Design, synthesis, and biological evaluation of potent anti-fatigue compounds based on 1-BCP targeting the AMPA receptor

Xin Yang; Yaowen Cui; Zhaolun He; Jing Xu; Tingting Chen; Shuchen Liu; Lin Wang; Shouguo Zhang

doi:10.25259/AJC_89_2025

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Original Article

2025

:18;

892025

doi:

10.25259/AJC_89_2025

Design, synthesis, and biological evaluation of potent anti-fatigue compounds based on 1-BCP targeting the AMPA receptor

Xin Yang^a,b, Yaowen Cui^b, Zhaolun He^b, Jing Xu^b, Tingting Chen^b, Shuchen Liu^b, Lin Wang^a,b,*, Shouguo Zhang^b,*

aSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China

bBeijing Institute of Radiation Medicine, Beijing, 100850, PR China

* Corresponding authors: E-mail addresses: zhangsg@bmi.ac.cn (S. Zhang), wanglin@bmi.ac.cn (L. Wang).

Received: 2025-01-28, Accepted: 2025-03-18, Epub ahead of print: 2025-06-25, Published: 2025-07-03

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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

Fatigue is a prevalent issue in modern fast-paced lifestyles, exerting numerous adverse effects. Piperonylic acid piperidide (1-BCP), an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor modulator, has demonstrated efficacy in alleviating sleep deprivation-induced fatigue. Furthermore, it significantly extends forced swimming time in mice and inhibits fatigue-induced changes in biochemical indices, validating its anti-fatigue effects. In this paper, a series of small-molecule compounds were designed with 1-BCP as the lead, from which the target compounds were screened by molecular dynamics simulation and molecular docking with the AMPA receptor. The target compounds were synthesised and evaluated for their anti-fatigue activity. The results of in vivo experiments showed that some compounds exhibited better anti-fatigue activity, among which compound A1 demonstrated the most significant activity, surpassing that of 1-BCP. The results of molecular dynamics simulation showed that A1 was able to bind to the AMPA receptor, preliminarily verifying the feasibility of the strategy for structural modification of 1-BCP. Taken together, A1 is a promising modulator of AMPA receptors. It is a potential candidate for further research on anti-fatigue drug development.

Keywords

1-BCP

AMPA receptor

Anti-fatigue

Molecular dynamics simulation

Structural modification

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1. Introduction

Fatigue is a state in which the organism is unable to sustain its functions at a specific level or maintain a predetermined work intensity [1], often accompanied by weakness, drowsiness, exhaustion, and lack of energy, which can seriously affect the quality of life, work efficiency, and even endanger human health [2,3]. Fast-paced lifestyles have exacerbated the prevalence of sub-healthy states, with millions of people around the world suffering from fatigue. It is commonly believed that nutritional supplements, including nucleotides [4], vitamins [5], as well as traditional Chinese medicines and natural products, such as ginseng [6], rhodiola rosea [7] and polyphenols [8], can improve athletic performance, have anti-fatigue effects and fewer side effects. However, most of these substances require long-term intake to be effective. Peptides and proteins have been found to be effective antioxidants that can enhance exercise performance and promote fatigue recovery [9,10], but their inherently less Table property limit their widespread use. In addition, caffeine [11], methylphenidate [12], and modafinil [13,14], as special military drugs, can be used to alleviate fatigue and maintain wakefulness, but their central excitability may lead to potential side effects. For people living in a fast-paced society, it is crucial to eliminate fatigue quickly and maintain a high level of energy. Therefore, it has become a priority to find safe, efficient, and stable substances to help the human body eliminate fatigue.

Glutamate is the major excitatory neurotransmitter in the hippocampus of the cerebral cortex and exerts its effects by binding to glutamate receptors. The α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is one subtype of ionic glutamate receptors [15], which play a pivotal role in enhancing excitatory synaptic transmission. They can compensate for synaptic or receptor loss linked to neurological disorders and improve processing efficiency in higher brain regions, thereby enhancing perception, motor, and cognitive performance [16]. Related studies have demonstrated that AMPA receptor modulators enhance neuronal activity in the frontal lobe while suppressing activity in the temporal cortex, thereby promoting cortical arousal and alleviating fatigue induced by sleep deprivation [17]. Research has demonstrated that Piperonylic acid piperidide (1-BCP), an AMPA receptor modulator of benzamide, exhibits no Table anti-fatigue properties by extending exercise duration in mice, mitigating the buildup of fatigue-associated metabolites, facilitating the rapid elimination of oxygen free radicals, and reducing glycogen depletion during exercise, thereby accelerating fatigue recovery [18]. Furthermore positive modulators of AMPA receptors may improve cognitive function and anti-fatigue through multiple mechanisms: counteracting the decline in glutamatergic synapses, enhancing synaptic plasticity, upregulating the synthesis of neurotrophic factors, and promoting cellular recovery within the central nervous system [17].

In vitro experiments demonstrated that 1-BCP could modulate the gating current of AMPA receptor and increase the current intensity into glutamate-gated ion channels [19]. In vivo, 1-BCP rapidly crossed the blood-brain barrier and modulated excitatory postsynaptic potentials [20], leading to long-term potentiation of synaptic activity in the rat hippocampus [21]. Clinical trials have shown that 1-BCP can alleviate fatigue symptoms caused by sleep deprivation without serious adverse effects and exhibits certain anti-fatigue effects [17,22]. However, existing benzamide-based AMPA receptor modulators, such as aniracetam, 1-BCP, and ampalex (CX516), have been reported to exhibit limitations, including low potency and short half-life [23].

Therefore, we designed, modified and synthesized novel molecules using 1-BCP as the lead compound and the AMPA receptor as the target, aiming to identify potent AMPA receptor modulators with significant anti-fatigue properties. The structural design of 1-BCP as an AMPA receptor modulator adheres to a goal-directed strategy, emphasizing the preservation of key pharmacophores and the optimization of structural substitutions to enhance binding affinity for the target receptor. Critical functional groups were retained to ensure efficient interaction with the ligand-binding domain of the AMPA receptor. Furthermore, computer-aided design was integrated with experimental data to design the scaffold and modify its structure, thereby improving bioactivity.

2. Materials and Methods

2.1 Materials and Characterization

Unless otherwise noted, all commercially available compounds were used as provided without further purification. Solvents for chromatography were technical grade. Flash column chromatography was done using silica gel (100–200 mesh). Melting points were determined by an electrothermal melting apparatus and were uncorrected. Chemical yields refer to isolated pure substances.

2.2. General method for synthesis of intermediates m

Under anhydrous and oxygen-free condition, TEA (43.8 mmol) was added to DCM (45.0 mL) containing substituted aniline (14.6 mmol). Then in an ice bath, 2-chloroacetyl chloride (17.5 mmol) was slowly added dropwise to the reaction solution. After the mixture was continuously stirred at room temperature for 3 hrs, residue was concentrated under reduced pressure and purified by rapid column chromatography to afford the intermediate m [30].

2.3. General method for synthesis of intermediates n

To a solution of DIPEA (45.0 mmol) in dichloromethane, 1-Boc-piperazine (30.0 mmol) was added, followed by the slow and dropwise addition of chlorides (30.0 mmol) at low temperature. The reaction mixture was stirred at room temperature for 2 hrs. It was then sequentially washed with 1mol/L hydrochloric acid and water. The organic layer was concentrated under reduced pressure, and the residue was dissolved in a 1:1 (v/v) mixture of DCM and TFA. The solution was stirred at room temperature until the reaction was completed, after which the pH was adjusted to 8-9. The mixture was extracted three times with DCM (30.0 mL each), and the combined organic extracts were concentrated under reduced pressure. The resulting mixture was used directly in subsequent synthetic steps without further purification [31].

2.4. Synthesis of compounds A and B

Intermediate m (5.0 mmol) was added to a solution of DMF (5.0 mL) containing TEA (7.5 mmol) and N-containing heterocyclic ring (6.0 mmol). The mixture was then heated at 100°C for 3 hrs. Upon completion of the reaction, water was added to the mixture. If the precipitate appeared, it was collected by filtration. If no solid formed, the products were separated using flash chromatography to yield compounds A and B.

2.5. Synthesis of compounds C and D

EDCI and HOBt were added to a stirred solution of carboxylic acids (6 mmol) in DMF (5.0 mL). After being stirred at room temperature for 30 mins, intermediate n was added to the mixture. After the reaction was completed at room temperature, the mixture was poured into ethyl acetate (20.0 mL). The organic phase was washed once with 20.0 mL 1 mol/L hydrochloric acid, saturated sodium bicarbonate solution, and water in turn, and then concentrated under reduced pressure. The resulting residue was subjected to rapid column chromatography to afford compound C. A small amount of methanol was added to the concentrated residue, the solid was precipitated after standing and filtered to give compound D.

2.5.1. N-(benzo[d] [1,3] dioxol-5-yl)-2-(piperidin-1-yl) acetamide (A00)

Brown solid; yield 1.10 g (83.6%); m.p. 98.5-99.8°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 9.19 (s, 1H), 7.32 (s, 1H), 6.87 (s, 1H), 6.75 (d, J = 8.3 Hz, 1H), 5.95 (s, 2H), 3.09 (s, 2H), 2.56 (s, 4H), 1.66 (s, 4H), 1.50 (s, 2H) ppm. ¹³C NMR (151 MHz, Chloroform- d₆) δ 168.62, 147.82, 143.99, 132.19, 112.33, 108.04, 102.14, 101.20, 62.67, 54.90, 26.29, 23.63 ppm. HRMS (ESI): exact mass calculated for C₁₄H₁₈N₂O₃ [M + H]⁺, 263.1396. Found: 263.1524.

2.5.2. N-(benzo[d][1,3]dioxol-5-yl)-2-morpholinoacetamide (A01)

Brown solid; yield 1.15 g (87.1%); m.p. 120.5-121.3°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.95 (s, 1H), 7.31 (d, J = 2.1 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 5.95 (s, 2H), 3.78 (t, J = 4.6 Hz, 4H), 3.13 (s, 2H), 2.63 (s, 4H) ppm. ¹³C NMR (151 MHz, Chloroform- d₆) δ 167.62, 147.90, 144.24, 131.85, 112.47, 108.10, 102.26, 101.29, 67.04, 62.37, 53.81 ppm. HRMS (ESI): exact mass calculated for C₁₃H₁₆N₂O₄ [M + H]⁺, 265.1188. Found: 265.1186.

2.5.3. N-(benzo[d][1,3]dioxol-5-yl)-2-(4-phenylpiperazin-1-yl)acetamide (A02)

Brown solid; yield 1.49 g (88.0%); m.p. 140.1-141.3°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 9.02 (s, 1H), 7.33 (s, 1H), 7.29 (dd, J = 8.7, 7.2 Hz, 2H), 6.95 (d, J = 7.6 Hz, 2H), 6.91 (t, J = 7.3 Hz, 1H), 6.86 (s, 1H), 6.76 (d, J = 8.3 Hz, 1H), 5.95 (s, 2H), 3.25 (d, J = 44.3 Hz, 6H), 2.81 (s, 4H) ppm. ¹³C NMR (151 MHz, Chloroform- d₆) δ 167.76, 150.95, 147.89, 144.19, 131.93, 129.20, 120.18, 116.25, 112.41, 108.10, 102.21, 101.26, 61.88, 53.52, 49.46 ppm. HRMS (ESI): exact mass calculated for C₁₉H₂₁N₃O₃ [M + H]⁺, 340.1661. Found: 340.1656.

2.5.4. 2-(piperidin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (B00)

Light brown solid; yield 1.22 g (79.1%); m.p. 54.8-56.9°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 9.18 (s, 1H), 6.88 (s, 2H), 3.87 (s, 6H), 3.82 (s, 3H), 3.07 (s, 2H), 2.55 (t, J = 5.3 Hz, 4H), 1.66 (p, J = 5.6 Hz, 4H), 1.50 (d, J = 12.0 Hz, 2H) ppm. ¹³C NMR (151 MHz, Chloroform- d₆) δ 168.89, 153.37, 133.92, 97.12, 97.10, 62.82, 60.94, 56.16, 54.91, 26.24, 23.59 ppm. HRMS (ESI): exact mass calculated for C₁₆H₂₄N₂O₄ [M + H]⁺, 309.1814. Found: 309.1810.

2.5.5. 2-morpholino-N-(3,4,5-trimethoxyphenyl)acetamide (B01)

Grey green solid; yield 1.27 g (81.6%); m.p. 86.9-88.7°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.94 (s, 1H), 6.87 (s, 2H), 3.87 (s, 6H), 3.82 (s, 3H), 3.81 – 3.78 (m, 4H), 3.14 (s, 2H), 2.64 (t, J = 4.6 Hz, 4H) ppm. ¹³C NMR (151 MHz, Chloroform- d₆) δ 167.83, 153.41, 133.63, 97.21, 66.97, 62.53, 60.95, 56.18, 53.79 ppm. HRMS (ESI): exact mass calculated for C₁₅H₁₂N₂O₅ [M + H]⁺, 311.1607. Found: 311.1603.

2.5.6. 2-(4-phenylpiperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (B02)

White solid; yield 1.64 g (85.3%); m.p. 170.0-171.1°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 9.02 (s, 1H), 7.29 (dd, J = 8.7, 7.3 Hz, 2H), 6.96 (d, J = 7.6 Hz, 2H), 6.90 (t, J = 7.3 Hz, 2H), 6.87 (s, 2H), 3.87 (s, 6H), 3.82 (s, 3H), 3.29 (t, J = 5.0 Hz, 4H), 3.21 (s, 2H), 2.81 (s, 4H) ppm. ¹³C NMR (151 MHz, Chloroform- d₆) δ 168.03, 153.43, 150.93, 134.71, 133.71, 129.21, 120.19, 116.17, 97.06, 62.03, 60.97, 56.18, 53.53, 49.41 ppm. HRMS (ESI): exact mass calculated for C₂₁H₂₈N₃O₄ [M + H]⁺, 386.2080. Found: 386.2072.

2.5.7. N-(benzo[d] [1,3] dioxol-5-yl)-2-(4-(1-phenylvinyl) piperazin-1-yl) acetamide (A1)

Brownish-yellow solid; yield 1.04 g (56.6%); m.p. 142.9-144.0°C. ¹H NMR (400 MHz, Chloroform-d₆) δ 8.85 (s, 1H), 7.42 (s, 5H), 7.29 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.3, 2.1 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 5.95 (s, 2H), 3.68 (d, J = 100.3 Hz, 4H), 3.16 (s, 2H), 2.63 (s, 4H) ppm. ¹³C NMR (101 MHz, Chloroform- d₆) δ 170.47, 167.32, 147.95, 144.36, 135.43, 131.75, 129.96, 128.60, 127.06, 112.58, 108.12, 102.34, 101.32, 61.93, 53.54 ppm. HRMS (ESI): exact mass calculated for C₂₀H₂₁N₃O₄ [M + H]⁺, 368.1610. Found: 368.1605.

2.5.8. N-(benzo[d][1,3]dioxol-5-yl)-2-(4-(4-methylbenzoyl) piperazin-1-yl) acetamide (A2)

Brownish-gray solid; yield 1.09 g (57.2%); m.p. 164.4-165.3°C. ¹H NMR (400 MHz, Chloroform-d₆) δ 8.86 (s, 1H), 7.33 – 7.24 (m, 4H), 7.21 (d, J = 7.8 Hz, 2H), 6.82 (dd, J = 8.3, 2.1 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 5.95 (s, 2H), 3.68 (s, 4H), 3.16 (s, 2H), 2.63 (s, 4H), 2.38 (s, 3H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 165.92, 162.59, 143.17, 135.45, 127.63, 126.97, 124.43, 122.45, 107.82, 103.37, 97.58, 96.57, 57.14,48.69, 16.66 ppm. HRMS (ESI): exact mass calculated for C₂₁H₂₃N₃O₄ [M + H]⁺, 382.1767. Found:382.1762.

2.5.9. N-(benzo[d][1,3]dioxol-5-yl)-2-(4-(4-methoxybenzoyl) piperazin-1-yl) acetamide (A3)

Milky white solid; yield 1.21 g (61.0%); m.p. 135.3-136.6°C. ¹H NMR (500 MHz, Chloroform-d₆) δ 8.92 (s, 1H), 7.31 (d, J = 2.1 Hz, 1H), 6.94 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 5.97 (s, 2H), 3.85 (s, 3H), 3.73 (s, 4H), 3.21 (s, 2H), 2.68 (s, 4H) ppm. ¹³C NMR (126 MHz, Chloroform- d₆) δ 170.46, 161.01, 131.71, 129.21, 127.31, 113.84, 112.58, 108.12, 102.32, 101.32, 61.82, 55.39, 53.46 ppm. HRMS (ESI): exact mass calculated for C₂₁H₂₁N₃O₅ [M + H]⁺, 398.1716. Found:398.1711.

2.5.10. N-(benzo[d][1,3]dioxol-5-yl)-2-(4-(4-(trifluoromethyl) benzoyl) piperazin-1-yl) acet-amide (A4)

Milky white solid; yield 1.16 g (53.5%); m.p. 86.3-88.7°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.81 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.3, 2.1 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 5.96 (s, 2H), 3.88 (s, 2H), 3.49 (s, 2H), 3.19 (s, 2H), 2.66 (d, J = 80.1 Hz, 4H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 168.91, 167.22, 147.91, 144.37, 138.95, 131.65, 127.43, 125.73, 124.56, 122.76, 112.63, 108.09, 102.36, 101.33, 61.82, 53.55, 53.13, 47.61, 42.14 ppm. HRMS (ESI): exact mass calculated for C₂₁H₂₀F₃N₃O₄ [M + H]⁺, 436.1484. Found:436.1478.

2.5.11. 4-(2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoethyl)-N,N-diethylpiperazine-1-carboxa-mide (A5)

Brown solid; yield 0.74 g (40.7%); m.p. 133.7-135.0°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.96 (s, 1H), 7.31 (d, J = 2.1 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 5.95 (s, 2H), 3.30 – 3.15 (m, 8H), 2.73 – 2.52 (m, 4H), 1.13 (t, J = 7.1 Hz, 6H) ppm. ¹³C NMR (101 MHz, Chloroform-d₆) δ 167.68, 164.38, 147.93, 144.25, 131.91, 112.44, 108.12, 102.23, 101.28, 62.01, 53.30, 47.34, 41.79, 13.23 ppm. HRMS (ESI): exact mass calculated for C₁₈H₂₆N₄O₄ [M + H]⁺, 363.2033. Found:363.2031.

2.5.12. 2-(4-benzoylpiperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (B1)

White solid; yield 1.16 g (56.2%); m.p. 158.6-160.0°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.85 (s, 1H), 7.46 – 7.39 (m, 5H), 6.85 (s, 2H), 3.84 (d, J = 31.4 Hz, 13H), 3.20 (s, 2H), 2.67 (s, 4H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 170.54, 153.47, 135.31, 134.92, 133.46, 130.03, 128.63, 127.07, 97.29, 62.05, 61.00, 56.22 ppm. HRMS (ESI): exact mass calculated for C₂₂H₂₇N₃O₅ [M + H]⁺, 414.2029. Found: 414.2023.

2.5.13. 2-(4-(4-methylbenzoyl) piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl) acetamide (B2)

Brown solid; yield 1.38 g (64.4%); m.p. 146.8-148.0°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.79 (s, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 7.8 Hz, 2H), 6.78 (s, 2H), 3.77 (d, J = 30.6 Hz, 13H), 3.12 (s, 2H), 2.58 (s, 4H), 2.31 (s, 3H) ppm. ¹³C NMR (101 MHz, Chloroform-d₆) δ 170.52, 167.51, 153.51, 135.39, 135.02, 133.49, 130.00, 128.62, 127.08, 97.39, 62.11, 60.98, 56.24, 53.52 ppm. HRMS (ESI): exact mass calculated for C₂₃H₂₉N₃O₅ [M + H]⁺, 428.2185. Found: 428.2183.

2.5.14. 2-(4-(4-methoxybenzoyl) piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl) acetamide (B3)

Brown solid; yield 1.19 g (53.6%); m.p. 132.6-133.9°C. ¹H NMR (500 MHz, Chloroform-d₆) δ 8.89 (s, 1H), 7.41 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 6.87 (s, 2H), 3.89 – 3.83 (m, 12H), 3.74 (s, 4H), 3.21 (s, 2H), 2.68 (s, 4H) ppm. ¹³C NMR (126 MHz, Chloroform-d₆) δ 170.51,167.60, 153.45, 133.51, 129.21, 127.27, 113.85, 97.27, 62.02, 60.99, 56.21, 55.40, 53.48 ppm. HRMS (ESI): exact mass calculated for C₂₃H₂₉N₃O₆ [M + H]⁺, 444.2135. Found: 444.2129.

2.5.15. 2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl) acetam-ide (B4)

Brown solid; yield 1.51 g (62.9%); m.p. 135.2-135.8°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.72 (s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 6.78 (s, 2H), 3.80 (s, 6H), 3.75 (s, 3H), 3.53 (d, J = 108.3 Hz, 4H), 3.14 (s, 2H), 2.61 (d, J = 71.3 Hz, 4H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 168.95, 167.42, 153.42, 138.88, 134.86, 133.45, 132.20, 131.98, 131.76, 131.55, 130.24, 127.74, 127.43, 125.75, 125.73, 125.71, 125.68, 124.54, 122.73, 114.20, 97.34, 61.96, 60.94, 53.48, 53.08, 47.54, 42.11 ppm. HRMS (ESI): exact mass calculated for C₁₈H₂₆N₄O₄ [M + H]⁺, 482.1903. Found: 482.1898.

2.5.16. N,N-diethyl-4-(2-oxo-2-((3,4,5-trimethoxyphenyl) amino) ethyl) piperazine-1-carbo-xamide (B5)

Light yellowish-brown solid; yield 0.72 g (35.3%); m.p. 157.8-159.2°C. ¹H NMR (500 MHz, Chloroform-d₆) δ 9.01 (s, 1H), 6.87 (s, 2H), 3.89 (s, 6H), 3.82 (s, 3H), 3.30 (s, 4H), 3.24 (q, J = 7.1 Hz, 4H), 3.18 (s, 2H), 2.67 (s, 4H), 1.14 (t, J = 7.1 Hz, 6H) ppm. ¹³C NMR (126 MHz, Chloroform-d₆) δ 167.82,164.45, 153.43, 134.72, 133.64, 97.05, 62.04, 60.99, 56.18, 53.20, 47.33, 41.74, 13.22 ppm. HRMS (ESI): exact mass calculated for C₂₀H₃₂N₂O₅ [M + H]⁺, 409.2451. Found: 409.2447.

2.5.17. (4-benzoyl piperazin-1-yl) (quinolin-2-yl) methanone (C1)

White solid; yield 1.75 g (84.4%); m.p. 129.3-131.2°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.29 (d, J = 8.5 Hz, 1H), 8.09 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.44 (s, 5H), 4.14 – 3.52 (m, 8H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 170.67, 167.65, 152.96, 146.39, 137.41, 135.30, 130.25, 130.04, 129.68, 128.65, 128.16, 127.86, 127.71, 127.09, 120.97,47.42, 42.61 ppm. HRMS (ESI): exact mass calculated for C₂₁H₁₉N₃O₂ [M + H]⁺, 346.1556. Found: 346.1550.

2.5.18. (4-(4-methylbenzoyl) piperazin-1-yl)(quinolin-2-yl) methanone (C2)

White solid; yield 1.36 g (62.9%); m.p. 181.0-182.6°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.31 (d, J = 8.4 Hz, 1H), 8.11 (s, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 7.6 Hz, 2H), 7.26 (s, 2H), 3.76 (d, J = 91.1 Hz, 8H), 2.40 (s, 3H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 170.85, 167.62, 152.96, 146.36, 140.24, 137.38, 132.29, 130.22, 129.65, 129.20, 128.14, 127.82, 127.68, 127.21, 120.95, 47.37, 42.92, 21.38 ppm. HRMS (ESI): exact mass calculated for C₂₂H₂₁N₃O₂ [M + H]⁺, 360.1712. Found: 360.1707.

2.5.19. (4-(4-methoxybenzoyl) piperazin-1-yl)(quinolin-2-yl) methanone (C3)

White solid; yield 0.85 g (37.7%); m.p. 165.4-166.6°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.30 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 6.8 Hz, 1H), 7.81 – 7.74 (m, 2H), 7.63 (t, J = 6.9 Hz, 1H), 7.42 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 3.82 (m, J = 23.8 Hz, 11H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 170.67, 167.62, 161.03, 152.96, 146.35, 137.43, 130.26, 129.64, 129.23, 128.16, 127.85, 127.69, 127.23, 120.95, 113.86, 55.37, 47.39, 42.67 ppm. HRMS (ESI): exact mass calculated for C₂₂H₂₁N₃O₃ [M + H]⁺, 376.1661. Found: 376.1655.

2.5.20. quinolin-2-yl(4-(4-(trifluoromethyl) benzoyl) piperazin-1-yl) methanone (C4)

Milky white solid; yield 1.00 g (40.4%); m.p. 175.8-177.0°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.31 (s, 1H), 8.10 (d, J = 52.5 Hz, 1H), 7.93 – 7.49 (m, 8H), 4.04 – 3.50 (m, 8H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 169.17, 167.67, 152.79, 146.36, 138.88, 137.43, 132.27, 132.05, 131.83, 131.61, 130.28, 129.68, 128.21, 127.92, 127.72, 127.50, 126.36, 124.55, 122.75, 121.03, 48.01, 47.58, 47.36, 47.06, 42.79, 42.67, 42.35, 42.08 ppm. HRMS (ESI): exact mass calculated for C₂₂H₁₈F₃N₃O₂ [M + H]⁺, 414.1429. Found: 414.1425.

2.5.21. N,N-diethyl-4-(quinoline-2-carbonyl) piperazine-1-carboxamide (C5)

Light yellow solid; yield 0.42 g (20.8%); m.p. 69.0-71.4°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 8.31 (s, 1H), 8.10 (d, J = 52.5 Hz, 1H), 7.93 – 7.49 (m, 8H), 4.04 – 3.50 (m, 8H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 167.70, 164.22, 153.39, 146.51, 137.28, 130.13, 129.71, 128.08, 127.69, 127.67, 120.81, 47.33, 47.15, 47.10, 42.25, 41.80, 13.19 ppm. HRMS (ESI): exact mass calculated for C₁₉H₂₄N₄O₂ [M + H]⁺, 341.1978. Found: 341.1972.

2.5.22. (4-benzoylpiperazin-1-yl)(4-(chloromethyl) phenyl) methanone (D1)

White solid; yield 0.55 g (26.9%); m.p. 138.2-139.5°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 7.43 (d, J = 14.9 Hz, 8H), 4.60 (s, 2H), 3.64 (d, J = 134.9 Hz, 8H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 170.63, 169.71, 143.22, 136.52, 135.05, 134.91, 130.15, 128.67, 127.83, 127.05, 124.50, 120.12, 108.60, 81.67, 50.58, 47.45, 45.46, 42.39 ppm. HRMS (ESI): exact mass calculated for C₁₉H₁₉N₂O₂Cl [M + H]⁺, 343.1213. Found: 343.1208.

2.5.23. (4-(4-(chloromethyl) benzoyl) piperazin-1-yl)(p-tolyl) methanone (D2)

White solid; yield 0.93 g (43.3%); m.p. 138.2-139.5°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 7.50 – 7.38 (m, 4H), 7.31 (d, J = 7.8 Hz, 2H), 7.22 (d, J = 7.8 Hz, 2H), 4.60 (s, 2H), 3.70 (s, 8H), 2.38 (s, 3H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 170.84, 170.03, 140.39, 139.43, 135.20, 132.11, 129.24, 128.83, 127.57, 127.22, 47.62, 45.47, 42.53, 21.41 ppm. HRMS (ESI): exact mass calculated for C₂₀H₂₁N₂O₂Cl [M + H]⁺, 357.1370. Found: 357.1362.

2.5.24. (4-(4-(chloromethyl) benzoyl) piperazin-1-yl)(4-methoxyphenyl) methanone (D3)

Light yellow solid; yield 1.06 g (47.2%); m.p. 199.2-200.8°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 7.27 – 7.19 (m, 6H), 6.73 (d, J = 8.3 Hz, 2H), 4.40 (s, 2H), 3.64 (s, 12H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 170.65, 170.04, 161.11, 139.44, 135.20, 129.24, 128.82, 127.57, 127.04, 113.88, 55.39, 45.45 ppm. HRMS (ESI): exact mass calculated for C₂₀H₂₁N₂O₃Cl [M + H]⁺, 373.1319. Found: 373.1314.

2.5.25. (4-(4-(chloromethyl) benzoyl) piperazin-1-yl)(4-(trifluoromethyl) phenyl) methanone (D4)

Light yellow solid; yield 1.07 g (43.5%); m.p. 177.0-178.2°C. ¹H NMR (600 MHz, Chloroform-d₆) δ 7.71 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 7.8 Hz, 2H), 7.49 – 7.34 (m, 4H), 4.60 (s, 2H), 3.63 (d, J = 168.8 Hz, 8H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 170.09, 169.15, 139.61, 138.65, 134.99, 132.42, 132.21, 131.99, 131.77, 128.87, 127.58, 127.48, 126.31, 125.85, 125.83, 125.80, 125.78, 124.51, 122.70, 120.90, 47.54, 45.42, 42.24 ppm. HRMS (ESI): exact mass calculated for C₂₀H₁₈F₃N₂O₂Cl [M + H]⁺, 411.1088. Found: 411.1088.

2.5.26. 4-(4-(chloromethyl)benzoyl)-N,N-diethylpiperazine-1-carboxamide (D5)

Yellowish oily liquid; yield 0.42 g (20.7%). ¹H NMR (600 MHz, Chloroform-d₆) δ 7.38 (d, J = 8.2 Hz, 2H), 7.34 – 7.32 (m, 2H), 4.53 (s, 2H), 3.71 (s, 2H), 3.38 (s, 2H), 3.16 (q, J = 7.1 Hz, 8H), 1.06 (t, J = 7.1 Hz, 6H) ppm. ¹³C NMR (151 MHz, Chloroform-d₆) δ 169.97, 164.16, 139.20, 135.56, 128.77, 127.54, 47.29, 45.52, 41.78, 13.19 ppm. HRMS (ESI): exact mass calculated for C₁₇H₂₄N₃O₂Cl [M + H]⁺, 338.1636. Found: 338.1631.

2.6. Animal experiments

Healthy Kunming mice, half male and half female, that were supplied by SPF Biotechnology Co. Ltd. (Beijing). Ethical statement: All animal procedures were performed according to a protocol approved by the Committee on the Ethics of Animal Experiments of the Animal Center at the Beijing Institute of Radiation Medicine (IACUC of DWZX 2024-P548).

2.8. In vivo anti-fatigue evaluation

The mice were randomly divided into control, 1-BCP, and compound administration groups (n=10 for each group, half male and half female). The compounds in each group were dissolved in 0.5% sodium carboxymethyl cellulose physiological saline with a concentration of 0.02 mmol/mL. Administration was taken by continuous gavage at a body weight-dependent dose of 0.01 mL/g for 7 days. The mice were acclimatized for 3 days before the formal experiments. The rotational speed of the rotary rod was set to 30, 40, and 50 rpm in succession, with each training session lasting for 5 mins. For swimming training, the mice were required to swim freestyle for 10 mins per day. The experiment was conducted 30 mins after the last administration of the compounds, with the rotating rod speed set to 60 rpm. The time it took for the mice to fall off the rod was recorded [32]. In the swimming experiment, mice were weighed with a load equivalent to 5% of their body weight. Mice were considered fatigued when they sank and failed to resurface within 10 seconds, and swimming time was recorded [33]. The maximum duration for both experiments was set at 30 mins.

2.7. Effect of biochemical indexes related to fatigue

The mice were randomly divided into blank control, model, 1-BCP, and preferred compound groups based on the results of mouse behavioral experiments. Thirty mins after the last administration, the mice were allowed to free-swim for 120 mins, then removed, blown dry, and rested for 30 mins. Blood samples, liver, and gastrocnemius muscle were collected, frozen and stored at -80°C for further analysis [34].

2.9. Central excitability test

Male and female Kunming mice were randomly divided into blank control and A1 groups (n = 10), and the dose and time of administration consistent with behavioral experiments. The central nervous system excitability was evaluated using the open-field test. The test was performed as previously reported with some modifications [35]. The mice were placed in a closed box in a quiet environment, and the total track distance of the mice during 10 mins was automatically measured using the infrared light-track tracking method.

2.10. Safety test

Male and female Kunming mice were randomly divided into 5 groups, with 10 mice in each group. The single gavage doses of 0, 1, 2, 4, and 8 mmol/kg, i.e., 5, 10, 20, and 40 times the normal dose, were administered and animals were observed for 72 hrs.

2.11. Molecular dynamics simulation

Molecular dynamics simulations were performed using GROMACS system. The initial structure was obtained through www.rcsb.org and CHARMM 36 force field was chosen to describe the intermolecular interactions. The system was placed in a dodecahedral box and SPC 216 was used to solvate the system. Appropriate Na⁺/Cl^- ions were added to maintain electrical neutrality. The system was subjected to energy minimization, using the Steepest Descent algorithm to converge the maximum force of the system to within 10 kcal/mol. Then, in canonical ensemble (NVT) equilibrium, the system was heated to 300 K and the pressure was maintained at 1 bar. Subsequently, flat molecular dynamics simulations were carried out for 100 ns, and the resulting parameters were used for subsequent analyses.

2.12. Molecular docking

AutoDock Vina has gained widespread recognition as an open-source molecular docking software, In this study, the three-dimensional structure of the target protein (PDB ID: 3O2A) was retrieved from the RCSB Protein Data Bank and subsequently prepared using the Discovery Studio software. Concurrently, the ligand structure was energy-minimized employing the MM2 force field in Chem 3D to ensure optimal molecular geometry. Both the processed protein and ligand structures underwent hydrogen addition and charge assignment using AutoDock Tools. The docking simulations performed by AutoDock Vina typically yield negative binding energy values, where more negative values indicate stronger molecular interactions and higher binding affinity. The resulting molecular interactions and binding conformations were further analysed and visualized through Discovery Studio and Pymol, providing detailed insights into the intermolecular forces and binding patterns at the atomic level.

2.13. Statistical analysis

The differences among variables were evaluated using a two-tailed Student’s t-test or one-way analysis of variance (ANOVA). Statistical analyses were conducted with SPSS 13.0 and GraphPad Prism 8.0 software. The statistical results were presented as the mean ± standard deviation (mean ± SD). P-value of less than 0.05 was deemed statistically significant in all experiments.

3. Results and Discussion

3.1. Structural design

Molecular dynamics (MD) is a classical simulation and computational method for exploring the interaction patterns between proteins and ligands. The simulation results of the lead compound 1-BCP with the AMPA receptor (PDB ID:3O2A) has been shown in Figure 1. The root mean square deviation (RMSD) of the protein skeleton exhibited a rapid increase between 20 and 40 ns, after which it stabilized. This suggested that the initially relaxed protein structure maintained stability throughout the simulation period. Additionally, no significant deviations in the protein skeleton were observed during the dynamic behavior of the 1-BCP and AMPA receptor complex. The root mean square fluctuation (RMSF) values of the protein bound to 1-BCP exhibited a pattern similar to that of the protein skeleton, indicating that ligand binding did not significantly affect the stability of the receptor protein. The analysis of the radius of gyration (Rog) indicated that the protein exhibited structural stability, maintaining a well-folded conformation throughout the simulation period. As illustrated in Figure 2(a), the AMPA receptor bound to 1-BCP retained its original conformation during the molecular dynamics simulation, with minimal fluctuations or shifts. In summary, 1-BCP demonstrated Table binding to the AMPA receptor without perturbing its natural conformation. Subsequently, an analysis of the key amino acids involved in receptor-ligand interactions (Figure 2b) revealed that ARG149 exhibited the lowest total binding energy, indicating the strongest binding affinity to 1-BCP, followed by ASP216, GLU145, and LYS218. Therefore, these were identified as the key amino acids for binding. Subsequently, the binding mode of 1-BCP to the protein was analysed by molecular docking, and it was found that (Figure 3) 1-BCP had interactions with key amino acids analysed in the molecular dynamics simulation and the key structural parts were revealed, including the carbonyl that bound to the hinge hydrogen bond, groups linked to key amino acids and other substituents that could be directed to the solvent region. Since molecular dynamics was time-consuming and demanding, molecular docking was used to participate in the subsequent structural design.

Synthetic routes for the target compounds. Reagents and conditions: (i) 1-boc-piperazine, DIPEA, DCM, r.t.,2 h; (ii) DCM: TFA=1:1, r.t., 0.5 h; (iii) 2-chloroacetyl chloride, TEA, DCM, r.t., 3 h; (iv) TEA, DMF, 100°C, 3 h; (v) EDCI, HOBt, DMF, r.t.

Molecular dynamics simulation results of AMPA protein and 1-BCP-AMPA protein system (a) RMSD values of backbone atoms for both systems (b) The RMSF values of the side-chain atoms for both systems (c) Radius of Gytration of both system.

(a) Changes in AMPA receptor bound to 1-BCP before and after simulation. (b) Amino acid of 1-BCP binding to the AMPA receptor by molecular dynamics analysis.

Interaction of 1-BCP with the AMPA (PDB ID:3O2A) protein from the docking study.

To enhance binding between the small molecule and protein cavity residues, the amide structure of lead compound 1-BCP was reversed, and a carbon atom was introduced to extend the skeleton, yielding compound A00. Docking analysis of A00 with the AMPA receptor (Figure 4) showed that while the key amino acid interactions were maintained, the van der Waals force between VAL95, SER142, and the ligand of the small molecule compound contributed to enhanced structural stability and a slight reduction in affinity. Animal behavioral experiments (Table 1) confirmed that A00 retained anti-fatigue activity comparable to 1-BCP, establishing its skeleton as a fragment II structure for subsequent partial compounds (Figure 5). Since the amide carbonyl structure could form Table hydrogen bonds with AMPA receptor, the piperidine of A00 was replaced by piperazine and a carbonyl group was introduced to the other nitrogen of piperazine to form a bis-amide structure.

Molecular docking of A00 to the AMPA receptor.

Table 1. Virtually prediction affinity to AMPA receptor and results of anti-fatigue behavioural experiments on A00.

Group

Affinity

(kcal/mol)

Rotation time (min)

Forced swimming time (min)

Control

8.29±1.60

10.94±2.59

1-BCP

-5.7

14.37±4.98^*

18.32±7.19^*

A00

-5.8

18.83±6.66^**

18.75±7.01^*

Note: The values are expressed as mean ± SD (n = 10). ^**P<0.01, ^*P<0.05 compared with the those of control group in which the mice were administrated with physiological saline.

Design strategy of target molecules including three segments of Ⅰ, Ⅱ, and Ⅲ.

The specific structural modifications have been shown in Figure 5. Series A compounds retained fragment Ⅰ of 1-BCP, fragment Ⅱ was consistent with A00, and fragment Ⅲ was the N-containing heterocyclic ring. B series was based on A with Ⅰ modified to a ring-opened structure. The 3,4,5-trimethoxy ring-opened structure of Ⅰ has been reported to exercise longer in weight-loaded swimming test relative to the 3,4-dimethoxy structure [24]. Therefore, it is considered that modification to 3,4,5-trimethoxy is more beneficial for anti-fatigue activity. Both series C and D retained the original benzamide skeleton of 1-BCP while modifying the substituents to increase structural diversity. CX516, a derivative of 1-BCP, was also an AMPA receptor modulator with an anti-fatigue effect [25], so we introduced quinoline in fragment Ⅰ based on the CX516 structure. Chlorine substituents increased the lipophilicity of a molecule, leading to higher partitioning into the lipophilic domain of proteins, and the introduction of chlorine atoms at one or more specific positions in a biologically active molecule has been found to significantly improve intrinsic biological activity [26,27]. Therefore, based on molecular docking, an attempt was made to introduce chloromethyl into fragment Ⅰ to explore this structure’s anti-fatigue activity.

The structures of the above four classes of compounds were designed, synthesized, and evaluated for anti-fatigue activity subsequently.

3.2. Chemistry

The amide derivatives were prepared by acyl halide substitution and acid-amine condensation reactions, and the stepwise synthesis scheme has been described in Scheme 1. Under alkaline conditions provided by triethylamine (TEA), substituted anilines reacted with 2-chloroacetyl chloride in dichloromethane (DCM) to form intermediates m. Substituted chloride reacted with 1-Boc-piperazine in the presence of N,N-diisopropylethylamine (DIPEA), and then Boc was removed in a mixed solvent of trifluoroacetic acid (TFA) and DCM to give intermediate n. m reacted with N-containing heterocyclic ring in N,N-dimethylformamide (DMF) to give compounds A and B. Substituted carboxylic acids were condensed with n in the presence of 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) to give compounds C and D.

2.3. Anti-fatigue activity assay

3.3.1. Animal behavior experiments

The duration of exercise performance is an important indicator for anti-fatigue activity to screen potential agents. The rotation time from the rotarod test was used to measure the ability of the mice to maintain balanced continuous movement on a rotary rod, and the time of forced swimming from the weight-loaded swimming test was used to evaluate the ability of mice to maintain the continuous exercise. The two tests provided reliable quantitative methods for determining the efficacy of candidate compounds in improving physical strength and eliminating fatigue [28].

The results of the rotarod test in mice, as depicted in Figure 6, indicated that compounds A01, A1, A2, B1, B2, B3, B4, C5, D2, D4, and D5 significantly prolonged the rotation time compared to the control group. It was seen they could enhance exercise tolerance and reduce fatigue in mice. Notably, A1 showed the most significant effect, stronger than that of 1-BCP.

Exhaustion time in the rotarod test. The values are expressed as mean ± SD (n=10). **P<0.01, *P<0.05 compared with the control; #P<0.05 compared with 1-BCP.

The results of the weight-loaded swimming test have been shown in Figure 7. They reveal that most compounds exhibited extended swimming time compared to the control group, and compounds A01, A1, A2, B00, B01, B02, B1, B2, B3, B4, B5, C5, D3, D4, and D5 showed the equivalent swimming time to 1-BCP.

Exhaustion time in the weight-loaded swimming test. The values are expressed as mean ± SD (n = 10). **P<0.01, *P<0.05 compared with the control.

The results indicated that the anti-fatigue activity of most compounds was either superior or equivalent to that of 1-BCP, validating the reliability of the design strategy of these compounds. Notably, compound A1 demonstrated a 3.5-fold rotation time and 1.8-fold swimming time compared to the control group, while it also showed a 1.5-fold rotation time and 1.2-fold swimming time compared to 1-BCP, respectively.

In terms of molecular structure, the results of mice behavioral experiments suggested that the benzamide bridge in the lead 1-BCP might not be an essential group for anti-fatigue activity. This bridge could be replaced by a flipped amide bridge while retaining its anti-fatigue efficacy. Specifically, the exercise duration for compounds A1 and A2 in series A, as well as B1 and B3 in series B, was significantly prolonged compared to the control group. The rotation time of A1 was longer than that of 1-BCP, and the rotation and swimming time of the remaining groups were comparable to 1-BCP. These findings indicated that the phenyl (piperazin-1-yl) ketone in fragment III enhances the anti-fatigue activity of A and B compounds, and the 3,4-methylenedioxy group in fragment I also contributes positively to activity. For compounds C and D, the introduction of N,N-diethylpiperazine-1-carboxamide in fragment III augmented biological activity.

3.3.2. Content of index related to fatigue

The results of behavioral experiments in mice showed that compounds A00, A01, A1, B1, B3, C5, D4, and D5 had relatively good anti-fatigue effects. Therefore, the contents of the biochemical index, including liver glycogen (LG), muscle glycogen (MG), blood urea nitrogen (BUN), and blood lactic acid (BLA) in these compound groups were measured [29].

The results of biochemical index contents have been shown in Table 2. In terms of BLA and BUN, all compounds were lower than the model and comparable to the blank control group and 1-BCP. Compounds A00, A01, A1, B1, and D5 reduced the depletion of LG, and its content was equivalent to 1-BCP. All compounds except A01 showed higher MG content than the model, especially A1 and B3, which were higher than 1-BCP.

Table 2. Effect of compounds on the contents of BLA, BUN, LG and MG.

Group	BLA	BUN	LG	MG
Group	(mmol/L)	(mmol/L)	(mg/g)	(mg/g)
Blank control	12.4±1.26	13.49±3	6.21±1.31	1.11±0.1
Model	17±1.81	26.12±4.47	1.21±0.18	0.65±0.04
1-BCP	15.4±1.56^*	16.63±2.44^**	2.09±0.81^*	0.86±0.08^**
A00	15.14±0.8^*	18.53±0.72^**	2.8±0.76^**	0.85±0.04^**
A01	13.88±1.23^**	15.57±1.6^**	2.33±0.34^*	0.73±0.04
A1	14.77±2^**	14.6±1.18^**	2.1±0.69^*	1.00±0.06^**##
B1	14.08±0.81^**	13.69±0.7^**#	2.25±0.72^*	0.82±0.09^**
B3	11.41±0.91^**	14.04±1.13^**	1.95±0.34	0.97±0.11^**#
C5	12.38±1.19^**##	19.81±2.55^**	2.05±0.17	0.76±0.07^*
D4	12.42±1.28^**##	23.37±1.56^*	1.77±0.51	0.87±0.11^**
D5	12.20±1.00^**##	20.62±2.46^**	2.48±0.27^**	0.83±0.13^**

Note: The values are expressed as mean ± SD (n = 10). ^**P<0.01, ^*P<0.05 compared with the model; ^##P<0.01, ^#P<0.05 compared with the 1-BCP.

The content results of these biochemical indexes verified the anti-fatigue activity of the compounds from another angle and verified the results of the behavioral experiments.

Based on the findings of the aforementioned experiments, compound A1 exhibits remarkable anti-fatigue activity, effectively overcoming the limitations observed in the lead compound 1-BCP. In this study, we focused on improving its biological activity mainly based on the better binding affinity data from molecular docking simulation. The result for A1 showing significant anti-fatigue activity was consistent with its high affinity to the AMPA receptor simulated by molecular docking. It is worth noting that the half-life of A1 (1.454 hrs) predicted by the website (https://admet.scbdd.com) was slightly better than that of 1-BCP (1.197 hrs). These results indicate that the structural modifications implemented in A1 have successfully extended its half-life, suggesting the anti-fatigue effect of A1 was better than 1-BCP from another perspective.

3.4. Central excitability of A1

The anti-fatigue effect can be achieved by excitation of the central nervous system. To assess whether A1 exerted its effect through this mechanism, the open field test was conducted at 0.5 hrs after A1 being administered to mice by gavage for 7 days. The total distance travelled by mice in the A1 group was not different from that of the blank control group (Figure 8), suggesting that A1 did not enhance locomotor activity of the mice. Therefore, the anti-fatigue mechanism of A1 did not involve the excitation of the central nervous system.

Trajectory distances of mice in the open-field test.

3.5. Safety of A1

A1 did not exhibit acute toxicity to mice at doses of 1 mmol/kg (5 times dose of functional testing), 2 mmol/kg (10 times), 4 mmol/kg (20 times), and 8 mmol/kg (40 times). The high-dose groups (8 and 4 mmol/kg) began to get drowsy without eating or drinking 10 to 20 mins after administration to mice, whereas the other groups did not differ from the control group. Locomotor activity of the mice was normal in all groups 24 hrs after administration and all groups had finished their rations. No signs of death or poisoning were observed during the 72-hour observation period.

In summary, compound A1 demonstrates significant anti-fatigue effects at a dose of 0.2 mmol/kg, with no signs of toxicity observed following either single administration or continuous administration for 7 days, confirming the safety of this dose. Even at 5-fold and 10-fold concentrations of the single administration dose, A1 did not exhibit any toxicity, indicating its favorable safety profile. However, the high-dose A1 group exhibited drowsiness at doses 20 and 40 times the effective dose. It was only at these elevated concentrations that A1 induced drowsiness, suggesting potential risks associated with the long-term use of A1 at such high doses. This observation further confirms the safety of the effective dose of A1 (0.2 mmol/kg). The results suggest that precise effective dose range of A1 will be key focuses of our future research.

3.6. Molecular dynamics simulation of A1

A1 was then analysed by molecular dynamics. The result (Figure 9) showed that A1 bound tightly to three key amino acids (ARG149, GLU145, and LYS218) of the protein receptor, overlapping with the binding of 1-BCP. This initially established that the modification of the structure did not induce a change in the binding mode of the ligand to the protein, and A1 might have the same biological effect as 1-BCP.

The results of RMSD have been shown in Figure 10(a). It was observed that all three reached a steady state after 40 ns, and the average protein skeleton bound to A1 and 1-BCP at RMSD of 0.16 nm and 0.15 nm, respectively, compared with 0.21 nm for the protein skeleton. The results showed that the A1-AMPA complex had good stability in the dynamic state.

Figure 9.

Amino acid profile of A1 binding to the AMPA receptor by molecular dynamics analysis.

Molecular dynamics simulation results of the systems of AMPA, 1-BCP-AMPA complex and A1-AMPA complex (a) RMSD values of all backbone atoms for these three systems (b) The RMSF values of the side-chain atoms for these three systems (c) Radius of Gyration of the 1-BCP-AMPA system and A1-AMPA system (d) Number of hydrogen bonds in the 1-BCP-AMPA system and A1-AMPA system.

The RMSF simulation results (Figure 10b) indicated the amino acid fluctuations of the protein skeleton fluctuated in roughly the same way as those in the complex, with most deviations not exceeding 0.300 nm, indicating that the protein was able to successfully maintain its natural conformational state during the simulation.

The RoG calculation revealed the compactness of the overall density of the complex over the time of the simulation. As shown in Figure 10(c), there was no significant deviation from steady state to the end of the simulation, with maximum values of 2.01 nm, 2.02 nm, and 1.96 nm for the skeleton protein, A1-AMPA complex, and 1-BCP-AMPA complex, respectively, indicating that binding of the ligand did not change the compactness of the protein.

Simulated interaction plot Figure 10(d) examined the number of hydrogen bonds formed by the protein-ligand complex during simulation time, and the hydrogen bond interactions were similar for both complexes, indicating that the force of binding to the protein through hydrogen bonds was also approximately the same.

The Molecular Mechanics Generalized Born Surface Area (MMGBSA) method was used to calculate the affinity of ligands to the receptor, and an increase in negative affinity enhanced the strength of binding. The results of the study have been presented in Table 3. They indicate that A1 binds with AMPA protein slightly more strongly than 1-BCP.

Table 3. The binding free energy of AMPA with ligands calculated by the MM-GBSA approach.

Molecule	Total ΔG_bind (kcal/mol)	Standard Deviation
A1	-19.37	0.87
1-BCP	-17.89	0.52

The results of the molecular dynamics parameters theoretically validated our design idea that A1 acted on the AMPA receptor, and the binding mechanism needs to be further explored.

4. Conclusions

Based on 1-BCP as the lead compound, 26 derivatives were designed and synthesized. Preliminary evaluation of their anti-fatigue activity revealed that compound A1 exhibited superior efficacy compared to 1-BCP. Molecular dynamics simulations further confirmed that A1 exhibits optimal binding affinity for the AMPA receptor, and the key amino acid binding of A1 to AMPA receptors is consistent with that of 1-BCP, theoretically proving that A1 and 1-BCP act in the same mechanism. In conclusion, A1 represents a promising novel AMPA receptor modulator and is worth further investigation as a potential anti-fatigue drug. Future studies will focus on elucidating the specific mechanism of its anti-fatigue effects mediated through the AMPA receptor.

Acknowledgment

This study was supported by the Beijing Natural Science Foundation of China (Project No.7242207).

CRediT authorship contribution statement

Xin Yang: Design, Literature search, Experimental studies, Data analysis, Manuscript editing. Yaowen Cui: Design, Statistical analysis. Zhaolun He: Data acquisition. Jing Xu: Manuscript preparation. Tingting Chen: Experimental studies, Data acquisition. Shuchen Liu: The definition of intellectual content. Lin Wang: Manuscript editing, Manuscript review, Funding acquisition. Shouguo Zhang: Design, Manuscript review, Funding acquisition. All the authors have read the article and agree to its contents.

Declaration of competing interest

The authors declare no competing interest.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Supplementary data

Supplementary material to this article can be found online at https://dx.doi.org/10.25259/AJC_89_2025.

Supplementary Material

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References

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Show Sections

[1] Matura L.A., Malone S., Jaime-Lara R., Riegel B.. A systematic review of biological mechanisms of fatigue in chronic illness. Biological Research for Nursing. 2018;20:410-421. https://doi.org/10.1177/1099800418764326
[Google Scholar]

[2] Wan J.J., Qin Z., Wang P.Y., Sun Y., Liu X.. Muscle fatigue: General understanding and treatment. Experimental & Molecular Medicine. 2017;49:e384. https://doi.org/10.1038/emm.2017.194
[Google Scholar]

[3] Zhu J., Yi J., Kang Q., Huang J., Cui Y., Zhang G., Wang Z., Zhang L., Zheng Z., Lu J., Hao L.. Anti-fatigue activity of hemp leaves water extract and the related biochemical changes in mice. Food and Chemical Toxicology : An International Journal published for the British Industrial Biological Research Association. 2021;150:112054. https://doi.org/10.1016/j.fct.2021.112054
[Google Scholar]

[4] Xu M., Liang R., Li Y., Wang J.. Anti-fatigue effects of dietary nucleotides in mice. Food & Nutrition Research. 2017;61:1334485. https://doi.org/10.1080/16546628.2017.1334485
[Google Scholar]

[5] Shimizu T., Hoshino H., Nishi S., Nozaki S., Watanabe Y.. Anti-fatigue effect of dicethiamine hydrochloride is likely associated with excellent absorbability and high transformability in tissues as a vitamin b(1) European Journal of Pharmacology. 2010;635:117-123. https://doi.org/10.1016/j.ejphar.2010.02.053
[Google Scholar]

[6] Yang Q.Y., Lai X.D., Ouyang J., Yang J.D.. Effects of ginsenoside Rg3 on fatigue resistance and SIRT1 in aged rats. Toxicology. 2018;409:144-151. https://doi.org/10.1016/j.tox.2018.08.010
[Google Scholar]

[7] Lu Y., Deng B., Xu L., Liu H., Song Y., Lin F.. Effects of rhodiola rosea supplementation on exercise and sport: A systematic review. Frontiers in Nutrition. 2022;9:856287. https://doi.org/10.3389/fnut.2022.856287
[Google Scholar]

[8] Luan G., Wang Y., Wang Z., Zhou W., Hu N., Li G., Wang H.. Flavonoid glycosides from fenugreek seeds regulate glycolipid metabolism by improving mitochondrial function in 3T3-L1 adipocytes in vitro. Journal of Agricultural and Food Chemistry. 2018;66:3169-3178. https://doi.org/10.1021/acs.jafc.8b00179
[Google Scholar]

[9] Liu J., Wang X., Zhao Z.. Effect of whey protein hydrolysates with different molecular weight on fatigue induced by swimming exercise in mice. Journal of the Science of Food and Agriculture. 2014;94:126-130. https://doi.org/10.1002/jsfa.6220
[Google Scholar]

[10] Bao L., Cai X., Wang J., Zhang Y., Sun B., Li Y.. Anti-fatigue effects of small molecule oligopeptides isolated from panax ginseng C A Meyer in mice. Nutrients. 2016;8:807. https://doi.org/10.3390/nu8120807
[Google Scholar]

[11] van Dam R.M., Hu F.B., Willett W.C.. Coffee, caffeine, and health. The New England Journal of Medicine. 2020;383:369-378. https://doi.org/10.1056/NEJMra1816604
[Google Scholar]

[12] Gong S., Sheng P., Jin H., He H., Qi E., Chen W., Dong Y., Hou L.. Effect of methylphenidate in patients with cancer-related fatigue: A systematic review and meta-analysis. PLoS One. 2014;9:e84391. https://doi.org/10.1371/journal.pone.0084391
[Google Scholar]

[13] Mignot E., Nishino S.. Emerging therapies in narcolepsy-cataplexy. Sleep. 2005;28:754-763. https://doi.org/10.1093/sleep/28.6.754
[Google Scholar]

[14] Greenblatt K., Adams N.. Structure-based discovery and development of novel O-GlcNAcase inhibitors for the treatment of Alzheimer’s disease. European Journal of Medicinal Chemistry. ;238:114444. https://doi.org/10.1016/j.ejmech.2022.114444
[Google Scholar]

[15] Goff D.C., Coyle J.T.. The emerging role of glutamate in the pathophysiology and treatment of schizophrenia. The American Journal of Psychiatry. 2001;158:1367-1377. https://doi.org/10.1176/appi.ajp.158.9.1367
[Google Scholar]

[16] Bigge C.F., Nikam S.S.. AMPA receptor agonists, antagonists and modulators: Their potential for clinical utility. Expert Opinion on Therapeutic Patents. 1997;7:1099-1114. https://doi.org/10.1517/13543776.7.10.1099
[Google Scholar]

[17] Lynch G.. Glutamate-based therapeutic approaches: Ampakines. Current Opinion in Pharmacology. 2006;6:82-8. https://doi.org/10.1016/j.coph.2005.09.005
[Google Scholar]

[18] Fan W., Wu X., Pan Y., Li C., Niu Y., Zhai Y., Mei Q.. 1-(1,3-benzodioxol-5-yl-carbo-nyl) piperidine, a modulator of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, ameliorates exercise-induced fatigue in mice. Biological & Pharmaceutical Bulletin. 2014;37:13-17. https://doi.org/10.1248/bpb.b13-00399
[Google Scholar]

[19] Arai A., Kessler M., Xiao P., Ambros-Ingerson J., Rogers G., Lynch G.. A centrally active drug that modulates AMPA receptor gated currents. Brain Research. 1994;638:343-346. https://doi.org/10.1016/0006-8993(94)90669-6
[Google Scholar]

[20] Black M.D., Wotanis J., Schilp D.E., Hanak S.E., Sorensen S.M., Wettstein J.G.. Effect of AMPA receptor modulators on hippocampal and cortical function. European Journal of Pharmacology. 2000;394:85-90. https://doi.org/10.1016/s0014-2999(00)00120-5
[Google Scholar]

[21] Stäubli U., Perez Y., Xu F.B., Rogers G., Ingvar M., Stone-Elander S., Lynch G.. Centrally active modulators of glutamate receptors facilitate the induction of long-term potentiation in vivo. Proceedings of the National Academy of Sciences of the United States of America. 1994;91:11158-11162. https://doi.org/10.1073/pnas.91.23.11158
[Google Scholar]

[22] Sanacora G., Zarate C.A., Krystal J.H., Manji H.K.. Targeting the glutamatergic system to develop novel, improved therapeutics for mood disorders. Nature Reviews. Drug Discovery. 2008;7:426-437. https://doi.org/10.1038/nrd2462
[Google Scholar]

[23] Mueller R., Li Y.X., Hampson A., Zhong S., Harris C., Marrs C., Rachwal S., Ulas J., Nielsson L., Rogers G.. Benzoxazinones as potent positive allosteric AMPA receptor modulators: Part I. Bioorganic & Medicinal Chemistry Letters. 2011;21:3923-6. https://doi.org/10.1016/j.bmcl.2011.05.026
[Google Scholar]

[24] Wu X., Fan W., Pan Y., Zhai Y., Niu Y., Li C., Mei Q.. Synthesis, crystal structure and anti-fatigue effects of some benzamide derivatives. Molecules (Basel, Switzerland). 2014;19:1034-1046. https://doi.org/10.3390/molecules(1901)1034
[Google Scholar]

[25] Yao H., Zhang D., Yu H., Shen H., Lan X., Liu H., Chen X., Wu X., Zhang G., Wang X.. AMPAkine CX516 alleviated chronic ethanol exposure-induced neurodegeneration and depressive-like behavior in mice. Toxicology and Applied Pharmacology. 2022;439:115924. https://doi.org/10.1016/j.taap.2022.115924
[Google Scholar]

[26] Müller G., Nkusi, Gérard, Schöler H.F.. Natural organohalogens in sediments. Journal fur Praktische Chemie/Chemiker-Zeitung. 1996;338:23-9. https://doi.org/10.1002/prac.19963380104
[Google Scholar]

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[Google Scholar]

[28] Armario A.. The forced swim test: Historical, conceptual and methodological considerations and its relationship with individual behavioral traits. Neuroscience and Biobehavioral Reviews. 2021;128:74-86. https://doi.org/10.1016/j.neubiorev.2021.06.014
[Google Scholar]

[29] Li Z., Zhu H., Hua H., Liu C., Cheng Y., Guo Y., Du P., Qian H.. Anti-fatigue activity of brassica rapa l. extract and correlation among biochemical changes in forced swimming mice. Food Bioscience. 2022;47:101633. https://doi.org/10.1016/j.fbio.2022.101633
[Google Scholar]

[30] Li X., Han J., Bujaranipalli S., He J., Kim E.Y., Kim H., Im J.H., Cho W.-J.. Structure-based discovery and development of novel o-glcNAcase inhibitors for the treatment of alzheimer’s disease. European Journal of Medicinal Chemistry. 2022;238:114444. https://doi.org/10.1016/j.ejmech.2022.114444
[Google Scholar]

[31] Mancuso F., Di Fiore A., De Luca L., Angeli A., Monti S.M., De Simone G., Supuran C.T., Gitto R.. Looking toward the rim of the active site cavity of druggable human carbonic anhydrase isoforms. ACS Medicinal Chemistry letters. 2020;11:1000-5. https://doi.org/10.1021/acsmedchemlett.0c00062
[Google Scholar]

[32] Wang J., Li C., Hu W., Li L., Cai G., Liu Y., Wang D.. Studies on the anti-fatigue activities of irpex lacteus polysaccharide-enriched extract in mouse model. Pakistan Journal of Pharmaceutical Sciences. 2019;32:1011-8.
[Google Scholar]

[33] Morihara N., Ushijima M., Kashimoto N., Sumioka I., Nishihama T., Hayama M., Takeda H.. Aged garlic extract ameliorates physical fatigue. Biological & Pharmaceutical Bulletin. 2006;29:962-6. https://doi.org/10.1248/bpb.29.962
[Google Scholar]

[34] Wu C., Chen R., Wang X.S., Shen B., Yue W., Wu Q.. Antioxidant and anti-fatigue activities of phenolic extract from the seed coat of euryale ferox salisb. and identification of three phenolic compounds by LC-ESI-MS/MS. Molecules (Basel, Switzerland). 2013;18:11003-11021. https://doi.org/10.3390/molecules180911003
[Google Scholar]

[35] Zhu H., Zeng W., Zhao T., Shi W., Dong X., Zhang A., Li X., Xu L.. Synthesis and evaluation of 5-aminimidazole-4-carboxamide riboside derivatives as anti-fatigue agents. Arabian Journal of Chemistry. 2022;15:104313. https://doi.org/10.1016/j.arabjc.2022.104313
[Google Scholar]

Design, synthesis, and biological evaluation of potent anti-fatigue compounds based on 1-BCP targeting the AMPA receptor

Abstract

Keywords

1-BCP

AMPA receptor

Anti-fatigue

Molecular dynamics simulation

Structural modification

1. Introduction

2. Materials and Methods

2.1 Materials and Characterization

2.2. General method for synthesis of intermediates m

2.3. General method for synthesis of intermediates n

2.4. Synthesis of compounds A and B

2.5. Synthesis of compounds C and D

2.5.1. N-(benzo[d] [1,3] dioxol-5-yl)-2-(piperidin-1-yl) acetamide (A00)

2.5.2. N-(benzo[d][1,3]dioxol-5-yl)-2-morpholinoacetamide (A01)

2.5.3. N-(benzo[d][1,3]dioxol-5-yl)-2-(4-phenylpiperazin-1-yl)acetamide (A02)

2.5.4. 2-(piperidin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (B00)

2.5.5. 2-morpholino-N-(3,4,5-trimethoxyphenyl)acetamide (B01)

2.5.6. 2-(4-phenylpiperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (B02)

2.5.7. N-(benzo[d] [1,3] dioxol-5-yl)-2-(4-(1-phenylvinyl) piperazin-1-yl) acetamide (A1)

2.5.8. N-(benzo[d][1,3]dioxol-5-yl)-2-(4-(4-methylbenzoyl) piperazin-1-yl) acetamide (A2)

2.5.9. N-(benzo[d][1,3]dioxol-5-yl)-2-(4-(4-methoxybenzoyl) piperazin-1-yl) acetamide (A3)

2.5.10. N-(benzo[d][1,3]dioxol-5-yl)-2-(4-(4-(trifluoromethyl) benzoyl) piperazin-1-yl) acet-amide (A4)

2.5.11. 4-(2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoethyl)-N,N-diethylpiperazine-1-carboxa-mide (A5)

2.5.12. 2-(4-benzoylpiperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (B1)

2.5.13. 2-(4-(4-methylbenzoyl) piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl) acetamide (B2)

2.5.14. 2-(4-(4-methoxybenzoyl) piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl) acetamide (B3)

2.5.15. 2-(4-(4-(trifluoromethyl)benzoyl)piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl) acetam-ide (B4)

2.5.16. N,N-diethyl-4-(2-oxo-2-((3,4,5-trimethoxyphenyl) amino) ethyl) piperazine-1-carbo-xamide (B5)

2.5.17. (4-benzoyl piperazin-1-yl) (quinolin-2-yl) methanone (C1)

2.5.18. (4-(4-methylbenzoyl) piperazin-1-yl)(quinolin-2-yl) methanone (C2)

2.5.19. (4-(4-methoxybenzoyl) piperazin-1-yl)(quinolin-2-yl) methanone (C3)

2.5.20. quinolin-2-yl(4-(4-(trifluoromethyl) benzoyl) piperazin-1-yl) methanone (C4)

2.5.21. N,N-diethyl-4-(quinoline-2-carbonyl) piperazine-1-carboxamide (C5)

2.5.22. (4-benzoylpiperazin-1-yl)(4-(chloromethyl) phenyl) methanone (D1)

2.5.23. (4-(4-(chloromethyl) benzoyl) piperazin-1-yl)(p-tolyl) methanone (D2)

2.5.24. (4-(4-(chloromethyl) benzoyl) piperazin-1-yl)(4-methoxyphenyl) methanone (D3)

2.5.25. (4-(4-(chloromethyl) benzoyl) piperazin-1-yl)(4-(trifluoromethyl) phenyl) methanone (D4)

2.5.26. 4-(4-(chloromethyl)benzoyl)-N,N-diethylpiperazine-1-carboxamide (D5)

2.6. Animal experiments

2.8. In vivo anti-fatigue evaluation

2.7. Effect of biochemical indexes related to fatigue

2.9. Central excitability test

2.10. Safety test

2.11. Molecular dynamics simulation

2.12. Molecular docking

2.13. Statistical analysis

3. Results and Discussion

3.1. Structural design

3.2. Chemistry

2.3. Anti-fatigue activity assay

3.3.1. Animal behavior experiments

3.3.2. Content of index related to fatigue

3.4. Central excitability of A1

3.5. Safety of A1

3.6. Molecular dynamics simulation of A1

4. Conclusions

Acknowledgment

CRediT authorship contribution statement

Declaration of competing interest

Declaration of Generative AI and AI-assisted technologies in the writing process

Supplementary data

References

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