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Flavonoids from the roots and rhizomes of Sophora tonkinensis and their in vitro anti-SARS-CoV-2 activities

  • Acute respiratory infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have caused a global pandemic since 2019, and posed a serious threat to the world public health security. Traditional Chinese medicine (TCM) has played an indispensable role in the battle against the epidemic. Many components original from the TCMs were found to inhibit the production of SARS-CoV-2 3C-like protease (3CLpro) and papain-like protease (PLpro), which are two promising therapeutic targets to inhibit the SARS-CoV-2. This study describes a systematic investigation of the roots and rhizomes of Sophora tonkinensis, which resulted in the characterization of 12 new flavonoids, including seven prenylated flavanones ( 17 ), one prenylated flavonol ( 8 ), two prenylated chalcones ( 910 ), one isoflavanone ( 11 ), and one isoflavan dimer ( 12 ), together with 43 known compounds ( 1355 ). Their structures including the absolute configurations were elucidated by comprehensive analysis of MS, 1D and 2D NMR data, and time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculations. Compounds 12 and 51 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC50 values of 34.89 and 19.88 μmol·L−1, and compounds 9 , 43 and 47 exhibited inhibitory effects against PLpro with IC50 values of 32.67, 79.38, and 16.74 μmol·L−1, respectively.
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Flavonoids from the roots and rhizomes of Sophora tonkinensis and their in vitro anti-SARS-CoV-2 activities

    Corresponding author: E-mails: suhaixia1@simm.ac.cn (Su Haixia); yaosheng@simm.ac.cn (YAO Sheng); yye@simm.ac.cn (YE Yang)
  • 1. School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
  • 2. State Key Laboratory of Drug Research, and Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
  • 3. School of Pharmacy, University of Chinese Academy of Sciences, Beijing 100049, China
  • 4. Discovery and Design Center, CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
  • 5. School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

Abstract: Acute respiratory infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have caused a global pandemic since 2019, and posed a serious threat to the world public health security. Traditional Chinese medicine (TCM) has played an indispensable role in the battle against the epidemic. Many components original from the TCMs were found to inhibit the production of SARS-CoV-2 3C-like protease (3CLpro) and papain-like protease (PLpro), which are two promising therapeutic targets to inhibit the SARS-CoV-2. This study describes a systematic investigation of the roots and rhizomes of Sophora tonkinensis, which resulted in the characterization of 12 new flavonoids, including seven prenylated flavanones ( 17 ), one prenylated flavonol ( 8 ), two prenylated chalcones ( 910 ), one isoflavanone ( 11 ), and one isoflavan dimer ( 12 ), together with 43 known compounds ( 1355 ). Their structures including the absolute configurations were elucidated by comprehensive analysis of MS, 1D and 2D NMR data, and time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculations. Compounds 12 and 51 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC50 values of 34.89 and 19.88 μmol·L−1, and compounds 9 , 43 and 47 exhibited inhibitory effects against PLpro with IC50 values of 32.67, 79.38, and 16.74 μmol·L−1, respectively.

    • Coronavirus disease-2019 (COVID-19) [1] is a pandemic disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high contagious, high human-to-human transmissibility, and significant mortality. The ongoing outbreak of 2019-nCoV has been declared a public health emergency of international concern by the World Health Organization. Two cysteine proteases, a chymotrypsin-like cysteine protease called 3C-like protease (3CLpro) and a papain-like protease (PLpro), responsible for proteolysis of new virions, were believed to be essential for the design of therapeutic targets [2, 3]. 3CLpro is also referred as the main protease (Mpro) because it plays a key role in mediating viral replication and transcription [4]. Thus, it is crucial to disclose more structurally diverse small molecules as 3CLpro and PLpro inhibitors in the drug discovery for COVID-19.

      Traditional Chinese medicines have provided effective and continuous prevention and treatment in the fight against the epidemic. Many natural compounds original from the medicinal herbs showed inhibitory activity against SARS-CoV-2 (Fig. 1) [5-7], and the inhibitory activity against SARS-CoV-2 3CLpro and PLpro are the most studied. For example, baicalin and baicalein, two natural flavonoids isolated from Scutellaria baicalensis[8], and four polyphenols from the fermentation products of Sanghuangporus sanghuang[9] exhibited promising SARS-CoV-2 3CLpro inhibitory activity. Eight natural compounds found in rhizomes of Alpinia officinarum and ginger were identified as potential inhibitors of SARS-CoV-2 PLpro[10]. Additionally, chloroquine[11] and glycyrrhizin[12], two natural compounds obtained from Cinchona officinalis and Glycyrrhiza glabra, respectively, are both efficient to block the interaction of RBD of SARS-CoV to ACE-2.

      Figure 1.  Representative natural products showing inhibitory activity against SARS-CoV-2 3CLpro and PLpro. Compounds were isolated from a: Scutellaria baicalensis, b: Sanghuangporus sanghuang, c: Alpinia officinarum, d: Cinchona officinalis

      In our previous research, the oral liquid and the lyophilized powder of the Shuang-Huang-Lian injection, and their bioactive components were found to dose-dependently inhibit SARS-CoV-2 3CLpro, and the replication of SARS-CoV-2 in Vero E6 cells as well. Another investigation showed that the pyrogallol group of a natural flavonoid named myricetin emerged as a warhead that could covalently linked to cysteine under the condition of oxidation, which provides the vital clue to understand the diverse bioactivities of natural products and identifying the phenolic natural products as covalent ligands [13]. In addition, a native MS-based affinity-selection method named bio-affinity-MS (BA-MS) was established in our lab, and was used successfully to discover three flavonoids, baicalein, baicalein and glycyrrhizin from the herbal extracts as potential noncovalent inhibitors against SARS-CoV-2 3CLpro [14].

      In the high-throughput screening of natural products in our in-house compound library, we found that the extract of a plant named Sophora tonkinensis showed inhibitory effects against 3CLpro. Sophora tonkinensis Gagnep. (Leguminosae), known as “Shan-Dou-Gen” in Chinese, widely distributed in Southern China and Vietnam. Its rhizomes and roots have long been used as a traditional Chinese medicine for the treatment of throat-swelling and acute pharyngolaryngeal infections [15]. Previous investigations revealed that the plant is abundant in prenylated flavonoids [16-18] and quinolizidine alkaloids [19-21]. To search for new flavonoids with anti-SARS-CoV-2 activities, the EtOH extract of this plant was investigated, and the active fractions with inhibitory effects on 3CLpro and PLpro were selected for isolation, resulting in the characterization of 12 undescribed flavonoids (Fig. 2), including seven prenylated flavanones (17), one prenylated flavonol (8), two prenylated chalcones (910), one isoflavanone (11), and one isoflavan dimer (12), together with 43 known analogs (1355) (Fig. S1, Supporting Information). Their planar structures were elucidated by extensive analysis of spectroscopic data and DFT NMR calculations. The absolute configurations were further substantiated by TDDFT ECD calculations. It is noteworthy that 2 and 11 are two pairs of enantiomers, and their racemic nature were resolved by chiral HPLC, which permitting online stereochemical characterization, when in hyphenation with circular dichroism (CD) spectroscopy (LC-CD coupling). All the compounds were evaluated for the anti-SARS-CoV-2 activities by suppressing the 3CLpro and PLpro production.

      Figure 2.  Structures of compounds 112

    Materials and Methods
    • IR spectra were measured with a Thermo Nicolet FTIR IS5 spectrometer (Thermo Fisher, USA). UV spectra were measured on an Agilent Cary 300 Series UV-Vis spectrophotometer (Agilent, USA). Optical rotations were recorded with a Rudolph Research Analytical Autopol VI 90079 polarimeter (Hackettstown, NJ). ECD spectra were recorded on a Jasco-815 CD spectropolarimeter (JASCO, Japan). HR-ESI-MS spectra were obtained on a Waters Synapt G2-Si Q-TOF Mass Spectrometer. NMR spectra were collected on a Bruker AVANCE III 500 or 600 MHz instrument (Bruker Biospin AG, Switzerland). Chemical shifts were reported in ppm (δ) with coupling constants (J) in Hertz. Analytical HPLC was performed on a Waters 2695 instrument with a Waters 2998 PDA detector, a Waters 2424 ELSD, and Waters 3100 MS detectors. Preparative HPLC was run on a Waters system with an Alltech 2424 ELSD and 2489 PDA using a Waters Sunfire RP C18 column (5 μm, 30 mm × 150 mm, flow rate 30 mL·min−1, acetonitrile−water). MCI gel CHP20P (75−150 μm, Mitsu bishi Chemical Industries, Japan). Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), and Silica gel (100–200, 200–300 and 300–400 mesh, Qingdao Marine Chemical Co., China) were used for column chromatography (CC). TLC analyses were carried out on precoated GF254 silica gel plates (Yantai Chemical Industrials, Yantai, China), and the TLC spots were observed at 254 nm and visualized by 5% sulfuric acid in alcohol containing 10 mg·mL−1 of vanillin followed by heating. All solvents were of analytical grade (Sinopharm Chemical Reagents Co., Ltd., Shanghai, China) for column chromatography, and of HPLC grade (Merck KGaA, Darmstadt, Germany and Ourchem, Shanghai, China) for HPLC and preparative HPLC.

    • The roots and rhizomes of Sophora tonkinensis Gapnep. were purchased from Shanghai Tongrentang Pharmaceutical Co., Ltd. (Shanghai, China), and authenticated by Mr. ZHANG Xiaoming of Shanghai Kangqiao Medicine Yinpian Co., Ltd. (Shanghai, China). A voucher specimen (No. 20210401) was deposited at the herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

    • The dried roots and rhizomes of S. tonkinensis (10 kg) were extracted with 95% EtOH at room temperature three times (7 days each). The percolates were combined and evaporated under reduced pressure to give a crude extract (680 g). The extract was then suspended in water, and partitioned successively with petroleum ether (PE) and ethyl acetate (EA) for three times, yielding fractions PE (Fr. A, 120.0 g), EA (Fr. B, 244.0 g), and water-soluble fraction (Fr. C). Fraction B was chromatographed over an MCI column eluted with aqueous MeOH (30%, 50%, 70% and 100%) to give Frs. B1−B4. Fr. B4 (126 g) was then subjected to MCI column chromatography (CC), eluting with EtOH in water (70%, 75%, 80%, 85%, 90%, 95%) to afford Frs. B4A to B4J. Fr. B4C (14.0 g) was separated by a Sephadex LH-20 gel column (CHCl3 : MeOH, 1 : 1, V/V) to afford Frs. B4C1−B4C7. All fractions were evaluated by the inhibitory effects against the SARS-CoV-2 3CLpro and PLpro with IC50 values, among them, Fr. B4C6 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC50 values of 6.22 ± 0.36 μg·mL−1, Fr. B4G and Fr. B4H exhibited inhibitory effects against PLpro with IC50 values of 17.52 ± 0.01, 36.10 ± 0.45 μg·mL−1, respectively. Fr. B4C6 (1.6 g) was then subjected to silica gel CC using a solvent mixture of PE/EA (5 : 1 to 1 : 1, V/V), and further purified by preparative HPLC (30%−50% CH3CN in H2O, 25 min), yielding seven compounds 11 (3.8 mg), 12 (6.5 mg), 41 (3.5 mg), 42 (1.4 mg), 44 (9.2 mg), 54 (98.2 mg), and 55 (20.2 mg). Fr. B4H (24.1 g) was fractionated using a Sephadex LH-20 gel (MeOH) column to provide Frs. B4H1−B4H6. Fr. B4H4 (345.0 mg) was applied to a Sephadex LH-20 gel column (CHCl3 : MeOH, 1 : 1, V/V) to give subfractions B4H4A−B4H4F. Fr. B4H4A (10.0 mg) was purified by preparative HPLC (70%−90% CH3CN in H2O, 25 min), yielding compound 46 (2.1 mg). Frs. B4H4C (40.9mg) and B4H4D (18.3 mg) were further purified using preparative HPLC (55%−75% CH3CN in H2O, 25 min), respectively, to yield compounds 16 (1.9 mg), 18 (2.1 mg), 27 (2.0 mg), and 29 (8.5 mg). Fr. B4G (14.3 g) was combined with Fr. B4H6 (4.1 g), and then purified by Sephadex LH-20 column eluting with MeOH to give subfractions B4G1−B4G6. Fr. B4G2 (3.8 g) was treated using a Sephadex LH-20 gel (MeOH) column to yield Frs. B4G2A−B4G2F. Fr. B4G2E (2.4 g) was disposed repeatedly with silica gel CC using CH2Cl2/MeOH (200 : 1 to 50 : 1, V/V), and finally purified by preparative HPLC (60%−80% CH3CN in H2O, 25 min), yielding compounds 3 (44.0 mg), 7 (3.5 mg), 17 (20.5 mg), 20 (4.1 mg), 21 (4.7 mg), 28 (9.7 mg), 33 (178.8 mg), 34 (123.5 mg), 37 (14.2 mg), and 48 (11.1 mg). Fr. B4G3 (2.1 g) was separated repeatedly by silica gel CC using a solvent mixture of CH2Cl2/acetone (150 : 1 to 50 : 1, V/V), and finally by preparative HPLC (65%−85% CH3CN in H2O, 25 min), affording compounds 1 (22.6 mg), 4 (5.6 mg), 5 (2.4 mg), 6 (17.5 mg), 14 (24.0 mg), 19 (5.1 mg), 22 (23.4 mg), 35 (3.6 mg) and 38 (8.9 mg). Fr. B4G4 (4.4 g) was fractionated using a Sephadex LH-20 gel column (CHCl3 : MeOH, 1 : 1, V/V) to provide Frs. B4G4A−B4G4C. Fr. B4G4C (785.7 mg) was separated repeatedly by silica gel CC eluting with CH2Cl2/MeOH (200 : 1 to 50 : 1, V/V), and finally purified by preparative HPLC (60%−80% CH3CN in H2O, 25 min) to give compounds 15 (3.3 mg), 30 (31.5 mg), 31 (21.9 mg), and 36 (8.7 mg). Fr. B4G4D (638.2 mg) and B4G4E (1.1 g) were treated with the similar procedures to yield compounds 8 (11.0 mg), 23 (22.9 mg), 24 (11.3 mg), 25 (2.2 mg), 26 (16.9 mg), 32 (75.1 mg) and 2 (2.4 mg), 13 (7.1 mg), 43 (1.3 mg), 52 (55.5 mg), and 53 (15.1 mg).

      Sophotonin A (1). Yellow powder; $[\text{α} ]_{\rm{D}}^{20}$ −77 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (5.78), 283 (5.36) nm; ECD (MeOH) (Δε) 305 (−5.66), 335 (+2.61) nm; IR (KBr) νmax 3337, 2967, 2913, 1655, 1594, 1437, 1374, 1272, 1215, 1174, 1118, 1085 cm−1; 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z 423.2174 [M + H]+ (Calcd. for C26H31O5, 423.2171).

      No.1b2b3a4a5a6a
      25.64, dd (12.4, 3.2)5.62, dd (13.1, 3.0)5.50, dd (13.1, 3.0)5.36, dd (13.2, 3.0)5.35, dd (13.3, 2.9)5.33, dd (13.2, 3.0)
      32.94, dd
      (17.0, 12.4), α
      3.08, dd
      (17.2, 13.1), α
      3.05, dd
      (16.9, 13.1), α
      2.98, dd
      (16.9, 13.2), α
      3.02, dd
      (16.9, 13.3), α
      2.99, dd
      (16.9, 13.2), α
      2.79, dd
      (17.0, 3.2), β
      2.76, dd,
      17.2, 3.0), β
      2.86, dd
      (16.9, 3.0), β
      2.79, dd
      (16.9, 3.0), β
      2.81, dd
      (16.9, 2.9), β
      2.80, dd
      (16.9, 3.0), β
      57.75, d (8.8)7.76, d (8.6)7.75, d (8.6)7.76, d (8.6)7.73, d (8.6)
      66.73, d (8.8)6.54, d (8.6)6.55, d (8.6)6.54, d (8.6)6.54, d (8.6)
      2'7.16, d (1.8)7.29, d (2.4)7.42, d (2.2)6.95, d (2.2)
      3'6.35, s6.33, d (2.3)
      5'6.35, dd (8.3, 2.3)
      6'7.12, s7.26, d (8.3)7.48, d (1.8)7.73, d (2.4)6.96, d (2.2)7.09, d (2.2)
      1''3.34, d (7.3)6.54, d (10.0)3.42, d (7.4)3.40, d (7.3)3.43, d (7.3)3.41, d (7.2)
      2''5.17, t (7.3)5.53, d (10.0)5.26, t (7.4)5.24, t (7.3)5.26, t (7.3)5.26, t (7.2)
      4''1.62, s1.44, s1.74, s1.73, d (1.3)1.75, d (1.4)1.76, s
      5''1.67, s1.41, s1.74, s1.73, d (1.3)1.74, d (1.4)1.73, s
      6''3.20, d (7.3)3.21, d (7.3)3.63, d (7.4)6.38, d (10.0)6.33, d (9.8)6.47, d (9.9)
      7''5.27, t (7.3)5.17, t (7.3)5.41, t (7.4)5.77, d (10.0)5.68, d (9.8)5.61, d (9.9)
      9''1.70, s1.66, d (1.3)1.76, d (1.4)1.53, s1.48, s3.70, d (11.8)
      3.53, d (11.8)
      10''1.64, s1.78, d (1.3)1.79, d (1.4)1.53, s1.48, s1.42, s
      11''6.60, s10.46, s6.83, d (16.4)3.41, d (15.4)
      3.18, dd (15.4, 7.2)
      12''6.93, d (16.4)5.26, t (7.2)
      14''1.69, s5.09, each d (16.5)1.74, d (1.4)
      15''1.69, s2.00, s1.72, d (1.4)
      7-OCH33.90, s
      a Measured in CDCl3; b Measured in CD3OD

      Table 1.  1H NMR (500 MHz) data of compounds 16 (δ in ppm, J in Hz)

      No.1b2b3a4a5a6a
      276.5, CH76.2, CH80.2, CH79.1, CH79.8, CH79.5, CH
      344.2, CH243.2, CH244.8, CH244.0, CH244.3, CH244.1, CH2
      4195.2, C199.1, C191.7, C191.1, C191.6, C192.0, C
      5127.2, CH157.2, C126.7, CH126.7, CH126.6, CH126.6, CH
      6105.9, CH103.0, C110.7, CH110.9, CH110.7, CH110.6, CH
      7164.9, C160.8, C161.5, C161.6, C161.4, C161.7, C
      8118.9, C110.3, C114.7, C114.8, C114.7, C115.0, C
      9162.4, C162.1, C160.9, C160.7, C160.9, C161.1, C
      10116.3, C103.4, C115.2, C115.1, C115.2, C115.0, C
      1'118.0, C117.9, C134.2, C131.6, C131.2, C131.5, C
      2'154.2, C156.9, C122.1, CH129.8, CH124.1, CH122.6, CH
      3'103.3, CH103.5, CH125.6, C122.9, C121.8, C120.9, C
      4'56.7, C159.8, C153.3, C156.5, C150.6, C150.3, C
      5'120.4, C107.8, CH128.4, C124.1, C125.8, C129.4, C
      6'128.4, CH128.7, CH116.4, CH125.0, CH123.4, CH128.0, CH
      1''23.0, CH2117.0, CH22.4, CH222.4, CH222.5, CH222.4, CH2
      2''123.2, CH127.2, CH121.2, CH121.2, CH121.2, CH121.4, CH
      3''132.5, C79.1, C135.3, C135.4, C135.5, C134.7, C
      4''26.0, CH328.7, CH325.9, CH325.9, CH326.0, CH326.0, CH3
      5''18.0, CH328.4, CH318.0, CH318.0, CH318.1, CH318.0, CH3
      6''28.6, CH221.7, CH228.4, CH2121.4, CH122.4, CH125.2, CH
      7''124.5, CH123.8, CH121.4, CH132.1, CH131.4, CH127.3, CH
      8''132.3, C131.7, C133.7, C78.3, C77.0, C79.7, C
      9''26.0, CH325.9, CH326.0, CH328.5, CH328.3, CH368.3, CH2
      10''17.8, CH318.0, CH318.1, CH328.5, CH328.3, CH322.9, CH3
      11''100.8, CH189.1, CH122.8, CH29.1, CH2
      12''163.9, C132.7, CH122.8, CH
      13''69.6, C142.7, C133.2, C
      14''28.9, CH3117.3, CH225.8, CH3
      15''28.9, CH318.7, CH318.0, CH3
      7-OCH356.4, CH3
      a Measured in CDCl3; b Measured in CD3OD

      Table 2.  13C NMR (126 MHz) data of compounds 16 (δ in ppm)

      Sophotonin B (2). Brown solid; $[\text{α} ]_{\rm{D}}^{20}$ −2 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (5.53), 274 (5.32) nm; ECD (2a; hexane/isopropanol) (Δε) 303 (+0.22), 333 (−0.04) nm; ECD (2b; hexane/isopropanol) (Δε) 303 (−0.35), 333 (+0.06) nm; IR (KBr) νmax 3371, 2975, 1624, 1444, 1410, 1383, 1305, 1167, 1117 cm−1; 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z 423.1795 [M + H]+ (Calcd. for C25H27O6, 423.1808).

      Sophotonin C (3). White powder; $[\text{α} ]_{\rm{D}}^{20}$ −76 (c 0.2, MeOH); UV (MeOH) λmax (log ε): 216 (5.48), 241 (5.13), 287 (4.91) nm; ECD (MeOH) (Δε) 297 (−10.27), 330 (+6.40) nm; IR (KBr) νmax 3271, 2980, 2915, 1661, 1588, 1444, 1286, 1169, 1135, 1106, 1046 cm−1; 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z 475.2480 [M + H]+ (Calcd. for C30H35O5, 475.2484).

      Sophotonin D (4). Yellow gum; $[\text{α} ]_{\rm{D}}^{20}$ −90 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 218 (5.58), 236 (5.46), 277 (5.19) nm; ECD (MeOH) (Δε) 288 (−8.29), 320 (+4.99) nm; IR (KBr) νmax 3282, 2974, 2925, 1683, 1589, 1443, 1375, 1337, 1286, 1208, 1150, 1109, 1070, 1045 cm−1; 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z 419.1859 [M + H]+ (Calcd. for C26H27O5, 419.1858).

      Sophotonin E (5). White powder; $[\text{α} ]_{\rm{D}}^{20}$ −80 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (5.56), 281 (5.41) nm; ECD (MeOH) (Δε) 297 (−7.81), 329 (+4.10) nm; IR (KBr) νmax 3269, 2973, 2925, 1660, 1588, 1442, 1378, 1336, 1282, 1151, 1107, 1045 cm−1; 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z 457.2372 [M + H]+ (Calcd. for C30H33O4, 457.2379).

      Sophotonin F (6). White powder; $[\text{α} ]_{\rm{D}}^{20}$ −69 (c 0.2, MeOH); UV (MeOH) λmax (log ε): 228 (5.45), 281 (4.92) nm; ECD (MeOH) (Δε) 297 (−9.81), 330 (+4.33) nm; IR (KBr) νmax 3272, 2969, 2923, 1660, 1588, 1441, 1374, 1337, 1284, 1209, 1150, 1106, 1046 cm−1; 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z 475.2842 [M + H]+ (Calcd. for C30H35O5, 475.2484).

      Sophotonin G (7). White powder; $[\text{α} ]_{\rm{D}}^{20}$ −67 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 223 (5.54), 279 (5.03) nm; ECD (MeOH) (Δε) 214 (+22.67), 230 (−10.34), 240 (+2.87), 300 (−6.48), 330 (+1.64) nm; IR (KBr) νmax 3408, 2973, 2926, 1668, 1607, 1457, 1376, 1308, 1258, 1211, 1150, 1096, 1070 cm−1; 1H and 13C NMR data see Tables 3 and 4; HR-ESI-MS m/z 491.2437 [M + H]+ (Calcd. for C30H35O6, 491.2434).

      No.7a, e8c, e9b, e10a, e11b, d12b, f
      α7.98, d (15.4)8.07, d (15.3)
      β7.73, d (15.4)7.61, d (15.3)
      25.37, dd (13.3, 2.9)4.52, dd (10.9, 10.9)4.14, ddd (10.1, 3.3, 1.9)
      4.39, dd (10.9, 5.5)3.87, dd (10.1, 10.1)
      33.09, dd (16.9, 13.3), α6.37, d (2.4)6.34, s4.24, dd (10.9, 5.5)3.42, dddd (11.0, 10.1, 5.4, 3.3)
      2.78, dd (16.9, 2.9), β
      42.78, dd (15.9, 11.0)
      2.66, ddd (15.9, 5.4, 1.9)
      57.90, d (8.6)7.87, d (8.6)6.35, dd (8.6, 2.4)6.55, s
      66.57, d (8.6)7.04, d (8.6)7.41, d (8.6)7.28, s5.90, d (2.2)
      85.88, d (2.2)6.37, s
      2'6.92, d (2.3)8.01, s
      3'6.41, s6.24, s
      5'6.40, d (8.9)
      6'7.04, d (2.3)8.01, s7.57, s7.72, d (8.9)6.55, s6.54, s
      1''5.52, d (4.7)3.71, d (7.2)3.37, d (7.1)3.30, d (7.2)
      2''4.43, d (4.7)5.40, t (7.2)5.20, t (7.1)5.29, t (7.2)4.17, dd (10.7, 3.3)
      4.02, dd (10.7, 7.1)
      3''3.62–3.56, m
      4''1.37, s1.83, s1.67, d (1.4)1.78, s4.45, d (7.3)
      5''1.27, s1.66, s1.79, d (1.4)1.78, s6.74, s
      6''6.30, d (9.8)3.46, d (7.3)3.28, d (7.4)3.47, d (7.2)
      7''5.64, d (9.8)5.40, t (7.3)5.35, t (7.4)5.29, t (7.2)
      8''6.32, s
      9''1.44, s1.76, s1.82, d (1.3)1.76, s
      10''1.44, s1.75, s1.75, d (1.3)1.84, s
      11''3.29, d (7.4)3.46, d (7.3)
      12''5.28, t (7.4)5.40, t (7.3)
      14''1.73, s1.76, s
      15''1.74, s1.75, s
      3'''6.28, s
      6'''6.22, s
      2'-OH13.96, s
      4'-OCH2O5.84, each d (1.2)
      6-OCH2O5.79, each d (1.2)
      6''-OCH2O5.76, each d (1.3)
      a Measured in CDCl3; b Measured in CD3OD; c Measured in acetone-d6; d Recorded at 400 MHz; e Recorded at 500 MHz; f Recorded at 600 MHz

      Table 3.  1H NMR data of compounds 712 (δ in ppm, J in Hz)

      No.7a8c9b10a11b12b
      α 142.2, CH 140.5, CH
      β 118.5, CH 118.5, CH
      β' 194.6, C 193.2, C
      1 115.7, C 115.4, C
      2 80.5, CH 145.6, C 161.0, C 156.1, C 71.4, CH2 71.0, CH2
      3 44.2, CH2 137.9, C 103.7, CH 104.1, CH 48.1, CH 33.4, CH
      4 190.5, C 173.4, C 162.7, C 158.1, C 199.0, C 31.4, CH2
      5 131.3, CH 124.4, CH 109.0, CH 120.3, C 165.9, C 107.8, CH
      6 105.2, CH 114.8, CH 133.2, CH 131.6, CH 97.1, CH 142.2, C
      7 166.9, C 160.2, C 168.3, C 147.7, C
      8 115.9, C 116.3, C 96.0, CH 98.7, CH
      9 159.9, C 155.7, C 165.2, C 150.7, C
      10 115.7, C 115.4, C 103.8, C 120.9, C
      1' 130.1, C 124.4, C 114.6, C 114.4, C 114.3, C 125.2, C
      2' 122.5, CH 127.8, CH 163.3, C 163.9, C 151.6, C 154.6, C
      3' 121.3, C 128.9, C 116.7, C 114.2, C 98.9, CH 103.5, CH
      4' 151.3, C 154.9, C 160.9, C 161.6, C 149.0, C 155.1, C
      5' 129.8, C 128.9, C 120.7, C 107.9, CH 142.1, C 114.7, C
      6' 127.7, CH 127.8, CH 129.1, CH 129.6, CH 110.3, CH 131.6, CH
      1'' 71.2, CH 22.9, CH2 22.8, CH2 29.2, CH2
      2'' 97.9, CH 122.8, CH 123.5, CH 121.8, CH 69.0, CH2
      3'' 71.4, C 132.7, C 132.4, C 135.5, C 39.1, CH
      4'' 26.1, CH3 18.2, CH3 26.0, CH3 26.0, CH3
      5'' 24.4, CH3 25.9, CH3 18.0, CH3 18.1, CH3 108.7, CH
      6'' 122.3, CH 29.4, CH2 29.0, CH2 21.9, CH2 142.0, C
      7'' 131.3, CH 123.0, CH 123.2, CH 121.4, CH 147.6, C
      8'' 76.7, C 133.8, C 134.5, C 135.9, C 98.4, CH
      9'' 28.3, CH3 25.9, CH3 26.0, CH3 26.0, CH3 150.8, C
      10'' 28.3, CH3 17.9, CH3 17.9, CH3 18.1, CH3 120.8, C
      11'' 28.4, CH2 29.4, CH2
      12'' 122.3, CH 123.0, CH
      13'' 132.7, C 133.8, C
      14'' 18.0, CH3 25.9, CH3
      15'' 25.9, CH3 17.9, CH3
      1''' 117.1, C
      2''' 149.2, C
      3''' 104.0, CH
      4''' 145.7, C
      5''' 140.2, C
      6''' 117.5, CH
      6-OCH2O 102.0, CH2
      4'-OCH2O 102.3, CH2
      6''-OCH2O 101.9, CH2
      a Measured in CDCl3; b Measured in CD3OD; c Measured in acetone-d6

      Table 4.  13C NMR (126 MHz) data of compounds 712 (δ in ppm)

      Sophotonin H (8). Yellow powder; $[\text{α} ]_{\rm{D}}^{20}$ +1 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 206 (5.81), 251 (5.32), 363 (5.40) nm; IR (KBr) νmax 3330, 2971, 2914, 1602, 1560, 1443, 1282, 1181, 1123, 1049 cm−1; 1H and 13C NMR data see Tables 3 and 4; HR-ESI-MS m/z 475.2480 [M + H]+ (Calcd. for C30H35O5, 475.2484).

      Sophotonin I (9). Yellow gum; $[\text{α} ]_{\rm{D}}^{20}$ −4 (c 0.1, MeOH); UV (MeOH) λmax (logε): 203 (5.53), 391 (5.30) nm; IR (KBr) νmax 3378, 2912, 1617, 1542, 1471, 1371, 1310, 1257, 1148 cm−1; 1H and 13C NMR data see Tables 3 and 4; HR-ESI-MS m/z 409.2010 [M + H]+ (Calcd. for C25H29O5, 409.2015).

      Sophotonin J (10). Yellow gum; $[\text{α} ]_{\rm{D}}^{20}$ −10 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 203 (5.60), 315 (4.94) nm; IR (KBr) νmax 3381, 2921, 1615, 1549, 1441, 1373, 1244, 1091, 1040 cm−1; 1H and 13C NMR data see Tables 3 and 4; HR-ESI-MS m/z 409.2013 [M + H]+ (Calcd. for C25H29O5, 409.2015).

      Sophotonin K (11). White powder; $[\text{α} ]_{\rm{D}}^{20}$ +1 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 201 (5.61), 292 (5.34) nm; ECD (11a; hexane/isopropanol) (Δε) 210 (−0.05), 280 (+0.08), 310 (−0.10) nm; ECD (11b; hexane/isopropanol) (Δε) 210 (+0.05), 280 (−0.05), 310 (+0.07) nm; IR (KBr) νmax 3346, 1637, 1503, 1483, 1447, 1385, 1262, 1163, 1037 cm−1; 1H and 13C NMR data see Tables 3 and 4; HR-ESI-MS m/z 317.0664 [M + H]+ (Calcd. for C16H13O7, 317.0661).

      Sophotonin L (12). Brown solid; $[\text{α} ]_{\rm{D}}^{20}$ −110 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (5.90), 298 (5.15) nm; ECD (MeOH) (Δε) 216 (−14.14), 298 (−1.92) nm; IR (KBr) νmax 3447, 1618, 1501, 1438, 1383, 1169, 1037 cm−1; 1H and 13C NMR data see Tables 3 and 4; HR-ESI-MS m/z 585.1412 [M + H]+ (Calcd. for C32H25O11, 585.1397).

    • HPLC separations were performed on a Jasco HPLC system on a Chiralcel AD-3 column (3 μm, 250 mm × 4.6 mm). The enantiomers of 2 [retention time (tR) 6.6 min for (2R)-2a, 7.6 min for (2S)-2b] were separated with a solvent mixture of hexane/isopropanol (80 : 20, V/V, 0.8 mL·min−1) as mobile phase, and the ECD spectra were recorded at 270 nm with a Jasco J-1500 CD spectrometer. The enantiomers of 11 [tR 11.5 min for (3S)-11a, 17.0 min for (3R)-11b] were run with a solvent mixture of hexane/isopropanol (75 : 25, V/V, 0.8 mL·min−1) as mobile phase, and the ECD spectra were recorded at 290 nm. The chromatogram was zeroed right after the start of recording, and hence relative absorbance was measured. The on-line CD spectra were recorded simultaneously at the maxima of the UV absorptions.

    • The DFT NMR and TDDFT ECD calculations for compounds 7 and 12 were performed with the Gaussian 16 program (Revision A.03, Gaussian Inc., Wallingford CT, 2016)[22]. The conformational searching was conducted by the Conflex 8.0 software (CONFLEX Corporation, Tokyo, Japan, 2017) using the MMFF force field within an energy window of 5.0 kcal·mol−1 [23]. The conformers with the Boltzmann population above 0.1% were re-optimized at the level of M06-2X/6-31G(d) in vacuo or with the SMD solvent model for methanol. TDDFT ECD calculations were run at the M06-2X/def2-TZVP level with the SMD solvent model for methanol. Calculated ECD spectra were generated using the SpecDis Version 1.71 with gaussian broadening after UV correction [24, 25]. DFT NMR calculations were run at the mPW1PW91/6-311G(d, p) level with the PCM solvent model for chloroform or the SMD solvent model for methanol. The possible configuration was specified by DP4+ probability [26, 27].

    • SARS-CoV-2 3CLpro and SARS-CoV-2 PLpro expression and purification were performed as described in the previous report [8]. The fractions or compounds against SARS-CoV-2 3CLpro was measured by A fluorescence resonance energy transfer (FRET) protease assay as previously reported [8]. In briefly, the fluorogenic substrate Dacyl-KTSAVLQSGFRKME-Edans was obtained from GenScript (Nanjing, China). SARS-CoV-2 3CLpro (50 nmol·L−1 final concentration) was mixed with different fractions or compounds in 80 µL assay buffer (50 mmol·L−1 Tris-HCl, pH 7.3, 1 mmol·L−1 EDTA) and incubated for 10 min. The reaction was triggered by adding fluorogenic substrate (40 µL) at a final concentration of 5 µmol·L−1. Then, the fluorescence signal at 340 nm (excitation)/490 nm (emission) was measured every 35 s for 3.5 min using a Bio-Tek SynergyH1plate reader. The inhibition of SARS-CoV-2 PLpro by the fractions or compounds was measured similar to that described previously [28] with a fluorogenic peptide (RLRGG-AMC) synthesized by GenScript (Nanjing, China). The reactions were performed in a total volume of 120 μL. Firstly, 50 nmol·L−1 PLpro was incubated with the indicated concentrations of the tested fractions or compounds in the condition of 50 mmol·L−1 HEPES, pH 7.5, 0.1 mg·mL−1 BSA, 5 mmol·L−1 DTT for 20 min. The reactions were initiated by the addition of 10 µmol·L−1 fluorogenic peptide. After that, the fluorescence signal at 360 nm (excitation)/460 nm (emission) was measured immediately every 1 min for 5 min with a Bio-Tek SynergyH1plate reader. Compounds PF-07321332 [29] and GRL0617 [30] were used as positive controls for SARS-CoV-2 3CLpro and PLpro, respectively.

      The initial velocities of reactions with fractions or compounds added at various concentrations compared to the reaction added with DMSO were calculated.

    Results and Discussion
    • Sophotonin A (1) was obtained as a yellow powder. Its molecular formula was established as C26H30O5 with 12 indices of hydrogen deficiency on the basis of its HR-ESI-MS analysis at m/z 423.2174 [M + H]+ (Calcd. for C26H31O5, 423.2171). The UV spectrum exhibited maximum absorptions at 230 and 283 nm, which implied a flavanone skeleton [31]. The IR spectrum exhibited absorption bands for hydroxy (3337 cm−1), conjugated carbonyl (1655 cm−1), and aromatic (1500, 1437 cm−1) functionalities.

      The 1H NMR spectrum (Table 1) displayed characteristic signals for a flavanone moiety at δH 5.64 (1H, dd, J = 12.4, 3.2 Hz), 2.94 (1H, dd, J = 17.0, 12.4 Hz), and 2.79 (1H, dd, J = 17.0, 3.2 Hz), two chemically inequivalent isoprenyl groups at δH 3.34 (2H, d, J = 7.3 Hz), 5.17 (1H, t, J = 7.3 Hz), 1.62 (3H, s), 1.67 (3H, s) and 3.20 (2H, d, J = 7.3 Hz), 5.27 (1H, t, J = 7.3 Hz), 1.70 (3H, s), 1.64 (3H, s), a methoxy group at δH 3.90 (3H, s), a set of ortho-coupled aromatic signals at δH 7.75 (1H, d, J = 8.8 Hz), 6.73 (1H, d, J = 8.8 Hz), and a pair of para-coupled ones at δH 6.35 (1H, s) and 7.12 (1H, s). The 13C NMR (Table 2) and DEPT spectra resolved 26 carbon signals ascribed to one carbonyl, 10 sp2 quaternary carbons, six sp2 methines, one sp3 methine, three sp3 methylenes, one methoxy, and four methyls. The 1H and 13C NMR spectroscopic data indicated that 1 is a prenylated flavanone related structurally to shandougenine D [32], isolated also from S. tonkinensis, except that one additional methoxy group was observed for 1.

      The structure of 1 was determined by the comprehensive analysis of 1H–1H COSY, HSQC, and HMBC correlations (Fig. 3). The ring A was established from the HMBC correlations from H-5 to C-4/C-7/C-9, and from H-6 to C-8/C-10. The methoxy group was located at C-7 in view of the HMBC correlation from the methoxy (δH 3.90) to C-7. One isoprenyl group was designated at C-8 by the HMBC correlations from H2-1" to C-7/C-8/C-9. The ring B was confirmed from the HMBC correlations from H-3' to C-2'/C-4', and from H-6' to C-2. The other isoprenyl group was assigned to C-5' by the HMBC correlations from H2-6'' to C-5'/C-6'.

      Figure 3.  Key 1H-1H COSY and HMBC correlations (H→C) for compounds 112

      The ECD spectrum of 1 gave a positive Cotton effect at 335 nm and a negative one at 305 nm, indicating the absolute configuration of C-2 being S. Typical 2S-flavanones are well known to show a positive CE at the n→π* transition (320−330 nm) and a negative CE at the π→π* transition (270−290 nm), respectively [33]. Therefore, the full structure of compound 1 was proposed, and named sophotonin A.

      Compound 2 was isolated as a brown solid. Its molecular formula was determined to be C25H26O6 with 13 indices of hydrogen deficiency. Its UV and IR data were very similar to those of 1. The 1H NMR spectrum (Table 1) showed signals for a flavanone skeleton at δH 5.62 (1H, dd, J = 13.1, 3.0 Hz), 3.08 (1H, dd, J = 17.2, 13.1 Hz), and 2.76 (1H, dd, J = 17.2, 3.0 Hz), an isoprenyl group at δH 3.21 (2H, d, J = 7.3 Hz), 5.16 (1H, t, J = 7.3 Hz), 1.78 (3H, d, J = 1.3 Hz), and 1.66 (3H, d, J = 1.3 Hz), a set of 1,2,4-trisubstituted phenyl signals at δH 7.26 (1H, d, J = 8.3 Hz), 6.35 (1H, dd, J = 8.3, 2.3 Hz), and 6.33 (1H, d, J = 2.3 Hz), and a gem-dimethylpyran moiety at δH 6.54 (1H, d, J = 10.0 Hz), 5.53 (1H, d, J = 10.0 Hz), 1.44 (3H, s), and 1.41 (3H, s). The 13C NMR (Table 2) and DEPT spectra illustrated 25 resonances including one carbonyl, 10 sp2 quaternary, one sp3 quaternary, six sp2 methine, one sp3 methine, two sp3 methylene, and four methyl carbons. The 1H and 13C NMR spectra of 2 were similar to those of kushenol E [34], isolated from Sophora flavescens, except for a gem-dimethylpyran moiety rather than an isoprenyl group was observed for 2.

      The structure of 2 was further confirmed by the comprehensive analysis of 1H–1H COSY, HSQC, and HMBC correlations (Fig. 3). In the 1H−1H COSY spectrum, fragments of H-2/H2-3, H-5'/H-6', H-1''/H-2'' and H2-6''/H-7'' were established. The ring A was constructed from the HMBC correlations from H-1'' to C-5/C-7, and from H-2'' to C-3''. The isoprenyl group was assigned to C-8 by the HMBC correlation from H2-6'' to C-7/C-8/C-9. The ring B was established from the HMBC correlations from H-3' to C-4', from H-5' to C-4', and from H-6' to C-2/C-2'/C-4'.

      The optical rotation value of compound 2 was close to zero, suggesting that it might exist as a racemic mixture. Chiral HPLC separation of 2 afforded two anticipated peaks of 2a and 2b in an approximate ratio of 1 : 1, which were evidenced as a pair of enantiomers by the opposite Cotton effects at 303 and 333 nm (Fig. 4). By comparing their experimental ECD spectra, the absolute configurations of 2a and 2b were assigned to be (2R)- and (2S)- configurated, respectively [33]. Thus, 2 was determined to be a racemic mixture, and named sophotonin B.

      Figure 4.  A, The chiral HPLC separation of 2a and 2b in a solvent mixture of hexane/isopropanol (80 : 20, V/V); B, Experimental ECD spectra of 2a (blue line) and 2b (red line) recorded by on-line LC-CD coupling in a solvent mixture of hexane/isopropanol

      Compound 3, obtained as a white powder, had a molecular formula of C30H34O5 with 14 indices of hydrogen deficiency. Its UV and IR spectra were similar to those of 2. The 1H and 13C NMR data (Tables 1 and 2) indicated that 3 is a prenylated flavanone, structurally related to the known compound 2-[{2'-(1-hydroxy-1-methylethyl)-7'-(3-methyl-2-butenyl)-2',3'-dihydrobenzofuran}-5'-yl]-7-hydroxy-8-(3-methyl-2-butenyl) chroman-4-one [35], also isolated in this study. The major differences were observed being the signals for a 5-(1-hydroxy-1,1-dimethylmeth-1-yl)-2,3-dihydrofuran moiety [δH 6.60 (1H, s), 1.69(6H, s) and δC 163.9, 100.8, 69.6, 28.9, 28.9] in 3, which was connected with C-4' inferred from the HMBC correlations from H-6' to C-11'', and from H-11'' to C-5'/C-12''. Compound 3 was optically active, and the absolute configuration at C-2 was determined as S by the ECD spectrum, showing a positive Cotton effect at 330 nm and a negative one at 297 nm [33]. Therefore, the structure of compound 3 was proposed, and named sophotonin C.

      Compound 4 was obtained as a yellow gum. Its molecular formula was determined as C26H26O5 with 14 indices of hydrogen deficiency. Its UV and IR spectra were indicative of a flavanone skeleton. The 1H NMR data of 4 (Table 1) revealed characteristic signals for a flavanone moiety, a isoprenyl group, two ortho-coupled aromatic protons at δH 7.75 (1H, d, J = 8.6 Hz), 6.55 (1H, d, J = 8.6 Hz), two meta-coupled aromatic protons at δH 7.73 (1H, d, J = 2.4 Hz), 7.29 (1H, d, J = 2.4 Hz), and a gem-dimethylpyran ring at δH 6.38 (1H, d, J = 10.0 Hz), 5.77 (d, J = 10.0 Hz, 1H), and 1.53 (6H, s), and a formyl group at δH 10.46 (1H, s). The 13C NMR (Table 2) and DEPT spectra resolved 26 carbon resonances ascribed to one carbonyl, one formyl, nine sp2 quaternary, one sp3 quaternary carbon, seven sp2 methine, one sp3 methine, two sp3 methylene, and four methyl carbons.

      The planar structure of 4 was finally established by the comprehensive analysis of 1H–1H COSY, HSQC, and HMBC correlations (Fig. 3). The isoprenyl group was attached to C-8 of ring A by the HMBC correlations from H2-1'' to C-7/C-8/C-9. The gem-dimethylpyran ring was specified to be fused to C-3' and C-4' of ring B deduced by the HMBC correlation from H-6'' to C-3'/C-4', and from H-9'' and H-10'' to C-8''. The position of formyl group was placed at C-5' by the HMBC correlations from H-11'' to C-5'. In the same way, the absolute configuration at C-2 was determined as S from its ECD spectrum [33]. Therefore, the full structure of 4 was proposed, and named sophotonin D.

      The molecular formula of compound 5 was established as C30H32O4 with 15 indices of hydrogen deficiency. Its UV and IR spectra were very similar to those of 4. Its 1H and 13C NMR data (Tables 1 and 2) showed high similarities to those of the known prenylated flavanone sophoranochromene [35], which was isolated previously from the same plant. The major difference was observed being a trans-2-methyl-1,3-pentadiene moiety [δH 6.83 (1H, d, J = 16.4 Hz), 6.93 (1H, d, J = 16.4 Hz), 5.09 (2H, each d, J = 16.5 Hz), 2.00 (3H, s) ; δC 142.7, 132.7, 122.8, 117.3, 18.7] in 5, which was supported by the 1H−1H COSY correlations between H-11"/H-12", and the HMBC correlations from H-6' to C-11'', H-12'' to C-5' and C-14'', and both H2-14'' and H3-15'' to C-13'' as well (Fig. 3). The 2S-configurtaion of 5 was determined the ECD spectrum [33]. Therefore, the full structure of 5 was established, and named sophotonin E.

      Compound 6 had a molecular formula of C30H34O5 determined by its HR-ESI-MS data, corresponding to 14 indices of hydrogen deficiency. Its UV and IR spectra were similar to those of 4 and 5. The 1H and 13C NMR data (Tables 1 and 2) indicated that 6 shared a similar skeleton with sophoranochromene [35], except that one hydroxymethyl group [δH 3.70 (1H, d, J = 11.8 Hz), 3.53 (1H, d, J = 11.8 Hz); δC 68.3 ppm] rather than an methyl was observed in the structure of 6. The hydroxymethyl group was further fixed at C-8'' by the HMBC correlations from H-7'' to C-9'' (Fig. 3). The absolute configuration at C-2 was determined as S from its ECD spectrum, showing a positive Cotton effect at 330 nm, and a negative one at 297 nm [33]. Therefore, the structure of 6 was defined, and named sophotonin F.

      Compound 7 was obtained as a white powder. Its molecular formula was established as C30H34O6 with 14 indices of hydrogen deficiency. Its UV and IR spectra were similar to those of 6. Compared with 6, the 1H and 13C NMR data (Tables 3 and 4) of 7 showed that a 4-hydroxy-5-(2-hydroxyisopropyl) dihydrofuran moiety [δH 5.52 (1H, d, J = 4.7 Hz), 4.43 (1H, d, J = 4.7 Hz), 1.37 (3H, s), 1.27 (3H, s) and δC 97.9, 71.4, 71.2, 26.1, 24.4 ppm] rather than a normal perylene group located at C-8 was present in 7. HMBC correlations from H-1'' to C-7 and C-8 suggested that the dihydrofuran moiety was fused at C-7 and C-8 (Fig. 3).

      The absolute configuration of C-2 was determined to be S from its Cotton effects in the ECD spectrum [33]. However, the stereochemistry of C-1'' and C-2'' remained unclear. The NOESY correlation of H-1''/H3-5'' suggested that H-1'' and H-2'' were in a trans form (Fig. 5). Thus, there are two possible configurations at C-1'' and C-2'' for 7, which having a certain 2S configuration. To figure out the relative configuration, DFT NMR calculations for two structures, rel-(2S, 1''S, 2''S)-7 and rel-(2S, 1''R, 2''R)-7, were carried out. To simplify the calculations, truncated structures with the isoprenyl group replaced by an ethyl were used. Conformational search was conducted using the Conflex in a 5.0 kcal·mol−1 energy window [23]. All conformers with the Boltzmann percentage above 0.1% were taken out for re-optimization at the level of M06-2X/6-31G(d) in vacuo. After removal of the duplicated conformers, the 1H and 13C NMR chemical shifts of the left optimized conformers were calculated at the level of mPW1PW91/6-311G(d, p) with the PCM solvent mode for chloroform. The NMR calculation method at the level of mPW1PW91/6-311G(d, p)/SMD (chloroform)//M062X/6-31G(d) was established in our previous study [36], so the specific set of statistical parameters [μ, σ, ν] was directly used for the custom DP4+ analysis in this study [26, 27]. The result gave 100% (H data, all data) and 94.28% possibilities (C data) for the (2S,1''R,2''R)-7, suggesting that both H-1'' and H-2'' were R*-configurated. To further substantiate its absolute configuration, the TDDFT ECD calculation was performed on the (2S,1''R,2''R)-7 at the level of M06-2X/def2TZVP with the SMD solvent model for methanol. The ECD spectrum was generated by SpecDis Version 1.71 with 0.25 sigma/gamma (eV) broadening after UV correction (+15 nm) [24, 25]. The result showed that the calculated curve matched well with the experimental one (Fig. 6), both having positive Cotton effects around 215 and 240 nm, and negative ones around 230 and 300 nm. Accordingly, the full structure of 7 was constructed, and named sophotonin G.

      Figure 5.  Key NOESY correlations (H↔H) for compound 7

      Figure 6.  Experimental ECD spectrum of compound 7 in MeOH compared with the Boltzmann-weighted M06-2X/def2TZVP SMD/MeOH ECD spectrum of the truncated structure (2S,1''R,2''R)-7 computed for the M06-2X/6-31G(d) optimized conformers

      Compound 8 was obtained as a yellow powder. Its molecular formula was established as C30H34O5 with 14 indices of hydrogen deficiency. The IR spectrum exhibited vibration bands for hydroxy (3330 cm−1), conjugated carbonyl (1602 cm−1), and aromatic (1560, 1500, 1443 cm−1) functionalities. The UV maximum absorptions at 251 and 363 nm, in combination with the characteristic carbons at δC 145.6 (C-2), 137.9 (C-3) and 173.4 (C-4), was indicative of a flavonol skeleton [31]. Comparing the 1H and 13C NMR data (Tables 3 and 4) of 8 with the known compound sophoranone [35], isolated previously from S. tonkinensis, revealed that they had the same substituent pattern. Therefore, the structure of compound 8 was established, and named sophotonin H.

      Compound 9, obtained as a yellow gum, possessed a molecular formula of C25H28O5 with 12 indices of hydrogen deficiency. The IR spectrum exhibited vibration bands for hydroxy (3378 cm−1), conjugated carbonyl (1617 cm−1), and aromatic (1542, 1471 cm−1) functionalities. The UV spectrum exhibited maximum absorptions at 260 and 391 nm, suggesting the presence of a chalcone ring [31]. The 1H and 13C NMR data (Tables 3 and 4) indicated that 9 is a prenylated chalcone, which structure was closely related to the previously reported compound 6-[3-(2',4'-dihydroxyphenyl)acryloyl]-7-hydroxy-2,2-dimethyl-8-(3-methyl-2-butenyl)-2H-benzopyran [37], isolated previously from the same plant, except that an isoprenyl group [δH 3.28 (2H, d, J = 7.4 Hz), 5.35 (1H, t, J = 7.4 Hz), 1.82 (3H, d, J = 1.3 Hz), 1.75 (3H, d, J = 1.3 Hz); δC 134.5, 123.2, 29.0, 26.0, 17.9 ppm] rather than the gem-dimethylpyran moiety was present in 9. Such elucidation was further supported by the 1H-1H COSY correlations between H2-6"/H-7", and the HMBC correlations from H2-6'' to C-4'' and C-6' (Fig. 3). Therefore, the structure of compound 9 was fully established, and named sophotonin I.

      Compound 10 had a molecular formula of C25H28O5 with 12 indices of hydrogen deficiency, the same with that of 9. Its UV and IR spectra were similar to those of 9. Comparing their 1H and 13C NMR data (Tables 1 and 2) revealed that they had the same skeleton and substituent groups, but different substituent patterns. The HMBC correlations from H2-1'' to C-4 and C-6, and from H2-6'' to C-2' and C-4' suggested that two isoprenyl groups were linked to C-5 and C-3', respectively. Therefore, the structure of compound 10 was proposed, and named sophotonin J.

      Compound 11 was obtained as a white powder. Its molecular formula was established as C16H12O7 with 11 indices of hydrogen deficiency. The UV spectrum exhibited maximum absorptions at 201, 235, and 292 nm. The IR absorption bands indicated the presence of hydroxy (3346 cm−1), conjugated carbonyl (1637 cm−1), aromatic (1503, 1447 cm−1), and methylenedioxy (-OCH2O-) (1037 and 940 cm−1) groups.

      The 1H NMR (Table 3) spectrum showed typical signals of an isoflavanone skeleton at δH 4.52 (1H, dd, J = 10.9, 10.9 Hz), 4.39 (1H, dd, J = 10.9, 5.5 Hz), and 4.24 (1H, dd, J = 10.9, 5.5 Hz), a set of meta-coupled aromatic signals at δH 5.90 (1H, d, J = 2.2 Hz), and 5.88 (1H, d, J = 2.2 Hz), a pair of para-coupled signals at δH 6.55 (1H, s), and 6.41 (1H, s), and a doublet signal for a methylenedioxy group at δH 5.84 (2H, d, J = 1.2 Hz). The 13C NMR (Table 4) and DEPT data illustrated 16 carbon signals ascribed to one carbonyl, eight sp2 quaternary, four sp2 methine, one sp3 methine, and two sp3 methylene carbons. The structure of 11 was evidenced by comprehensive analysis of its 1H–1H COSY, HSQC, and HMBC correlations (Fig. 3). The methylenedioxy group was assigned to be fused to C-4' and C-5' by the HMBC correlations from the oxygenated methine at δH 5.84 to C-4'/C-5'. Therefore, the full structure of compound 11 was proposed, and named sophotonin K.

      The optical rotation value of 11 was close to zero, suggesting that it might exist as a racemic mixture like compound 2. Using the same method as describe for 2, the anticipated enantiomers of 11a and 11b with an approximate ratio of 1 : 1 were measured by chiral HPLC, and their enantiomeric natures were evidenced by the opposite Cotton effects at 210, 280 and 310 nm (Fig. 7). Thus, the absolute configurations of 11a and 11b were assigned to be (3S)- and (3R)-configurated, respectively, by comparing their experimental ECD spectra with the literature data [33].

      Figure 7.  A, The chiral HPLC separation of 11a and 11b in a solvent mixture of hexane/isopropanol (75 : 25, V/V); B, Experimental ECD spectra of 11a (blue line) and 11b (red line) recorded by on-line LC-CD coupling in a solvent mixture of hexane/isopropanol

      Compound 12 was obtained as a brown solid. Its molecular formula was established as C32H26O11 with 20 indices of hydrogen deficiency, based on its HR-ESI-MS analysis at m/z 585.1412 [M − H] (Calcd. for C32H25O11, 585.1397). The UV spectrum exhibited maximum absorptions at 202, 235, and 298 nm. The IR spectrum indicated the presence of hydroxy (3347 cm−1), aromatic (1618, 1501, 1438 cm−1), and methylenedioxy (-OCH2O-) (1037 cm−1) groups.

      The 1H NMR data of 12 (Table 3) displayed characteristic signals for two isoflavan moiety [δH 4.14 (1H, ddd, J = 10.1, 3.3, 1.9 Hz), 3.87 (1H, dd, J = 10.1, 10.1 Hz), 3.42 (1H, dddd, J = 11.0, 10.1, 5.4, 3.3 Hz), 2.78 (1H, dd, J = 15.9, 11.0 Hz), 2.66 (1H, ddd, J = 15.9, 5.4, 1.9 Hz), and 4.17 (1H, dd, J = 10.7, 3.3 Hz), 4.02 (1H, dd, J = 10.7, 7.1 Hz), 3.62−3.56 (m, 1H), 4.45 (1H, d, J = 7.3 Hz)], four pairs of para-coupled aromatic signals [δH 6.55 (1H, s), 6.37 (1H, s); 6.54 (1H, s), 6.24 (1H, s); 6.74 (1H, s), 6.32 (1H, s); 6.27 (1H, s), 6.22 (1H, s)], and two methylenedioxy groups [δH 5.79 (2H, each d, J = 1.2 Hz), 5.76 (2H, each d, J = 1.3 Hz)]. The 13C NMR (Table 4) and DEPT spectra resolved only 31 carbon signals, ascribed to 16 sp2 quaternary carbons, eight sp2 methines, two sp3 methines and five sp3 methylenes. One sp3 methine, absent in the 13C NMR spectrum, was discovered from the HMBC spectrum. All these data suggested the presence of an isoflavan dimer.

      The planar structure of 12 was further evidenced by the comprehensive analysis of 1H–1H COSY, HSQC, and HMBC correlations (Fig. 3). The two methylenedioxy groups were assigned to be fused to C-6 and C-7, and C-6'' and C-7'', respectively, from the HMBC correlation from one oxygenated methine at δH 5.79 to C-6/C-7, and from the other one at δH 5.76 to C-6''/C-7''. The unambiguous HMBC correlations from H-4'' to C-4' and C-6' revealed a unique C-5'/C-4'' linkage between two isoflavan moieties.

      Compound 12 was optically active, and eight possible configurations were generated from three chiral carbons C-3, C-3'', and C-4''. To figure out the relative configuration, DFT NMR calculations were performed on four structures, namely, rel-(3R,3''R,4''R)-12, rel-(3R,3''R,4''S)-12, rel-(3R,3''S,4''R)-12, and rel-(3R,3''S,4''S)-12. The similar method as described for compound 7 was carried out at the level of M06-2X/6-31G(d)/SMD(methanol)//mPW1PW91/6-311G(d, p)/SMD(methanol). A set of new statistical parameters [μ, σ, ν] was also calculated at the same level, which was further used for the custom DP4+ analysis [26, 27]. The result gave 99.98% (H data) and 99.96% (all data) possibilities for rel-(3R,3''S,4''R)-12.

      The arbitrarily chosen enantiomer (3R,3''S,4''R)-12 was further taken for TDDFT ECD calculation at the level M06-2X/def2TZVP with the SMD solvent model for methanol. The ECD spectrum was generated by SpecDis Version 1.71 with 0.45 sigma/gamma (eV) broadening without UV correction [24, 25]. The mirror image of the calculated curve showed a good agreement with the experimental one (Fig. 8), suggesting that compound 12 possessed a (3S,3''R,4''S)-configuration. Therefore, the full structure of compound 12 was proposed, and named sophotonin L.

      Figure 8.  Experimental ECD spectrum of compound 12 in MeOH compared with the Boltzmann-weighted M06-2X/def2TZVP SMD/MeOH ECD spectrum of the truncated structure (3R,3''S,4''R)-12 and its mirror image computed for the M06-2X/6-31G(d) SMD/MeOH optimized conformers

      In addition to new compounds, a total of 43 known compounds were characterized from S. tonkinensis in this study as 2-(2,4-dimethoxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4-one (13) [38], glabrol (14) [39], euchrenone α8 (15) [40], (2S)-7,4'-dihydroxy-5'-aldehyde-8,3'-(3''-methyl-but-2''-enyl)-flavanone (16) [41], tonkinochromane F (17) [35], (−)-sophoranone (18) [35], (2S)-7,2',4'-trihydroxy-8,3',5'-tri(3-methyl-but-2-enyl)-flavanone (19) [41], tonkinochromane G (20) [35], tonkinochromane I (21) [42], 6,8-di(γ,γ-dimethylallyl)-4',7-dihydroxyflavanone (22) [17], 6,8-diprenylnaringenin (23) [43], kushenol E (24) [34], Lespedezaflavanone H (25) [44], paratocarpin L (26) [45], euchrenone α5 (27) [45], tonkinochromane H (28) [35], 5-dehydroxyupinifolin (29) [46], lupinifolin (30) [47], 2-(2',4'-dihydroxyphenyl)-8,8-dimethyl-10-(3-methyl-2-butenyl)-8H-pyrano[2,3-d]chroman-4-one (31) [48], flemichin D (32) [49], 2-[{3'-hydroxy-2',2'-dimethyl-8'-(3-methyl-2-butenyl)}chroman-6'-yl]-7-hydroxy-8-(3-methyl-2-butenyl)chroman-4-one (33) [50], 2-[(2'-(1-hydroxy-1-methyle-thyl)-7'-(3-methyl-2-butenyl)-2',3'-dihydrobenzofuran)-5'-yl]-7-hydroxy-8-(3-methyl-2-butenyl) chroman-4-one (34) [50], lonchocarpol (35) [51], exiguaflavanone B (36) [52], (2R,3R)-7,4'-dihydroxy-8,3',5'-triprenyldihydroflavanol (37) [53], lupinifolinol (38) [54], tonkinensisol (39) [55], 6,8-diprenyl-kaempferol (40) [49], genistein (41) [56], 3',5,7-trihydroxy-4'-methoxyisoflavone (42) [57], 7-O-methylluteone (43) [57], isoliquiritigenin (44) [58], kanzonol C (45) [59], sophoradin (46) [60], 6-[3-(2',4'-dihydroxyphenyl)acryloyl]-7-hydroxy-2,2-dimethyl-8-(3-methyl-2-butenyl)-2H-benzopyran (47) [37], sophoratonin H (48) [18], maackiain (49) [34], (−)-4-methoxymaackiain (50) [61], sophotokin (51) [41], maackiapterocarpan B (52) [62], maackiapterocarpan A (53) [62], 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan (54) [63], and bolusanthin IV (55) [64].

    • 3CLpro and PLpro are essential for SARS-CoV-2 transcription and replication. To investigate the inhibitory activities of the isolated compounds against the SARS-CoV-2 3CLpro and PLpro, all compounds were tested by the FRET-based protease assay. The results are shown in Table 5. Compounds 12 and 51 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC50 values of 34.89, and 19.88 μmol·L−1, and compounds 9, 43 and 47 exhibited inhibitory effects against PLpro with IC50 values of 32.67, 79.38, and 16.74 μmol·L−1, respectively. Compounds PF-07321332 [29] and GRL0617 [30] were used as positive controls for SARS-CoV-2 3CLpro and PLpro, respectively. The determined IC50 value of PF-07321332 against SARS-CoV-2 3CLpro was 27.36 nmol·L−1, and the measured IC50 value of GRL0617 against SARS-CoV-2 PLpro was 1.77 μmol·L−1.

      CompoundIC50/(μmol·L−1)
      SARS-CoV-2 3CLproSARS-CoV-2 PLpro
      9-32.67 ± 4.34
      1234.89 ± 4.48-
      43-79.38 ± 1.56
      47-16.74 ± 1.40
      5119.88 ± 5.71-
      PF-0732133227.36 nmol·L−1-
      GRL0617-1.77 μmol·L−1

      Table 5.  Inhibition of SARS-CoV-2 3CLpro and PLpro by compounds 9, 12, 43, 47, and 51

      Among the active compounds, sophotokin (51) is one of the major constituents in the rhizomes and roots of S. tonkinensis, which, to some extent, could explain for the inhibitory effect against 3CLpro of the plant extract. At the same time, we did not observe the common structural features of the active compounds that could distinguish them from the inactive ones. More work on the structure-activity relationship is needed in the future investigation.

    Conclusion
    • In summary, a total of 12 undescribed flavonoids were separated from S. tonkinensis, along with 43 known compounds. The full structures including the absolute configurations for all new compounds were proposed by extensive analysis of spectroscopic data, DFT NMR calculations coupled with DP4+ statistical analysis, and TDDFT ECD calculations. It is noteworthy that 2 and 11 are racemic mixtures, and their racemic natures were resolved by chiral HPLC sparation, which permitting online stereochemical characterization, when in hyphenation with circular dichroism (CD) spectroscopy (LC-CD coupling). Some compounds exhibited inhibitory effects against SARS-CoV-2 3CLpro and PLpro with IC50 values ranging from 16.74 to 79.38 μmol·L−1, but no enough data could support a structure-activity relationship. Our finding enriches the chemical diversity of S. tonkinensis, and provides evidence to support its usage as an anti- SARS-CoV-2 agent.

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