Synthesis and Anticholinesterase Evaluation of Cassine, Spectaline and Analogues

: In this work, twelve analogues of piperidine alkaloids (-)-cassine and (-)-spectaline were synthesized, as well as the racemic forms of these natural products. The compounds were evaluated for their inhibition of electric eel acetylcholinesterase (AChE ee ) and human butyrylcholinesterase (BChE hu ) by on-ﬂow mass-spectrometry-based dual-enzyme assay, and the inhibition mechanisms for the most potent analogues were also determined. Our results showed a preference for BChE hu inhibition with compounds 10c (Ki = 5.24 µ M), 12b (Ki = 17.4 µ M), 13a (Ki = 13.2 µ M) and 3 (Ki = 11.3 µ M) displaying the best inhibitory activities.

An estimated 40-50 million people live with dementia worldwide, and Alzheimer's disease (AD) is the most common cause [15,16].AD has complex pathophysiology, and knowledge about this disease is constantly evolving.However, some of its characteristics are well-established, including: (i) decreased acetylcholine levels in synaptic clefts of most regions of the hippocampus (cholinergic hypothesis); (ii) amyloid beta peptide (Aβ) accumulation; and (iii) tau protein hyperphosphorylation.In addition, other mechanisms such as oxidative stress, energy metabolism dysregulation and inflammation play a role in the disease process [16,17].Acetylcholinesterase (AChE) inhibition prevents acetylcholine (ACh) conversion into choline (Ch), thus increasing ACh levels in the synaptic clefts.In fact, AChE inhibition is the targeted mechanism currently available to treat AD [16].Although selective AChE inhibition restores the cholinergic system to some extent, studies have demonstrated that butyrylcholinesterase (BChE) can rescue the cholinesterasic function when AChE is absent.Therefore, dual AChE and BChE inhibition has been described as a more-beneficial treatment for AD patients [18].Besides playing a role in ACh cleavage, AChE could participate in the amyloidogenic pathway through the interaction of Aβ with a hydrophobic environment that is close to the AChE peripheral anionic site [19,20].BChE is potentially a better target than the well-known AChE for the treatment of later-stage cognitive decline in AD [21].
The anticholinesterase activity of 3 and its derivatives was first identified in 2005 [11], and a deeper investigation identified the mechanism of action and in vivo effects of these compounds [12].Although (-)-cassine (1) is the main piperidine alkaloid in the S. spectabilis flower, its anticholinesterase activity was only evaluated by bioautography and microplate screening assays [22], while the activity of (-)-spectaline (3) and the corresponding O-acetyl derivatives (2 and 4) were separately also assessed (Figure 1A).The authors suggested that the 3-OH group had a role in establishing more important interactions with the enzyme than the acetyl group in compounds 2 and 4, and the docking studies pointed out that the length of the side chain had an effect in the inhibition of AChE.[23].
with a hydrophobic environment that is close to the AChE peripheral anionic site [19 BChE is potentially a better target than the well-known AChE for the treatment of la stage cognitive decline in AD [21].
The anticholinesterase activity of 3 and its derivatives was first identified in 2005 and a deeper investigation identified the mechanism of action and in vivo effects of th compounds [12].Although (-)-cassine (1) is the main piperidine alkaloid in the S. spec lis flower, its anticholinesterase activity was only evaluated by bioautography and mi plate screening assays [22], while the activity of (-)-spectaline (3) and the correspond O-acetyl derivatives (2 and 4) were separately also assessed (Figure 1A).The authors gested that the 3-OH group had a role in establishing more important interactions w the enzyme than the acetyl group in compounds 2 and 4, and the docking studies poin out that the length of the side chain had an effect in the inhibition of AChE.[23].Encouraged by these previous studies which suggested that the size of the side c may play a role in AChE inhibition and that the methyl group in the piperidine ring d not interact with the active site, here we investigate the potential biological effects of thetic (±)-cassine (1), (±)-spectaline (3) and analogues thereof on cholinesterase inhibit and evaluate the impact of structural simplification by removal of the methyl group sent at C-2 in the structure of these natural products, as well as the length and the prese of unsaturation in the alkyl side chain and in the piperidine ring, as depicted in Figure

Synthetic Methods
General: Dichloromethane (DCM) and triethylamine (Et3N) were pretreated with cium hydride and distilled before use.Ethyl acetate, acetonitrile, methanol, chlorof and toluene were treated with 4 Å molecular sieves for at least 24 h before use and sto under nitrogen-purged atmosphere.BF3•OEt was distilled prior to use.All other solv and commercial reagents were used as supplied without further purification unless st otherwise.Reactions were monitored by thin-layer chromatography (silica gel 60 F25 aluminum foil), and visualization was achieved under UV light (254 nm) followed staining in potassium permanganate (KMnO4), Dragendorff stain or p-anisaldehyde s (p-ASD).Silica gel 60 (200-400 Mesh) was used for purifications by standard flash colu chromatography.NMR spectra were recorded on a Bruker Avance DPX 250 MHz MHz 1 H, 63 MHz 13 C), Bruker Avance III 400 (400 MHz 1 H, 101 MHz 13 C) or Bruker Ava III 500 (500 MHz 1 H, 126 MHz 13 C) unit (Bruker Co., Billerica, Massachusetts, USA).chemical shifts are expressed in parts per million (ppm) relative to the residual sol signal as an internal reference: (1) CDCl3 1 H RMN = 7.26, 13 C RMN = 77.16;(2) metha d4: 1 H RMN = 3.31, 13 C RMN = 49.00.Multiplicities are reported with the following s bols: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and multiples ther High-resolution mass spectra (ESI) were acquired on an Xevo Q-Tof mass spectrom (Waters, Manchester, UK) equipped with a nanoESI-type ionization source.IR spe were recorded using a Thermo Scientific Nicolet IS5 spectrometer, using Thermo Scien Encouraged by these previous studies which suggested that the size of the side chain may play a role in AChE inhibition and that the methyl group in the piperidine ring does not interact with the active site, here we investigate the potential biological effects of synthetic (±)-cassine (1), (±)-spectaline (3) and analogues thereof on cholinesterase inhibition, and evaluate the impact of structural simplification by removal of the methyl group present at C-2 in the structure of these natural products, as well as the length and the presence of unsaturation in the alkyl side chain and in the piperidine ring, as depicted in Figure 1B.

Synthetic Methods
General: Dichloromethane (DCM) and triethylamine (Et 3 N) were pretreated with calcium hydride and distilled before use.Ethyl acetate, acetonitrile, methanol, chloroform and toluene were treated with 4 Å molecular sieves for at least 24 h before use and stored under nitrogen-purged atmosphere.BF 3 •OEt was distilled prior to use.All other solvents and commercial reagents were used as supplied without further purification unless stated otherwise.Reactions were monitored by thin-layer chromatography (silica gel 60 F254 in aluminum foil), and visualization was achieved under UV light (254 nm) followed by staining in potassium permanganate (KMnO 4 ), Dragendorff stain or p-anisaldehyde stain (p-ASD).Silica gel 60 (200-400 Mesh) was used for purifications by standard flash column chromatography.NMR spectra were recorded on a Bruker Avance DPX 250 MHz (250 MHz 1 H, 63 MHz 13 C), Bruker Avance III 400 (400 MHz 1 H, 101 MHz 13 C) or Bruker Avance III 500 (500 MHz 1 H, 126 MHz 13 C) unit (Bruker Co., Billerica, Massachusetts, USA).The chemical shifts are expressed in parts per million (ppm) relative to the residual solvent signal as an internal reference: (1) CDCl 3 1 H RMN = 7.26, 13 C RMN = 77.16;(2) methanol-d4: 1 H RMN = 3.31, 13 C RMN = 49.00.Multiplicities are reported with the following symbols: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and multiples thereof.High-resolution mass spectra (ESI) were acquired on an Xevo Q-Tof mass spectrometer (Waters, Manchester, UK) equipped with a nanoESI-type ionization source.IR spectra were recorded using a Thermo Scientific Nicolet IS5 spectrometer, using Thermo Scientific ID3 ATR (Thermo Fisher Scientific, Waltham, Massachusetts, USA) Melting points were recorded on a MP50 Mettler Toledo (Columbus, Ohio, USA) melting point apparatus and are uncorrected.
TLC: (hexanes: EtOAc = 7:3), Rf = 0,4 (UV, KmnO 4 or p-ASD) Commercially available furfurylamine (1.00 mL, 12.0 mmol, 1.20 eq) and benzophenone (1.80 g, 10.0 mmol, 1.00 eq) were dissolved in toluene (24.0 mL).Then, BF 3 •OEt 2 (123 µL, 1.00 mmol, 0.10 eq) was added, the round bottom flask was adapted with a Dean-Stark trap and the reaction mixture was heated under reflux overnight.After this period, the solvent was removed under reduced pressure, producing a brown-yellow solid.Recrystallization with methanol (heat to 45 • C and cool to 0 • C) furnished N-(diphenylmethylene)-1-(furan-2-yl)methanamine (S-I, CAS: 56542-90-6) as white crystals (1.85 g, 7.00 mmol, 71%).S-I (1.0 g, 3.8 mmol, 1.0 eq) was solubilized in THF (38 mL, 0.1 M) under a nitrogen atmosphere and the mixture was cooled to −78 • C. A solution of n-BuLi 1.6 M in hexanes (2.50 mL, 5.75 mmol, 1.50 eq) was added dropwise and the solution was stirred for 40 min.Iodomethane (360 µL, 5.75 mmol, 1.50 eq) was added to the dark red solution which was allowed to stir for 1 h at 0 • C.After this period, the reaction turned a dark yellow color and it was quenched with saturated aqueous NaHCO 3 solution and extracted with Et 2 O.The combined organic phases were washed with saturated NaCl solution, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.The crude residue was used without purification in the next step.
The residue from the previous step was dissolved in a mixture of acetone (19 mL) and HCl 1M (19 mL) at 0 • C. The mixture was stirred overnight and then extracted with Et 2 O.The aqueous phase was neutralized with solid K 2 CO 3 until pH 7-8 and THF (19 mL), NaHCO 3 (968 mg, 11.5 mmol, 3.00 eq) and 2-nitrobenzenesulfonyl chloride (1.0 g, 4.6 mmol, 1.2 eq) were added.The reaction was stirred for 5 h or until TLC showed complete conversion of the starting material.The reaction mixture was extracted with EtOAc (3 × 20.0 mL) and the combined organic phases were washed with saturated NaCl solution, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.The material was purified by flash column chromatography (SiO 2 , hexanes/EtOAc 0% to 40%, 10% increases) yielding 5b as a white solid (795 mg, 2.68 mmol, 70% yield).
The material obtained previously was dissolved in dry MeCN (50 mL, 0,1 M) under an N 2 atmosphere and cooled to −30 • C.Then, allyltrimethylsilane (2.45 mL, 15.0 mmol, 3.00 eq) was added followed by Sn(oTf) 2 (322 mg, 0.750 mmol, 0.150 eq), and the reaction was kept at this temperature under magnetic stirring for 60 min or until total consumption of starting material was achieved (TLC analysis).After this period, the reaction was quenched by the addition of saturated NaHCO 3 solution (20 mL) and extracted with EtOAc (3 × 20 mL).The combined organic phases were washed with saturated NaCl solution, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.The material was purified by column chromatography (SiO 2 , hexanes/EtOAc 0% to 50%, 10% increases) yielding 7a as a light yellow solid (972 mg, 3.00 mmol, 60% yield, 2 steps).
The material obtained in the previous step was solubilized in dry MeCN (20 mL, 0,1 M) under an N 2 atmosphere and cooled to −30 • C.Then, allyltrimethylsilane (1.3 mL, 8.0 mmol, 4.0 eq) was added followed by Sn(oTf) 2 (125 mg, 0.30 mmol, 0.15 eq), and the reaction was kept at this temperature under magnetic stirring for 60 min or until total consumption of starting material was achieved (TLC analysis).After this period, the reaction was quenched by addition of saturated NaHCO 3 solution (20 mL) and extracted with EtOAc (3 × 20 mL).

Methyl ketones synthesis General procedure A (alkylation)
Acetyl acetoacetate (0.61 mL, 4.8 mmol, 1.2 eq) was added to a solution of NaOEt, prepared from ethanol (4.0 mL) and Na (110 mg, 4.80 mmol, 1.20 eq).Bromo alkene (4.0 mmol, 1.0 eq) was added to the solution and kept stirring under reflux for 12 h.After this period, the reaction was allowed to reach room temperature, neutralized with HCl 6 M and, after addition of water, extracted with EtOAc (3×).The combined organic phases were washed with saturated NaCl solution, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.The material was purified by column chromatography (SiO 2 , hexanes/EtOAc 0% to 4%, 2% increases).

General procedure B (Krapcho decarboxylation)
A solution of ketoester (1.00 eq) in DMSO (sufficient for 0.05 M) was treated with ground NaCl (3 eq) and H 2 O (32 eq) and heated between 170-180 • C for 18 h.After this period, the reaction mixture was cooled, water was added and extracted with EtOAc.The combined organic phases were washed with saturated NaCl solution, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.The material was purified by column chromatography (SiO 2 , hexanes/EtOAc 5% to 10%, 1% increases).
TLC: (hexanes: EtOAc = 7:3), Rf = 0,33 (UV or p-ASD) To a solution of N-nosylpiperidine in MeCN (sufficient for 0.05 M) was added K 2 CO 3 (5 eq) and benzenethiol (3 eq).The resulting yellow solution was stirred for 45 min at room temperature or until total consumption of starting material, then filtered.The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (SiO 2 , hexanes 100% to eliminate yellow compounds, then DCM:MeOH 0% to 10%, with 0,5% Et 3 N as additive).
TLC: (DCM: MeOH: NH 4 OH (27%) = 88:10:2), Rf = 0,5 (p-ASD) To a solution of compound 10a-c (1.0 eq) in EtOAc (sufficient for 0.1 M) at 0 • C, was added Boc 2 O (1.3 eq), and it was allowed to reach room temperature.After total consumption of starting material, according to TLC, Ac 2 O (2.0 eq), Et 3 N (4.00 eq) and DMAP (0.05 eq) were added.The reaction mixture was stirred for 2 h, then diluted with EtOAc and washed with a citric acid 5% solution.The aqueous phase was extracted with EtOAc and washed with saturated NaCl solution, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.To the residue was added a HCl 4 M solution in EtOAc.After total consumption of starting material, the reaction was treated with saturated NaHCO 3 solution and extracted with EtOAc (3×).The combined organic phases were washed with saturated NaCl solution, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.The material was purified by column chromatography (SiO 2 , DCM/MeOH 0% to 10%, 2% increases).
TLC: (DCM:MeOH = 8:2), Rf = 0,4 (p-ASD) MP: 66.6-67.9To a solution of compound 12a-c in EtOAc (sufficient for 0.1 M) was added 0.1 mL HCl 4 M in dioxane.After 18 h the solvent was removed under reduced pressure, the residue was suspended in 1 mL of DCM and acetyl chloride (1.8 eq), freshly distilled, was added.The mixture was kept under reflux for 18 h.After this period was added NaHCO 3 saturated solution and extracted with EtOAc.The combined organic phases were washed with saturated NaCl solution, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.The material was purified by column chromatography (SiO 2 , DCM/MeOH 0% to 10%, 2% increases).

Biological Assays
Di-and trisubstituted piperidine derivatives (10a-c, 11a-c, 12a-c, 13a-c, 1 and 3) were submitted to cholinesterase-inhibition screening assays based on the simultaneous on-flow dual parallel enzyme assay system.The approach included immobilization of AChE from Electrophorus Electricus (AChE ee , Sigma-Aldrich, S. Louis, MO, USA) and BChE from human serum (BChE hu ) in order to obtain AChE ee -ICER and BChE hu -ICER, respectively.The LC-MS configuration and the mass spectrometer (MS) parameters have been previously described [27,28].The on-flow dual parallel enzyme assay was carried out on an Nexera LC system (Shimadzu, Kyoto, Japan) system consisting of three LC 20AD pumps, an SIL-20A auto-sampler, a DGU-20A degasser, a CTO-20A oven and a CBM-20A system controller.
The LC system was coupled with an AmaZon Speed Ion Trap (IT) mass spectrometry (MS) instrument (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) interface source, operating in the positive mode (scan 50-250 m/z).
The two immobilized capillary enzyme reactors (ICERs) and the MS instrument were interfaced through two 10-port two-position high-pressure switching valves (Valco Instruments Co. Inc., Houston, USA) [27].
The dual-system assay comprised three steps.Briefly, after the sample was injected, with both valves (A and B) in position 1, the reactive content of each ICER was transferred to the storage (step 1).In step 2, with both valves (A and B) in position 2, pump B directed the AChE ee -ICER enzymatic reaction for analysis in the MS.Meanwhile, the BChE hu -ICER reactive content was held in storage.In Step 3, while valve A was switched to position 1 again, valve B was kept in position 2. In this position, the BChE hu -ICER enzymatic reaction content that had been held in storage was flushed by Pump B and finally analyzed in the MS [27].
The data were acquired by using the Bruker Data Analysis Software (version 4.3, Bruker Daltonics Inc., Billerica, United States).All the analyses were performed at room temperature (21 • C).The enzymatic reaction was monitored by directly quantifying the acetylcholine hydrolysis product, choline (Ch) [M + H]+ m/z 104 [27,28].
Initially, the inhibition assay was conducted with the compounds at a fixed concentration of 100 µM, prepared with 10 µL of stock solution of the tested compound, 20 µL of ACh solution (final concentration of 70 µM) and 70 µL of ammonium acetate solution (15.0 mM, pH 8.0).The solutions were prepared in duplicate and vortex-mixed for 10 s, and 20-µL aliquots were used for injection.The negative (absence of ACh) and positive (presence of ACh and absence of the tested compound) controls were analyzed between each sample.The percentage of inhibition provided by each sample was calculated by comparing the area of enzymatic activity in the presence (Pi) and absence (P 0 ) of the inhibitor, according to the equation below, where (P i ) is the peak area of Ch that was produced in the presence of the tested compound and in the absence of the tested compound (P 0 ), and Sb corresponds to Ch that was quantified during spontaneous ACh hydrolysis.
Sb was determined by injecting the reaction mixture into an empty open tubular silica capillary (blank analysis to quantify spontaneous ACh hydrolysis).
The half maximum inhibitory concentration (IC 50 ), the mechanism of action, and its steady-state inhibition constant (Ki) were determined for the compounds with %I ≥ 65% at 100 µM.
To verify the inhibition mechanism, reciprocal plots of 1/[product choline] versus 1/[ACh] were constructed, and Ki was determined from the re-plots of the primary reciprocal plot data.

Chemistry
Due to its varied biological properties and the difficulties associated with its isolation in pure-form from natural sources, several different synthetic methodologies have been reported for the alkaloid (-)-cassine (1) [29].To achieve the construction of the piperidine core, we explored the approach reported by Zhou and colleagues [30,31], and later employed by Padwa and colleagues, in the synthesis of epi-indolizidine 223A [32] and (±)-cassine (1) [33] which allowed the synthesis of natural products 1 and 3 as well as their analogues 10a-3, 11a-c, 12a-c and 13a-c.

Chemistry
Due to its varied biological properties and the difficulties associated with its isolation in pure-form from natural sources, several different synthetic methodologies have been reported for the alkaloid (-)-cassine (1) [29].To achieve the construction of the piperidine core, we explored the approach reported by Zhou and colleagues [30,31], and later employed by Padwa and colleagues, in the synthesis of epi-indolizidine 223A [32] and (±)cassine (1) [33] which allowed the synthesis of natural products 1 and 3 as well as their analogues 10a-3, 11a-c, 12a-c and 13a-c.

Scheme 1. Synthesis of key intermediates 8a and 8b.
The entirely cis configuration of 8a was initially assigned by 1 H NMR with 3 JH5-H6ax = 9.9 Hz consistent with axial orientation of H-5, and further confirmed by NOESY correlations between H6ax and the allyl substituent, as well as between H2 and the ortho hydrogen of the nosyl (Ns) protecting group (Figure S1A).This assignment was later corroborated by X-ray diffraction crystallography analysis of 8a (Figure S1B).The disfavored A 1,3 -strain involving the nosyl group and the C-2 substituent in 1,2,3,6-tetrahydropyridine 8b explains the pseudo-axial orientation of the allyl substituent at C-2.The exceptional stereospecificity in the reduction of 7a is rationalized by the axial attack of the incoming hydride reagent controlled by the steric hindrance of the substituents at C-2 and C-6.(Figure S1C) [33].
The entirely cis configuration of 8a was initially assigned by 1 H NMR with 3 J H5-H6ax = 9.9 Hz consistent with axial orientation of H-5, and further confirmed by NOESY correlations between H6 ax and the allyl substituent, as well as between H2 and the ortho hydrogen of the nosyl (Ns) protecting group (Figure S1A).This assignment was later corroborated by X-ray diffraction crystallography analysis of 8a (Figure S1B).The disfavored A 1,3 -strain involving the nosyl group and the C-2 substituent in 1,2,3,6-tetrahydropyridine 8b explains the pseudo-axial orientation of the allyl substituent at C-2.The exceptional stereospecificity in the reduction of 7a is rationalized by the axial attack of the incoming hydride reagent controlled by the steric hindrance of the substituents at C-2 and C-6.(Figure S1C) [33].

Scheme 3. Final steps in the synthesis of compounds 12a-c, 13a-c, 1 and 3.
A total of 12 analogues were synthesized in six to eight steps, with overall yields ranging from 9 to 28% for the analogues bearing the 5-hydroxypiperidine moiety and from 2% to 13% for the acetylated analogues.
The preliminary inhibition data at 100 μM showed that the compounds tended to have higher affinity for BChEhu than for AChEee, including the racemic form of the natural products cassine (1) and spectaline (3), with both exhibiting a mixed-type mechanism of inhibition.These results are in accordance with those reported by Suciati and colleagues who also observed higher BChEhu inhibition, compared to AChEee, for the ethanolic extract of S. spectabilis [41].Piperidine derivatives 12b and 12c, lacking the methyl group, displayed a reduction in the % of inhibition of AChEee in comparison to 1 and 3, which was less significant for BChEhu.The presence of a methyl group did not seem to be essential for the inhibitory activity of BChEhu when the percentage of inhibition was considered, but its presence enhanced the anti-AChEee activity (Table 1).To prepare the remaining analogues, intermediates 10a-c were subjected to a stepwise procedure to achieve selective O-acetylation, which yielded analogues 11a-c.In parallel, intermediates 10a-e underwent catalytic hydrogenation to provide saturated analogues 12a-c and natural products (±)-cassine (1) and (±)-spectaline (3).Finally, analogues 13a-c were obtained by selective O-acetylation of compounds 12a-c, respectively (Scheme 3).

Scheme 3. Final steps in the synthesis of compounds 12a-c, 13a-c, 1 and 3.
A total of 12 analogues were synthesized in six to eight steps, with overall yields ranging from 9 to 28% for the analogues bearing the 5-hydroxypiperidine moiety and from 2% to 13% for the acetylated analogues.
The preliminary inhibition data at 100 μM showed that the compounds tended to have higher affinity for BChEhu than for AChEee, including the racemic form of the natural products cassine (1) and spectaline (3), with both exhibiting a mixed-type mechanism of inhibition.These results are in accordance with those reported by Suciati and colleagues who also observed higher BChEhu inhibition, compared to AChEee, for the ethanolic extract of S. spectabilis [41].Piperidine derivatives 12b and 12c, lacking the methyl group, displayed a reduction in the % of inhibition of AChEee in comparison to 1 and 3, which was less significant for BChEhu.The presence of a methyl group did not seem to be essential for the inhibitory activity of BChEhu when the percentage of inhibition was considered, but its presence enhanced the anti-AChEee activity (Table 1).A total of 12 analogues were synthesized in six to eight steps, with overall yields ranging from 9 to 28% for the analogues bearing the 5-hydroxypiperidine moiety and from 2% to 13% for the acetylated analogues.

Cholinesterase-Inhibition Screening Assays Results
In this study, the capacity for cholinesterase inhibition (AChE ee and BChE hu ) of compounds 10a-c, 11a-c, 12a-c, 13a-c, 1 and 3 was investigated by the recently developed on-flow mass-spectrometry-based dual-enzyme assay detailed in supporting information [27,28].
The preliminary inhibition data at 100 µM showed that the compounds tended to have higher affinity for BChE hu than for AChE ee , including the racemic form of the natural products cassine (1) and spectaline (3), with both exhibiting a mixed-type mechanism of inhibition.These results are in accordance with those reported by Suciati and colleagues who also observed higher BChE hu inhibition, compared to AChE ee , for the ethanolic extract of S. spectabilis [41].Piperidine derivatives 12b and 12c, lacking the methyl group, displayed a reduction in the % of inhibition of AChE ee in comparison to 1 and 3, which was less significant for BChE hu .The presence of a methyl group did not seem to be essential for the inhibitory activity of BChE hu when the percentage of inhibition was considered, but its presence enhanced the anti-AChE ee activity (Table 1).
On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChE hu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that the O-acetylated analogue 13a with a shorter side chain at C-2 displayed higher inhibition than the corresponding O-acetylated nor-cassine (13b) and nor-spectaline (13c) analogues regarding both AChE ee and BChE hu , with a striking difference being observed for the former.As for the other derivatives bearing a seven-carbon side chain, i.e., 10a, 11a and 12a, their inhibitory activity was shown to be lower (or at most, equipotent) when compared to the other analogues in the same series.On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that  On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that  On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that  On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that  On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that  On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that On the basis of these preliminary results, the effect of the alkyl chain length on the inhibition activity was unclear, although five out of six compounds with inhibition ≥65% for BChEhu have longer alkyl chains (n = 12 or 14; compounds 10c, 12b,c, 1 and 3) with only 13a displaying the same range of inhibition with a shorter side chain.It is noteworthy that The incorporation of two unsaturated compounds in these piperidine derivatives (10a-c compared to 12a-c) seems to be detrimental to their anti-AChE ee properties, while the picture for the anti-BChE hu activity is much less clear, as within the O-acetylated series (11a-c vs. 13a-c).
Comparison of the bis-unsaturated piperidine derivatives 10a-c and 11a-c shows that O-acetylation appears to be beneficial regarding anticholinesterase activity, with 11a-c inhibiting AChE ee more extensively than 10a-c; however, the same does not hold true for the inhibition of BChE hu .The O-acylation in the series of saturated piperidine derivatives (12a-c vs. 13a-c) does not translate into a significant increase in the anti-cholinesterase activity for both enzymes (12b vs. 13b and 12c vs. 13c), except when one compares the % of inhibition of 13a and 12a.
Furthermore, the combined characteristics of unsaturation and hydroxyl-group acetylation at C-3 reduced the activity of the compounds.For example, compounds 11a-c had both modifications and did not reach the minimum inhibition of 65% for either AChE ee or BChE hu.(Table 1).
the O-acetylated analogue 13a with a shorter side chain at C-2 displayed higher inhibition than the corresponding O-acetylated nor-cassine (13b) and nor-spectaline (13c) analogues regarding both AChEee and BChEhu, with a striking difference being observed for the former.As for the other derivatives bearing a seven-carbon side chain, i.e., 10a, 11a and 12a, their inhibitory activity was shown to be lower (or at most, equipotent) when compared to the other analogues in the same series.
The incorporation of two unsaturated compounds in these piperidine derivatives (10a-c compared to 12a-c) seems to be detrimental to their anti-AChEee properties, while the picture for the anti-BChEhu activity is much less clear, as within the O-acetylated series (11a-c vs. 13a-c).
Comparison of the bis-unsaturated piperidine derivatives 10a-c and 11a-c shows that O-acetylation appears to be beneficial regarding anticholinesterase activity, with 11ac inhibiting AChEee more extensively than 10a-c; however, the same does not hold true for the inhibition of BChEhu.The O-acylation in the series of saturated piperidine derivatives (12a-c vs. 13a-c) does not translate into a significant increase in the anti-cholinesterase activity for both enzymes (12b vs. 13b and 12c vs. 13c), except when one compares the % of inhibition of 13a and 12a.
Furthermore, the combined characteristics of unsaturation and hydroxyl-group acetylation at C-3 reduced the activity of the compounds.For example, compounds 11a-c had both modifications and did not reach the minimum inhibition of 65% for either AChEee or BChEhu.(Table 1).
To further understand the inhibition activity of these compounds, the type of inhibition mechanism was determined (Figures 2-5B and Figures S72B and S73B).To further understand the inhibition activity of these compounds, the type of inhibition mechanism was determined (Figures 2, 3, 4 and 5B and Figures S72B and S73B).
The results of our studies on the inhibition mechanisms indicate that cassine (1) and spectaline (3) inhibit butyrylcholinesterase via a mixed mechanism, the same pattern observed for 5-hydroxy piperidines 10c and 12c both displaying a 14-carbon alkyl chain at C-2.A non-competitive mechanism was observed for compounds 12b and 13a, the latter a 5-hydroxy piperidine bearing a seven-carbon side chain.
For mixed-type inhibition, V Max and K M values are affected.K M increases and V Max decreases since the inhibitor binds to the enzyme at a location distinct from the substrate binding site.Binding affinity for the substrate is decreased when the inhibitor is present.For non-competitive inhibition, K M value remains unchanged but V Max decreases.Here, the inhibitor binds to a site other than the active site.Binding causes a change in the structure of the enzyme so the substrate cannot bind, and no catalysis occurs [42,43].
The inhibitor constant, Ki, relates to the binding affinity, and the values for each compound were determined by replotting the primary reciprocal plot data.The slope and 1/v-axis intercept of each complex can be replotted against its corresponding inhibitor concentration.
Compounds 10c (K i = 5.24 µM), 3 (K i = 11.3 µM), 12b (K i = 17.4 µM) and 13a (K i = 15.2 µM) substantially reduced the rate of the enzymatic reaction and showed higher binding activity, which illustrates a characteristic behavior of non-competitive and mixedtype inhibitors as observed in the double-reciprocal plots (Figures 1, 2, 3 and 4B, Figures S72B and S73B).In the early stages of drug discovery studies, the evaluation of inhibition mode is a significant assessment since the interaction mode could be affected by the physiological environments to which the enzyme is exposed.Competitive inhibitors bind exclusively to the free enzyme form, while non-competitive or mixed-type inhibitors bind with some affinity to both forms, e.g., the free enzyme and the enzyme-substrate complex.While mixed-type inhibitors bind to the enzyme and the enzyme-substrate complex with different affinity, non-competitive inhibitors bind equally well to the enzyme and enzyme-substrate complex.
Therefore, the non-competitive and mixed-type mechanisms of inhibition can be a significant advantage in vivo when the physiological environment exposes the enzyme to high substrate concentrations.Although the clinical advantage of non-competitive inhibition has been recognized, the historical approaches for drug discovery have been focused on active-site-directed inhibitors as is the principal model of drugs in clinical use today [44].

Conclusions
As BChE hu is potentially a better target than the well-known AChE for the treatment of later-stage cognitive decline in AD, the discovery of BChE hu inhibitors that can act selectively and reversibly or pseudo-irreversibly in vivo is desirable because they will provide not only drug candidates, but also chemical probes to investigate the potential of BChE hu to serve as a therapeutic target.Our results indicate that none of the analogues of cassine (1) and spectaline (3) prepared performed better than the parent compounds in the inhibition assay of AChE ee at 100 µM.On the other hand, compounds 10c, 12b and 13a displayed smaller inhibition constants than cassine (1) while only 10c stood as a more potent inhibitor than spectaline (3) for BChE hu , pointing to the fact that deletion of the methyl group at C-2 (spectaline numbering) and the unsaturation in the side chain are beneficial for BChE hu inhibition, a feature that should be taken into consideration for future development of BChE hu inhibitors and structure-activity relationship studies.

Figure 2 .
Figure 2. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 10c BChEhu-ICER using the on-flow dual parallel enzyme assay.Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.

Figure 3 .
Figure 3. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 1 BChE hu -ICER using the on-flow dual parallel enzyme assay.Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.

Figure 3 .
Figure 3. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 1 BChEhu-ICER using the on-flow dual parallel enzyme assay.Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.

Figure 4 .
Figure 4. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 12b BChEhu-ICER using the on-flow dual parallel enzyme assay.Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.

Figure 4 .
Figure 4. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 12b BChE hu -ICER using the on-flow dual parallel enzyme assay.Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.

Figure 4 .
Figure 4. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 12b BChEhu-ICER using the on-flow dual parallel enzyme assay.Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.

Figure 5 .
Figure 5. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 13a BChE hu -ICER using the on-flow dual parallel enzyme assay.Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.

Table 1 .
Results of the studies about the inhibition of AChE ee -ICER and BChE hu -ICER activities by heterocyclic compounds 10a

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.

Table 1 .
Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by [6]ChEee and BChEhu standard inhibitor ** value reported in reference[6]; 1 SEM: standard error of the mean; IC50: required compound concentration to achieve 50%; ND: not determined.