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Article

Excavating Precursors from Herb Pairs Polygala tenuifolia and Acori tatarinowii: Synthesis and Anticonvulsant Activity Evaluation of 3,4,5-Trimethoxycinnamic Acid (TMCA) Piperazine Amide Derivatives

by
Zefeng Zhao
1,*,
Mengchen Lei
1,
Yongqi Wang
1,
Yujun Bai
2 and
Haifa Qiao
1,*
1
Institute for Chinese Medicine Frontier Interdisciplinary Science and Technology, Shaanxi University of Chinese Medicine, Xianyang 712046, China
2
Biomedicine Key Laboratory of Shaanxi Province, Northwest University, 229 Taibai Road, Xi’an 710069, China
*
Authors to whom correspondence should be addressed.
Pharmaceuticals 2025, 18(9), 1312; https://doi.org/10.3390/ph18091312
Submission received: 10 July 2025 / Revised: 26 August 2025 / Accepted: 27 August 2025 / Published: 1 September 2025
(This article belongs to the Section Medicinal Chemistry)
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Abstract

Background: Epilepsy is a cluster of central nervous system (CNS) disorders identified by recurrent seizures, which affects about 60 million people around the world. In this research, a total of 40 types of 3,4,5-trimethoxycinnamic acid (TMCA) piperazine amide derivatives were designed and synthesized, inspired by the traditional Chinese medicine (TCM) herb pair drugs Polygala tenuifolia and Acori tatarinowii, followed by determination of their anticonvulsant potency. Methods: All the TMCA analogues were tested for their anticonvulsant potential through two acute models of seizures induced in mice: the maximal electroshock (MES) and sc-pentylenetetrazole (PTZ) models. In addition, the lactate dehydrogenase (LDH) inhibitory activity was determined in vitro. Results: The results showed that compounds A3, A9, A12, A14, B9, and B12 exhibited preferable anticonvulsant activity in the primary evaluation. In addition, the molecular docking results predicted good interactions of screened analogues with the LDH. Molecular dynamic simulation was used to reveal the consensual binding affinity between the most promising compound (B9) and active site interactions with LDH. Electroencephalogram (EEG) analysis and silver and immunofluorescence staining were performed to illustrate the anti-epilepsy potential of compound B9. Conclusions: Novel derivatives in this study provide new cores for the further design and optimization inspired by TCM herb pair drugs P. tenuifolia and A. tatarinowii, with the aim to explore new anticonvulsant agents.

1. Introduction

Epilepsy is a severe chronic neurological disorder affecting 0.5–1.0% of the global population [1,2]. Anti-epileptic drugs (AEDs) remain the primary therapeutic approach. Natural products offer promising avenues for novel interventions in this field [3,4]. Traditional Chinese medicine (TCM), recognized as a valuable natural resource, demonstrates favorable safety and efficacy profiles while mitigating treatment-associated side effects [5]. Building upon prior research [6,7,8], we identified 3,4,5-trimethoxycinnamic acid (TMCA) from P. tenuifolia as a precursor exhibiting significant anticonvulsant potential, providing a rationale for exploring TMCA derivatives (Figure 1).
In this study, we synthesized a series of TMCA piperazine amide derivatives (Scheme 1, A1–17, B1–17). The synthetic derivatives were classified into two categories according to the linker of butyryl moiety (A1–17) and butanedioic amide (B1–17), and the substitutions mainly constituted five kinds of A. tatarinowii component derivatives (substitutions S2–6) and nine kinds of arylpiperazine derivatives (S8–16). TMCA is an established central nervous system (CNS) agent targeting the GABAA/BZ receptor [9], 5-HT (5-hydroxytryptamine) [10], AChE (acetylcholine) [11] and EP2 [12]. Its role as the functional core in marketed drugs, such as cinepazide and cinepaxadil, underscores its potential as a novel drug precursor.
The rationale for selecting the residual groups for amide/ester bond formation comprised three elements: (1) TCM-inspired neural actives—incorporation of A. tatarinowii constituents/derivatives, including methyl eugenol (S2), isoeugenol (S3), and asaronol (S5), to explore anticonvulsant potential and leverage TCM herb-pairing principles [13,14,15]. Borneol (S7), a recognized CNS-active TCM compound, was also included. (2) Market-drug-derived arylpiperazines—inclusion of arylpiperazine moieties from established drugs (cinnarizine (S8), flunarizine (S9), lurasidone (S10), aripiprazole (S12), known for neural activity [16,17,18]; (3) integration of piperidine analogue tetrahydroisoquinoline (S17) to broaden structure–activity relationship (SAR) exploration. All synthesized derivatives were fully characterized. Their anticonvulsant potency was evaluated using classical seizure models. The article details results including inhibition of lactate dehydrogenase (LDH), neurotoxicity assessment, electroencephalogram (EEG) analysis, silver and immunofluorescence staining, molecular docking studies and SAR investigations.

2. Results

2.1. Chemistry

All titled compounds A1–17 and B1–17 were successfully prepared through the synthetic protocols presented in Scheme 1, and the structurally simple intermediates A1 and B1 can be characterized by the subsequent derivatives. The spectra are listed in the Supplementary Materials.
4-allyl-2-methoxyphenyl (E)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (A2): yellow liquid; yield: 80%; Rf = 0.7 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 15.8 Hz, 1H), 7.03 (d, J = 7.9 Hz, 1H), 6.87–6.76 (m, 4H), 6.75–6.66 (m, 1H), 6.58 (d, J = 15.9 Hz, 1H), 6.05–5.89 (m, 1H), 5.17–5.01 (m, 3H), 3.90 (d, J = 1.1 Hz, 9H), 3.87 (d, J = 2.1 Hz, 2H), 3.82 (d, J = 10.2 Hz, 3H), 3.36 (dd, J = 32.9, 6.8 Hz, 3H), 2.68–2.34 (m, 2H), 1.41 (s, 1H), 1.27 (d, J = 13.6 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 165.47, 165.25, 153.57, 151.10, 146.54, 140.47, 139.14, 138.08, 137.18, 129.90, 122.77, 120.84, 116.29, 115.63, 112.88, 105.56, 105.13, 61.12, 56.34, 56.29, 56.01, 55.97, 55.92, 40.25, 31.75, 29.77; HRMS (ESI+) calcd for C30H38N2O7 (m/z [M + H]+): 539.2757; found: 539.2725.
methoxy-4-((E)-prop-1-en-1-yl)phenyl 4-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (A3): white liquid; yield: 73%; Rf = 0.65 (DCM/Me = 10:1, v/v);1H NMR (400 MHz, CDCl3) δ 6.95 (d, J = 4.0 Hz, 1H), 6.77–6.71 (m, 1H), 6.64–6.55 (m, 2H), 5.91–5.83 (m, 1H), 5.05–4.93 (m, 2H), 3.78 (s, 3H), 3.65 (s, 9H), 3.34 (s, 14H);13C NMR (101 MHz, CDCl3) δ 171.59, 165.26, 153.39, 153.35, 150.95, 146.48, 143.87, 139.00, 137.85, 137.08, 131.84, 122.63, 121.12, 120.68, 115.46, 111.13, 105.39, 105.04, 60.92, 60.89, 56.11, 56.06, 55.77, 55.75, 40.08, 39.85, 39.62; HRMS (ESI+) calcd for C30H38N2O7 (m/z [M + H]+): 539.2757; found: 539.2757.
2-allylphenyl (E)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (A4): yellow solid; yield: 75%; Rf = 0.8 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.28 (s, 1H), 7.08 (s, 1H), 6.82 (s, 2H), 6.05–5.87 (m, 2H), 5.09–5.06 (m, 2H), 5.05–5.02 (m, 3H), 3.89 (d, J = 3.8 Hz, 9H), 3.84 (s, 1H), 3.39 (s, 1H), 3.37 (s, 8H), 3.09 (s, 6H);13C NMR (101 MHz, CDCl3) δ 154.20, 139.68, 136.51, 130.57, 128.04, 125.38, 121.08, 116.64, 115.93, 35.26, 29.78; HRMS (ESI+) calcd for C29H36N2O6 (m/z [M + H]+): 509.2652; found: 509.2621.
(E)-3-(2,4,5-trimethoxyphenyl)allyl 4-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (A5): yellow solid; yield: 64%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 15.9 Hz, 1H), 6.97 (t, J = 8.0 Hz, 2H), 6.73 (s, 2H), 6.48 (s, 1H), 6.37 (d, J = 15.9 Hz, 1H), 6.28–6.20 (m, 1H), 4.86–4.83 (m, 2H), 3.87 (s, 3H), 3.85 (s, 9H), 3.84 (s, 2H), 3.81 (s, 3H), 2.88 (d, J = 27.8 Hz, 1H), 1.93 (d, J = 68.8 Hz, 1H), 1.30–1.16 (m, 7H), 0.94–0.79 (m, 3H); 13C NMR (101 MHz, CDCl3) δ 166.83, 162.59, 153.47, 151.78, 150.04, 144.93, 143.36, 140.15, 129.98, 129.28, 121.37, 117.39, 116.94, 110.10, 105.27, 97.56, 66.00, 60.99, 56.61, 56.56, 56.17, 56.10, 31.46, 29.74, 22.73; HRMS (ESI+) calcd for C33H42N2O9 (m/z [M + H]+): 599.2969; found: 599.1808.
cinnamyl 4-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (A6): yellow solid; yield: 68%; Rf = 0.8 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 15.9 Hz, 1H), 7.41–7.32 (m, 7H), 6.75 (s, 2H), 6.60 (dt, J = 15.9, 1.6 Hz, 2H), 4.87 (dd, J = 6.5, 1.3 Hz, 2H), 3.87 (s, 9H), 3.70 (q, J = 7.0 Hz, 2H), 2.93 (s, 1H), 2.87 (s, 1H), 1.89 (s, 6H), 1.23 (t, J = 7.0 Hz, 4H); 13C NMR (101 MHz, CDCl3) δ 173.40, 162.74, 153.51, 153.48, 140.23, 136.81, 136.30, 134.43, 129.96, 128.71, 128.67, 128.20, 127.74, 123.30, 117.21, 105.36, 105.11, 65.27, 61.06, 58.48, 57.52, 56.30, 56.24, 31.56, 22.04; HRMS (ESI+) calcd for C29H36N2O6 (m/z [M + H]+): 509.2652; found: 509.2553.
rel-(1R,2S,4R)-1,7,7-trimethylbicyclo [2.2.1]heptan-2-yl 4-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (A7): yellow solid; yield: 75%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.5 Hz, 1H), 8.06–7.96 (m, 2H), 7.58 (d, J = 15.5 Hz, 2H), 3.93 (d, J = 11.4 Hz, 9H), 2.31–2.26 (m, 1H), 2.26 (s, 1H), 2.11 (s, 5H), 1.92–1.88 (m, 1H), 1.88–1.84 (m, 2H), 1.74–1.68 (m, 3H), 1.61 (t, J = 4.6 Hz, 2H), 1.29–1.17 (m, 4H), 1.01 (s, 2H), 0.89 (s, 2H), 0.86 (s, 4H), 0.81 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 161.86, 153.70, 153.56, 149.52, 141.58, 133.11, 129.40, 127.07, 106.40, 105.35, 80.06, 61.19, 61.10, 56.50, 56.29, 51.86, 49.62, 49.09, 48.15, 45.21, 45.15, 40.50, 39.13, 34.04, 28.40, 26.04, 20.60, 20.31, 20.23, 18.80; HRMS (ESI+) calcd for C30H44N2O6 (m/z [M + H]+): 529.3278; found: 529.3278.
(E)-1-(4-benzhydrylpiperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A8): white solid; yield: 78%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 15.3 Hz, 1H), 7.45–7.37 (m, 4H), 7.32–7.23 (m, 5H), 7.19 (t, J = 7.3 Hz, 2H), 6.69 (s, 2H), 4.26 (s, 1H), 3.86 (d, J = 2.3 Hz, 9H), 3.79–3.55 (m, 5H), 2.50–2.29 (m, 5H), 1.91–1.61 (m, 2H), 1.41 (d, J = 5.6 Hz, 1H), 1.26 (d, J = 12.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 165.38, 153.54, 153.51, 142.83, 142.23, 139.66, 131.00, 128.75, 128.73, 128.01, 127.31, 116.45, 105.12, 105.03, 76.08, 61.08, 56.34, 52.32, 51.74, 46.19, 42.50, 31.77, 29.77; HRMS (ESI+) calcd for C37H46N4O5 (m/z [M + H]+): 627.3546; found: 627.3509.
(E)-1-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A9): white solid; yield: 69%; Rf = 0.9 (DCM/Me = 10:1, v/v);1H NMR (400 MHz, CDCl3) δ 7.60–7.54 (m, 1H), 7.35 (s, 5H), 7.01 (s, 2H), 6.97 (s, 2H), 6.72 (d, J = 13.6 Hz, 3H), 3.87 (q, J = 2.1 Hz, 9H), 3.73 (s, 2H), 3.66 (s, 3H), 2.41 (s, 4H), 2.03 (d, J = 17.2 Hz, 1H), 1.59 (t, J = 1.5 Hz, 11H); 13C NMR (101 MHz, CDCl3) δ 165.39, 153.52, 143.04, 139.64, 137.74, 130.93, 129.39, 129.31, 122.41, 116.24, 115.85, 115.64, 104.97, 92.30, 74.43, 61.12, 56.29, 51.65, 34.48, 28.81; HRMS (ESI+) calcd for C37H44F2N4O5 (m/z [M]+): 662.3280; found: 662.3478.
(E)-1-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A10): white solid; yield: 74%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.62 (d, J = 15.3 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 3.88 (d, J = 5.3 Hz, 9H), 3.86 (s, 4H), 3.85 (s, 1H), 3.69 (pd, J = 7.8, 4.3 Hz, 1H), 3.57 (t, J = 5.1 Hz, 5H), 3.50 (dd, J = 7.8, 4.3 Hz, 1H), 2.45 (dd, J = 17.4, 10.2 Hz, 2H), 1.99–1.47 (m, 1H), 1.43–1.19 (m, 3H); 13C NMR (101 MHz, CDCl3) δ 171.42, 165.72, 163.36, 153.44, 153.40, 152.83, 143.37, 139.63, 130.77, 127.84, 127.82, 124.23, 123.64, 123.41, 120.73, 105.02, 104.94, 61.02, 61.00, 57.67, 56.24, 56.21, 53.43, 50.39, 50.07, 49.89, 45.63, 45.29, 42.04, 30.76, 22.17; HRMS (ESI+) calcd for C31H39N5O5S (m/z [M + H]+): 594.2750; found: 594.2787.
(E)-1-(4-phenylpiperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A11): yellow solid; yield: 75%; Rf = 0.6 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 15.4 Hz, 1H), 7.24 (s, 1H), 6.95 (s, 1H), 6.94 (s, 6H), 3.90 (s, 6H), 3.88 (s, 3H), 3.24 (s, 2H), 3.15 (s, 5H), 3.14 (s, 2H), 3.03 (d, J = 4.5 Hz, 1H), 3.00 (s, 5H), 2.23 (s, 7H); 13C NMR (101 MHz, CDCl3) δ 165.58, 154.53, 153.55, 149.23, 143.32, 139.81, 130.88, 129.40, 129.19, 120.70, 119.94, 116.76, 116.29, 106.69, 105.18, 61.09, 57.79, 56.35, 51.67, 50.43, 49.27, 46.16, 31.77, 22.31; HRMS (ESI+) calcd for C30H40N4O5 (m/z [M + H]+): 537.3077; found: 537.3027.
(E)-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A12): white solid; yield: 77%; Rf = 0.9 (PE/EA = 2:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 15.3 Hz, 1H), 7.20–7.18 (m, 1H), 6.93 (dd, J = 7.7, 1.8 Hz, 1H), 6.80 (d, J = 15.3 Hz, 1H), 6.75 (s, 3H), 3.89 (d, J = 3.0 Hz, 9H), 3.87 (d, J = 2.4 Hz, 11H), 3.07 (t, J = 4.9 Hz, 5H), 2.50 (s, 1H), 1.26 (d, J = 13.8 Hz, 5H); 13C NMR (101 MHz, CDCl3) δ 171.41, 165.69, 153.53, 153.50, 150.65, 143.40, 139.72, 134.33, 130.85, 127.84, 127.72, 125.37, 118.89, 116.14, 105.07, 105.02, 61.11, 57.79, 56.32, 51.99, 51.24, 46.26, 45.88, 42.53, 41.93, 32.04, 29.82; HRMS (ESI+) calcd for C30H38Cl2N4O5 (m/z [M + H]+): 605.2298; found: 605.2329.
(E)-1-(4-(3-chlorophenyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A13): yellow solid; yield: 73%; Rf = 0.8 (DCM/Me = 10:1, v/v);1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 15.3 Hz, 1H), 7.21–7.15 (m, 1H), 6.88 (t, J = 2.1 Hz, 1H), 6.85 (ddd, J = 7.8, 1.9, 0.9 Hz, 1H), 6.81–6.78 (m, 1H), 6.75 (s, 1H), 6.75 (s, 2H), 3.89 (s, 6H), 3.87 (s, 3H), 3.84 (d, J = 15.3 Hz, 6H), 3.81 (s, 3H), 3.25 (s, 1H), 3.23 (s, 3H), 3.23 (s, 4H), 3.22 (s, 1H), 2.55–2.36 (m, 1H), 1.77 (s, 2H), 1.43–1.21 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 171.32, 165.62, 153.56, 153.53, 152.01, 143.51, 139.89, 135.19, 130.80, 130.32, 120.27, 116.44, 116.02, 114.51, 105.23, 105.14, 61.09, 57.76, 56.35, 56.34, 49.31, 45.64, 45.35, 31.75, 23.15; HRMS (ESI+) calcd for C30H39ClN4O5 (m/z [M + H]+): 571.2687; found: 571.2643.
(E)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A14): yellow solid; yield: 67%; Rf = 0.8 (DCM/Me = 10:1, v/v);1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 15.3 Hz, 1H), 7.52 (s, 1H), 7.50 (s, 2H), 6.95 (s, 1H), 6.75 (s, 3H), 3.90 (s, 8H), 3.88 (s, 1H), 3.36 (s, 2H), 3.35 (s, 4H), 3.34 (s, 1H), 3.31–3.24 (m, 1H), 2.61–2.37 (m, 1H), 1.68 (s, 8H), 1.41 (s, 1H), 1.25 (s, 4H); 13C NMR (101 MHz, CDCl3) δ 165.64, 153.57, 143.68, 139.86, 130.77, 126.68, 115.87, 115.12, 105.16, 61.14, 56.35, 48.23, 41.85, 29.83; HRMS (ESI+) calcd for C29H35F3N4O5 (m/z [M + H]+): 577.2638; found: 577.2661.
(E)-1-(4-(2-methoxyphenyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A15): yellow solid; yield: 75%; Rf = 0.8 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 15.2 Hz, 1H), 7.05–7.03 (m, 1H), 6.94–6.88 (m, 3H), 6.81 (d, J = 15.3 Hz, 1H), 6.74 (d, J = 6.9 Hz, 2H), 3.96–3.90 (m, 2H), 3.89 (s, 6H), 3.88–3.87 (m, 12H), 3.87 (s, 3H), 3.84–3.62 (m, 1H), 3.50–3.31 (m, 1H), 3.21–2.99 (m, 5H), 2.57–2.39 (m, 2H), 1.45–1.21 (m, 2H);13C NMR (101 MHz, CDCl3) δ 171.21, 165.55, 165.49, 153.49, 152.33, 143.10, 140.66, 139.63, 130.95, 123.73, 121.16, 118.55, 116.35, 115.94, 111.38, 105.04, 61.08, 57.77, 56.29, 55.59, 55.54, 51.26, 51.12, 50.69, 47.88, 46.29, 45.91, 44.10, 42.53, 31.74, 22.18; HRMS (ESI+) calcd for C31H42N4O6 (m/z [M + H]+): 567.3183; found: 567.3197.
(E)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A16): white solid; yield: 71%; Rf = 0.9 (DCM/Me = 10:1, v/v);1H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 8.24 (s, 1H), 7.53 (d, J = 15.3 Hz, 1H), 6.75 (d, J = 15.3 Hz, 1H), 6.69 (s, 2H), 6.47 (t, J = 4.8 Hz, 1H), 3.82 (s, 6H), 3.78 (s, 3H), 3.71 (t, J = 9.1 Hz, 6H), 3.62 (d, J = 7.0 Hz, 2H), 3.58 (d, J = 7.0 Hz, 2H), 3.35 (s, 1H), 2.47 (s, 11H); 13C NMR (101 MHz, CDCl3) δ 165.68, 162.55, 161.40, 157.76, 153.34, 143.17, 139.57, 130.72, 116.15, 110.45, 105.05, 60.87, 57.97, 56.17, 50.33, 45.61, 43.55, 41.95, 36.45, 31.35; HRMS (ESI+) calcd for C28H38N6O5 (m/z [M + H]+): 539.2982; found: 539.2891.
(E)-1-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butan-1-one (A17): white solid; yield: 76%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.63 (s, 1H), 7.59 (s, 1H), 7.27 (d, J = 2.2 Hz, 1H), 7.08 (d, J = 2.2 Hz, 1H), 6.75 (s, 4H), 3.90 (s, 11H), 3.87 (s, 5H), 2.21 (d, J = 3.6 Hz, 1H), 2.18 (s, 2H), 2.03 (s, 5H), 1.67 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 165.69, 164.16, 163.10, 160.30, 153.57, 143.23, 139.80, 130.95, 122.38, 117.19, 116.50, 112.93, 112.68, 105.18, 97.89, 61.11, 56.37, 45.99, 42.23, 34.51, 30.48; HRMS (ESI+) calcd for C32H39FN4O6 (m/z [M + H]+): 595.2932; found: 595.2935.
4-allyl-2-methoxyphenyl (E)-4-oxo-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (B2): yellow liquid; yield: 77%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 15.3 Hz, 1H), 6.98 (d, J = 7.9 Hz, 1H), 6.80–6.64 (m, 5H), 5.95 (ddt, J = 16.9, 10.1, 6.7 Hz, 1H), 5.15–5.01 (m, 2H), 3.89 (d, J = 9.0 Hz, 9H), 3.81 (s, 3H), 3.71 (q, J = 5.9 Hz, 4H), 3.60 (d, J = 5.3 Hz, 2H), 3.34 (dd, J = 20.6, 6.7 Hz, 2H), 3.05–2.93 (m, 2H), 2.77 (t, J = 6.8 Hz, 2H);13C NMR (101 MHz, CDCl3) δ 171.57, 165.78, 153.56, 150.90, 143.94, 139.89, 139.16, 138.01, 137.16, 130.66, 122.71, 120.85, 116.31, 115.67, 112.82, 105.16, 61.13, 56.35, 55.99, 40.22, 40.02, 31.76, 29.24, 28.05; HRMS (ESI+) calcd for C30H36N2O8 (m/z [M + H]+): 553.2550; found: 553.2551.
2-methoxy-4-((E)-prop-1-en-1-yl)phenyl 4-oxo-4-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (B3): yellow liquid; yield: 78%; Rf = 0.8 (DCM/Me = 10:1, v/v) 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 15.3 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.84 (s, 2H), 6.82 (s, 3H), 6.77 (s, 1H), 6.75 (s, 2H), 3.87 (s, 9H), 3.79 (s, 3H), 3.69 (d, J = 7.1 Hz, 4H), 3.46–3.34 (m, 3H), 3.04–2.95 (m, 2H), 2.74 (t, J = 6.8 Hz, 2H), 2.24–2.14 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 143.99, 137.95, 121.29, 115.68, 114.34, 111.18, 105.17, 56.36, 55.98, 40.03, 26.93, 14.99; HRMS (ESI+) calcd for C30H36N2O8 (m/z [M + H]+): 553.2550; found: 553.2551.
2-allylphenyl (E)-4-oxo-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (B4): white solid; yield: 69%; Rf = 0.8 (DCM/Me = 10:1, v/v);1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 15.3 Hz, 1H), 7.14 (d, J = 1.4 Hz, 1H), 6.95 (s, 2H), 6.67 (s, 2H), 5.96–5.89 (m, 2H), 4.99 (s, 2H), 4.93 (s, 2H), 3.79 (d, J = 11.3 Hz, 9H), 3.30 (s, 3H), 3.28 (s, 4H), 2.87 (s, 4H), 2.78 (s, 2H); 13C NMR (101 MHz, CDCl3) δ 170.21, 166.02, 162.94, 154.51, 153.17, 148.66, 147.00, 144.02, 139.48, 137.71, 136.86, 135.79, 129.69, 126.91, 119.40, 114.99, 114.93, 111.55, 105.05, 60.65, 57.52, 57.15, 55.94, 55.51, 49.34, 45.30, 33.98, 31.37, 29.27; HRMS (ESI+) calcd for C29H34N2O7 (m/z [M + H]+): 523.2444; found: 523.2444.
(E)-3-(2,4,5-trimethoxyphenyl)allyl 4-oxo-4-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (B5): yellow solid; yield: 72%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 15.9 Hz, 1H), 7.03–6.97 (m, 2H), 6.76 (s, 2H), 6.50 (s, 1H), 6.39 (dd, J = 15.9, 0.9 Hz, 1H), 6.26 (dt, J = 16.0, 6.7 Hz, 1H), 4.87 (d, J = 6.7 Hz, 2H), 3.91 (s, 3H), 3.89–3.88 (m, 9H), 3.87 (s, 3H), 3.85 (d, J = 0.9 Hz, 3H), 1.56 (s, 10H), 1.25 (s, 2H);13C NMR (101 MHz, CDCl3) δ 173.98, 168.46, 158.98, 153.56, 148.27, 145.04, 140.31, 129.40, 121.42, 110.06, 105.28, 97.54, 61.13, 56.72, 56.27, 47.90, 41.81, 36.81, 27.42, 21.27; HRMS (ESI+) calcd for C32H39N2NaO10 (m/z [M + Na]+): 635.2581; found: 635.2593.
(E)-3-(2,4,5-trimethoxyphenyl)allyl 4-oxo-4-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (B6): yellow solid; yield: 75%; Rf = 0.8 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 15.3 Hz, 1H), 7.32 (s, 3H), 7.24 (s, 1H), 7.18 (s, 1H), 6.76–6.72 (m, 3H), 6.54 (s, 1H), 6.34 (s, 1H), 4.74 (s, 1H), 4.72 (s, 2H), 3.85 (d, J = 5.0 Hz, 9H), 3.73–3.69 (m, 3H), 3.51 (s, 2H), 3.38 (s, 2H), 2.73–2.67 (m, 2H), 2.63 (t, J = 5.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 172.73, 165.73, 162.64, 153.22, 143.66, 139.58, 136.77, 133.99, 130.14, 128.97, 128.92, 128.43, 128.35, 127.27, 122.87, 115.45, 105.05, 65.18, 60.73, 57.68, 56.02, 51.69, 49.92, 45.22, 41.96, 31.27, 29.06; HRMS (ESI+) calcd for C29H34N2O7 (m/z [M + H]+): 523.2444; found: 523.2458.
(1R,2S,4R)-1,7,7-trimethylbicyclo [2.2.1]heptan-2-yl 4-oxo-4-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butanoate (B7): white solid; yield: 78%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 15.3 Hz, 1H), 6.73 (s, 1H), 6.72 (s, 3H), 6.70 (d, J = 1.9 Hz, 1H), 4.87 (dd, J = 10.1, 2.8 Hz, 1H), 3.87 (d, J = 1.9 Hz, 7H), 3.85 (s, 3H), 2.65 (s, 3H), 2.05 (s, 5H), 1.59 (s, 1H), 1.21 (s, 2H), 0.99 (d, J = 2.0 Hz, 3H), 0.87 (d, J = 2.1 Hz, 4H), 0.79 (s, 2H); 13C NMR (101 MHz, CDCl3) δ 173.34, 165.79, 162.72, 153.49, 143.93, 139.80, 130.59, 115.58, 105.11, 80.29, 61.06, 56.29, 49.54, 49.01, 48.86, 48.07, 47.86, 46.40, 45.11, 39.01, 33.96, 29.54, 27.30, 27.17, 19.76, 18.90, 13.42; HRMS (ESI+) calcd for C30H42N2O7 (m/z [M + H]+): 543.3070; found: 543.3071.
(E)-1-(4-benzhydrylpiperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B8): white solid; yield: 76%; Rf = 0.8 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 15.3 Hz, 1H), 7.39 (d, J = 7.6 Hz, 5H), 7.28 (s, 2H), 7.24 (s, 1H), 7.18 (d, J = 7.2 Hz, 1H), 6.75–6.67 (m, 3H), 4.22 (s, 1H), 3.87 (d, J = 8.3 Hz, 9H), 3.69–3.64 (m, 3H), 3.59 (t, J = 5.1 Hz, 3H), 3.51 (t, J = 5.0 Hz, 2H), 2.89 (d, J = 28.1 Hz, 3H), 2.66 (s, 4H), 2.37 (dt, J = 14.6, 5.0 Hz, 5H); 13C NMR (101 MHz, CDCl3) δ 170.26, 165.76, 162.66, 153.53, 143.75, 142.22, 139.88, 130.66, 128.71, 127.95, 127.25, 115.79, 105.20, 76.02, 61.06, 56.33, 51.98, 51.62, 45.56, 42.06, 31.53, 27.97; HRMS (ESI+) calcd for C37H44N4O6 (m/z [M + H]+): 641.3339; found: 641.3338.
(E)-1-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B9): white solid; yield: 77%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 15.3 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 7.32 (s, 1H), 7.31 (s, 2H), 6.99 (s, 1H), 6.97 (s, 2H), 6.95 (s, 2H), 6.72 (d, J = 13.2 Hz, 3H), 3.88 (d, J = 8.5 Hz, 9H), 3.79–3.65 (m, 5H), 3.60 (s, 4H), 3.52 (s, 2H), 2.94 (s, 1H), 2.87 (s, 1H), 2.67 (s, 4H), 2.38–2.35 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 165.77, 163.25, 160.81, 153.56, 143.79, 139.93, 137.76, 130.68, 129.37, 129.29, 115.80, 115.58, 105.22, 74.35, 61.09, 56.35, 51.87, 51.51, 46.52, 45.53, 42.01, 31.55, 27.98; HRMS (ESI+) calcd for C37H42F2N4O6 (m/z [M + H]+): 677.3151; found: 677.3121.
(E)-1-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B10): white solid; yield: 74%; Rf = 0.9 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.4 Hz, 2H), 7.81 (t, J = 8.2 Hz, 2H), 7.62 (d, J = 15.3 Hz, 1H), 7.48 (ddd, J = 8.4, 7.0, 1.2 Hz, 1H), 7.42–7.31 (m, 2H), 3.88 (d, J = 9.2 Hz, 9H), 3.78 (s, 2H), 3.70 (s, 3H), 3.64 (s, 2H), 3.56 (s, 5H), 3.52 (d, J = 5.4 Hz, 3H), 2.91 (d, J = 29.1 Hz, 1H), 2.78 (s, 4H); 13C NMR (101 MHz, CDCl3) δ 170.67, 165.77, 163.49, 153.56, 152.97, 152.85, 143.81, 139.93, 130.68, 127.94, 127.88, 124.27, 124.07, 123.73, 120.79, 105.22, 61.09, 56.36, 50.26, 49.95, 45.24, 41.72, 28.10; HRMS (ESI+) calcd for C31H37N5O6S (m/z [M + H]+): 608.2543; found: 608.2543.
(E)-1-(4-phenylpiperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B11): yellow solid; yield: 68%; Rf = 0.7 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 15.3 Hz, 1H), 7.30–7.26 (m, 2H), 6.94 (s, 2H), 6.92 (s, 2H), 6.74 (s, 2H), 3.89 (d, J = 9.2 Hz, 9H), 3.77 (t, J = 5.3 Hz, 3H), 3.70 (s, 8H), 3.67–3.61 (m, 4H), 3.20 (s, 1H), 2.75 (s, 4H); 13C NMR (101 MHz, CDCl3) δ 171.00, 170.44, 165.79, 153.55, 151.03, 143.81, 139.91, 130.67, 129.35, 120.65, 116.75, 115.77, 105.22, 61.08, 56.34, 49.72, 49.45, 45.41, 41.84, 29.79, 28.05; HRMS (ESI+) calcd for C30H38N4O6 (m/z [M + H]+): 551.2870; found: 551.2868.
(E)-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B12): white solid; yield: 75%; Rf = 0.8 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 15.3 Hz, 1H), 7.19–7.12 (m, 2H), 6.90 (dd, J = 7.7, 1.9 Hz, 1H), 6.76–6.70 (m, 3H), 3.87 (d, J = 9.6 Hz, 9H), 3.78 (s, 4H), 3.70 (s, 7H), 3.63 (s, 1H), 2.99 (s, 1H), 2.94 (s, 1H), 2.86 (s, 1H), 2.74 (s, 5H); 13C NMR (101 MHz, CDCl3) δ 170.97, 170.53, 165.76, 150.70, 143.78, 139.90, 134.27, 130.66, 127.83, 127.66, 125.29, 118.87, 115.76, 105.20, 61.07, 56.33, 51.62, 51.21, 45.69, 42.10, 31.53, 28.04; HRMS (ESI+) calcd for C30H36Cl2N4O6 (m/z [M + H]+): 619.2090; found: 619.2091.
(E)-1-(4-(3-chlorophenyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B13): yellow solid; yield: 74%; Rf = 0.8 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 15.3 Hz, 1H), 7.18 (t, J = 8.1 Hz, 1H), 6.87–6.86 (m, 2H), 6.78 (dd, J = 8.5, 2.4 Hz, 2H), 6.74 (s, 2H), 3.88 (d, J = 9.0 Hz, 9H), 3.77 (s, 1H), 3.76 (s, 3H), 3.70 (s, 2H), 3.65–3.62 (m, 4H), 3.21 (s, 2H), 3.16 (t, J = 5.3 Hz, 3H), 2.91 (d, J = 29.1 Hz, 1H), 2.75 (s, 4H); 13C NMR (101 MHz, CDCl3) δ 170.62, 170.51, 165.80, 153.59, 152.06, 143.84, 139.99, 135.19, 130.69, 130.30, 120.24, 120.20, 116.47, 115.79, 114.52, 105.28, 61.11, 56.38, 51.96, 49.16, 48.96, 45.23, 41.65, 36.60, 29.82, 28.12, 28.07; HRMS (ESI+) calcd for C30H37ClN4O6 (m/z [M + H]+): 585.2480; found: 585.2480.
(E)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B14): yellow solid; yield: 71%; Rf = 0.8 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 15.3 Hz, 1H), 7.50 (s, 1H), 7.48 (s, 1H), 6.93 (s, 1H), 6.91 (s, 1H), 6.75–6.70 (m, 3H), 3.89 (s, 6H), 3.87 (s, 3H), 3.78 (t, J = 5.3 Hz, 3H), 3.72 (t, J = 5.2 Hz, 1H), 3.69 (s, 9H), 3.63 (s, 2H), 3.29 (d, J = 12.4 Hz, 1H), 2.74 (s, 4H); 13C NMR (101 MHz, CDCl3) δ 165.78, 154.80, 153.57, 139.90, 130.67, 126.69, 126.00, 121.18, 115.37, 105.17, 61.14, 56.36, 48.54, 48.38, 45.03, 41.45, 28.11; HRMS (ESI+) calcd for C31H37F3N4O6 (m/z [M + H]+): 619.274.
(E)-1-(4-(2-methoxyphenyl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B15): white solid; yield: 73%; Rf = 0.7 (DCM/Me = 10:1, v/v); 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 15.3 Hz, 1H), 7.03 (s, 1H), 6.90 (s, 3H), 6.89 (s, 1H), 6.74 (s, 2H), 3.89 (s, 6H), 3.87 (s, 6H), 3.79 (s, 3H), 3.70 (s, 8H), 3.70 (s, 5H), 2.74 (d, J = 6.3 Hz, 4H); 13C NMR (101 MHz, CDCl3) δ 170.32, 165.75, 153.55, 152.34, 143.81, 140.76, 139.84, 130.69, 123.70, 121.16, 118.55, 115.76, 111.38, 105.14, 61.11, 56.34, 55.56, 51.01, 50.67, 45.72, 42.11, 29.82, 28.06; HRMS (ESI+) calcd for C31H40N4O7 (m/z [M + H]+): 581.2975; found: 581.2964.
(E)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B16): white solid; yield: 76%; Rf = 0.9 (DCM/Me = 10:1, v/v);1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.31 (s, 1H), 7.61 (d, J = 15.3 Hz, 1H), 6.77–6.68 (m, 3H), 6.53 (td, J = 4.8, 2.3 Hz, 1H), 3.89 (s, 6H), 3.87 (s, 3H), 3.82 (dd, J = 6.6, 4.1 Hz, 2H), 3.69 (s, 6H), 3.62 (dd, J = 6.6, 4.1 Hz, 2H), 2.75 (dd, J = 7.8, 3.8 Hz, 4H), 1.75 (d, J = 3.7 Hz, 4H), 1.26 (d, J = 12.2 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 165.80, 161.62, 157.91, 153.56, 143.83, 139.93, 130.69, 115.78, 110.58, 105.23, 61.10, 56.36, 45.29, 43.75, 43.61, 41.74, 31.75, 29.81; HRMS (ESI+) calcd for C28H36N6O6 (m/z [M + H]+): 553.2775; found: 553.2775.
(E)-1-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-4-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperazin-1-yl)butane-1,4-dione (B17): white solid; yield: 71%; Rf = 0.8 (DCM/Me = 10:1, v/v);1H NMR (400 MHz, CDCl3) δ 7.67 (dd, J = 8.7, 5.1 Hz, 1H), 7.60 (d, J = 15.3 Hz, 1H), 7.25–7.21 (m, 1H), 7.05 (td, J = 8.8, 2.1 Hz, 1H), 6.76–6.69 (m, 3H), 3.88 (s, 6H), 3.86 (s, 3H), 3.70 (s, 3H), 3.63 (s, 1H), 3.33 (s, 2H), 3.31 (s, 1H), 3.28 (d, J = 12.9 Hz, 1H), 2.98–2.83 (m, 2H), 2.74 (s, 4H), 2.09 (dt, J = 16.2, 4.0 Hz, 2H), 1.87 (s, 4H); 13C NMR (101 MHz, CDCl3) δ 170.41, 165.79, 165.54, 164.08, 163.04, 153.53, 143.79, 139.89, 130.66, 122.51, 117.14, 115.78, 112.58, 105.21, 97.52, 61.07, 56.33, 45.39, 41.87, 34.41, 31.53, 30.47, 30.20, 28.15; HRMS (ESI+) calcd for C32H37FN4O7 (m/z [M + H]+): 609.2725; found: 609.2725.

2.2. Pharmacology

2.2.1. Maximal Electroshock (MES) Test

In the MES test at 100 mg/kg via intraperitoneal administration, several TMCA piperazine amide derivatives demonstrated protective effects against tonic–clonic seizures at 0.5 h post-administration (Table 1). Compounds achieving complete protection (4/4 mice) included A3, A9, A12, A14, B9, and B12, indicating their potent activity in the early phase. While efficacy generally diminished over time, compounds A3, A12, and A14 exhibited sustained activity at 3 h (1/4 protection), with A14 uniquely maintaining complete protection at both 0.5 h and 1 h (4/4). Compound B12 also showed residual activity at 3 h (1/4). The initial drug design, inspired by traditional Chinese medicine (TCM), revealed that compounds with moieties derived from A. tatarinowii and conjugated to TMCA piperazine exhibited anticonvulsant activity. Analogues B3, A5 and B5 exhibited favorable results (3/4 in both 0.5 and 1 h), suggesting this research preliminarily developed novel potential derivatives reflecting the function of initial pair herbs. Compounds A7 and B7 (3/4) introduced borneol, which partially elucidated the concept of compatibility in TCM. Despite the passage of time, natural products, including TCM, continue to provide valuable references for the development of antiepileptic drugs (AEDs).
Compounds demonstrating complete protection at 0.5 h were advanced to quantitative ED50 determination. As shown in Table 2, compound B9 displayed the highest potency (ED50: 23.58 mg/kg), followed by compound A9 (ED50: 37.46 mg/kg) and A14 (ED50: 52.39 mg/kg). Crucially, the most promising candidate B9 exhibited the highest protective index (PI: 9.97), significantly exceeding the stiripentol (STP) established PI threshold of 1.74 for standard therapeutic products (STPs). While compound A9 also demonstrated a favorable PI value (8.14), compounds A3 (PI: 6.05) and A12 (PI: 5.20) exhibited lower safety margins compared to B9.

2.2.2. Pentylenetetrazole (PTZ)-Induced Seizure Test

The anticonvulsant effects of selected TMCA piperazine amide derivatives A3, A9, A12, A14, B9, and B12 were further evaluated against scPTZ-induced seizures (Table 2). Compounds demonstrating complete protection (4/4) in the MES model at 0.5h were prioritized for scPTZ assessment. Notably, compound B9 exhibited the highest protective ratio (3/4 mice), followed by derivative A14 (both 2/4), while analogue A12 showed no protection (0/4) in this model. Regarding the latency period of epileptic seizures, most tested compounds exhibited activity in extending this latency. As the most promising compound, B9 significantly prolonged seizure latency to 274.3 s, exceeding that of the reference drug STP and indicating robust suppression of seizure initiation. Initial structural analysis revealed that derivatives featuring arylpiperazine pharmacophores (e.g., bis(4-fluorophenyl)methylpiperazine moiety of compound B9) and rigid heterocyclic elements demonstrated enhanced efficacy in this model. The extended latency observed with compounds B9 compared to A12 further underscores the critical influence of para-fluorinated aryl groups on elevating seizure thresholds, potentially through enhanced receptor affinity or pharmacokinetic optimization [19].

2.2.3. Acute Neurotoxicity Screening

In the neurotoxicity evaluation, heterocyclic substituted analogues A3, A9, A12, A14, B9, and B12 showed clear neurotoxicity with the TD50 values ranging from 235 to 443 mg/kg, and the ester compound A3 displayed weak neurotoxicity (Table 2). All the tested compounds exhibited higher PI values than the reference standard, among which compound B9 was considered to be the most promising candidate according to the perspective of anticonvulsant activity and protective index.

2.2.4. Validation of Anti-Seizure Potential of B9 Through EEG

The EEG results revealed that compound B9 showed a coherent neurophysiological anticonvulsant profile: (1) early spike delay—similar to the effect of positive standard STP, compound B9 extended the latency to the first PTZ-induced epileptic spike (Figure 2E); (2) spike density reduction—compound B9 significantly reduced spike frequency within 60 s post-initial spike compared to PTZ controls (Figure 2D,F). Animal EEG spike density is a validated predictor of clinical seizure severity [20]. Dual efficacy of compound B9 in suppressing both EEG spikes and behavioral convulsions positions it as a high-priority candidate for further development.

2.2.5. Silver Staining Results

From the silver staining, we observed that the neuronal cell bodies in the CA1 and CA3 regions of the hippocampus in the PTZ group mice appeared blurry, with shorter axons and a significant reduction in the number of neurons compared with the control group (Figure 3). Conversely, the treatments of STP group and the B9 group alleviated symptoms.

2.2.6. Immunofluorescence Staining

The immunofluorescence analysis for LDH (Figure 4) showed in the CA1 a 1.6-fold significant increase in labeling in the PTZ group compared to NS (p < 0.05). The immunolabelling intensity in both SPT and B9 groups was lower than the values found in the PTZ group (Figure 4). Similar data were also seen in the CA3 area, in which a respective 1.7-fold (p < 0.05) increase was present. Values were similar to those of the LDH level in both SPT and B9 groups, providing evidence for potential of B9 mediating LDH in PTZ-induced epilepsy mice.

2.2.7. In Vitro LDH Assay

In order to investigate more potential mechanisms about LDH modulatory action of TMCA piperazine amide derivatives, in vitro enzymatic assay was performed. The active compounds A3, A9, A12, A14, B9, and B12 were evaluated for the LDH inhibitory potency at the dose of 0.1 mM (Table 3). The result showed that compounds A9 and B9 exhibited a more than 50% inhibitory ratio while other derivatives were weaker inhibitors. Compared with the marketed anti-epilepsy drug STP targeting LDH and the original ligand for the LDH co-crystal complex GEN-140 (IC50: 0.003 μM) [21], the test analogues exhibited moderate inhibitory potency, representing a feasible route to develop promising LDH inhibitors with a novel skeleton.

2.2.8. Molecular Docking Analysis

Based on comprehensive pharmacological evaluation, the potential TMCA piperazine amide derivatives A3, A9, A12, A14, B9, and B12 were systematically analyzed for their binding mechanisms with LDH through molecular docking studies, comparing with the positive drug STP. As quantified in Table 3, these compounds exhibited binding affinities ranging from −6.3 kcal/mol (A3) to −9.3 kcal/mol (B9), with all potential hydrogen bonds (<3 Å) indicating stable protein–ligand interactions (Figure 4). Crucially, the affinity ranking (A3 < A14 < A12 < B12 < A9 < B9) demonstrated a direct correlation with their established anticonvulsant potency in MES and PTZ models, where B9 showed exceptional activity surpassing the reference drug STP. Structural analysis revealed that the TMCA core anchors to LDH’s catalytic cleft through conserved interactions: the carbonyl group formed hydrogen bonds with key residues ASN 137 (A9, A14) and ARG 98 (B9, B12), while aromatic moieties engaged TYR 238 via π-stacking (A3, STP) and hydrophobic sub-pockets lined by ILE 241, ILE 251, and VAL 30 (Figure 5). Notably, B9 achieved its superior affinity (−9.3 kcal/mol vs. STP’s −7.3 kcal/mol) by simultaneously occupying the NAD+-binding site (ARG 98 hydrogen bond) and the adjacent hydrophobic cavity (VAL 30/ALA 29 contacts). These findings establish the TMCA–piperazine scaffold as a novel chemotype for LDH modulation, with future optimization focusing on enhancing interactions with the defined pharmacophoric anchors to improve target engagement and anticonvulsant efficacy.

2.2.9. MD Simulation

Based on comprehensive MD simulations and MM/GBSA analysis, derivative B9 demonstrated superior binding stability and energetic favorability against LDH compared to the reference drug STP. Analysis of the trajectory revealed that compound B9 maintained exceptional conformational stability within the enzyme’s catalytic cleft, as evidenced by ligand root-mean-square deviation (RMSD) values converging to 1.5 Å after 20 ns and persisting within this narrow threshold throughout the simulation. This remarkable structural fidelity substantially exceeded the stability profile of the reference compound STP, which exhibited RMSD fluctuations up to 2.8 Å. Critically, compound B9 preserved its dual-residue binding mode: persistent hydrogen bonding interactions with ARG 98 anchored the ligand to the NAD+ cofactor binding site, while extensive hydrophobic contacts with residues VAL 30 and ALA 29 stabilized its occupation of the adjacent hydrophobic sub-pocket.
Complementary MM/GBSA free energy calculations (Table 4) quantified the thermodynamic drivers of this superior binding, yielding a net binding free energy (ΔGbind) of −58.45 kcal/mol for B9-a threefold enhancement over STP (−19.16 kcal/mol). Deconvolution of energy components demonstrated that van der Waals interactions (ΔEVDW: −72.55 kcal/mol) constituted the dominant stabilizing force, primarily mediated through optimal shape complementarity with the VAL 30/ALA 29 hydrophobic cavity. Electrostatic contributions (ΔEelec: −19.91 kcal/mol), largely attributable to the high-fidelity hydrogen bond with ARG 98, provided substantial additional stabilization despite partial compensation by polar solvation penalties (Figure 6). This energetically favorable binding profile directly correlated with exceptional functional performance of compound B9, including its >50% inhibition of LDH activity at 0.1 mM concentration in biochemical assays. The simultaneous occupation of both the NAD+-binding site and hydrophobic cavity explained the superior anticonvulsant efficacy of B9 in MES and PTZ models. Collectively, these computational and experimental results established the TMCA–piperazine scaffold as a privileged chemotype for LDH modulation, with the B9 binding paradigm informing future structure-based optimization strategies targeting residues 98, 30, and 29 to enhance hydrophobic packing and electrostatic complementarity.

2.2.10. In Silico ADMET Predictions

Given that compound B9 exhibited the highest potential for pharmacological effects, computational instruments (ADMET lab 3, VNN-ADMET, PreADMET, and PkCSM) were employed to forecast its pharmacokinetic characteristics. Based on the ADMET properties of compound B9 summarized in Table 5, compound B9 showed reduced solubility in water and compromised skin permeability, indicating difficulties in formulation for either parenteral or transdermal administration. Nonetheless, the observation of moderate Caco-2 permeability and significant absorption in the human intestines underscores its potential for oral bioavailability. This substance regularly acted as an inhibitor of P-gp, potentially affecting its distribution and the pattern of interactions between drugs. Research on its distribution indicated a minimal distribution volume (VDss) and successful penetration into the blood–brain barrier, despite the presence of contradictory computational data (PkCSM: log BB = −1.872). From a metabolic standpoint, compound B9 demonstrated an inhibition effect on CYP2C9 and CYP3A4, exhibited an unclear level of inhibition on CYP1A2 (with some agreement), and showed no suppression of CYP2C19 or CYP2D6. The minimal overall clearance suggested a possibility for extended half-life. From a toxicological standpoint, compound B9 showed no mutations (AMES negative) or skin sensitivity, yet it exhibited notable hERG inhibition, suggesting a cardiotoxicity risk that defies initial beliefs. Consequently, despite B9’s advantageous intestinal uptake and metabolic steadiness, its growth capabilities were offset by challenges in solubility, inconsistent evidence of CNS penetration, and significant hERG vulnerability. Furthermore, we studied the ADMET properties of compounds A14 and STP as well (details in Supplementary Materials). The results revealed that active compounds screened from this study showed worse pharmaceutical properties compared to STP, especially in cardiac toxicity and water solubility.

2.2.11. Structure–Activity Relationships (SAR)

In general, we acquired hopeful anticonvulsant derivatives after our chemical modification. The SAR study can be summarized as follows (Figure 7): (1) TMCA moiety from P. tenuifolia provides essential anticonvulsant activity; (2) a piperazine fragment provides drug-like properties inspired by cinepazide, with a difference in the linker fragment, i.e., the butyryl group gives more potential metabolic stability in vivo and the succinoyl group interacts better with potential targets, indistinctive in anticonvulsant tests; (3) both groups derived from A. tatarinowii and aryl-piperzine provide favorable anticonvulsant activity in initial MES tests, where aryl-piperzine derivatives exhibited better effects in the PTZ model. The fluoro-biphenyl group performed the best in the evaluations. Specifically, both the electron-withdrawing group (the para-fluoro group) and the electron-donating group (the ortho-methoxy group) promote anticonvulsant activity.

3. Discussion

In search of better anticonvulsant agents, a cluster of novel derivatives of TMCA piperazine amide derivatives were synthesized through nucleophilic addition and amide coupling reactions. Cinnamyl piperazine has been disclosed as a hopeful bioactive motif in our previous article [22]; herein, the piperazine moiety is introduced to the analogues inspired by marketed drugs cinepazide and cinnarizine. Moreover, structural modification with TMCA from P. tenuifolia [8] and constituents from A. tatarinowii for anti-epilepsy agent development complies with our consistent previous research strategy. In this study, all the derivatives were screened for their anticonvulsant activities in vivo, among which compounds A3, A9, A12, A14, B9, and B12 were found to exhibit significant anticonvulsive potential in the MES model; furthermore, using the scPTZ model, in silico docking and in vitro LDH inhibition, neurotoxicity of the mentioned compounds was investigated. The most promising candidate (B9) emerged and then EEG and silver staining were carried out to illustrate the anti-epilepsy property of compound B9. In addition, immunofluorescence staining and MD simulation were executed to prove the effects of B9 modulating LDH. MES, PTZ and EEG models have been widely used as a primary drug screening test for anticonvulsant efficacy [23]. The MES test has been the most important model for clinical drug activity against tonic–clonic seizures because of its feasibility in the laboratory. The PTZ model is the most useful chemoconvulsant in the predictivity of clinical efficacy for seizures of the petit mal type according to its block inhibition or enhanced excitation of neurons.
As for the potential target, LDH was reported to play an important role in electrically regulating astrocyte–neuron lactate shuttle, suggesting the potential to identify metabolic enzymes that control epilepsy. Inhibition of the enzyme LDH hyperpolarized neurons, which was reversed by the downstream metabolite pyruvate. STP, along with its structurally modified derivative isosafrole with a necessary methylenedioxy-substituted allyl benzene skeleton, was revealed as novel group of antiepileptic drugs because of its unique antiepileptic mechanism of inhibiting LDH. Our previous research [13] proved that TMCA-asaronol ester extracted from P. tenuifolia and A. tatarinowii showed similar antiepileptic activity to STP, which provided the rationality of drug design of this article. In this study, we prepared potential derivative B9 with superior activities to STP in both in vivo anti-epilepsy models and in vitro LDH inhibitory models. Above all, it could be concluded that the most promising compound (B9) is a worthwhile scaffold for further investigation in the development of anticonvulsant agents inspired by TCM.

4. Materials and Methods

4.1. Chemistry

The chemicals and reagents were purchased from several chemical companies such as Alfa-Aesar (Ward Hill, MA, USA) and Macklin (Shanghai, China). The progress of the reaction was monitored by thin layer chromatography analysis using silica gel G plates through a UV chamber at 256 nm for visualization of TLC spots. The mixtures were purified by flash column chromatography using silica gel. 1H NMR and 13C NMR spectra were obtained by a Bruker 400 NMR spectrometer (Bruker Corporation, Billerica, MA, USA) using CDCl3 as a solvent and TMS as an internal standard. The chemical shifts were expressed in ppm. Mass spectral ESI measurements were executed on SCIEX 5600 LC/MS instruments (SCIEX, Framingham, MA, USA).

General Procedure for the Synthesis of TMCA Peptide Derivatives

All titled compounds, including A1–17 and B1–17, were successfully prepared through the synthetic protocols presented in Scheme 1. Core scaffold TMCA (1.0 equiv) was activated with EDCI (1.3 equiv) and DMAP (0.15 equiv) in DCM at rt (Step a) [24] to trigger condensation reaction conditions with piperazine (1.2 equiv). The intermediate underwent O-alkylation with alkyl halides (1.5 equiv) using K2CO3 (2.0 equiv) and catalytic KI in DMF at 75 °C (Step b) [25]. Subsequent hydrolysis with NaOH (2.0 M, 3.0 equiv) in MeOH/H2O (1:1) at rt (Step c) yielded carboxylic acid intermediates. EDCI (1.5 equiv)/DMAP (0.2 equiv) activation in DCM (rt, Step d), followed by coupling with amines (1.2 equiv) under EDCI/DMAP conditions (Step e), afforded targets A2–A17/B2–B17. The obtained product was washed with sodium bicarbonate water solution and diluted hydrochloric acid. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness to give the crude product. The mixtures were purified by flash column chromatography using silica gel in the system DCM/MeOH from 80:1 to 30:1.

4.2. Biological Activity

4.2.1. Animals and Experimental Conditions

Male Kunming mice (23–28 g) were maintained under controlled conditions with ad libitum access to standard pellet diet and water. Test compounds were formulated immediately prior to administration in normal saline containing 0.5% Tween-80. Our experiment was authorized by the Institutional Animal Care and Use Committee of Shaanxi University of Chinese Medicine (animal ethics certificate number: SUCMDL20210309027).

4.2.2. Maximal Electroshock (MES) Test

The MES test carried out following the method of Swinyard [26]. A 60 Hz current (50 mA; 0.25 s duration) was delivered via corneal electrodes using a YSD-4G electro convulsion meter (Zhenghua Instruments, Huaibei, China, SN: ZH0056232). Stiripentol (STP, 100 mg/kg in 1% v/v Tween-80/0.9% NaCl) served as the positive control. Test compounds were suspended in an identical vehicle. Protection was defined as abolition of tonic hindlimb extension. Test groups received intraperitoneal (i.p.) injections of compounds at 100 mg/kg for preliminary screening, followed by 75, 50, 25, and 12.5 mg/kg for ED50 determination.

4.2.3. Pentylenetetrazole (PTZ)-Induced Seizures

Seizures were induced by subcutaneous (s.c.) administration of PTZ (85 mg/kg in 1% v/v Tween-80/0.9% NaCl), 30 min after compound treatment. This dose produces clonic seizures (≥5 s duration) in 97% of naive mice. Latency to seizure onset was recorded during continuous observation [27]. Compounds demonstrating complete protection at 100 mg/kg (i.p.) were evaluated for latent period prolongation.

4.2.4. Acute Neurotoxicity Screening

Neurotoxicity was assessed via a rotarod test [28]. Mice were trained to maintain balance on a rotating rod (diameter: 4 cm; 24 rpm). Trained animals received i.p. injections of test compounds or reference drugs. At 30 min post-administration, neurologic impairment was defined as failure to remain on the rod for 3 min (≥3 falls within the testing period). Data were used to calculate TD50 values.

4.2.5. Electroencephalogram (EEG)

Mice sedated with urethane were subjected to aseptic craniotomy, involving the stereotaxic insertion of cortical screw electrodes aligned with bregma coordinates (anterior–posterior: +3.0 mm; mediolateral: ±1.5 mm) and a reference electrode (anterior–posterior: −2.0 mm; mediolateral: −1.5 mm). The electrodes were secured using dental acrylic, followed by a recovery period of 5–7 days for the animals before the instrumentation. The continuous EEG data were captured through using 8-channel bioamplifiers linked to a PowerLab 8/30 system (AD Instruments, Shanghai, China). After a 30 min acclimatization period in the recording chambers, initial activity levels were documented for 15–20 min. The experimental substances (STP and B9, 100 mg/kg) were delivered intraperitoneally. After 60 min, pentylenetetrazol (PTZ, 85 mg/kg i.p.) was administered, initiating seizure. Sampling of EEG signals occurred at a frequency of 200 Hz, utilizing a bandpass filter ranging from 0.1 to 60 Hz [29]. Electrographic convulsions were characterized by spikes surpassing double the baseline amplitude, quantified by (1) the delay before the initial spike (seconds after PTZ); (2) the frequency of spikes (counts per 60 s period from the first spike) through established peak-detection methods.

4.2.6. Silver Staining

Following the creation of the PTZ model, a modified version of the silver impregnation staining technique was applied for silver staining, enabling the observation of changes in neuronal fibers and synapses throughout diseases [30]. Brain tissue slices from each mouse, encased in paraffin, underwent deparaffinization using xylene and absolute ethanol, followed by a treatment in acidic formaldehyde, and then incubation in a glycine–silver mixture. Following the application of a reducing solution for staining, the samples were analyzed using an optical microscope. Against a yellow backdrop, nerve cells’ axons, dendrites, collagen fibers, and neurofibrillary tangles were colored in dark brown or black.

4.2.7. Immunofluorescence Staining

Brain samples were extracted, preserved in 4% paraformaldehyde for a day, and subsequently immersed in a 30% sucrose solution to dehydrate. Post OCT embedding, slices of brain tissue measuring 16 μm in thickness were sliced using a cryostat. Following the permeabilization using 0.5% TritonX-100 and subsequent blocking with 10% goat serum at ambient temperature to remove nonspecific staining, the tissue samples were subjected to overnight incubation at 4 °C with the primary antibody, LDHA (Proteintech (Rosemont, IL, USA), Cat No. 19987-1-AP, Lot: 00106920, rabbit anti-rat 1:200). On the next day, the slides underwent incubation using a secondary antibody. The slides underwent counterstaining using DAPI. Ovarian slices were examined and visualized utilizing the VS200-BU Slide Scanner (Olympus Corporation, Tokyo, Japan).

4.2.8. Statistical Analysis

All data were presented as mean ± standard deviation. The results were analyzed by a one-way ANOVA, followed by Student’s two-tailed t-test for comparison between test and control, and Dunnett’s test when the data involved three or more groups. The level of significance for all tests was set at p < 0.05.

4.2.9. In Vitro LDH Assay

Employing the commercial LDH enzyme (Solarbio Life Science, Beijing, China, CAS:9001-60-9, Lot. Document No. 14240222001) includes a colorimetric assay kit designed to assess the synthesized substances’ capacity to suppress the LDH enzyme, using established techniques with slight alterations [31,32]. In summary, a 96-well microplate was filled with 30 μL of LDH enzyme (300 U/mL) and 70 μL of the inhibitor mixture (0.1 mM in final concentration). Following a 10 min pre-incubation process at ambient temperature, 110 μL of NADH (0.2 mM) was introduced. Subsequently, each well received 40 μL of phosphate buffer (20 mM, pH 6.8), reaching a total volume of 250 μL, followed by a 10 min incubation of the assay plate at 28 °C. Subsequently, dopachrome’s absorbance was gauged at 340 nm utilizing a SpectraMax 190 Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA). STP (0.1 mM) served as a benchmark inhibitor, while phosphate buffer functioned as the negative control. In the initial screening, the level of suppression by the test substances was denoted as a percentage (%). The calculation for the LDH inhibition ratio was as follows:
Inhibition (%) = [(Asample − Ablank)/Acontrol] × 100%
Asample denotes the OD340 absorbance of test compound and Ablank is the OD340 absorbance for the blank. Acontrol represents the OD340 absorbance of control.

4.2.10. Molecular Modeling

The crystal structure of lactate dehydrogenase (LDH; PDB: 4ZVV) was retrieved from the Protein Data Bank [21]. Flexible docking simulations were performed using AutoDock Vina 1.1.2 to analyze interactions between the LDH active site and seven compounds (STP, A3, A9, B9, A12, B12, A14) [33]. The previous preparatory work for the complex was completed by the Amdock tool 4.2.6 [34]. The docking grid center was defined by the original ligand GEN-140 (X: 41.554545, Y: 14.328606, Z: 25.699061) with a 15 Å search radius [35]. Key active site residues (ASN 137, ASP 165, ARG 168, HIS 192, GLY 193, ASP 194, ALA 237, TYR 238, ILE 241, GLY 245, THR 247)—known to interact with native ligand GEN-140—were set as rotatable to enable conformational sampling. To ensure the rationality of the docking results, we also performed the docking of the original ligand GEN-140 and compared the RMSD value of the conformation obtained from the docking with that of the original ligand conformation (results shown in Supplementary Materials). Binding affinity (kcal/mol) and intermolecular interactions (hydrogen/hydrophobic bonds) were quantified through Discovery Studio Visualizer.

4.2.11. Molecular Dynamic (MD) Simulation

The optimal docking pose of compounds B9 and STP was selected for molecular dynamics (MD) simulations using GROMACS 2022.3. The ligand–protein complex was solvated in a TIP3P water box, energy-minimized, and equilibrated in NVT/NPT ensembles prior to 100 ns production MD simulation (2 fs timestep, 300 K, NPT ensemble). Simulations employed the AMBER force field with conformations sampled at discrete time points to assess ligand binding stability via VMD visualization. Binding free energies were calculated by MM/PBSA analysis with energy decomposition in GROMACS [36,37].

4.2.12. In Silico ADMET Predictions

To evaluate the pharmacokinetic properties of the most promising compounds (B9, A14 and STP), in silico ADMET predictions were conducted using several reliable prediction servers, including Preadmet (http://preadmet.bmdrc.kr/ accessed on 16 June 2025), vNN-ADMET (https://vnnadmet.bhsai.org/ accessed on 16 June 2025), pKCSM (http://biosig.unimelb.edu.au/pkcsm/ accessed on 16 June 2025), and ADMETlab 3.0 (https://admetmesh.scbdd.com/ accessed on 16 June 2025). A consensus prediction approach was employed, where a property was considered valid if it was indicated by more than two servers [38]. This method ensures higher confidence in the predicted pharmacokinetic profiles of the compounds. In brief, the chemical structures of the compounds were prepared in SMILES format and uploaded to each server. ADMET properties, including absorption, distribution, metabolism, excretion, and toxicity, were selected across all tools. Key pharmacokinetic properties evaluated include Caco-2 permeability, skin permeability, blood–brain barrier (BBB) penetration, volume of distribution (VDss), cytochrome P450 (CYP) inhibition, AMES toxicity and hERG inhibition.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ph18091312/s1. HRMS and NMR spectra of derivatives A2–17 and B2–17.

Author Contributions

The individual contributions of the authors are as follows: Conceptualization, methodology, writing—original draft preparation, funding Acquisition, Z.Z.; Investigation, M.L. and Y.W.; Formal Analysis, Y.B.; Resources, Project Administration, H.Q. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (no. 8210142829).

Institutional Review Board Statement

Our experiment was authorized by the Institutional Animal Care and Use Committee of Shaanxi University of Chinese Medicine (animal ethics certificate number: SUCMDL20210309027, approved date: 2021.03.09).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We thank the Institute for Chinese Medicine Frontier Interdisciplinary Science and Technology, Shaanxi University of Chinese Medicine and Shaanxi Provincial Key Laboratory of Chinese Medicine for Neurological Disorders for providing us with the facilities to perform the experiments.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Design of TMCA piperazine amide derivatives as anticonvulsant agents inspired by TCM herb pairs P. tenuifolia and A. tatarinowii.
Figure 1. Design of TMCA piperazine amide derivatives as anticonvulsant agents inspired by TCM herb pairs P. tenuifolia and A. tatarinowii.
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Scheme 1. Synthesis of compounds A1–17 and B1–17.
Scheme 1. Synthesis of compounds A1–17 and B1–17.
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Figure 2. After pretreatment with STP and B9 to the respective groups (n = 4), the animals were injected with PTZ. Typical EEG traces recorded during the experiment: (A) Representative portion of baseline EEG before administration of the convulsant; 60 s of electrical activity recorded after appearance of first epileptic spike post-PTZ injection, showing EEG of (B) PTZ control (PTZ only), (C) standard control (PTZ + STP) and (D) treatment group (B9 + PTZ). All data are represented as mean ± SEM and ** indicates significance at p < 0.01. (E) Latency to the first epileptic spike and (F) the number of spikes in post-first spike 60 s were monitored with EEG.
Figure 2. After pretreatment with STP and B9 to the respective groups (n = 4), the animals were injected with PTZ. Typical EEG traces recorded during the experiment: (A) Representative portion of baseline EEG before administration of the convulsant; 60 s of electrical activity recorded after appearance of first epileptic spike post-PTZ injection, showing EEG of (B) PTZ control (PTZ only), (C) standard control (PTZ + STP) and (D) treatment group (B9 + PTZ). All data are represented as mean ± SEM and ** indicates significance at p < 0.01. (E) Latency to the first epileptic spike and (F) the number of spikes in post-first spike 60 s were monitored with EEG.
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Figure 3. Silver staining of hippocampal tissue CA1, CA3, DG and temporal lobe in the control group, PTZ-induced seizure mice and treatment groups with STP and B9 intervention (scale bar: 100 μm).
Figure 3. Silver staining of hippocampal tissue CA1, CA3, DG and temporal lobe in the control group, PTZ-induced seizure mice and treatment groups with STP and B9 intervention (scale bar: 100 μm).
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Figure 4. Immunofluorescence staining of hippocampal tissue CA1 and CA3 areas in the control group, PTZ-induced seizure mice and treatment groups intervened with STP and B9 (scale bar: 50 μm). (A) Representative LDH immunofluorescence staining images and (B) quantification of fluorescence density of LDH in each group. All data are represented as mean ± SEM and ** indicates significance at p < 0.01.
Figure 4. Immunofluorescence staining of hippocampal tissue CA1 and CA3 areas in the control group, PTZ-induced seizure mice and treatment groups intervened with STP and B9 (scale bar: 50 μm). (A) Representative LDH immunofluorescence staining images and (B) quantification of fluorescence density of LDH in each group. All data are represented as mean ± SEM and ** indicates significance at p < 0.01.
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Figure 5. Docking mode of compounds A3, A9, A12, A14, B9, and B12 at LDH (PDB cod: 4ZVV). The left columns are 3D interaction patterns, and the right columns are 2D interaction patterns in the binding pocket. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein, and the important H-bonds.
Figure 5. Docking mode of compounds A3, A9, A12, A14, B9, and B12 at LDH (PDB cod: 4ZVV). The left columns are 3D interaction patterns, and the right columns are 2D interaction patterns in the binding pocket. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein, and the important H-bonds.
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Figure 6. The 100 ns MD simulation results for STP and B9: (A) gyrate; (B) H-bond number; (C) SASA; (D) RMSD plot for the backbone atoms; (E) RMSF; (F) MM/GBSA free energy calculations for STP; (G) MM/GBSA free energy calculations for B9.
Figure 6. The 100 ns MD simulation results for STP and B9: (A) gyrate; (B) H-bond number; (C) SASA; (D) RMSD plot for the backbone atoms; (E) RMSF; (F) MM/GBSA free energy calculations for STP; (G) MM/GBSA free energy calculations for B9.
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Figure 7. Preliminary SAR analysis of TMCA piperazine amide derivatives.
Figure 7. Preliminary SAR analysis of TMCA piperazine amide derivatives.
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Table 1. Anticonvulsant activity in the MES model (100 mg/kg) of compounds administered i.p. to mice.
Table 1. Anticonvulsant activity in the MES model (100 mg/kg) of compounds administered i.p. to mice.
Com.0.5 h1 h2 h3 hCom.0.5 h1 h2 h3 h
Ctrl0/4 a0/40/40/4
STP4/42/41/40/4
A11/41/40/40/4B11/41/40/40/4
A22/42/41/40/4B23/43/41/40/4
A34/43/42/41/4B33/43/41/40/4
A42/42/41/40/4B43/42/42/40/4
A53/43/42/40/4B53/43/42/40/4
A62/42/41/40/4B62/41/41/40/4
A73/42/41/40/4B73/42/41/40/4
A83/42/41/40/4B83/43/41/40/4
A94/43/42/40/4B94/43/41/40/4
A103/41/41/40/4B103/43/41/40/4
A111/41/41/40/4B112/42/41/40/4
A124/43/43/41/4B124/43/41/41/4
A131/41/40/40/4B132/41/40/40/4
A144/44/43/41/4B143/43/42/40/4
A153/43/41/40/4B153/43/41/40/4
A163/43/42/40/4B163/42/42/40/4
A172/42/41/40/4B172/42/41/40/4
Notes: a protected mice/total mice.
Table 2. Further screening for some active compounds.
Table 2. Further screening for some active compounds.
Com.MES ED50SCPTZTD50PI a
(MES)
Protective Ratio (100 mg/kg)Latent Time (100 mg/kg, s)
NS-0/4150.8 ± 16.3--
STP b277 (1182)2/4269.5 ± 19.8 **161.7 (146.2–256.2)1.41
A373.31 (41.96–92.14)1/4222.0 ± 14.7 **443.42 (327.60–626.48)6.05
A937.46 (16.51–78.97)1/4204.5 ± 12.6 **304.79 (188.99–474.43)8.14
B923.58 (10.33–71.72)3/4274.3 ± 17.2 **235.04 (107.18–373.21)9.97
A1254.71 (29.90–85.28)0/4161.5 ± 24.0284.38 (173.91–448.98)5.20
B1260.27 (38.71–84.89)1/4230.3 ± 17.4 **417.16 (293.63–614.75)6.92
A1452.39 (28.48–83.43)2/4233.5 ± 18.3 **358.26 (231.95–528.49)6.84
Notes: a PI: protective index (TD50/ED50); b reference drug, data from [13]; **: p < 0.01 vs. control; 95% confidence intervals given in parentheses.
Table 3. Molecular docking score and LDH inhibitory evaluation for active compounds in vitro.
Table 3. Molecular docking score and LDH inhibitory evaluation for active compounds in vitro.
Com.Binding Affinity (Kcal/mol)Key InteractionsLDH Inhibition Ratio (0.1 mM)
STP−6.5ARG 168 (H-bond), TYR 238 (Pi-Sigma), GLY 193 (Amide-Pi Stacked), VAL 234, VAL 237 (Pi-Alkyl), VAL 233, HIS 192 (Carbon Hydrogen Bond)46.1 ± 6.2%
A3−6.3ASN 137 (H-bond), TYR 238 (Pi-Pi T-shaped), SER 136, VAL 135, THR 94 (Carbon Hydrogen Bond)37.6 ± 8.1%
A9−8.5ASN 137 (H-bond), VAL 90, ILE241, ILE 251 (Pi-Alkyl), GLY96, ALA 97, GLN 99 (Halogen), ARG 98, VAL 135, HIS 192 (Carbon Hydrogen Bond)52.6 ± 8.5%
B9−9.3VAL 30, ALA 29, ARG 98 (H-bond), TYR 238 (Pi-Sigma), VAL 233 (Halogen), GLY 193 (Amide-Pi Stacked), THR 247, GLY 28, VAL 234 (Carbon Hydrogen Bond)54.9 ± 9.7%
A12−7.6GLN 99 (H-bond), ILE 241, ILE 251, VAL 30 (Alkyl)41.9 ± 9.2%
B12−7.7ARG 98 (H-bond), ALA 237, VAL 30, ILE 251 (Alkyl)44.3 ± 6.5%
A14−6.9ASN 137 (H-bond), VAL 30, ILE 251, ILE 241 (Alkyl), SER 136, HIS 192, GLY 96, GLY 28 (Carbon Hydrogen Bond)42.6 ± 7.3%
Table 4. MM/GBSA free energy calculations for STP and B9.
Table 4. MM/GBSA free energy calculations for STP and B9.
Contribution ComponentsLDH-STPLDH-B9
ΔVDWAALS−29.93 ± 0.04−72.55 ± 1.86
ΔEelec−4.87 ± 0.01−19.91 ± 0.77
ΔEGB19.63 ± 0.1443.11 ± 1.25
ΔEsurf−3.99 ± 0.01−9.09 ± 0.19
ΔGgas−34.81 ± 0.04−92.47 ± 2.02
ΔGsolvation15.65 ± 0.1434.02 ± 1.26
ΔTotal−19.16 ± 0.15−58.45 ± 2.38
Table 5. ADMET properties of compound B9.
Table 5. ADMET properties of compound B9.
Category Property ADMET Lab 3VNN-ADMET PreADMETPkCSM Consensus
AbsorptionWater solubility -389.712 mg/L−4.246 log mol/LLow
Caco-2 permeability−5.454 log unit-52.768 nm/s0.856 log Papp in 10−6 cm/sModerate
Human intestinal absorption (%)--97.68365.214 High
Skin permeability (log Kp)--−2.563−2.735Low
P-gp inhibitorYesNoYesYesYes
DistributionVDss (human, log L/kg)---0.674Low
BBB permeability (log BB)Yes-Yes−1.872Yes
MetabolismCYP1A2 inhibitorNoYes-NoPartial
CYP2C9 inhibitorYesNoYesYesYes
CYP2C19 inhibitorYesNoNoNoNo
CYP2D6 inhibitorYesNoNoNoNo
CYP3A4 inhibitorYesNoYesYesYes
ExcretionTotal clearance (log ml/min/kg)---0.367Low
ToxicityAMES toxicityNo-NoNoNo
hERG blockerYesYesMedium riskNoYes
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Zhao, Z.; Lei, M.; Wang, Y.; Bai, Y.; Qiao, H. Excavating Precursors from Herb Pairs Polygala tenuifolia and Acori tatarinowii: Synthesis and Anticonvulsant Activity Evaluation of 3,4,5-Trimethoxycinnamic Acid (TMCA) Piperazine Amide Derivatives. Pharmaceuticals 2025, 18, 1312. https://doi.org/10.3390/ph18091312

AMA Style

Zhao Z, Lei M, Wang Y, Bai Y, Qiao H. Excavating Precursors from Herb Pairs Polygala tenuifolia and Acori tatarinowii: Synthesis and Anticonvulsant Activity Evaluation of 3,4,5-Trimethoxycinnamic Acid (TMCA) Piperazine Amide Derivatives. Pharmaceuticals. 2025; 18(9):1312. https://doi.org/10.3390/ph18091312

Chicago/Turabian Style

Zhao, Zefeng, Mengchen Lei, Yongqi Wang, Yujun Bai, and Haifa Qiao. 2025. "Excavating Precursors from Herb Pairs Polygala tenuifolia and Acori tatarinowii: Synthesis and Anticonvulsant Activity Evaluation of 3,4,5-Trimethoxycinnamic Acid (TMCA) Piperazine Amide Derivatives" Pharmaceuticals 18, no. 9: 1312. https://doi.org/10.3390/ph18091312

APA Style

Zhao, Z., Lei, M., Wang, Y., Bai, Y., & Qiao, H. (2025). Excavating Precursors from Herb Pairs Polygala tenuifolia and Acori tatarinowii: Synthesis and Anticonvulsant Activity Evaluation of 3,4,5-Trimethoxycinnamic Acid (TMCA) Piperazine Amide Derivatives. Pharmaceuticals, 18(9), 1312. https://doi.org/10.3390/ph18091312

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