Abstract
Alzheimer's disease (AD) is a neurodegenerative disease that is characterized as the main dementia in the elderly. Eighteen pyrazolines were synthesized and evaluated for their inhibitory effects against acetylcholinesterase (AChE) in vitro. Possible interactions between pyrazolines and the enzyme were explored by in silico experiments. Compound 2B of the series was the most active pyrazoline with an IC50 value of 58 nM. Molecular docking studies revealed two important π–π interactions with residues Trp 286 and Tyr 341. A correlation between the HOMO-1 surface and AChE inhibition was observed. ADMET assays demonstrated a good profile for compound 2B. From the abovementioned findings, a new avenue of compound 2B analogues could be explored to develop anti-AD agents.
We report the synthesis and investigation of the anticholinesterase potential of pyrazolines, using experimental and theoretical techniques.
1. Introduction
Alzheimer's disease (AD), described in 1906 by the psychiatrist Alois Alzheimer, is a multifactorial complex neural disorder which results in cognitive impairment, loss of cholinergic neurons in synapses of the basal forebrain and neuronal death, with multifactorial pathogenesis.1,2 The current treatment of the disease, essentially symptomatic, is based on inhibitors of the enzyme acetylcholinesterase and memantine, which act on the glutamatergic system. A multipotent drug for AD has the potential to bind or inhibit multiple targets.3–5
AD has an estimated prevalence of 10 to 30% in the population over 65 years of age, with an incidence of 1 to 3%. Most patients with Alzheimer's disease (>95%) have the sporadic form, which is characterized by a late onset, between 80 and 90 years of age.6
Since AD is a disorder, whose main characteristic is the progressive loss of neurons, in the initial stage of the disease, the affected neurons act in the cholinergic pathways, extending to other areas of the brain as the disease progresses. From a macroscopic view of the disease, brain atrophy, narrower gyri, enlarged sulci, reduced brain weight, and enlarged ventricles can be observed.7 From a histological perspective, there are amyloid plaques in the extracellular space, especially around neuronal connections and in the walls of blood vessels, which are related to inflammatory processes and neuronal dysfunction. There are also changes in nerve cells, such as neurofibrillary tangles, that relate to changes in dendrites and axons.8
The pathophysiology is still not fully understood, as it is characterized as a multifactorial and highly complex disease. In the brain parenchyma of AD patients, there are histopathological aspects, such as amyloid fibrils, which are nothing more than fibrillar deposits on the walls of blood vessels, linked to a variety of types of senile plaques, phosphorylation of the TAU protein or accumulation of anomalous filaments of the TAU protein, which leads to the formation of neurofibrillary tangles, neuronal and synaptic damage, glial activation, and the inflammatory process.9
In the current scenario, anticholinesterases, whose mechanism of action is the inhibition of the enzyme acetylcholinesterase (AChE), are the most successful drugs developed, since they can potentiate cholinergic function, inducing improvement in the cognitive profile and also of some behavioural effects from the disease.10
The increasing impact of AD, together with the few safe therapeutic agents available, has determined a great effort to discover new AChE inhibitor drugs. Because of their several biological activities, pyrazolines have attracted increasing interest in the field of medicinal chemistry. Their antioxidant,11 anti-inflammatory,12 antimicrobial,13,14 antituberculosis,13 antibacterial15,16 and antitumoral17–20 properties are due to the presence of a heterocyclic ring and their structural variety.19,21
Pyrazolines are five-membered heterocyclic compounds containing two nitrogen atoms.22 The most widely used method for obtaining this class of compounds starts from an α-β-unsaturated ketone, commonly a chalcone, which is cyclized with a hydrazine derivative.23 There are a variety of techniques for synthesizing pyrazolines from chalcones, either in basic or acidic medium, using microwave radiation or ultrasonication.24
Although anticholinesterase agents have been exhaustively reported in the literature, in this present work we adopt modern strategies in Medicinal Chemistry, based on the integrated use of SBDD (structure-based drug design) and LBDD (ligand-based drug design) strategies,25 for which several correlation studies were carried out between experimental and theoretical methodologies, which will be described in the following sections.
2. Experimental
2.1. Synthesis and characterization of pyrazolines 1a–i and 2a–i
All reagents were obtained commercially (Sigma-Aldrich), and all solvents were of analytical grade and used without further purification. To obtain pyrazolines (Table 1, Fig. 1), we first synthesized their precursor chalcones by Claisen–Schmidt condensation of different benzaldehydes with p-phenyl acetophenone. Acetophenone (5 mmol) and benzaldehyde (5 mmol) were added to a round-bottom flask containing 20 mL of ethanol. The mixture was kept under magnetic stirring as 20 to 30 drops of 50% (w/v) sodium hydroxide were added. Then, the reaction was left under stirring at room temperature for 24 h. After this period, the reaction mixture was poured into distilled water and neutralized with 10% (v/v) hydrochloric acid. The chalcone was recovered as a precipitate by vacuum filtration.26 Shortly after, the synthesized chalcone was cyclized to pyrazoline. Briefly, 1 mmol chalcone and 3 mmol hydrazine in 10 mL of acetic acid were added to a flask and refluxed for 6 h. After this period, the reaction was poured onto ice, and the precipitate was filtered in vacuo. The product was purified by recrystallization using ethanol as solvent.17
Chemical structure of pyrazolines 1a–i and 2a–i.
|
|
||
|---|---|---|---|
| Compound | R′ | Compound | R′′ |
| 1a | Ph | 2a | 4-CH3–Ph |
| 1b | 2-NO2–Ph | 2b | 2-NO2–Ph |
| 1c | 3-NO2–Ph | 2c | 4-Br–Ph |
| 1d | 4-NO2–Ph | 2d | 3,4,5-triOCH3–Ph |
| 1e | 4-COOH–Ph | 2e | 2,4-diOCH3–Ph |
| 1f | 2,5-diOCH3–Ph | 2f | 2,5-diOCH3–Ph |
| 1g | 3,4-diOCH3–Ph | 2g | 3,4-OCH2O–Ph |
| 1h | 3,4-OCH2O–Ph | 2h | 1-Naphthyl |
| 1i | 2-Naphthyl | 2i | 2-Naphthyl |
Fig. 1. Synthesis of intermediate chalcones and pyrazolines 1a–i and 2a–i. Reagents and conditions: (i) ethanol, sodium hydroxide, r.t.; (ii) hydrazine, acetic acid, reflux.
The obtained compounds were analyzed by thin-layer chromatography using aluminum plates coated with silica gel. All synthesized compounds were also characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and melting point determination. Melting points were determined on a Microquimica MGAPF-301 apparatus. 1H and 13C NMR spectra were recorded on a Bruker Ac-200F (operating at 200 MHz for 1H and 50 MHz for 13C) or a Bruker Avance DRX 400 spectrometer (operating at 400 MHz for 1H and 100 MHz for 13C) using tetramethylsilane as internal standard. The structures were confirmed by mass spectrometry. High-resolution mass spectra (HRMS) were recorded on a micrOTOF-QII mass spectrometer (Bruker Daltonics) equipped with an automatic syringe pump (KD Scientific) for sample injection (constant flow of 3 μL min−1) in positive mode of electrospray ionization (ESI) (4.5 kV and 180 °C) using acetonitrile as solvent. The instrument was calibrated in the m/z range of 50 to 3000 using a low concentration tuning mix solution (Agilent Technologies). Data were processed using Bruker Data Analysis software version 4.0. When the calculated and experimental masses were compared, the error was within 2 ppm.
2.1.1. Data on the characterization of compounds 1a–i and 2a–i are presented below
(1a) 1-(3-([1,1′-Biphenyl]-4-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 20%; white solid; mp 167.3–169.0 °C; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.83 (2H, d, J = 8.0 Hz), 7.68–7.63 (4H, m), 7.49 (2H,m), 7.42–7.25 (6H, m), 5.63 (1H, dd, J = 12.0, 4.0 Hz), 3.80 (1H, dd, J = 20.0, 12.0 Hz), 3.21 (1H, dd, J = 16.0, 4.0 Hz), 2.46 (3H, s); 13C NMR (100 MHz, CDCl3) δ (ppm): 168.8, 153.5, 143.0, 141.9, 140.1, 130.3, 128.9 (2), 127.9, 127.7, 127.4, 127.0, 125.6, 60.0, 42.4, 22.0; HRMS (ESI-TOF) m/z: 341.1646 [M + H]+, calculated for C23H20N2O, 341.1648.
(1b) 1-(3-([1,1′-Biphenyl]-4-yl)-5-(2-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 23%; light-green solid; mp 196.7–198.1 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 8.13 (1H, d, J = 8.1 Hz), 7.81–7.85 (2H, m), 7.69–7.61 (5H, m), 7.48–7.27 (5H, m), 6.17 (1H, dd, J = 12.0, 6.0 Hz), 4.10 (1H, dd, J = 18.0, 12.0 Hz), 3.21 (1H, dd, J = 18.0, 6.0 Hz), 2.50 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.9, 154.0, 147.1, 143.3, 140.0, 137.1, 134.3, 129.9, 128.9, 128.4, 127.9, 127.4, 127.1, 126.4, 125.5, 57.1, 42.6, 21.8; HRMS (ESI-TOF) m/z: 386.1503 [M + H]+, calculated for C23H19N3O3, 386.1499.
(1c) 1-(3-([1,1′-Biphenyl]-4-yl)-5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 68%; yellow solid; mp 145.5–147.9 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 8.14 (1H, d, J = 8.0 Hz), 7.80 (2H, m), 7.69–7.42 (10H, m), 5.71 (1H, dd, J = 12.0, 6.0 Hz), 3.89 (1H, dd, J = 18.0, 12.0 Hz), 3.21 (1H, dd, J = 18.0, 6.0 Hz), 2.47 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 169.1, 153.3, 148.7, 143.9, 143.4, 140.0, 131.9, 129.9, 129.8, 128.9, 128.0, 127.4, 127.1, 127.0, 122.8, 120.9, 59.4, 42.2, 21.9; HRMS (ESI-TOF) m/z: 386.1498 [M + H]+, calculated for C23H19N3O3, 386.1499.
(1d) 1-(3-([1,1′-Biphenyl]-4-yl)-5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 71%; yellow solid; mp 153.6–156.8 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 8.21 (2H, d, J = 10.0 Hz), 7.82 (2H, d, J = 10.0 Hz), 7.70–7.66 (4H, m), 7.46–7.40 (5H, m), 5.69 (1H, dd, J = 12.0, 4.0 Hz), 3.87 (1H, dd, J = 18.0, 12.0 Hz), 3.19 (1H, dd, J = 18.0, 5.0 Hz), 2.47 (3H, s);13C NMR (50 MHz, CDCl3) δ (ppm): 169.0, 153.3, 148.8, 147.4, 143.4, 140.0, 129.7, 129.0, 128.0, 127.5, 127.1, 127.1, 126.7, 124.3, 59.5, 42.1, 21.9; HRMS (ESI-TOF) m/z: 386.1504 [M + H]+, calculated for C23H19N3O3, 386.1499.
(1e) 4-(3-([1,1′-Biphenyl]-4-yl)-1-acetyl-4,5-dihydro-1H-pyrazol-5-yl)benzoic acid. Yield: 48%; white solid; mp 205.7–208.0 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 8.03 (2H, d, J = 8.1 Hz), 7.83 (2H, d, J = 8.1 Hz), 7.69–7.62 (4H, m), 7.52–7.32 (5H, m), 5.68 (1H, dd, J = 12.0, 4.0 Hz), 3.84 (1H, dd, J = 18.0, 12.0 Hz), 3.20 (1H, dd, J = 18.0, 5.0 Hz), 2.50 (3H, s); HRMS (ESI-TOF) m/z: 385.1549 [M + H]+, calculated for C24H20N2O3, 385.1547.
(1f) 1-(3-([1,1′-Biphenyl]-4-yl)-5-(2,5-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 35%; yellow solid; mp 147.3–148.6 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.82–7.42 (10H, m, overlapping with CDCl3), 6.86–6.61 (3H,m), 5.85 (1H, dd, J = 12.0, 4.0 Hz), 3.83 (3H, s), 3.70 (1H, dd, J = 17.7 Hz, overlapping peak), 3.73 (3H, s), 3.06 (1H, dd, J = 18.0, 4.0 Hz), 2.50 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.8, 154.3, 153.8, 150.2, 142.8, 140.2, 130.6, 128.9, 127.8, 127.3, 127.0, 112.5, 112.2, 112.0, 56.1, 55.6 (2), 41.5, 22.0; HRMS (ESI-TOF) m/z: 401.18595 [M + H]+, calculated for C25H24N2O3, 401.18597.
(1g) 1-(3-([1,1′-Biphenyl]-4-yl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 57%; yellow solid; mp 115.5–117 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.83 (2H, d, J = 8.0 Hz), 7.69–7.62 (4H, m), 7.48–7.35 (4H, m), 6.81 (2H, s), 5.58 (1H, dd, J = 12.0, 4.0 Hz), 3.86 (3H, s), 3.84 (3H, s), 3.74 (1H, dd, J = 16.0, 12.0 Hz), 3.20 (1H, dd, J = 17.6, 4.4 Hz), 2.47 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.8, 153.5, 149.2, 148.4, 142.9, 140.0, 134.5, 130.3, 128.8, 127.9, 127.2, 126.9, 117.5, 111.5, 109.1, 59.7, 55.8(2), 42.3, 21.9; HRMS (ESI-TOF) m/z: 401.1858 [M + H]+, calculated for C25H24N2O3, 401.1860.
(1h) 1-(3-([1,1′-Biphenyl]-4-yl)-5-(benzo[d][1,3]dioxol-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 45%; yellow solid; mp 193.4–195.0 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.84–7.43 (9H, m), 6.73 (3H, m), 5.93 (2H, s), 5.54 (1H, dd, J = 12.0, 4.0 Hz), 3.76 (1H, dd, J = 18.0, 12.0 Hz), 3.18 (1H, dd, J = 18.0, 4.0 Hz), 2.45 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.8, 153.5, 148.1, 147.0, 143.0, 140.1, 135.9, 130.3, 128.9, 127.9, 127.3, 127.0, 119.1, 108.5, 106.0, 101.1, 59.8, 42.4, 22.0; HRMS (ESI-TOF) m/z: 385.1548 [M + H]+, calculated for C24H20N2O3, 385.1547.
(1i) 1-(3-([1,1′-Biphenyl]-4-yl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 58%; white solid; mp 145.4–146.3 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.88–7.27 (18H, m, overlapping with CDCl3), 5.80 (1H, dd, J = 12.0, 4.0 Hz), 3.86 (1H, dd, J = 18.0, 12.0 Hz), 3.28 (1H, dd, J = 18.0, 4.0 Hz), 2.49 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.9, 153.6, 143.1, 140.1, 139.1, 133.3, 132.9, 130.3, 129.1, 128.9, 128.0, 127.9, 127.6, 127.4, 127.1, 126.3, 125.9, 124.6, 123.4, 60.2, 42.4, 22.0; HRMS (ESI-TOF) m/z: 391.1807 [M + H]+, calculated for C27H22N2O, 391.1805.
(2a) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 86%; white solid; mp 109.1–110 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.66–7.20 (13H, m),5.61 (1H, dd, J = 11.5, 4.0 Hz), 3.73 (1H, dd, J = 18.0, 12.0 Hz), 3.18 (1H, dd, J = 18.0, 4.1 Hz), 2.44 (3H, s), 2.39 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.8, 154.0, 140.9, 140.8, 140.7, 140.6, 129.4, 128.7, 127.6, 127.2, 127.0, 126.5, 126.0, 59.6, 42.3, 21.9, 21.4; HRMS (ESI-TOF) m/z: 355.1804 [M + H]+, calculated for C24H22N2O, 355.1805.
(2b) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(2-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 89%; beige solid; mp 180.8–182.5 °C; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.88–7.35 (13H, m), 5.65 (1H, dd, J = 12.1, 4.0 Hz), 3.78 (1H, dd, J = 16.0, 12.1 Hz), 3.13 (1H, dd, J = 16.0, 4.0 Hz), 2.36 (3H, s); 13C NMR (100 MHz, CDCl3) δ (ppm):169.2, 150.6, 140.8, 140.6, 140.2, 132.4, 130.5, 130.0, 128.7, 127.7, 127.2, 127.0, 126.1, 126.0, 124.3, 60.0, 43.8, 21.9; HRMS (ESI-TOF) m/z: 386.1502 [M + H]+, calculated for C23H19N3O3, 386.1499.
(2c) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 41%; white solid; mp 175.5–176.7 °C; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.64–7.29 (13H, m), 5.65 (1H, dd, J = 12.1, 4.6 Hz), 3.77 (1H, dd, J = 16.0, 12.0 Hz), 3.19 (1H, dd, J = 17.9, 5.0 Hz), 2.45 (3H, s); 13C NMR (100 MHz, CDCl3) δ (ppm): 168.9, 152.7, 140.7, 140.6, 140.6, 131.9, 130.2, 128.7, 128.0, 127.7, 127.2, 127.0, 125.9, 124.6, 59.8, 42.1, 21.9; HRMS (ESI-TOF) m/z: 419.0753 [M + H]+, calculated for C23H19BrN2O, 419.0754.
(2d) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 33%; white solid; mp 177.0–178.4 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.57–7.29 (9H, m), 6.99 (2H, s), 5.66 (1H, dd, J = 12.0, 4.0 Hz), 3.93 (6H, s), 3.91 (3H, s), 3.80 (1H, dd, J = 18.0, 12.0 Hz), 3.21 (1H, dd, J = 17.8, 4.3 Hz), 2.47 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.8, 153.6, 153.4, 140.8, 140.7, 140.3, 128.7, 127.7, 127.3, 127.0, 126.8, 126.0, 104.0, 60.9, 59.8, 56.2, 42.4, 22.0; HRMS (ESI-TOF) m/z: 431.1966 [M + H]+, calculated for C26H26N2O4, 431.1965.
(2e) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 35%; beige solid; mp 143.2–144.1 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.93 (1H, d, J = 8.0 Hz), 7.56–7.53 (4H, m), 7.45–7.25 (5H, m), 6.60–6.48 (2H, m), 5.57 (1H, dd, J = 12.0, 4.0 Hz), 3.86 (3H, s), 3.82 (3H, s), 3.84 (1H, overlapping peak), 3.35 (1H, dd, J = 18.0, 4.0 Hz), 2.43 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.7, 162.7, 159.7, 154.0, 141.4, 140.9, 140.4, 130.2, 128.7, 127.6, 127.1, 126.2, 113.6, 105.6, 98.7, 59.5, 55.5, 55.4, 45.6, 22.0; HRMS (ESI-TOF) m/z: 401.1861 [M + H]+, calculated for C25H24N2O3, 401.1860.
(2f) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(2,5-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 37%; white solid; mp 156.0–157.0 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.57–7.31 (10H, m), 6.93 (2H, m), 5.60 (1H, dd, J = 12.0, 6.0 Hz), 3.94 (1H, dd, J = 20.0, 12.0 Hz), 3.85 (3H, s), 3.80 (3H, s), 3.38 (1H, dd, J = 20.0, 4.0 Hz), 2.44 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.9, 154.0, 153.6, 152.8, 141.2, 140.8, 140.4, 128.7, 127.6, 127.1(2), 126.1, 121.1, 117.3, 113.4, 113.0, 59.8, 56.0, 55.8, 45.5, 22.0; HRMS (ESI-TOF) m/z: 401.1862 [M + H]+, calculated for C25H24N2O3, 401.1860.
(2g) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(benzo[d][1,3]dioxol-5-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 80%; beige solid; mp 171.0–173.0 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.55–7.27 (10H, m), 7.10 (1H, d, J = 8.0 Hz), 6.83 (1H, d, J = 8.0 Hz), 6.02(2H, s), 5.62 (1H, dd, J = 12.0, 4.0 Hz), 3.72 (1H, dd, J = 18.0, 12.0 Hz), 3.14 (1H, dd, J = 18.0, 4.0 Hz), 2.43 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.8, 153.6, 149.6, 148.2, 140.8, 140.7, 140.6, 128.7, 127.7, 127.3, 127.1, 126.0, 125.7, 121.6, 108.2, 106.2, 101.5, 59.7, 42.2, 21.9; HRMS (ESI-TOF) m/z: 385.1549 [M+H]+, calculated for C24H20N2O3, 385.1547.
(2h) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(naphthalen-1-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 86%; white solid; mp 173.3–175.5 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 9.30 (1H, d, J = 8.0 Hz), 7.92–7.34 (15H, m), 5.63 (1H, dd, J = 11.3, 3.4 Hz), 3.98 (1H, dd, J = 18.0, 12.0 Hz), 3.39 (1H, dd, J = 18.0, 4.0 Hz), 2.54 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 169.0, 154.4, 140.8, 140.7, 134.1, 131.3, 130.6, 128.8, 128.7, 128.4, 127.7, 127.3, 127.1, 126.7, 126.4, 126.1, 124.8, 58.6, 44.9, 22.2; HRMS (ESI-TOF) m/z: 391.1806 [M + H]+, calculated for C27H22N2O, 391.1805.
(2i) 1-(5-([1,1′-Biphenyl]-4-yl)-3-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone. Yield: 83%; white solid; mp 163.6–164.6 °C; 1H NMR (200 MHz, CDCl3) δ (ppm): 8.12 (1H, d, J = 8.0 Hz), 7.96–7.84 (4H, m), 7.57–7.27 (11H, m), 5.71 (1H, dd, J = 12.0, 4.0 Hz), 3.91 (1H, dd, J = 18.0, 12.0 Hz), 3.36 (1H, dd, J = 18.0, 4.0 Hz), 2.52 (3H, s); 13C NMR (50 MHz, CDCl3) δ (ppm): 168.9, 153.9, 140.9, 140.8, 140.7, 134.2, 133.0, 129.0, 128.7, 128.5, 128.4, 127.9, 127.7, 127.3, 127.1, 126.7, 126.1, 123.3, 59.9, 42.3, 22.0; HRMS (ESI-TOF) m/z: 391.1806 [M + H]+, calculated for C27H22N2O, 391.1805.
The spectra of pyrazolines 1a–i and 2a–i can be viewed in the Supplementary Information section.
2.2. Acetylcholinesterase inhibitory activity
The enzymatic inhibition was measured using the method described previously.27 Briefly, 90 μL of 50 mmol L−1 Tris-HCl buffer pH 8.0, 30 μL of a buffer solution containing the compound (0.1 mg mL−1) dissolved in MeOH and 15 μL of an AChE solution containing 0.25 U mL−1, dissolved in 50 mmol L−1 Tris-HCl pH 8 buffer, containing 0.1% of bovine serum albumin were incubated for 15 min. Then, 25 μL of an acetylthiocholine iodide solution (15 mmol in water) and 140 μL of Ellman's reagent (3 mmol L−1 in Tris-HCl pH 8.0 buffer containing 0.1 mol L−1 NaCl and 0.02 mol L−1 MgCl2) were added, and the final mixture was incubated for another 30 min at 28 °C. The absorbance of the mixture was measured at 405 nm. The same solvent in which the sample was dissolved, considered to have 100% AChE activity, was used as negative control. The inhibition (%) was calculated as follows, eqn (1), in which Asample is the absorbance of the sample and Acontrol is the absorbance without the sample:
| I(%) = (100 − Asample/Acontrol)·100 | 1 |
2.3. Parallel artificial membrane permeability assays (PAMPA)
PAMPA was performed according to a previously described method.28 The filters of a 96-well filter plate were coated with 10 μL of a 1% (w/v) dodecane solution of phosphatidylcholine to mimic permeation through the gastrointestinal tract. Donor solutions of compounds 1a–i and 2a–i were prepared by diluting the DMSO stock solutions (1000 ppm) in 50% (v/v) phosphate buffered saline and stirring overnight. Then, 150 μL of donor solution was added to the filter plate wells, and 300 μL of acceptor solution (50% DMSO in phosphate buffer saline) was added to the receiver plate wells. The filter plate was coupled to the receiver plate, and samples were incubated in the dark for 5 h under agitation and then for a further 20 min at room temperature. Meanwhile, 150 μL of donor solution was added to 300 μL of acceptor solution to obtain the respective equilibrium solutions. After incubation, the filter plate and the receiver plate were separated, and donor, acceptor, and equilibrium solutions were analyzed by high-performance liquid chromatography with diode array detection. Experiments were performed in quadruplicate.
The apparent permeability (Papp) was calculated using eqn (2)29
 |
2 |
where Vd is the volume of the donor well (cm3), Va is the volume of the acceptor well (cm3), A is the area of the membrane (cm2), t is the incubation time (s), and r is the ratio of the acceptor solution concentration to the equilibrium solution concentration.
Membrane retention (MR) was calculated by the following equation (eqn (3)):
 |
3 |
where Ce is the compound concentration in the equilibrium solution, Ca is the compound concentration in the acceptor well, and Cd is the compound concentration in the donor well.
2.4. Computational studies
2.4.1. Molecular docking
Molecular docking simulations were performed with GOLD30 v.2020.2.0 and the ChemPLP scoring function.31 The receptor, human acetylcholinesterase (PDB 4M0E, 2.00 Å),32 was treated as rigid, and the compounds were treated as fully flexible. The preparation of the receptors was made within the GOLD suite. Only was used the chain A of both. No crystallographic water molecules were considered. The binding site was defined as all the receptor atoms up to 6 Å of the reference crystallographic inhibitor. At least 10 poses were generated for each ligand using the default parameters of the genetic algorithm. For analysis, the top-scoring conformations of the most populated clusters of poses/ligands were selected. Prior to the simulations, the ligands were optimized with a steepest descent algorithm (100 steps. FF: AM1BCC. The atomic charges were assigned with Ammp-Mom) in VEGA ZZ v.3.2.33 The propensity maps were generated with SuperStar, a module for knowledge-based pharmacophore generation and prediction of intermolecular interactions available within the GOLD suite. We worked with the default parameters for cavity detection and the PDB data, allowing R–H rotation and [O, N, S]–H bonds. The propensity map figure was generated with Hermes v.1.10.5 (also available within the GOLD suite). The receptor-ligand figures were generated with PyMOL v.1.8.34
2.4.2. Quantum studies
Molecular structure optimization and vibrational harmonic frequencies were calculated using DFT on Gaussian v.09 program package with B3LYP level and 6–311++G(d,p) basis sets.35
2.4.3. Density by Kernel (EDK)
A kernel function is created with the datum at its centre this ensures that the kernel is symmetric about the datum. Kernel density estimation smooths the contribution of data points to give overall picture of the density of data points, using the software Origin 2021b.36
2.4.4. In silico pharmacokinetics prediction
The structure of compounds was drawn using ChemDraw software (version Ultra 12.0, PerkinElmer Informatics, Waltham, MA, USA) and were converted into a single database file SMILES. In silico physicochemical and pharmacokinetics prediction were made using two different pkCSM37 and SwissADME.38
2.5. Statistical analysis
The IC50 data were obtained from a nonlinear regression fit curve of concentration log versus normalized response. The values were expressed as mean ± S.D. Analyses were performed with the software GraphPad Prism® version 7 (GraphPad Software Inc., San Diego, CA, USA), and differences were considered significant when p < 0.05 by one-way analysis of variance (ANOVA) and Mann–Whitney test.
3. Results and discussion
3.1. Synthesis
Synthesis of pyrazolines 1a–i and 2a–i was performed in two steps. First, a Claisen–Schmidt condensation afforded the precursor chalcone. Subsequently, a cyclization reaction was carried out with the pyrazoline, as shown in Fig. 1. Although hydrazine was used to perform cyclization, the final products were acetylated, as acetic acid was used as solvent. Acetylation occurred on the secondary nitrogen, forming acetyl pyrazolines.
This is the first report of compounds 1c, 1d, 1f, 1g, 1i, 2a, 2b, 2c, 2e, 2g, 2h, and 2i. Pyrazolines were obtained in 20–89% yields and were characterized according to their melting point, 1H and 13C NMR, and HRMS spectra.
1H NMR spectra of the obtained pyrazolines showed double doublet signals at region 3.06–3.39, 3.70–4.10, and 6.17–5.40 ppm, attributed to the characteristic Ha, Hb, and Hc of the heterocyclic ring (Table 1). The first signal, Ha, showed coupling constants of 8 and 5 Hz with Hb and Hc, respectively; the methylenic hydrogen Ha couples with Hb (geminal coupling) and Hc (vicinal coupling) with different intensities because they are diasterotopic hydrogens. Hb, in turn, couples with Ha, with a Jba of 18 Hz, and with Hc, with a Jbc of 12 Hz. Hc has the following coupling constants: Jcb of 12 Hz and Jca of 5 Hz. For all pyrazolines, there is a signal resonating as a singlet at 2.45 ppm, corresponding to an acetyl moiety. Moreover, aromatic hydrogens were observed at 7.0–9.0 ppm.
13C NMR spectra showed signals between 100.0 and 154.0 ppm, attributed to the resonance of aromatic carbons and non-aromatic sp2-hybridized carbons. The signals at 152.0, 59.0, and 44.0 ppm were consistent with carbon shifts of the pyrazoline ring. HMRS spectra gave expected cationic ions corresponding to the molecular formula of the obtained pyrazolines.
3.2. Inhibitory effect of the pyrazolines and structure–activity relationship studies
Over the decades, important efforts have been made to better understand the pathogenesis of AD, mainly in search for potential drugs for pharmacological treatment. The chemotherapy of this disease relies on AChE inhibitors. The developed drugs including tacrine, rivastigmine, donepezil, galantamine cause changes in central cholinergic function, by inhibiting enzymes that degrade ACh (AChE and butyrylcholinesterase), and subsequently causing an increase in the ability of acetylcholine to stimulate brain receptors. AChE inhibitors are the symptomatic treatment of choice for mild to moderate stages. Treatment with anticholinesterase agents consists of improving the transmission of nerve impulses in the synapse. The impediment of the hydrolysis of acetylcholine by AChE, by inhibition of the enzyme, is done by drugs that interact with the enzyme, allowing the maintenance of the concentration of the neurotransmitter (acetylcholine), during the processes of signal conduction to other neurons.
Inhibition of the enzyme acetylcholinesterase was verified for pyrazolines 1a–1i and 2a–2i (Table 2). Four drugs used in the pharmacological treatment of AD were used as standards. All the tested compounds showed inhibitory effects against AChE with IC50 values ranging between 0.052 and 1.947 μM. Compared to the most active standard drug, donepezil (IC50 value of 0.087 μM), two compounds (2b and 2i) of the series showed significant inhibitory effect with IC50 values of 0.052 and 0.068 μM, respectively. Tacrine and physostigmine, two other standard drugs inhibited AChE with IC50 values of 0.17 and 0.19 μM, respectively. None of the remaining pyrazolines were more potent than these two drugs, in contrast, their inhibitory effects were more potent than that of galantamine.
Comparison between the IC50 values of compounds related to inhibition of AChE and the scores obtained through the ChemPLP score function.
| Compound | IC50−inhibition of AchE (μmol L−1) | Score |
|---|---|---|
| 1a | 1328 | 82.422 |
| 1b | 0.640 | 87.822 |
| 1c | 1028 | 84.851 |
| 1d | 1556 | 79.831 |
| 1e | 1541 | 80.692 |
| 1f | 1421 | 81.686 |
| 1g | 1426 | 81.599 |
| 1h | 0.988 | 85.133 |
| 1i | 0.799 | 86.394 |
| 2a | 0.845 | 86.924 |
| 2b | 0.052 | 92.519 |
| 2c | 0.903 | 85.943 |
| 2d | 1947 | 77.492 |
| 2e | 1128 | 84.523 |
| 2f | 1705 | 79.426 |
| 2g | 0.913 | 85.720 |
| 2h | 1015 | 84.949 |
| 2i | 0.068 | 91.242 |
| Donepezil | 0.087 | 90.608 |
| Galantamine | 17 250 | 60.244 |
| Physostigmine | 0.190 | 63.616 |
| Tacrine | 0.178 | 62.143 |
Molecular docking studies were carried out to better understand the IC50 results and to properly conduct discussions on the structure–activity relationship., four different scoring functions of the GOLD program30 were used, namely: GoldScore, ChemScore, ChemPLP and ASP. Through the analysis of the results of the interaction calculation, it was evident that the ChemPLP function (Table 1) better represents the experimental results, and for this reason, this function was selected for the respective analyses. Factors such as the redocking result (Fig. 2) and the correlation between the experimental and computational results (Fig. 3), Adj.R-Square of 0.9901, support this choice.
Fig. 2. Overlays of molecular docking calculation results for the co-crystallized ligand (1YL604). The figure was created using the PyMol program.
Fig. 3. Correlation between experimental (IC50) and computational (score) results. Graphic obtained through the Origin program.
In this redocking pose (Fig. 2), three structures of the co-crystallized ligand (1YL604) are present: in “deep teal” it is the one coming from the crystallographic image, in “dirty violet” the structure coming from the calculation of the series 1a–1i in “deep olive” the structure derived from the calculation of series 2a–2i. The alignment between these structures is evident. Such alignment helps in understanding how well the computational results represent the experimental results.
Through the analysis of the 2D interaction diagram (Fig. 4), it is evident that, in addition to the interactions of the hydrogen bond type, a series of aromatic structures and hydrophobic side chains are present in the enzymatic cavity. Above all, it is important to mention the π–π interactions that occur between the structure of residue Trp 286 and the aromatic portion of the co-crystallized ligand. According to the computational results (Table 2), the compounds that presented the best scores, from both series, are: 1b (Fig. 5) and 2b (Fig. 6). It is interesting to note the similarity between the interactions of both compounds (1b and 2b) and the active site of the protein. In both situations, two π–π interactions are performed with the same residues Trp 286 and Tyr 341.
Fig. 4. 2D diagram of the interaction between the cofactor (1YL604) and the 4M0E protein enzymatic cavity. Diagram obtained through the maestro program.
Fig. 5. Intermolecular interactions of compound 1b in the enzymatic cavity of the AChE enzyme. The figure was prepared using PyMol.
Fig. 6. Intermolecular interactions of compound 2b in the enzymatic cavity of the AChE enzyme. The figure was prepared using PyMol.
In addition, each of these compounds performs a hydrogen bonding interaction and, in both cases, uses the nitro group portion of the compound. However, for compound 1b, residue Tyr 72 was used in this interaction and in the case of compound 2b the residue used was Ser 293.
Based on preliminary analyzes performed on the 2D interaction diagrams and on the fact that all compounds have the same base structure, an interaction pattern was evidenced in which at least one π–π interaction is established. This premise can be verified by observing the interaction of the least active compound of the two series, 2d. It is worth mentioning that the mentioned interaction pattern happens preferentially with the aromatic portion of residue Trp 286.
To rationalize the data obtained experimentally, the compounds were structurally studied through their physicochemical properties by the SwissADME platform (https://www.swissadme.ch), which consists of a simple and free quantitative correlation tool. The physicochemical properties are fundamental in the development of new drugs, since from these data it is possible to predict the behavior and the difficulties of the potential drug in transit to the active binding site.
Oral drugs with a good pharmacological profile generally obey Lipinski's five rules,39 a rule that establishes the following characteristics as ideal: molecular weight (MW) under 500; milogP less than 5; up to 5 hydrogen bond donors (nOHNH); less than 10 hydrogen bond acceptors (nON). According to Veber et al. (2002),40 the characteristics of a good drug candidate would be: number of rotatable bonds (nRotb) ≤10, (vi) Polar surface area (PSA) <140 Å2. Some of these physicochemical properties of the compounds are shown in Tables 3 and 4. To correlate the physical–chemical parameters obtained by the SwissADME platform, as molecular weight, num. heavy atoms, consensus Log P, num. aromatic heavy atoms and fraction of Csp3 were generated Kernel density maps (KDE).
First set of physicochemical properties of pyrazolines (1a–1i and 2a–2i) and anticholinesterase drugs.
| Compound | Molecular weight (g mol−1) | Num. heavy atoms | Num. aromatic heavy atoms | Fraction Csp3 | Num. rotatable bonds |
|---|---|---|---|---|---|
| 1a | 340.42 | 26 | 18 | 0.13 | 4 |
| 1b | 385.42 | 29 | 18 | 0.13 | 5 |
| 1c | 385.42 | 29 | 18 | 0.13 | 5 |
| 1d | 385.42 | 29 | 18 | 0.13 | 5 |
| 1e | 384.43 | 29 | 18 | 0.12 | 5 |
| 1f | 400.47 | 30 | 18 | 0.20 | 6 |
| 1g | 400.47 | 30 | 18 | 0.20 | 6 |
| 1h | 384.43 | 29 | 18 | 0.17 | 4 |
| 1i | 390.48 | 30 | 22 | 0.11 | 4 |
| 2a | 354.44 | 27 | 18 | 0.17 | 4 |
| 2b | 385.42 | 29 | 18 | 0.13 | 5 |
| 2c | 419.31 | 27 | 18 | 0.13 | 4 |
| 2d | 430.50 | 32 | 18 | 0.23 | 7 |
| 2e | 400.47 | 30 | 18 | 0.20 | 6 |
| 2f | 400.47 | 30 | 18 | 0.20 | 6 |
| 2g | 384.43 | 29 | 18 | 0.17 | 4 |
| 2h | 390.48 | 30 | 22 | 0.11 | 4 |
| 2i | 390.48 | 30 | 22 | 0.11 | 4 |
| Donepezil | 379.49 | 28 | 12 | 0.46 | 6 |
| Galantamine | 287.35 | 21 | 6 | 0.53 | 1 |
| Physostigmine | 276.35 | 20 | 6 | 0.53 | 3 |
| Tacrine | 276.35 | 20 | 6 | 0.53 | 3 |
Second set of physicochemical properties of pyrazolines (1a–1i and 2a–2i) and anticholinesterase drugs.
| Compound | Num. H-bond acceptors | Num. H-bond donors | Molar refractivity | TPSA (Å2) | Consensus log Po/w |
|---|---|---|---|---|---|
| 1a | 2 | 0 | 112.47 | 32.67 | 4.19 |
| 1b | 4 | 0 | 121.29 | 78.49 | 3.46 |
| 1c | 4 | 0 | 121.29 | 78.49 | 3.44 |
| 1d | 4 | 0 | 121.29 | 78.49 | 3.43 |
| 1e | 4 | 1 | 119.43 | 69.97 | 3.74 |
| 1f | 4 | 0 | 125.45 | 51.13 | 4.17 |
| 1g | 4 | 0 | 125.45 | 51.13 | 4.14 |
| 1h | 4 | 0 | 118.53 | 51.13 | 4.01 |
| 1i | 2 | 0 | 129.97 | 32.67 | 5.07 |
| 2a | 2 | 0 | 117.43 | 32.67 | 4.52 |
| 2b | 4 | 0 | 121.29 | 78.49 | 3.28 |
| 2c | 2 | 0 | 120.17 | 32.67 | 4.79 |
| 2d | 5 | 0 | 131.94 | 60.36 | 4.12 |
| 2e | 4 | 0 | 125.45 | 51.13 | 4.14 |
| 2f | 4 | 0 | 125.45 | 51.13 | 4.17 |
| 2g | 4 | 0 | 118.53 | 51.13 | 4.02 |
| 2h | 2 | 0 | 129.97 | 32.67 | 5.06 |
| 2i | 2 | 0 | 129.97 | 32.67 | 5.08 |
| Donepezil | 4 | 0 | 115.31 | 38.77 | 4.00 |
| Galantamine | 4 | 1 | 84.05 | 41.93 | 1.92 |
| Physostigmine | 2 | 2 | 85.90 | 46.01 | 0.64 |
| Tacrine | 2 | 2 | 85.90 | 46.01 | 0.64 |
Kernel density estimation is used to estimate the probability density in a dataset in a non-parameterized way. Kernel-estimated density fundamentally smooths a finite sample of data and follows the following formula:
 |
4 |
where X is an element of a sample set (i = 1…N), K is a Kernel function and h > 0 is the bandwidth that controls smoothing, in other words the influence of neighbors.
Most of approved small molecule CNS drugs have 27–37 heavy atoms,41 and among all oral drugs invented post-1950, 44% have molecular weight < 400.42 The vast majority of pyrazolines investigated in this work have molecular weight and number of heavy atoms in that range. In this sense, when analyzing Fig. 7, it is possible to observe that the pyrazolines investigated in this work have mostly between 29–30 heavy atoms and 18 heavy aromatic atoms.
Fig. 7. Kernel density map for correlation between “number of aromatic heavy atoms” and “numbers of heavy atoms” of pyrazolines (1a–1i and 2a–2i) and anticholinesterase drugs.
A good candidate for the inhibition of the acetylcholinesterase enzyme for the treatment of AD should include log P values between 0.8 and 4.9, polar surface area between 30.0 and 60.0 Å2, preferably be an aromatic system and have enough hydrogen acceptors and a small amount of hydrogen donors.43 In the series of pyrazolines investigated in this work, the compounds are very close to this ideality. The compounds have between 0 and 2 hydrogen donors, between 2–5 acceptors, 3.28–5.08 for consensus Log P and between 32.67–78.49 Å2 of polar topological area (TPSA) (Table 4).
All compounds in this work have 0 alerts for PAINS and compounds 1a–1h, 2b, 2d–2g respect all Lipinski rules (rule of five). All compounds comply with Veber's criteria regarding the number of rotatable links and topological area restrictions (<140 Å2).
Another common feature of acetylcholinesterase inhibitors is to have an HOMO-1 orbital energy between −8.60 and −6.00 eV.43 For the pyrazolines under analysis, the data for the HOMO-1 orbital, obtained through B3LYP/6-31 + G(d,p), are in agreement with the values observed in the literature, for 1b (Fig. 8), 2b and 2i (Fig. 9), which showed higher AChE inhibition activity and values for HOMO-1 in the range described by Nascimento et al. 2011.43
Fig. 8. HOMO-1 surface and energies for 1a–1i. Aqua represents positive contributions and fuchsia represents negative contributions.
Fig. 9. HOMO-1 surface and energies for 2a–2i. Aqua represents positive contributions and fuchsia represents negative contributions.
The ability of the synthesized compounds to cross gastrointestinal membranes was assessed in a PAMPA model (Table 5). Pyrazoline 2b had the best permeability result, with a Papp of 1.22 × 10−5 cm s−1 and an MR of 6.6%, followed by compound 2e (Papp of 1.00 × 10−5 cm s−1 and MR of 18.4%). Compound 2e (miLogP of 4.76) was more lipophilic than compound 2b (miLogP of 4.63), suggesting that it has a greater chance to be retained in the membrane.
Apparent permeability (Papp) and membrane retention (MR) of pyrazolines 1a–i and 2a–i in a parallel artificial membrane permeability assay–gastrointestinal tract absorption model.
| Compound | P app (cm s−1) | MR (%) |
|---|---|---|
| 1a | 9.15 × 10−6 | 30.7 |
| 1b | 6.90 × 10−6 | 47.6 |
| 1c | 9.42 × 10−6 | 27.7 |
| 1d | 7.85 × 10−6 | 32.6 |
| 1e | 2.13 × 10−8 | 15.0 |
| 1f | 6.94 × 10−6 | 38.3 |
| 1g | 9.02 × 10−6 | 9.2 |
| 1h | 8.75 × 10−6 | 33.7 |
| 1i | 3.36 × 10−6 | 59.7 |
| 2a | 4.27 × 10−6 | 44.3 |
| 2b | 1.22 × 10−5 | 6.6 |
| 2c | 4.90 × 10−6 | 49.0 |
| 2d | 7.89 × 10−6 | 9.5 |
| 2e | 1.00 × 10−5 | 18.4 |
| 2f | 6.87 × 10−6 | 36.2 |
| 2g | 8.44 × 10−6 | 17.0 |
| 2h | 5.47 × 10−6 | 35.8 |
| 2i | 4.43 × 10−6 | 56.9 |
The presence of an electron-withdrawing substituent (nitro group) in pyrazoline 2b is likely associated with its higher permeability compared to that of other compounds with electron-donor substituents, such as 2a and 2d–f containing methyl and methoxy groups. The permeability results of pyrazolines 2d (Papp of 7.89 × 10−6 cm s−1), 2e (Papp of 1.00 × 10−5 cm s−1), and 2f (Papp of 6.87 × 10−6 cm s−1) show that the number and position of methoxyl groups in the aromatic ring also influence permeability. Although compounds 2e and 2f had the same miLogP value (4.76), 2e showed better permeability and lower membrane retention.
Pyrazolines 2h and 2i were also composed of electron-withdrawing substituents (naphthyl groups), but their Papp values were lower than those of 2b. Such results can be attributed to the higher lipophilicity of 2h and 2i. Like the observed for pyrazolines 2a–i, among compounds 1a–i, 1c (which contains a nitro as the electron-withdrawing group) had the highest permeability value (Papp of 9.42 × 10−6 cm s−1). However, it is possible to observe that the position of the nitro group influenced permeability, as compounds 1b and 1d did not show the same permeability as compound 1c. Compound 1a showed the second-highest permeability value (Papp of 9.15 × 10−6 cm s−1). Pyrazoline 1g (Papp of 9.02 × 10−6 cm s−1) also showed good permeability, and, because of the positions of the methoxy groups, had a better permeability than compound 1f (Papp of 6.94 × 10−6 cm s−1). Of all tested compounds, pyrazoline 1e (Papp of 2.13 × 10−8 cm s−1) had the lowest permeability, which may be related to the presence of a carboxyl group.
In addition to the previously mentioned tests, a comparative study was carried out with ADMET with the most active compound (2b), and donepezil, the reference drug for the treatment of Alzheimer's disease, the results of which can be seen in Table 6.
Pharmacokinetics parameters for 2b and donepezil.
| Property | Model name | Compounds | |
|---|---|---|---|
| 2b | Donepezil | ||
| Absorption | Water solubility (log mol L−1) | −6.521 | −4.495 |
| Caco-2 permeability (log Papp in 10−6 cm s−1) | 0.336 | 1.584 | |
| Intestinal absorption – human (% absorbed) | 93.487 | 94.775 | |
| Skin permeability (log Kp) | −2.731 | −2.9 | |
| P-Glycoprotein substrate | Yes | Yes | |
| P-Glycoprotein I inhibitor | Yes | Yes | |
| P-Glycoprotein II inhibitor | Yes | Yes | |
| Distribution | Human ssVD (log L kg−1) | 0.043 | 0.938 |
| BBB permeability (log BB) | −0.453 | 0.428 | |
| CNS permeability (log PS) | −1.646 | −1.466 | |
| Metabolism | CYP2D6 substrate | No | No |
| CYP3A4 substrate | Yes | Yes | |
| CYP1A2 inhibitor | Yes | No | |
| CYP2C19 inhibitor | Yes | No | |
| CYP2C9 inhibitor | Yes | No | |
| CYP2D6 inhibitor | No | Yes | |
| CYP3A4 inhibitor | Yes | No | |
| Excretion | Total clearance (log mL min−1 kg−1) | 0.671 | 1.008 |
| Renal OCT2 substrate | No | Yes | |
| Toxicity | AMES toxicity | Yes | No |
| Human max. tolerated dose (log mg kg−1 per day) | 0.386 | −0.036 | |
| hERG I inhibitor | No | No | |
| hERG II inhibitor | Yes | Yes | |
| Oral rat acute toxicity LD50 (mol kg−1) | 2.722 | 2.999 | |
| Oral rat chronic toxicity LOAEL (log mg kg−1 bw per day) | 0.808 | 1.514 | |
| Hepatotoxicity | Yes | No | |
| Skin sensitization | No | No | |
| T pyriformis toxicity (log μg L−1) | 0.323 | 0.526 | |
| Minnow toxicity (log mM) | −2.027 | −2.194 | |
The two compounds have some properties in common, for example they are likely substrates of P-glycoprotein, an important biological barrier against toxins and xenobiotics in cells, and inhibitors I and II of P-glycoprotein, that is, they can be transported by it. Both have high permeability to CaCO2 (log Papp > 0.90), which means that the absorption potential of these two compounds orally is high. In addition, both have relatively low skin permeability, which occurs when log Kp > −2.5. Donepezil has a high steady-state volume of distribution (DVss) (log DVss > 0.45), while 2b is intermediate (log DVss between −0.15 and 0.45). Compound 2b as well as donepezil can penetrate the central nervous system, as they both have a log PS > −2.
Both compounds can act as a substrate and being metabolized by the CYP2D5 and CYP3A4 isoforms of cytochrome P450, however, they differ in terms of inhibition or not of the 5 isoforms of this cytochrome, which may influence interaction and toxicity parameters.
In reference to excretion parameters, 2b is unlikely to be an OCT2 renal substrate. Compound 2b has a drug clearance or total Clarence lower than the reference, which indicates that compound 2b remains in the body for a longer time. Regarding toxicity, 2b shows positive signalling for the AMES test, which indicates the mutagenic and consequently carcinogenic potential. However, the same compound has a higher recommended maximum tolerated dose window. Despite its potential promising result of enzymatic activity, compound 2b needs structural adjustments that enable the optimization of its pharmacokinetic parameters.
Conclusions
In this article we report the synthesis of a series of 18 pyrazolines, obtained by Claisen–Schmidt condensation and, subsequently, by reaction of cyclization in variable yields (20 to 89%). The anticholinesterase profile of this series of compounds was evaluated, and all the tested compounds showed inhibitory effects against AChE with IC50 values ranging between 0.052 and 1.947 μM. In vitro investigations of AChE inhibition showed a high correlation with molecular docking studies, especially for the ChemPLP scoring function (r2 = 0.9901). DFT studies were applied, which showed a correlation between the results of enzymatic inhibition and the energy of the HOMO-1 orbital, which are consistent with data in the literature.
Additionally, the physicochemical properties, the ADMET profile and the membrane permeability (PAMPA) of pyrazolines were investigated. The results encourage the use of compound 2b in molecular studies of optimization that can result in promising compounds in the development of drugs with anticholinesterase properties that can be used in the treatment of AD.
Author contributions
Valkiria Machado: investigation. Arthur R. Cenci: investigation. Kerolain F. Teixeira: investigation. Larissa Sens: investigation. Tiago Tizziani: investigation. Ricardo J. Nunes: supervision. Leonardo L. G. Ferreira: writing – review & editing. Rosendo A. Yunes: conceptualization, resources. Louis P. Sandjo: writing – review & editing. Adriano D. Andricopulo: supervision, writing – review & editing. Aldo S. de Oliveira: conceptualization, methodology, supervision, writing – review & editing.
Conflicts of interest
There are no conflicts to declare.
Supplementary Material
Acknowledgments
The authors thank the Department of Chemistry of the Federal University of Santa Catarina (UFSC), Brazil, and the Central Laboratory of Structural Biology (CEBIME) for providing equipment and allowing the use of some facilities. We also thank the Brazilian National Council for Scientific and Technological Development (CNPq) and the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES) for the scholarship and financial support.
Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2md00262k
References
- Kumar B. Dwivedi A. R. Arora T. Raj K. Prashar V. Kumar V. Singh S. Prakash J. Kumar V. ACS Chem. Neurosci. 2022;13:2122–2139. doi: 10.1021/acschemneuro.2c00132. [DOI] [PubMed] [Google Scholar]
- Kumar N. Kumar V. Anand P. Kumar V. Dwivedi A. R. Kumar V. Bioorg. Med. Chem. 2022;61:116742. doi: 10.1016/j.bmc.2022.116742. [DOI] [PubMed] [Google Scholar]
- Kumar B. Kumar V. Prashar V. Saini S. Dwivedi A. R. Bajaj B. Mehta D. Parkash J. Kumar V. Eur. J. Med. Chem. 2019;177:221–234. doi: 10.1016/j.ejmech.2019.05.039. [DOI] [PubMed] [Google Scholar]
- Ibrahim M. M. Gabr M. T. Neural Regener. Res. 2019;14:437–440. doi: 10.4103/1673-5374.245463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mangialasche F. Solomon A. Winblad B. Mecocci P. Kivipelto M. Lancet Neurol. 2010;9:702–716. doi: 10.1016/S1474-4422(10)70119-8. [DOI] [PubMed] [Google Scholar]
- Hayden E. Y. Teplow D. B. Alzheimer’s Res. Ther. 2013;5(60):1–11. doi: 10.1186/alzrt226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cenini G. Dowling A. L. S. Beckett T. L. Barone E. Mancuso C. Murphy M. P. LeVine H. Lott I. T. Schmitt F. A. Butterfield D. A. Head E. Biochim. Biophys. Acta, Mol. Basis Dis. 2012:130–138. doi: 10.1016/j.bbadis.2011.10.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen X. Q. Mobley W. C. Front. Neurosci. 2019:13. [Google Scholar]
- Knopman D. S. Amieva H. Petersen R. C. Chételat G. Holtzman D. M. Hyman B. T. Nixon R. A. Jones D. T. Nat. Rev. Dis. Primers. 2021;7:33. doi: 10.1038/s41572-021-00269-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Araújo C. R. M. Santos V. L. A. Gonsalves A. A. Rev. Virtual Quim. 2016;8(6):1818–1834. doi: 10.21577/1984-6835.20160122. [DOI] [Google Scholar]
- Raut D. G. Lawand A. S. Kadu V. D. Hublikar M. G. Patil S. B. Bhosale D. G. Bhosale R. B. Polycyclic Aromat. Compd. 2022;42(1):70–79. doi: 10.1080/10406638.2020.1716028. [DOI] [Google Scholar]
- Abdellatif K. R. A. Elshemy H. A. H. Azoz A. A. Bioorg. Chem. 2015;63:13–23. doi: 10.1016/j.bioorg.2015.09.002. [DOI] [PubMed] [Google Scholar]
- Harikrishna N. Isloor A. M. Ananda K. Obaid A. Fun H. K. New J. Chem. 2016;40:73–76. doi: 10.1039/C5NJ02237A. [DOI] [Google Scholar]
- Nisa S. Yusuf M. J. Heterocycl. Chem. 2020:1–13. [Google Scholar]
- Khan S. A. Asiri A. M. Kumar S. Sharma K. Eur. J. Chem. 2014;5(1):85–90. doi: 10.5155/eurjchem.5.1.85-90.789. [DOI] [Google Scholar]
- Viveka S. Shama P. Nagaraja G. K. Ballav S. Kerkar S. Eur. J. Med. Chem. 2015;101:442–451. doi: 10.1016/j.ejmech.2015.07.002. [DOI] [PubMed] [Google Scholar]
- Nepali K. Kadian K. Ojha R. Dhiman R. Garg A. Singh G. Buddhiraja A. Bedi P. M. S. Dhar K. L. Med. Chem. Res. 2012;21:2990–2997. doi: 10.1007/s00044-011-9824-9. [DOI] [Google Scholar]
- Karabacak M. Altintop M. D. Çiftçi H. I. Koga R. Otsuka M. Fujita M. Özdemir A. Molecules. 2015;20:19066–19084. doi: 10.3390/molecules201019066. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chouiter M. I. Boulebd H. Pereira D. M. Valentão P. Andrade P. B. Belfaitah A. Silva A. M. S. Future Med. Chem. 2020;12(6):493–509. doi: 10.4155/fmc-2019-0342. [DOI] [PubMed] [Google Scholar]
- Nawaz F. Alam O. Perwez A. Rizvi M. A. Naim M. J. Siddiqui N. Pottoo F. H. Jha M. Arch. Pharm. 2020:1–18. doi: 10.1002/ardp.201900262. [DOI] [PubMed] [Google Scholar]
- Lévai A. Silva A. M. S. Pinto D. C. G. A. Cavaleiro J. A. S. Alkorta I. Elguero J. Jekö J. Eur. J. Org. Chem. 2004;22:4672–4679. doi: 10.1002/ejoc.200400465. [DOI] [Google Scholar]
- Mathew B. Suresh J. Anbazhagan S. Mathew G. Cent. Nerv. Syst. Agents Med. Chem. 2013;13(3):195–206. doi: 10.2174/1871524914666140129122632. [DOI] [PubMed] [Google Scholar]
- Mantzanidou M. Pontiki E. Hadjipavlou-Litina D. Molecules. 2021;26(11):3439. doi: 10.3390/molecules26113439. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Safaei-Ghomi J. Bamoniri A. H. Soltanian-Telkabadi M. Chem. Heterocycl. Compd. 2006;42(7):892–896. doi: 10.1007/s10593-006-0176-1. [DOI] [Google Scholar]
- De Oliveira A. S. Mello L. D. S. Ogihara C. H. Döring T. H. Palomino-Salcedo D. L. Michelan-Duarte S. Ferreira L. L. G. Souza J. M. Dávila-Rodríguez M. J. Da Cruz Júnior J. W. Dockal E. R. Andricopulo A. D. Future Med. Chem. 2021;13:2167–2183. doi: 10.4155/fmc-2021-0194. [DOI] [PubMed] [Google Scholar]
- Borchhardt D. M. Mascarello A. Chiaradia L. D. Nunes R. J. Oliva G. Yunes R. A. Andricopulo A. D. J. Braz. Chem. Soc. 2010;21(1):142–150. doi: 10.1590/S0103-50532010000100021. [DOI] [Google Scholar]
- Oliveira A. S. Meier L. Zapp E. Brondani D. Brighente I. M. C. Sá M. M. J. Braz. Chem. Soc. 2019;30(5):1045–1054. [Google Scholar]
- Venzke D. Carvalho F. K. Ruani A. P. Oliveira A. S. Brighente I. M. C. Micke G. A. Barison A. Pizzolatti M. G. J. Braz. Chem. Soc. 2013;24(12):1991–1997. [Google Scholar]
- Wohnsland F. Faller B. J. Med. Chem. 2001;44:923–930. doi: 10.1021/jm001020e. [DOI] [PubMed] [Google Scholar]
- Jones G. Willett P. Glen R. C. Leach A. R. Taylor R. J. Mol. Biol. 1997;4, 267(3):727–748. doi: 10.1006/jmbi.1996.0897. [DOI] [PubMed] [Google Scholar]
- Korb O. Stützle T. Exner T. E. J. Chem. Inf. Model. 2009;49(1):84–96. doi: 10.1021/ci800298z. [DOI] [PubMed] [Google Scholar]
- Cheung J. Gary E. N. Shiomi K. Rosenberry T. L. ACS Med. Chem. Lett. 2013;23, 4(11):1091–1096. doi: 10.1021/ml400304w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pedretti A. Villa L. Vistoli G. J. Comput.-Aided Mol. Des. 2004;18:167–173. doi: 10.1023/B:JCAM.0000035186.90683.f2. [DOI] [PubMed] [Google Scholar]
- DeLano W. L., CCP4 Newsl. Protein Crystallogr, 2002 [Google Scholar]
- Nageswari G. George G. Ramalingam S. Govindarajan M. J. Mol. Struct. 2018;1166:422–441. doi: 10.1016/j.molstruc.2018.04.014. [DOI] [Google Scholar]
- Origin, Origin: Data Analysis and Graphing Software, 2018 [Google Scholar]
- Pires D. E. V. Blundell T. L. Ascher D. B. J. Med. Chem. 2015;58:4066–4072. doi: 10.1021/acs.jmedchem.5b00104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Daina A. Michielin O. Zoete V. Sci. Rep. 2017;7:1–13. doi: 10.1038/s41598-016-0028-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barret R., Medicinal Chemistry, Fundamentals, 2018, ch. 6 [Google Scholar]
- Veber D. F. Johnson S. R. Cheng H.-Y. Smith B. R. Ward K. W. Kopple K. D. J. Med. Chem. 2002;45(12):2615–2623. doi: 10.1021/jm020017n. [DOI] [PubMed] [Google Scholar]
- Wang Y., Michael S., Huang R., Zhao J., Recabo K., Bougie D., Shu Q., Shinn P. and Sun H., bioRxiv Prepr. Serv. Biol., bioRxiv, 2021, preprint, 10.1101/2021.05.11.442656 [DOI]
- Leeson P. Nature. 2012;481(7382):455–456. doi: 10.1038/481455a. [DOI] [PubMed] [Google Scholar]
- Nascimento É. C. M. Martins J. B. L. J. Mol. Model. 2011;17:1371–1379. doi: 10.1007/s00894-010-0838-x. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.