Mohd Abdullaha,Razia Banoo,Vijay K. Nuthakki,Mohit Sharma,Sukhleen Kaur,Shikha Thakur,Ajay Kumar,Hemant R. Jadhav,Sandip B. Bharate
Methoxy-naphthyl-Linked N-Benzyl Pyridinium Styryls as Dual Cholinesterase Inhibitors: Design, Synthesis, Biological Evaluation, and Structure–Activity Relationship
The multifaceted nature of Alzheimer’s disease (AD) indicates the need for multitargeted agents as potential therapeutics. Both cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play a vital role in disease progression. Thus, inhibiting both ChEs is more beneficial than only one for effectively managing AD. The present study provides a detailed lead optimization of the e-pharmacophore-generated pyridinium styryl scaffold to discover a dual ChE inhibitor. A structure–activity relationship analysis indicated the importance of three structural fragments, methoxy-naphthyl, vinyl-pyridinium, and substituted-benzyl, in a dual ChE inhibitor pharmacophore. The optimized 6-methoxy-naphthyl derivative, 7av (SB-1436), inhibits EeAChE and eqBChE with IC50 values of 176 and 370 nM, respectively. The kinetic study has shown that 7av inhibits AChE and BChE in a non-competitive manner with ki values of 46 and 115 nM, respectively. The docking and molecular dynamics simulation demonstrated that 7av binds with the catalytic and peripheral anionic sites of AChE and BChE. Compound 7av also significantly stops the self-aggregation of Aβ. The data presented herein indicate the potential of 7av for further investigation in preclinical models of AD.
Methoxy-naphthyl-Linked N-Benzyl Pyridinium Styryls as Dual Cholinesterase Inhibitors: Design, Synthesis, Biological Evaluation, and Structure–Activity Relationship

The multifaceted nature of Alzheimer’s disease (AD) indicates the need for multitargeted agents as potential therapeutics. Both cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play a vital role in disease progression. Thus, inhibiting both ChEs is more beneficial than only one for effectively managing AD. The present study provides a detailed lead optimization of the e-pharmacophore-generated pyridinium styryl scaffold to discover a dual ChE inhibitor. A structure–activity relationship analysis indicated the importance of three structural fragments, methoxy-naphthyl, vinyl-pyridinium, and substituted-benzyl, in a dual ChE inhibitor pharmacophore. The optimized 6-methoxy-naphthyl derivative, 7av (SB-1436), inhibits EeAChE and eqBChE with IC50 values of 176 and 370 nM, respectively. The kinetic study has shown that 7av inhibits AChE and BChE in a non-competitive manner with ki values of 46 and 115 nM, respectively. The docking and molecular dynamics simulation demonstrated that 7av binds with the catalytic and peripheral anionic sites of AChE and BChE. Compound 7av also significantly stops the self-aggregation of Aβ. The data presented herein indicate the potential of 7av for further investigation in preclinical models of AD.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by memory loss and cholinergic deficit. The World Health Organization has forecasted that AD will dominate AIDS and cancer over the next century. The primary hallmarks of AD are central cholinergic depletion and Aβ plaque formation. Several neurochemical factors are associated with AD initiation and progression. To date, the exact cause of AD is not well-ascertained; however, various proposed hypotheses rest on experimental pathophysiological factors. The cholinergic theory is the most widely investigated, postulating that acetylcholine (ACh) deficiency causes AD. The ACh deficiency occurs either due to decreased production or increased enzymatic degradation by acetylcholinesterase (AChE). Besides, AChE also plays a vital role in forming Aβ plaques. Consequently, the inhibition of AChE is considered an important therapeutic target in the drug discovery program for AD. The inhibition of AChE helps increase ACh concentrations in the brain and reduces the AChE-assisted deposition of toxic Aβ plaques. However, in the advanced stage of AD, the activity of AChE is surrogated by the BChE (pseudo-ChE). As a result, the inhibition of both AChE and BChE concurrently is equally essential. Four FDA-approved AChE inhibitors are tacrine, donepezil, rivastigmine, and galantamine. However, tacrine has been discontinued because of its high hepatotoxicity, while other three AChE inhibitors offer merely a symptomatic relief. Enormous efforts have been made to discover selective AChE inhibitors in the past few years; however, there are not many scaffolds with dual AChE/BChE inhibition. Rivastigmine is the known dual ChE inhibitor but has high micromolar inhibitory activity. Continuing our previous efforts, we conducted the medicinal chemistry of the pyridinium styryl scaffold, which was identified previously via the e-pharmacophore approach. Thus, herein, we report the synthesis and structure–activity relationship studies of the benzylpyridinium styryl series for dual ChE inhibition.

We recently optimized the e-pharmacophore-generated N-benzyl pyridinium benzamide scaffold to find new dual ChE inhibitors that provided a methoxy-naphthyl derivative SB-1389 as a dual ChE inhibitor. Continuing this study, we synthesized and optimized another e-pharmacophore-generated N-benzyl pyridinium styryl scaffold. The docking study demonstrates the interaction of pyridinium styryl with the peripheral anionic site (PAS) and catalytic anionic sites (CASs) of AChE in the same way as donepezil. The phenyl moiety shows π–π stacking interactions with the PAS residue Trp 286, whereas the quaternary nitrogen builds π-cation contacts with CAS residues Trp 86 and Tyr 337. Furthermore, the benzyl moiety attains a position to stack with Trp 86 (Figure 1).

The synthesis of pyridinium styryls 6a–k started with aryl aldehydes 1a–k, which were reduced to the corresponding benzyl alcohols 2a–k using NaBH4 in methanol at 0 °C. Benzyl alcohols 2a–k on treatment with triphenylphosphine (PPh3) and N-bromosuccinamide (NBS) afforded the corresponding benzyl bromides, 3a–k. This was followed by synthesizing Wittig’s salt, 4a–k, by treating benzyl bromides 3a–k with PPh3 in THF for 3 h. Different pyridine styryls, 6a–k, were synthesized by treating Wittig’s salt, 4a–k, with pyridine-4-carboxaldehyde (5) under a reflux condition at 80 °C. Finally, the pyridinium styryls 7aq–ax were prepared by refluxing pyridine styryls 5a–k and benzyl halides in acetonitrile (ACN) at 80 °C for 3 h (Schemes 1 and 2). Interestingly, the integration of aryl proton signals of unsubstituted and Me- or halogen-substituted benzyl in the 1H NMR for pyridinium styryls is lower. This could be because of the partial exchange of these protons with deuterium from CD3OD. In particular, the fluoro-substituted benzyls have shown much-pronounced aryl proton exchange. For the structure–activity relationship, the central vinyl pyridine region was also replaced with an amino-piperidinyl unit, and three representative analogues, 9a, 10a, 10b, were synthesized, as depicted in Scheme 3.

All synthesized compounds were tested in the Ellman assay to inhibit EeAChE and eqBChE at 1 μM. Compounds showing >50% inhibition of AChE and BChE were considered for IC50 determination. Most compounds exhibited IC50 < 1 μM for AChE, and few have shown IC50 < 1 μM for BChE. During lead optimization studies, phenyl and benzyl rings were substituted with different electron-donating and electron-withdrawing groups, and the phenyl moiety was also replaced with naphthalene. The compounds with R1 = OMe at the 3rd and 5th positions (i.e., 7a, 7b, 7c, and 7g) of phenyl display higher inhibition of AChE than compounds carrying R1 = OMe at the 3rd and 4th positions of phenyl (i.e., 7j, 7k, 7l, and 7m). Furthermore, increasing the number of methoxy groups to three on the phenyl ring decreases the activity, which is evident by comparing 7b, 7c, and 7g with 7o, 7p, and 7q, respectively (Table 1). Compounds bearing methoxy and ethoxy groups at the 4th position of phenyl also display an excellent anti-AChE profile. The substitution of a powerful electron-withdrawing group −NO2 on the benzyl group (e.g., 7a vs 7h and 7i) reduces the activity. The number of −OMe groups on ring A directly impacts BChE inhibition. Trimethoxy-pyridinium styryls (e.g., 7p) show significantly superior inhibition of BChE compared to the dimethoxy counterpart, 7c (Table 1). Furthermore, we prepared compounds bearing phenyl (ring A) substituted with moderate electron-withdrawing groups, −Cl and −Br. These groups did not significantly increase potency against AChE, and none of the compounds showed >50% inhibition of BChE at the tested concentration (Table 2). As observed in Table 1 and based on the literature precedence, the bulkier hydrophobic functionalities at the PAS binding pharmacophore helps to boost BChE inhibition. Thus, we synthesized naphthalene-linked N(1-benzyl pyridinium) styryls and substituted the benzyl moiety (ring C) with different EDGs and EWGs. Replacing the phenyl moiety (ring A) with naphthalene ameliorated the inhibitory potential against BChE. This is evident by comparing the activity potential of 7a and 7aq (20 vs 60% inhibition of BChE at 1 μM). The Cl moiety at the 2nd position and −F at the 4th position of benzyl (ring C) play a vital role in attaining dual potency. This series’s most potent analogue, 7av, exhibited dual anti-AChE/BChE activity with IC50 values of 0.176 and 0.37 μM, respectively (Table 3). Compound 7av also inhibits recombinant human AChE with an IC50 value of 0.08 μM.

The shift of the methoxy group from 3,4 to 3,5 positions on the phenyl ring (ring A) of N(1-benzyl pyridinium) styryl framework considerably increases the efficacy against AChE (7j vs 7a). The substitution of methyl on the benzyl moiety decreases the activity (compound 7b). In contrast, the −Cl group (7c) at the same position surges the action for both enzymes. Furthermore, the tri-methoxy-substituted N(1-benzyl pyridinium) styryl with the chloro at the 2nd position of benzyl considerably enhances anti-BChE potential (compound 7p). Removing a methoxy group from the third position of 7j decreases activity against AChE (7t). The exchange of methoxy with the ethoxy functionality at the 4th position increases the activity against BChE (7w). Furthermore, substituting the phenyl ring with electron-withdrawing groups such as −NO2 (compound 7an) decreases the efficacy against AChE and BChE. The substitution of −Cl at the phenyl (ring A) drastically reduces the therapeutic effectiveness against AChE (7ag); however, the activity is re-gained on further substitution of −Cl on the benzyl (ring C) (7ai). Bromo-substitution on phenyl decreases the activity potential against AChE (7ac), but further substituting the fluoro (7ae) or chloro (7ad) group on ring C ameliorates the anti-ChE activity potential. Furthermore, the exchange of phenyl with naphthyl results in a considerable surge in the activity against AChE and BChE. Additionally, the substitution of chloro (7as) or fluoro (7av) on benzyl of naphthyl N(1-benzyl pyridinium) further improves the dual inhibition potency. In a nutshell, the position of −Cl and −F on the benzyl moiety of the N(1-benzyl pyridinium) styryl layout is crucial for accomplishing dual AChE/BChE inhibition competence (Figure 2). A selected set of derivatives were synthesized by replacing the central vinyl-pyridinyl unit with the amino-piperidinyl moiety to understand the structure–activity relationship. The AChE and BChE inhibition data of these series of vinyl-pyridinyl-region modified analogues, 9a, 10a, 10b, are depicted in Figure 3A. Replacing vinyl-pyridinyl with flexible piperidinyl units (e.g., 9a and 10a) without varying the linker length yielded compounds with significantly reduced ChE inhibition. The analogue 10b with the elongated linker is slightly better than 10a; however, its activity is still in the micromolar range. Interestingly, the replacement of benzylic −CH2 in 10b with −C=O has drastically improved ChE inhibition activity (compound 10c(20)). This simple variation of −CH2 vs −C=O impacts AChE inhibition by 20-fold and BChE inhibition by 10-fold. To understand the significantly different ChE inhibition by 10b and 10c, the docking with AChE was compared (Figure 3B,C). The −C=O in 10c participates in two vital H-bonding interactions with Phe 295 and Arg 296 residues at PAS. These interactions are not present in 10b. Removal of −C=O from 10c also results in a slight shift in the naphthyl position in the active site, with less tight π–π stacking with Trp 286. These vital differences in interactions of 10b vs 10c could be accounting for their different ChE inhibition activities. The naphthyl derivative with the 4 F-benzyl moiety, 7av, is the most active dual AChE/BChE inhibitor with IC50 values of 176 and 370 nM, respectively. The compound 7av also exhibits 60 and 85% inhibition of self-induced Aβ42-aggregation at 5 and 10 μM, respectively.

To understand the type of inhibition and mode of interaction of 7av with ChEs, enzyme kinetics and in silico studies were performed. Based on the Lineweaver–Burk double reciprocal plot, compound 7av inhibits AChE in a non-competitive manner with an inhibition rate constant (ki) of 0.046 μM (Figure 4A). The in silico docking of 7av with AChE [Protein Data Bank (PDB) ID: 4EY7] further supported the non-competitive mode of inhibition. The compound 7av occupies an active-site gorge and is engaged in various hydrophobic interactions with both PAS and CAS domain residues of the AChE protein (Figure 4B). The naphthalene ring is oriented toward the PAS, while the N-benzyl moiety faces the CAS. In the PAS region, the naphthalene moiety stacks with Trp 286, while in the CAS region, the quaternary nitrogen constructs a cation−π interaction with Tyr 337 and Trp 86. Furthermore, the N-benzyl moiety reproduces a π–π stacking interaction with Trp 86. The nitrogen of the pyridinium ring displays a π–cation interaction with Tyr 337 and Trp 86 residues of the anionic subsite at the CAS. The eqBChE inhibition is also non-competitive, with a rate constant (ki) of 0.115 μM (Figure 4C). The docking study validated the kinetic study results. The compound 7av accommodates the active site with pyridinium ring stacking with Tyr 332 of PAS of BChE (Figure 4D). The elongated structure of the ligand allowed it to occupy the entire active-site gorge.

In the quest to find the stability of the protein–ligand (PL) complex, we performed molecular dynamics (MD) studies on the ligand 7av with two protein systems hAChE (PDB ID: 4EY7) and hBChE (PDB: 6EP4). Initially, we performed the root mean square deviation (RMSD) analysis to foresee if there are any perturbations upon ligand binding within the protein cavity. The analysis revealed that both proteins remained almost stable throughout the simulation duration of 100 ns. However, a slight fluctuation for hAChE and hBChE in the range of 5.6–1.8 and 1.25–3.5 Å, respectively, occurred (Figures 5A and 6A). Second, a similar stable pattern was observed for the ligand 7av throughout the simulation duration. Nevertheless, a little drift around 85 ns was observed in ligand RMSD in the case of hAChE (Figure 5A). Next, we performed root mean square fluctuation (RMSF) analysis to forecast perturbations that occur in the protein chain throughout the simulation upon ligand binding and lead to a plausibly unstable PL complex. The protein RMSF for hAChE analysis revealed that the fluctuations were observed in the backbone residues of Thr 109 (3.1 Å), Gly 263 (4.8 Å), and Trp 385 (2.4 Å) (Figure 5B). Furthermore, the P-L contact diagram revealed that residual amino acids Trp 86, Trp 286, Tyr 337, and Tyr 341 were involved in the interactions with 7av (Figure 5D). The major interaction (86%) was observed with the Trp 86 residue that was involved in the π–π stacking with the aromatic ring of 7av. A similar interaction (π–π stacking) was observed with the Trp 286 residue (50%) and Tyr 341 residue (79%). Residues Trp 86 (30%) and Tyr 341 (60%) besides π–π stacking were also involved in the π–cationic interaction with Tyr 337 (38%), allowing the stability through the simulation time, as shown in Figure 5D. Moreover, Phe 338 is also involved in hydrophobic interactions over 80% of simulation that also aids in 7av-AChE complex stabilization. Next, the protein RMSF analysis for hBChE revealed the fluctuations over the backbone residues 250–300 (1.6 Å) and 350–400 (2 Å), as portrayed in Figure 6B. Furthermore, the P-L contact diagram illustrated that the Asp 70 residue was involved in the ionic interaction (56%) with nitrogen present in the heterocyclic ring of the 7av, while the Tyr 332 residue was found to be involved in the π–π stacking (42%) with the aromatic ring, allowing the stability to PL complex through the simulation time. Moreover, 40% of the simulation was shown by hydrophobic interaction involving Trp 82, Pro 285, Tyr 430, and Tyr 440 residues aiding in additional complex stabilization (Figure 6D). The time-stamp analysis of both complexes is shown in the Supporting Information (Figures S54 and S55). Notably, most of the interactions found during the docking study (Figure 4B,D) were retained after 100 ns MD simulation, indicating both PL complexes’ stable nature.

The toxicity of 7av in J774A.1 cells and primary astrocytes was assessed for 48 h incubation time. Results are shown in Figure 7. Astrocytes are the major glial cells in the central nervous system and are involved in regulating various processes in the brain. These cells along with murine macrophages (J774A.1 cells) are used as model systems to study neuroinflammation. Therefore, cytotoxicity was assessed in these cells. The concentrations of 7av from 0.78 to 100 μM were tested, and it was found that the test compound reduces the cell viability in both cell lines. The compound is comparatively less toxic in primary astrocytes; however, its GI50 in both cell types is ∼6.25 μM. The compound 7av was further tested for its effect on neuroinflammation via measuring the IL-1β levels against nigericin-induced NLRP3 inflammasome activation; however, no statistically significant effect was observed. The pyridinium compound, 7av, has a CLogP of 1.5, primarily because of the pyridinium salt. The investigation of its blood–brain barrier (BBB) permeability via PAMPA assay also revealed its low BBB permeability (Pe < 3.0 × 10–6 cm/s). However, pyridinyl compounds, e.g., pralidoxime and obidoxime, are known to reach the brain as their pharmacokinetic studies have shown that BBB is not entirely impermeable to quaternary pyridinium compounds. Thus, a detailed pharmacokinetic study for 7av would be required to understand its brain exposure and the permeation mechanism.

The “pyridinium styryl” scaffold identified via e-pharmacophore modeling was investigated in detail for lead optimization toward identifying a dual ChE inhibitor. The synthesis of fifty-three N-benzyl pyridinium styryl derivatives followed by their in vitro ChE inhibition assays led to the identification of 7av (SB-1436) as a dual AChE and BChE inhibitor showing half-maximal inhibitory concentration values of 0.176 and 0.37 μM, respectively. The enzyme kinetics and docking studies have demonstrated that SB-1436 exhibits a non-competitive mode of inhibition for both AChE and BChE with ki values of 0.046 and 0.115 μM, respectively. In addition to its dual ChE inhibition, it also stops the self-aggregation of the Aβ42 peptide. The SAR studies on pyridinium styryls indicate that chloro or fluoro on the benzylic ring (ring C) boosts the BChE inhibition activity. The presence of naphthyl ring vs. phenyl (ring A) also helps in a drastic boost in BChE inhibition potency. Further replacement of the “pyridinium” ring (ring B) with non-quaternary “piperidine” is still able to maintain a dual AChE/BChE inhibition potency. These SAR studies provide vital insights into further ChE-targeted medicinal chemistry on this scaffold. Overall, the results presented herein advocate the detailed preclinical investigation of the identified dual ChE inhibitor, 7av.

All chemicals were purchased from Sigma-Aldrich and TCI Companies and were used as such as received without further purifications. The instruments used to record the analytical data, such as 1H, 13C, IR, and melting point data, were the same as the previous work.

To the solution of aryl aldehyde, 1a–k, in methanol was added slowly NaBH4 under an ice bath with continuous stirring. The mixture was concentrated on a rotary evaporator and partitioned between ethyl acetate and water. The ethyl acetate layer was evaporated on a vacuo rotavapor to dryness to obtain the corresponding benzyl alcohols, 2a–j, which were used as such for the next step.

To a solution of N-bromosuccinamide in methylene chloride was slowly added PPh3 at 0 °C with continuous stirring. The resulting mixture was refluxed at 60 °C for 15 min. The reaction mixture was cooled to room temperature. Benzyl alcohol was added to the cooled reaction mixture, and it was then refluxed at 60 °C for 30 min. The reaction mixture was concentrated to dryness, and the obtained product was used as such for the next step.

To a solution of benzyl bromide in THF was added PPh3 (1.2 equiv) slowly at 0 °C with continuous stirring. The resulting mixture was refluxed at 85 °C for 2 h. A white solid is formed, indicating the formation of Wittig salt. The reaction mixture was allowed to cool to room temperature and then filtered. The solid residue was washed with ethyl acetate or THF, dried under vacuum, and used for the next step.

To the solution of triphenylphosphine halide salts 4a–k (2 mmol) in dry THF, potassium tert-butoxide (2.5 mmol) was added at room temperature. The resulting mixture was refluxed for 30 min. The color of the reaction mixture turned orange after which it was allowed to cool to room temperature. The pyridine aldehyde 5 (2 mmol) was added, and the resulting reaction mixture was refluxed at 80 °C for 30 min. THF was evaporated on a vacuo rotavapor, and the resultant solid mass was partitioned between ethyl acetate and water. The ethyl acetate layer was dried over anhydrous sodium sulfate, which was further evaporated on a rotary evaporator to obtain the crude product. Purification was done using routine silica gel (mesh 100–200) column chromatography using ethyl acetate–hexane as a mobile phase to obtain (E)-4-styrylpyridine 6a-k in 80–95% yield.

To the solution of (E)-4-styrylpyridines, 6a–k (0.5 mmol) in ACN (10 mL) was added benzyl halides (0.5 mmol), and the resulting mixture was stirred at 80 °C for 1 h. The formation of white/yellow precipitates was seen in the reaction mixture. The precipitates were filtered and washed with acetone (10 mL × 3). The resulting residue was dried to obtain the desired N-benzyl pyridinium styryls, 7a–7ax, in 85–95% yield.

Yield: 88%; yellow solid; mp 250–252 °C; IR (νmax): 3416, 3034, 2920, 1740, 1622, 1554, 1495, 1470, 1455, 1427, 1336, 1305, 1286, 1207, 1056, 972 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.78 (d, J = 6.8 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H), 7.76 (d, J = 16.3 Hz, 1H), 7.42–7.32 (m, 6H), 6.82 (s, 2H), 6.51 (s, 1H), 5.66 (s, 2H), 3.75 (s, 6H); 13C NMR (126 MHz, CDCl3): δ 165.33, 158.45, 147.85, 146.06, 140.85, 137.54, 133.46, 133.24, 132.51, 128.09, 126.78, 109.84, 106.47, 67.13, 58.52. ESI-MS m/z: 332.0 [M – Br]+; HR-ESIMS m/z: 332.1654 calcd for C22H22NO2+ (332.1651).

Yield: 91%; yellow solid; mp 261–263 °C; IR (νmax): 3584, 2919, 1641, 1617, 1592, 1514, 1461, 1426, 1257, 1063, 986 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.63 (d, J = 6.8 Hz, 2H), 8.09 (d, J = 6.8 Hz, 2H), 7.77 (d, J = 16.3 Hz, 1H), 7.35–7.27 (m, 3H), 6.81 (d, J = 2.1 Hz, 2H), 6.49 (d, J = 2.0 Hz, 1H), 5.71 (s, 2H), 3.74 (s, 6H) 2.24 (s, 3H); 13C NMR (126 MHz, DMSO-d6): δ 161.27, 153.76, 141.69, 137.54, 137.33, 133.02, 131.38, 129.75, 129.42, 127.19, 124.69, 124.39, 106.60, 103.03, 61.01, 55.92, 19.29. ESI-MS m/z: 346.0 [M – Br]+; HR-ESIMS m/z: 346.1808 calcd for C23H24NO2+, (346.1807).

Yield: 87%; yellow solid; mp 250–252 °C; IR (νmax): 3446, 3383, 3013, 2964, 2920, 2850, 1700, 1695, 1642, 1624, 1586, 1472, 1451, 1428, 1356, 1331, 1316, 1294, 1283, 979 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.71 (d, J = 6.8 Hz, 2H), 8.09 (d, J = 6.8 Hz, 2H), 7.78 (d, J = 16.3 Hz, 1H), 7.53–7.39 (m, 5H), 6.81 (d, J = 2.1 Hz, 2H), 6.48 (t, J = 2.1 Hz, 1H), 5.79 (s, 2H), 3.74 (s, 6H); 13C NMR (126 MHz, CD3OD): δ 161.37, 154.72, 144.07, 142.34, 136.92, 134.36, 132.00, 131.64, 130.79, 130.26, 128.01, 124.02, 122.85, 105.95, 102.64, 60.92, 54.62. ESI-MS m/z: 366.0 [M – Cl]+; HR-ESIMS m/z: 366.1261 calcd for C22H21ClNO2+, (366.1261).

Yield: 90%; yellow solid; mp 149–151 °C; IR (νmax): 3387, 3038, 2922, 2847, 1658, 1641, 1620, 1594, 1456, 1427, 1354, 1289, 1208, 1159, 973 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.77 (d, J = 6.7 Hz, 2H), 8.09 (d, J = 6.8 Hz, 2H), 7.78 (d, J = 16.3 Hz, 1H), 7.49 (s, 1H), 7.39–7.32 (m, 4H), 6.81 (d, J = 2.1 Hz, 2H), 6.48 (s, 1H), 5.64 (s, 2H), 3.74 (s, 6H); 13C NMR (126 MHz, CD3OD): δ 161.36, 154.71, 143.90, 142.29, 136.91, 135.62, 135.02, 130.92, 129.64, 128.61, 126.97, 124.31, 122.88, 105.99, 102.61, 62.38, 54.68. ESI-MS m/z: 366.0 [M – Cl]+; HR-ESIMS m/z: 366.1262 calcd for C22H21ClNO2+ (366.1261).

Yield: 93%; yellow solid; IR (νmax): 3387, 2921, 2850, 1620, 1592, 1556, 1517, 1454, 1425, 1351, 1287, 1208, 1159, 1095, 975 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.75 (d, J = 6.8 Hz, 2H), 8.09 (d, J = 6.7 Hz, 2H), 7.77 (d, J = 16.3 Hz, 1H), 7.40–7.33 (m, 5H), 6.81 (d, J = 2.1 Hz, 2H), 6.49 (t, J = 2.0 Hz, 1H), 5.63 (s, 2H), 3.74 (s, 6H); 13C NMR (126 MHz, CD3OD): δ 161.37, 154.60, 143.88, 142.21, 136.92, 135.53, 132.35, 130.32, 129.37, 124.23, 122.86, 105.93, 102.60, 62.28, 54.61. ESI-MS m/z: 366.0 [M – Cl]+; HR-ESIMS m/z: 366.1270 calcd for C22H21ClNO2+ (366.1261).

Yield: 89%; yellow solid; mp 257–259 °C; IR (νmax): 3416, 3010, 2921, 2849, 1625, 1562, 1518, 1492, 1356, 1285, 1257, 1214, 1197, 1181, 977 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.75 (d, J = 6.3 Hz, 2H), 8.09 (d, J = 6.2 Hz, 2H), 7.77 (d, J = 16.2 Hz, 1H), 7.53–7.24 (m, 5H), 6.81 (br s, 2H), 6.48 (br s, 1H), 5.73 (s, 2H), 3.74 (s, 6H); 13C NMR (126 MHz, CD3OD): δ 161.40, 154.74, 143.97, 142.34, 132.35, 132.28, 131.31, 125.18, 124.12, 122.81, 120.81, 120.69, 115.96, 115.80, 105.92, 102.56, 57.64, 54.57. ESI-MS m/z: 350.0 [M – Br]+; HR-ESIMS m/z: 350.1558 calcd for C22H21FNO2+ (350.1556).

Yield: 90%; white solid; mp 220–222 °C; IR (νmax): 3359, 2839, 1618, 1591, 1528, 1470, 1367, 1286, 1224, 1207, 1158, 1067, 925 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.75 (d, J = 6.8 Hz, 2H), 8.08 (d, J = 6.8 Hz, 2H), 7.77 (d, J = 16.3 Hz, 1H), 7.49–7.12 (m, 5H), 6.80 (d, J = 2.1 Hz, 2H), 6.49 (t, J = 2.1 Hz, 1H), 5.63 (s, 2H), 3.74 (s, 6H); 13C NMR (126 MHz, CD3OD): δ 164.50, 162.52, 161.36, 154.52, 143.78, 142.13, 136.93, 131.12 (3JCF = 8.8 Hz), 129.68, 124.20, 122.86, 116.19 (2JCF = 22.7 Hz), 105.91, 102.58, 62.32, 54.60. ESI-MS m/z: 350.0 [M – Cl]+; HR-ESIMS m/z: 350.1556 calcd for C22H21FNO2+ (350.1556).

Yield: 93%; yellow solid; mp 251–253 °C; IR (νmax): 3384, 2918, 2849, 1341, 1616, 1592, 1539, 1521, 1464, 1456, 1425, 1342, 1287, 1156, 973 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.70 (d, J = 6.7 Hz, 2H), 8.22 (d, J = 8.1 Hz, 1H), 8.12 (d, J = 6.7 Hz, 2H), 7.82–7.76 (m, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 16.3 Hz, 1H), 6.82 (d, J = 1.8 Hz, 2H), 6.50 (s, 1H), 6.02 (s, 2H), 3.74 (s, 6H); 13C NMR (126 MHz, DMSO-d6): δ 161.27, 154.04, 148.30, 145.22, 142.03, 141.95, 137.51, 130.41, 124.85, 124.63, 124.32, 106.63, 103.10, 61.59, 55.93. ESI-MS m/z: 377.0 [M – Br]+; HR-ESIMS m/z: 377.1505 calcd for C22H21N2O4+ (377.1501).

Yield: 95%; orange solid; mp 184–186 °C; IR (νmax): 3356, 2921, 2349, 2334, 1616, 1555, 1517, 1469, 1347, 1205, 1161, 987 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.98 (d, J = 6.4 Hz, 2H), 8.18 (dd, J = 10.1, 7.8 Hz, 4H), 7.87 (d, J = 16.3 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 16.3 Hz, 1H), 6.83 (s, 2H), 6.50 (s, 1H), 5.82 (s, 2H), 3.70 (s, 6H); 13C NMR (126 MHz, DMSO-d6): δ 161.27, 154.04, 148.31, 145.21, 142.02, 141.95, 137.51, 130.40, 124.85, 124.63, 124.32, 106.63, 103.10, 61.60, 55.93. ESI-MS m/z: 377.0 [M – Br]+; HR-ESIMS m/z: 377.1505 calcd for C22H21N2O4+ (377.1501).

Yield: 87%; yellow solid; mp 263–265 °C; IR (νmax): 3328, 2948, 2869, 1703, 1626, 1558, 1522, 1421, 1401, 1223, 1188, 1150, 1128, 970 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.95 (d, J = 6.8 Hz, 2H), 8.11 (d, J = 6.8 Hz, 2H), 7.90 (d, J = 16.2 Hz, 1H), 7.45 (d, J = 6.3 Hz, 2H), 7.40–7.32 (m, 5H), 7.21 (dd, J = 8.3, 1.7 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 5.65 (s, 2H), 3.76 (s, 3H), 3.74 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 154.20, 151.75, 149.56, 144.59, 142.17, 135.12, 129.70, 129.10, 128.43, 124.07, 123.86, 121.26, 112.20, 110.39, 62.54, 56.12, 56.08. ESI-MS m/z: 332.0 [M – Br]+; HR-ESIMS m/z: 332.1651 calcd for C22H22NO2+ (332.1651).

Yield: 87%; yellow solid; mp 255–257 °C; IR (νmax): 3356, 2936, 2858, 1622, 1598, 1517, 1497, 1422, 1223, 1175, 1126, 1022 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.76 (d, J = 5.9 Hz, 2H), 8.09 (d, J = 5.9 Hz, 2H), 7.89 (d, J = 16.3 Hz, 1H), 7.37 (d, J = 16.2 Hz, 1H), 7.32 (s, 1H), 7.27–7.17 (m, 4H), 7.05 (d, J = 7.5 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 5.69 (s, 2H), 3.75 (s, 3H), 3.73 (s, 3H), 2.21 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 154.97, 152.08, 149.57, 143.52, 142.41, 137.37, 131.28, 131.24, 131.10, 129.81, 129.72, 128.23, 126.79, 123.45, 120.11, 111.39, 111.31, 110.20, 110.06, 61.05, 55.22, 55.08, 17.79. ESI-MS m/z: 346.0 [M – Br]+; HR-ESIMS m/z: 346.1809 calcd for C23H24NO2+ (346.1807).

Yield: 88%; yellow solid; mp 156–158 °C; IR (νmax): 3352, 2927, 2856, 1632, 1586, 1477, 1423, 1236, 1184, 1022 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.63 (d, J = 6.2 Hz, 2H), 8.02 (d, J = 6.2 Hz, 2H), 7.80 (d, J = 16.1 Hz, 1H), 7.52–7.47 (m, 5H), 7.44–7.28 (m, 2H), 6.94 (d, J = 8.4 Hz, 1H), 5.75 (s, 2H), 3.81 (s, 3H), 3.80 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 143.74, 142.67, 135.38, 133.82, 131.88, 131.61, 130.94, 130.28, 129.86, 129.45, 127.99, 127.12, 123.56, 123.35, 120.03, 111.30, 110.03, 60.70, 55.15, 55.10. ESI-MS m/z: 366.0 [M – Cl]+; HR-ESIMS m/z: 366.1263 calcd for C22H21ClNO2+ (366.1261).

Yield: 90%; yellow solid; mp 157–159 °C; IR (νmax): 3348, 2950, 2866, 1622, 1598, 1533, 1494, 1453, 1241, 1182, 1123, 1056 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.68 (d, J = 6.4 Hz, 2H), 8.02 (d, J = 6.4 Hz, 2H), 7.79 (d, J = 16.2 Hz, 1H), 7.46 (dd, J = 7.9, 5.4 Hz, 2H), 7.27 (br s, 2H), 7.24–7.19 (m, 2H), 6.94 (d, J = 8.3 Hz, 2H), 5.60 (s, 2H), 3.81 (s, 3H), 3.80 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 164.45 (1JCF = 248 Hz), 154.91, 151.93, 149.50, 143.46, 142.33, 131.07 (3JCF = 8.8 Hz), 129.83, 128.23, 123.57, 123.46, 120.11, 116.16 (2JCF = 21.4 Hz), 111.31, 110.07, 62.09, 55.18, 55.10. ESI-MS m/z: 350.0 [M – Cl]+; HR-ESIMS m/z: 350.1555 calcd for C22H21FNO2+ (350.1556).

Yield: 88%; yellow solid; mp 199–201 °C; IR (νmax): 3336, 2955, 2893, 1629, 1597, 1522, 1498, 1443, 1235, 1183, 1154, 1106, 1031, 956 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.90 (d, J = 6.8 Hz, 2H), 8.41 (d, J = 8.7 Hz, 2H), 8.25 (d, J = 6.8 Hz, 2H), 8.01 (d, J = 16.2 Hz, 1H), 7.78 (d, J = 8.6 Hz, 2H), 7.47–7.340 (m, 3H), 7.13 (d, J = 8.4 Hz, 1H), 5.95 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H); 13C NMR (126 MHz, DMSO-d6): δ 154.55, 151.82, 149.57, 148.28, 144.89, 142.50, 142.15, 130.34, 128.44, 124.64, 124.17, 123.96, 121.25, 112.22, 110.47, 61.39, 56.14, 56.11. ESI-MS m/z: 377.0 [M – Br]+; HR-ESIMS m/z: 377.1507 calcd for C22H21N2O4+ (377.1501).

Yield: 86%; yellow solid; mp 225–227 °C; IR (νmax): 3420, 3040, 2921, 2849, 1636, 1581, 1537, 1466, 1422, 1355, 1337, 1276, 1211, 1152 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.60 (d, J = 6.8 Hz, 2H), 8.05 (d, J = 6.8 Hz, 2H), 7.79 (d, J = 16.2 Hz, 1H), 7.28 (m, 5H), 6.98 (br s, 2H), 5.70 (s, 2H), 3.82 (s, 6H), 3.72 (s,3H), 2.24 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 154.64, 154.60, 153.55, 153.53, 143.66, 142.16, 142.13, 137.42, 131.18, 131.12, 130.94, 129.87, 129.81, 126.82, 123.81, 123.79, 121.79, 105.71, 105.62, 61.19, 59.87, 55.44, 55.41, 17.83. ESI-MS m/z: 376.0 [M – Br]+; HR-ESIMS m/z: 376.1910 calcd for C24H26NO3+ (376.1913).

Yield: 88%; yellowish solid; mp 210–212 °C; IR (νmax): 3368, 2954, 1616, 1580, 1503, 1466, 1421, 1359, 1276, 1241, 1182, 1055 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.68 (d, J = 6.6 Hz, 2H), 8.06 (d, J = 6.7 Hz, 2H), 7.80 (d, J = 16.2 Hz, 1H), 7.51–7.47 (m, 2H), 7.45–7.39 (m, 2H), 7.31 (d, J = 16.2 Hz, 1H), 6.98 (s, 2H), 5.77 (s, 2H), 3.82 (s, 6H), 3.73 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 154.89, 153.57, 143.97, 142.42, 134.50, 131.90, 131.63, 130.85, 130.82, 130.28, 128.00, 123.68, 121.72, 105.62, 60.82, 59.84, 55.34. ESI-MS m/z: 396.0 [M – Cl]+; HR-ESIMS m/z: 396.1366 calcd for C23H23ClNO3+ (396.1366).

Yield: 90%; yellow solid; mp 182–184 °C; IR (νmax): 3416, 2918, 2848, 1616, 1641, 1581, 1510, 1467, 1423, 1356, 1327, 1228, 1151, 1125, 997 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.73 (d, J = 6.7 Hz, 2H), 8.05 (d, J = 6.7 Hz, 2H), 7.78 (d, J = 16.2 Hz, 2H), 7.49–7.45 (m, 2H), 7.30 (d, J = 16.2 Hz, 2H), 6.98 (s, 2H), 5.62 (s, 2H), 3.81 (s, 6H), 3.72 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 153.61, 143.66, 142.26, 130.94, 123.90, 121.72, 116.20, 116.03, 105.72, 62.30, 59.85, 55.39. ESI-MS m/z: 380.0 [M – Cl]+; HR-ESIMS m/z: 380.1664 calcd for C23H23FNO3+ (380.1662).

Yield: 91%; yellow solid 217–219 °C; IR (νmax): 3386, 3096, 2928, 2855, 1620, 1594, 1558, 1471, 1427, 1367, 1354, 1289, 1208, 1159, 1097, 1082, 1068, 1057, 973 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.67 (d, J = 6.8 Hz, 2H), 8.23 (d, J = 8.1 Hz, 1H), 8.08 (d, J = 6.7 Hz, 2H), 7.82–7.69 (m, 4H), 7.42 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 16.2 Hz, 1H), 6.99 (s, 1H), 6.00 (s, 2H), 3.82 (s, 6H), 3.73 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 154.96, 153.56, 148.08, 144.13, 142.46, 140.42, 134.69, 131.94, 131.05, 130.89, 127.93, 125.86, 123.67, 121.77, 105.65, 60.24, 59.84, 55.37. ESI-MS m/z: 407.0 [M – Br]+; HR-ESIMS m/z: 407.1613 calcd for C23H23N2O5+ (407.1607).

Yield: 93%; Red solid; mp 200–202 °C; IR (νmax): 3357, 2924, 2853, 1612, 1516, 1456, 1419, 1377, 1345, 1328, 1277, 1241, 1210, 1155, 962 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.96 (d, J = 5.7 Hz, 2H), 8.20 (d, J = 8.4 Hz, 2H), 8.13 (d, J = 6.0 Hz, 2H), 7.90 (d, J = 16.2 Hz, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 16.2 Hz, 1H), 7.00 (s, 2H), 5.81 (s, 2H), 3.75 (s, 6H), 3.62 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 155.08, 149.77, 146.49, 143.72, 143.35, 132.53, 131.80, 126.05, 125.91, 124.36, 107.85, 63.06, 62.12, 57.99. ESI-MS m/z: 407.0 [M – Br]+; HR-ESIMS m/z: 407.1607 calcd for C23H23N2O5+ (407.1607).

Yield: 94%; yellow solid; mp 214–216 °C; IR (νmax): 3428, 3032, 2960, 2925, 2860, 2363, 1615, 1596, 1519, 1436, 1393, 1289, 1258, 1157, 1042, 985 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.69 (d, J = 6.9 Hz, 2H), 8.02 (d, J = 6.9 Hz, 2H), 7.81 (d, J = 16.2 Hz, 1H), 7.61 (m, 3H), 7.39–7.36 (m, 5H), 6.92 (d, J = 8.8 Hz, 2H), 5.61 (s, 2H), 3.76 (s, 3H). 13C NMR (126 MHz, CD3OD): δ 162.23, 154.95, 143.55, 142.11, 133.73, 130.05, 129.45, 129.28, 128.54, 127.77, 123.54, 119.83, 114.29, 62.96, 54.61. ESI-MS m/z: 302.0 [M – Br]+; HR-ESIMS m/z: 302.1548 calcd for C21H20NO+ (302.1545).

Yield: 88%; yellow solid; mp 222–227 °C; IR (νmax): 3405, 3034, 2922, 2351, 1618, 1595, 1572, 1556, 1517, 1493, 1468, 1425, 1329, 1308, 1259, 1214, 1024, 988 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.78 (d, J = 6.8 Hz, 2H), 8.12 (d, J = 6.9 Hz, 2H), 7.92 (d, J = 16.2 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.61–7.50 (m, 5H), 7.02 (d, J = 8.7 Hz, 2H), 5.79 (s, 2H), 3.86 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 162.32 (1JCF = 259 Hz), 155.22, 143.64, 142.38, 132.27, 132.20, 131.25, 130.31, 130.07, 127.75, 127.11, 125.18, 125.15, 123.49, 120.76, 119.79, 115.93 (2JCF = 20 Hz), 114.31, 114.18, 57.39, 54.61. ESI-MS m/z: 320.0 [M – Cl]+; HR-ESIMS m/z: 320.1450 calcd for C21H19FNO+(320.1451).

Yield: 87%; yellow solid; mp 205–207 °C; IR (νmax): 3425, 2928, 2836, 1645, 1522, 1452, 1345, 1256, 1157, 1022 cm–1;1H NMR (400 MHz, CD3OD): δ 8.98 (d, J = 6.4 Hz, 1H), 8.74 (d, J = 6.6 Hz, 2H), 8.32–8.30 (m, 1H), 8.20 (d, J = 6.3 Hz, 2H), 7.90–7.60 (m, 5H), 7.55 (dd, J = 21.7, 7.4 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 6.21 (s, 1H), 6.09 (s, 2H), 3.87 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 162.27, 160.73, 155.27, 148.04, 144.89, 143.97, 142.42, 134.74, 131.98, 131.00, 130.18, 127.78, 125.83, 125.58, 123.42, 114.32, 60.10, 54.66. ESI-MS m/z: 347.0 [M – Br]+; HR-ESIMS m/z: 347.1401 calcd for C21H19N2O3+ (347.1396).

Yield: 95%; yellow solid; mp 251–253 °C; IR (νmax): 3416, 3031, 2979, 2924, 2851, 2354, 1619, 1595, 1572, 1517, 1455, 1393, 1330, 1307, 1289, 1258, 1176, 1157, 1042, 985 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.68 (d, J = 6.2 Hz, 2H), 8.01 (d, J = 6.7 Hz, 2H), 7.80 (d, J = 16.2 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 3.6 Hz, 4H), 6.90 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 8.7 Hz, 1H), 6.53 (d, J = 12.1 Hz, 1H), 5.60 (s, 2H), 4.01 (q, J = 14.0, 7.0 Hz, 2H), 1.32 (t, J = 5.6 Hz, 3H); 13C NMR (126 MHz, CD3OD): δ 161.58, 154.98, 143.54, 142.18, 133.74, 130.06, 129.45, 129.28, 128.52, 127.61, 123.49, 119.69, 114.74, 63.44, 62.93, 13.65. ESI-MS m/z: 316.0 [M – Br]+; HR-ESIMS m/z: 316.1705 calcd for C22H22NO+ (316.1701).

Yield: 94%; yellow solid; mp 232–234 °C; IR (νmax): 3030, 2978, 2928, 1643, 1616, 1570, 1556, 1471, 1391, 1332, 1311, 1286, 1216 982 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.56 (d, J = 6.7 Hz, 2H), 8.02 (d, J = 6.7 Hz, 2H), 7.81 (d, J = 16.2 Hz, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.31–7.20 (m, 5H), 6.90 (d, J = 8.7 Hz, 2H), 5.68 (s, 2H), 4.01 (q, J = 7.0 Hz, 2H), 2.23 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CD3OD): δ 161.57, 155.81, 143.73, 143.54, 141.44, 141.28, 131.14, 130.35, 130.23, 130.10, 129.95, 129.40, 129.37, 129.02, 127.17, 127.08, 126.84, 123.58, 123.44, 122.55, 119.70, 114.79, 114.66, 63.34, 62.53, 17.83, 13.65; ESI-MS m/z: 330.0 [M – Br]+; HR-ESIMS m/z: 330.1855 calcd for C23H24NO+ (330.1858).

Yield: 93%; yellow solid; mp 219–221 °C; IR (νmax): 3036, 2980, 2932, 1620, 1572, 1469, 1393, 1331, 1309, 1291, 1204, 1175, 989 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.63 (d, J = 6.9 Hz, 2H), 8.02 (d, J = 6.9 Hz, 2H), 7.82 (d, J = 16.2 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.50–4.40 (m, 5H), 6.90 (d, J = 8.7 Hz, 2H), 5.75 (s, 2H), 4.01 (q, J = 7.0 Hz, 2H), 1.31 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CD3OD): δ 161.63, 155.22, 144.01, 143.76, 142.44, 134.31, 132.11, 131.93, 131.57, 130.91, 130.35, 130.24, 130.13, 127.99, 127.61, 126.99, 123.34, 122.58, 119.69, 114.77, 114.63, 63.47, 60.67, 13.65. ESI-MS m/z: 350.0 [M – Cl]+; HR-ESIMS m/z: 350.1315 calcd for C22H21ClNO+ (350.1312).

Yield: 95%; yellow solid; mp 208–210 °C; IR (νmax): 3034, 2981, 2931, 1642, 1596, 1572, 1558, 1517, 1472, 1426, 1413, 1329, 1207, 977 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.77 (d, J = 6.9 Hz, 2H), 8.12 (d, J = 6.9 Hz, 2H), 7.91 (d, J = 16.2 Hz, 1H), 7.70 (m, 3H), 7.49 (br s, 4H), 7.00 (d, J = 8.8 Hz, 2H), 5.69 (s, 2H), 4.11 (q, J = 7.0 Hz, 2H), 1.41 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CD3OD): δ 161.61, 155.12, 143.78, 143.57, 142.32, 141.40, 135.46, 132.48, 130.39, 130.33, 130.24, 130.09, 129.35, 127.61, 127.15, 123.56, 122.54, 119.69, 114.77, 114.64, 63.46, 62.04, 13.65. ESI-MS m/z: 350.0 [M – Cl]+; HR-ESIMS m/z: 350.1313 calcd for C22H21ClNO+ (350.1312).

Yield: 90%; yellow solid; mp 232–234 °C; IR (νmax): 3386, 3028, 2919, 2849, 2353, 1643, 1570, 1470, 1455, 1333, 1306, 1216, 1117, 985 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.67 (d, J = 6.7 Hz, 2H), 8.01 (d, J = 6.9 Hz, 2H), 7.81 (d, J = 16.2 Hz, 1H), 7.76 (d, J = 6.7 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.52–7.42 (m, 2H), 6.90 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 8.7 Hz, 1H), 6.53 (d, J = 12.0 Hz, 1H), 5.68 (s, 2H), 4.01 (q, J = 7.0 Hz, 2H), 1.32 (t, J = 5.5 Hz, 3H); 13C NMR (126 MHz, CD3OD): δ 162.24 (1JCF = 252 Hz), 161.65, 155.21, 143.63 (3JCF = 30 Hz), 132.20, 131.26, 130.34, 130.10, 127.09, 125.18, 123.46, 122.52, 119.66, 115.95 (2JCF = 21.4 Hz), 114.76, 63.46, 57.82 (3JCF = 55 Hz), 13.63. ESI-MS m/z: 334.0 [M – Br]+; HR-ESIMS m/z: 334.1613 calcd for C22H21FNO+ (334.1607).

Yield: 87%; yellow solid; mp 209–211 °C; IR (νmax): 3416, 2921, 2852, 2328, 1729, 1695, 1641, 1616, 1595, 1516, 1468, 1308, 1257, 1171, 1042 cm–1; 1H NMR (400 MHz, CD3OD) 8.62 (d, J = 6.7 Hz, 2H), 8.21 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 6.7 Hz, 2H), 7.84–7.68 (m, 4H), 7.62 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 7.6 Hz, 1H), 6.91 (d, J = 8.6 Hz, 2H), 5.98 (s, 2H), 4.02 (q, J = 6.9 Hz, 2H), 1.32 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, DMSO-d6): δ 161.20, 154.68, 147.98, 145.22, 142.19, 135.42, 130.82, 130.73, 130.53, 130.17, 128.11, 126.07, 124.09, 121.14, 115.58, 63.92, 59.88, 15.03. ESI-MS m/z: 361.0 [M – Br]+; HR-ESIMS m/z: 361.1556 calcd for C22H21N2O3+ (361.1552).

Yield: 88%; light yellow sticky solid; IR (νmax): 3416, 3033, 2922, 2851, 2345, 1652, 1641, 1620, 1583, 1558, 1514, 1467, 1455, 1404, 1336, 1241, 1156 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.79 (d, J = 6.8 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H), 7.80 (d, J = 16.3 Hz, 1H), 7.57–7.51 (m, 5H), 7.43–7.33 (m, 5H), 5.66 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 154.21, 143.96, 140.51, 134.23, 133.57, 132.00, 130.41, 129.66, 129.53, 129.31, 128.75, 128.64, 127.43, 125.21, 124.46, 124.30, 123.23, 63.26. ESI-MS m/z: 350.0 [M – Br]+; HR-ESIMS m/z: 350.0550 calcd for C20H17BrN+ (350.0544).

Yield: 86%; yellowish sticky solid; IR (νmax): 3422, 3037, 2941, 2863, 2322, 1625, 1633, 1571, 1566, 1456, 1331, 1210, 1156, 1071 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.72 (d, J = 6.7 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H), 7.82 (d, J = 16.3 Hz, 1H), 7.60–7.33 (m, 9H), 5.79 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 154.46, 144.20, 140.76, 134.38, 134.22, 132.07, 132.02, 131.67, 130.76, 130.27, 129.70, 128.02, 124.53, 124.15, 123.21, 61.00. ESI-MS m/z: 385.0 [M – Cl]+; HR-ESIMS m/z: 386.0308 calcd for C20H16BrClN+ (386.0311).

Yield: 87%; yellow solid; mp 255–257 °C; IR (νmax): 3424, 3032, 2930, 2853, 2322, 1667, 1522, 1482, 1407, 1331, 1250, 1212, 1157, 1065, 1022 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.77 (d, J = 6.6 Hz, 2H), 8.70 (d, J = 6.6 Hz, 2H), 8.10 (d, J = 6.7 Hz, 2H), 7.81 (d, J = 16.3 Hz, 1H), 7.73 (d, J = 6.5 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 16.4 Hz, 1H), 6.73 (d, J = 12.1 Hz, 1H), 5.71 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 162.26 (1JCF = 249 Hz), 154.97, 154.43, 144.19 (3JCF = 13.9 Hz), 140.73, 139.70, 134.22, 132.35, 132.01, 130.42, 129.70, 127.44, 125.20, 124.29, 123.21, 115.95 (2JCF = 21.4 Hz), 58.02 (3JCF = 41.6 Hz). ESI-MS m/z: 368.0 [M – Br]+; HR-ESIMS m/z: 368.0450 calcd for C20H16BrFN+ (368.0450).

Yield: 89%; white solid; mp 258–260 °C; IR (νmax): 3425, 3026, 2933, 2846, 2331, 1625, 1593, 1522, 1461, 1332, 1215, 1166, 1063 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.77 (d, J = 6.6 Hz, 2H), 8.10 (d, J = 6.7 Hz, 2H), 7.81 (d, J = 16.3 Hz, 1H), 7.55–7.45 (m, 8H), 7.36 (d, J = 16.3 Hz, 1H), 5.63 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 164.51 (1JCF = 249.5 Hz), 154.30, 143.90, 140.59, 134.22, 132.02, 131.12, 131.05, 129.63, 124.50, 124.32, 123.21, 116.20 (2JCF = 22.7 Hz),62.40. ESI-MS m/z: 368.0 [M – Cl]+; HR-ESIMS m/z: 368.0457 calcd for C20H16BrFN+ (368.0450).

Yield: 88%; yellow solid; IR (νmax): 3420, 2924, 2354, 1642, 1621, 1517, 1455, 1343, 1218, 1157, 1088, 1012 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.78 (d, J = 6.8 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H), 7.82 (d, J = 16.3 Hz, 1H), 7.64 (m, 3H), 7.43–7.36 (m, 7H), 5.66 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 154.28, 140.47, 136.21, 133.84, 133.56, 129.53, 129.46, 129.31, 128.99, 128.58, 124.25, 123.13, 63.25. ESI-MS m/z: 306.0 [M – Cl]+; HR-ESIMS m/z: 306.1054 calcd for C20H17ClN+ (306.1050).

Yield: 86%; white solid; mp 253–255 °C; IR (νmax): 3415, 2924, 2853, 2351, 1621, 1589, 1516, 1491, 1409, 1214, 1151, 1086 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.65 (d, J = 6.8 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H), 7.83 (d, J = 16.3 Hz, 1H), 7.65 (m, 3H), 7.39–7.21 (m, 6H), 5.72 (s, 2H), 2.24 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 154.28, 143.92, 140.50, 137.45, 136.19, 133.87, 131.12, 129.88, 129.49, 128.98, 127.40, 124.22, 123.17, 61.35, 17.85. ESI-MS m/z: 320.0 [M – Br]+; HR-ESIMS m/z: 320.1206 calcd for C21H19ClN+(320.1206).

Yield: 87%; white solid; mp 146–148 °C; IR (νmax): 3383, 2922, 2852, 2343, 1620, 1588, 1491, 1468, 1222, 1155, 1088, 1021 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.77 (d, J = 6.7 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H), 7.83 (d, J = 16.3 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.49–7.45 (m, 2H), 7.36–7.12 (m, 5H), 5.63 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 154.30, 143.89, 140.50, 136.19, 133.85, 131.13, 131.06, 129.67, 129.48, 128.98, 124.30, 123.14, 116.19, 116.02, 62.38. ESI-MS m/z: 340.0 [M – Cl]+; HR-ESIMS m/z: 340.0663 calcd for C20H16Cl2N+ (340.0660).

Yield: 93%; white solid; mp 123–125 °C; IR (νmax): 3391, 3120, 3088, 3041, 2095, 1621, 1589, 1567, 1515, 1492, 1470, 1410, 1330, 1207, 1157, 1012, 980 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.78 (d, J = 6.0 Hz, 2H), 8.11 (d, J = 6.7 Hz, 2H), 7.83 (d, J = 16.3 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.43–7.33 (m, 7H), 5.65 (s, 2H); 13CNMR (126 MHz, CD3OD): δ 154.37, 143.99, 140.57, 136.21, 135.54, 133.84, 132.32, 130.34, 129.50, 129.37, 128.99, 124.33, 123.14, 62.34. ESI-MS m/z: 340.0 [M – Cl]+; HR-ESIMS m/z: 340.0661 calcd for C20H16Cl2N+ (340.0660).

Yield: 88%; white solid; mp 237–239 °C; IR (νmax): 2921, 2351, 1692, 1659, 1621, 1555, 1469, 1453, 1157 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.72 (d, J = 6.8 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H), 7.84 (d, J = 16.3 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.52–7.33 (m, 7H), 5.79 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 154.50, 144.18, 140.70, 136.24, 134.37, 133.84, 132.03, 131.66, 130.76, 130.26, 129.52, 128.99, 128.01, 124.12, 123.13, 60.98. ESI-MS m/z: 324.0 [M – Cl]+; HR-ESIMS m/z: 324.0959 calcd for C20H16ClFN+ (324.0955).

Yield: 87%; white solid; mp 233–235 °C; IR (νmax): 2921, 2341, 1642, 1653, 1623, 1529, 1351, 1165, 1085 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.72 (d, J = 6.7 Hz, 2H), 8.22 (d, J = 8.1 Hz, 1H), 8.13 (d, J = 6.7 Hz, 2H), 7.86 (d, J = 16.3 Hz, 2H), 7.78 (t, J = 7.5 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 7.6 Hz, 1H), 7.38 (dd, J = 12.4, 3.8 Hz, 2H), 6.02 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 158.54, 152.04, 148.35, 144.71, 143.62, 140.20, 138.68, 137.79, 136.11, 135.06, 134.19, 133.49, 132.94, 131.71, 131.22, 129.81, 129.14, 128.06, 127.11, 64.34. ESI-MS m/z: 351.0 [M – Br]+; HR-ESIMS m/z: 351.0908 calcd for C20H16ClN2O2+ (351.0900).

Yield: 89%; white solid; mp 283–285 °C; IR (νmax): 2921, 2851, 2399, 1694, 1679, 1627, 1564, 1535, 1502, 1468, 1453, 1430, 1203, 994 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.80 (d, J = 6.8 Hz, 2H), 8.11 (d, J = 6.8 Hz, 2H), 7.80 (d, J = 16.3 Hz, 1H), 7.69 (s, 1H), 7.59–7.57 (m, 1H), 7.41–7.35 (m, 8H), 5.66 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 154.06, 144.02, 140.16, 137.16, 134.75, 133.56, 130.30, 130.03, 129.54, 129.31, 128.64, 127.62, 126.39, 124.46, 124.00, 63.31. ESI-MS m/z: 306.0 [M – Br]+; HR-ESIMS m/z: 306.1052 calcd for C20H17ClN+ (306.1050).

Yield: 86%; white solid; mp 126–128 °C; IR (νmax): 3416, 2922, 2850, 2351, 1658, 1621, 1556, 1511, 1454, 1336, 1214, 1106 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.86 (d, J = 6.3 Hz, 2H), 8.20 (t, J = 7.6 Hz, 4H), 7.95–7.87 (m, 4H), 7.55 (d, J = 16.4 Hz, 1H), 7.42–7.38 (m, 4H), 5.70 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 153.53, 148.51, 144.25, 141.25, 138.88, 133.45, 129.58, 129.32, 128.84, 128.69, 126.61, 124.90, 123.79, 63.49. ESI-MS m/z: 317.0 [M – Br]+; HR-ESIMS m/z: 317.1290 calcd for C20H17N2O2+ (317.1290).

Yield: 85%; white solid; mp 260–262 °C; IR (νmax): 3256, 2918, 2355, 2327, 1653, 1641, 1622, 1558, 1512, 1453, 1340, 1102, 1005 cm–1;1H NMR (400 MHz, CD3OD): δ 8.72 (d, J = 6.8 Hz, 2H), 8.23–8.17 (m, 4H), 7.94 (m, 4H), 7.56 (d, J = 16.4 Hz, 1H), 7.33–7.22 (m, 3H), 5.76 (s, 2H), 2.24 (s, 3H); 13C NMR (126 MHz, DMSO-d6): δ 161.91, 154.56, 148.31, 144.92, 142.11, 140.68, 131.10, 130.67, 130.42, 130.27, 127.56, 124.65, 124.27, 121.15, 115.20, 61.46, 55.94. ESI-MS m/z: 331.0 [M – Br]+; HR-ESIMS m/z: 331.1449 calcd for C21H19N2O2+ (331.1447).

Yield: 86%; white solid; mp126–128 °C; IR (νmax): 2919, 2351, 1700, 1695, 1684, 1653, 1646, 1616, 1558, 1540, 1506, 1336 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.85 (d, J = 6.2 Hz, 2H), 8.22 (m, 4H), 7.93–7.88 (m, 4H), 7.54–7.47 (m, 4H), 5.67 (s, 2H); 13C NMR (126 MHz, CD3OD): δ 164.54, 162.56, 153.58, 148.52, 144.20, 141.23, 138.93, 131.20, 131.13, 129.55, 128.82, 126.59, 124.91, 123.81, 116.23 (2JCF = 21.4 Hz), 62.63. ESI-MS m/z: 335.0 [M – Cl]+; HR-ESIMS m/z: 335.1199 calcd for C20H16FN2O2+ (335.1196).

Yield: 88%; white solid; mp 281–283 °C; IR (νmax): 3407, 2922, 2852, 2345, 1642, 1608, 1556, 1516, 1481, 1454, 1391, 1264, 1219, 1157, 1022 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.73 (d, J = 6.9 Hz, 2H), 8.09 (d, J = 6.8 Hz, 2H), 8.00–7.96 (m, 2H), 7.79–7.73 (m, 5H), 7.41–7.36 (m, 6H), 5.63 (s, 2H), 3.84 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 159.45, 154.77, 143.67, 142.57, 136.26, 133.70, 130.44, 130.34, 129.96, 129.49, 129.30, 128.79, 128.52, 127.55, 127.28, 123.78, 123.65, 121.31, 119.33, 105.72, 63.05, 54.54. ESI-MS m/z: 352.0 [M – Br]+; HR-ESIMS m/z: 352.1705 calcd for C25H22NO+ (352.1701).

Yield: 88%; white solid; mp 265–267 °C; IR (νmax): 2924, 2341, 1641, 1482, 1463, 1416, 1349, 1265, 1218, 1192, 1024, 973 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.71 (d, J = 5.4 Hz, 2H), 8.21 (d, J = 5.6 Hz, 2H), 8.12–8.07 (m, 2H), 7.91–7.86 (m, 4H), 7.50–7.29 (m, 6H), 5.81 (s, 2H), 3.96 (s, 3H), 2.36 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 159.35, 154.97, 143.77, 137.38, 131.12, 130.44, 130.28, 129.96, 129.90, 129.42, 128.81, 128.54, 127.55, 127.32, 127.26, 126.81, 126.01, 123.85, 123.73, 121.33, 119.30, 61.16, 54.55, 17.78. ESI-MS m/z: 366.0 [M – Br]+; HR-ESIMS m/z: 366.1859 calcd for C26H24NO+ (366.1858).

Yield: 90%; brown solid; mp 256–258 °C; IR (νmax): 3356, 2930, 2859, 2343, 1703, 1650, 1632, 1539, 1526, 1469, 1341, 1223, 1171, 1025, 977 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.68 (d, J = 6.8 Hz, 2H), 8.09 (d, J = 6.8 Hz, 2H), 8.01–7.97 (m, 2H), 7.79–7.73 (m, 4H), 7.52–7.49 (m, 2H), 7.45–7.39 (m, 2H), 7.37 (d, 2H), 5.77 (s, 2H), 3.84 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 159.45, 154.98, 143.85, 142.80, 136.28, 134.34, 131.94, 130.43, 130.27, 130.01, 129.46, 128.01, 127.56, 127.17, 126.04, 123.71, 123.63, 121.30, 119.30, 105.79, 60.79, 54.58. ESI-MS m/z: 386.0 [M – Cl]+; HR-ESIMS m/z: 386.1316 calcd for C25H21ClNO+ (386.1312).

Yield: 88%; brown solid; mp 153–155 °C; IR (νmax): 3389, 2919, 2850, 2353, 1670, 1642, 1609, 1555, 1516, 1454, 1350, 1263, 1192, 985 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.72 (d, J = 6.8 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H), 8.01–7.97 (m, 2H), 7.79–7.73 (m, 5H), 7.40 (m, 5H), 5.62 (s, 2H), 3.84 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 159.45, 154.88, 143.68, 142.68, 136.27, 135.51, 132.42, 130.44, 130.38, 130.31, 130.24, 129.99, 129.37, 128.78, 127.55, 127.33, 123.84, 123.68, 121.30, 119.30, 105.76, 62.15, 54.55. ESI-MS m/z: 386.0 [M – Cl]+; HR-ESIMS m/z: 386.1312 calcd for C25H21ClNO+ (386.1312).

Yield: 93%; yellow solid 241–243 °C; IR (νmax): 2916, 2848, 2351, 1695, 1659, 1642, 1608, 1555, 1482, 1454, 1349, 1235, 1024 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.71 (d, J = 6.3 Hz, 2H), 8.09 (d, J = 6.3 Hz, 2H), 7.99 (br s, 1H), 7.95 (s, 1H), 7.76 (t, J = 10.8 Hz, 4H), 7.65–7.01 (m, 6H), 5.72 (s, 2H), 3.84 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 159.44, 154.93, 143.75, 142.74, 136.27, 132.30, 132.23, 131.33, 130.42, 129.99, 128.77, 127.54, 125.20, 125.17, 123.75, 123.69, 121.28, 120.89, 120.78, 119.30, 115.95, 115.78, 105.73, 57.46, 54.55. ESI-MS m/z: 370.0 [M – Br]+; HR-ESIMS m/z: 370.1608 calcd for C25H21FNO+ (370.1607).

Yield: 95%; yellow solid; mp 261–263 °C; IR (νmax): 3387, 2920, 2850, 2344, 1642, 1609, 1556, 1508, 1482, 1468, 1390, 1264, 1230, 1193, 1025, 984 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.72 (d, J = 6.8 Hz, 2H), 8.09 (d, J = 6.9 Hz, 2H), 8.00–7.96 (m, 2H), 7.79–7.73 (m, 4H), 7.49–7.35 (m, 6H), 5.61 (s, 2H), 3.84 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 159.45, 154.82, 143.60, 142.62, 136.27, 131.02 (3JCF = 8.8 Hz), 130.43 (3JCF = 8.8 Hz), 129.96, 128.78, 127.55, 123.80, 123.64, 121.29, 119.33, 116.20 (2JCF = 22.7 Hz), 105.72, 62.18, 54.54. ESI-MS m/z: 370.0 [M – Cl]+; HR-ESIMS m/z: 370.1606 calcd for C25H21FNO+ (370.1607).

Yield: 85%; yellow solid; mp 251–253 °C; IR (νmax): 3363, 2951, 2362, 1644, 1605, 1585, 1463, 1357, 1263, 1239, 1186, 1158, 1135, 1023, 985 cm–1; 1H NMR (400 MHz, CD3OD): δ 8.67 (d, J = 6.5 Hz, 2H), 8.23 (d, J = 8.1 Hz, 1H), 8.12 (d, J = 6.6 Hz, 2H), 8.03–8.00 (m, 3H), 7.80–7.76 (m, 5H), 7.71–7.67 (m, 1H), 7.43–7.39 (m, 2H), 6.00 (s, 2H), 3.85 (s, 3H); 13C NMR (126 MHz, CD3OD): δ 159.50, 155.14, 148.08, 144.09, 142.93, 136.33, 134.69, 131.87, 131.04, 130.47, 129.99, 128.79, 127.58, 125.87, 123.66, 123.61, 121.32, 119.35, 105.73, 60.20, 54.55. ESI-MS m/z: 397.0 [M – Br]+; HR-ESIMS m/z: 397.1548 calcd for C25H21N2O3+ (397.1552).

Yield: 94%; yellow solid; mp 278–280 °C; IR (νmax): 3362, 3011, 2921, 2352, 1642, 1607, 1555, 1518, 1484, 1346, 1265, 1234, 1196, 1157, 1104, 972 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.96 (d, J = 6.8 Hz, 2H), 8.21–8.19 (m, 4H), 8.10–8.04 (m, 3H), 7.81 (t, J = 4.4 Hz, 3H), 7.66 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 16.2 Hz, 1H), 7.29 (d, J = 2.3 Hz, 1H), 5.81 (s, 2H), 3.80 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 159.24, 148.38, 144.83, 142.48, 130.79, 130.54, 130.26, 128.72, 128.21, 124.79, 124.66, 124.60, 122.72, 119.90, 106.92, 55.89. ESI-MS m/z: 397.0 [M – Br]+; HR-ESIMS m/z: 397.1551 calcd for C25H21N2O3+ (397.1552).

To the solution of 6-methoxy-2-naphthaldehyde (1, equiv) and 1-benzylpiperidin-4-amine (1.5, equiv) in ethanol, few drops of glacial acetic acid were added. The resulting mixture was stirred at room temperature for 6–7 h. The solvent was evaporated on a rotary evaporator to obtain the imine product, 9a, in 80% yield. White solid; mp 143–145 °C; IR (νmax): 3436, 2949, 2851, 2800, 1628, 1602, 1553, 1480, 1384, 1246, 1169, 1024 cm–1. 1H NMR (400 MHz, CDCl3): δ 8.46 (s, 1H), 8.01–7.94 (m, 2H), 7.84–7.73 (m, 2H), 7.43–7.37 (m, 3H), 7.37–7.33 (m, 1H), 7.32–7.29 (m, 1H), 7.22–7.14 (m, 2H), 3.96 (s, 3H), 3.60 (s, 2H), 3.37–3.25 (m, 1H), 3.00 (d, J = 12.2 Hz, 2H), 2.28–2.15 (m, 2H), 2.04–1.90 (m, 2H), 1.81 (t, J = 15.2 Hz, 2H); 13C NMR (101 MHz, CDCl3): δ 159.36, 158.60, 136.04, 132.13, 130.12, 129.45, 129.19, 128.49, 128.22, 127.24, 126.99, 124.71, 119.19, 105.96, 67.73, 63.19, 55.37, 52.18, 33.56. ESI-MS m/z: 359.0 [M + H]+; HR-ESIMS m/z: 359.2122 calcd for C24H26N2O + H+ (359.2123).

To the solution of 1-benzyl-N-((2-methoxynaphthalen-6-yl)methylene)piperidin-4-amine (9a) in methanol was added sodium borohydride (2 equiv), and the mixture was stirred at room temperature for 4 h. The solvent was evaporated, and the resulting crude residue was separated in dichloromethane and water. The dichloromethane layer was dried over a rotary evaporator, and the obtained residue was purified using silica gel column chromatography using the dichloromethane: methanol mobile phase to afford 10a as a white solid in 68% yield. mp 112–114 °C; IR (νmax): 3435, 3052, 2932, 2796, 1604, 1553, 1451, 1384, 1263, 1173, 1028 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.58 (s, 1H), 7.58–7.51 (m, 2H), 7.34–7.25 (m, 1H), 7.18 (s, 2H), 7.17 (s, 2H), 7.13–7.08 (m, 1H), 7.04–6.97 (m, 2H), 3.80 (d, J = 6.7 Hz, 2H), 3.78 (s, 3H), 3.36 (s, 2H), 2.84–2.66 (m, 2H), 2.48–2.36 (m, 1H), 1.94–1.83 (m, 2H), 1.83–1.72 (m, 2H), 1.41–1.32 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 157.46, 138.42, 135.71, 133.70, 129.20, 128.91, 128.20, 127.17, 126.99, 126.35, 118.81, 105.67, 63.09, 55.32, 54.18, 52.37, 50.82, 32.63. ESI-MS m/z: 361.0 [M + H]+; HR-ESIMS m/z: 361.2284 calcd for C24H28N2O + H+ (361.2280).

To the solution of 6-methoxy-2-naphthaldehyde (1, equiv) and 2-(1-benzylpiperidin-4-yl)ethanamine (1.5, equiv) in ethanol, few drops of glacial acetic acid were added. The resulting mixture was stirred at room temperature for 6 h. The solvent was evaporated on a rotary evaporator to obtain the imine, 9b, which was directly used for the next step. The methanolic solution of imine 9b and sodium borohydride (2 equiv) was stirred at room temperature for 4 h. The solvent was evaporated, and the resulting crude residue was separated in dichloromethane and water. The dichloromethane layer was dried over a rotary evaporator, and the obtained residue was purified using silica gel column chromatography using the dichloromethane: methanol mobile phase to afford 10b as a white solid in 56% yield. mp 102–104 °C; IR (νmax): 3437, 3051, 2919, 2344, 1602, 1566, 1452, 1384, 1265, 1152 cm–1 .1H NMR (400 MHz, CDCl3): δ 7.63 (s, 1H), 7.63–7.56 (m, 2H), 7.34–7.32 (m, 1H), 7.22 (s, 2H), 7.17 (d, J = 7.2 Hz, 2H), 7.09–7.02 (m, 3H), 3.83 (s, 3H), 3.83 (d, 2H), 3.40 (s, 2H), 2.93–2.70 (m, 2H), 2.64–2.55 (m, 2H), 1.89–1.79 (m, 2H), 1.58–1.50 (m, 2H), 1.42–1.35 (m, 2H), 1.24–1.16 (m, 3H). 13C NMR (101 MHz, CDCl3): δ 157.46, 138.48, 135.54, 133.71, 129.30, 129.20, 128.90, 128.15, 127.18, 126.95, 126.92, 126.43, 118.81, 105.67, 63.56, 55.32, 54.16, 53.87, 46.94, 36.89, 33.84, 32.42, 29.75. ESI-MS m/z: 389.0 [M + H]+; HR-ESIMS m/z: 389.2592 calcd for C26H32N2O + H+ (389.2593).

The assay followed here is the same as described in our previous work. Briefly, the enzyme solution (2.5 U/mL AChE/BChE; 20 μL) and test compound solution (inhibitor; 20 μL) were incubated at 25 °C for 20 min. DTNB (140 μL of 0.3 mM) followed by AtChI (20 μL of 10 mM) solution were added. The developed yellow color was measured at 412 nm. The percent inhibition of the enzyme activity was calculated using the formula [1 – (Sample absorbance/Controlabsorbance)] × 100, and the IC50 was determined graphically using GraphPad Prism software. The enzyme kinetics for cholinesterase inhibition was carried out as described earlier. The enzyme was incubated with different substrate and inhibitor concentrations, and the Lineweaver–Burk double-reciprocal plot was plotted between reaction velocity and substrate concentration. The inhibition rate constant (ki) was calculated by replotting the slopes of the Lineweaver–Burk plot versus inhibitor concentrations.

The assay is the same as described in our previous publication. Briefly, the Aβ1-42 rat peptide (Sigma-Aldrich) was dissolved in 10% w/v NH4OH at 0.5 mg/ml. The solution was incubated for 10 min at room temperature, followed by removing NH4OH under vacuum to obtain fluffy white powder. The powder is then reconstituted in a mixture of CH3CN/0.3 mM Na2CO3/250 mM NaOH (48.4:48.4:3.2) to obtain a 200 μM solution. The test compound was incubated with peptide solution at 10 μM. After the incubation period, 40 μM Thioflavin T was added to the assay solution, and fluorescence intensity was recorded with 446/490 nm excitation/emission filters set for 300 s at an interval of 30 s. The % inhibition of aggregation by the test compound was calculated using the formula 100 – (IFi/IFo × 100), where IFi and IFo are the respective fluorescence intensities obtained in the presence and the absence of the inhibitor, respectively.

The cell viability was determined by MTT assay in primary astrocytes and J774A.1 cells. Primary astrocytes and J774A.1 were seeded at a density of 4 × 103 and 104 cells per well of a 96-well plate, respectively. Cells were treated with various concentrations of 7av (0.78 to 100 μM) for 24 h. After drug incubation, MTT was added at a concentration of 2.5 mg/mL for 4 h. The supernatants were discarded, and DMSO was added into the wells for dissolving formazan crystals. The absorbance was measured at 570 nm.

NLRP3 inflammasome assay was done in J774A.1 cells, which were activated by LPS (1 μg/mL) and nigericin (10 μM). Cells were treated with various concentrations (5, 10, 20, and 40 μM) of 7av 1 h prior to treatment with nigericin. MCC950 at 100 nM was used as the standard and added to cells 1 h before treatment with nigericin. After the treatments, the supernatant was collected for analysis of IL-1β through ELISA. Furthermore, the remaining cells were collected and lysed with lysis buffer (0.2 N NaOH + 1% Triton X-100) for protein estimation. The level of pro-inflammatory cytokine IL-1β in the supernatant was assessed by using an Invitrogen mIL-1β ELISA kit, and the OD values were normalized by the total protein present in the sample.

The crystal structures of human AChE (PDB ID: 4EY7) and human BChE (PDB ID: 6EP4) were retrieved from a PDB and were used for molecular modeling studies under default settings from Glide. The docking was performed as described earlier. Briefly, the protein was made ready for docking using the protein preparation wizard tool. The bond angle and bond orders were assigned in Prep wizard; subsequently, hydrogen atoms, missing loops, and side chains were included. The restrained minimization of protein conformation was performed with an RMSD cutoff of 0.30 Å and the OPLS-2005 force field. Ligands were drawn in ChemDraw and saved in the mol2 format. LigPrep was used for preparing these ligands ready for docking. The grid was generated on the protein using a co-crystalized ligand as a centroid. Docking was performed in the XP mode under default settings of Glide. The docked poses were analyzed, and the pose with highest dock score was used for interaction analysis.

The stability of docked complexes was assessed via performing MD simulation using Desmond, as described earlier. The protein–ligand docked complex (.pv file) retrieved from XP docking was imported into the Maestro terminal of Desmond. Next, the system build-up was performed using the system builder, followed by minimization of the protein–ligand complex using a minimization module. The MD simulation was run for 100 ns trajectory, and results were obtained using the simulation interaction diagram module.