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. 2022 Sep 19;45(9):631–643. doi: 10.1007/s12272-022-01401-6

Structures, biomimetic synthesis, and anti-SARS-CoV-2 activity of two pairs of enantiomeric phenylpropanoid-conjugated protoberberine alkaloids from the rhizomes of Corydalis decumbens

Jing-Guang Lu 1, Yingwei Wang 1, Ming-Rong Yang 1, Cai-Yun Wang 1, Jieru Meng 1, Jiazheng Liu 1, Zifeng Yang 2, Kongsong Wu 3, Li-Ping Bai 1,✉, Guo-Yuan Zhu 1,✉, Zhi-Hong Jiang 1,✉
PMCID: PMC9484358  PMID: 36121609

Abstract

(±)-Decumicorine A (1) and (±)-epi-decumicorine A (2), two pairs of enantiomeric isoquinoline alkaloids featuring a novel phenylpropanoid-conjugated protoberberine skeleton, were isolated and purified from the rhizomes of Corydalis decumbens. The separation of (±)-1 and (±)-2 was achieved by chiral HPLC to produce four optically pure enantiomers. The structures and absolute configurations of compounds (−)-1, (+)-1, (−)-2, and (+)-2 were elucidated by spectroscopic analysis, ECD calculations, and X-ray crystallographic analyses. The two racemates were generated from a Diels-Alder [4 + 2] cycloaddition between jatrorrhizine and ferulic acid in the proposed biosynthetic pathways, which were fully verified by a biomimetic synthesis. Moreover, compound (+)-1 exhibited an antiviral entry effect on SARS-CoV-2 pseudovirus by blocking spike binding to the ACE2 receptor on HEK-293T-ACE2h host cells.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12272-022-01401-6.

Keywords: Corydalis decumbens, Isoquinoline alkaloid, Biomimetic synthesis, COVID-19, Anti-SARS-CoV-2

Introduction

Decumbent Corydalis Rhizome (DCR) is the dried tuber of Corydalis decumbens (Thunb.) Pers. (“Xia-tian-wu” in Chinese) from the Papaveraceae family. Flora of China states that the geographical distribution of C. decumbens mainly includes the Chinese provinces of Jiangxi, Jiangsu, Zhejiang, Anhui, Fujian, Hubei, Hunan, Shanxi, and Southern Japan. According to the latest Chinese Pharmacopoeia (ChP 2020), DCR and the therapeutic drugs, which were developed from its total alkaloids in various dosage forms (tablets, eye drops, and so on), are clinically used in China for treating hemiplegia caused by wind-stroke, headache, traumatic injuries, and other painful diseases. Isoquinoline alkaloids are well known, rich in species from the genus Corydalis and are considered principal bioactive constituents (Iranshahy et al. 2014; Deng et al. 2021). At present, more than 70 isoquinoline alkaloids (which are summarized at https://www.nmrdata.com) have been isolated from DCR, including the typical isoquinoline alkaloids palmatine, berberine, and l-tetrahydropalmatine. The objective of this research was to discover more isoquinoline alkaloids in DCR that are structurally unique and biologically active. In this study, LC‒MS analysis indicated that obvious alkaloids were still observed in the aqueous layer of total alkaloids after adequate partitioning was achieved. Finally, (±)-decumicorine A (1) and (±)-epi-decumicorine A (2), two pairs of enantiomeric isoquinoline alkaloids that contain a novel carbon framework of phenylpropanoid-conjugated protoberberine, were obtained (Fig. 1) (Tantillo 2021) from the aqueous layer of total alkaloids after partitioning with the organic solvent. Herein, we report the isolation, structural determination, plausible biosynthetic pathways, biomimetic synthesis, and anti-SARS-CoV-2 activity of these compounds.

Fig. 1.

Fig. 1

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Structures of compounds 1 and 2

Materials and methods

General experimental procedures

Optical rotations were performed on a Rudolph Research Autopol I automatic polarimeter (Rudolph Research Analytical, NJ, USA). UV and CD spectra were measured on a JASCO High performance J-1500 CD spectrometer (JASCO, Tokyo, Japan). IR spectra were recorded on an Agilent Cary 600 series FT-IR spectrometer (KBr) (Agilent Technologies, CA, USA). The melting point was obtained on a BA-350 melting point apparatus (BenAng, Shanghai, China). 1D and 2D NMR spectra were acquired on a Bruker Ascend™ 600 MHz NMR spectrometer (Bruker, Fällanden, Switzerland). LC-MS and HR-ESI-MS spectra were determined using an Agilent 1290 Infinity II UHPLC combined with a 6230 high-resolution TOF-MS system with an Agilent Poroshell 120 EC-C18 column (2.7 μm, 100 × 4.6 mm, i.d.) (Agilent Technologies, CA, USA). Semipreparative HPLC was carried out on an Agilent 1200 series HPLC system (Agilent Technologies, CA, USA) using Waters Xselect CSH Phenyl-Hexyl (5 μm, 250 × 10 mm, i.d.) column (Waters, MA, USA) with a constant flow of 3.0 mL/min. Preparative-HPLC was conducted on an LabAlliance HPLC system (LabAlliance, NY, USA) using Grace VisionHT C18 (5 μm, 250 × 22 mm, i.d.) column (Grace Davision Discovery Sciences, Lokeren, Belgium) under an isocratic flow of 15.0 mL/min. MPLC was performed on a Büchi Sepacore flash chromatography system (Büchi, Flawil, Switzerland) using a self-prepared C18 column (Unisil 10–120 μm C18 Ultra Plus, 460 × 36 mm, i.d., Suzhou NanoMicro Technology Co., Ltd., China) with a gradient eluted program. The above-utilized mobile phases were composed of MeCN and H2O containing either 0.1% formic acid (FA), or 10 mM ammonium acetate (AA), or a buffer of 0.1% FA and10 mM AA. Column chromatography (CC) was performed with a microporous adsorbent resin (D101, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China; 2.8 L, 610 × 76 mm, i.d.) as packing material. Chiral-LC separation was carried out on a CHIRALCEL® OZ-H chiral column (5 μm, 250 × 10 mm, i.d.) (Chiral Technologies, PA, USA), with a mobile phase of n-hexane and absolute EtOH containing a buffer of 0.1% glacial acetic acid (GAA) and 0.1% diethyl ammonium (DEA), under an isocratic flow of 3.0 mL/min. Reference compounds of jatrorrhizine hydrochloride and ferulic acid were purchased from Chengdu MUST Bio-Technology Co., Ltd (Chengdu, China). All solvents and reagents were in either spectroscopic, or HPLC, or ACS grade.

Plant materials

The rhizomes of Corydalis decumbens (Decumbent Corydalis Rhizome, DCR) were collected in May 2018 from the culture field of Jiangxi HERBISKY Chinese Medicine Co., Ltd., Jiangxi Province, China, and were authenticated by the author, Prof. Zhi-Hong Jiang. The voucher specimen (DCR-2018-05) was deposited at the State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology.

Extraction and isolation

The air-dried DCR (6 kg) was powdered and extracted with 80% EtOH (24 L × 3) under reflux. The extracted solutions were concentrated under reduced pressure to obtain a total extract, which was subsequently dissolved in 1% HCl (8 L) and centrifuged under 3500 rpm for 30 min to remove non-alkaloid fraction (Zhu et al. 2014). The supernatant was adjusted to pH 10.0 with ammonia water to precipitate alkaloids, and then partitioned by CH2Cl2 and water-saturated n-BuOH, successively. The aqueous layer (W, 762 g) was subjected to the D101 resin CC eluting with H2O, 10%, 20%, and 95% EtOH (each 8 L) successively to afford the corresponding fractions W-a–W-d. After removal of the solvent, fraction W-d (20 g) was isolated by MPLC system using gradient program of MeCN‒H2O (10∶90 → 60∶40, v/v, H2O containing 10 mM AA) to afford 9 fractions (W-d-1–W-d-9). W-d-4 (2.3 g) was further isolated with preparative HPLC and eluted with MeCN‒H2O (23∶77, v/v, H2O containing 0.1% FA and 10 mM AA) to obtain 4 subfractions (W-d-4-1–W-d-4-4). W-d-4-2 (610 mg) was separated and purified by semipreparative HPLC with MeCN‒H2O (22∶78, v/v, H2O containing 0.1% FA) to afford compound 1 (58 mg, tR = 10.0 min). In a similar manner, W-d-6 (2.0 g) was finally isolated and purified by semipreparative HPLC with MeCN‒H2O (23∶77, v/v, H2O containing 0.1% FA) to afford compound 2 (180 mg, tR = 13.5 min). Compound 1 (11.7 mg) was further separated by chiral HPLC and eluted with n-hexane‒EtOH (65∶35, v/v, containing 0.1% GAA and 0.1% DEA) to afford (-)-1 (tR 41.2 min, 5.3 mg) and (+)-1 (tR 51.9 min, 5.6 mg). Compound 2 (22 mg) was further isolated by chiral HPLC and eluted with n-hexane‒EtOH (55∶45, v/v, containing 0.1% GAA and 0.1% DEA) to obtain (−)-2 (tR 19.3 min, 10.2 mg) and (+)-2 (tR 27.8 min, 10.0 mg). Compounds (−)-1, (+)-1, (−)-2 and (+)-2 were finally purified with the same semipreparative HPLC conditions mentioned above to remove the remaining salt derived from GAA and DEA.

(±)-Decumicorine A (1): yellowish brown crystal (MeOH/H2O, 5∶1, v/v); m.p. 177–178 °C; [α]20D 1.7 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 232.0 (4.04), 255.3 (3.84), 276.0 (3.78), 317.0 (3.52) nm; IR (KBr) νmax 3564, 3389, 3019, 2964, 2937, 2837, 1614, 1574, 1516, 1493, 1402, 1325, 1302, 1261, 1215, 1163, 1134, 1078, 1003, 964, 874, 849 cm− 1. 1H and 13C NMR data, see Table 1; HRMS (ESI-TOF) m/z 532.1945 [M + H]+ (calcd. for C30H29NO8, 532.1966); (−)-1: light yellow crystal (MeOH/H2O, 5∶1, v/v); m.p. 164–165 °C; [α]20D -110.4 (c 0.5, MeOH); ECD (MeOH) λmax (∆ε) 207.1 (− 20.2), 234.5 (5.5), 251.5 (− 2.7), 278.2 (− 3.0) nm. (+)-1: yellowish brown crystal (MeOH/H2O, 5∶1, v/v); m.p. 171–172 °C; [α]20D 118.1 (c 0.5, MeOH); ECD (MeOH) λmax (∆ε) 206.5 (21.1), 233.0 (− 5.1), 251.8 (3.1), 275.5 (2.9) nm.

Table 1.

NMR spectroscopic data for 1 and 2 in CD3OD#

Position 1 2
δH(J, Hz) δC δH(J, Hz) δC
1 6.83, s 113.1 7.69, s 113.5
1a 115.8 115.3
2 149.9 149.4
3 162.0 158.9
4 6.69, s 117.4 6.86, s 117.2
4a 135.1 135.8
5 3.10, m; 3.03, m 27.2 3.31, overlap; 2.98, dt (16.8, 6.0) 26.8
6 4.40, m; 4.15, m 49.5 4.32, m; 4.04, m 51.0
8 6.00, d (3.6) 64.7 6.03, d (1.2) 64.8
8a 129.1 131.5
9 146.4 145.1
10 153.6 153.9
11 7.01, d (8.1) 113.9 7.03, d (7.8) 113.8
12 7.29, d (8.1) 120.9 7.05, d (7.8) 124.3
12a 130.6 127.2
13 5.27, d (2.4) 51.2 5.21, d (2.4) 50.0
14 176.5 176.6
1ʹ 133.1 132.4
2ʹ 6.66, d (1.8) 112.6 5.88, d (1.8) 112.3
3ʹ 149.1 148.6
4ʹ 147.0 147.1
5ʹ 6.64, d (8.1) 116.4 6.61, d (8.4) 116.0
6ʹ 6.62, dd (8.1, 1.8) 121.5 6.31, dd (8.4, 1.8) 122.4
7ʹ 3.89, dd (6.0, 2.4) 49.4 3.65, dd (5.4, 2.4) 46.5
8ʹ 3.53, dd (6.0, 3.6) 54.6 2.92, dd (5.4, 1.2) 56.0
9ʹ 174.5 176.7
2-OCH3 3.66, s 56.6 3.99, s 57.2
9-OCH3 3.96, s 62.1 4.02, s 62.4
10-OCH3 3.87, s 56.6 3.90, s 56.8
3ʹ-OCH3 3.63, s 56.3 3.56, s 55.6
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#Data were assigned by the DEPT, HSQC, HMBC, 1H−1H COSY, and NOESY spectra.

(±)-Epi-decumicorine A (2): light-yellow crystal (absolute EtOH/H2O, 30∶1, v/v); m.p. 187–188 °C; [α]20D 2.2 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 252.1 (4.11), 276.0 (3.80), 313.9 (3.93), 375.9 (4.06) nm; IR (KBr) νmax 2999, 2941, 2835, 1717, 1670, 1609, 1556, 1518, 1493, 1464, 1431, 1398, 1373, 1313, 1273, 1227, 1161, 1080, 1001, 851, 804 cm−1. 1H and 13C NMR data, see Table 1; HRMS (ESI-TOF) m/z 532.1979 [M + H]+ (calcd. for C30H29NO8, 532.1966); (−)-2: light yellow powder; [α]20D − 139.1 (c 0.5, MeOH); ECD (MeOH) λmax (∆ε) 203.5 (− 23.8), 231.1 (6.0), 247.8 (− 2.2) nm. (+)-2: light yellow powder; [α]20D 148.4 (c 0.5, MeOH); ECD (MeOH) λmax (∆ε) 203.5 (26.7), 230.5 (− 7.1), 247.2 (2.8) nm.

ECD calculations

In general, conformational analyses were carried out via random searching in the Sybyl-X 2.0 (Tripos Associates Inc., St. Louis, MO, USA) using the MMFF94S force field with an energy cutoff of 2.5 kcal/mol. The results showed ten lowest energy conformers for both compounds. Subsequently, the conformers were re-optimized using DFT at the PBE0-D3(BJ)/def2-SVP level in MeOH using the polarizable conductor calculation model (SMD) by the ORCA4.2.1 program (Neese 2012, 2018). The energies, oscillator strengths, and rotational strengths (velocity) of the first 30 electronic excitations were calculated using the TDDFT methodology at the PBE0-D3(BJ)/def2-TZVP level in MeOH. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, sigma = 0.30 for all) (Stephens and Harada 2010). To obtain the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G). By comparing the experiment spectra with the calculated model molecules, the absolute configuration of the only chiral center was finally determined.

X-ray crystallography

Crystal Data for 1 with C30H29NO8 (M = 531.54 g/mol): monoclinic, space group P21/c (no. 14), a = 14.1060(2) Å, b = 13.1956(2) Å, c = 15.7116(2) Å, β = 100.3350(10)°, V = 2877.06(7) Å3, Z = 4, T = 150.00(10) K, µ(Cu Kα) = 0.740 mm−1, Dcalc = 1.227 g/cm3, 27,805 reflections measured (6.37° ≤ 2Θ ≤ 147.004°), 5734 unique (Rint = 0.0416, Rsigma = 0.0235) which were used in all calculations. The final R1 was 0.0491 (I > 2σ(I)) and wR2 was 0.1359 (all data), Goodness-of-fit on F2 = 1.041.

Crystal Data for (−)-1 with C30H38NO12.5 (C30H29NO8·4.5H2O, M = 612.61 g/mol): orthorhombic, space group P212121 (no. 19), a = 12.8256(4) Å, b = 15.2527(3) Å, c = 30.4200(7) Å, V = 5950.9(3) Å3, Z = 8, T = 149.99(10) K, µ(Cu Kα) = 0.900 mm−1, Dcalc = 1.368 g/cm3, 20,280 reflections measured (5.81° ≤ 2Θ ≤ 147.336°), 11,274 unique (Rint = 0.0292, Rsigma = 0.0424) which were used in all calculations. The final R1 was 0.1152 (I > 2σ(I)) and wR2 was 0.3400 (all data), Goodness-of-fit on F2 = 1.419, Flack/Hooft parameter = 0.03(9)/0.06(9).

Crystal Data for (+)-1 with C30H36NO12 (C30H29NO8·4H2O, M = 602.60 g/mol): orthorhombic, space group P212121 (no. 19), a = 12.7247(2) Å, b = 15.0980(3) Å, c = 30.3866(5) Å, V = 5837.76(19) Å3, Z = 8, T = 149.99(10) K, µ(Cu Kα) = 0.896 mm−1, Dcalc = 1.371 g/cm3, 24,829 reflections measured (5.816° ≤ 2Θ ≤ 147.72°), 11,219 unique (Rint = 0.0223, Rsigma = 0.0294) which were used in all calculations. The final R1 was 0.1249 (I > 2σ(I)) and wR2 was 0.3664 (all data), Goodness-of-fit on F2 = 1.611, Flack/Hooft parameter = 0.08(7)/0.11(6).

Crystal Data for 2 with C30H29NO8·H2O (M = 549.56 g/mol): monoclinic, space group I2/a (no. 15), a = 18.2529(7) Å, b = 9.8066(4) Å, c = 28.5267(11) Å, β = 91.824(4)°, V = 5103.7(3) Å3, Z = 8, T = 100.0(3) K, µ(Cu Kα) = 0.882 mm− 1, Dcalc = 1.430 g/cm3, 16,816 reflections measured (6.2° ≤ 2Θ ≤ 147.502°), 5069 unique (Rint = 0.0863, Rsigma = 0.0645) which were used in all calculations. The final R1 was 0.0682 (I > 2σ(I)) and wR2 was 0.1935 (all data), Goodness-of-fit on F2 = 1.079.

Compounds 1, (−)-1, (+)-1, and 2 were separately collected at 150(10), 149.99(10), 149.99(10), and 100(3) K on a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero equipped with an AtlasS2 CCD using Cu Kα radiation. The data were processed using CrysAlisPro (Oxford Diffraction Ltd., Abingdon, England, UK, 201024). The structures were solved by direct methods using Olex2 software (Dolomanov et al. 2009), and the non-hydrogen atoms were located from the trial structure and then refined anisotropically with SHELXL-2018 (Kratzert et al. 2015) using a full-matrix least squares procedure based on F2. The weighted R factor, wR and goodness-of-fit S values were obtained based on F2. The hydrogen atom positions were fixed geometrically at the calculated distances and allowed to ride on their parent atoms. Crystallographic data for the structures of 1, (−)-1, (+)-1, and 2 reported in this paper have been deposited at the Cambridge Crystallographic Data Center and allocated with the deposition numbers: CCDC 2106787, 2106788, 2106791, and 2106792 for compounds 1, (−)-1, (+)-1, and 2, respectively. The ORTEP diagrams of (−)-1, (+)-1, and 2 described below are derived from their crystals without hydration.

Biomimetic synthesis

A mixture of jatrorrhizine hydrochloride (3.73 mg, 0.01 mmol) and excessive ferulic acid (3.88 mg, 0.02 mmol) was dissolved in 1 mL of anhydrous methanol, and then anhydrous potassium carbonate (2.76 mg, 0.02 mmol) was added to the above solution under temperature of 38 °C. The reaction was monitored by LC-MS for a complete conversion of jatrorrhizine hydrochloride (6 days). The reaction mixture was first neutralized by FA (1.5 µL, 0.04 mmol), and then was isolated by semipreparative HPLC using MeCN‒H2O (20∶80, v/v, H2O containing 0.1% FA) to give two compounds syn-1 (1.07 mg) and syn-2 (3.10 mg), of which MS spectra, 1 H/13C NMR data, and chiral-HPLC chromatograms were identical to those of compounds 1 and 2 (see the supporting information for details). Other experimental conditions (Bradsher and Day 1971; Cavé et al. 1980; Gupta and Franck 1987; Mallamo et al. 1994) were also investigated as shown in Table 2.

Table 2.

Experimental conditions studied for Diels-Alder [4 + 2] reaction

No. Reaction conditions Products and isolated yields
1 Anhydrous MeOH and K2CO3, 38 °C, 6 days (±)-1 and (±)-2 (1∶3), ~ 78.3% yield
2 Anhydrous MeOH and CaCO3, N2, 38–50 °C, 6 days No reaction
3 Bromobenzene, 100 °C, 10–24 h No reaction
4 ACN, hydroquinone, 5 days No reaction
5 ACN, reflux heat, N2, 16 h No reaction
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Inhibition of entry of SARS-CoV-2 pseudovirus into HEK-293T-ACE2h cells (Guo et al. 2021; Zhang et al. 2021)

Firstly, the HEK-293T-ACE2h cells (Sino Biological, Beijing, China) were seeded in 96-well plates at 1 × 104 cells in 100 µL DMEM per well. The cells were cultured in a 37 °C incubator containing 5% CO2 for 24 h. 30 µL of SARS-CoV-2 spike pseudovirus (Sino Biological, Beijing, China, PSV001) diluted in 70 µL of medium was mixed with 100 µL either compounds-containing or SARS-CoV-2 antibody-containing media. The above 200 µL of pseudovirus and compound (antibody) mixture solution was then immediately added into each well to infect cells for 2 h incubation in the 37 °C incubator. After the inoculum was disposed, cells were then overlaid with 100 µL fresh DMEM media, and incubated for 48 h at 37 °C with 5% CO2. After that, the culture medium was removed and 100 µL of cell lysate was added into each well. After 10 min, the luciferase luminescence was measured at 568 nm by a microplate reader immediately after 100 µL of luminescence solution was added into each well according to the instruction from the Firefly Luciferase Reporter Gene Assay Kit (Beyotime, RG005). HEK293T/hACE2 cells infected only with SARS-CoV-2 pseudovirus were utilized as control group, and the luciferase luminescence value of the pseudovirus control group was defined as 100%, and the values of luminescence of either compound-treated or antibody-treated groups were normalized accordingly. Data were from three independent experiments.

Bio-layer interferometry (BLI)

The binding kinetics of the isolated compounds and ACE2 protein with the protein of SARS-CoV-2 spike RBD were analyzed by bio-layer interferometry on an Octet system (ForteBio, Octet RED96). A histidine-tagged SARS-CoV-2 spike RBD protein (25 µg/mL aqueous solution, Sino Biological, Cat 40592-V08H) was immobilized on a nickel charged nitrilotriacetic acid (Ni-NTA) biosensors (Fortebio, Inc) by a protein-loading program of the instrument. Each stock solution of samples (50 mM in DMSO) was serially diluted by PBS buffer with a final DMSO concentration of 0.2%. Protein-immobilized biosensors were equilibrated in PBS buffer for 10 min at room temperature before preceding data acquisition, and all experiments were performed at 30 °C. The Spike RBD-coated biosensors were dipped in wells containing serially diluted samples, along with ACE2 protein (positive control). The signal of background was subtracted from all samples by dipping a protein-immobilized biosensor in the blank buffer. The subtracted sensorgrams were then fitted to a 1:1 binding model by using Octet Data Analysis Software v11.1 (ForteBio) to calculate the equilibrium dissociation constant (KD) values.

Molecular docking study

CDOCKER of Discovery Studio software was utilized to conduct molecular docking study on the structure of viral spike RBD from SARS-CoV-2 (PDB ID: 6M0J) with compound (+)-1. The active contact sites were determined to be GLN493, GLN498, TYR453, and SER494 of the spike RBD with ACE2 receptor according to the reported literature (Ding et al. 2020; Shang et al. 2020). The binding mode was analyzed with the Discovery Studio software.

Results

Structure elucidation

(±)-Decumicorine A (1) was obtained as a yellowish-brown crystal (MeOH/H2O, 5∶1, v/v; m.p. 177–178 °C). Its molecular formula was determined to be C30H29NO8 by HR-ESI-TOF-MS analysis (m/z 532.1945 [M + H]+, calcd 532.1966), revealing 17 unsaturated degrees in 1. Analysis of the 1H NMR data (Table 1) showed the presence of an ABX spin system at δH 6.66 (d, J = 1.8 Hz, H-2′), 6.64 (d, J = 8.1 Hz, H-5′), and 6.62 (dd, J = 8.1, 1.8 Hz, H-6′), an AB spin system at δH 7.01 (d, J = 8.1 Hz, H-11) and 7.29 (d, J = 8.1 Hz, H-12), two aromatic singlets at δH 6.69 (s, H-4) and 6.83 (s, H-1), four methoxy groups (each s, δH 3.66, 3.96, 3.87, and 3.63), two coupled methylenes at δH 3.10 and 3.03 (both m, H2-5), and δH 4.40 and 4.15 (both m, H2-6), and four continuously linked methines at δH 6.00 (d, J = 3.6 Hz, H-8), 3.53 (dd, J = 6.0, 3.6 Hz, H-8′), 3.89 (dd, J = 6.0, 2.4 Hz, H-7′), and 5.27 (d, J = 2.4 Hz, H-13). Comprehensive interpretation of the 13 C NMR, DEPT, and HSQC spectra revealed four methoxy groups, two methylenes, four aliphatic methines, eighteen aromatic carbons (eleven aromatic non-protonated carbons), one carbonyl unit (δC 174.5, C-9ʹ), and one characteristic carbon (δC 176.5, C-14) for the conjugated iminium nitrogen-carbon double bonds (Cavé et al. 1980) in 1. According to the HR-TOF-MS and NMR data, compound 1 has one trisubstituted benzene unit, two (ortho- and para-) tetrasubstituted benzene units, one carbonyl group, and one conjugated iminium nitrogen-carbon double bond. These functional groups occupied 14 degrees of unsaturation, indicating the presence of three additional cyclic groups in the skeleton of 1. The 1H–1H COSY spectrum of 1 displayed homonuclear correlations for H-11/H-12, H-5′/H-6′, H2-5/H2-6, and H-8/H-8′/H-7′/H-13, which corresponded to the aforementioned coupling peaks in its 1H NMR spectrum, revealing that compound 1 contains four vicinal coupling fragments (Fig. 2, bold lines). The HMBC spectrum of 1 (Fig. 2) showed correlations from H2-5 to C-1a/C-4, H-4 to C-1a/C-5, and H-1 to C-4a/C-14, indicating that C-5 is linked with C-4a, and C-14 is connected to C-1a. The HMBC correlations between H2-6 and C-4a/C-8/C-14 as well as their chemical shift values indicated that the nitrogen atom was shared by three carbons, C-6/C-8/C-14, and formed a conjugated iminium nitrogen-carbon double bond with C-14, which produced an isoquinoline skeleton. A protoberberine skeleton fragment (Fig. 2 and 1F) was deduced from the HMBC correlations from H-8 to C-6/C-9/C-12a/C-14, H-13 to C-1a/C-8a/C-12, and H-12 to C-8a/C13. C-7′ linked with C-1′ from the trisubstituted benzene ring was assigned based on the HMBC correlations between H-7′/H-8′/H-13 and C-1′ and between H-7′ and C-2′/C-6′. Considering the molecular formula of 1, the HMBC correlation from H-7′/H-8′ to C-9′ indicated that C-8′ should be connected to the carbonyl C-9′ of a carboxylic acid group. Thus, a structural fragment containing a phenylpropanoid skeleton (Fig. 2, 2F) was established in compound 1. Furthermore, HMBC correlations between H-7′ and C-12a/C-13/C-14 and between H-8′ and C-8/C-8a suggested that the two fragments (1 F and 2 F) were connected by C-7′–C-13 and C-8′–C-8 to form a rare ring skeleton (Cavé et al. 1980; Chang et al. 2015; Santos et al. 2019; Yin et al. 2020). Therefore, the plain architectural structure of 1 was established to be a zwitterionic phenylpropanoid-conjugated protoberberine alkaloid.

Fig. 2.

Fig. 2

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Key HMBC and 1H–1H COSY correlations of compounds 1 and 2

The NOESY spectrum of 1 revealed NOE correlations between H-8ʹ and H-2′/H-6ʹ, indicating that the ABX coupling pattern at C-7ʹ and the carboxylic acid group at C-8ʹ remained in the opposite directions. The NOE correlations between H-6a and H-2ʹ/H-6ʹ suggested that the ABX spin system existed towards the isoquinoline moiety, and the carboxylic acid group occurred facing the AB coupling system (Figs. 3, S19). Thus, the relative configuration of the ABX coupling pattern at C-7ʹ was 7ʹα, and that of the carboxylic acid group at C-8ʹ was determined to be 8ʹβ. The relative configurations of C-8 and C-13 in 1 could not be determined because the NOE correlations of H-8/H-7ʹ and those of H-13/H-8′ were absent. This may be because both H-8 and H-13 coexist on the plain surface of C-8-C-8ʹ-C-7ʹ-C13, which was supported by the actual 3D structure of 1 in Fig. 4. Fortunately, a crystal that was appropriate for crystallographic analysis was generated with a mixed solvent of methanol-water (5∶1). The X-ray crystallographic analysis revealed that the relative configuration of 1 was a zwitterion, and an ORTEP diagram of 1 is shown in Fig. 4. The optical rotation of 1 is near zero, and no Cotton effect was observed in the ECD spectrum of 1. Moreover, no flack parameter was found from the crystallographic analysis of 1, indicating that 1 is a racemic mixture. Chiral resolution of 1 was performed by chiral HPLC separation (Figs. S36, S37, S38) to afford the anticipated enantiomers of (-)-1 and (+)-1, which produced the same NMR data, opposite optical rotations between (−)-1 ([α]20D -110.4) and (+)-1 ([α]20D +118.1), and mirror image-like ECD curves (Fig. 5). According to the crystallographic analysis of 1, the ECD curves of the two enantiomers (8R,13S,7′R,8ʹS-1 and 8S,13R,7ʹS,8ʹR-1) were calculated using the TDDFT theory method for the absolute configuration of (−)-1 and (+)-1 (Zhu et al. 2014; Liu et al. 2019). A comparison between the theoretically calculated data and those of the experiment could assign the absolute configurations of (−)-1 and (+)-1 as 8R,13S,7′R,8′S and 8S,13R,7′S,8′R, respectively.

Fig. 3.

Fig. 3

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Selective NOESY correlations of compounds 1 and 2

Fig. 4.

Fig. 4

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ORTEP diagrams of compounds 1 and 2

Fig. 5.

Fig. 5

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Experimental and theoretical ECD spectra of compounds (−)-1 and (+)-1 and (−)-2 and (+)-2

Luckily, the (−)-1 and (+)-1 enantiomers offered suitable crystals for crystallographic analysis under the same solvent system of methanol-water (5∶1). The absolute stereochemical structures of (−)-1 and (+)-1 were further demonstrated by X-ray crystallographic analyses with corresponding flack parameters of 0.03(9) and 0.08(7), respectively. The ORTEP diagrams of (−)-1 and (+)-1 are shown in Fig. 6.

Fig. 6.

Fig. 6

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ORTEP diagrams of compounds (−)-1 and (+)-1

(±)-Epi-decumicorine A (2) was obtained as a light-yellow crystal (absolute EtOH/H2O, 30:1, v/v; m.p. 187–188 °C). The same molecular formula of that for compound 1 (C30H29NO8) was observed by HR-ESI-TOF-MS analysis (m/z 532.1979 [M + H]+, calcd 532.1966). The 1H and 13C NMR data of 2 (Table 1) are similar to those of 1 with minor variations, in which the chemical shift difference of H-1/H-2′/H-8′ in the 1H NMR spectra is more than 0.5 ppm between 1 and 2, and those of C-3/C-8a/C-12/C-12a/C-7′/C-9′ in the 13 C NMR spectra are larger than 2 ppm between them. In addition, a slight difference in the coupling constant of H-8 was observed between 1 (δH 6.00, d, J = 3.6) and 2 (δH 6.03, d, J = 1.2). After 2D NMR (1H–1H COSY, HSQC, and HMBC) analysis, the plain structure of 2 was found to be the same as that of 1. The small differences in the 1H and 13C NMR data indicated that the chiral carbons of C-8/C-13/C-7′/C-8′ were different between 1 and 2. Thus, compound 2 was named (±)-epi-decumicorine A. Likewise, according to the NOE correlations from H-11 to H-2′/H-6ʹ, H-12 to H-2′/H-6ʹ, H-8ʹ to H-2′/H-6ʹ, and H-1 to H-7′ in 2 (Figs. 3, S35), the relative configuration of the ABX spin system at C-7ʹ occurred as 7ʹβ, and those of the carboxylic acid group at C-8ʹ existed as 8ʹα. The single crystal of 2 was produced under a mixed solvent of absolute ethanol-water (30 ∶ 1), and the ORTEP diagram of 2 is shown in Fig. 4, which exhibited opposite chiral carbons with C-7′ and C-8′ than those of 1. Similarly, compound 2 is also a racemic mixture, which was subjected to chiral HPLC (Figs. S40, S41, S42) separation to afford the two enantiomers of (−)-2 ([α]20D − 139.1) and (+)-2 ([α]20D + 148.4), and these enantiomers exhibit the same NMR data and mirror image-like ECD curves (Fig. 5). Similar to compound 1, the absolute configurations of (−)-2 and (+)-2 were confirmed to be 8R,13S,7′S,8′R and 8S,13R,7′R,8′S, respectively.

Biomimetic synthesis

A plausible biosynthetic pathway for (±)-1 and (±)-2 is shown in Fig. 7. First, based on dopamine and p-hydroxyphenylacetaldehyde derived from the precursor l-tyrosine (Weber and Opatz 2019), the key intermediate (S)-reticuline could be produced from a Mannich reaction and subsequent oxidation and methylation reactions (Grycova et al. 2007). Second, transformations from (S)-reticuline to berberine were conducted with several enzymes, including berberine bridge enzyme (BBE), methyltransferase, and (S)-tetrahydroprotoberberine oxidase (STOX) (Grycova et al. 2007). Third, berberine could be converted to jatrorrhizine by reopening its methylenedioxy group (Beecher and Kelleher 1983; Rueffer et al. 1983). In addition, p-coumaric acid, which is also derived from the precursor l-tyrosine via tyrosine ammonia-lyase (TAL), could be transformed to ferulic acid through oxidation, hydrogen transfer with nicotinamide adenine dinucleotide phosphate (NADPH), and transmethylation via S-adenosyl-l-methionine (SAM) (Zhao and Moghadasian 2008). Finally, compounds (±)-1 and (±)-2 were generated from a Diels-Alder [4 + 2] cycloaddition reaction between the intermediates of jatrorrhizine and ferulic acid (Cavé et al. 1980; Chang et al. 2015; Santos et al. 2019; Yin et al. 2020), which is critical for the occurrence of (±)-1 and (±)-2. Based on our improved method (Gupta and Franck 1987), a one-step reaction for the biomimetic synthesis of racemates (±)-1 and (±)-2 was developed using jatrorrhizine and ferulic acid as starting materials (Fig. 8). The ratio of (±)-1 versus (±)-2 was 1:3, and the isolated yield was 78.3%, as shown in Table 2.

Fig. 7.

Fig. 7

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Proposed biosynthetic pathways for compounds (±)-1 and (±)-2. BBE berberine bridge enzyme, STOX (S)-tetrahydroprotoberberine oxidase, TAL tyrosine ammonia-lyase, NADPH nicotinamide adenine dinucleotide phosphate, SAM S-adenosyl-l-methionine

Fig. 8.

Fig. 8

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Biomimetic synthesis of compounds (±)-1 and (±)-2

Anti-SARS-CoV-2 activity evaluation

Compounds (-)-1, (+)-1, (-)-2, and (+)-2 were evaluated for their antiviral activity against viral entry into HEK-293T-ACE2h cells by a SARS-CoV-2 pseudovirus inhibition assay (Guo et al. 2021; Zhang et al. 2021) within their maximum nontoxic concentration of 100 µM. Both SARS-CoV-2 antibody (IC50 = 0.031 µM) and a reported Evans blue inhibitor (IC50 = 26.95 µM) (Bojadzic et al. 2021) were employed as positive controls. As a potential anti-SARS-CoV-2 component derived from traditional Chinese medicines (TCMs), glycyrrhizic acid was also examined for its antiviral entry activity in this assay (Yu et al. 2021). Anti-SARS-CoV-2 activity screening was performed with compounds (−)-1, (+)-1, (−)-2, and (+)-2, and the results are summarized in Table 3; Fig. 9.

Table 3.

Inhibitory activities of compounds (-)-1, (+)-1, (-)-2, and (+)-2 against SARS-CoV-2 pseudotyped virus’s entry into the HEK-293T-ACE2h cells

Compounds Inhibition rate (%)
25 µM 50 µM 100 µM
(−)-1 − 3.92 ± 8.25 7 ± 5.93 17.23 ± 6.22
(+)-1 17.06 ± 6.30 19.89 ± 7.24 28.19 ± 3.61
(−)-2 − 17.20 ± 3.63 1.02 ± 7.47 23.76 ± 6.54
(+)-2 13.02 ± 2.06 10.83 ± 8.27 17.71 ± 2.83
Glycyrrhizic acid − 16.85 ± 9.88 4.01 ± 7.03 11.23 ± 6.96
Evans blue IC50 value of 26.95 ± 4.34 µM
Antibody inhibitor IC50 value of 0.031 ± 0.003 µM
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Fig. 9.

Fig. 9

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The inhibitory activities of compounds (−)-1, (+)-1, (−)-2, and (+)-2 against the entry of SARS-CoV-2 pseudovirus into the host cell of HEK-293T-ACE2h highly expressing ACE2 receptor. Both antibody (IC50 = 0.031 µM) and Evans blue (IC50 = 26.95 µM) inhibitors were used as positive controls in this assay. Glycyrrhizic acid was also examined as an antiviral entry component from TCMs. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with the group of pseudovirus control

Discussion

Currently, only four Diels-Alder [4 + 2] protoberberine cycloadducts have been discovered from C. decumbens (Chang et al. 2015; Yin et al. 2020), Duguetia staudtii (Cavé et al. 1980)d furfuracea (Santos et al. 2019), in which heterodienes (jatrorrhizine, 5,6-dehydrocoptisine, and 5,6-dehydro-13-methylcoptisine) and dienophiles (fumaric acid, 5-(hydroxymethyl)-2(3H)-furanone, and 2,4,5-trimethoxystyrene) are involved. In contrast to staudine from D. staudtii and D. furfuracea, compounds 1 and 2 contained one additional carboxylic acid group at C-8ʹ and a 4-hydroxy-3-methoxyphenyl group instead of a 2,4,5-trimethoxyphenyl moiety at C-7ʹ, increasing the number of chiral carbons in compounds 1 and 2. Therefore, ferulic acid was found to be conjugated with protoberberine alkaloid to form zwitterionic [4 + 2] cycloadducts for the first time.

Based on the biomimetic synthesis of compounds (±)-1 and (±)-2 (Fig. 8), Diels-Alder [4 + 2] cycloaddition occurs under ferulic acid (dienophile in red) and jatrorrhizine (heterodiene in red) to form a tricyclic system that contained four chiral centres and an iminium ion, and this reaction is also known as the inverse-electron-demand Diels-Alder reaction (Wang 2010; Wu and Devaraj 2016). The reaction (Fig. 8) should occur via a concerted but nonsynchronous cycloaddition that utilizes a weak charge-transfer complex along the reaction pathway (Bradsher et al. 1979). Theoretically, four racemic compounds could be formed via two types of connections (C-8/C-8ʹ, C-13/C-7ʹ, and C-8/C-7ʹ, C-13/C-8ʹ) in the Diels-Alder reaction of jatrorrhizine and ferulic acid. However, merely two racemates, 1 and 2 with C-8/C-8ʹ and C-13/C-7ʹ connections, were discovered in DCR, which was verified by a biomimetic synthesis, suggesting that electronic and steric effects might occur for their paired selectivity (Brocksom et al. 2001). Compounds 1 and 2 should not be artefacts during the isolation process because the compounds were directly detected by LC‒MS analysis with the methanol extract of DCR without heating (sonicate for 30 minutes at room temperature) (Fig. S44), and they were not produced via the reaction of jatrorrhizine and ferulic acid in the other four experimental conditions presented in Table 2. Jatrorrhizine is among the main constituents in DCR (Mao et al. 2017). Ferulic acid was also detected in the methanol extract from the crude drug (Fig. S45). As shown in Fig. S44, the amount of compound 2 is more dominant than that of compound 1 in the crude DCR, which is consistent with the biomimetic synthesis result. These results may because less steric effects occur in 2 than in 1 between the ABX spin system at C-7ʹ and the isoquinoline group.

Based on the results of the bioactivity screening (Table 3; Fig. 9), compound (+)-1 reduced the pseudovirus’s entry ratio from 17.06 to 28.19% under the tested concentrations (25, 50, 100 µM) in a dose-dependent manner. Under the treatment of 100 µM, compounds (−)-1, (−)-2, and (+)-2 also showed antiviral entry activity with inhibitory rates of 17.23%, 23.76%, and 17.71%, respectively. The antiviral entry activities of the four alkaloids were more potent than that of glycyrrhizic acid (inhibition rate of 11.23% at 100 µM). The binding interaction of compound (+)-1 with the receptor binding domain (RBD) of the viral spike protein was subsequently determined by a biolayer interferometry (BLI) binding assay with human ACE2 protein as a positive control (KD = 19.39 nM). Compound (+)-1 bound to the SARS-CoV-2 spike RBD protein with a KD value of 22.2 µM (Fig. 10). A molecular docking study further confirmed that compound (+)-1 could bind well to the spike RBD with a calculated interaction energy of − 31.8204 kcal/mol (Fig. 11). This indicated that compound (+)-1 exhibited a specific stereochemical structure-activity relationship for targeting the spike protein to perform antiviral entry activity against SARS-CoV-2.

Fig. 10.

Fig. 10

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Dose-response curves of A human ACE2 protein and B compound (+)-1 binding to SARS-CoV-2 spike RBD protein. The concentrations were 0.03125 ~ 0.5 nM for human ACE2 protein, and 3.12 ~ 100 µM for Compound (+)-1, respectively

Fig. 11.

Fig. 11

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Docking simulation of compound (+)-1 in the active sites of SARS-CoV-2 spike RBD protein (PDB code: 6M0J). A 3D interaction model of (+)-1 with spike RBD protein. B 2D interaction model of (+)-1 with spike RBD protein. C CDOCKER interaction energy of (+)-1 with spike RBD protein. The result revealed that compound (+)-1 interacts with the amino residues of GLN493, GLN498, TYR453, and SER494 in spike RBD protein with a calculated CDOCKER interaction energy of − 31.8204 kcal/mol

In this study, phenylpropanoid-conjugated protoberberine alkaloids were shown to suppress SARS-CoV-2 viral entry by targeting the spike RBD. During the SARS-CoV-2 pandemic, targeting the SARS-CoV-2 spike was considered a safe and promising strategy for the research and development of anti-SARS-CoV-2 reagents/drugs. The phenylpropanoid-conjugated protoberberines could be a new type of chemical with antiviral effects by targeting spike protein, which might be helpful for the rational design and synthesis of more potent antiviral molecules that target spike protein.

In summary, we reported two pairs of enantiomeric isoquinoline alkaloids that possess a novel carbon skeleton of phenylpropanoid-conjugated protoberberine from the rhizomes of C. decumbens. The absolute configurations of compounds (−)-1, (+)-1, (−)-2, and (+)-2 were determined by NMR, ECD calculations and X-ray crystallographic analyses. The biomimetic syntheses of (±)-decumicorine A and (±)-epi-decumicorine A were validated by a one-step Diels-Alder [4 + 2] cycloaddition reaction. Compound (+)-1 demonstrated potential anti-SARS-CoV-2 activity by preventing viral spikes from attaching to the ACE2 receptor on host cells.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 16444 KB) (16.1MB, docx)
Supplementary file2 (RAR 798 KB) (797.6KB, rar)

Acknowledgements

The authors would like to acknowledge the undergraduate students of Li Y.-L. and Zhao M. from the Faculty of Chinese Medicine (Macau University of Science and Technology) for their assistance in sample preparation. This research was financially supported by The Science and Technology Development Fund, Macau SAR (File No. 0065/2020/A2). This work was also supported by the Emergency Key Program of Guangzhou Laboratory (Grant No. EKPG21-06). The authors also thank the Department of Science and Technology of Guangdong Province for the support of Guangdong-Hong Kong-Macao Joint Laboratory of Respiratory Infectious Disease.

Declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Footnotes

Publisher’s Note

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Contributor Information

Li-Ping Bai, Email: lpbai@must.edu.mo.

Guo-Yuan Zhu, Email: gyzhu@must.edu.mo.

Zhi-Hong Jiang, Email: zhjiang@must.edu.mo.

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