Design, synthesis and evaluation of N-arylmethylamide derivatives as cholinesterase inhibitors

doi:10.21203/rs.3.rs-3240453/v1

Download PDF

Research Article

Design, synthesis and evaluation of N-arylmethylamide derivatives as cholinesterase inhibitors

https://doi.org/10.21203/rs.3.rs-3240453/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

A series of N-arylmethylamide derivatives were designed and synthesized as cholinesterase inhibitors (ChEIs) for the treatment of Alzheimer’s disease (AD). Furthermore, the compounds were assayed for their inhibitory activity to cholinesterase in vitro, and the results indicated that most of the compounds had moderate inhibitory activity to cholinesterase. Among them, compound 22j showed the best inhibitory activity against BuChE (IC₅₀ = 0.46 µM) and moderate inhibitory activity against AChE (IC₅₀ = 6.52 µM); remarkably, compound 22c was found to be a dual inhibitor of AChE (IC₅₀ = 1.11 µM) and BuChE (IC₅₀ = 1.14 µM). In addition, the results of molecular docking studies exhibited that 22j could simultaneously bind to both CAS and PAS of BuChE, which was consistent with the mixed mode of inhibition shown by enzyme kinetic studies of 22j. Moreover, the molecular properties of all compounds were predicted by the molinspiration server, and the compounds 22j and 22c matched the most properties of orally administered drugs. All these suggested that 22j and 22c could be considered as a lead compound for the development of AD drugs.

Alzheimer’s disease

Cholinesterase inhibitor

Kinetic study

Molecular docking study

Alzheimer's disease (AD) is a severe and irreversible progressive neurodegenerative disease of the central nervous system characterized by memory loss, disorientation, cognitive deficits and behavioral abnormalities [1]. Currently, AD affects more than 50 million people worldwide, and this number will continue to increase with the aging population growing [2]. In addition, the annual healthcare costs associated with AD amount to $820 billion, which will place a huge social and economic burden on patients and their families [3].

Researchers have made many efforts to treat AD, and various hypotheses have been proposed by researchers, such as the cholinergic hypothesis [4], the amyloid (Aβ) toxicity hypothesis [5], the Tau protein abnormality hypothesis [6], the neuroinflammatory hypothesis [7], the free radical damage hypothesis [8] and so on. Of these, the cholinergic hypothesis is one of the widely accepted hypotheses by researchers [9]. This hypothesis suggests that AD patients lose acetylcholine (ACh) in the brain during the onset of the disease, which in turn affects the patient’s ability to focus and memory [10]. It has been confirmed that the decreasing of ACh in the brain is mainly caused by acetylcholinesterase (AChE) hydrolyzing. Therefore, the classical treatment of AD is to increase the concentration of ACh by inhibiting AChE activity, thereby improving the patient’s cognitive level [11, 12]. Up to now, most of the drugs approved by the U.S. Food and Drug Administration (FDA) for the treatment of AD are acetylcholinesterase inhibitors (AChEIs), including tacrine, rivastigmine, donepezil, and galantamine [13] (Fig. 1). Currently, AChEIs are still one of the research hotspots, and many new AChEIs for the treatment of AD have been reported, such as compounds 5–7 [14–16] (Fig. 2).

With the in-depth study on AD, it was found that AChE is the main enzyme responsible for ACh metabolism in the normal brain and in the early stages of AD [17], but the AChE is 67% lower than normal level in stages of advanced AD, which lead to lose efficacy of AChEIs for treating advanced AD. However, butyrylcholinesterase (BuChE) in the brain of advanced AD was found to 165% higher than normal level [18, 19], which can also hydrolyze ACh [20]. Thereby, butyrylcholinesterase inhibitors (BuChEIs) may be used to treat advanced AD, and by now, many novel BuChEIs for the treatment of AD have been reported, such as compounds 8–10 [22–24] (Fig. 2).

Therefore, simultaneous inhibition of AChE and BuChE could be a beneficial treatment approach for AD. Based on this, compounds with AChE and BuChE inhibitory activities were designed for the treatment of AD in this paper, and their design philosophy was as follows: in a previous study, it was shown that compound 11 (Fig. 3) could inhibit AChE and BuChE with IC₅₀ value of 4.61 µM and 0.94 µM separately [25]. And structural analysis showed that the A region could enhance the inhibitory activity against BuChE. Therefore, the A region of compound 11 is the core molecule of its BuChE inhibitory activity. In addition, it was indicated that compound 12 (Fig. 3) possessed good ChE inhibitory activity (AChE: IC₅₀ = 0.22 µM; BuChE:

IC₅₀ = 0.0016 µM) [26]. Moreover, molecular docking analysis showed that the B region could be associated with hydrogen bonding as well as π-π stacking with the PAS site of BuChE. Meanwhile, the B region could also produce hydrogen bonding as well as π-π stacking with the PAS site of AChE. Therefore, the A region of compound 11 was combined with the B region of compound 12 through an amide chain to produce a new compound 13 for inhibiting AChE and BuChE simultaneously; then under the guidance of molecular docking evaluation, the compound 13 was optimized and the target compounds was designed (Fig. 3).

Chemical synthesis

The synthetic strategy for target compounds 22a-22v was depicted in Scheme 1. Commercially available compound 14a-14d was reacted with diamines protected by a Boc group to afford the intermediates 15a-15i. Then, compounds 15a-15i were reacted with different acyl chlorides to give compounds 16a-16n. After purification by column chromatography, Boc removal was achieved by dissolving 16a–16n in MeOH solution of HCl gas at 25°C for 6 hours, then the resulted free amines were immediately reacted with the monomethyl terephthalate to give target compounds 19a. After hydrolysis, compounds 20a were converted into compounds 22a-22i by reacting with different amines in the presence of O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and N,N-Diisopropylethylamine (DIEA). In addition, monomethyl terephthalate was reacted with 3-(trifluoromethyl)-benzylamine, which was hydrolyzed to afford compound 21a. The condensation reactions of compound 21a with compounds 17b-17n in the presence of TBTU and DIEA yielded the target compounds 22j-22v. The structures of new compounds confirmed by the ¹H NMR, ¹³C NMR, HRMS and IR spectra, and the purity of all the target compounds was determined to be over 95.0% by high-performance liquid chromatography (HPLC) analysis.

AChE and BuChE assay

To evaluate the eeAChE and eqBuChE inhibitory activity of the target compounds, Ellman's method [27] was performed using Huperzine-A and tacrine as reference compounds, and the results were shown in Table 1. In general, all of the target compounds 22a-22v showed a certain inhibitory activity against eeAChE and eqBuChE with IC₅₀ values ranging from 0.46 to 38.79 µM. Among them, compound 22j (eeAChE, IC₅₀ = 0.46 µM; eqBuChE, IC₅₀ = 6.52 µM) was found to have the best inhibitory activity to eqBuChE and moderate inhibitory activity to eeAChE. In addition, it was found that compound 22c (eeAChE, IC₅₀ = 1.11 µM; eqBuChE, IC₅₀ = 1.14 µM) showed good inhibitory activity to both eeAChE and eqBuChE. For eqBuChE, when the substituent on benzene ring of R₃ is an electron-withdrawing group, the activity is significantly higher than that of the electron-donating group. For example, the activity of compound 22c with trifluoromethyl on benzene ring of R₃ is significantly higher than that of compound 22d with methoxy on benzene ring of R₃. However, the inhibitory activity against eqBuChE was significantly reduced when the R₃ group was changed to pyrazin-2-methyl and 2-(pyrrolidin-1-yl)ethyl. Moreover, when R₂ was propyl, the inhibitory effect on eqBuChE was the best; and the inhibitory effect on eqBuChE decreased with the increasing of n value. Furthermore, the inhibitory activity of eqBuChE was significantly reduced when the R₁ group was changed from the naphthalene to the biphenyl, anthracene and indole. For eeAChE, when the R₃ was benzyl or benzyl with substituents, the inhibitory activity is better than that of pyrazin-2-methyl or 2-(pyrrolidin-1-yl)ethyl. In addition, when R₂ was isopropyl and n is 1, the inhibitory effect on eeAChE was the best.

Kinetic analysis

In order to understand the inhibition mechanism of BuChE, kinetic analysis of BuChE inhibition was carried out for compound 22j. Lineweaver-Burk plots were plotted for three different concentrations of compound 22j with six different concentrations of substrates. As shown in Fig. 4, double reciprocal curves for different concentrations of 22j intersected in the second quadrant, which indicates that compound 22j inhibited BuChE in competitive and non-competitive manner simultaneously.

ADMET prediction

The properties of the synthesized compounds 22a-22v were predicted online using SwissADME (http://www.swissadme.ch). As shown in Table 3, the synthesized compounds were found to match the most properties of orally administered drugs.

Unfortunately, the most of the tested compounds could not cross the blood-brain barrier, thus they could not be used directly as oral drugs and needed optimizing further.

Molecular docking study

In order to explore the potential binding modes of compound 22j with AChE (PDB code: 4EY7) and BuChE (PDB code: 5K5E), molecular docking studies were performed using Autodock 4.2, and 3D docking results were outputted using the software PyMOL (https://www.pymol.org/pymol.html). As shown in Fig. 5, the benzene ring of the F region of compound 22j could interact π-π with Tyr-341 and the benzene ring of the R₃ group could interact π-π with Trp-86. At the same time, hydrogen bonding between the amide bond in the E region of the benzene ring and Phe-295 was also observed. Therefore, compound 22j was tightly bound to the optimal conformation of AChE and stabilized in the cavity. As shown in Fig. 6, the F region of the benzene ring of compound 22j could interact π-π with Tyr-332. Meanwhile, the naphthalene ring of R₁ group could interact with Phe-329 and Trp-231. In addition, a hydrogen bond was observed between the amide bond in the G region and Pro-285. Thus, compound 22j was tightly bound to the optimal conformation of BuChE and stabilized in the cavity.

In summary, a series of N-arylmethylamide derivatives were designed, synthesized, and evaluated as potential ChEIs against AD. Biological assays demonstrated that all synthesized compounds possessed a certain inhibitory activity to AChE and BuChE. Among them, compound 22j was the most potent BuChE inhibitor (IC₅₀ = 0.46 µM) and possessed AChE inhibitory activity (IC₅₀ = 6.52 µM). In addition, the inhibitory activity of compound 22c to AChE (IC₅₀ = 1.11 µM) and BuChE (IC₅₀ = 1.14 µM) was good and equal. Furthermore, molecular docking studies showed that 22j was able to bind to the CAS and PAS of BuChE, which is consistent with the mixed mode of inhibition shown by enzyme kinetic studies. Moreover, the predicted ADMET results indicate that the designed compounds conform to most of the drug properties. Taken together, compounds 22j and 22c as the dual inhibitors of AChE and BuChE may be a promising lead compounds for the development of new anti-AD drugs.

Chemistry

All experiments were carried out under air atmosphere, 2-naphthaldehyde, tert-butyl N-(2-aminoethyl)carbamate, mono-methyl terephthalate, 3-(trifluoromethyl)benzy-lamine and other reagent materials were commercially available analytically pure or chemically pure, unless stated otherwise. Reaction progress was observed by thin layer chromatography on the glass-backed silica gelsheets (Silica Gel 60 GF254) and visualized under UV light (254 nm). High Resolution Mass Spectrometry was determined by Bruker Maxis 4G Mass Spectrometer, and the nuclear magnetic resonance spectrum was measured by Bruker Avance III 600 MHz nuclear magnetic resonance spectrometer. The infrared (IR) spectra were run as KBr disk on FTIR-850 spectrophotometer (Tianjin Gangdong Sci. & Tech Co., Ltd.). The purity of the target compounds was determined by LC-3000 HPLC system (Beijing Chuangxin tongheng Technology Co., Ltd.). The melting point was determined by SGW X-4 micro melting point apparatus (Shanghai Precision Scientific Instrument Co., Ltd.). The 96 plate was read by 1420 Victor Microplate Reader. eeAChE and eqBuChE were purchased from Sigma, Huperzine-A and tacrine was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

General procedure for the synthesis of target compounds 22a–22i

To a solution of compound 20a (2.0 mmol, 0.836 g) and 3, 5-bis(trifluorometh-yl)benzylamine (2.0 mmol, 0.486 g) in CH₂Cl₂ (10 mL) at 0 °C were added TBTU (3.0 mmol, 0.963 g) and DIEA (3.0 mmol, 0.387 g). The reaction mixture was stirred at 25 °C for 4h. The completion of the reaction n was assessed by TLC. Then washed with 5% aqueous HCl (15.0 mL), 5% aqueous NaOH (20.0 mL) and brine (20.0 mL). The organics was dried over Na₂SO₄, filtered, and concentrated in vacuo to give crude compound 22a. The crude compound 22a was purified by column chromatography on silica gel and recrystallized from CH₂Cl₂/MeOH (1:50) to afford target compound 22a (1.080 g, 84%). Compounds 22b-22i were prepared by the same method produces.

N¹-(3,4-bis(trifluoromethyl)benzyl)-N⁴-(2-(N-(naphthalen-2-ylmethyl) isobutyr-amido)ethyl)terephthalamide (22a)

Chemical Formula: C₃₄H₃₁F₆N₃O₃; white solid; Yield: 84%; mp 167.1-167.6 °C; Purity: 95.15% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.86 (d, J = 5.9 Hz, -ArH, 5H), 7.82 (s, -ArH, 4H), 7.81 – 7.74 (m, -ArH, 3H), 7.57 (s, -NH-, 1H), 7.53 – 7.42 (m, -ArH, 2H), 7.14 (t, J = 6.0 Hz, -NH-, 1H), 4.77 (s, -CH₂-, 2H), 4.74 (d, J = 5.8 Hz, -CH₂-, 2H), 3.76 – 3.72 (m, -CH₂-, 2H), 3.62 – 3.58 (m, -CH₂-, 2H), 2.89 – 2.80 (m, -CH-, 1H), 1.11 (s, -CH₃, 3H), 1.10 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 180.73, 167.02, 166.51, 141.03, 136.96, 136.31, 133.60, 133.38, 132.92, 132.36, 132.14, 131.92, 131.70, 129.12, 127.97, 127.79, 127.69, 127.44, 127.38, 126.75, 126.34, 124.82, 124.10, 124.02, 122.30, 121.56, 53.40, 51.47, 44.93, 43.26, 40.42, 30.70, 19.75; ESI-MS cacld for 644.2349 [M+H]⁺, found 644.2340 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3292 (-CONH-), 1643 (C=O), 1633 (C=O).

N¹-(2-(N-(naphthalen-2-ylmethyl)isobutyramido)ethyl)-N⁴-(3-(trifluorometh-yl)benzyl)terephthalamide(22b)

Chemical Formula: C₃₃H₃₂F₃N₃O₃; white solid; Yield: 79%; mp 109.5-110.4 °C; Purity: 96.41% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.86 (d, J = 4.2 Hz, -ArH, 4H), 7.84 (d, J = 11.5 Hz, -ArH, 2H), 7.77 (s, -ArH, 2H), 7.60 (s, -ArH, 1H), 7.57 (s, -ArH, 1H), 7.56 (s, -ArH, 1H), 7.54 (s, -NH-, 1H), 7.52 – 7.49 (m, -ArH, 2H), 7.47 (s, -ArH, 1H), 7.46 (s, -ArH, 1H), 6.82 (t, J = 6.0 Hz, -NH-, 1H), 4.77 (s, -CH₂-, 2H), 4.70 (d, J = 5.8 Hz, -CH₂-, 2H), 3.79 – 3.71 (m, -CH₂-, 2H),3.65 – 3.56 (m, -CH₂-, 2H), 2.89 – 2.80 (m, -CH-, 1H), 1.10 (s, -CH₃, 3H), 1.09 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 180.67, 166.81, 166.49, 139.22, 136.82, 136.62, 133.65, 133.39, 132.92, 131.28, 131.22, 131.01, 129.30, 129.10, 127.70, 127.41, 127.35, 127.30, 127.16, 126.31, 124.81, 124.58, 124.55, 124.50, 124.48, 124.04, 51.41, 44.89, 43.67, 40.35, 34.70, 30.69, 19.76; ESI-MS cacld for 576.2475 [M+H]⁺, found 576.2480 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3318 (-CONH-), 1644 (C=O), 1639 (C=O).

N¹-(2-(N-(naphthalen-2-ylmethyl)isobutyramido)ethyl)-N⁴-(4-(trifluorometh-yl)benzyl)terephthalamide(22c)

Chemical Formula: C₃₃H₃₂F₃N₃O₃; white solid; Yield: 87%; mp 168.5-169.1 °C; Purity: 96.19% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.87 (d, J = 5.2 Hz, -ArH, 4H), 7.86 – 7.83 (m, -ArH, 2H), 7.78 (d, J = 2.7 Hz, -ArH, 2H), 7.62 – 7.56 (m, -ArH, 3H), 7.51 – 7.47 (m, -ArH, 4H), 7.28 (s, -NH-, 1H), 6.76 (t, J = 6.1 Hz, -NH-, 1H), 4.78 (s, -CH₂-, 2H), 4.71 (d, J = 5.8 Hz, -CH₂-, 2H), 3.78 – 3.72 (m, -CH₂-, 2H), 3.63 (d, J = 5.5 Hz, -CH₂-, 2H), 2.90 – 2.79 (m, -CH-, 1H), 1.11 (s, -CH₃, 3H), 1.10 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 180.70, 166.79, 166.45, 142.21, 136.85, 136.58, 133.63, 133.39, 132.93, 130.05, 129.84, 129.11, 128.06, 127.44, 127.28, 126.75, 126.32, 125.77, 125.74, 125.72, 125.69, 124.96, 124.82, 124.04, 123.16, 51.43, 44.89, 43.64, 40.38, 30.70, 26.92, 19.76; ESI-MS cacld for 576.2475 [M+H]⁺, found 576.2482 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3318 (-CONH-), 1662 (C=O), 1642 (C=O).

N¹-(3,4-dimethoxybenzyl)-N⁴-(2-(N-(naphthalen-2-ylmethyl)isobutyramido)- ethyl)terephthalamide(22d)

Chemical Formula: C₃₄H₃₇N₃O₅; white solid; Yield: 75%; mp 118.3-118.9 °C; Purity: 99.42% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.86 (d, J = 3.2 Hz, -ArH, 1H), 7.85 (d, J = 3.4 Hz, -ArH, 2H), 7.84 (s, -ArH, 2H), 7.83 (s, -ArH, 1H), 7.83 – 7.76 (m, -ArH, 2H), 7.76 – 7.71 (m, -ArH, 1H), 7.57 (s, -ArH, 1H), 7.53 – 7.47 (m, -ArH, 2H), 6.90 (d, J = 8.3 Hz, -ArH, 2H), 6.84 (d, J = 7.9 Hz, -NH-, 1H), 6.56 (t, J = 5.6 Hz, -NH-, 1H), 4.77 (s, -CH₂-, 2H), 4.58 (d, J = 5.5 Hz, -CH₂-, 2H), 3.87 (s, -CH₃, 3H), 3.86 (s, -CH₃, 3H), 3.77 – 3.72 (m, -CH₂-, 2H), 3.65 – 3.61 (m, -CH₂-, 2H), 2.87 – 2.80 (m, -CH-, 1H), 1.10 (s, -CH₃, 3H), 1.09 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 180.61, 166.55, 166.52, 149.31, 148.72, 136.98, 136.65, 133.69, 133.40, 130.61, 127.36, 127.22, 126.72, 124.81, 124.05, 120.35, 111.50, 111.41, 55.98, 55.96, 53.40, 51.38, 44.88, 44.13, 40.28, 30.69, 19.76; ESI-MS cacld for 568.2812 [M+H]⁺, found 568.2815 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3316 (-CONH-), 1657 (C=O), 1636 (C=O).

N¹-(4-methoxybenzyl)-N⁴-(2-(N-(naphthalen-2-ylmethyl)isobutyramido)eth-yl)terephthalamide(22e)

Chemical Formula: C₃₃H₃₅N₃O₄; white solid; Yield: 77%;; mp 181.4-181.9 °C; Purity: 99.16% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.87 (d, J = 3.3 Hz, -ArH, 1H), 7.85 (d, J = 6.6 Hz, -ArH, 4H), 7.83 (d, J = 2.3 Hz, -ArH, 1H), 7.79 (d, J = 2.6 Hz, -ArH, 1H), 7.73 (s, -ArH, 1H), 7.57 (s, -ArH, 1H), 7.52 – 7.47 (m, -ArH, 2H), 7.30 – 7.26 (m, -ArH, 2H), 6.95 (d, J = 7.6 Hz, -ArH, 1H), 6.91 (s, -NH-, 1H), 6.86 – 6.82 (m, -ArH, 1H), 6.57 (d, J = 5.6 Hz, -NH-, 1H), 4.78 (s, -CH₂-, 2H), 4.63 (d, J = 5.6 Hz, -CH₂-, 2H), 3.80 (s, -CH₃, 3H), 3.77 – 3.72 (m, -CH₂-, 2H), 3.66 – 3.60 (m, -CH₂-, 2H), 2.88 – 2.79 (m, -CH-, 1H) 1.11 (s, -CH₃, 3H), 1.10 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 180.63, 166.61, 166.52, 160.01, 139.58, 136.92, 136.65, 133.70, 133.40, 132.92, 129.89, 129.09, 127.78, 127.37, 127.29, 127.11, 126.73, 124.81, 124.06, 120.17, 113.63, 113.12, 113.02, 55.27, 51.37, 44.86, 44.22, 40.28, 30.69, 26.92, 19.77; ESI-MS cacld for 538.2707 [M+H]⁺, found 538.2719 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3301 (-CONH-), 1656 (C=O), 1628 (C=O).

N¹-benzyl-N⁴-(2-(N-(naphthalen-2-ylmethyl)isobutyramido)ethyl)terephthala-mide(22f)

Chemical Formula: C₃₂H₃₃N₃O₃; white solid; Yield: 76%; mp 130.5-131.1 °C; Purity: 96.06% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.87 (d, J = 2.7 Hz, -ArH, 1H), 7.86 – 7.85 (m, -ArH, 2H), 7.84 (s, -ArH, 2H), 7.83 (d, J = 2.3 Hz, -ArH, 1H), 7.78 (d, J = 2.8 Hz, -ArH, 1H), 7.73 (d, J = 6.2 Hz, -ArH, 1H), 7.57 (s, -NH-, 1H), 7.53 – 7.47 (m, -ArH, 2H), 7.37 (s, -ArH, 2H), 7.36 (d, J = 1.6 Hz, -ArH, 2H), 7.30 (d, J = 2.7 Hz, -ArH, 2H), 6.60 (t, J = 5.7 Hz, -NH-, 1H), 4.77 (s, -CH₂-, 2H), 4.65 (d, J = 5.6 Hz, -CH₂-, 2H), 3.77 – 3.72 (m, -CH₂-, 2H), 3.66 – 3.60 (m, -CH₂-, 2H), 2.88 – 2.79 (m, -CH-, 1H), 1.10 (s, -CH₃, 3H), 1.09 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) ) δ 180.62, 166.63, 166.54, 138.01, 136.94, 136.64, 133.69, 133.40, 132.92, 129.09, 128.84, 127.97, 127.78, 127.71, 127.36, 127.28, 127.24, 126.73, 126.30, 124.81, 124.06, 51.37, 44.86, 44.25, 40.26, 30.69, 26.92, 19.76; ESI-MS cacld for 508.2601 [M+H]⁺, found 508.2593 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3318 (-CONH-), 1662 (C=O), 1634 (C=O).

N¹-benzhydryl-N⁴-(2-(N-(naphthalen-2-ylmethyl)isobutyramido)ethyl)terepht-halamide(22g)

Chemical Formula: C₃₈H₃₇N₃O₃; white solid; Yield: 77%; mp 145.3-145.8 °C; Purity: 99.22% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.86 (d, J = 2.8 Hz, -ArH, 4H), 7.85 (d, J = 3.2 Hz, -ArH, 2H), 7.78 (d, J = 2.6 Hz, -ArH, 1H), 7.74 (s, -ArH, 1H), 7.58 (s, -ArH, 1H), 7.50 (t, J = 2.8 Hz, -ArH, 2H), 7.37 (d, J = 2.0 Hz, -ArH, 1H), 7.36 (s, -ArH, 2H), 7.34 (s, -ArH, 2H), 7.32 (d, J = 1.7 Hz, -ArH, 2H), 7.31 – 7.30 (m, -ArH, 2H), 7.29 (s, -NH-, 1H), 7.28 (d, J = 1.7 Hz, -ArH, 1H), 6.84 (d, J = 7.9 Hz, -ArH, 1H), 6.46 (d, J = 7.8 Hz, -CH-, 1H), 4.77 (s, -CH₂-, 2H), 3.79 – 3.71 (m, -CH₂-, 2H), 3.67 – 3.59 (m, -CH₂-, 2H), 2.89 – 2.77 (m, -CH-, 1H), 1.11 (s, -CH₃, 3H), 1.10 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 180.62, 166.47, 165.81, 141.31, 136.78, 133.70, 133.41, 132.92, 129.09, 128.79, 128.62, 127.79, 127.71, 127.65, 127.53, 127.39, 127.33, 127.14, 126.73, 126.30, 124.81, 124.06, 57.58, 53.40, 51.42, 44.92, 40.32, 30.70, 19.78; ESI-MS cacld for 584.2914 [M+H]⁺, found 584.2925 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3302 (-CONH-), 1652 (C=O), 1634 (C=O).

N¹-(2-(N-(naphthalen-2-ylmethyl)isobutyramido)ethyl)-N⁴-(pyrazin-2-ylmeth-yl)terephthalamide(22h)

Chemical Formula: C₃₀H₃₁N₅O₃; white solid; Yield: 78%; mp 123.8-124.3 °C; Purity: 96.35% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 8.70 (s, -ArH, 1H), 8.59 – 8.50 (m, -ArH, 2H), 7.90 (d, J = 2.3 Hz, -ArH, 4H), 7.85 (d, J = 3.1 Hz, -ArH, 2H), 7.78 (s, -ArH, 1H), 7.76 (d, J = 4.5 Hz, -ArH, 1H), 7.58 (s, -NH-, 1H), 7.50 (t, J = 3.7 Hz, -ArH, 2H), 7.45 (t, J = 5.2 Hz, -ArH, 1H), 7.29 (d, J = 1.8 Hz, -NH-, 1H), 4.83 (d, J = 5.1 Hz, -CH₂-, 2H), 4.79 (s, -CH₂-, 2H), 3.82 – 3.71 (m, -CH₂-, 2H), 3.69 – 3.61 (m, -CH₂-, 2H), 2.93 – 2.77 (m, -CH-, 1H), 1.11 (s, -CH₃, 3H), 1.10 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 201.56, 180.62, 166.77, 166.50, 152.14, 144.22, 143.67, 143.63, 136.87, 136.48, 133.70, 133.40, 132.92, 129.09, 127.78, 127.71, 127.40, 127.35, 127.16, 126.73, 126.30, 124.82, 124.06, 51.39, 44.89, 42.71, 40.28, 30.69, 29.68, 19.76; ESI-MS cacld for 510.2506 [M+H]⁺, found 510.2511 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3318 (-CONH-), 1672 (C=O), 1645 (C=O).

N¹-(2-(N-(naphthalen-2-ylmethyl)isobutyramido)ethyl)-N⁴-(2-(pyrrolidin-1-yl)-ethyl)terephthalamide(22i)

Chemical Formula: C₃₁H₃₈N₄O₃; white solid; Yield: 80%; mp 119.5-121.3 °C; Purity: 97.71% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 8.71 (s, -ArH, 1H), 8.03 (s, -NH-, 1H), 8.01 (s, -ArH, 1H), 7.84 (d, J = 8.7 Hz, -ArH, 2H), 7.81 (d, J = 8.2 Hz, -ArH, 2H), 7.78 (d, J = 9.0 Hz, -ArH, 2H), 7.64 (s, -ArH, 1H), 7.57 (s, -NH-, 1H), 7.48 (t, J = 3.2 Hz, -ArH, 2H), 4.78 (s, -CH₂-, 2H), 3.87 (t, J = 5.6 Hz, -CH₂-, 2H), 3.77 – 3.71 (m, -CH₂-, 2H), 3.67 – 3.60 (m, -CH₂-, 2H), 3.37 – 3.26 (m, -CH₂-, 6H), 2.87 – 2.78 (m, J = 6.1, 5.5 Hz, -CH-, 1H), 2.09 – 2.07 (m, -CH₂-, 4H), 1.09 (s, -CH₃, 3H), 1.07 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 180.29, 167.18, 166.74, 136.82, 135.91, 133.90, 133.40, 132.89, 129.02, 127.76, 127.72, 127.12, 126.22, 125.90, 124.80, 124.12, 118.88, 55.81, 54.49, 51.34, 44.91, 39.92, 36.61, 31.91, 30.66, 29.67, 23.30, 19.75, 14.08; ESI-MS cacld for 515.3023 [M+H]⁺, found 515.3015 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3336 (-CONH-), 1662 (C=O), 1547 (C=O).

General procedure for the synthesis of target compounds 22j–22v

To a solution of compound 21a (2.0 mmol, 0.646 g) and compound 17b (2.0 mmol, 0.540 g) in CH₂Cl₂ (10.0 mL) at 0 °C were added TBTU (3.0 mmol, 0.963 g) and DIEA (3.0 mmol, 0.387 g). The reaction mixture was stirred at 25 °C for 4h. The completion of the reaction was assessed by TLC. Then washed with 5% aqueous HCl (15.0 mL), 5% aqueous NaOH (20.0 mL) and brine (20.0 mL). The organics was dried over Na₂SO₄, filtered, and concentrated in vacuo to give crude compound 22j. The crude compound 22j was purified by column chromatography on silica gel and recrystallized from CH₂Cl₂/MeOH (1:45) to afford target compound 22j (1.007 g, 77%). Compounds 22k-22v were prepared by the same method produces.

N¹-(2-(N-(naphthalen-2-ylmethyl)butyramido)ethyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22j)

Chemical Formula: C₃₃H₃₂F₃N₃O₃; white solid; Yield: 77%; mp 159.0-159.7 °C; Purity: 97.00% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.82 (d, J = 8.0 Hz, -ArH, 2H), 7.79 (d, J = 9.7 Hz, -ArH, 3H), 7.59 (s, -ArH, 1H), 7.56 (s, -ArH, 1H), 7.53 (s, -ArH, 1H), 7.52 (d, J = 1.7 Hz, -ArH, 2H), 7.51 – 7.50 (m, -ArH, 1H), 7.50 (s, -ArH, 1H), 7.49 – 7.48 (m, -ArH, 1H), 7.45 (d, J = 7.7 Hz, -ArH, 1H), 7.18 (d, J = 8.4 Hz, -ArH, 1H), 6.67 (s, -NH-, 1H), 6.34 (s, -NH-, 1H), 4.69 – 4.66 (m, -CH₂-, 4H), 3.71 (t, J = 5.7 Hz, -CH₂-, 2H), 3.57 – 3.49 (m, -CH₂-, 2H), 2.16 (t, J = 7.6 Hz, -CH₂-, 2H), 1.67 – 1.62 (m, -CH₂-, 2H), 0.94 (t, J = 7.3 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 174.44, 172.58, 166.55, 139.10, 138.96, 135.38, 133.31, 132.91, 131.25, 131.01, 129.30, 129.05, 127.77, 127.73, 127.44, 126.97, 126.70, 126.36, 125.70, 124.51, 124.43, 52.84, 44.08, 43.64, 38.70, 37.96, 19.08, 13.84; ESI-MS cacld for 576.2475 [M+H]⁺, found 576.2469 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3297 (-CONH-), 1665 (C=O), 1626 (C=O).

N¹-(2-(N-(naphthalen-2-ylmethyl)pentanamido)ethyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22k)

Chemical Formula: C₃₄H₃₄F₃N₃O₃; white solid; Yield: 80%; mp 131.7-132.3 °C; Purity: 99.23% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.86 (d, J = 6.9 Hz, -ArH, 5H), 7.83 (d, J = 2.4 Hz, -NH-, 1H), 7.79 (d, J = 2.8 Hz, -ArH, 2H), 7.60 (s, -ArH, 1H), 7.58 (s, -NH-, 1H), 7.56 (s, 2H), 7.55 (s, -ArH, 1H), 7.50 (dd, J = 6.4, 3.4 Hz, -ArH, 2H), 7.47 (s, -ArH, 1H), 6.80 (s, -ArH, 1H), 4.75 (s, -CH₂-, 2H), 4.71 (d, J = 5.1 Hz, -CH₂-, 2H), 3.74 (s, -CH₂-, 2H), 3.60 (s, -CH₂-, 2H), 2.42 (t, J = 7.5 Hz, -CH₂-, 2H), 1.61 (t, J = 7.4 Hz, -CH₂-, 2H), 1.28 (q, J = 7.5 Hz, -CH₂-, 2H), 0.82 (t, J = 7.3 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 176.57, 166.79, 166.50, 139.21, 136.87, 136.61, 133.40, 132.94, 131.27, 131.03, 129.30, 129.13, 127.78, 127.72, 127.45, 127.30, 126.74, 126.32, 124.96, 124.56, 124.54, 124.51, 124.48, 124.15, 123.09, 51.95, 45.10, 43.68, 40.52, 33.14, 27.53, 22.44, 13.76; ESI-MS cacld for 590.2631 [M+H]⁺, found 590.2639 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3310 (-CONH-), 1652 (C=O), 1636 (C=O).

N¹-(2-(3-methyl-N-(naphthalen-2-ylmethyl)butanamido)ethyl)-N⁴-(3-(trifluor-omethyl)benzyl)terephthalamide(22l)

Chemical Formula: C₃₄H₃₄F₃N₃O₃; white solid; Yield: 81%; mp 167.5-168.3 °C; Purity: 99.51% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.88 (d, J = 8.1 Hz, -ArH, 3H), 7.85 (d, J = 8.3 Hz, -ArH, 4H), 7.81 – 7.77 (m, -NH-, 1H), 7.61 (s, -ArH, 2H), 7.58 – 7.54 (m, -ArH, 3H), 7.52 – 7.45 (m, -ArH, 3H), 6.80 (s, -NH-, 1H), 4.75 (s, -CH₂-, 2H), 4.70 (d, J = 5.6 Hz, -CH₂-, 2H), 3.76 – 3.72 (m, -CH₂-, 2H), 3.59 (t, J = 5.2 Hz, -CH₂-, 2H), 2.31 (d, J = 7.0 Hz, -CH₂-, 2H), 2.25 – 2.17 (m, -CH-, 1H), 0.91 (s, -CH₃, 3H), 0.90 (s, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 175.93, 166.79, 166.43, 139.23, 136.82, 136.61, 133.39, 133.35, 132.95, 131.27, 129.30, 129.12, 127.79, 127.33, 127.28, 126.74, 126.33, 124.96, 124.57, 124.55, 124.50, 124.47, 124.45, 124.13, 123.09, 51.90, 45.01, 43.67, 42.14, 40.69, 26.92, 25.87, 22.57; ESI-MS cacld for 590.2631 [M+H]⁺, found 590.2623 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3310 (-CONH-), 1668 (C=O), 1636 (C=O).

N¹-(2-(N-(naphthalen-2-ylmethyl)hexanamido)ethyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22m)

Chemical Formula: C₃₅H₃₆F₃N₃O₃; white solid; Yield: 78%; mp 155.4-155.8 °C; Purity: 98.11% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.99 – 7.81 (m, -ArH, 5H), 7.81 – 7.71 (m, -ArH, 2H), 7.60 (s, -ArH, 2H), 7.55 (d, J = 7.6 Hz, -ArH, 2H), 7.51 (s, -NH-, 1H), 7.50 (d, J = 6.1 Hz, -ArH, 4H), 6.83 (s, -NH-, 1H), 5.11 – 4.43 (m, -CH₂-, 4H), 4.01 – 2.99 (m, -CH₂-, 4H), 2.52 – 2.10 (m, -CH₂-, 2H), 1.64 (s, -CH₂-, 2H), 1.30 (s, -CH₂-, 2H), 1.23 (s, -CH₂-, 2H), 0.78 (d, J = 6.1 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 177.15, 166.83, 166.59, 139.28, 136.84, 136.64, 133.46, 133.40, 132.94, 131.33, 131.19, 130.98, 129.31, 129.16, 127.80, 127.77, 127.48, 126.76, 126.33, 124.90, 124.58, 124.46, 124.26, 123.10, 43.72, 40.42, 33.57, 33.48, 31.54, 26.79, 25.19, 22.43, 13.87; ESI-MS cacld for 604.2788 [M+H]⁺, found 604.2779 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3305 (-CONH-), 1663 (C=O), 1636 (C=O).

N¹-(2-(N-(naphthalen-2-ylmethyl)heptanamido)ethyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22n)

Chemical Formula: C₃₆H₃₈F₃N₃O₃; white solid; Yield: 79%; mp 163.4-163.9 °C; Purity: 96.48% (HPLC); ¹H NMR (600 MHz, CDCl₃) ) δ 7.85 (d, J = 9.5 Hz, -ArH, 6H), 7.80 (s, -ArH, 1H), 7.60 (s, -ArH, 1H), 7.57 (d, J = 9.0 Hz, -ArH, 2H), 7.55 (d, J = 8.3 Hz, -ArH, 2H), 7.51 – 7.48 (m, -ArH, 3H), 7.45 (d, J = 7.7 Hz, -NH-, 1H), 6.89 (s, -NH-, 1H), 4.74 (s, -CH₂-, 2H), 4.70 (s, -CH₂-, 2H), 3.74 (s, -CH₂-, 2H), 3.59 (s, -CH₂-, 2H), 2.41 (t, J = 7.5 Hz, -CH₂-, 2H), 1.77 (s, -CH₂-, 2H), 1.61 (d, J = 7.4 Hz, -CH₂-, 2H), 1.27 – 1.25 (m, -CH₂-, 2H), 1.17 (t, J = 3.8 Hz, -CH₂-, 2H), 0.81 – 0.74 (m, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 176.56, 166.79, 166.52, 139.26, 136.83, 136.60, 133.45, 133.39, 133.33, 132.93, 131.29, 130.99, 129.30, 129.28, 129.13, 127.79, 127.35, 126.74, 126.32, 124.96, 124.54, 124.48, 124.46, 124.19, 123.09, 45.14, 43.67, 40.57, 33.45, 31.52, 29.69, 29.03, 25.44, 22.43, 13.94; ESI-MS cacld for 618.2944 [M+H]⁺, found 618.2933 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3305 (-CONH-), 1647 (C=O), 1634 (C=O).

N¹-(2-(N-(naphthalen-2-ylmethyl)octanamido)ethyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22o)

Chemical Formula: C₃₇H₄₀F₃N₃O₃; white solid; Yield: 80%; mp 132.4-132.6 °C; Purity: 97.35% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.88 – 7.80 (m, -ArH, 3H), 7.79 (d, J = 7.1 Hz, -ArH, 2H), 7.59 (s, -NH-, 1H), 7.56 (s, -ArH, 1H), 7.55 – 7.48 (m, -ArH, 5H), 7.44 (t, J = 7.6 Hz, -ArH, 2H), 7.17 (d, J = 8.3 Hz, -ArH, 1H), 6.68 (s, -NH-, 1H), 6.32 (s, -ArH, 1H), 4.67 (d, J = 6.6 Hz, -CH₂-, 4H), 3.80 – 3.19 (m, -CH₂-, 4H), 2.16 (d, J = 7.8 Hz, -CH₂-, 2H), 1.61 (t, J = 7.3 Hz, -CH₂-, 2H), 1.35 – 1.15 (m, -CH₂-, 8H), 0.85 (t, J = 6.9 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 173.88, 172.54, 166.54, 139.11, 138.96, 135.38, 133.32, 132.90, 131.24, 131.22, 131.01, 129.29, 129.04, 127.77, 127.43, 127.00, 126.70, 126.35, 125.71, 124.50, 52.87, 44.13, 43.64, 37.95, 36.81, 31.67, 29.29, 28.99, 25.70, 22.59, 14.04; ESI-MS cacld for 632.3101 [M+H]⁺, found 632.3110 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3307 (-CONH-), 1642 (C=O), 1627 (C=O).

N¹-(3-(N-(naphthalen-2-ylmethyl)butyramido)propyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22p)

Chemical Formula: C₃₄H₃₄F₃N₃O₃; white solid; Yield: 76%; mp 145.1-145.7 °C; Purity: 98.19% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 8.08 (t, J = 6.1 Hz, -NH-, 1H), 7.96 (d, J = 8.0 Hz, -ArH, 2H), 7.86 (d, J = 8.1 Hz, -ArH, 4H), 7.80 (d, J = 2.2 Hz, -ArH, 1H), 7.61 (s, -NH-, 1H), 7.59 – 7.53 (m, -ArH, 3H), 7.50 (t, J = 2.8 Hz, -ArH, 2H), 7.46 (t, J = 7.8 Hz, -ArH, 2H), 6.92 (t, J = 5.9 Hz, -ArH, 1H), 4.70 (d, J = 4.8 Hz, -CH₂-, 4H), 3.59 (t, J = 6.0 Hz, -CH₂-, 2H), 3.44 – 3.37 (m, -CH₂-, 2H), 2.42 (t, J = 7.5 Hz, -CH₂-, 2H), 1.77 – 1.68 (m, -CH₂-, 4H), 0.94 (t, J = 7.4 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 175.17, 166.92, 166.16, 139.30, 137.50, 136.50, 133.56, 133.40, 132.90, 131.24, 131.20, 129.27, 129.03, 127.69, 127.47, 127.28, 126.71, 126.27, 124.91, 124.76, 124.48, 124.44, 123.11, 51.03, 43.64, 42.35, 35.93, 35.32, 26.94, 19.04, 13.93; ESI-MS cacld for 590.2631 [M+H]⁺, found 590.2627 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3302 (-CONH-), 1665 (C=O), 1639 (C=O).

N¹-(4-(N-(naphthalen-2-ylmethyl)butyramido)butyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22q)

Chemical Formula: C₃₅H₃₆F₃N₃O₃; white solid; Yield: 78%; mp 116.5-117.2 °C; Purity: 98.87% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.86 (d, J = 8.1 Hz, -ArH, 2H), 7.83 (d, J = 5.1 Hz, -ArH, 2H), 7.80 (d, J = 8.0 Hz, -ArH, 4H), 7.74 (d, J = 2.5 Hz, -NH-, 1H), 7.61 (s, -ArH, 1H), 7.55 (s, -ArH, 2H), 7.53 (s, -ArH, 1H), 7.48 (t, J = 2.3 Hz, -NH-, 1H), 7.45 (d, J = 3.5 Hz, -ArH, 1H), 7.39 (s, -ArH, 1H), 7.11 (s, -ArH, 1H), 4.70 (s, -CH₂-, 2H), 4.67 (s, -CH₂-, 2H), 3.50 – 3.41 (m, -CH₂-, 4H), 2.36 (t, J = 7.5 Hz, -CH₂-, 2H), 1.71 – 1.66 (m, -CH₂-, 2H), 1.66 – 1.63 (m, -CH₂-, 2H), 1.59 – 1.56 (m, -CH₂-, 2H), 0.91 (t, J = 7.4 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 173.96, 173.23, 166.65, 139.34, 139.24, 137.39, 136.54, 135.37, 134.12, 133.37, 132.83, 131.25, 129.25, 128.88, 127.76, 127.48, 127.21, 126.55, 126.17, 124.79, 124.52, 124.49, 124.39, 124.29, 51.52, 45.46, 43.64, 43.61, 39.47, 35.22, 25.85, 18.79, 13.91; ESI-MS cacld for 604.2788 [M+H]⁺, found 604.2781 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3290 (-CONH-), 1652 (C=O), 1632 (C=O).

N¹-(5-(N-(naphthalen-2-ylmethyl)butyramido)pentyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22r)

Chemical Formula: C₃₆H₃₈F₃N₃O₃; white solid; Yield: 79%; mp 107.5-107.9 °C; Purity: 95.25% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.82 (d, J = 6.8 Hz, -ArH, 3H), 7.80 (d, J = 2.1 Hz, -ArH, 3H), 7.75 (d, J = 5.3 Hz, -ArH, 1H), 7.60 (s, -NH-, 1H), 7.55 (s, -ArH, 1H), 7.54 (s, -ArH, 1H), 7.52 (s, -ArH, 1H), 7.49 (t, J = 2.5 Hz, -ArH, 2H), 7.43 (d, J = 3.4 Hz, -ArH, 2H), 7.13 (d, J = 6.1 Hz, -ArH, 1H), 6.88 (d, J = 5.8 Hz, -NH-, 1H), 4.69 (s, -CH₂-, 2H), 4.67 (s, -CH₂-, 2H), 3.44 (t, J = 7.0 Hz, -CH₂-, 2H), 3.40 – 3.37 (m, -CH₂-, 2H), 2.31 (t, J = 7.5 Hz, -CH₂-, 2H), 1.85 (s, -CH₂-, 2H), 1.64 (t, J = 7.9 Hz, -CH₂-, 2H), 1.60 – 1.58 (m, -CH₂-, 2H), 1.33 (t, J = 7.8 Hz, -CH₂-, 2H), 0.84 (t, J = 7.4 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 174.07, 173.22, 166.91, 139.33, 137.53, 136.55, 135.42, 134.21, 133.39, 132.80, 131.25, 129.23, 128.85, 128.43, 127.75, 127.69, 127.42, 127.21, 126.60, 126.18, 126.13, 124.66, 124.54, 124.26, 123.11, 51.11, 45.28, 43.61, 40.11, 35.29, 28.45, 27.15, 23.95, 18.85, 13.88; ESI-MS cacld for 618.2944 [M+H]⁺, found 618.2949 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3304 (-CONH-), 1642 (C=O), 1632 (C=O).

N¹-(6-(N-(naphthalen-2-ylmethyl)butyramido)hexyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22s)

Chemical Formula: C₃₇H₄₀F₃N₃O₃; white solid; Yield: 77%; mp 107.8-108.3 °C; Purity: 98.46% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.83 (d, J = 2.9 Hz, -ArH, 2H), 7.80 (d, J = 2.6 Hz, -ArH, 4H), 7.75 (s, -ArH, 1H), 7.72 (d, J = 7.8 Hz, -ArH, 1H), 7.59 (s, -ArH, 2H), 7.53 (d, J = 5.8 Hz, -ArH, 2H), 7.48 (d, J = 1.9 Hz, -ArH, 1H), 7.43 (d, J = 2.7 Hz, -ArH, 2H), 7.32 (d, J = 1.6 Hz, -NH-, 1H), 7.07 (d, J = 6.7 Hz, -NH-, 1H), 4.72 (s, -CH₂-, 2H), 4.66 (d, J = 4.8 Hz, -CH₂-, 2H), 3.42 (t, J = 7.2 Hz, -CH₂-, 2H), 3.36 (d, J = 6.3 Hz, -CH₂-, 2H), 2.33 (t, J = 7.5 Hz, -CH₂-, 2H), 1.64 (q, J = 7.4 Hz, -CH₂-, 2H), 1.56 (d, J = 7.2 Hz, -CH₂-, 2H), 1.54 (d, J = 7.3 Hz, -CH₂-, 2H), 1.37 (t, J = 7.5 Hz, -CH₂-, 2H), 1.27 – 1.24 (m, -CH₂-, 2H), 0.88 (t, J = 7.4 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 173.87, 173.30, 166.82, 139.38, 136.54, 135.43, 134.28, 133.39, 132.80, 131.24, 129.22, 129.20, 128.84, 128.42, 127.75, 127.70, 127.67, 127.38, 127.28, 126.59, 126.11, 124.67, 124.50, 124.29, 123.13, 51.08, 48.39, 45.39, 43.60, 40.04, 39.51, 35.27, 29.09, 27.22, 18.82, 13.90; ESI-MS cacld for 632.3101 [M+H]⁺, found 632.3107 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3302 (-CONH-), 1648 (C=O), 1620 (C=O).

N¹-(2-(N-((1H-indol-4-yl)methyl)butyramido)ethyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22t)

Chemical Formula: C₃₁H₃₁F₃N₄O₃; white solid; Yield: 79%; mp 132.9-133.4 °C; Purity: 98.90% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 8.54 (s, -NH-, 1H), 7.89 (s, -ArH, 1H), 7.83 (s, -ArH, 3H), 7.76 (s, -ArH, 1H), 7.60 (s, -ArH, 1H), 7.56 (t, J = 7.4 Hz, -ArH, 3H), 7.48 (d, J = 7.8 Hz, -NH-, 1H), 7.37 (d, J = 8.2 Hz, -ArH, 1H), 7.18 (t, J = 7.7 Hz, -ArH, 1H), 6.86 (d, J = 7.2 Hz, -ArH, 1H), 6.79 (d, J = 6.3 Hz, -ArH, 1H), 6.50 (t, J = 2.5 Hz, -NH-, 1H), 4.87 (s, -CH₂-, 2H), 4.70 (d, J = 5.8 Hz, -CH₂-, 2H), 3.74 (d, J = 5.0 Hz, -CH₂-, 2H), 3.58 – 3.51 (m, -CH₂-, 2H), 2.42 (t, J = 7.4 Hz, -CH₂-, 2H), 1.68 (s, -CH₂-, 2H), 0.89 (t, J = 7.3 Hz, -CH₂-, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 176.57, 166.90, 166.48, 139.22, 136.88, 136.49, 135.93, 131.26, 129.29, 127.46, 127.36, 127.26, 125.70, 124.83, 124.46, 122.22, 116.57, 110.97, 99.73, 50.14, 45.26, 43.64, 40.82, 35.20, 18.83, 13.87; ESI-MS cacld for 565.2427 [M+H]⁺, found 565.2420 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3287 (-CONH-), 1655 (C=O), 1624 (C=O).

N¹-(2-(N-(anthracen-9-ylmethyl)butyramido)ethyl)-N⁴-(3-(trifluoromethyl)-benzyl)terephthalamide(22u)

Chemical Formula: C₃₇H₃₄F₃N₃O₃; white solid; Yield: 84%; mp 118.6-119.3 °C; Purity: 99.63% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 8.41 (s, -NH-, 1H), 8.39 – 8.30 (m, -ArH, 1H), 8.03 (s, -ArH, 2H), 7.96 (s, -ArH, 1H), 7.85 (s, -ArH, 2H), 7.78 (s, -ArH, 1H), 7.60 (s, -ArH, 1H), 7.59 (s, -NH-, 1H), 7.57 (s, -ArH, 1H), 7.53 (d, J = 7.8 Hz, -ArH, 2H), 7.51 – 7.47 (m, -ArH, 1H), 7.47 – 7.41 (m, -ArH, 3H), 7.38 (s, -ArH, 1H), 6.98 (s, -ArH, 1H), 5.91 (s, -CH-, 1H), 5.50 (s, -CH-, 1H), 4.84 – 4.62 (m, -CH₂-, 2H), 3.35 – 2.77 (m, -CH₂-, 4H), 2.24 – 2.00 (m, -CH₂-, 2H), 1.61 (s, -CH₂-, 2H), 0.87 (t, J = 6.9 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, DMSO-d₆) δ 172.28, 170.10, 166.56, 141.02, 131.40, 130.99, 130.71, 129.36, 129.30, 129.28, 129.09, 128.88, 127.47, 126.72, 126.56, 125.26, 125.12, 123.75, 123.54, 123.32, 68.15, 54.85, 45.57, 42.29, 36.41, 18.40, 13.62; ESI-MS cacld for 626.2631 [M+H]⁺, found 626.2628 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3294 (-CONH-),1636 (C=O), 1614 (C=O).

N¹-(2-(N-([1,1'-biphenyl]-4-ylmethyl)butyramido)ethyl)-N⁴-(3-(trifluorometh-yl)benzyl)terephthalamide(22v)

Chemical Formula: C₃₅H₃₄F₃N₃O₃; white solid; Yield: 82%; mp 123.9-124.3 °C; Purity: 95.05% (HPLC); ¹H NMR (600 MHz, CDCl₃) δ 7.86 (s, -ArH, 2H), 7.85 (s, -ArH, 3H), 7.60 (d, J = 1.8 Hz, -ArH, 2H), 7.59 (s, -NH-, 1H), 7.57 (d, J = 1.1 Hz, -ArH, 4H), 7.55 – 7.54 (m, -ArH, 1H), 7.47 (s, -ArH, 1H), 7.44 (d, J = 6.1 Hz, -ArH, 2H), 7.36 (d, J = 2.1 Hz, -ArH, 1H), 7.23 (s, -ArH, 1H), 6.79 (t, J = 6.0 Hz, -NH-, 1H), 4.70 (d, J = 5.9 Hz, -CH₂-, 2H), 4.63 (s, -CH₂-, 2H), 3.76 – 3.66 (m, -CH₂-, 2H), 3.64 – 3.56 (m, -CH₂-, 2H), 2.38 (t, J = 7.5 Hz, -CH₂-, 2H), 1.67 (q, J = 7.4 Hz, -CH₂-, 2H), 0.91 (t, J = 7.4 Hz, -CH₃, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 176.29, 166.80, 166.49, 141.01, 140.31, 139.20, 136.85, 136.58, 134.94, 131.29, 131.00, 129.31, 129.29, 128.88, 128.83, 127.82, 127.72, 127.58, 127.49, 127.41, 127.30, 127.05, 127.03, 126.74, 124.57, 124.55, 124.51, 77.23, 77.02, 76.81, 51.40, 43.67, 40.52, 35.24, 18.94, 18.82, 13.88; ESI-MS cacld for 602.2631 [M+H]⁺, found 602.2634 [M+H]⁺; IR (KBr), υ (cm⁻¹): 3302 (-CONH-),1662 (C=O), 1637 (C=O).

AChE and BuChE Inhibition Assay

The assay was performed according to our previous reports [28] based on the Ellman’s method.

Kinetic analysis of 22j

To determine the inhibition type of these compounds against electroporation BuChE, a kinetic study was carried out with inhibitor 22j as the representative BuChEI. In the process, the used concentrations of inhibitor 0.5 × IC₅₀, IC₅₀, and 2 × IC₅₀ were 0.23, 0.46, and 0.92μM, respectively. The type of inhibition was established from the analysis of Lineweaver-Burk reciprocal plots.

Molecular docking study

Molecular docking studies were performed using AutoDock 4.2. X-ray structures of AChE (PDB ID: 4EY7) and BuChE (PDB ID: 5K5E) were downloaded from the Protein Data Bank (https://www.rcsb.org/). The downloaded proteins were then prepared by adding hydrogen atoms, removing water, and assigning Kollman atomic charges, and the prepared proteins were converted to pdbqt files using AutoDock, while the ligands were prepared in the pdbqt files. The processed proteins and ligands were then docked using AutoDock. Finally, the docking results are prepared by PyMOL.

Acknowledgements

This work was supported by Hebei Natural Science Foundation (S&T Program of Hebei, NO. B2020201056, H2023201020).

Compliance with ethical standards

Conflict of interest The authors declare no competing interests.

Khan BA, Hamdani SS, Alsfouk BA, Ejaz SA, Aziz M, Channar PA et al (2023) Synthesis, biological evaluation and computational investigations of S-benzyl dithiocarbamates as the cholinesterase and monoamine oxidase inhibitors. J Mol Struct 1271:134–138
Li X, Li T, Zhang P, Li X, Lu L, Sun Y et al (2022) Discovery of novel hybrids containing clioquinol – 1-benzyl-1,2,3,6-tetrahydropyridine as multi-target-directed ligands (MTDLs) against Alzheimer’s disease. Eur J Med Chem 244:114841
Han C, Wei B-B, Shang P-P, Guo X-Y, Bai L-G, Ma Z-Y (2022) Design, synthesis and evaluation of 2-(2-oxoethyl)pyrimidine-5-carboxamide derivatives as acetylcholinesterase inhibitors. Bioorg Med Chem Lett 72:128873
Zhou Y, Fu Y, Yin W, Li J, Wang W, Bai F et al (2021) Kinetics-Driven drug design strategy for Next-Generation acetylcholinesterase inhibitors to clinical candidate. J Med Chem 64(4):1844–1855
Yue N, Fu H, Chen Y, Gao X, Dai J, Cui M (2022) Rational design of molecular rotor-based fluorescent probes with bi-aromatic rings for efficient in vivo detection of amyloid-β plaques in Alzheimer’s disease. Eur J Med Chem 243:114715
Morris M, Knudsen GM, Maeda S, Trinidad JC, Ioanoviciu A, Burlingame AL et al (2015) Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nat Neurosci 18(8):1183–1189
Tang Y, Zhang D, Gong X, Zheng J (2022) A mechanistic survey of Alzheimer’s disease. Biophys Chem 281:106735
Gibson GE, Huang H-M (2005) Oxidative stress in Alzheimer’s disease. Neurobiol Aging 26(5):575–578
Zhou Y, Sun W, Peng J, Yan H, Zhang L, Liu X et al (2019) Design, synthesis and biological evaluation of novel copper-chelating acetylcholinesterase inhibitors with pyridine and N-benzylpiperidine fragments. Bioorg Chem 93:103322
Jiang Y, Gao H (2019) Pharmacophore-based drug design for the identification of novel butyrylcholinesterase inhibitors against Alzheimer’s disease. Phytomedicine 54:278–290
Ahmad S, Iftikhar F, Ullah F, Sadiq A, Rashid U (2016) Rational design and synthesis of dihydropyrimidine based dual binding site acetylcholinesterase inhibitors. Bioorg Chem 69:91–101
Jiang Y, Gao H (2019) Pharmacophore-based drug design for the identification of novel butyrylcholinesterase inhibitors against Alzheimer’s disease. Phytomedicine 54:278–290
Khan S, Iqbal S, Taha M, Rahim F, Shah M, Ullah H et al (2022) Synthesis, in vitro biological evaluation and in silico molecular docking studies of indole based thiadiazole derivatives as dual inhibitor of cetylcholinesterase and butyrylchloinesterase. Molecules 27(21):7368
Suwanhom P, Saetang J, Khongkow P, Nualnoi T, Tipmanee V, Lomlim L (2021) Synthesis, biological evaluation, and in silico studies of new acetylcholinesterase inhibitors based on quinoxaline scaffold. Molecules 26(16):4895
Alım Z, Tunç T, Demirel N, Günel A, Karacan N (2022) Synthesis of benzimidazole derivatives containing amide bond and biological evaluation as acetylcholinesterase, carbonic anhydrase I and II inhibitors. J Mol Struct 1268:133647
Hasan AH, Abdulrahman FA, Obaidullah AJ, Alotaibi HF, Alanazi MM, Noamaan MA et al (2023) Discovery of novel coumarin-schiff base hybrids as potential acetylcholinesterase inhibitors: design, synthesis, enzyme inhibition, and computational studies. Pharmaceuticals 16(7):971
Su J-b, Wu W-l, Dong C-E, Yang S, Feng Y-y, Qin T et al (2023) Synthesis, characterization, crystal structure and biological evaluation of 1,3,5-triazine-quinoline derivatives as butyrylcholinesterase inhibitors. J Mol Struct 1274:1–15
Li Q, Xiong B, Wang Y, Lyu W, Xing S, Chen Y et al (2022) A highly effective and stable butyrylcholinesterase inhibitor with multi-faceted neuroprotection and cognition improvement. Eur J Med Chem 239:114510
Yu D, Yang C, Liu Y, Lu T, Li L, Chen G et al (2023) Synthesis and biological evaluation of substituted acetamide derivatives as potential butyrylcholinestrase inhibitors. Sci Rep 13(1):4877
Košak U, Brus B, Knez D, Žakelj S, Trontelj J, Pišlar A et al (2017) The magic of crystal structure-based inhibitor optimization: development of a butyrylcholinesterase inhibitor with picomolar affinity and in vivo activity. J Med Chem 61(1):119–139
Guo Y, Yang H, Huang Z, Tian S, Li Q, Du C et al (2020) Design, synthesis, and evaluation of acetylcholinesterase and butyrylcholinesterase dual-target inhibitors against Alzheimer’s diseases. Molecules 25(3):489
Sarfraz M, Sultana N, Rashid U, Akram MS, Sadiq A, Tariq MI (2017) Synthesis, biological evaluation and docking studies of 2,3-dihydroquinazolin-4(1H)-one derivatives as inhibitors of cholinesterases. Bioorg Chem 70:237–244
Jaipea S, Saehlim N, Sutcharitruk W, Athipornchai A, Ingkaninan K, Saeeng R (2023) Synthesis of piperine analogues as AChE and BChE inhibitors for the treatment of Alzheimer’s disease. Phytochem Lett 53:216–221
Su J-b, Wu W-l, Dong C-E, Yang S, Feng Y-y, Qin T et al (2023) Synthesis, characterization, crystal structure and biological evaluation of 1,3,5-triazine-quinoline derivatives as butyrylcholinesterase inhibitors. J Mol Struct 1274:134391
Wei B-B, Han C, Shang P-P, Guo X-Y, Bai L-G, Ma Z-Y (2022) Design, synthesis and assay of 2-(4-phenylpiperazin-1-yl)pyrimidine-5- carboxamide derivatives as acetylcholinesterase inhibitors. Med Chem Res 31(11):1901–1915
Lu X, Qin N, Liu Y, Du C, Feng F, Liu W et al (2022) Design, synthesis, and biological evaluation of aromatic tertiary amine derivatives as selective butyrylcholinesterase inhibitors for the treatment of Alzheimer’s disease. Eur J Med Chem 243:114729
Ellman GL, Courtney KD, Andres V, Featherstone RM A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 19617; (2): 88–95
Zhang X-Z, Xu Y, Jian M-M, Yang K, Ma Z-Y, Synthesis (2019) in vitro assays, molecular docking, theoretical ADMET prediction, and evaluation of 4-methoxy-phenylthiazole-2-amine derivatives as acetylcholinesterase inhibitors. Med Chem Res 28(10):1683–1693

Table 1 is available in the Supplementary Files section.

Table 3

ADMET prediction of the compound **22a-22v**.
CODE	MW	LogP	HBA	HBD	TPSA	n_violations	BBB permeant
Rule	< 500	< 5	< 10	< 5	< 130Ǻ²	≤ 1	-
19a	643.62	5.50	9	2	78.51	2	No
19b	575.62	4.79	6	2	78.51	2	No
19c	575.62	4.79	6	2	78.51	2	No
19d	567.67	3.38	5	2	96.97	1	No
19e	537.65	3.72	4	2	87.74	1	No
19f	507.62	4.06	3	2	78.51	1	No
19g	583.72	4.94	3	2	78.51	2	No
19h	509.60	1.70	5	2	104.29	1	No
19i	514.66	2.89	4	2	81.75	1	No
19j	575.62	4.79	6	2	78.51	2	No
19k	589.65	4.97	6	2	78.51	2	No
19l	589.65	4.97	6	2	78.51	2	No
19m	603.67	5.15	6	2	78.51	2	No
19n	617.70	5.32	6	2	78.51	2	No
19o	631.73	5.50	6	2	78.51	2	No
19p	589.65	4.97	6	2	78.51	2	No
19q	603.67	5.15	6	2	78.51	2	No
19r	617.70	5.32	6	2	78.51	2	No
19s	631.73	5.50	6	2	78.51	2	No
19t	564.60	3.53	6	3	94.30	1	No
19u	625.68	5.33	6	2	78.51	2	No
19v	601.66	5.11	6	2	78.51	2	No

MW: 150g/mol < MV < 500g/mol, LIPO(Lipophility): -0.7 < LOGP < + 5.0, HBA(H-bond acceptors): 0 < Num. H-bond acceptors < 10, HBD(H-bond donors): 0 < Num. H-bond donors < 5, POLAR(Polarity): 20 Å² < TPSA < 130 Å², n_violation(number violations from Lipinski’s rule): n_violation ≤ 1, BBB: The ability of crossing BBB blood brain barrier.

Scheme 1 is available in the Supplementary Files section

Table1.docx
Scheme1.png
Scheme 1 The synthetic route of target compounds 22a-22v. Reagents and conditions: (a) MeOH, Boc-NH-(CH₂)_n-NH₂, NaBH₃CN, 3h, rt; (b) CH₂Cl₂, R₂-COCl, K₂CO₃, 5h, rt;(c) MeOH solution of HCl gas, 6h, rt; (d) CH₂Cl₂, 17a, DIEA, TBTU, 4h, rt; (e) ⅰ) CH₂Cl₂, Corresponding amines, DIEA, TBTU, 4h, rt; ⅰi) MeOH/H₂O, NaOH, 5h, rt; (f) MeOH/H₂O, NaOH, 5h, rt; (g) CH₂Cl₂, 17b-17n, DIEA, TBTU, 4h, rt; (h) CH₂Cl₂, Corresponding amines, DIEA, TBTU, 4h, rt.
Supportinginformation.doc

Download PDF

Version 1

posted

You are reading this latest preprint version

Design, synthesis and evaluation of N-arylmethylamide derivatives as cholinesterase inhibitors

Status:

Version 1

Abstract

Figures

Introduction

Results and discussion

Chemical synthesis

AChE and BuChE assay

Kinetic analysis

ADMET prediction

Molecular docking study

Conclusion

Experimental Section

Declarations

References

Tables

Schemes

Supplementary Files

Status:

Version 1