Bioactive isopimarane and 3,4-seco isopimarane diterpenoids from Isodon amethystoides

Isodon amethystoides (Lamiaceae) is a popular plant in folk medicine in the southern provinces of China. Our phytochemical investigation of the twigs and leaves of this plant led to the discovery of five new diterpenoids with isopimarane and 3,4-seco isopimarane scaffolds [isoamethinols A–E (1–5)], along with the known compound 3,4-seco isopimara-4(18),7,15-triene-3-oic acid methylester (6). The chemical structures of these compounds, including the absolute configurations of the new diterpenoids, were determined by comprehensive spectroscopic analyses and single crystal X-ray diffraction measurements. These compounds were evaluated for their biological activities against a panel of human cancer cell lines, gram-positive bacterial strains and HIV. Notably, the 3,4-seco-isopimarane isoamethinol D (4) showed toxicity to the cervical Hela cancer (Hela) cells with an IC50 value of 27.21 μM and the lung (A549) cancer cells with an IC50 value of 21.47 μM. Compound 4 also exhibited mild antimicrobial activity against the oral bacterial strain Streptococcus mutans. These findings suggested that the diterpenoids with a 3,4-seco-isopimarane diterpenoids isolated from I. amethystoides could provide a novel structure scaffold for the discovery of anticancer and antimicrobial compounds. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13065-022-00880-4.


Introduction
Plants are known to produce abundant small molecules of secondary metabolites with novel and unique chemical structures and a wide range of bioactivities, thus providing an important source for the discovery and development of new drugs.
The plant genus Isodon (Lamiaceae), mainly distributed in the tropical and subtropical regions of Asia, has been increasingly studied in the recent years due to its structurally diverse constituents with medical applications [1][2][3]. As popular Chinese folk medicines, some species in the genus Isodon have been traditionally used for the treatment of gastritis, arthritis, hepatitis, cholecystitis, enteritis, and amenorrhea in the southern provinces of China [4][5][6][7][8]. Diterpenoids are a major class of natural compounds that are abundantly found in Isodon plants. They have been discovered to have a broad-spectrum of pharmacological activities, including anticancer, antiviral, and antibacterial properties [1-3, 5, 7].
Isodon amethystoides is a medicinal plant belonging to the genus Isodon. Its leaf extract has been patented as a commercial drug with the trade-name "Wei Fu Chun Pian" for the treatment of precancerous lesions of gastric cancer lesions and also serves as an adjuvant therapy after gastric cancer surgery, as recorded in the Chinese Pharmacopoeia 2020 edition [9]. Previous phytochemical investigations of this species revealed significant differences in the chemical profiles of the secondary metabolites among the plants collected from various geographical areas [4][5][6]. Our recent research on a sample of this species collected from Libo County in Guizhou Province also led to the identification of a novel bioactive diterpene with 6/5/7 carbon skeleton, which showed inhibitory activity against the autoimmune disease-associated RORγt [10]. These prior findings encouraged us to carry out further phytochemical studies to discover additional new bioactive diterpenoids from this plant. As a result, a total of five new isopimarane and 3,4-seco isopimarane diterpenes [isoamethinols A-E (1-5)], along with the known compound 3,4-seco isopimara-4(18),7,15-triene-3-oic acid methylester (6) [11], were isolated from the leaves of I. amethystoides (Fig. 1). The newly isolated compounds were subsequently tested for their cytotoxic, antibacterial, and antiviral activities. Herein, we describe the isolation, structural determination, and biological activity evaluation of the compounds obtained from I. amethystoides.    (10) from the refinement of the X-ray crystallographic data on CuKα radiation allows the determination of the absolute configuration of 1 to be the same as an isopimarane diterpenoid (Fig. 4). The structure of compound 1 was thus assigned as isopimara-7α-hydroxy-8,15-diene-3-one, and given the trivial name isoamethinol A. Compound 2, colorless oil, was found to possess a molecular formula of C 20 H 28 O by the HR-ESI-MS data [m/z 285.2206 [M + H] + (calcd for 285.2213)]. The similar coupling pattern and chemical shifts of the 1 H and 13 C NMR spectroscopic data to 1 were observed for 2 (Tables 1 and 2). Compound 2 differs from 1 by lacking the hydroxyl group at C-7 but having three olefinic double bonds in the structure. The three double bonds in 2 are assigned as Δ 7,8 , Δ 9,11 and Δ 15,16 , respectively, by analyzing the 2D correlation data of the 1 H-1 H COSY, HMQC and HMBC NMR spectra (Fig. 2). Compound 2 shared the similar key NOE correlation pattern (H-1/H-20, H-18/H-5, H-19/H-20, H-12β/H-17) to 1, revealing that 2 is also an isopimarane diterpenoid. The structure of 2 was thus established as Table 2 13 C NMR data for compounds 1-5 (δ in ppm)

Result and discussion
The 13 C NMR (150 MHz) spectral data of 1 and 3-5 were measured in CDCl 3 , and the 13 C NMR (100 MHz) spectral data of 2 were measured in CDCl 3 isopimara-7,9(11),15-triene-3-one, and given the trivial name isoamethinol B. The key NOESY correlations (red arrows) of 1-4 spectra ( Fig. 2), 3 was elucidated to be a ring-expanded ω lactone congener of 2. Specifically, the absence of the HMBC correlations of H 3 -18 to C-3 and H 3 -19 to C-3 in 3 (Additional file 1: Figs. S25 and S26 combined with the observation of the upfield chemical shift of C-3 (δ C 175.8) and a downfield chemical shift of C-4 (δ C 86.4) in 3 in comparison of 2, revealed that the ω lactone is formed via an oxygen insertion at the position between C-3 carbonyl carbon and C-4. Compared with 2, compound 3 contains a less olefinic double bond, which is determined to occur at C-8 and C-9. These structure features in 3 are evidenced by the disappearance of the characteristic double bond Δ 9,11 (δ C 117.1, 145.2) resonance signals (Table 2), the upfield shift of the carbonyl carbon (from δ C 216.6 to 175.8) and the downfield shift of the gem methyl group connected quaternary carbon (from δ C 47.8 to 86.4) in comparison with 2. It is worthy to mention that a pimarane diterpene with ring A being oxidated to a lactone like 3 is rarely discovered in nature.
The relative stereochemistry of 3 was determined by a NOESY experiment. The presence of the NOE crosspeaks of H 3 -20/H-11α, H 3 -20/H 3 -19 and H-11α/H 3 -17 demonstrated that the three methyl groups of C-17, C-19 and C-20 are co-facial and β-oriented. H-5 and H-9 were established as α-oriented by the observation of the NOE correlations between H-5 and H 3 -18 and between H-9 and H 3 -18. In addition, the X-ray diffraction results further confirmed the structure elucidated from the above deduction (Fig. 4). As a result, 3 was identified as isopimara-7,15-diene-3,4-lactone and given the trivial name isoamethinol C.  Finally, a single-crystal X-ray diffraction study (CuKα radiation) with a Flack parameter of − 0.01(6) determined the absolute configuration of 4 shown in Fig. 4. Accordingly, 4 was determined as 3,4-seco-isopimara-4-hydroxy-7,15-diene-3-oic acid methylester, and given the trivial name isoamethinol D.
Compound 5 was obtained as yellow oil. The analysis of the molecular ion peak [M + H] + at m/z 333.2420 (calcd 333.2424) measured in the HR-ESI-MS spectrum rendered the molecular formula of 5 as C 21 H 32 O 3 , which has 6 degrees of unsaturation and two protons less than that of 4. Compound 5 showed very similar 1 H and 13 C NMR spectral data to 4. The one more degree of unsaturation of 5 than 4 suggested that additional ring could be formed in 5. Through the total analysis of the 1D and 2D NMR data (Tables 1 and 2, and Fig. 2), the additional ring was found to be formed by the bond connection between C-4 and C-9. Moreover, 5 shared high similarity of the proton NMR spectral data to our previously identified compound fladin A, except for an additional methoxy group (3H, δ H 3.66, s, δ C 51.7) observed in 5 [17]. Herein, compound 5 is a methyl ester congener of fladin A. The absolute configuration of 5 was assigned the same as 4, which was deduced by the consideration of the biosynthetic origins and the similar NOE correlation patterns observed between the two compounds. Compound 5 was thus identified as 3,4-seco isopimara-4,9β-epoxy-7,15diene-3-oic acid methylester, and given the trivial name isoamethinol E.
Compounds 1-5 were evaluated for their in vitro cytotoxic activities against a panel of human cancer cell lines (HL-60 promyelocytic leukemia cells, SMMC-7721 hepatocellular cancer cells, MCF-7 breast cancer cells, SW480 colon cancer cells, and Hela cervical carcinoma cells). Among these compounds, only 4 showed moderate cytotoxicities against Hela and A549 cells with IC 50 values of 27.21 and 21,47 μM, respectively (Fig. 5). These compounds were also evaluated for their activities against the gram-positive bacteria including methicillin-resistant strain Staphylococcus aureus (MRSA), Streptococcus sobrinus, and S. mutans. As a result, only 4 was demonstrated with mild antibacterial effect on S. mutans with an inhibitory rate of 42.1% at the concentration of 149.48 μM. In addition, the anti-HIV activities of these compounds were further explored for their antiviral potential using our previously established "One-Stone-Two-Birds" protocol [12]. However, no significant antiviral activities were observed for these compounds at a concentration of 25 μM.

Conclusion
Five new isopimarane and 3,4-seco isopimarane diterpenes, along with a known 3,4-seco isopimarane diterpene, were isolated from the leaves of I. amethystoides. Their chemical structures and absolute configurations were determined by analysing their spectroscopic and X-ray crystallographic data. To the best of our knowledge, isoamethinol C (3) is the first naturally occurring 3,4-seco isopimarane diterpenoid containing a A-ring ω lactone ring, which expands the chemical diversity of isopimarane diterpenoid family. Isoamethinol D (4) showed moderate cytotoxicity and mild antibacterial activity. The present findings through the phytochemical investigation including the absolute structure confirmation and the bioactivity evaluation of the new isolates suggested that the structurally diversified isopimarane diterpenes derived from I. amethystoides could serve as a class of lead molecules for further drug discovery study.

General
Optical rotation was measured at 28.6 °C for compounds 1-5 with a WZZ-3 automatic polarimeter (INESA Analytical Instrument, Shanghai, People's Republic of China). An ICAN-9 Fourier transform infrared spectrometer was used for scanning IR spectroscopy using KBr discs. Nuclear magnetic resonance (NMR) experiments were carried out on a Bruker Advance NEO (600 MHz, Brucker, Karlsruhe, Germany) spectrometer and an INOVA (400 MHz, Varian, Palo Alto, USA) spectrometer using TMS as an internal standard. X-ray crystallographic data were obtained on a Bruker SMART APEX CCD instrument using MoKα radiation for compound 3 and on a Bruker D8 VENTURE instrument using CuKα radiation for compound 1 and 4 (Brucker, Karlsruhe, Germany). HR-ESI-MS data were recorded on a Bruker Daltonics Compact Q-TOF spectrometer (Brucker, Karlsruhe, Germany) equipped with an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm; Waters, Milford, MA, USA) under the column heater fixed at 40 °C. Silica gel (100-200 and 200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China) was used for flash chromatography. Sephadex LH-20 gel (Shanghai EKEAR Bio@Tech, Shanghai, People's Republic of China) was used for chromatographic separation. Thin-layer chromatography (TLC) analyses were carried out on precoated silica gel GF 254 plates (Qingdao Marine Chemical Inc., Qingdao, People's Republic of China) using various solvent system, and spots were visualized by heating the silica gel plates sprayed with 95-98% H 2 SO 4 -EtOH (v/v = 10:90). All solvents were commercially purchased and distilled prior to use.

Plant material
The leaves of I. amethystoides were collected in October 2018 from Libo County in Guizhou Province of China. The collected plant was authenticated by Professor Junhua Zhao from Guizhou University of Traditional Chinese Medicine. A voucher plant specimen (GZLB20181003004) was deposited at Guizhou University of Traditional Chinese Medicine.

X-ray crystallographic data of 1, 3 and 4
Crystals of 1, 3, and 4 were obtained from MeOH. Single-crystal X-ray crystallographic analyses of 1 and 4 were obtained on a Bruker D8 VENTURE diffractometer (Cu Kα radiation, λ = 1.54178 Å). The crystal structure of 3 was determined on a Bruker APEX-II CCD diffractometer (Mo Kα radiation, λ = 0.71076 Å). The crystals of 1, 3, and 4 were kept at 160.0 K, 293.0 (2) K, and 170.0 K during data collection, respectively. Using Olex2, the structures were solved with the ShelXT structure solution program using direct methods, dual space, or intrinsic phasing, and refined with the ShelXL refinement package using least-squares minimization [13][14][15].

Cytotoxicity assay
A panel of human cancer cell lines (HL-60, SMMC-7721, MCF-7, SW480, and Hela) were utilized to determine the cytotoxic effects of compounds 1-5 using MTS method as previously reported [16]. HL-60, MCF-7, and Hela cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), where the SMMC-7721 and SW480 cells were cultured in RPMI-1640. Cells were seeded at 4000 cells/190 µL per well in 96-well plates in their corresponding medium before adding 10 µL test samples. Serial concentrations (1.25-40 µM) of each tested compound were added in wells, and the plates were incubated for 48 h. Spent media was decanted after 48 h incubation. To each well, 100 µL of fresh media and 20 µL of 2 mg/mL MTS stock solution were added and the cells were incubated for 3 h at 37 °C. Afterwards, the optical density was measured at 515 nm using an ELISA plate reader. Both cisplatin and paclitaxel were used as positive controls.

Antibacterial assay
The bacteriostatic activity assay for compounds 1-5 was carried out using the standard microdilution method in 96-well plates as previously described [17]. Each well contained 50 μL two-fold serially diluted test agents, 50 μL growth medium (Tryptic Soy Broth Medium for MRSA, Brain Heart Infusion for S. mutans and S. sobrinus, respectively), and 10 μL of an overnight culture of strains, representing approximately 5 × 10 7 CFU mL −1 . The controls comprised inoculated growth medium without test agents and sample blanks in growth medium only. The plates were incubated overnight at 200 rpm, 37 °C. The OD 600 values were obtained using the microplate reader.

Anti-HIV assay
Compounds 1 to 5 were also evaluated for their anti-HIV activity using our previously established "One-Stone-Two-Birds" protocol [18]. Human embryonic kidney cells 293 T were transiently transfected with 0.5 μg VSV-G envelope expression plasmid and 2 μg Env-deficient HIV vector (pNL4.3. Luc-R-E-) in 10 cm plates via PEI (polyethyleneimine). Sixteen hours posttransfection, all media was replaced with fresh, complete DMEM. Forty-eight hours post-transfection, the supernatants were collected and filtered through a 0.45 μm-pore size filter, and the pseudovirions were directly used for infection. Targeted A549 cells were seeded at 4000 cells/190 µL per well in 96-well plate in DMEM supplemented with 10% FBS. The tested samples were added, 10 μL in each well, with a final concentration of 5 and 25 μM. Forty-eight hours postinfection, cells were lysed and prepared for luciferase assay.