Isolation, total synthesis, and biological evaluation of dearomatized isoprenylated acylphloroglucinols from Hypericum przewalskii

1 Introduction

Natural phloroglucinols are widely distributed in plants of families such as Myrtaceae, Euphorbiaceae, Clusiaceae, Dryopteridaceae, Asteraceae, Fabaceae, and Rutaceae, as well as in marine and microbial sources [1]. Among them, prenylated phloroglucinols are a special type of hybrid natural products derived from polyketide combined with isoprenylation biosynthetic pathways, and mainly reported from plants of the genera Hypericum and Garcinia in the family of Guttiferae [2–4]. We have isolated and identified two new dearomatized isoprenylated acylphloroglucinols hyperprzewones A and B (1 and 2) from the Hypericum przewalskii. However, there are relatively few reports on the synthesis of such natural products. Guan [5]first achieved the asymmetric total synthesis of hyperbeanol A through a bioinspired alkylation dearomatization reaction. We adopted a concise and effective synthetic route to synthesize two new dearomatized isoprenylated natural acylphloroglucinols, and synthesize a series of derivatives based on 1 and 2. They provide a reference for subsequent synthesis research and development of DIAPs-type natural products [6, 7].

T-type calcium channels (TTCCs), as a key subtype of voltage-gated calcium channels (VGCCs), are renowned for their unique electrophysiological characteristics, including low activation threshold, brief opening, and rapid inactivation [8, 9]. Its functional abnormalities are closely related to various diseases, including neuropathic pain, epilepsy, hypertension, and cardiovascular diseases, making it an important direction for the development of new targeted drugs [10, 11]. In this study, we targeted Ca_v3.2 channel heterologously expressed in HEK-293 T cells, and screened two natural products 1 and 2, as well as a series of their synthetic derivatives, using the whole-cell patch-clamp recording method [12, 13]. The results showed that derivatives 8, 10, and 14 exhibit inhibitory effects on T-type calcium channels Ca_v3.2. Fig. 1 structures of hyperprzewones A (1) and B (2).

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2 Results and discussion

Compound 1 was acquired as a brown oil. Combining the negative-ion HRESIMS peak at m/z 411.2549 [M–H]⁻ and ¹³C NMR revealed a molecular formula of C₂₆H₃₆O₄. The IR spectrum implied absorption bands for hydroxyl (3433 cm⁻¹) and carbonyl (1634 cm⁻¹) functionalities. The ¹H NMR data (Table 1) illustrated four olefinic protons (δ_H 4.8, 4.8, 5.5, 6.4) and an isobutyl group (δ_H 0.95, d, J = 6.8 Hz; 2.07, dq, J = 13.4, 6.4 Hz; 2.91, d, J = 7.0 Hz). The ¹³C NMR (Table 1) and DEPT spectra showed 26 carbons including eight methyls, five methylenes, three methines, and 10 quaternary carbons. particularly, obvious signals for a typical dearomatized phloroglucinol core (δ_C 107.6, C–1; 187.5, C–2; 109.2, C–3; 173.4, C–4; 58.2, C–5; 197.3, C–6). Two isoprenyls and an isopropyl of DIAPs-type metabolites can be distinguished [14]. In the HMBC spectrum, the key correlations (Fig. 2) from H₂–12/H₂–17 to C–4, C–5 and C–6 confirmed that two isoprenyls were both attached to C–5, the HMBC correlation from H–1' to C–2, C–3 and C–4 indicates that C–1' is connected to the benzene ring's C–3 (Fig. 2). The ether linkage of C–4 and C–3' was evidenced by indices of hydrogen deficiency, the downfield chemical shift of C–3' (δ_C 82.8) (Fig. 2). Based on the key ¹H–¹H COSY correlations of H–1' and H–2', as well as the HMBC correlations between Me–5' and C–3', C–2', combined with the ¹³C NMR signals (δ_C 114.9, C–1'; 125.1, C–2'), the C₁₀ unit was inferred. (Fig. 2). Additionally, the remained isobutyryl can only be attached at C–1.

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2 was acquired as a pale brown oil. Combining the positive-ion HRESIMS peak at m/z 413.2690 [M + H]⁺ and ¹³C NMR revealed a molecular formula of C₂₆H₃₆O₄. The IR spectrum implied absorption bands for hydroxyl (3440 cm⁻¹) and carbonyl (1634 cm⁻¹) functionalities. NMR spectra of 2 showed a close resemblance to those of 1 except for the replacements of the iso-butyl group at C–7 in 1 by a sce–butyl in 2, respectively. The substituent at C–1 included a methine (δ 3.87, H–8), a methylene (δ 1.34, 1.74, H₂–10), a methyl triplet (δ 0.90, H₃–11), and a methyl doublet (δ 1.09, H₃–9) (Table 1). In the COSY spectrum, the H₂–10 was correlated with H₃–11, and H–8 was correlated with H₃–9. The HMBC spectrum showed that H₃–11 was correlated with C–8, C–10, and H₃–9 is correlated with C–7, C–8, which confirmed that the 2–methylbutyryl side chain was connected to C–1 (Fig. 2).

As outlined in the retrosynthetic analysis (Scheme 1), we envisioned oxidizing triisoprenyl acylphloroglucinol via selectively oxidative [4 + 2] cyclization of intermediates 6a/6b, thereby enabling the effective synthesis of the bicyclic natural products 1 and 2. At the same time, we envisioned synthesizing intermediates 6a/6b by first undergoing Friedel–Crafts alkylation and followed by dearomatization via intermediates 4a/4b, or by simultaneously undergoing Friedel–Crafts alkylation and dearomatization using a certain method. The synthesis of intermediates 4a/4b only requires phloroglucinol as the raw material, and can be carried out under conventional Friedel–Crafts acylation reaction conditions. This proposed route is more efficient, concise, and low-cost for synthesizing natural products 1 and 2 (Scheme 1).

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Our synthesis began with the Friedel–Crafts acylation of ordinary phloroglucinol 3 under the conditions of acyl chloride/AlCl₃ in nitrobenzene to give the intermediate 4a (88% yield) and 4b (84% yield), respectively [15]. It was found that intermediate 6a or 6b can be simultaneously synthesized in one step from intermediate 4a or 4b through an alkylation/dearomatization cascade [16]. However, this method gave 6a/6b in dissatisfactory yields (< 15%) in this work due to the instable and tautomeric nature of 4a/4b. Therefore, we adopted stepwise alkylation and C-5 dearomatization as the ideal conditions to improve the yields of 6a/6b. Intermediates 4a/4b underwent an alkylation reaction with prenyl bromide using DBU as the base at 45 ℃ to synthesize intermediate 5a (47% yield) and 5b (49% yield), respectively [17]. Based on Lee's protocol, attempt to extend the reaction time to 2 h in KOH aqueous solution at 0 ℃ gave 6a (61% yield) and 6b (59% yield) [16] (Scheme 2). Subsequently, oxidative [4 + 2] cyclization of 6a/6b in the presence of PhI(OAc)₂/TEMPO furnished natural products 1 in 69% yield and 2 in 72% yield, respectively. This transformation might be resulted from the o-quinone methide intermediates i and ii (Scheme 2), which formed by selective hydrogen abstraction of in situ generated TEMPO cation [18]. It is worth noting that the 4–phenol selectivity of this oxidative [4 + 2] cyclization could be well controlled due to the hydrogen bonding interactions between 2–phenol group with C–7 carbonyl group giving 1 or 2 as the single product. However, both products 1 and 2 were prone to enol-ketone tautomeric mixture (3:1), which were proved in ¹H and ¹³C NMR spectral data as their natural samples. Finally, the syntheses of compounds 1 and 2 were completed in four steps, in overall yields of 17% (Scheme 2).

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Next, we prepared a series of derivatives based on 1 and 2. Initially, treatment 5a with PSTA (2.0 eq.) gave bicyclic compounds 7a (49% yield) and 7b (33% yield) through 6-endo-trig cyclization, respectively [19]. We believed that stoichiometric PTSA destroyed the hydrogen bonding interactions between 2– and 6–phenols with C–7 carbonyl enabling the corresponding cyclization through the proposed intermediate iii (Scheme 3), which promoted weaker nucleophilic 2-phenol to cyclization to 7a as a major product [20]. A similar C–5 alkylation/dearomatization cascade of 7a using prenyl bromide/KOH (aq.) generated 8 in 61% yield. It was found that C–4 phenol group ensured the dissolubility of such bicyclic compounds in KOH aqueous solution, as 7a but 7b was dissolvable. These observations provided a valuable reference for the synthesis of subsequent DIAPs natural products. Furthermore, we also transformed 5a to 9 (53% yieid) by shortening the reaction time to 15 min in KOH aqueous solution (Scheme 3). Subsequently, 4a underwent an alkylation reaction with geranyl bromide using DIPEA as the base at 90 ℃ to furnish derivative 10 in 47% yield [21]. Moreover, we synthesized tetraisopentenyl acylphloroglucinol derivative 11 albeit in 17% yield through C–3/C–5 alkylation/dearomatization cascade of 4a under the conditions of prenyl bromide in an aqueous ammonia solution [22] (Scheme 3). Finally, a similar two-step synthesis of acylated derivative 14 (32% yield over two steps) was carried out using prenyl bromide/DBU and PhI(OAc)₂/TEMPO from substrate 12. Interestingly, 12 seemed more effective than 4a/4b to generate the C–3 prenylated and C–5 di–prenylated product like 13 in 47% yield due to its less steric hindrance.

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With all synthetic 1/2-derivatives in hands, their Ca_v3.2 channel inhibition were tested. As a result, natural products 1 and 2 exhibited no inhibitory effects on the Ca_v3.2 channel at a concentration of 10 μM. However, their derivatives 8, 10, and 14 exhibited a good inhibitory effect on the Ca_v3.2 channel (Fig. 3). At a concentration of 10 µM, the inhibitory rates of these derivatives were 55.29% (8), 36.55% (10), and 37.80% (14), respectively. Among them, derivative 8 showed the highest inhibitory activity, suggesting tautomeric enol-ketone moiety might be unfavorable for Ca_v3.2 inhibition.

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3 Conclusions

In summary, two new natural products hyperprzewones A (1) and B (2) were isolated from Hypericum przewalskii. Structurally, these compounds were characterized by a dearomatized isoprenylated acylphloroglucinol core combined a functionalized cyclohexene skeleton. In addition, we have successfully achieved the total syntheses of 1 and 2 through Friedel–Crafts acylation, alkylation, dearomatization, and oxidative [4 + 2] cyclization in four steps. Biological evaluation of synthetic 1/2-derivatives showed that 8, 10, and 14 exhibit inhibitory effects on the Ca_v3.2 channel. Our finding provided a new direction for the development of Ca_v3.2 channel drugs for the treatment of diseases such as neuropathic pain and epilepsy.

4 Experimental section

4.1 General experimental procedures

Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were detected on a Shmadzu UV-2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. All 1D and 2D NMR spectra were recorded on Bruker AVANCE Ⅲ 400 MHz and Bruker DRX–600 MHz spectrometers using TMS as an internal standard. Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference to the solvent signals. ESIMS and HRESIMS analysis were carried out on Waters Xevo TQS and Aglient G6230 TOF mass spectrometers, respectively. MCI gel (7–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography. Synthetic field unless otherwise mentioned, all reactions were carried out under an argon atmosphere under anhydrous conditions, and all reagents were purchased from commercial suppliers without further purification. Silica gel (100–200, 200–300 mesh, Qingdao Marine Chemical Co., Ltd., People's Republic of China). Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd.), and spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in EtOH.

4.2 Plant materials

The dried aerial parts of Hypericum przewalskii were collected at Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province, China, in September 2024. The plant was identified by Dr. Ruizhu Bai, Kunming Institute of Botany, Kunming, P. R. China. A voucher specimen was deposited with Kunming Institute of Botany with identification number (2024H01).

4.3 Extraction and isolation

The air-dried aerial parts of Hypericum przewalskii (10.0 kg) were powered and extracted with MeOH (4 × 50 L) at room temperature for 24 h and filtered. After evaporating in vacuo, the crude extract (1.8 kg) was subjected to silica gel column chromatography and eluted with CHCl₃ to afford a fraction (0.18 kg). This fraction was separated by MCI column chromatography (MeOH–H₂O, 75:25 to 100:0) to provide seven sub-fractions (Fr. A1–A7). Fr. A3 (18.5 g) was separated into eight sub-fractions (Fr. A3.1–3.8) on silica gel column chromatography using a gradient of petroleum ether‒EtOAc (500:0 to 0:1). The obtained sub-fraction Fr. A3.1.5 (154.5 mg) was further fractionated by using an ODS column eluted with CH₃OH–H₂O (90:10, v/v) to give 1 (9.8 mg, t_R 15.7 min, CH₃OH–H₂O, 90%) and 2 (11.3 mg, t_R 14.3 min, CH₃OH–H₂O, 90%).

4.4 Syntheses

3-methyl-1-(2,4,6-trihydroxy-3-(3-methylbut-2-en-1-yl)phenyl)butan-1-one (5a)

2-methyl-1-(2,4,6-trihydroxy-3-(3-methylbut-2-en-1-yl)phenyl)butan-1-one (5b)

To a solution of 4a/4b (200 mg, 0.95 mmol, 1.0 eq.) and prenyl bromide (196 μL, 1.9 mmol, 2.0 eq.) in dry THF (3 mL) was added DBU (284 μL, 1.9 mmol, 2.0 eq.). The resultant suspension was heated at 45 ℃ for 24 h. The reaction mixture allowed to cool to room temperature, then acidified with 1 N HCl solution (1 mL) and extracted with EtOAc (3 × 10 mL). The combined extracts were washed with brine (3 × 10 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel to give 5a (124 mg, 47%) and 5b (129 mg, 49%) as a yellow oil. Date for 5a: R_f 0.6 (petrol/EtOAc, 1:1); ¹H NMR (400 MHz, CD₃OD) δ 5.89 (s, 1H), 5.21–5.11 (m, 1H), 3.18 (d, J = 7.1 Hz, 2H), 2.91 (d, J = 6.8 Hz, 2H), 2.21 (dt, J = 13.5, 6.8 Hz, 1H), 1.74 (s, 3H), 1.67–1.62 (m, 3H), 0.95 (d, J = 6.7 Hz, 6H). ¹³C NMR (150 MHz, CD₃OD) δ 205.67, 163.65, 162.17, 159.96, 129.65, 123.17, 106.62, 104.06, 93.45, 52.40, 25.44, 24.59, 21.82, 20.78, 16.48; HRMS (C₁₆H₂₂O₄, ESI): calculated [M-H]⁻ 277.1445, found 277.1446. Date for 5b: R_f 0.6 (petrol/EtOAc, 1:1); ¹H NMR (400 MHz, CD₃OD) δ 5.88 (s, 1H), 5.15 (s, 1H), 3.87 (h, J = 6.7 Hz, 1H), 3.17 (d, J = 7.2 Hz, 2H), 1.86–1.76 (m, 1H), 1.76–1.72 (m, 3H), 1.67–1.62 (m, 3H), 1.36 (dt, J = 13.8, 7.1 Hz, 1H), 1.11 (d, J = 6.7 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 210.10, 163.85, 162.01, 159.70, 129.62, 123.19, 106.70, 103.70, 93.54, 45.29, 26.81, 24.57, 20.81, 16.45, 15.87, 10.99; HRMS (C₁₆H₂₂O₄, ESI): calculated [M-H]⁻ 277.1445, found 277.1446.

3,5-dihydroxy-4,6,6-tris(3-methylbut-2-en-1-yl)-2-(3-methylbutanoyl)cyclohexa-2,4-dien-1-one (6a)

3,5-dihydroxy-4,6,6-tris(3-methylbut-2-en-1-yl)-2-(2-methylbutanoyl)cyclohexa-2,4-dien-1-one (6b)

To a solution of 5a/5b (100 mg, 0.36 mmol, 1.0 eq.) in H₂O (2 mL) under a nitrogen atmosphere at 0 ℃ was added KOH (40 mg, 0.72 mmol, 2.0 eq.) in one portion, then prenyl bromide (70 μL, 0.72 mmol, 2.0 eq.) was added dropwise over 20 min. The reaction mixture was stirred at 0 ℃ for a further 2 h, during which time a thick orange precipitate was formed. The reaction mixture was then acidified with 1 N HCl solution (1 mL) and then extracted with EtOAc (3 × 10 mL). The combined extracts were washed with brine (3 × 10 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (petrol/EtOAc, 60:1 → 30:1) to give 6a (90 mg, 61%) as a pale yellow oil and 6b (88 mg, 59%) as a white oil. Date for 6a: R_f 0.5 (petrol/EtOAc, 2:1); ¹H NMR (400 MHz, CD₃OD) δ 5.25–5.09 (m, 1H), 4.85–4.73 (m, 2H), 3.20 (d, J = 7.3 Hz, 2H), 2.92 (d, J = 7.0 Hz, 2H), 2.67–2.61 (m, 2H), 2.51 (dd, J = 13.8, 7.8 Hz, 2H), 2.19–2.09 (m, 1H), 1.79 (d, J = 5.0 Hz, 6H), 1.56 (s, 12H), 0.98–0.95 (m, 6H). ¹³C NMR (150 MHz, CD₃OD) δ 203.97, 198.19, 191.00, 174.44, 135.67, 132.59, 123.22, 119.21, 112.39, 109.36, 58.7, 50.10, 38.77, 27.05, 26.03, 23.11, 21.61, 17.94; HRMS (C₂₆H₃₈O₄, ESI): calculated [M-H]⁻ 413.2697, found 413.2703. Date for 6b: R_f 0.5 (petrol/EtOAc, 2:1); ¹H NMR (400 MHz, CD₃OD) δ 5.01 (d, J = 7.2 Hz, 1H), 4.77 (d, J = 7.4 Hz, 2H), 3.89 (q, J = 7.0 Hz, 1H), 3.10 (d, J = 7.0 Hz, 2H), 2.66–2.51 (m, 4H), 1.73 (d, J = 5.3 Hz, 4H), 1.67 (s, 3H), 1.58–1.53 (m, 12H), 1.38–1.33 (m, 1H), 1.08 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 7.4 Hz, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 208.30, 198.02, 191.21, 174.23, 135.63, 132.56, 123.24, 119.20, 112.35, 108.82, 58.73, 43.72, 39.05, 38.78, 27.7, 26.05, 21.65, 18.10, 16.99, 12.36; HRMS (C₂₆H₃₈O₄, ESI): calculated [M-H]⁻ 413.2697, found 413.2707.

hyperprzewone A (1) and hyperprzewone B (2)

To a solution of 6a/6b (50 mg, 0.12 mmol, 1.0 eq.) in dry THF (2 mL) at −78 ℃ under a nitrogen atmosphere was added TEMPO (38 mg, 0.24 mmol, 2.0 eq.) followed by PhI(OAc)₂ (45 mg, 0.14 mmol, 1.2 eq.). The reaction mixture was stirred at −78 ℃ for 5 min, then allowed to warm to room temperature over 30 min. The reaction mixture was quenched with H₂O (1 mL), then extracted with EtOAc (3 × 10 mL). The combined organics were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was then purified by flash chromatography on silica gel (petrol/EtOAc, 100:1 → 40:1) to give 1 (33 mg, 69%) and 2 (36 mg, 72%) as a brown oil. Date for 1: R_f 0.6 (petrol/EtOAc, 10:1); ¹H NMR (400 MHz, CD₃OD) δ 6.44 (d, J = 10.1 Hz, 1H), 5.45 (dd, J = 22.8, 10.1 Hz, 1H), 4.80–4.68 (m, 2H), 2.91 (d, J = 7.0 Hz, 2H), 2.65 (dd, J = 13.8, 7.8 Hz, 2H), 2.51 (dd, J = 13.9, 7.3 Hz, 2H), 2.07 (dq, J = 13.4, 6.4 Hz, 1H), 1.57 (d, J = 8.8 Hz, 12H), 1.44 (d, J = 11.8 Hz, 6H), 0.95 (d, J = 6.8, , 6H). ¹³C NMR (150 MHz, CD₃OD) δ 203.52, 197.30, 187.49, 173.38, 136.00, 125.06, 119.16, 118.66, 114.98, 109.27, 107.61, 82.85, 58.24, 49.39, 38.66, 37.35, 29.08, 27.27, 25.98, 22.96, 18.31; HRMS (C₂₆H₃₆O₄, ESI): calculated [M-H]⁻ 411.2541, found 411.2549; [α] −1.17 (c 0.12, MeOH); UV (MeOH)λ_max (log ε) 195 (4.34), 226 (3.80), 267 (3.97), 326 (3.59), 337 (3.60) nm; IR (KBr)v_max 3433, 2962, 2928, 2871, 1654, 1634, 1527, 1466, 1381. Date for 2:R_f 0.6 (petrol/EtOAc, 10:1); ¹H NMR (400 MHz, CD₃OD) δ 6.44 (d, J = 10.1 Hz, 1H), 5.48 (d, J = 10.2 Hz, 1H), 4.82–4.77 (m, 2H), 3.95–3.79 (m, 1H), 2.67 (dt, J = 14.3, 7.4 Hz, 2H), 2.51 (dd, J = 13.9, 7.4 Hz, 2H), 1.76–1.70 (m, 1H), 1.57 (d, J = 9.0 Hz, 12H), 1.46 (s, 6H), 1.34 (s, 1H), 1.09 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 7.5 Hz, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 208.09, 197.18, 187.54, 173.34, 136.00, 125.07, 119.24, 114.99, 108.70, 107.49, 82.95, 58.43, 43.42, 38.84, 38.51, 29.07, 27.71, 26.01, 18.29, 16.94, 12.33; HRMS (C₂₆H₃₆O₄, ESI): calculated [M+H]⁺ 413.2686, found 413.2690; [α] −0.83 (c 0.17, MeOH); UV (MeOH)λ_max (log ε) 195 (4.01), 228 (3.36), 267 (3.57), 327 (3.04), 351 (3.08) nm; IR (KBr)v_max 3440, 2969, 2928, 2876, 1654, 1634, 1526, 1465, 1380.

1-(5,7-dihydroxy-2,2-dimethylchroman-8-yl)-3-methylbutan-1-one (7a)

1-(5,7-dihydroxy-2,2-dimethylchroman-6-yl)-3-methylbutan-1-one (7b)

To a solution of 5a (100 mg, 0.36 mmol, 1.0 eq.) in toluene (3 mL) at room temperature was added PSTA (124 mg, 0.72 mmol, 2.0 eq.). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with H₂O (1 mL), then extracted with EtOAc (3 × 10 mL). The combined organics were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was then purified by flash chromatography on silica gel (petrol/EtOAc, 40:1 → 15:1) to give 7a (49 mg 49%) as a white oil and on silica gel (petrol/EtOAc, 40:1 → 20:1) to give 7b (33 mg 33%) as a yellow oil. Date for 7a: R_f 0.4 (petrol/EtOAc, 4:1); ¹H NMR (400 MHz, CDCl₃) δ 13.92 (s, 1H), 5.93 (s, 1H), 2.90 (d, J = 7.0 Hz, 2H), 2.59 (t, J = 6.8 Hz, 2H), 2.21 (dq, J = 13.4, 6.7 Hz, 1H), 1.80 (t, J = 6.8 Hz, 2H), 1.40 (s, 6H), 0.97 (d, J = 6.6 Hz, 6H). ¹³C NMR (600 MHz, CDCl₃) δ 206.01, 165.07, 159.94, 157.14, 106.32, 99.36, 95.34, 76.04, 53.40, 31.51, 26.77, 25.39, 22.77, 16.35; HRMS (C₁₆H₂₂O₄, ESI): calculated [M-H]⁻ 277.1445, found 277.1442. Date for 7b: R_f 0.5 (petrol/EtOAc, 4:1); ¹H NMR (400 MHz, CDCl₃) δ 13.57 (s, 1H), 6.43 (s, 1H), 5.72 (s, 1H), 2.93 (d, J = 6.8 Hz, 2H), 2.58 (t, J = 6.8 Hz, 2H), 2.26 (dp, J = 13.4, 6.7 Hz, 1H), 1.78 (t, J = 6.8 Hz, 2H), 1.32 (s, 6H), 0.97 (d, J = 6.7 Hz, 6H). ¹³C NMR (150 MHz, CDCl₃) δ 205.87, 164.00, 160.40, 158.01, 104.46, 101.74, 95.72, 76.13, 52.91, 32.31, 26.89, 25.62, 23.04, 16.30; HRMS (C₁₆H₂₂O₄, ESI): calculated [M-H]⁻ 277.1445, found 277.1452.

5-hydroxy-2,2-dimethyl-6,6-bis(3-methylbut-2-en-1-yl)-8-(3-methylbutanoyl)-2,3,4,6-tetrahydro-7H-chromen-7-one (8)

To a solution of 7a (40 mg, 0.14 mmol, 1.0 eq.) in H₂O (1.5 mL) under a nitrogen atmosphere at 0 ℃ was added KOH (16 mg, 0.28 mmol, 2.0 eq.) in one portion, then prenyl bromide (27 μL, 0.28 mmol, 2.0 eq.) was added dropwise over 20 min. The resultant suspension was stirred at 0 ℃ for a further 2 h, then reaction mixture was acidified with 1 N HCl solution (1 mL) and extracted with EtOAc (3 × 10 mL). The combined extracts were washed with brine (3 × 10), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (petrol/EtOAC, 60:1 → 35:1) to give 8 (34 mg, 61%) as a pale yellow oil. Date for 8: R_f 0.6 (petrol/EtOAc, 4:1); ¹H NMR (400 MHz, CD₃OD) δ 4.78–4.70 (m, 2H), 2.78 (d, J = 7.3 Hz, 2H), 2.69–2.55 (m, 4H), 2.34 (t, J = 6.8 Hz, 2H), 2.10 (dt, J = 13.6, 6.8 Hz, 1H), 1.78 (t, J = 6.8 Hz, 2H), 1.60–1.46 (m, 12H), 1.40 (s, 6H), 0.96 (d, J = 6.7 Hz, 6H). ¹³C NMR (150 MHz, CD₃OD) δ 201.38, 198.09, 196.61, 166.61, 136.01, 119.17, 108.78, 107.39, 80.25, 61.01, 49.43, 39.67, 32.52, 28.58, 26.66, 26.03, 22.87, 17.98, 17.32; HRMS (C₂₆H₃₈O₄, ESI): calculated [M-H]⁻ 413.2770, found 413.2697.

(E)-1-(3-(3,7-dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)-3-methylbutan-1-one (10)

To a mixture solution of 4a (100 mg, 0.48 mmol, 1.0 eq.) and geranyl bromide (190 μL, 0.96 mmol, 2.0 eq.) in DMF (2 mL) was added DIPEA (171 μL, 0.96 mmol, 2.0 eq.). The resultant suspension was heated at 80 ℃ for 3 h, then gradually warmed to room temperature. The reaction mixture was quenched with H₂O (1 mL) and extracted with EtOAc (3 × 10 mL). The combined organic extracts were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was then purified by flash chromatography on silica gel (petrol/EtOAc, 20:1 → 5:1) to give 10 (78 mg 47%) as a yellow oil. Date for 10: R_f 0.4 (petrol/EtOAc, 2:1); ¹H NMR (400 MHz, CD₃OD) δ 5.88 (s, 1H), 5.17 (dt, J = 6.1, 4.3 Hz, 1H), 5.05 (dt, J = 7.3, 1.5 Hz, 1H), 3.18 (d, J = 7.1 Hz, 2H), 2.91 (d, J = 6.8 Hz, 2H), 2.21 (dt, J = 13.1, 6.6 Hz, 1H), 2.04 (q, J = 7.4 Hz, 2H), 1.93 (t, J = 7.5 Hz, 2H), 1.75–1.72 (m, 3H), 1.63– 1.56 (m, 3H), 1.55 (s, 3H), 0.95 (d, J = 6.7 Hz, 6H). ¹³C NMR (150 MHz, CD₃OD) δ 207.03, 165.37, 163.87, 161.63, 134.93, 130.94, 125.80, 124.94, 108.35, 105.75, 95.12, 53.80, 40.93, 28.01, 27.13, 26.13, 23.50, 22.09, 17.71, 16.48; HRMS (C₂₁H₃₀O₄, ESI): calculated [M + H]⁺ 347.2217, found 347.2217.

5-hydroxy-2,2,6,6tetrakis(3-methylbut-2-en-1-yl)-4-(3-methylbutanoyl)cyclohex-4-ene-1,3-dione (11)

To a solution of 4a (40 mg, 0.19 mmol, 1.0 eq.) and prenyl bromide (45 μL, 0.38 mmol, 2.0 eq.) in NH₃·H₂O (1.5 mL). The resultant suspension was stirred for 4 h at room temperature, then acidified with 1 N HCl solution (1.5 mL) and extracted with EtOAc (3 × 10 mL). The combined extracts were washed with brine (3 × 10 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (petrol/EtOAc, 100:1 → 60:1) to give 11 (13 mg, 17%) as a white oil. Data for 11: R_f 0.4 (petrol/EtOAc, 20:1); ¹H NMR (400 MHz, CDCl₃) δ 4.90 (s, 4H), 2.81 (s, 2H), 2.65 (dd, J = 13.8, 8.0 Hz, 2H), 2.53 (dt, J = 14.9, 7.9 Hz, 4H), 2.30 (dd, J = 14.6, 6.1 Hz, 2H), 2.23–2.14 (m, 1H), 1.63–1.54 (m, 24H), 0.99 (d, J = 6.7 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 207.55, 203.65, 197.41, 194.73, 136.16, 134.60, 118.89, 118.26, 113.57, 65.53, 60.94, 47.65, 36.82, 33.85, 25.96, 25.85, 25.77, 22.69, 17.88, 17.84; HRMS (C₃₁H₄₆O₄, ESI): calculated [M + H]⁺ 483.3469, found 483.3472.

6-acetyl-5-hydroxy-2,2-dimethyl-8,8-bis(3-methylbut-2-en-1-yl)-2,8-dihydro-7H-chromen-7-one (14) To a solution of 13 (200 mg, 0.54 mmol, 1.0 eq.) in dry THF (4 mL) at −78 ℃ under a nitrogen atmosphere was added TEMPO (169 mg, 1.08 mmol, 2.0 eq.) followed by PhI(OAc)₂ (209 mg, 0.65 mmol, 1.2 eq.). The reaction mixture was stirred at −78 ℃ for 5 min, then allowed to warm to room temperature over 30 min. The reaction mixture was quenched with H₂O (2 mL), then extracted with EtOAc (3 × 30 mL). The combined organics were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was then purified by flash chromatography on silica gel (petrol/EtOAc, 80:1 → 30:1) to give 14 (110 mg, 64%) as a yellow oil. Date for 14: R_f 0.4 (petrol/EtOAc, 10:1); ¹H NMR (400 MHz, CDCl₃) δ 6.48 (dd, J = 29.2, 10.0 Hz, 1H), 5.32 (dd, J = 14.7, 10.0 Hz, 1H), 4.77 (t, J = 7.1 Hz, 2H), 2.70 (d, J = 8.3 Hz, 2H), 2.58 (s, 3H), 2.48 (dd, J = 13.9, 7.6 Hz, 2H), 1.62 (d, J = 1.0 Hz, 12H), 1.41 (d, J = 13.9 Hz, 6H). ¹³C NMR (150 MHz, CDCl₃) δ 199.48, 195.47, 185.85, 172.21, 134.60, 123.10, 117.90, 114.31, 108.20, 105.70, 81.03, 56.80, 37.34, 28.50, 25.50, 17.91; HRMS (C₂₃H₃₀O₄, ESI): calculated [M-H]⁻ 369.2071, found 369.2075.

4.5 Cell preparation and expression

Human embryonic kidney (HEK) 293 T cells were grown in DMEM (cytiva) plus 10% newborn calf serum (Gibco) and penicillin (100 U/mL)/streptomycin (0.1 mg/mL) (Biological Industries). HEK 293 T cells were transiently co-transfected with pCDNA3.1-Ca_v3.2 and EGFP plasmids together using Lipofectamine™3000 (invitrogen) and used in 48 h.

Notes

Acknowledgements

This study was supported financially by Yunnan Key Laboratory Screening and Research on Anti-pathogenic Plant Resources from Western Yunnan (APR202301); Yunnan Revitalization Talent Support Program "Innovation Team" Project (202305AS350014), and Foundation of DR. PLANT.

Author contributions

Yong Li carried out the experiments and wrote original draft; Fei-Fei Xiong Carried out a separation and extraction experiment; Xiao-Yang Sun conducted activity screening. Li-Dong Shao and Xing-Ren Li revised the manuscript; Dao-Feng Chen, Yin Nian and Gang Xu designed the experiments and revised the manuscript. All authors read and approved the final manuscript.

Funding

Yunnan Key Laboratory Screening and Research on Anti-pathogenic Plant Resources from Western Yunnan, APR202301, Gang Xu, Yunnan Revitalization Talent Support Program Innovation Team Project, 202305AS350014, Gang Xu.

Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.