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

Abstract

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

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 [1,2,3, 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.

Fig. 1

The chemical structures of compounds 1–6

Result and discussion

Compound 1 was obtained as a colorless cubic crystal (MeOH). It has 6 degrees of unsaturation according to the deduced molecular formula of C₂₀H₃₀O₂ by analysis of the HR-ESI-MS data [m/z 325.2133 [M + Na]⁺ (calcd for 325.2138)]. By interpreting the chemical shift signals in the high and middle field regions of the ¹H-NMR spectrum (Table 1), 1 was found to possess four methyl groups (3H, δ_H 1.00, s; 1.01, s; 1.06, s; 1.12, s) and three olefinic groups (1H, δ_H 5.72, dd, J = 17.5, 10.8; 4.96, dd, J = 17.5, 1.3; 4.93, dd, J = 10.8, 1.3). Moreover, analyses of the 1D ¹³C and DEPT-135 NMR data (Table 2), combined with the HSQC correlation data, allowed to identify the 20 carbon resonances in 1 as one carbonyl carbon (δ_C 217.5), five quaternary carbons including two non-protonated olefinic carbons (δ_C 127.5 and 140.0), one methine (δ_C 45.3), one oxygenated methine (δ_C 68.8), six methylenes (δ_C, 34.7; 34.4; 29.5; 21.3; 34.6; 39.2), two protonated olefinic carbons (δ_C 145.8 and 111.6), and four methyl groups (δ_C 28.1; 26.7; 21.3; 17.9). The above NMR spectral data suggested that compound 1 belongs to a pimarane or an isopimarane diterpenoid. Based on the analysis of the correlations obtained from the ¹H-¹H COSY and HMBC spectra (Fig. 2), the ketone carbon at δ_C 217.5 is assigned at C-3, the four olefinic carbons at δ_C 127.5/140.0 and δ_C 111.6/145.8 are assigned as the two double bonds of Δ^8,9 and Δ^15,16 respectively, and the oxygenated methine carbon at δ_C 68.8 is assigned at C-7. The relative configuration of 1 was established through the analysis of the NOE correlations obtained from the NOESY spectrum of 1. First of all, the presence of the NOE correlations between H-5 and H₃-18 and between H₃-19 and H₃-20 indicated the possibility of a trans-fused A/B ring system (Fig. 3). In addition, the observed NOSEY cross-peaks between H-7 and H-14β and between H-14β and H₃-17 determined the co-facial relationship of the protons of H-7 and H₃-17. The resulted Flack number of 0.07 (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.

Table 1 ¹H NMR data for 1–5 (δ in ppm)

Table 2 ¹³C NMR data for compounds 1–5 (δ in ppm)

Fig. 2

The selected key ¹H-¹H COSY (blue bold) and HMBC (red arrows) correlations of 1–5

Fig. 3

The key NOESY correlations (red arrows) of 1–4

Fig. 4

X-ray crystallographic structures of 1, 3 and 4

Compound 2, colorless oil, was found to possess a molecular formula of C₂₀H₂₈O 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 ¹H and ¹³C 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 ¹H-¹H 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 isopimara-7,9(11),15-triene-3-one, and given the trivial name isoamethinol B.

Compound 3 was obtained as colorless crystals (MeOH). Given the HR-ESI–MS molecular ion peak at m/z 325.2129 [M + Na]⁺ (calcd 325.2138) and the molecular formula of C₂₀H₃₀O₂, 3 is calculated to have one less degree of unsaturation than 2. By interpretation of the correlation data of the HMBC and ¹H-¹H COSY spectra (Fig. 2), 3 was elucidated to be a ring-expanded ω lactone congener of 2. Specifically, the absence of the HMBC correlations of H₃-18 to C-3 and H₃-19 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 cross-peaks of H₃-20/H-11α, H₃-20/H₃-19 and H-11α/H₃-17 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 H₃-18 and between H-9 and H₃-18. 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.

Compound 4, colorless needle crystals (MeOH), was determined to possess 5 degrees of unsaturation calculated by the molecular formula of C₂₁H₃₄O₃, which was obtained through the analysis of its HR-ESI-MS data [m/z 357.2397 [M + Na]⁺ (calcd for C₂₁H₃₄O₃Na, 357.2400)]. It has one unsaturation degree less than 3. The complete assignments of the 1D and 2 D NMR signals (Tables 1 and 2, and Fig. 2) allowed us to identify a methyl propanoate ester group [δ_H 3.65 (3H, s, OCH₃), 1.75 (1H, ddd, J = 17.4, 11.2, 5.4, H-1α), 2.28 (1H, dt, J = 11.3, 2.7, H-1β), 2.22 (1H, ddd, J = 14.0, 11.9, 5.7, H-2α), 2.70 (1H, ddd, J = 14.3, 11.0, 2.0, H-2β); δ_C 51.7 (OCH₃), 32.3 (C-1), 29.3 (C-2), 175.7 (C-3, the ester carbonyl carbon)] linked to C-10. The analysis of the NMR data also assigned an oxygenated tertiary carbon at C-4, which is attached with two gem methyl groups to form a 2-hydroxy propanyl group. Collectively, compound 4 was elucidated as a ring A opened methyl ester derivative. The key NOE correlations observed between H-5 and H-9 and between H-20 and H-11β determined that the propanoate ester group and the 2-hydroxy propanyl group are trans-oriented with each other. Moreover, the presence of the cross-peaks of H₃-17/H-11β and H₃-20/H-11β revealed the β-orientation of the methyl groups at C-10 and C-13. 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 C₂₁H₃₂O₃, which has 6 degrees of unsaturation and two protons less than that of 4. Compound 5 showed very similar ¹H and ¹³C 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-7,15-diene-3-oic acid methylester, and given the trivial name isoamethinol E.

Compound 6 was identified as the known 3,4-seco isopimara-4(18),7,15-triene-3-oic acid methylester based on the comparison of the proton coupling patterns and chemical shifts of the ¹H and ¹³C NMR data (Additional file 1: Figs. S50 and S51) with the reported literature data [11].

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 IC₅₀ 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.

Fig. 5

Growth inhibitory effects of compound 4 on Hela and A549 cancer cells. The in vitro dose–response was evaluated in the cells at seven twofold serial dilution concentrations with 3 independent experiments. The IC₅₀ values were determined by fitting dose–response curves with the equation log (inhibitor) vs. normalized response–variable slope to the data by using GraphPad Prism software

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.

Experimental section

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 GF₂₅₄ 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% H₂SO₄-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.

Extraction and isolation

The air-dried leaves of I. amethystoides (29 kg) were ground into powder and further extracted by MeOH at room temperature (3 times, 7 days each time) to yield a crude extract (3.2 kg), which was suspended in water (8 L), and partitioned with petroleum ether (PE) (3 × 8 L, 48 h each time) and ethyl acetate (EtOAc) (3 × 8 L, 48 h each time). The PE fraction (1.5 kg) was subjected to a silica gel column separation with gradient elution of PE/EtOAc (1:0 to 1:1) to provide fractions A–D. Fraction B (234 g) was further separated on a silica gel column using a gradient elution of PE/dichloromethane (CH₂Cl₂) (99:1 to 1:1) to afford compound 6 (160 mg). Fraction C (345 g) was separated on a silica gel column, eluting with gradient PE/EtOAc (49:1 to 1:1) to obtain 2 (18 mg) and 5 (200 mg). The EtOAc fraction (880 g) was subjected to fractionation over an MCI gel column using gradient MeOH/H₂O (50:50 to 90:10) to yield fractions E–I. Fraction G (179 g) was subjected to a silica gel chromatographic separation using gradient (EtOAc/MeOH) (50:1 to 5:1) to produce sub-fractions G1–G4. G1 was subjected to a Sephadex LH-20 chromatographic column eluting with CH₂Cl₂/MeOH (1:1) to give 1 (23 mg). Fraction H (191 g) was further purified by a silica gel column, eluting with gradient CH₂Cl₂/MeOH (50:1 to 1:1) to afford 3 (156 mg) and 4 (90 mg).

Isoamethinol A (1)

Colorless cubic crystal, mp 88.6–89.1 °C; [α]^28.6_D + 46.67 (c 0.6, CH₂Cl₂); IR (KBr) υ_max 3522, 2935, 2921, 2873, 1690, 1458, 1454, 1383, 1245, 1040, 932, 909 cm^–1; ¹H and ¹³C NMR (CDCl₃), see Tables 1 and 2; HR-ESI-MS m/z 325.2133 [M + Na]⁺ (calcd. for C₂₀H₃₀O₂Na, 325.2138).

Isoamethinol B (2)

Colorless oil; [α]^28.6_D + 69.54 (c 0.3, CH₂Cl₂); IR (KBr) υ_max 3404, 2964, 1709, 1458, 1386, 1271, 1116, 1006, 910, 803 cm^–1; ¹H and ¹³C NMR (CDCl₃), see Tables 1 and 2; HR-ESI-MS m/z 285.2218 [M + H]⁺ (calcd. for C₂₀H₂₉O, 285.2218).

Isoamethinol C (3)

Colorless needle crystal, mp 83.3–83.6 °C; [α]^28.6_D-15.38 (c 0.26, CH₂Cl₂); IR (KBr) υ_max 3421, 2957, 2918, 1726, 1639, 1389, 1371, 1225, 1108, 986, 910 cm^–1; ¹H and ¹³C NMR (CDCl₃), see Tables 1 and 2; HR-ESI-MS m/z 325.2129 [M + Na]⁺ (calcd. for C₂₀H₃₀O₂Na, 325.2138).

Isoamethinol D (4)

Colorless needle crystal, mp 84.36–85.3 °C; [α]^28.6_D + 17.39 (c 0.23, CH₂Cl₂); IR (KBr) υ_max 3491, 3373, 2905, 2842, 1732, 1638, 1439, 1411, 1251, 1198, 1001, 912 cm^–1; ¹H and ¹³C NMR (CDCl₃), see Tables 1 and 2; HR-ESI-MS m/z 357.2397 [M + Na]⁺ (calcd. for C₂₁H₃₄O₃Na, 357.2400).

Isoamethinol E (5)

Yellow oil; [α]^28.6_D + 38.10 (c 0.53, CH₂Cl₂); IR (KBr) υ_max 2359, 2342, 1741, 1173, 913 cm^–1; ¹H and ¹³C NMR (CDCl₃), see Tables 1 and 2; HR-ESI-MS m/z 357.2397 [M + Na]⁺ (calcd. for C₂₁H₃₄O₃Na, 357.2400).

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].

Crystallographic data of 1

C₂₀H₃₀O₂, M = 302.44, a = 9.1730 (2) Å, b = 11.5454 (3) Å, c = 16.8349 (4) Å; α = 90^◦, β = 90^◦, γ = 90^◦, V = 1782.92 (6) ų, T = 160.0 K, space group P2₁2₁2₁, Z = 4, μ (Cu Kα) = 0.54 mm⁻¹; 49814 reflections collected, 3644 independent reflections (R_int = 0.0617, R_sigma = 0.0275). The final R₁ values were 0.0438 [I > 2σ (I)]. The final wR (F²) values were 0.1161 [I > 2σ (I)]. The final R₁ values were 0.0484 (all data). The final wR (F²) values were 0.1188 (all data). The goodness of fit on F² was 1.049. Flack parameter = 0.07 (10).

Crystallographic data of 3

C₂₀H₃₀O₂, M = 302.44, a = 6.238 (2) Å, b = 9.613 (4) Å, c = 29.043 (13) Å; α = 90^◦, β = 90^◦, γ = 90^◦, V = 1782.92 (6) ų, T = 293.0 (2) K, space group P2₁2₁2₁, Z = 4, μ (Mo Kα) = 0.07 mm⁻¹; 18262 reflections collected, 3072 independent reflections (R_int = 0.0203, R_sigma = 0.0133). The final R₁ values were 0.0290 [I > 2σ (I)]. The final wR (F²) values were 0.0720 [I > 2σ (I)]. The final R₁ values were 0.0297 (all data). The final wR (F²) values were 0.0725 (all data). The goodness of fit on F² was 1.055. Flack parameter = − 0.2 (3).

Crystallographic data of 4

C₂₁H₃₄O₃, M = 334.48, a = 13.8691 (10) Å, b = 10.3267 (7) Å, c = 14.8423 (11) Å; α = 90^◦, β = 107.992(3), γ = 90^◦, V = 2021.8 (3) ų, T = 170.0 K, space group P2₁, Z = 4, μ (Cu Kα) = 0.56 mm⁻¹; 44189 reflections collected, 8085 independent reflections (R_int = 0.0531, R_sigma = 0.0323). The final R₁ values were 0.0381 [I > 2σ (I)]. The final wR (F²) values were 0.1042 [I > 2σ (I)]. The final R₁ values were 0.0443 (all data). The final wR (F²) values were 0.1086 (all data). The goodness of fit on F² was 1.048. Flack parameter = − 0.01 (6).

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 × 10⁷ 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 OD₆₀₀ 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 post-transfection, 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 post-infection, cells were lysed and prepared for luciferase assay.