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Article

Synthesis and Anticholinesterase Evaluation of Cassine, Spectaline and Analogues

1
Institute of Chemistry, University of Campinas (UNICAMP), Campinas 13083-970, Brazil
2
Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, Brazil
*
Author to whom correspondence should be addressed.
Sci. Pharm. 2022, 90(4), 63; https://doi.org/10.3390/scipharm90040063
Received: 18 August 2022 / Revised: 25 September 2022 / Accepted: 27 September 2022 / Published: 13 October 2022

Abstract

:
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 (AChEee) and human butyrylcholinesterase (BChEhu) 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 BChEhu inhibition with compounds 10c (Ki = 5.24 μM), 12b (Ki = 17.4 μM), 13a (Ki = 13.2 μM) and 3 (Ki = 11.3 μM) displaying the best inhibitory activities.

Graphical Abstract

1. Introduction

(-)-Cassine (1) is a piperidine alkaloid first isolated from Cassia excelsa leaves in 1964, [1] and later from Senna spectabilis (syn Cassia spectabilis), species occurring mainly in tropical and subtropical areas of the planet [2]. Among the alkaloids obtained from S. spectabilis, (-)-cassine (1) was isolated as the major component, together with (-)-spectaline (3) [3]. Due to the difficulties associated with the separation of these homologous structures, many biological studies were initially undertaken on this alkaloid mixture or on their semi-synthetic analogues [3,4,5]. The reported biological activities of mixtures of (-)-cassine (1) and (-)-spectaline (3) include antimalarial, schistosomicidal, antiproliferative, antinociceptive, antiviral, anti-inflammatory, analgesic, leishmanicidal and cholinesterase-inhibitory [4,5,6,7,8,9,10,11,12,13,14].
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].
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.

2. Materials and Methods

2.1. Synthetic Methods

General: Dichloromethane (DCM) and triethylamine (Et3N) 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. BF3·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 (KMnO4), 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 1H, 63 MHz 13C), Bruker Avance III 400 (400 MHz 1H, 101 MHz 13C) or Bruker Avance III 500 (500 MHz 1H, 126 MHz 13C) 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) CDCl3 1H RMN = 7.26, 13C RMN = 77.16; (2) methanol-d4: 1H RMN = 3.31, 13C 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.
N-(furan-2-ylmethyl)-2-nitrobenzenesulfonamide (5a) [24]. Commercially available furfurylamine (0.70 mL, 8.0 mmol, 1.0 eq) was dissolved in THF:H2O (v/v 1:1, 80.0 mL), followed by the addition of NaHCO3 (2.00 g, 24.0 mmol, 3.00 eq) and 2-nitrobenzenesulfonyl chloride (NsCl) (2.20 g, 9.60 mmol, 1.20 eq) at room temperature. The reaction mixture was stirred for 2 h and extracted with EtOAc (3 × 50.0 mL). The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO2, hexanes/EtOAc 0% to 30%, 10% increases) yielding 5a as a white solid (2,0g, 7,3 mmol, 92% yield).
TLC: (hexanes: EtOAc = 7:3), Rf = 0,4 (UV, KmnO4 or p-ASD)
1H NMR (500 MHz, CDCl3) δ 8.07 - 7.99 (m, 1 H), 7.86 - 7.79 (m, 1 H), 7.72 - 7.63 (m, 2 H), 7.07 (dd, J = 0.9, 1.8 Hz, 1 H), 6.16 - 6.07 (m, 2 H), 5.86 (t, J = 5.8 Hz, 1 H), 4.35 (d, J = 6.1 Hz, 2 H)
13C NMR (126 MHz, CDCl3) δ 149.2, 147.8, 142.7, 134.1, 133.5, 132.9, 131.2, 125.4, 110.4, 108.7, 40.8
IR (cm−1, thin film, ATR) 3298, 1530, 1360, 1323, 1157, 1044, 856, 700
HRMS (ESI) calculated for C11H10N2O5Sna [M+Na]+: 305.0208; found 305.0226
N-(1-(furan-2-yl)ethyl)-2-nitrobenzenesulfonamide (5b):
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, BF3·OEt2 (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%).
1H NMR (400 MHz, CDCl3) δ 7.69 - 7.63 (m, 2 H), 7.53 - 7.44 (m, 3 H), 7.42 - 7.30 (m, 4 H), 7.24 (s, 2 H), 6.33 (br s, 1 H), 6.23 (br s., 1 H), 4.55 (s, 2 H)
13C NMR (101 MHz, CDCl3) δ 170.2, 153.9, 141.8, 139.7, 136.5, 130.4, 128.8, 128.7, 128.2, 128.0, 110.4, 106.5, 51.2
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 NaHCO3 solution and extracted with Et2O. The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, 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 Et2O. The aqueous phase was neutralized with solid K2CO3 until pH 7–8 and THF (19 mL), NaHCO3 (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 Na2SO4, filtered and concentrated under reduced pressure. The material was purified by flash column chromatography (SiO2, hexanes/EtOAc 0% to 40%, 10% increases) yielding 5b as a white solid (795 mg, 2.68 mmol, 70% yield).
TLC: (hexanes: EtOAc = 7:3), Rf = 0.5 (UV, p-ASD)
MP: 62.5–64.6 °C
1H NMR (250 MHz, CDCl3) δ 8.05 - 7.89 (m, 1 H), 7.86 - 7.75 (m, 1 H), 7.71 - 7.55 (m, 2 H), 7.01 - 6.91 (m, 1 H), 6.11 - 5.96 (m, 2 H), 5.81 (d, J = 8.8 Hz, 1 H), 4.83 - 4.60 (m, 1 H), 1.53 (d, J = 7.0 Hz, 3 H)
13C NMR (63 MHz, CDCl3) δ 153.4, 147.5, 142.0, 134.3, 133.3, 132.9, 131.0, 125.3, 110.1, 106.6, 48.3, 20.9
IR (cm−1, thin film, ATR) 1538, 1415, 1355, 1338, 1167, 1157, 735
HRMS (ESI) calculated for C12H12N2O5sNa [M+Na]+: 319.0365; found 319.0309
6-allyl-1-((2-nitrophenyl)sulfonyl)-1,6-dihydropyridin-3(2H)-one (7a): To a solution of compound 5a (1.4 g, 5.0 mmol, 1.0 eq) in THF:H2O (v/v 4:1, 50.0 mL), was added NaHCO3 (842 mg, 10.0 mmol, 2.00 eq), NaOAc (410 mg, 5.00 mmol, 1.00 eq) and N-bromosuccinimide (899 mg, 5.00 mmol, 1.00 eq) at 0 °C. The reaction was kept at this temperature under magnetic stirring for 30 min or until total consumption of starting material was achieved (TLC analysis). After this period, the reaction was quenched by addition of saturated NaHCO3 solution (20.0 mL), saturated with Na2S2O3 (20.0 mL) and extracted with EtOAc (3 × 20.0 mL). The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to the next step without purification.
The material obtained previously was dissolved in dry MeCN (50 mL, 0,1 M) under an N2 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 NaHCO3 solution (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The material was purified by column chromatography (SiO2, hexanes/EtOAc 0% to 50%, 10% increases) yielding 7a as a light yellow solid (972 mg, 3.00 mmol, 60% yield, 2 steps).
TLC: (hexanes: EtOAc = 7:3), Rf = 0,33 (UV, kMnO4)
MP: 108–111 °C
1H NMR (400 MHz, CDCl3) δ 8.00 (dd, J = 1.7, 7.6 Hz, 1 H), 7.69 (dquin, J = 1.7, 7.5 Hz, 2 H), 7.65 - 7.60 (m, 1 H), 7.04 (dd, J = 5.1, 10.5 Hz, 1 H), 6.03 - 5.93 (m, 1 H), 5.82 (tdd, J = 7.2, 10.0, 17.1 Hz, 1 H), 5.24 - 5.10 (m, 2 H), 4.80 - 4.71 (m, 1 H), 4.32 (d, J = 18.6 Hz, 1 H), 4.02 (d, J = 18.6 Hz, 1 H), 2.64 - 2.51 (m, 2 H)
13C NMR (126 MHz, CDCl3) δ 191.0, 149.8, 147.9, 134.3, 132.5, 132.4, 132.2, 131.2, 127.0, 124.6, 119.7, 54.0, 49.7, 37.4
IR (cm−1, thin film, ATR) 1693, 1542, 1439, 1358, 1261, 1165, 1126, 1048, 993, 920, 852, 778, 743, 730, 675
HRMS (ESI) calculated for C14H15N2O5S [M+H]+: 323.0702; found 323.0699
6-allyl-2-methyl-1-((2-nitrophenyl)sulfonyl)-1,6-dihydropyridin-3(2H)-one (7b): To a solution of compound 5b (593 mg, 2.00 mmol, 1.00 eq) in THF:H2O (v/v 4:1, 20 mL) was added NaHCO3 (337 mg, 4.00 mmol, 2.00 eq), NaOAc (164 mg, 2.00 mmol, 1.00 eq) and N-bromosuccinimide (360 mg, 2.00 mmol, 1.00 eq) at 0 °C. The reaction was kept at this temperature under magnetic stirring for 30 min or until total consumption of starting material was achieved according to TLC. After this period, the reaction was quenched by addition of saturated NaHCO3 solution (10 mL), saturated with Na2S2O3 (10mL) and extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to the next step without purification.
The material obtained in the previous step was solubilized in dry MeCN (20 mL, 0,1 M) under an N2 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 NaHCO3 solution (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The material was purified by column chromatography (SiO2, hexanes/EtOAc 0% to 50%, 10% increases) yielding 7b as a solid (414 mg, 1.23 mmol, 61% yield, 2 steps).
TLC: (hexanes: EtOAc = 7:3), Rf = 0,5 (UV, p-ASD)
MP: 90.8–91.8 °C
1H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 7.5 Hz, 1 H), 7.76 - 7.65 (m, 2 H), 7.62 (d, J = 8.8 Hz, 0 H), 7.06 (dd, J = 5.0, 10.7 Hz, 1 H), 5.98 (dd, J = 1.3, 10.7 Hz, 0 H), 5.94 - 5.84 (m, 1 H), 5.26 - 5.14 (m, 2 H), 4.77 - 4.64 (m, 1 H), 4.37 (q, J = 7.3 Hz, 1 H), 2.79 (td, J = 6.8, 13.4 Hz, 1 H), 2.65 - 2.50 (m, 1 H), 1.59 (d, J = 7.5 Hz, 4 H)
13C NMR (126MHz, CDCl3) δ 194.1, 148.9, 148.1, 134.2, 133.0, 132.6, 132.1, 131.4, 124.8, 124.8, 119.5, 57.4, 54.1, 42.1, 21.6
IR (cm−1, thin film, ATR) 1675, 1535, 1358, 1170
6-allyl-1-((2-nitrophenyl)sulfonyl)-1,2,3,6-tetrahydropyridin-3-ol (8a): Compound 7a (754 mg, 2.35 mmol, 1.00 eq) was dissolved in methanol (45 mL), then CeCl3·7H2O (1,2 g, 3.0 mmol, 1.3 eq) was added. After a homogeneous solution was formed, the reaction mixture was cooled to −78 °C and NaBH4 (148 mg, 3.50 mmol, 1.50 eq) was added. After 20 min, TLC analysis showed complete conversion of starting material and the reaction mixture was allowed to reach room temperature. The reaction was quenched by addition of HCl 0,5M solution (20 mL) and extracted with DCM (3 × 20 mL). The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The material was purified by column chromatography (SiO2, hexanes/EtOAc 0% to 50%, 10% increases) yielding 8a as a white solid (560 mg, 2,20 mmol, 74% yield).
TLC: (hexanes: EtOAc = 1:1), Rf = 0,46 (UV, kMnO4 or p-ASD)
MP: 82–85 °C
1H NMR (500 MHz, CDCl3) δ 8.04 (dd, J = 1.7, 7.5 Hz, 1 H), 7.69 (dquin, J = 1.7, 7.4 Hz, 2 H), 7.64 - 7.61 (m, 1 H), 5.84 - 5.78 (m, 1 H), 5.78 - 5.66 (m, 2 H), 5.06 (dd, J = 1.6, 17.1 Hz, 1 H), 5.04 - 4.97 (m, 1 H), 4.39 (br s., 1 H), 4.17 - 4.09 (m, 1 H), 4.05 (dd, J = 6.1, 13.7 Hz, 1 H), 2.98 (dd, J = 9.9, 13.7 Hz, 1 H), 2.45 - 2.35 (m, 2 H)
13C NMR (126 MHz, CDCl3) δ 147.9, 134.0, 133.8, 133.5, 132.0, 130.8, 130.3, 129.0, 124.4, 118.6, 62.9, 54.0, 45.3, 38.9
IR (cm−1, thin film, ATR) 3496, 1539, 1330, 1163, 1151, 939, 741
HRMS (ESI) calculated for C14H15N2O5SK [M+K]+: 363.0417; found 363.0411
6-allyl-2-methyl-1-((2-nitrophenyl)sulfonyl)-1,2,3,6-tetrahydropyridin-3-ol (8b): Compound 7b (414 mg, 1.23 mmol, 1.00 eq) was dissolved in methanol (25 mL), then CeCl3·7H2O (602 mg, 1.60 mmol, 1.30 eq) was added. After a homogeneous solution was formed, the reaction mixture was cooled to −78 °C and NaBH4 (78 mg, 1.8 mmol, 1.5 eq) was added. After 1 h, TLC analysis showed complete conversion of starting material and the reaction mixture was allowed to reach room temperature. The reaction was quenched by addition of HCl 0,5M solution (10 mL) and extracted with DCM (3 × 15 mL). The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The material was purified by column chromatography (SiO2, hexanes/EtOAc 0% to 70%, 10% increases) yielding 8b as an oil (302 mg, 0.89 mmol, 72% yield).
TLC: (hexanes: EtOAc = 1:1), Rf = 0,40 (UV and p-ASD)
1H NMR (250 MHz, CDCl3) δ 8.11 - 7.96 (m, 1 H), 7.75 - 7.65 (m, 2 H), 7.65 - 7.54 (m, 1 H), 5.97 - 5.74 (m, 2 H), 5.64 - 5.50 (m, 1 H), 5.20 - 5.11 (m, 1 H), 5.09 (s, 1 H), 4.40 - 4.24 (m, 1 H), 4.24 - 4.12 (m, 1 H), 4.09 (br s, 1 H), 2.78 - 2.57 (m, 1 H), 2.40 (ddd, J = 8.7, 9.5, 13.5 Hz, 1 H), 1.75 (d, J = 5.1 Hz, 1 H), 1.28 (d, J = 6.7 Hz, 3 H)
13C NMR (63 MHz, CDCl3) δ 148.0, 134.4, 133.8, 133.8, 131.9, 131.2, 127.2, 126.8, 124.5, 118.5, 65.6, 53.5, 50.6, 42.2, 14.8
IR (cm−1, thin film, ATR) 3530, 1542, 1371, 1169, 1139, 1125, 1020, 996, 757
HRMS (ESI) calculated for C15H19N2O5S [M+H]+: 339.1009; found 339.1001
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 Na2SO4, filtered and concentrated under reduced pressure. The material was purified by column chromatography (SiO2, hexanes/EtOAc 0% to 4%, 2% increases).
Ethyl 2-acetyldec-9-enoate (S-IV): The title compound was prepared according to general procedure A using 8-bromo-1-octene (0.7 mL, 4.0 mmol, 1.0 eq). Yield of 71%, colorless oil.
TLC: (hexanes: EtOAc = 9:1), Rf = 0,43 (p-ASD)
1H NMR (400 MHz, CDCl3) δ 5.78 (tdd, J = 6.7, 10.3, 17.0 Hz, 1 H), 4.97 (qd, J = 1.6, 17.1 Hz, 1 H), 4.91 (dd, J = 1.4, 10.2 Hz, 1 H), 4.23 - 4.14 (m, 2 H), 3.38 (t, J = 7.4 Hz, 1 H), 2.20 (s, 3 H), 2.07 - 1.96 (m, 2 H), 1.90 - 1.75 (m, 2 H), 1.39 - 1.28 (m, 6 H), 1.28 - 1.23 (m, 5 H).
13C NMR (101 MHz, CDCl3) δ 203.3, 169.9, 139.0, 114.2, 61.2, 59.9, 33.6, 29.1, 28.7 (3x) 28.1, 27.3, 14.1)
IR: (cm−1, thin film, ATR) 2928, 2856, 1740, 1716, 1241, 1148, 909
Ethyl 2-acetyldodec-11-enoate (S-V): The title compound was prepared according to general procedure A using 10-bromo-decene (0.83 mL, 4.00 mmol, 1.00 eq). Yield was 72%, colorless oil.
TLC: (hexanes: EtOAc = 9:1), Rf = 0,43 (p-ASD)
1H NMR 1H NMR (500MHz, CDCl3) δ 5.77 (tdd, J = 6.7, 10.3, 17.0 Hz, 1 H), 4.95 (qd, J = 1.7, 17.1 Hz, 1 H), 4.89 (td, J = 1.1, 10.1 Hz, 1 H), 4.21 - 4.12 (m, 2 H), 3.36 (t, J = 7.5 Hz, 1 H), 2.19 (s, 3 H), 2.04 - 1.95 (m, 2 H), 1.88 - 1.73 (m, 2 H), 1.39 - 1.29 (m, 2 H), 1.29 - 1.18 (m, 13 H)
13C NMR 13C NMR (126 MHz, CDCl3) δ 203.2, 169.9, 139.0, 114.1, 61.2, 59.9, 33.7, 29.2, 29.2, 29.2, 29.0, 28.8, 28.6, 28.1, 27.3, 14.0
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 H2O (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 Na2SO4, filtered and concentrated under reduced pressure. The material was purified by column chromatography (SiO2, hexanes/EtOAc 5% to 10%, 1% increases).
Undec-10-en-2-one (B) (CAS 36219-73-5) [25]: the title compound was prepared according to general procedure B using S-IV (103 mg, 0.43 mmol, 1.00). Yield was 69% (50.0 mg, 0.23 mmol), colorless oil.
TLC: (hexanes: EtOAc = 9:1), Rf = 0,53 (p-ASD)
1H NMR 1H NMR (500MHz, CDCl3) δ 5.79 (dd, J = 10.3, 17.1 Hz, 1 H), 5.01 - 4.89 (m, 2 H), 2.40 (t, J = 7.5 Hz, 2 H), 2.12 (s, 3 H), 2.05 - 1.99 (m, 2 H), 1.59 - 1.51 (m, 2 H), 1.39 - 1.34 (m, 2 H), 1.33 (br s., 1 H), 1.31 - 1.21 (m, 6 H)
13C NMR (126 MHz, CDCl3) δ 209.2, 139.0, 114.1, 43.7, 33.7, 29.8, 29.2, 29.1, 28.9, 28.8, 23.8
Tridec-12-en-2-one (C) (CAS 60437-21-0) [26]: the title compound was prepared according to general procedure B using S-V (609 mg, 2.57 mmol, 1.00). Yield was 80% (609 mg, 2.57 mmol), colorless oil.
TLC: (hexanes: EtOAc = 9:1), Rf = 0,50 (p-ASD)
1H NMR (400 MHz, CDCl3) δ 5.79 (tdd, J = 6.7, 10.3, 17.0 Hz, 1 H), 4.97 (qd, J = 1.7, 17.1 Hz, 1 H), 4.94 − 4.87 (m, 1 H), 2.40 (t, J = 7.5 Hz, 2 H), 2.12 (s, 3 H), 2.07 - 1.97 (m, 2 H), 1.61 - 1.49 (m, 2 H), 1.42 - 1.30 (m, 3 H), 1.26 (s, 11 H)
13C NMR (101MHz, CDCl3) δ 209.3, 139.2, 114.1, 43.8, 33.8, 29.8, 29.4, 29.4, 29.3, 29.1, 29.1, 28.9, 23.8
General Procedure C (Cross-metathesis reaction)
To a mixture of hydroxypiperidine (8a or 8b, 1.0 eq) and unsaturated methyl ketone (5.0 eq) in DCM (sufficient for 0.05 M) was added Hoveyda–Grubbs II catalyst 7.5 mol%, portion-wise. The reaction mixture was kept under reflux for 24 h, allowed to reach room temperature and treated with DMSO (3,75 eq) for 12 h under magnetic stirring. The solvent was removed under reduced pressure and the residue was purified by column chromatography (SiO2, hexanes/Et2O 0% to 100%, 10% increases).
7-(5-hydroxy-1-((2-nitrophenyl)sulfonyl)-1,2,5,6-tetrahydropyridin-2-yl)hept-5-en-2-one (E/Z mixture) (9a): the title compound was prepared according to general procedure C using 8a (130 mg, 0.40 mmol, 1.00 eq), commercially available 5-hexen-2-one (234 µL, 2.00 mmol, 5.00 eq) and Hoveyda–Grubbs II catalyst (19.40 mg, 0.030 mmol, 0.075 eq). Yield was 81% (128 mg, 0.324 mmol).
TLC: (hexanes: EtOAc = 1:1), Rf = 0,17 (UV or p-ASD)
1H NMR (400MHz, CDCl3) δ 8.06 - 8.00 (m, 1 H), 7.73 - 7.65 (m, 2 H), 7.65 - 7.60 (m, 1 H), 5.82 - 5.74 (m, 1 H), 5.74 - 5.67 (m, 1 H), 5.47 - 5.33 (m, 2 H), 4.41 - 4.33 (m, 1 H), 4.06 - 3.95 (m, 2 H), 2.56 - 2.44 (m, 2 H), 2.44 - 2.19 (m, 5 H), 2.16 - 2.10 (m, 3 H) (Major isomer)
13C NMR (101MHz, CDCl3) δ 209.2, 148.0, 134.1, 133.7, 132.6, 132.0, 130.9, 130.7, 128.8, 126.2, 124.4, 62.8, 54.1, 45.5, 42.7, 37.8, 30.3, 26.6
IR (cm−1, thin film, ATR) 3421, 1708, 1543, 1371, 1164, 971
HRMS (ESI) calculated for C18H22N2O6SK [M+K]+: 433.0836; found 433.0804
12-(5-hydroxy-1-((2-nitrophenyl)sulfonyl)-1,2,5,6-tetrahydropyridin-2-yl)dodec-10-en-2-one (E/Z mixture) (9b): the title compound was prepared according to general procedure C using 8a (114 mg, 0.35 mmol, 1.00 eq), methyl ketone B (294 mg, 1.75 mmol, 5.00 eq) and Hoveyda–Grubbs II catalyst (17.0 mg, 0.026 mmol, 0.075 eq). Yield was 68% (111.0 mg, 0.2380 mmol).
TLC: (Et2O, 100%), Rf = 0,43 (UV or p-ASD
1H NMR (400MHz, CDCl3) δ 8.03 - 7.97 (m, 1 H), 7.71 - 7.58 (m, 3 H), 5.82 - 5.68 (m, 2 H), 5.48 - 5.37 (m, 1 H), 5.36 - 5.24 (m, 1 H), 4.32 (br s, 1 H), 4.14 - 3.93 (m, 2 H), 3.04 - 2.89 (m, 1 H), 2.45 - 2.27 (m, 4 H), 2.12 (s, 3 H), 1.99 - 1.85 (m, 2 H), 1.61 - 1.45 (m, 2 H), 1.34 - 1.28 (m, 1 H), 1.25 (br s, 7 H)
13C NMR (101MHz, CDCl3) δ 210.0, 147.8, 134.7, 134.0, 133.6, 131.9, 130.6, 130.2, 128.9, 124.6, 124.3, 62.7, 54.2, 45.2, 43.8, 37.7, 32.4, 29.9, 29.1(×2), 29.0, 23.8, 23.8 (×2)
IR (cm−1, thin film, ATR) 3427, 2928, 2855, 1706, 1543, 1371, 1165, 970, 851, 745
HRMS (ESI) calculated for C23H32N2O6SNa [M+Na]+: 487.1879; found 487.1858
14-(5-hydroxy-1-((2-nitrophenyl)sulfonyl)-1,2,5,6-tetrahydropyridin-2-yl)tetradecadec-12-en-2-one (E/Z mixture) (9c): the title compound was prepared according to general procedure C using 8a (97 mg, 0.3 mmol, 1.0 eq), methyl ketone C (297 mg, 1.5 mmol, 5.0 eq) and Hoveyda–Grubbs II catalyst (9.700 mg, 0.015 mmol, 0.075 eq). Yield was 82% (121.0 mg, 0.246 mmol).
TLC: (Et2O, 100%), Rf = 0,43 (UV or p-ASD
1H NMR (400MHz, CDCl3) δ 8.03 - 7.97 (m, 1 H), 7.71 - 7.58 (m, 3 H), 5.82 - 5.68 (m, 2 H), 5.48 - 5.37 (m, 1 H), 5.36 - 5.24 (m, 1 H), 4.32 (br s, 1 H), 4.14 - 3.93 (m, 2 H), 3.04 - 2.89 (m, 1 H), 2.45 - 2.27 (m, 4 H), 2.12 (s, 3 H), 1.99 - 1.85 (m, 2 H), 1.61 - 1.45 (m, 2 H), 1.34 - 1.28 (m, 1 H), 1.25 (Br. s., 7 H)
13C NMR (101MHz, CDCl3) δ 210.1, 147.6, 134.6, 133.8, 133.5, 131.8, 130.4, 130.1, 128.6, 124.4, 124.1, 62.5, 54.1, 45.0, 43.6, 37.5, 32.3, 29.7 (×2), 29.2, 29.1, 29,0, 28.9 (×2), 23.7
IR (cm−1, thin film, ATR) 3417, 2923, 1706, 1544, 1370, 1165, 970, 744
HRMS (ESI) calculated for C25H37N2O6SK [M+H]+: 493.2372; found 493.2291
12-(5-hydroxy-6-methyl-1-((2-nitrophenyl)sulfonyl)-1,2,5,6-tetrahydropyridin-2-yl)dodec-10-en-2-one (E/Z mixture) (9d): the title compound was prepared according to general procedure C using 8b (102 mg, 0.30 mmol, 1.00 eq), methyl ketone B (255 mg, 1.50 mmol, 5.0 eq) and Hoveyda–Grubbs II catalyst (14.00 mg, 0.023 mmol, 0.075 eq). Brown oil, 57% yield (115.0 mg, 0.227 mmol).
TLC: (hexanes: EtOAc = 1:1), Rf = 0,23 (UV or p-ASD)
1H NMR (250 MHz, CDCl3) δ 8.09 - 8.00 (m, 1 H), 7.75 - 7.57 (m, 3 H), 5.87 - 5.76 (m, 1 H), 5.63 - 5.35 (m, 3 H), 4.33 - 4.20 (m, 1 H), 4.20 - 4.12 (m, 1 H), 4.09 (d, J = 4.9 Hz, 1 H), 2.68 - 2.55 (m, 1 H), 2.42 (t, J = 7.4 Hz, 2 H), 2.13 (s, 3 H), 2.08 - 1.92 (m, 2 H), 1.69 - 1.48 (m, 4 H), 1.42 - 1.17 (m, 11 H)
13C NMR (63 MHz, CDCl3) δ 209.9, 148.0, 134.7, 133.8, 133.7, 131.9, 131.1, 127.0, 126.8, 125.6, 124.4, 65.5, 54.0, 50.6, 43.9, 41.1, 32.6, 30.0, 29.3, 29.3, 29.2, 29.0, 23.9, 14.8
IR (cm−1, thin film, ATR) 2926, 2853, 1705, 1543, 1370, 1170, 1138, 757
HRMS (ESI) calculated for C24H35N2O6S [M+H]+: 479.2210; found 479.2207
14-(5-hydroxy-6-methyl-1-((2-nitrophenyl)sulfonyl)-1,2,5,6-tetrahydropyridin-2-yl)tetradec-12-en-2-one (E/Z mixture) (9e): the title compound was prepared according to general procedure C using 8b (117 mg, 0.350 mmol, 1.00 eq), methyl ketone C (343 mg, 1.73 mmol, 5.00 eq) and Hoveyda–Grubbs II catalyst (17.0 mg, 0.026 mmol, 0.075 eq). Yield was 66% (115 mg, 0.227 mmol).
TLC: (hexanes: EtOAc = 7:3), Rf = 0,33 (UV or p-ASD)
1H NMR (400 MHz, CDCl3) δ 8.04 - 8.00 (m, 1 H), 7.70 - 7.65 (m, 2 H), 7.62 - 7.58 (m, 1 H), 5.80 (td, J = 2.8, 10.6 Hz, 1 H), 5.60 - 5.36 (m, 3 H), 4.24 (m, 1 H), 4.15 (m, 1 H), 4.08 (br s, 1 H), 2.63 - 2.54 (m, 1 H), 2.40 (t, J = 7.5 Hz, 2 H), 2.32 (ddd, J = 7.9, 10.2, 13.2 Hz, 1 H), 2.12 (s, 3 H), 2.08 - 1.94 (m, 3 H), 1.55 (t, J = 6.8 Hz, 2 H), 1.39 - 1.18 (m, 15 H)
13C NMR (101 MHz, CDCl3) δ 209.8, 148.0, 134.8, 133.8, 133.7, 131.9, 131.1, 127.0, 126.8, 125.6, 124.4, 65.6, 54.0, 50.6, 43.9, 41.1, 32.6, 30.0, 29.5, 29.5, 29.3, 29.3, 29.2, 29.2, 24.0, 14.8
IR (cm−1, thin film, ATR) 3463, 2926, 2853, 1708, 1544, 1370, 1170, 1138, 1020, 778, 757
HRMS (ESI) calculated for C26H38N2O6SNa [M+Na]+: 529.2343; found 529.2339
General Procedure D (N-deprotection)
To a solution of N-nosylpiperidine in MeCN (sufficient for 0.05 M) was added K2CO3 (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 (SiO2, hexanes 100% to eliminate yellow compounds, then DCM:MeOH 0% to 10%, with 0,5% Et3N as additive).
7-(5-hydroxy-1,2,5,6-tetrahydropyridin-2-yl)hept-5-en-2-one (10a,E/Z mixture): the title compound was prepared according to general procedure D using 9a (66.00 mg, 0.096 mmol, 1.000 eq), K2CO3 (115 mg, 0.84 mmol, 5.00 eq) and benzenethiol (53 µL, 0.5 mmol, 3.0 eq). Yield was 82% (29.00 mg, 0.138 mmol).
TLC: (DCM:MeOH = 8:2), Rf 0.33= (KMnO4 or Dragendorff)
1H NMR (400 MHz, CDCl3) δ 5.97 −-5.91 (m, 1 H), 5.79 - 5.73 (m, 1 H), 5.57 - 5.46 (m, 1 H), 5.46 - 5.36 (m, 1 H), 3.98 - 3.91 (m, 1 H), 3.36 - 3.25 (m, 1 H), 3.13 (d, J = 12.8 Hz, 1 H), 3.01 (br s, 2 H), 2.92 (dd, J = 2.9, 12.8 Hz, 1 H), 2.51 (q, J = 6.8 Hz, 2 H), 2.29 (q, J = 6.9 Hz, 2 H), 2.25 - 2.15 (m, 2 H), 2.13 (s, 2 H)
13C NMR (101 MHz, CDCl3) δ 208.6, 133.6, 132.3, 128.1, 126.6, 62.2, 54.0, 50.4, 43.0, 38.2, 30.0, 26.7
IR (cm−1, thin film, ATR) 3353, 2917, 1708, 1436, 1362, 1041, 971, 734
HRMS (ESI) calculated for C12H20NO2 [M+H]+: 210. 1494; found 210. 1477
12-(5-hydroxy-1,2,5,6-tetrahydropyridin-2-yl)dodec-10-en-2-one (10b,E/Z mixture): the title compound was prepared according to general procedure D using 9b (110 mg, 0.24 mmol, 1.00 eq), K2CO3 (164 mg, 1.19 mmol, 5.00 eq) and benzenethiol (75 µL, 0.7 mmol, 3.0 eq). Yield was 84% (56.0 mg, 0.20 mmol).
TLC: (DCM: MeOH = 9:1), Rf = 0,33 (KMnO4 or Dragendorff)
1H NMR (500 MHz, CDCl3) δ 6.01 - 5.90 (m, 1 H), 5.77 (d, J = 10.1 Hz, 1 H), 5.60 - 5.51 (m, 1 H), 5.40 - 5.28 (m, 1 H), 4.69 (br s, 2 H), 4.01 (br s, 1 H), 3.54 (q, J = 7.2 Hz, 1 H), 3.44 - 3.33 (m, 1 H), 3.33 - 3.14 (m, 1 H), 3.05 - 2.91 (m, 1 H), 2.45 - 2.31 (m, 2 H), 2.11 (s, 3 H), 2.06 - 1.90 (m, 2 H), 1.62 - 1.49 (m, 2 H), 1.49 - 1.39 (m, 1 H), 1.37 - 1.14 (m, 7 H)
13C NMR (126 MHz, CDCl3) δ 209.4, 135.2, 131.2, 128.0, 124.5, 61.6, 53.9, 52.8, 50.0, 43.7, 37.4, 32.5, 29.8, 29.2, 29.0, 28.9, 23.7
IR (cm−1, thin film, ATR) 3330, 2925, 2853, 1711, 1438, 1361, 1038, 970, 749
HRMS (ESI) calculated for C17H30NO2 [M+H]+: 280.2277; found 280.2264
14-(5-hydroxy-1,2,5,6-tetrahydropyridin-2-yl)tetradec-12-en-2-one (10c,E/Z mixture): the title compound was prepared according to general procedure D using 9c (202 mg, 0.41 mmol, 1.00 eq), K2CO3 (283 mg, 2.05 mmol, 5.00 eq) and benzenethiol (130 µL, 1.2 mmol, 3.0 eq). Yield was 85% (107 mg, 0.35 mmol).
TLC: (DCM: MeOH = 9:1), Rf = 0,33 (KMnO4 or Dragendorff)
1H NMR (500 MHz, CDCl3) δ 5.94 - 5.84 (m, 1 H), 5.74 (d, J = 10.1 Hz, 1 H), 5.56 - 5.45 (m, 1 H), 5.39 - 5.26 (m, 1 H), 3.93 (br s, 1 H), 3.48 (br s, 2 H), 3.23 (t, J = 6.1 Hz, 1 H), 3.09 (d, J = 12.6 Hz, 1 H), 2.88 (dd, J = 2.8, 12.7 Hz, 1 H), 2.37 (t, J = 7.5 Hz, 2 H), 2.16 (t, J = 6.9 Hz, 1 H), 2.09 (s, 3 H), 2.03 - 1.90 (m, 2 H), 1.52 (t, J = 6.9 Hz, 2 H), 1.36 - 1.26 (m, 2 H), 1.22 (br s, 9 H)
13C NMR (126 MHz, CDCl3) δ 209.3, 134.6, 133.1, 127.8, 125.1, 77.3, 76.8, 62.1, 54.1, 50.2, 43.6, 38.2, 32.5, 29.7, 29.3, 29.2, 29.1, 29.0, 23.7
IR (cm−1, thin film, ATR) 3318, 2923, 2852, 1713, 1436, 1360, 1020, 969, 720
HRMS (ESI) calculated for C19H34NO2 [M+H]+: 308.2589; found 308.2502
12-(5-hydroxy-6-methyl-1,2,5,6-tetrahydropyridin-2-yl)dodec-10-en-2-one (10d,E/Z mixture): the title compound was prepared according to general procedure D using 9d (52.0 mg, 0.10 mmol, 1.00 eq), K2CO3 (70.0 mg, 0.51 mmol, 5.00 eq) and benzenethiol (33.0 µL, 0.31 mmol, 3.00 eq). Light yellow oil, 81% yield (26 mg, 0.09 mmol).
TLC: (DCM:MeOH = 9:1), Rf = 0.5 (p-ASD)
1H NMR (500 MHz, CDCl3) δ 5.96 (ddd, J = 2.3, 5.1, 9.9 Hz, 1 H), 5.74 (d, J = 9.9 Hz, 1 H), 5.55 −-5.46 (m, 1 H), 5.39 - 5.30 (m, 1 H), 3.66 (d, J = 5.0 Hz, 1 H), 3.35 (t, J = 6.6 Hz, 1 H), 2.85 (dq, J = 2.0, 6.5 Hz, 1 H), 2.39 (t, J = 7.5 Hz, 2 H), 2.29 - 2.12 (m, 3 H), 2.11 (s, 3 H), 2.05 - 1.95 (m, 2 H), 1.54 (t, J = 6.9 Hz, 2 H), 1.38 - 1.19 (m, 8 H), 1.19 - 1.11 (m, 3 H)
13C NMR (126 MHz, CDCl3) δ 209.5, 134.5, 133.8, 128.7, 125.7, 65.9, 55.8, 53.4, 43.9, 39.0, 32.7, 30.0, 29.5, 29.3, 29.2, 29.1, 23.9, 17.7
IR (cm−1, thin film, ATR) 337, 2925, 2853, 1713, 1359, 972
HRMS (ESI) calculated for C18H32NO2 [M+H]+: 294.2428; found 294.2426
14-(5-hydroxy-6-methyl-1,2,5,6-tetrahydropyridin-2-yl)tetradec-12-en-2-one (10e,E/Z mixture): the title compound was prepared according to general procedure D using 9e (90.0 mg, 0.18 mmol, 1.00 eq), K2CO3 (123 mg, 0.89 mmol, 5.00 eq) and benzenethiol (57.0 µL, 0.54 mmol, 3.00 eq). Light yellow solid, 92% yield (56 mg, 0.16 mmol).
TLC: (DCM: MeOH: NH4OH (27%) = 88:10:2), Rf = 0,5 (p-ASD)
1H NMR (250 MHz, CDCl3) δ 5.72 (dd, J = 1.4, 10.0 Hz, 1 H), 5.58 - 5.42 (m, 1 H), 5.41 - 5.23 (m, 1 H), 3.67 - 3.61 (m, 1 H), 3.32 (t, J = 6.7 Hz, 1 H), 2.82 (dq, J = 2.1, 6.5 Hz, 1 H), 2.38 (t, J = 7.4 Hz, 2 H), 2.22 - 2.06 (m, 6 H), 1.97 (q, J = 6.7 Hz, 2 H), 1.52 (t, J = 7.0 Hz, 2 H), 1.36 - 1.18 (m, 11 H), 1.14 (d, J = 6.6 Hz, 3 H)
13C NMR (63 MHz, CDCl3) δ 209.4, 134.4, 133.9, 128.7, 125.7, 77.7, 76.7, 65.9, 55.8, 53.4, 43.9, 39.0, 32.7, 29.9, 29.5, 29.4, 29.4, 29.2, 23.9, 17.7
IR (cm−1, thin film, ATR) 3402, 2923, 2852, 1714, 1462, 1359, 971, 718, 640
HRMS (ESI) calculated for C20H36NO2 [M+H]+: 322.2741; found 322.2736
General Procedure E (synthesis of compounds 11a, 11b, 11c)
To a solution of compound 10ac (1.0 eq) in EtOAc (sufficient for 0.1 M) at 0 °C, was added Boc2O (1.3 eq), and it was allowed to reach room temperature. After total consumption of starting material, according to TLC, Ac2O (2.0 eq), Et3N (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 Na2SO4, 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 NaHCO3 solution and extracted with EtOAc (3×). The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The material was purified by column chromatography (SiO2, DCM/MeOH 0% to 10%, 2% increases).
6-(6-oxohept-2-en-1-yl)-1,2,3,6-tetrahydropyridin-3-yl acetate (11a, E/Z mixture): the title compound was prepared according to general procedure E using 10a (21 mg, 0.1 mmol, 1.0 eq), Boc2O (30.0 µL, 0.13 mmol, 1.30 eq), Ac2O (19 µL, 0.2 mmol, 2.0 eq), Et3N (56 µL, 0.4 mmol, 4.0 eq) and DMAP (0.600 mg, 0.005 mmol, 0.050 eq). Yield was 48% (12 mg, 0.05 mmol).
1H NMR (400 MHz, CDCl3) δ 6.01 - 5.93 (m, 1 H), 5.93 - 5.86 (m, 1 H), 5.61 - 5.51 (m, 1 H), 5.50 - 5.39 (m, 1 H), 5.09 - 5.01 (m, 1 H), 3.32 (dt, J = 1.7, 6.5 Hz, 1 H), 3.25 - 3.18 (m, 1 H), 3.08 - 2.98 (m, 1 H), 2.90 (br s, 1 H), 2.56 - 2.48 (m, 2 H), 2.39 - 2.20 (m, 4 H), 2.17 - 2.12 (m, 3 H), 2.10 - 2.05 (m, 3 H)
13C NMR (101 MHz, CDCl3) δ 208.2, 170.7, 136.5, 132.6, 126.3, 123.7, 64.7, 53.5, 47.1, 43.1, 37.9, 29.9, 26.7, 21.3
IR (cm−1, thin film, ATR) 2920, 1728, 1715, 1370, 1238, 1024, 971
HRMS (ESI) calculated for C14H22NO3 [M+H]+: 252.1600; found 252.1616
6-(11-oxododec-2-en-1-yl)-1,2,3,6-tetrahydropyridin-3-yl acetate (11b,E/Z mixture): the title compound was prepared according to general procedure E using 10b (56.0 mg, 0.16 mmol, 1.0 eq), Boc2O (51.0 µL, 0.18 mmol, 1.30 eq), Ac2O (39 µL, 0.3 mmol, 2.0 eq), Et3N (113 µL, 0.65 mmol, 4.0 eq) and DMAP (1.200 mg, 0.008 mmol, 0.050 eq), resulting in 29% yield (18 mg, 0.06 mmol).
1H NMR (400 MHz, CDCl3) δ 6.00 (d, J = 10.3 Hz, 1 H), 5.92 - 5.84 (m, 1 H), 5.60 - 5.49 (m, 1 H), 5.45 - 5.31 (m, 1 H), 5.01 (br s, 1 H), 3.33 - 3.23 (m, 1 H), 3.17 (d, J = 13.9 Hz, 1 H), 2.99 (dd, J = 3.4, 14.0 Hz, 1 H), 2.40 (t, J = 7.5 Hz, 2 H), 2.32 −-2.16 (m, 2 H), 2.16 - 2.09 (m, 3 H), 2.09 - 2.03 (m, 4 H), 2.03 - 1.90 (m, 2 H), 1.62 - 1.48 (m, 2 H), 1.38 - 1.20 (m, 8 H)
13C NMR (101 MHz, CDCl3) δ 209.3, 170.7, 137.3, 134.5, 125.3, 123.5, 65.1, 53.7, 47.4, 43.7, 38.2, 32.5, 29.8, 29.3, 29.2, 29.1, 28.9, 23.8, 21.3
IR (cm−1, thin film, ATR) 2926, 2853, 1731, 1715, 1433, 1369, 1237, 1026, 968
HRMS (ESI) calculated for C19H32NO3 [M+H]+: 322.2382; found 322.2375
6-(13-oxotetradec-2-en-1-yl)-1,2,3,6-tetrahydropyridin-3-yl acetate (11c,E/Z mixture): the title compound was prepared according to general procedure E using 10c (60.0 mg, 0.16 mmol, 1.0 eq), Boc2O (50.0 µL, 0.18 mmol, 1.30 eq), Ac2O (38 µL, 0.3 mmol, 2.0 eq), Et3N (110 µL, 0.65 mmol, 4.0 eq) and DMAP (1.200 mg, 0.008 mmol, 0.050 eq), resulting in 34% yield (18 mg, 0.06 mmol).
1H NMR (400 MHz, CDCl3) δ 5.99 (d, J = 10.1 Hz, 1 H), 5.93 - 5.83 (m, 1 H), 5.61 - 5.49 (m, 1 H), 5.43 - 5.31 (m, 1 H), 5.00 (br s, 1 H), 3.32 - 3.21 (m, 1 H), 3.16 (d, J = 14.1 Hz, 1 H), 2.99 (dd, J = 3.3, 14.1 Hz, 1 H), 2.40 (t, J = 7.5 Hz, 2 H), 2.30 - 2.16 (m, 2 H), 2.12 (s, 3 H), 2.09 - 2.02 (m, 3 H), 1.99 (q, J = 7.0 Hz, 2 H), 1.92 - 1.80 (m, 1 H), 1.65 - 1.48 (m, 2 H), 1.39 - 1.29 (m, 2 H), 1.26 (br s, 10 H)
13C NMR (101 MHz, CDCl3) δ 209.3, 170.7, 137.5, 134.5, 125.4, 123.7, 123.5, 65.2, 53.7, 47.5, 43.7, 38.3, 32.6, 29.8, 29.4, 29.3, 29.2, 29.1, 29.1, 23.8, 21.3
IR (cm−1, thin film, ATR) 2918, 2849, 1731, 1716, 1369, 1238, 1025, 968, 719
HRMS (ESI) calculated for C21H36NO3 [M+H]+: 350.2695; found 350.2684
General Procedure F (catalytic hydrogenation)
To a solution of compound 10a–e (1 eq) in AcOEt (sufficient for 0.1 M) under N2 atmosphere was added Pd(OH)2 20%/C (20 mol%). Then, the atmosphere was changed to H2 (1 atm) and the reaction was left stirring overnight. After this period, the mixture was filtered through a pad of Celite and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (SiO2, isocratic DCM: MeOH: NH4OH, 88:10:2).
7-(5-hydroxypiperidin-2-yl)heptan-2-one (12a): the title compound was prepared according to general procedure F using 10a (31.0 mg, 0.15 mmol, 1.00 eq) and Pd(OH)2 (4.00 mg, 0.03 mmol, 0.20 eq). Isolated in 47% yield (15.0 mg, 0.07 mmol).
TLC: (DCM: MeOH = 8:2), Rf = 0,33 (p-ASD)
1H NMR (500 MHz, CDCl3) δ 3.89 (br s, 1 H), 3.77 (br s, 2 H), 3.12 (d, J = 12.1 Hz, 1 H), 2.81 (d, J = 12.1 Hz, 1 H), 2.55 (Br. s., 1 H), 2.42 (t, J = 7.3 Hz, 2 H), 2.16 - 2.08 (m, 3 H), 1.85 (br s, 1 H), 1.59 - 1.45 (m, 5 H), 1.43 - 1.24 (m, 5 H)
13C NMR (126 MHz, CDCl3) δ 209.2, 63.7, 56.7, 51.9, 43.5, 35.9, 30.6, 29.9, 29.1, 26.0, 25.4, 23.6
IR (cm−1, thin film, ATR) 3353, 2915, 2851, 1704, 1448, 1163, 1074
HRMS (ESI) calculated for C12H24NO2 [M+H]+: 214.1807; found 214.1793
12-(5-hydroxypiperidin-2-yl)dodecan-2-one (12b): the title compound was prepared according to general procedure F using 10b (62.0 mg, 0.22 mmol, 1.00 eq) and Pd(OH)2 (18.0 mg, 0.13 mmol). Isolated in 38% yield (24.0 mg, 0.08 mmol).
TLC: (CHCl3: MeOH = 9:1), Rf = 0,16 (p-ASD)
1H NMR (500 MHz, CDCl3) δ 3.83 (Br. s., 1 H), 3.03 (d, J = 12.1 Hz, 1 H), 2.77 (d, J = 11.9 Hz, 1 H), 2.51 - 2.44 (m, 1 H), 2.41 (t, J = 7.5 Hz, 2 H), 2.24 (d, J = 19.6 Hz, 2 H), 2.13 (s, 3 H), 1.87 - 1.79 (m, 1 H), 1.61 - 1.47 (m, 4 H), 1.47 - 1.38 (m, 1 H), 1.38 - 1.29 (m, 4 H), 1.27 (br s, 11 H)
13C NMR (126 MHz, CDCl3) δ 209.4, 63.9, 56.8, 52.0, 43.8, 36.3, 30.8, 29.8, 29.6, 29.5, 29.5, 29.4, 29.3, 29.1, 26.2, 25.6, 23.8
IR (cm−1, thin film, ATR) 3397, 2915, 2848, 1718, 1445, 1152, 962
HRMS (ESI) calculated for C17H34NO2 [M+H]+: 284.2589; found 284.2581
14-(5-hydroxypiperidin-2-yl)tetradecan-2-one (12c): the title compound was prepared according to general procedure F using 10c (107 mg, 0.35 mmol, 1.00 eq) and Pd(OH)2 (21.0 mg, 0.15 mmol). Isolated in 46% yield (50.0 mg, 0.16 mmol).
TLC: (DCM:MeOH = 8:2), Rf = 0,33 (p-ASD)
1H NMR (400 MHz, CDCl3) δ 3.80 (br s, 1 H), 3.01 (d, J = 12.1 Hz, 1 H), 2.75 (d, J = 11.9 Hz, 1 H), 2.54 (br s, 2 H), 2.49 - 2.42 (m, 1 H), 2.39 (t, J = 7.5 Hz, 2 H), 2.11 (s, 3 H), 1.81 (d, J = 13.3 Hz, 1 H), 1.57 - 1.40 (m, 4 H), 1.39 - 1.27 (m, 5 H), 1.23 (br s, 15 H)
13C NMR (126 MHz, CDCl3) δ 209.4, 64.3, 56.8, 52.4, 43.8, 36.8, 31.0, 29.8, 29.7, 29.5 (×3), 29.4, 29.3, 29.1, 26.8, 25.7, 23.8
IR (cm−1, thin film, ATR) 3329, 2923, 2852, 1715, 1439, 1358, 1163, 753
HRMS (ESI) calculated for C19H38NO2 [M+H]+: 312.2903; found 312.2885
12-(5-hydroxy-6-methylpiperidin-2-yl)dodecan-2-one [1, (±)-cassine]: the title compound was prepared according to general procedure F using 10d (25.0 mg, 0.08 mmol, 1.00 eq) and Pd(OH)2 (5.00 mg, 0.04 mmol). Light yellow solid, 87% yield (23.0 mg, 0.08 mmol).
TLC: (DCM:MeOH = 8:2), Rf = 0,4 (p-ASD)
MP: 66.6–67.9 °C
1H NMR (500 MHz, CDCl3) δ 3.53 (br s, 1 H), 2.75 (q, J = 6.4 Hz, 1 H), 2.57 - 2.49 (m, 1 H), 2.40 (t, J = 7.4 Hz, 2 H), 2.12 (s, 3 H), 1.91 - 1.85 (m, 1 H), 1.59 - 1.49 (m, 2 H), 1.49 - 1.46 (m, 1 H), 1.45 (dd, J = 2.2, 4.4 Hz, 1 H), 1.36 - 1.22 (m, 18 H), 1.09 (d, J = 6.4 Hz, 3 H)
13C NMR (126 MHz, CDCl3) δ 209.5, 68.1, 57.3, 55.9, 43.9, 37.1, 32.2, 30.0, 29.9, 29.7, 29.6, 29.5, 29.5, 29.3, 26.2, 25.9, 24.0, 18.8
IR (cm−1, thin film, ATR) 2919, 2850, 1708, 1472, 1425, 1357, 1161, 993
HRMS (ESI) calculated for C18H36NO2 [M+H]+: 298.2741; found 298.2739
14-(5-hydroxy-6-methylpiperidin-2-yl)tetradecan-2-one [3, (±)-spectaline]: the title compound was prepared according to general procedure F using 10e (53 mg, 0.16 mmol, 1.00 eq) and Pd(OH)2 (10.0 mg, 0.07 mmol). Light yellow solid, 90% yield (48.0 mg, 0.15 mmol).
TLC: (DCM:MeOH = 8:2), Rf = 0,4 (p-ASD)
1H NMR (500 MHz, MeOD(d4)) δ 3.60 - 3.57 (m, 1 H), 2.76 (dq, J = 1.4, 6.7 Hz, 1 H), 2.60 - 2.53 (m, 1 H), 2.47 (t, J = 7.4 Hz, 2 H), 2.13 (s, 1 H), 2.12 - 2.10 (m, 1 H), 1.94 - 1.87 (m, 1 H), 1.67 - 1.59 (m, 1 H), 1.59 −-1.46 (m, 4 H), 1.44 - 1.32 (m, 6 H), 1.30 (br s, 15 H), 1.11 (d, J = 6.6 Hz, 3 H)
13C NMR (126 MHz, MeOD(d4)) δ 212.4, 68.2, 57.9, 56.4, 44.5, 37.6, 32.8, 31.0, 30.9, 30.8, 30.8, 30.8, 30.7, 30.4, 29.9, 27.0, 26.2, 25.0, 18.4
IR (cm−1, thin film, ATR) 2917, 2849, 1712, 1470, 1261, 1090, 993
HRMS (ESI) calculated for C20H40NO2 [M+H]+: 326.3054; found 326.3049
General Procedure G (synthesis of compounds 13a-c)
To a solution of compound 12ac 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 NaHCO3 saturated solution and extracted with EtOAc. The combined organic phases were washed with saturated NaCl solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The material was purified by column chromatography (SiO2, DCM/MeOH 0% to 10%, 2% increases).
6-(6-oxoheptyl)piperidin-3-yl acetate (13a): the title compound was prepared according to general procedure G using 12a (24.0 mg, 0,06 mmol, 1.00 eq) and AcCl (8.0 µL, 0,1 mmol, 1.8 eq). Yield was 63% (10.0 mg, 0.04 mmol).
TLC: (DCM:MeOH = 9:1), Rf = 0,26 (p-ASD)
1H NMR (500 MHz, CDCl3) δ 4.85 (br s, 1 H), 3.22 - 3.08 (m, 1 H), 2.84 (d, J = 13.8 Hz, 1 H), 2.51 (br s, 1 H), 2.41 (t, J = 7.4 Hz, 2 H), 2.12 (s, 2 H), 2.11 - 2.02 (m, 3 H), 1.96 (d, J = 14.5 Hz, 1 H), 1.69 - 1.51 (m, 4 H), 1.45 - 1.27 (m, 6 H), 1.24 (s, 2 H)
13C NMR (101 MHz, CDCl3) δ 208.8, 170.3, 67.0, 55.6, 48.8, 43.3, 35.9, 29.5, 28.8, 27.9, 26.9, 25.3, 23.3, 21.1
IR (cm−1, thin film, ATR) 2915, 2850, 1738, 1716, 1465, 1376, 1235, 1087, 1022, 668
HRMS (ESI) calculated for C14H26NO3 [M+H]+: 256.1913; found 256.1910
6-(11-oxododecyl)piperidin-3-yl acetate (13b): the title compound was prepared according to general procedure G using 12b (15.0 mg, 0,05 mmol, 1.00 eq) and AcCl (6.0 µL, 0,08 mmol, 1.80 eq). Yield was 58% (15.0 mg, 0.03 mmol).
TLC: (DCM:MeOH = 9:1), Rf = 0,4 (p-ASD)
1H NMR (500 MHz, CDCl3) δ 4.88 (br s, 1 H), 3.20 (d, J = 13.8 Hz, 1 H), 2.87 (d, J = 13.7 Hz, 1 H), 2.55 (br s, 1 H), 2.42 (t, J = 7.5 Hz, 2 H), 2.14 (s, 3 H), 2.11 (s, 3 H), 2.02 - 1.94 (m, 1 H), 1.70 - 1.51 (m, 4 H), 1.51 - 1.31 (m, 5 H), 1.27 (br s, 13 H)
13C NMR (126 MHz, CDCl3) δ 209.4, 170.7, 67.4, 56.1, 49.2, 43.8, 36.5, 29.9, 29.7, 29.5, 29.5, 29.4, 29.4, 29.2, 28.2, 27.2, 25.9, 23.9, 21.5
IR (cm−1, thin film, ATR) 2923, 2850, 1733, 1716, 1372, 1240, 1022, 668
HRMS (ESI) calculated for C19H36NO3 [M+H]+: 326.2695; found 326.2712
6-(13-oxotetradecyl)piperidin-3-yl acetate (13c): the title compound was prepared according to general procedure G using 12c (23.0 mg, 0,07 mmol, 1.00 eq) and AcCl (10.0 µL, 0,14 mmol, 1.80 eq). Yield was 57% (15.0 mg, 0.04 mmol).
TLC: (DCM:MeOH = 9:1), Rf = 0,46 (p-ASD)
1H NMR (400 MHz, CDCl3) δ 4.88 - 4.81 (m, 2 H), 3.15 (td, J = 2.3, 13.8 Hz, 2 H), 2.84 (dd, J = 2.0, 13.8 Hz, 2 H), 2.59 - 2.44 (m, 2 H), 2.40 (t, J = 7.5 Hz, 3 H), 2.12 (s, 5 H), 2.10 - 2.07 (m, 5 H), 2.00 −-1.90 (m, 3 H), 1.73 - 1.61 (m, 2 H), 1.61 - 1.51 (m, 6 H), 1.47 - 1.30 (m, 8 H)
13C NMR (101 MHz, CDCl3) δ 209.5, 170.9, 67.9, 56.2, 49.7, 44.0, 37.0, 30.0, 29.9, 29.7(×3), 29.6 (×2), 29.5, 29.3, 28.6, 27.8, 26.1, 24.0, 21.6
IR (cm−1, thin film, ATR) 2924, 2852, 1734, 1717, 1436, 1373, 1240, 1022, 668
HRMS (ESI) calculated for C21H40NO3 [M+H]+: 354.3008; found 354.3005

2.2. Biological Assays

Di- and trisubstituted piperidine derivatives (10ac, 11ac, 12ac, 13ac, 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 (AChEee, Sigma-Aldrich, S. Louis, MO, USA) and BChE from human serum (BChEhu) in order to obtain AChEee-ICER and BChEhu-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 AChEee-ICER enzymatic reaction for analysis in the MS. Meanwhile, the BChEhu-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 BChEhu-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].
The racemic form of the piperidine derivatives 10ac, 11ac, 12ac, 13ac and (±)-cassine (1) and (±)-spectaline (3) were solubilized in methanol to a stock solution of 1.00 mM for each compound. Galantamine was used as standard cholinesterase inhibitor.
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 (P0) of the inhibitor, according to the equation below, where (Pi) is the peak area of Ch that was produced in the presence of the tested compound and in the absence of the tested compound (P0), and Sb corresponds to Ch that was quantified during spontaneous ACh hydrolysis.
%   i n h i b i t i o n = [ 1 ( P i S b P 0 S b ) ] × 100
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 (IC50), the mechanism of action, and its steady-state inhibition constant (Ki) were determined for the compounds with %I ≥ 65% at 100 µM.
To obtain the IC50 value of each compound, stock solutions of compounds 10c (2.5–1000 μM), 12b (2.5–1.500 μM), 12c (2.5–2000 μM), 13a (2.5–1000 μM), 1 (2.5–1.500 μM) and 3 (2.5–2000 μM) were prepared in methanol. The reaction solutions were prepared by mixing 10 μL of compound 10c (final concentration 0.25–100 μM), 12b (0.25–150 μM), 12c (0.25–200 μM), 13a (0.25–100 μM), 1 (0.25–150 μM) and 3 (0.25–200 μM) with 20 μL of 70 μM ACh; the final volume of 100 μL was reached with 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 injected into the system. The percentage of inhibition (%I) was calculated by using the equation above.
To determine Ki, 20 μL of different AChEee solutions (10, 20, 50, 60 or 100 µM) containing 10 μL of one of the tested compounds at a fixed concentration (10c at 5, 10 or 20 µM, 12b at 10, 20 or 30 µM, 12c at 50, 60 or 70 µM, 13a at 7, 15 or 20 µM, 1 at 10, 30 or 40 µM or 3 at 10, 30 or 40 µM) were mixed. The final volume of 100 μL was reached with 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 injected into the system. Positive controls (presence of ACh at 10, 20, 50, 60 or 100 µM and absence of compound) were also analyzed.
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.

3. Results and Discussion

3.1. 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.
Starting from protected furfurylamine 5a, an aza-Achmatowicz rearrangement [34,35,36,37] provided hemiaminal 6a, which was not isolated but immediately submitted to a Hosomi–Sakurai allylation reaction catalyzed by Sn(OTf)2 [38], to yield piperidinone 7a. Next, Luche reduction [39] of piperidinone 7a stereoselectively furnished the key intermediate 8a. The same sequence was employed to prepare intermediate 8b (Scheme 1).
The entirely cis configuration of 8a was initially assigned by 1H NMR with 3JH5-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 A1,3-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].
From hydroxypiperidine 8a, a cross-metathesis reaction catalyzed by Hoveyda–Grubbs II catalyst with different unsaturated methyl ketones [40] provided compounds 9ae. Under mild N-deprotection conditions, compounds 9ae yielded nor-cassine and nor-spectaline analogues 10ac with 7, 12 and 14 carbons in the alkyl side chain, respectively. Natural product precursors 10d,e were synthesized accordingly [Scheme 2].
To prepare the remaining analogues, intermediates 10ac were subjected to a stepwise procedure to achieve selective O-acetylation, which yielded analogues 11ac. In parallel, intermediates 10ae underwent catalytic hydrogenation to provide saturated analogues 12ac and natural products (±)-cassine (1) and (±)-spectaline (3). Finally, analogues 13ac were obtained by selective O-acetylation of compounds 12ac, respectively (Scheme 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.

3.2. Cholinesterase-Inhibition Screening Assays Results

In this study, the capacity for cholinesterase inhibition (AChEee and BChEhu) 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 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).
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 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 12ac) 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 (11ac vs. 13ac).
Comparison of the bis-unsaturated piperidine derivatives 10ac and 11ac shows that O-acetylation appears to be beneficial regarding anticholinesterase activity, with 11ac inhibiting AChEee more extensively than 10ac; however, the same does not hold true for the inhibition of BChEhu. The O-acylation in the series of saturated piperidine derivatives (12ac vs. 13ac) 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 11ac had both modifications and did not reach the minimum inhibition of 65% for either AChEee or BChEhu. (Table 1).
All the compounds that presented inhibition ≥65% (10c, 12bc, 13a, 1, 3) had their IC50 values determined for BChEhu. Compound 10c was the most active (IC50 3.89± 1 μM) (Figure 2A), followed by the racemic natural product (±)-cassine (1) (IC50 18.1 ± 3 μM) (Figure 3A), 12b (IC50 23.3 ± 3 μM) (Figure 4A) and 13a (IC50 29.0 ± 4 μM) (Figure 5A). The least-active compounds were (±)-spectaline (3) (Figure S73A) and its analogue 12c (IC50 111 ± 16 μM) (Figure S72A), which was surprising because analogue 10c, which also displayed 14 carbons in the alkyl side chain, was the most active, suggesting that unsaturation played a role for this compound.
To further understand the inhibition activity of these compounds, the type of inhibition mechanism was determined (Figure 2, Figure 3, Figure 4 and Figure 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, VMax and KM values are affected. KM increases and VMax 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, KM value remains unchanged but VMax 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 (Ki = 5.24 µM), 3 (Ki = 11.3 µM), 12b (Ki = 17.4 µM) and 13a (Ki = 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 mixed-type inhibitors as observed in the double-reciprocal plots (Figure 1, Figure 2, Figure 3 and Figure 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].

4. Conclusions

As BChEhu is potentially a better target than the well-known AChE for the treatment of later-stage cognitive decline in AD, the discovery of BChEhu 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 BChEhu 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 AChEee 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 BChEhu, 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 BChEhu inhibition, a feature that should be taken into consideration for future development of BChEhu inhibitors and structure–activity relationship studies.

Supplementary Materials

The following are available at: https://www.mdpi.com/article/10.3390/scipharm90040063/s1. Figure S1. NOESY correlations for 8a and 8b (S1A and S1C) and coupling constant H5–H6 (S1A) for 8a observed by 1H RMN spectroscopy. B: Crystal structure of intermediate 8a. 1H- and 13C-NMR spectra of compounds 5a, S-I, 5b, 7a, 7b, 8a, 8b, S-IV, S-V, B, C, 9a-9e, 10a-e, 11a-c, 12a-c, 1, 3 and 13a-c and HSQC, COSY and NOESY NMR spectra of compound 8a and 8b shown in Figure S2–S71. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 12c and 3, respectively, in Figures S72 and S73, respectively.

Author Contributions

Conceptualization: M.C.R.S., C.L.C. and R.A.P.; Methodology: M.C.R.S., A.F.L.V., C.L.C. and R.A.P.; Investigation: M.C.R.S., A.F.L.V., C.L.C. and R.A.P.; Writing: M.C.R.S., C.L.C. and R.A.P.; Funding acquisition: C.L.C. and R.A.P. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are grateful to FAPESP (2016/12541-4; 2019/13104-5; PROEM 2014/50299-5; 2016/12541-4 and 2019/13104-5), the Centre of Excellence for Research in Sustainable Chemistry—CERSusChem (FAPESP/GSK2014/50249-8) and CNPq (grants 307500/2015-2; 306747/2020-0; 130980/2016-1, 307500/2015-2, 306747/2020-0) for all the financial support provided. A.F.L.V. and C.L.C acknowledge FAPESP for Ph.D. scholarship (grant number 2016/02873-0).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this manuscript are available upon request to the authors.

Conflicts of Interest

No potential conflicts of interest were reported by the authors. The authors declare no conflict of interest.

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Figure 1. (A): Chemical structures of natural products (-)-cassine (1), (-)-spectaline (3), O-acetylspectaline (4) and semi-synthetic derivative O-acetylcassine (2). (B): General structures of 3-hydroxypiperidines investigated in this work.
Figure 1. (A): Chemical structures of natural products (-)-cassine (1), (-)-spectaline (3), O-acetylspectaline (4) and semi-synthetic derivative O-acetylcassine (2). (B): General structures of 3-hydroxypiperidines investigated in this work.
Scipharm 90 00063 g001
Scheme 1. Synthesis of key intermediates 8a and 8b.
Scheme 1. Synthesis of key intermediates 8a and 8b.
Scipharm 90 00063 sch001
Scheme 2. Final steps in the synthesis of compounds 10a–10e.
Scheme 2. Final steps in the synthesis of compounds 10a–10e.
Scipharm 90 00063 sch002
Scheme 3. Final steps in the synthesis of compounds 12a–c, 13a–c, 1 and 3.
Scheme 3. Final steps in the synthesis of compounds 12a–c, 13a–c, 1 and 3.
Scipharm 90 00063 sch003
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 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.
Scipharm 90 00063 g002
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 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.
Scipharm 90 00063 g003
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. 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.
Scipharm 90 00063 g004
Figure 5. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 13a BChEhu-ICER using the on-flow dual parallel enzyme assay. Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.
Figure 5. Dose-response inhibition curve (A) and Lineweaver−Burk reciprocal plots (B) for compound 13a BChEhu-ICER using the on-flow dual parallel enzyme assay. Results obtained from three independent experiments (n = 3) expressed by mean ± SEM.
Scipharm 90 00063 g005
Table 1. Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by heterocyclic compounds 10a-c, 11a-c, 12a-c, 13a-c, 1 and 3.
Table 1. Results of the studies about the inhibition of AChEee-ICER and BChEhu-ICER activities by heterocyclic compounds 10a-c, 11a-c, 12a-c, 13a-c, 1 and 3.
Chemical StructuresAChEeeBChEhu
% Inhibition at 100 μM% Inhibition at 100 μMIC50 ± SEM 1 (μM)Ki (μM)Mechanism Type
Scipharm 90 00063 i0011001000.227 ± 0.002 **0.25 **Competitive **
Scipharm 90 00063 i0023.6127.9NDNDND
Scipharm 90 00063 i00314.852.2NDNDND
Scipharm 90 00063 i00429.178.43.89 ± 15.24Mixed
Scipharm 90 00063 i00513.511.9NDNDND
Scipharm 90 00063 i00627.157.4NDNDND
Scipharm 90 00063 i00753.350.2NDNDND
Scipharm 90 00063 i00832.218.4NDNDND
Scipharm 90 00063 i00935.374.423.2 ± 317.4Non-competitive
Scipharm 90 00063 i01032.665.1111 ± 1654.7Mixed
Scipharm 90 00063 i01160.385.929 ± 415.2Non-competitive
Scipharm 90 00063 i0120.4224.0NDNDND
Scipharm 90 00063 i01319.263.8NDNDND
Scipharm 90 00063 i01458.278.718.1 ± 330.3Mixed
Scipharm 90 00063 i01542.469.849.8 ± 411.3Mixed
* AChEee 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.
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Silva, M.C.R.; Vilela, A.F.L.; Cardoso, C.L.; Pilli, R.A. Synthesis and Anticholinesterase Evaluation of Cassine, Spectaline and Analogues. Sci. Pharm. 2022, 90, 63. https://doi.org/10.3390/scipharm90040063

AMA Style

Silva MCR, Vilela AFL, Cardoso CL, Pilli RA. Synthesis and Anticholinesterase Evaluation of Cassine, Spectaline and Analogues. Scientia Pharmaceutica. 2022; 90(4):63. https://doi.org/10.3390/scipharm90040063

Chicago/Turabian Style

Silva, Marcela C. R., Adriana F. L. Vilela, Carmen L. Cardoso, and Ronaldo A. Pilli. 2022. "Synthesis and Anticholinesterase Evaluation of Cassine, Spectaline and Analogues" Scientia Pharmaceutica 90, no. 4: 63. https://doi.org/10.3390/scipharm90040063

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