..45
Figure 15. Plot of percentage specific binding of [125I]BTX to rat brain homogenates as a function of iodo-GTS-21 concentration (nM) (n = 3)…………………………..46
Figure 16. Plot of normalized percentage specific binding of [3H]cytisine to rat brain homogenates as a function of (-)-cytisine concentration (nM) (n = 3)……………...47
Figure 17. Plot of normalized percentage specific binding of [3H]cytisine to rat brain homogenates as a function of (-)-nicotine ditartrate concentration (nM) (n = 3)…...48
Figure 18. Plot of percentage specific binding of [3H]cytisine to rat brain homogenates as a function of GTS-21 concentration (nM) (n = 3)…………………………..49
Figure 19. Plot of normalized percentage specific binding of [3H]cytisine to rat brain homogenates as a function of iodo-GTS-21 concentration (nM) (n = 3)…………...50
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