Radiosynthesis and whole‐body distribution in mice of a 18F‐labeled azepino[4,3‐b]indole‐1‐one derivative with multimodal activity for the treatment of Alzheimer's disease

Recently, the azepino[4,3‐b]indole‐1‐one derivative 1 showed in vitro nanomolar inhibition against butyrylcholinesterase (BChE), the ChE isoform that plays a role in the progression and pathophysiology of Alzheimer's disease (AD), and protects against N‐methyl‐ d‐aspartate‐induced neuronal toxicity. Three 9‐R‐substituted (R = F, Br, OMe) congeners were investigated. The 9‐F derivative (2a) was found more potent as BChE inhibitors (half‐maximal inhibitory concentration value = 21 nM) than 2b (9‐Br) and 2c (9‐OMe), achieving a residence time (38 s), assessed by surface plasmon resonance, threefold higher than that of 1. To progress in featuring the in vivo pharmacological characterization of 2a, herein the 18F‐labeled congener 2a was synthesized, by applying the aromatic 18F‐fluorination method, and its whole‐body distribution in healthy mice, including brain penetration, was evaluated through positron emission tomography imaging. [18F]2a exhibited a rapid and high brain uptake (3.35 ± 0.26% ID g−1 at 0.95 ± 0.15 min after injection), followed by a rapid clearance (t1/2 = 6.50 ± 0.93 min), showing good blood–brain barrier crossing. After a transient liver accumulation of [18F]2a, the intestinal and urinary excretion was quantified. Finally, ex vivo pharmacological experiments in mice showed that the unlabeled 2a affects the transmitters' neurochemistry, which might be favorable to reverse cognition impairment in mild‐to‐moderate AD‐related dementias.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder, accounting for most elderly-related dementias.Recent World Alzheimer Reports predicted that people affected by AD will increase to more than 131 million by 2050. [1]The main histopathological hallmarks of AD are the generation and deposition of senile plaques, incorporating the extracellular neurotoxic amyloid-β (Aβ) peptide aggregates, along with intracellular neurofibrillary tangles of hyperphosphorylated τ-protein, which triggers oxidative stress, perturbation of cellular metabolism, and finally synaptic and neuronal loss, [2] and the impairment of the cholinergic neurotransmission. [3]beit the advances in understanding the multifactorial nature of AD, including the epigenetic mechanisms underlying the disease, [4] efforts to develop disease-modifying agents failed, and the available treatments only address the symptoms of mild-to-moderate AD.The approved drugs include three acetylcholinesterase (AChE) inhibitors (rivastigmine, galantamine, and donepezil [DNPZ]) and one N-methyl-D-aspartate receptor (NMDAR) antagonist (memantine) to counteract decreased acetylcholine (ACh) level and glutamate-induced excitotoxicity in AD brain, respectively. [5]The cholinergic system is a network of neurons, glia, and immune cells that use the excitatory neurotransmitter ACh in physiological processes. [6][10] ACh levels and cholinergic transmission in the brain are regulated by the two enzyme isoforms, namely, AChE hydrolyzing ACh in the synaptic cleft of the cholinergic neurons and butyrylcholinesterase (BChE), but at higher ACh concentrations. [11,12]ChEs' inhibition has been widely associated with cognitive and persistent benefits over time in AD, with reduced mortality risk. [13]BChE is associated with the CNS glial cells, vascular structures, and neurons, where it may serve as a coregulator of the cholinergic neurotransmission.BChE is primarily located in the CNS amygdala, HIPP, and thalamus, which are involved in cognition and behavior functions compromised in AD.Despite BChE activity is normally observed at higher levels in the white matter and CNS glial cells, in both animal models and postmortem brain samples of AD patients, an increase of BChE expression and activity has been observed [14] in association with Aβamyloid plaques and neurofibrillary tangles.Data from animal models would suggest an active role of BChE in the pathogenesis of AD, leading to consider BChE as a pharmacological (co)target, and/or a diagnostic marker of disease progression, and the BChE inhibitors (BChE-Is) are worthy of consideration as anti-AD pharmacological agents. [11,12]Interestingly, due to the lack of peripheral side effects (i.e., gastrointestinal, cramps), BChE-Is may be more tolerated than the AChE inhibitors.
In addition to BChE-selective inhibition, THAI derivatives showed protective effects against N-methyl-D-aspartate (NMDA)-induced excitotoxicity on neuronal SH-SY5Y cells.
Considering the in vitro biological profile, the low cytotoxicity, and the predicted good blood-brain barrier (BBB) crossing, to progress in featuring the in vivo pharmacological profile of 2a, we conducted a preliminary evaluation of its whole-body distribution in mice by positron emission tomography (PET) imaging, synthesizing a THAI-labeled tracer.THAI 1 could be potentially labeled with radioisotopes (e.g., 18 F, 76 Br, or 11 C) from suitable precursors, and we prepared three unlabeled analogs (2a-c), bearing 9-F, 9-Br, and 9-OCH 3 substituents.The preliminary evaluation of the anti-ChE activities of 2a-c guided the selection of the 9-F THAI congener (2a) that underwent the radiolabeling procedure.[ 18 F]2a was synthesized and characterized, and its in vivo distribution in healthy mice was investigated.The effects of the intraperitoneally (i.p.) administered single dose (20 mg kg −1 ) of 2a on different brain neurotransmitters involved in behavioral responses (i.e., serotonin [5-HT], dopamine [DA], noradrenaline [NA]) were studied in healthy mice through ex vivo neuropharmacological assays to forecast developments of 2a as an anti-AD agent.

| In vitro biological characterization of 9-substituted THAI derivatives
By applying the Ellman colorimetric assay, according to a previously reported protocol, [18,19] and using DNPZ and tacrine (TAC) as positive controls, the in vitro inhibitory activities of 1 and the 9-Xsubstituted analogs 2a-c were determined toward heterologous cholinesterases (electric eel [ee] AChE and horse serum [hs] BChE), and the human ChE isoforms.The values, expressed as the halfmaximal inhibitory concentration values (IC 50 ) or inhibition percentage at the maximum concentration tested (25 μM), are summarized in Table 1.
The THAI derivatives 1 and 2a confirmed to be almost equipotent as BChE-Is in the low nanomolar range of concentrations. [16]While no appreciable difference emerged between the inhibition data toward the species-related ChEs' isoforms, bulkiness of the R substituent at position 9 should affect the BChE inhibition potency.In fact, Br (2b) and OMe (2c), bulkier than F, resulted in congeners 10-and fivefold less potent than 2a, respectively.
The binding kinetics and affinity of 1 and 2a toward BChE was further featured by using a surface plasmon resonance (SPR) biosensor (Figure S1 in Supporting Information).The hs BChE, which shares high homology with the human isoform but higher stability, was first immobilized on a COOH-V sensor chip surface by amine coupling, resulting in an efficient biosensor, even after repeated regeneration cycles with a 3 M NaCl solution (details on protocols for immobilization and assay in the experimental section). [17]Briefly, assay solutions of tested compounds were prepared by dissolving 10 mM dimethyl sulfoxide (DMSO) solutions in 10 mM phosphate buffer (PB) (pH 8) up to a final 2% DMSO concentration, as matched in the running buffer.The assay solutions were injected over BChE functionalized surface, avoiding any limitation by mass transfer, and after completion of injection, the running buffer was flowed to allow a complete dissociation phase, without regeneration cycles.The results obtained with good approximation are summarized in Table 2.
The K D values of 1 and 2a, along with the reference drugs TAC and DNPZ, agreed with the inhibition potency (IC 50 ) data.The binding curves (Figure S1   Applying the thermodynamic equilibrium method, the experimental aqueous solubility of compound 2a was determined (Table 2 and Supporting Information Table S2).According to that predicted by SwissADME (3.80 × 10 −2 mM), the aqueous solubility of 2a was almost five times lower than that of 1 (Table 2), but yet ranking it as a moderately water-soluble compound (6 < Log S < 2).

| Ex vivo neurochemical assays
Besides the cholinergic hypothesis, multiple neurotransmitter abnormalities can affect the AD brain, and some other related neurological disorders, which may benefit from drugs with multiple effects. [20]We investigated the ability of compound 2a to affect brain neurotransmission in healthy mice, by quantitatively determining noradrenaline (NA), dopamine (DA), and serotonin (5-HT) content in the T A B L E 1 Inhibitory activities on cholinesterases of 9-X-substituted 2,3,4,5-tetrahydroazepino [4,3-b]indol-1(2H)-one derivatives.a IC 50 values determined by interpolation of the sigmoidal dose-response curves as obtained by regression with GraphPad Prism software (ver.5.01) of at least seven data points, or percent inhibition at 10 μM concentration in square brackets for compounds achieving less than 40% inhibition at 25 μM; data are mean ± SD of three independent measurements.b The previously reported compounds 1 and 2a [16] were retested in this study.
c TAC and DNPZ were used as positive controls.
T A B L E 2 Affinity kinetics data to BChE, affinity to HSA and aqueous solubility of 9-R-substituted 2,3,4,5-tetrahydroazepino [4,3-b]indol-1(2H)-one derivatives.b Binding to immobilized HSA, measured by SPR.Warfarin was reported as the reference, and its K D value resulted close to those previously reported. [18]ta are mean ± SD of at least three independent measurements.c Aqueous solubility determined in 0.05 M PBS, pH 7.40 (0.15 M KCl) at 25 ± 1°C.
prefrontal cortex (PFC) and HIPP of mice treated with 2a, at a dose of 20 mg kg −1 (or vehicle) i.p., 30 min after administration.The results are shown in Figure 2.
The quantification analysis of neurotransmitter distribution showed that NA and DA content in PFC brain area was not affected by administration of 2a (Figures 2a,b), while 5-HT levels were significantly increased in 2a-treated mice, in comparison to control (Figure 2c; unpaired Student's t test with Welch's correction).As for the HIPP area, after i.p. administration 2a induced a significant increase in NA level (Figure 2d) and a significant reduction in DA content in this brain area (Figure 2e).Finally, significant alteration in hippocampal 5-HT concentration was not evidenced after administration (Figure 2f).
After i.p. administration, compound 2a was, therefore, able to diversely alter PFC and HIPP neurochemistry.A significantly increased 5-HT content was determined in the cortical area, which could be particularly interesting considering that very often neuropsychiatric complications, such as depression, accompany or even precede neurological disorders.Therefore, the increase in cortical 5-HT may be beneficial, since drugs able to enhance serotonergic neurotransmission have been shown to facilitate Aβ clearance, [21] and influence APP processing toward Aβ production. [22,23]garding the hippocampal area, NA level was also found to be significantly increased in 2a-treated mice compared to control.Such an outcome is in line with previously reported increased NA release after the administration of AChE inhibitors. [24]This effect may result favorable in the AD treatment, as NA regulates cognitive and behavioral functions and can also protect neurons against a variety of insults that lead to inflammatory and oxidative unbalances, [25] such as the Aβ production and a reduction in NA concentration often occurring in AD patients. [26]Considering the indication of NA as an endogenous suppressor of brain inflammatory responses, [27] drugs able to increase NA release, along with ACh release, may be advantageous. [28]As far as the dopaminergic system is concerned, we found DA concentrations were significantly decreased in HIPP after 2a treatment.These results are in line with previous data indicating a reduction in DA levels after inhibition of AChE. [29]It is known that cholinergic and dopaminergic systems are interconnected and play a role in the modulation of memory processes.In good agreement, the DA system involved in cognitive functions and located in brain areas such as HIPP and cerebral cortex is under cholinergic control.In our experimental procedure, we recognized an area-dependent effect, and DA reduction was strongly evident only at hippocampal levels.This dramatic reduction in DA levels may be secondary to nicotinic receptor desensitization occurring after AChE inhibition as shown for other areas such as the striatum. [30]It has been reported that the BChE distribution is involved in the coregulation of cholinergic and noncholinergic neurotransmission at the hippocampal level in humans; therefore, the area-specific effect retrieved might overlap BChE distribution and specific function in that area. [31]ole physicochemical and neurochemical data, as well as the recent interest devoted to identification of PET tracers based on competitive inhibitors to study the BChE role in AD progression, [32,33] prompted us to investigate the in vivo ability of 2a to cross BBB and penetrate CNS, and its whole-body distribution.To do this, we undertook the radiosynthesis of [ 18 F]2a to study the distribution into the brain and the peripheral organs in mice.The 18 F isotope, which has a half-life of 109.8 min, showed favorable properties over other radioisotopes commonly used for nuclear imaging techniques, high positron yields, and a good spatial resolution, which is a result of relatively low positron energies. [6]4 | Radiosynthesis of 18 F-labeled compound 2a The 18 F-labeled 2a was synthesized by direct aromatic 18 F-fluorination of the boron ester precursor, [29] preferred to hypervalent iodine [34] and stannyl compounds, [35] following a consolidated in-house developed method (Scheme 2). [34]The precursor 7 was prepared starting from the 9-Br analog 2b.

| Stability and lipophilicity of [ 18 F]2a
Radio-thin layer chromatography (radio-TLC) was applied to study the in vitro stability of the radioligand [ 18 F]2a in human serum from healthy volunteers.The samples were incubated with human serum at 37°C, and aliquots were taken off at 0, 30, 60, 90, and 180 min, mixed with CH 3 CN, then centrifuged, and the resulting supernatants analyzed by a radio-TLC scanner.[ 18 F]2a did not undergo any noticeable degradation, such as defluorination or any kind of breakdown, within 180 min after incubation in human plasma (Supporting Information, Figure S6).
Compound 2a was predicted by admetSAR software [36] as a good brain-permeating compound (CNS+).The lipophilicity of [ 18 F]2a was determined by the micro shake-flask method (Supporting Information, Table S7).The distribution coefficient between pH 7.4 aqueous phosphate-buffered saline (PBS) and n-octanol (Log D 7.4 ) was found equal to 3.35 ± 0.04.The experimental Log D value falls within the range of 2.0-3.5, which is established as optimal to achieve high/good penetration across BBB and limit the target to nontarget ratios. [37]6 | Whole-body distribution study of [ 18

F]2a in healthy mice
The in vivo biodistribution study was assessed by intravenously administering a single dose of a solution of newly radiosynthesized compound [ 18 F]2a in healthy ICR (Institute of Cancer Research) mice to provide a quantitative evaluation of biodistribution levels in brain tissues, organs, and whole body, by using a small animal PET/CT scanner system to obtain dynamic images and generate time-activity curves of regions of interest (Figure 3).

PET measurements of the whole radioactivity related to [ 18 F]2a
were performed in 0-90 min intervals.A suitable radiotracer for the brain should have a good penetration of the BBB, that is, a high uptake after intravenous bolus injection (2%-4% ID g −1 in mice), low nonspecific binding in all brain areas, including those without target protein, and a rapid washout in 2-30 min in healthy mice. [38]th dynamic images and time-activity curves support the brain penetration ability of [ 18 F]2a.Based on PET images in mice, we expect that [ 18 F]2a may show high in vivo stability.The skull radio uptake, which originated in dissociated free 18 F from [ 18 F]2a, was not observed in the PET imaging within 90 min.The feature is according to analytical evaluation that showed high stability of 2a in mice brain homogenate (Supporting Information S1,Figure S8), which did not undergo apparent metabolism within 24 h.
Based on the PET signal, the radiolabeled [ 18 F]2a proved to enter readily in the brain in few seconds.Recently, some radiolabelled BChE competitive inhibitors, built on DNPZ-like sulfonamides, [32,39] or pseudoirreversible carbamate inhibitors have been reported and applied as potential PET tracers.These compounds retained enough potency of the parent lead compounds, also showing only a limited brain uptake, and were accumulated in internal organs such liver and kidneys.The presented radiolabeled [ 18 F]2a proved to improve the ability to enter the brain.However, the highest uptake in relevant brain areas (3.35 ± 0.26 ID g −1 ) was exhibited at 0.95 ± 0.15 min postinjection, followed by a rapid clearance (t 1/2 = 6.50 ± 0.93 min), which suggests that [ 18 F]2a can cross BBB, without permanent accumulation in the brain.
The gallbladder showed the greatest accumulation, as expressed by area under the curve in %ID g −1 min, followed by the urinary bladder, intestine, liver, kidney, heart, lung, and brain.The substantial accumulation in the gallbladder, intestine, and liver implies that [ 18 F] 2a may be excreted via the hepatobiliary route of excretion with a greater fraction than renal excretion.

| CONCLUSIONS
In the context of our medicinal chemistry program on small molecules as MTDLs in AD dementias, we recently discovered potent BChEselective inhibitors built up on the THAI scaffold and disclosed the 6-phenetyl derivative 1 and its 9-F congener 2a, which displayed in vitro low neuro-and organotoxicity, nanomolar inhibition potency against human BChE, and protective effects against NMDA-induced neurotoxicity.To progress in featuring its neuropharmacological profile and suitability as an anti-AD molecule, we determined further biochemical and biophysical parameters and carried out a wholebody distribution study in healthy mice of the newly synthesized labeled analog [ 18 F]2a.The PET imaging study proved the ability of [ 18 F]2a to cross the BBB, appearing in the brain within 1 min after intravenous administration, followed by rapid washout, a behavior comparable with those of general brain-targeted radiotracers in healthy subjects, [40] and suggest that future studies should aim to  In radiochemistry, H 2 18 O was purchased from Taiyo Nippon Sanso Corporation.[ 18 F]Fluoride was produced by 18 O(p,n) 18 F reaction through proton irradiation using a cyclotron.Radio-TLC was detected by an AR-2000 radio-TLC imaging scanner (Bioscan Inc.).All radioactivity counting was analyzed by a VDC-505 (Comecer) activity calibrator from Veenstra Instruments.PET imaging of mice was performed on a NanoPET/CT (Mediso).All compounds were obtained by following the reactions reported in Scheme 1.The synthesis of compound 2a was reported as an example.Complete descriptions of the intermediates for the synthesis of compounds 2b and 2c were reported in the Supplementary Information.The InChI codes of compounds 2a-c, together with some biological activity data, are also provided as Supporting Information.
6-Fluoro-1,2,3,9-tetrahydro-4H-carbazol-4-one oxime (5a): The oxime derivative 5a was synthesized starting from compound 4a. [18]dium acetate (1.The oxime derivative 5a (2.5 g, 12.5 mmol) was added portionwise to preheated (110°C) 25 g of PPA under vigorous stirring.The mixture was stirred 30 min at this temperature before being poured in 100 g of ice, and triturated until complete dissolution of PPA.The obtained gray precipitate was collected by filtration under reduced pressure, washed with 100 mL of water, 10 mL of 5% diluted ammonia, and further 100 mL of water.The solid was suspended in 50 mL of MeOH and refluxed for 1 h after the addition of 500 mg of vegetal carbon.
resulted in different half-lives of enzyme-ligand complex, as highlighted by the differences in residence times (12 and 38 s for 1 and 2a, respectively).The interactions of compounds 1 and 2a and human serum albumin (HSA) were also determined by SPR.HSA represents the most abundant protein in plasma, playing a key role in modulating the ADME profiles of xenobiotics.The assessment of the binding affinity to HSA can help to estimate the bioavailability in the early stage of drug design and development.Compounds 1 and 2a appeared as fast and reversible HSA binders, endowed with high affinity to the circulating protein.The higher affinity site of HSA was occupied at concentrations lower than 15 μM with a dissociation constant (K D ) of 0.85 and 0.24 μM for compounds 1 and 2a, respectively.Considering the physiological HSA concentration in plasma (680 μM), at 10 μM concentration, both compounds can be predicted bound to HSA for distribution to the peripheral tissues.

assess [ 18 F
]2a as a potential BChE target radioligand in AD animal models.Ex vivo neuropharmacological experiments in mice revealed diverse effects on neurotransmitters in cortical and HIPP areas 30 min after i.p. administration of 2a (20 mg kg −1 ).An increased level of 5-HT was observed in cortical brain areas, whereas increased NA and a reduced DA concentration appeared in HIPP.These changes in neurochemical transmission contribute to conceiving the potential of multimodal bioactive profile of 2a in neurodegenerative diseases, considering that a 5-HT level increase could contribute to enhancing the clearance of Aβ protein, whereas restoring the NA/DA balance could help in regulating cognitive and behavioral functions.In this light, screening of 2a in animal models of AD will be the logical prosecution of this work, which may provide a supporting proof of its effectiveness in the treatment of AD-related disorders.
PET-based assessment of in vivo distribution of [ 18 F]2a.Abbreviations: AUC, area under the curve; C max , peak concentration; PET, positron emission tomography; t 1/2 , half-life; T max , time to reach the C max .
Starting materials and all reagents and solvents were purchased from Sigma-Aldrich and Alfa Aesar, and were used without further purification.All reaction was monitored by precoated plates (silica gel 60F254; Merck).Melting points were determined by using the capillary method on a Stuart Scientific SMP3 electrothermal T A B L E 3 spectra were recorded at 500 MHz on a Varian instrument.Chemical shift data are expressed in δ and the coupling constants J are in hertz (Hz); the following abbreviations are used for multiplicity: s, singlet; d, doublet; dd, doublet-doublet; td, triplet of doublets; t, triplet; q, quartet; m, multiplet.Signals due to NH and OH protons were located by deuterium exchange with D 2 O. Chromatographic separations were performed on silica gel 60 for column chromatography (Merck 70-230 mesh).
temperature for 48 h and then diluted with 20 mL of DCM and 20 mL of water.The collected organic phase was washed twice with 20 mL of brine, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure.The residue was purified by chromatography on silica gel, eluting with ethyl acetate to afford 2a.White solid.Yield 51% (0.120 g).FT-IR (υ max , cm −1 ) 3437, 3027, 2921, 1634, 1523, 1470, 1384, 1132, (134 mg, 0.53 mmol) were dissolved in DMF (10 mL) and then stirred at 80°C for 5 h under argon atmosphere.After cooling, the reaction mixture was diluted by DCM (10 mL) and filtered through a Celite layer.The filtrate was washed with water (3 × 10 mL), dried over anhydrous