Semi-Synthesis and Biological Evaluation of Novel Sinomenine Derivatives

Abstract

Sinomenine has long been known as an anti-inflammatory drug, while poor efficiency and large-dose treatment had limited its further application. Five novel sinomenine 1-Br-4-cinnamic acid esters derivatives 2a–2e were designed and synthesized to improve its analgesic and anti-inflammatory activity. All synthesized sinomenine derivatives were structurally confirmed by NMR and ESI-MS. Molecular docking results showed that compounds 2a–2e had stable binding to the GBP5 protein. The compounds 2a–2e showed stable binding to the GBP5 protein by molecular docking Pre-preparing the druggability of compounds 2a–2e by ADEMT 3.0 showed that each derivative had similar druggability to sinomenine. The analgesic activity of compounds 2a–2e was preliminarily determined by hot plate and acetic acid writhing experiments, while anti-neuroinflammatory effects were evaluated by a xylene-induced mouse ear edema model. The results of the hot plate method showed that the synthesized sinomenine derivatives 2a–2e had some analgesic effects. The results of the acetic acid writhing test showed that the analgesic effects of 2a, 2c, 2e were better than that of sinomenine, and the other derivatives were equivalent to sinomenine. Compound 2b showed excellent anti-inflammatory properties in mouse ear edema.

1. Introduction

Sinomenine (SIN) is a bioactive isoquinoline alkaloid extracted from the medicinal plant Sinomenium acutum [1], which has a wide range of pharmacodynamic properties, including anti-inflammatory, antioxidative, antisynovitis, antineoplastic, immunosuppressive, antihypertensive, and antiarrhythmic effects, and exerts therapeutic effects on rheumatoid arthritis in clinical application [2,3,4,5]. Therefore, SIN was regarded as a lead compound in drug research, especially for anti-inflammatory medicines. However, some side effects, for example, hydrophilic–lipophilic balance (HLB), large dose allergies, and the induction of hypersusceptibility [6,7,8,9,10], prevent it from becoming a drug directly. In order to obtain sinomenine analogs with better therapeutic effects and fewer side effects, structural modifications of sinomenine have attracted significant research to improve its further applications [11,12,13].

Structurally, SIN is an isoquinoline alkaloid with a tetracyclic structure derived from the morphine backbone, featuring fused rings that include an isoquinoline core and structural characteristics typical of opioid alkaloids. It contains a number of functional groups, methoxyl, such as aryl, double bond, carbonyl, phenolic hydroxy, and N-methyl. Thus, it is very suitable for modification research [6]. Furthermore, SIN possesses a well-designed substitution pattern on ring A. The free sinomenine phenolic hydroxyl on ring A is readily oxidized by air and may be the main cause of hypersusceptibility [14]. To improve activity, the structural modification of sinomenine has attracted enormous interest in recent years [15,16].

The typical symptoms that can be induced by inflammatory stimuli mainly include fever, pain, redness, and swelling [17]. Despite the existence of studies on the relieving effect of sinomenine derivatives, the potential anti-inflammation effects of sinomenine derivatives in xylene-induced ear edema still remain poorly investigated and understood [15]. Xylene can promote the generation and transmittance of TNF-α, IL-1β, and inflammatory mediator PGE2, leading to acute exudative inflammatory edema in the mouse ear, with a significant increase in the rate of change in ear weight and ear thickness. To establish a xylene-induced mouse ear edema, an in vivo inflammatory model is beneficial to a more comprehensive study of the inhibitory mechanism of sinomenine derivatives on inflammation.

In this article, a series of sinomenine 1-Br-4-hydroxy esters with cinnamic acids are designed based on the prodrug principle and the synergistic effect for some cinnamic acids and their derivatives possessing good analgesic and anti-inflammatory activity [18]. Furthermore, the analgesic effects of the sinomenine derivatives were investigated by the hot plate method and the acetic acid-induced writhing test, while a mouse ear edema model induced by xylene was established to investigate the anti-inflammatory impact of various sinomenine 1-Br-4-cinnamic acid ester derivatives at certain dosages. These esters were expected to possess more efficient analgesic and anti-inflammatory activity effects with better bioavailability and HLB [19].

2. Results and Discussion

2.1. Chemical Synthesis

Compounds 2a–2e were prepared following the procedure presented in Scheme 1. SIN reacted with N-bromosuccinimide (NBS) in dry DCM to yield compounds 1. As the H-1 of SIN was easily halogenated by NBS, the SIN-1-Br was prepared. Then, it reacted with cinnamic acid in the presence of EDC and DMAP to yield compounds 2. NMR analysis confirmed the successful synthesis of compounds 2a–2e.

1-Br-4-cinnamic acid ester-sinomenine 2a: white solid, 80% yield; m.p. 35–38 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.02 (d, J = 16.3 Hz, 1H), 7.65 (d, J = 3.4 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.45–7.39 (m, 2H), 7.07 (s, 1H), 6.68 (d, J = 16.1 Hz, 1H), 5.46 (s, 1H), 3.92 (d, J = 15.1 Hz, 1H), 3.72 (s, 3H), 3.50 (s, 3H), 3.24 (t, J = 4.6 Hz, 1H), 3.06 (d, J = 18.8 Hz, 1H), 2.99 (s, 1H), 2.50 (dd, J = 17.8, 6.1 Hz, 3H), 2.43 (s, 3H), 2.10 (t, J = 12.4 Hz, 1H), 1.86 (td, J = 12.6, 4.4 Hz, 1H), 1.62 (d, J = 10.3 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 192.47, 152.72, 150.44, 148.12, 138.95, 134.22, 132.45, 130.97, 129.86, 129.37, 129.07, 128.71, 115.29, 114.74, 77.48, 77.16, 76.84, 56.28, 56.25, 55.06, 50.41, 46.46, 46.14, 42.91, 41.50, 37.13, 26.24; MS (ESI) m/z C₂₈H₂₉BrNO₅⁺: [M + H]⁺_calcd =538.12236, and [M + H]⁺_measured =538.12183.

1-Br-4-(3-methyl)-cinnamic acid ester-sinomenine 2b: white solid, 83% yield; m.p. 35–38 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.18 (d, J = 16.0 Hz, 1H), 7.69–7.60 (m, 2H), 7.47 (dt, J = 25.4, 7.6 Hz, 2H), 6.86 (d, J = 16.0 Hz, 1H), 5.65 (d, J = 2.1 Hz, 1H), 4.12 (d, J = 15.8 Hz, 1H), 3.91 (s, 3H), 3.69 (s, 3H), 3.43 (t, J = 4.7 Hz, 1H), 3.30–3.15 (m, 2H), 2.75–2.66 (m, 3H), 2.60 (d, J = 17.4 Hz, 6H), 2.29 (t, J = 11.7 Hz, 1H), 2.04 (td, J = 12.5, 4.5 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 192.50, 164.60, 152.70, 150.45, 148.33, 138.97, 138.76, 134.14, 132.43, 131.81, 129.34, 129.01, 128.94, 125.88, 121.16, 116.37, 115.28, 114.74, 77.48, 77.16, 76.84, 56.27, 55.05, 50.38, 46.46, 46.12, 42.90, 41.48, 37.10, 26.22, 21.41; MS (ESI) m/z C₂₉H₃₁BrNO₅⁺: [M + H]⁺_calcd = 552.13801, and [M + H]⁺_measured = 552.13721.

1-Br-4-(3-methoxy)-cinnamic acid ester-sinomenine 2c: white solid, 81% yield; m.p. 35–38 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.97 (dd, J = 16.2, 5.4 Hz, 1H), 7.63 (s, 1H), 7.52 (d, J = 7.3 Hz, 1H), 7.41–7.30 (m, 2H), 7.07 (s, 1H), 6.67 (d, J = 16.1 Hz, 1H), 5.46 (d, J = 2.1 Hz, 1H), 3.86 (s, 1H), 3.72 (s, 3H), 3.50 (s, 3H), 3.24 (t, J = 4.6 Hz, 1H), 3.09–2.96 (m, 2H), 2.51 (dd, J = 14.8, 4.5 Hz, 3H), 2.43 (s, 3H), 2.10 (t, J = 12.2 Hz, 1H), 1.87 (dt, J = 15.2, 7.5 Hz, 1H), 1.61 (s, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 192.47, 164.07, 152.76, 150.35, 138.83, 136.05, 135.14, 132.42, 130.78, 130.31, 130.07, 129.08, 128.45, 126.78, 115.33, 114.77, 77.48, 77.16, 76.84, 56.29, 56.26, 55.53, 55.07, 50.51, 50.36, 46.48, 46.42, 46.15, 42.91, 41.54, 37.16, and 26.25; MS (ESI) m/z C₂₉H₃₁BrNO₆⁺: [M + H]⁺_calcd = 568.13292, and [M + H]⁺_measured = 568.13202.

1-Br-4-(3-Cl)-cinnamic acid ester-sinomenine 2d: white solid, 77% yield; m.p. 35–38 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 16.1 Hz, 1H), 7.63 (s, 1H), 7.52 (d, J = 7.1 Hz, 1H), 7.41–7.34 (m, 2H), 7.07 (s, 1H), 6.67 (d, J = 16.0 Hz, 1H), 5.46 (d, J = 2.1 Hz, 1H), 3.88 (d, J = 15.8 Hz, 1H), 3.72 (s, 3H), 3.50 (s, 3H), 3.24 (t, J = 4.6 Hz, 1H), 3.09–2.96 (m, 2H), 2.55–2.51 (m, 1H), 2.51–2.46 (m, 2H), 2.43 (s, 3H), 2.06 (s, 1H), 1.86 (td, J = 12.6, 4.6 Hz, 1H), 1.59 (d, J = 12.7 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 192.50, 152.72, 150.43, 148.09, 146.50, 135.55, 135.12, 130.79, 130.32, 130.07, 128.45, 126.78, 121.55, 118.13, 117.36, 116.87, 115.29, 114.76, 77.48, 77.16, 76.84, 56.28, 55.53, 55.07, 50.50, 50.36, 46.47, 46.11, 42.92, 41.49, 37.11, 26.23; MS (ESI) m/z C₂₈H₂₈BrClNO₅⁺: [M + H]⁺_calcd = 572.08338, and [M + H]⁺_measured = 572.08240.

1-Br-4-(3-nitro)-cinnamic acid ester-sinomenine 2e: yellow solid, 79% yield; m.p. 35–38 °C, ¹H NMR (400 MHz, CDCl₃) δ 8.49 (t, J = 1.9 Hz, 1H), 8.27 (dd, J = 8.2, 2.1 Hz, 1H), 8.08 (d, J = 16.0 Hz, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.08 (s, 1H), 6.79 (d, J = 16.0 Hz, 1H), 5.48 (d, J = 2.1 Hz, 1H), 3.86 (d, J = 15.6 Hz, 1H), 3.73 (s, 3H), 3.51 (s, 3H), 3.25 (t, J = 4.7 Hz, 1H), 3.13–2.93 (m, 2H), 2.57–2.46 (m, 3H), 2.44 (s, 3H), 2.10 (t, J = 12.2 Hz, 1H), 1.88 (td, J = 12.6, 4.6 Hz, 1H), 1.61 (s, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 192.53, 163.62, 152.77, 150.24, 148.84, 145.14, 138.70, 136.01, 134.24, 132.37, 130.16, 129.15, 125.11, 122.98, 121.42, 119.92, 115.37, 114.87, 77.48, 77.16, 76.84, 56.29, 56.22, 55.09, 50.59, 46.37, 46.15, 42.90, 41.58, 37.19, 26.25; MS (ESI) m/z C₂₈H₂₈BrN₂O₇⁺: [M + H]⁺_calcd = 583.10744, and [M + H]⁺_measured = 583.10669.

2.2. Molecular Docking Test

The results of molecular docking are shown in

Table 1. Molecular docking score of sinomenine and sinomenine derivatives.

Model	Binding Energy (kcal/mol)
SIN	−6.65
2a	−7.61
2b	−7.17
2c	−9.31
2d	−7.54
2e	−8.57

. Using MOE software, the binding energy of the GBP5 protein to sinomenine was −6.6513 kcal/mol. Five derivatives were docked with GBP5, and their binding energy was less than that of SIN.

GBP5 is a potential target for sinomenine in inflammatory cells. Sinomenine directly binds to GBP5, inhibits its activity, regulates P2X7R, and inhibits downstream NLRP3-induced inflammatory pathways. Molecular docking showed that sinomenine could bind to the active pocket of GBP5 with a strong binding energy of −7.72 kcal/mol [20,21]. The 2a–2e were docked with GBP5 protein (PDB no. 7ckf) by the molecular docking software MOE, with the binding site of GDP set as the docking pocket and one strand of the protein retained for docking. The docking results showed that 2c has the lowest binding score, indicating that the energy required for binding to the protein is low and it is easier to bind. The highest docking score of 2a is −7.61 kcal/mol, which is also relatively stable compared to the −6.65 kcal/mol of SIN. Meanwhile, 2a–2e have a more stable binding with GBP5.

The binding mode between the molecule and the target protein was analyzed using the MOE software. The molecular docking diagram of SIN is shown in Figure 1.

2.3. Druggability Analysis

ADMET prediction refers to the prediction of a series of key characteristics of drugs in vivo. The predicted LogP of SIN was 0.67, the F50% was between 0.7 and 0.9, the plasma protein binding was 39.2%, the blood–brain barrier was between 0.1 and 0.3, The HLM stability was between 0.5 and 0.7, the T_1/2 was 3.604, the HH was 0.583 and the DIN was 0.255. Compared with SIN, the fat solubility increased, and the logP of each derivative increased. F50% decreased by 0.2–0.8. The plasma protein binding increased by 39.2–58.4% compared to SIN, of which 2a, 2b, 2c, 2d, 2e were 96%, 96.5%, 95.7%, 97.3%, 97.6%, respectively, and the plasma protein binding rate was relatively high. The blood–brain barrier of 2a, 2b, 2d increased by 0.4–0.9 compared with SIN, demonstrating that the permeability of the blood–brain barrier was relatively high. The HLM stability was 0.2–0.7 lower than that of SIN, and the probability of each derivative being metabolized in the liver was more stable than that of SIN. The T_1/2 of 2a–2e decreased by 1.72–2.29 compared to SIN, which was shorter half-life in the body. The HH of 2a–2e was 0.04–0.10 higher than that of SIN, indicating that the derivatives had certain hepatotoxicity, unlike SIN. The DIN of 2a–2e was 0.088–0.346 higher than that of SIN, indicating that the SIN derivatives had certain nephrotoxicity, unlike SIN. The results of druggability analysis are shown in

Table 2. Analysis of druggability of SIN and SIN derivatives.

Drug	logP	F50%	PPB	BBB	HLM Stability	T1/2	HH	DIN
SIN	0.67	0.7–0.9	39.2%	0.1–0.3	0.5–0.7	3.60	0.583	0.255
2a	3.75	0.3–0.5	96.0%	0.7–0.9	0–0.1	1.88	0.675	0.425
2b	4.01	0.3–0.5	96.5%	0.7–0.9	0.1–0.3	1.52	0.685	0.448
2c	3.78	0.5–0.7	95.7%	0.1–0.3	0–0.1	1.70	0.621	0.524
2d	4.06	0.3–0.5	97.3%	0.9–1.0	0–0.1	1.31	0.685	0.621
2e	3.57	0.1–0.3	97.6%	0.1–0.3	0–0.1	1.57	0.676	0.343

.

2.4. Hot Plate Method

The pain threshold before high-dose administration of 2a was 7.59 ± 1.67, and it was 9.92 ± 2.66 at 15 min. The pain threshold before high-dose administration of 2b was 7.2 ± 2.19, and it was 10.30 ± 2.51 at 15 min and 9.67 ± 3.19 at 30 min. The pain threshold before the high-dose administration of 2c was 8.72 ± 1.86, and it was 9.92 ± 2.66 at 30 min and 10.29 ± 1.57 at 60 min. The pain threshold before the high-dose administration of 2d was 8.30 ± 2.71, and it was 10.88 ± 3.10 at 15 min. The pain threshold before the high-dose administration of 2e was 8.36 ± 1.30, and it was 11.17 ± 2.53 at 15 min. The hot plate results show that the pain threshold of 2a–2e was significantly increased compared to that before administration and compared to the saline group, indicating that it may have a significant analgesic effect. The results of the hot plate method for SIN and SIN derivatives are shown in

Table 3. Results of the hot plate method for SIN and SIN derivatives.

Compounds	Dose (mg/kg)	Pain Threshold Before Administration $\stackrel{-}{\mathrm{X}}$ ± s (s)	Pain Threshold at Different Times After Administration (s)
			15 min	30 min	60 min
SIN	15	9.91 ± 2.62	10.39 ± 3.26 ▲	11.63 ± 2.36 *▲	10.72 ± 3.07
2aC1	30	7.59 ± 1.67	9.92 ± 2.66 *▲	9.26 ± 3.19 Δ	8.85 ± 2.08 *
2aC2	15	8.34 ± 3.14	8.33 ± 1.87	6.88 ± 3.17 *Δ	8.15 ± 2.49 Δ
2aC3	7.5	7.62 ± 2.35	8.74 ± 3.46	9.96 ± 2.79 ▲	8.42 ± 3.26
2bC1	30	7.25 ± 2.19	10.30 ± 2.51 *▲	9.67 ± 3.19 *▲	7.66 ± 2.95 Δ
2bC2	15	6.36 ± 1.24	7.63 ± 1.43 *Δ	6.57 ± 1.61 Δ	7.18 ± 2.25 Δ
2bC3	7.5	6.10 ± 1.04	7.52 ± 2.51 *Δ	7.92 ± 2.19 *Δ	6.97 ± 2.20 Δ
2cC1	30	8.72 ± 1.86	8.05 ± 2.27	9.85 ± 1.20 *▲Δ	10.29 ± 1.57 *▲
2cC2	15	8.40 ± 2.44	7.85 ± 1.85 Δ	7.63 ± 1.22 Δ	7.39 ± 0.68 Δ
2cC3	7.5	7.72 ± 1.30	7.77 ± 1.52 Δ	8.76 ± 1.62 Δ	6.71 ± 2.24 Δ
2dC1	30	8.30 ± 2.71	10.88 ± 3.10 *▲	8.17 ± 3.60 Δ	10.07 ± 3.57 *
2dC2	15	6.40 ± 2.76	7.67 ± 3.02 *	7.57 ± 2.00 Δ	9.21 ± 2.75 *
2dC3	7.5	8.20 ± 2.05	9.31 ± 2.58	8.36 ± 2.56 Δ	8.72 ± 1.79
2eC1	30	8.36 ± 1.30	11.17 ± 2.53 *▲	8.26 ± 2.12 Δ	10.96 ± 3.48 ▲
2eC2	15	7.50 ± 2.80	9.30 ± 2.52	9.19 ± 4.32	10.26 ± 3.63 *
2eC3	7.5	6.56 ± 0.99	7.43 ± 2.09 Δ	7.65 ± 2.56 Δ	8.17 ± 2.48 Δ
saline		7.70 ± 1.56	7.53 ± 1.59	7.07 ± 1.65	8.26 ± 1.79

*, p < 0.05 compared with the pain threshold before administration; ▲, p < 0.05 compared with saline; Δ, p < 0.05 compared with the sinomenine group. Results are expressed as means ± SD (n = 10 per group).

.

2.5. Acetic Acid-Induced Writhing Test

The results of the acetic acid-induced writhing test are shown in

Table 4. Results of acetic acid-induced writhing test for sinomenine and sinomenine derivatives.

Compounds	Dose (mg/kg)	The Prolonged Rate of Latency (%)	Writhing Times Inhibition Rate (%)	Percentage of Analgesia (%)
SIN	15	39.10	29.47	0
2aC1	30	31.33	43.72	0
2aC2	15	32.25	36.59	0
2aC3	7.5	24.99	46.85	0
2bC1	30	35.50	33.07	0
2bC2	15	33.05	25.12	0
2bC3	7.5	17.64	18.36	0
2cC1	30	11.44	57.13	0
2cC2	15	27.77	29.95	0
2cC3	7.5	38.31	28.14	0
2dC1	30	11.56	35.27	0
2dC2	15	23.08	39.61	0
2dC3	7.5	28.51	37.20	0
2eC1	30	55.56	57.00	0
2eC2	15	20.85	45.65	0
2eC3	7.5	19.79	24.64	0
saline		18.10		0

. At the dose of 30 mg/kg, the writhing latency at each dose of 2a was longer than the saline group and the inhibition rates of 2a showed advantages compared to the SIN group. The inhibition rate of the writhing times of 2c was higher in the high-dose group compared to the SIN group. The writhing latency of the high-dose 2e was longer than the saline group and the SIN group. The inhibition rate of the medium and high dose 2e was higher compared to the SIN group. The reason why the latency of writhing, the inhibition rate of writhing times, and the percentage of analgesia in each dose of the same derivative were significantly different may be related to the individual differences in mice.

2.6. Evaluation of Anti-Inflammatory Capacity in Mouse Ear Edema Model

In

Table 5. Results of the mouse ear swelling experiment.

Compounds	Dose (mg/kg)	Auricular Swelling Degree (X ± s)	Swelling Inhibition Rate (%)
SIN	30	0.0043 ± 0.0016 ▲	32.26
2a	30	0.0049 ± 0.0018	20.98
2b	30	0.0040 ± 0.0016 ▲	35.48
2c	30	0.0047 ± 0.0026	24.19
2d	30	0.0061 ± 0.0028	1.61
2e	30	0.0044 ± 0.0017 ▲	29.03
saline		0.0062 ± 0.0024 Δ

▲, Compared with the saline group, p < 0.05; Δ, compared with the SIN group, p < 0.05.

, the auricular swelling degree of 2b was 0.0040 ± 0.0016 after stimulating the xylene, which was different from the saline group. The swelling inhibition rate of the mouse given 2b was 35.48%. It was shown that 2b could alleviate the inflammatory reaction caused by xylene. However, 2a, 2c, 2d, 2e had no significant difference from the saline group and SIN group.

3. Materials and Methods

3.1. Materials and Reagents

Sinomenine, pure 98%, Lot No. C15533222, Shanghai McLean Biochemical Technology Co. Ltd., Shanghai, China; p-Bromobenzoic acid, AR, Lot No.Lf1025177438, Shanghai Haohong Bio-medical Technology Co. Ltd., Shanghai, China; Dimethoxypyridine, AR, Lot No. RH626903, Shanghai Een Chemical Technology Co. Ltd., Shanghai, China; all other reagents were obtained from Shanghai Titan Technology Co, Shanghai, China, AR. SPF grade Kunming mice [SCXK (Xiang) 2019-0011], female, body mass (20 ± 4 g), Hunan Slike Jingda Laboratory Animal Co., Ltd., Changsha, China.

3.2. Synthesis and Chemical Characterization

SIN (6.0 mmol, 2 g) was dissolved in 20 mL of CH₂Cl₂, and N-bromosuccinimide (NBS) (6.66 mmol, 1.2 g) was dissolved in CH₂Cl₂ and slowly added to sinomenine. After stirring at room temperature for 12 h, saturated salt water was added. The water layer was extracted with CH₂Cl₂ (20 mL × 3), dried with anhydrous sodium sulfate, and the organic layer was merged, which was concentrated in vacuum. The residuals were purified by silica gel column of CH₂Cl₂ and CH₃OH (30:1) to obtain 1-Br-SIN1 [22].

Subsequently, 1 (0. 6 g, 1.47 mmol), cinnamic acid (0.22 g, 2.21 mmol), EDC (0.46 g, 2.94 mmol), DMAP (0.27 g, 2.21 mmol), and 10 mL CH₂Cl₂ were stirred at room temperature for 10 h, and saturated salt water (3 × 10 mL) was added for extraction [23]. The organic layer was dried over by Na₂SO₄ and concentrated in vacuum. The residuals were purified by silica gel column of CH₂Cl₂ and CH₃OH (50:1) to yield 1-Br-4-hydroxy ester derivatives 2a–2e; the yields were above 77% (Scheme 1). All structures of these compounds were confirmed by ESI-MS, ¹H NMR, and ¹³C NMR.

¹H and ¹³C-NMR spectra were run on a Bruker Avance 400 MHz NMR spectrometer using tetramethylsilane (TMS) as the internal standard and CDCl₃ as the solvent (Chemical shifts in δ, ppm). Splitting patterns were designated as follows: s—singlet; d—doublet; m—multiplet. The mass spectra were performed at the Q-Exactive Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray ionization source.

3.3. Molecular Docking Test

Molecular docking studies were conducted to predict the binding affinity and interaction modes of sinomenine and its derivatives (2a–2e) with the guanylate-binding protein 5 (GBP5), a reported inflammatory regulator (PDB ID: 7CKF). Docking was performed using a Molecular Operating Environment (MOE, version 2019, Chemical Computing Group, Montreal, Canada) with the following validated protocol. The crystal structure was downloaded from the RCSB Protein Data Bank. All co-crystallized ligands, water molecules, and ions were removed. Hydrogen atoms were added, protonation states were assigned at physiological pH (7.4), and the protein was energy-minimized using the AMBER10:EHT force field to relieve steric clashes. The 2D structures of SIN and its derivatives were drawn in ChemDraw (version 2019) and converted into 3D geometries using MOE (version 2019). Ligands were protonated at pH 7.4, energy-minimized with the MMFF94x force field, and multiple low-energy conformations were generated (up to 30 per ligand). The binding site was defined around the co-crystallized GDP binding pocket, which has been implicated in GBP5 regulation. The Triangle Matcher algorithm was employed for pose generation, and the London dG scoring function was used for initial ranking. The top 30 poses were retained for each ligand, refined using force field-based minimization, and rescored with GBVI/WSA dG to estimate binding free energies. The best-ranked poses were selected for analysis. To assess docking reliability, the co-crystallized GDP ligand was re-docked into the binding pocket. The RMSD between the experimental and predicted binding conformations was 1.62 Å, indicating good agreement and validating the docking protocol.

Overall, these docking results suggest that the cinnamic acid esterification of sinomenine improves the binding affinity to GBP5, potentially enhancing its inhibitory effect on GBP5-mediated inflammatory signaling pathways.

3.4. Druggability Analysis

A Tencent AI Lab-iDrug platform was applied to predict the absorption distribution metabolism and drug formation of 2a–2e in vivo.

3.5. Bioassay for In Vivo Analgesic and Anti-Inflammatory Activity

3.5.1. Hot Plate Method

The mice were placed on a hot plate with a cut-off time of 30 s to avoid damaging the claws of the animals. The mice were randomly divided into 30 groups, 10 mice in each group. The mice in the blank group were intragastrically administered the same dose of normal saline, the SIN group was intragastrically administered 15 mg/kg of SIN, and the experimental group was intragastrically administered various doses of SIN derivatives (7.5 mg/kg in the low dose group, 15 mg/kg in the middle dose group, and 30 mg/kg in the high dose group). Gavage was applied for 3 days. After the last administration, the mice were recorded at 15 min, 30 min, 60 min intervals for licking the hind foot, as the reaction time (pain threshold) [24].

3.5.2. Acetic Acid-Induced Writhing Test

Young Kunming mice were intraperitoneally injected with 0.6% acetic acid at a dose of 0.1 mL/kg before administration (10 mice in each group) [25]. The first writhing time (latency period) was recorded. Then, the sinomenine (15 mg/kg), normal saline (10 mL/kg) and various doses of SIN derivatives (7.5 mg/kg in the low dose group, 15 mg/kg in the middle dose group, and 30 mg/kg in the high dose group) were administered orally 3 days (once a day) prior to treatment with acetic acid. Half an hour after the last administration, the mice were intraperitoneally injected with 0.6% acetic acid. The first writhing time and the number of writhings in 15 min were observed and recorded immediately.

$$\mathrm{The}\mathrm{prolonged}\mathrm{rate} \mathrm{of} \mathrm{latency} (\%)={\displaystyle \frac{\mathrm{time} \mathrm{after} \mathrm{administration}-\mathrm{time}\mathrm{before}\mathrm{administration}}{\mathrm{time}\mathrm{before}\mathrm{administration}}} \times 100\%$$

(1)

$$\mathrm{Percentage}\mathrm{of}\mathrm{analgesia}(\%)={\displaystyle \frac{\mathrm{number}\mathrm{of}\mathrm{twisted}\mathrm{rats}\mathrm{in}\mathrm{control}\mathrm{group}-\mathrm{number}\mathrm{of}\mathrm{twisted}\mathrm{rats}\mathrm{in}\mathrm{experimental}\mathrm{group}}{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{w}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}} \times 100\%$$

(2)

$$\begin{gathered} \mathrm{Writhing}\mathrm{times}\mathrm{inhibition}\mathrm{rate}(\%)= \\ {\displaystyle \frac{\mathrm{mean}\mathrm{number}\mathrm{of}\mathrm{body}\mathrm{torsions}\mathrm{in}\mathrm{the}\mathrm{control}\mathrm{group}-\mathrm{mean}\mathrm{number}\mathrm{of}\mathrm{body}\mathrm{torsions}\mathrm{in}\mathrm{the}\mathrm{experimental}\mathrm{group}}{\mathrm{mean}\mathrm{number}\mathrm{of}\mathrm{body}\mathrm{torsions}\mathrm{in}\mathrm{the}\mathrm{control}\mathrm{group}}} \times 100\% \end{gathered}$$

(3)

3.5.3. Evaluation of Anti-Inflammatory Capacity in Mouse Ear Edema Model

Thirty young male Kunming mice (20–25 g) were classified into the following three groups at random. The three groups are the experimental group, the positive control group, and the blank group. The experimental group were given sinomenine 4-cinnamic acid ester derivatives at 30 mg/kg. The positive control sample received 30 mg/kg SIN. The blank sample were given saline. Each sample was treated with the medicine for 7 days continuously. A total of 30 min after the last administration, 40 μL of xylene was evenly coated on the two sides of the mouse [26].

The mice were cervically dislocated and killed 40 min after xylene was applied, and an 8 mm round piece of ear was taken at the same position in the right and left ears using a perforator and weighed with a precision analytical balance. The absolute values of the differences in mass and thickness between the two ears were taken as the degree of ear edema.

3.6. Statistical Analysis

Results were analyzed using SPSS 20.0. Multiple comparisons were performed using one-way ANOVA followed by an LSD t-test. Results were considered statistically significant with a p < 0.05. Results are expressed as mean ± SD.

4. Conclusions

In conclusion, sinomenine 1-Br-4-cinnamic acid ester derivatives were highly selectively synthesized. The reaction occurred readily with sinomenine and cinnamic acid. The yields ranged from good to excellent. The structures of the target compounds were established by ¹H NMR, ¹³C NMR, and MS spectra. It may be widely used for modifying these polyfunctional natural products.

Five derivatives had good binding activity with the GBP5 protein by molecular docking software MOE. Pre-preparing the druggability of the derivatives by ADEMT 3.0 showed that each derivative had similar druggability to SIN. The analgesic activities of five derivatives were evaluated by the hot plate method and the acetic acid writhing method. The results showed that the analgesic effects of 2a, 2c, 2e were better than that of sinomenine, and the other derivatives were equivalent to sinomenine. Furthermore, the auricular swelling degree of 2b was lower than SIN and the swelling percentages of 2b were higher than SIN in the xylene-induced ear edema assay. Therefore, 2b was also confirmed to have a potent anti-inflammatory effect. This research established a basis theory for the development and application of 1-Br-4-cinnamic acid ester derivatives as natural analgesic and anti-inflammatory agents. Altogether, the results related to the series of SIN derivatives support the potential value of analgesia and its anti-inflammatory properties and have practical significance for the expansion of the clinical applications of SIN.