The amide derivative of anticopalic acid induces non

Dec 13, 2023

Scientific Reports volume 13, Article number: 13456 (2023) Cite this article

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Anticopalic acid (ACP), a labdane type diterpenoid obtained from Kaempferia elegans rhizomes, together with 21 semi-synthetic derivatives, were evaluated for their cancer cytotoxic activity. Most derivatives displayed higher cytotoxic activity than the parent compound ACP in a panel of nine cancer cell lines. Among the tested compounds, the amide 4p showed the highest cytotoxic activity toward leukemia cell lines, HL-60 and MOLT-3, with IC50 values of 6.81 ± 1.99 and 3.72 ± 0.26 µM, respectively. More interestingly, the amide derivative 4l exhibited cytotoxic activity with an IC50 of 13.73 ± 0.04 µM against the MDA-MB-231 triple-negative breast cancer cell line, which is the most aggressive type of breast cancer. Mechanistic studies revealed that 4l induced cell death in MDA-MB-231 cells through non-apoptotic regulated cell death. In addition, western blot analysis showed that compound 4l decreased the phosphorylation of FAK protein in a concentration-dependent manner. Molecular docking simulations elucidated that compound 4l could potentially inhibit FAK activation by binding to a pocket of FAK kinase domain. The data suggested that compound 4l could be a potential FAK inhibitor for treating triple-negative breast cancer and worth being further investigated.

Cancer is the world’s devastating disease, and breast cancer is the second highest cause of death in all types of cancers1. According to the World Health Organization (WHO), around 7.8 million women worldwide were diagnosed with breast cancer in the year 20202. Although uncommon, breast cancer also occurs in male and transgender people3. Breast cancer is a highly complex disease classified into many subtypes, each with its own biological features, gene expression profiles, and clinical behaviors4. Among breast cancer subtypes, triple-negative breast cancer (TNBC) is the most aggressive and has the highest recurrence rate, metastatic spread, and mortality risks5. TNBC is characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This subtype accounts for approximately 15% of all diagnosed breast cancers and 25% of all deaths related to breast cancer4. Due to the lack of suitable targeted receptors, common breast cancer treatments such as hormone therapy and targeted therapy against ER, PR, and HER2 are ineffective in TNBC patients. As a result, TNBC has limited treatment options, and is known to have a worse clinical outcome than other breast cancer subtypes. Chemotherapy remains a standard treatment for TNBC, but chemoresistance frequently occurs5. These problems indicate the importance of finding new drugs or efficacious treatment options for TNBC.

Natural products have long made major contributions to drug discovery, especially for cancer and infectious diseases6,7. They serve as good sources of new drugs since they are typically affordable materials, having various scaffolds, pharmacophores, and accessibility. In addition, combined with chemical synthesis, natural products can be used as starting points to access diverse molecular structures with amplified bioactivity and desired bioavailability. For these reasons, the concept of isolating a natural product for lead optimization studies or the generation of screening libraries has been regularly reported in the literature8. However, to make this strategy effective requires (1) access to adequate supplies for the large-scale isolation of the desired natural product, (2) the high abundance of the natural product of interest from the natural product source, and (3) the presence of chemical functionalities in the natural product scaffold that allows for the simple and high yielding generation of derivatives. Therefore, natural products that could meet these criteria are considered as good sources for a natural product-based drug discovery program.

Anticopalic acid (ACP) known as (+)-copalic acid (Fig. 1), is a labdane-type bicyclic diterpenoid previously isolated from several plants including Vitex hemsley9, Eperua purpurea10, Eperua leucantha11, Pinus monticola12,13, Pinus strobus13, and Oxystigma oxyphyllum14,15. In 2018, our group isolated ACP in large amounts (~ 23%) from the crude extract of the rhizomes of Kaempferia elegans16,17. Since Kaempferia elegans is a plant in the ginger family, which can be easily cultivated, and ACP is deemed to have a particularly attractive natural product scaffold due to its low MW (304 Da), multiple stereogenic centers (n = 3, conferring a unique 3D shape), and containing a potential chemical handle, such as carboxylic acid in the molecule. Therefore, ACP can be a good candidate as a starting point for optimization into novel natural product-based bioactive agents. It is worth mentioning that anticopalic acid is one of the rare natural products that have both enantiomeric forms found in nature18. (−)-Copalic acid (COPA) (Fig. 1), the opposite enantiomeric form of ACP, is the major diterpenoid present in folk medicine, copaiba oil19.

(−)-Copalic acid (COPA) and (+)-copalic acid (anticopalic acid, ACP).

Based on the comprehensive application of copaiba oil in folk medicine and its recent popularity in the cosmetics and pharmaceutical industries, COPA and its derivatives have been subjected to various scientific studies aiming to prove their biological activities. Previous studies revealed that COPA possesses interesting bioactivities, including antibacterial20,21, antifungal22, anti-inflammatory23,24, anti-tuberculosis25 and cancer cytotoxic26 properties. Studies of COPA derivatives that offered superior bioactivities than that of the parent COPA have also been reported. For example, the hydrogenated COPA derivative and the COPA derivatives with epoxide, diketone, or sodium carboxylate moieties provided much higher anti-tuberculosis activity21,25. Some COPA amide analogues improved their activities in downregulating the expression of androgen receptor for the treatment of prostate cancer27.

In contrast to COPA, little is known about the bioactivities of ACP. To date, there have only been reports of the antimicrobial activity of ACP16,28 and antifeedant activity against Spodoptera frugiperda of ACP and derivatives9. In view of the limited existing studies of the bioactivities of ACP and the potential use of ACP in natural product-based drug discovery, we embarked on the synthesis and cytotoxic evaluation of ACP derivatives. Herein, the cytotoxic activity of ACP derivatives, mainly the amide analogs, against nine cancer cell lines and the normal cell line MRC-5 was evaluated. Additionally, the molecular mechanisms and biological targets of ACP derivative 4l in MDA-MB-231 triple-negative breast cancer cells were elucidated.

The formation of the ACP derivatives 1–3 and 4a–4s was performed as illustrated in Fig. 2. These included the reduction to the alcohol 2, the aldehyde 3, amidation to the amide analogs 4a–4r, and the further hydrolysis of the amide methyl ester 5q to the acid 4s. It is noteworthy that most derivatives are amide analogs due to their suitability for quickly performing studies on structure–activity relationship (SAR). In fact, amide formation tends to be the most commonly used by medicinal chemists, and amide bonds are widely present in pharmaceutically active substances, estimated to be found in 25% of marketed drugs29.

Synthesis of ACP derivatives.

The yield of the synthesized derivatives was moderate to excellent (20–100%). Of these derivatives, nineteen compounds of the amide derivatives, were reported for the first time. The structures of the synthesized compounds were confirmed by analysis of NMR spectroscopic data and MS. The physical characteristics and spectroscopic data of the synthesized compounds are reported in the Supplementary Information.

The synthetic compounds were screened for their cytotoxic activity using standard MTT or XTT methods30 against a panel of nine human cancer cell lines and one normal cell line, including MDA-MB-231 (triple-negative breast cancer), T-47D (hormone-dependent breast cancer), HepG2 (hepatocellular carcinoma), A549 (lung adenocarcinoma), H69AR (multidrug-resistant small cell lung carcinoma), HuCCA-1 (cholangiocarcinoma derived from a Thai patient), HeLa (cervical carcinoma), and two leukemia cell lines, HL-60 (acute promyelocytic leukemia) and MOLT-3 (T cell acute lymphoblastic leukemia). MRC-5 normal embryonic lung fibroblast cell line was used to represent normal cells. Chemotherapeutic drugs, doxorubicin and etoposide, were used as positive controls. IC50 values of the tested compounds are given in Table 1.

The results showed that the cytotoxic activity of almost all compounds in this series was higher than that of the parent compound ACP. Several derivatives demonstrated a selective cytotoxic activity towards the leukemia cell lines, HL-60 and MOLT-3, whereby compound 4p exhibited the most potent activity with IC50 of 6.81 ± 1.99 µM for HL-60 and 3.72 ± 0.26 µM for MOLT-3. Additionally, a derivative compound 4l exhibited notable cytotoxic activity against the TNBC cell line MDA-MB-231, with an IC50 of 13.73 ± 0.04 µM.

TNBC is the most aggressive type of human breast cancer, and there are currently no effective treatments5. From the screening, among the tested compounds, compound 4l has demonstrated the most promising cytotoxicity and the highest selectivity index toward MDA-MB-231 cells (Table 1, Supplementary Table S1). Moreover, this compound showed a superior selectivity index (SI = 2.9) compared to the positive drug doxorubicin (SI = 0.3), (see Supplementary Table S1). Given this potential, compound 4l was chosen for further investigation of its cytotoxic mechanism in MDA-MB-231 cells in order to develop new drugs for TNBC treatment.

Our cytotoxic screening includes multiple cancer cell lines derived from different organ origins (breast, liver, bile duct, lung, cervical, and lymphocytes), while only one normal cell line (MRC-5 lung fibroblast) was used as a representative of normal cells. MRC-5 cell line is a well-known normal cell line that has been widely used in cytotoxic screening31,32. This cell line can be easily cultured in standard medium without the use of any special supplements. Fibroblasts are a common cell type present in connective tissue of many organs. The selectivity index (SI) evaluation based on cytotoxicity against MRC-5 cell line reflects the degree of side effect of the tested compounds against a widely distributed cell type in the body, but it does not imply to the side effect in the specific organs, which is a limitation of our study.

Cell death modality has been categorized into accidental non-regulated cell death (necrosis) and 12 types of regulated cell death by the Nomenclature Committee on Cell Death, based on morphological features, biochemical events during initiation of cell death, and signaling pathways involved33. Regulated cell death can be categorized into apoptotic cell death modes (apoptosis and anoikis) and non-apoptotic cell death modes, which are further divided into 2 groups based on the presence of vacuole accumulation such as autophagy, or the absence of vacuoles such as necroptosis34. The majority of reported cytotoxic natural products induce cancer cell death through apoptosis. Still, recent findings demonstrated that several natural products exerted their cytotoxic activity through induction of non-apoptotic cell death modes, such as resveratrol-induced autophagy and shikonin-induced necroptosis35,36.

Mode of cell death induced by compound 4l was explored using MDA-MB-231 cells by observing change in cell morphology and flow cytometric analysis of annexin V/7-AAD double stained cells. The cells were treated with 4l for 24–48 h, at concentrations of 25–50 µM, which are higher than the IC50 value in this cell line. Microscopically, the 4l treatment did not result in the immediate formation of bubbles on cell surface (Fig. 3a). Accidental cell death necrosis is typically indicated by the immediate production of bubbles on the cell surface after treatment37. However, this morphological change was not observed in the 4l-treated cells, indicating that 4l treatment did not induce necrosis. Additionally, vacuole accumulation was not observed in the treated cells until 48 h after treatment (Fig. 3a). Taken together, we hypothesized that 4l-induced cell death in MDA-MB-231 cells was not processed through necrosis or vacuole-presenting regulated cell death such as autophagy.

Mode of cell death in MDA-MB-231 breast cancer cells treated with compound 4l. The cells were treated with 4l, doxorubicin (DOX), shikonin (SKN) at indicated concentrations for 24–48 h. Then photographs were taken and the cells were subjected to annexin V/7-AAD double staining and analyzed by the flow cytometric technique. (a) Morphology of 4l-treated cells, original magnification of × 400. (b,d) Representative dot plots of flow cytometric analysis of the treated cells showing the percentages of live cells (lower left), early apoptotic cells (lower right), late apoptotic cells (upper right), and dead cells (upper left). (c,e) Bar graphs displaying percentages of early apoptotic cells, late apoptotic cells, and dead cells in a total cell population. Data are expressed as mean ± SD from three independent experiments. Significant difference between treatment vs control at corresponding time points are shown by *p < 0.05, **p < 0.01, and ***p < 0.001, and significant difference between 24 h vs 48 h are indicated by &p < 0.05, &&p < 0.01, and &&&p < 0.001.

Interestingly, flow cytometric analysis with annexin V/7-AAD double staining showed that 4l treatment markedly increased the late apoptotic cell population in MDA-MB-231 cells, in a dose- and time-dependent manner. In comparison, the percentage of early apoptotic cell population did not change significantly with time (Fig. 3b,c). At 48 h, the late apoptotic cell population increased significantly from 5.8% for the control group to 19.0–26.3% for 4l at 25–50 µM (Fig. 3c). In contrast, the early apoptotic cell populations of 4l treatments ranged from 5.3 to 5.9%, which was not statistically different from the 4.2% found in the control group (Fig. 3c), indicating that the process of 4l-induced cell death did not progress through early stage apoptosis.

To compare annexin V/7-AAD double staining profiles of apoptotic and non-apoptotic cell death modes, doxorubicin (DOX) and shikonin (SKN) were employed as inducers of apoptosis and necroptosis, respectively. As shown in Fig. 3d,e, in DOX-induced cell death, the early apoptotic cell population was greater than the late apoptotic cell population at 24 h. Then it decreased at 48 h, concomitant with a time-dependent increase in the late apoptotic cell population (Fig. 3e). SKN, on the other hand, showed a time-dependent increase in the late apoptotic cell population during 24–48 h treatment, whereas the early apoptotic cell population was not elevated throughout the time course (Fig. 3e). The annexin V/7-AAD double staining profile of 4l-treated cells differs from that of the apoptosis inducer and resembles that of the necroptosis inducer.

Taken together, the results suggested that 4l-induced cell death in MDA-MB-231 cells was mediated by non-apoptotic regulated cell death, as indicated by a time-dependent increase in late apoptotic population without an increase in the early apoptotic population. Furthermore, the morphology of 4l-treated cells did not display an accumulation of autophagic vacuoles, excluding the possibility of autophagic cell death. Necroptosis, ferroptosis, mitoptosis, parthanatos, NETosis, and pyroptosis are non-apoptotic cell death modes that do not result in vacuole accumulation32. The precise mode of cell death induced by compound 4l, however, will be investigated further using multiple biochemical analyses.

Most chemotherapeutic drugs currently in use get rid of tumors by inducing apoptosis in cancer cells. However, apoptosis tolerance caused by dysregulation of apoptotic machinery is a mechanism that contributes to the development of cancer multidrug resistance, which is a major cause of failure in chemotherapeutic treatment38. Induction of non-apoptotic cell death mode by small molecules is an attractive strategy for overcoming the problem of apoptosis evasion39. Therefore, the ability of compound 4l to induce non-apoptotic cell death is of interest, suggesting that the compound has promise for future anticancer drug development.

We further investigated targets of compound 4l on cell survival signaling pathways including EGFR, FAK, Akt, and ERK in MDA-MB-231 cells. After treatment with 4l at concentrations of 25–50 µM for 24 h, activation (phosphorylation) of the signaling pathways was determined by using western blot analysis. As shown in Fig. 4, phosphorylation of FAK protein was selectively reduced by 4l in a dose-dependent manner, while phosphorylation of EGFR, Akt, and ERK was not affected by the treatment, suggesting that FAK inhibition might be the cytotoxic mechanism of compound 4l.

Inhibitory effect of compound 4l on cell survival signaling pathways in MDA-MB-231 cells. After 24 h treatment with compound 4l, phosphorylation levels of selected signaling proteins in the treated cells was detected by western blot analysis. Results are representative blots of three independent experiments.

To determine whether FAK inhibition is a mechanism involved in the induction of non-apoptotic cell death by compound 4l in MDA-MB-231 cells, the cells were treated with FAK specific inhibitor (1,2,4,5-benzenetetraamine or FAKi) at a cytotoxic concentration (16 μM), followed by analysis of the mode of cell death. As shown in Fig. 5a, after 24 h treatment, FAK phosphorylation was clearly inhibited in FAKi-treated cells. Throughout the 48-h treatment period, a time-dependent increase in the late apoptotic cell population was observed in FAKi-treated cells, which was always greater than the early apoptotic cell population (Fig. 5b,c). The results indicated that FAK inhibition in MDA-MB-231 cells caused non-apoptotic cell death, similar to that observed in 4l-treated cells.

Mode of cell death in MDA-MB-231 cells exposed to FAK specific inhibitor (FAKi). The cells were treated with FAKi (16 μM) for 24–48 h. (a) Western blot analysis of FAK phosphorylation at 24 h after treatment. (b) Representative dot plots of flow cytometric analysis of the treated cells. (c) Bar graphs displaying percentages of early apoptotic cells, late apoptotic cells, and dead cells in total cell population. Data are expressed as mean ± SD from three independent experiments. Significant difference between treatment vs control at corresponding time point are shown by *p < 0.05, **p < 0.01, and ***p < 0.001, and significant difference between 24 vs 48 h are indicated by &&p < 0.01.

Several lines of evidence indicate that FAK regulates cell survival as well as several malignant characteristics of cancer cells and is frequently overexpressed in a wide range of tumors40. FAK signaling has been reported to be involved in the radio/chemo resistance of cancers. Downregulation of FAK increases the cytotoxic effect of radiation in colon cancer cells41, and also enhances cisplatin sensitivity in TNBC cells42. In vivo evaluation of a small molecule FAK inhibitor BI 856520 in mouse models of breast cancer yields promising results on suppression of primary tumor growth and outgrowth of metastatic tumors, through impairing cell proliferation both in vitro and in vivo43. Several FAK inhibitors are currently being tested in clinical trials in various cancer types, either as single therapy or in combination with other anticancer drugs44. Therefore, the compound 4l might be a potential compound for development as a chemotherapeutic agent to treat TNBC.

To explore the binding between FAK and compound 4l, the molecular docking was conducted using iGEMDOCK v2.1 software. Two possible binding sites for small molecule inhibitor on FAK protein, FERM domain and kinase domain, were used as receptor. Unlike FAKi which has been shown to bind to the FERM domain at a pocket closed to Tyr39745, the binding site of 4l on the FERM domain appears to be far from the Tyr397 pocket (Fig. 6a). On the other hand, the binding site of 4l on the kinase domain was located in an ATP binding pocket (Fig. 6b), which was identical to the binding site of another FAK specific inhibitor, TAE22646, indicating that FAK catalytic activity would be inhibited upon binding of 4l. These results suggested that compound 4l reduced Tyr397 autophosphorylation of FAK by interfering with FAK kinase activity via binding to the kinase domain rather than the FERM domain.

The binding positions of 4l on FAK protein. (a) FAK FERM domain (PDB:2AL6), the phosphorylation site Tyr397 are indicated, three subdomains are labeled with colors violet (F1), green (F2), and red (F3), and compound 4l is shown in pink stick. (b) FAK kinase domain (PDB ID: 2JKK) with compound 4l (pink stick) and TAE226 (yellow stick) in the ATP binding pocket (grey).

As illustrated in Fig. 7a, redock TAE226 was located to the same position as co-crystallized TAE226, indicating that our docking method was acceptable. The binding energy of TAE226 was − 134.05 kcal/mol, and TAE226 formed two hydrogen bonds with Asp564 and Cys502 (Fig. 7b). Moreover, as shown in Table 2, 4l had the binding energy of − 80.44 kcal/mol. Figure 7c demonstrates that 4l formed one hydrogen bond with Cys502 of FAK kinase domain. Fused-cyclohexane rings, methyl carbon and methylene carbon of 4l also made hydrophobic contacts with Ile428 and Leu501 (Fig. 7d). It can also be noticed that there were hydrophobic interactions between the tetrahydropyran ring of 4l and four amino acid side chains including Ala452, Val484, Met499, and Leu553 (Fig. 7d). This indicates that fused-cyclohexane rings and tetrahydropyran ring of 4l can act as the key moieties in binding to the catalytic site of FAK, making 4l a potential FAK inhbitor.

Superposition of redocked TAE226 (red) and co-crystallized TAE226 (yellow) (a), hydrogen bond interaction of TAE226 (b), hydrogen bond interaction of 4l (pink) (c) and 2D diagram representing interactions of 4l (d) in the ATP binding pocket of FAK kinase domain (PDB ID: 2JKK).

To evaluate the druggability47,48 of compound 4l, the SwissADME website service was used to study the physicochemical properties and analysis by Lipinski’s and Veber’s rules. The calculation of the parent compound ACP was also performed to compare with 4l. The calculated parameters are presented in Table 3. Considering the Lipinski’s rule of five, the parameters of these two compounds, including the molecular weight, the number of hydrogen bond acceptors and the number of hydrogen bond donors abided by the rule. However, the MLogP values, representing the lipophilicity of the compound, are slightly greater than the reported requirement (4.15)49. These MLogP values are consistent with the LogS values, which indicated the lower water solubility of these compounds. Nevertheless, compound 4l and ACP have high degree of fraction sp3 (Fsp3), indicating high degree of saturation and chirality content, which could promote a significant preference for binding and selectivity to proteins. Lastly, compound 4l and ACP have TPSA values much less than 140 Å2 indicating good cell membrane permeability as molecules with a TPSA value more than 140 (Å2) tend to be poor at permeating cell membranes50. Moreover, the introduction of the amide substituents leads to an increase in the TPSA of 4l, making the TSPA value of 4l close to the range that attributed to most successful drug (≤ 60–70 Å2). According to the physiochemical parameters, both 4l and ACP exhibited parameters suitable for appropriate drug ability, except for the solubility of the compounds. The chemical modification of the parent compounds in drug discovery generally aims to improve either physiochemical properties or potency of the drugs, or both. In this study, eventhough compound 4l did not show improvement in terms of the physiochemical parameters compared to the parent compound ACP, compound 4l exhibited much higher potency than ACP, indicating improvement in the potency of the drugs from the modification. Further derivatization of the compounds could allow for better physiochemical values that conform to the drug likeness guideline.

1H- and 13C-NMR spectra were recorded in CDCl3 or DMSO-d6 using a Bruker AVANCE 300 NMR or a Bruker AVANCE 400 NMR spectrometer. 1H-NMR and 13C-NMR chemical shifts (δ) Chemical shifts were expressed in ppm and referenced to the residual solvent signals. Coupling constants (J) were reported in Hertz (Hz). IR spectra were recorded on a PerkinElmer Spectrum One Spectrometer using a universal attenuated reflectance (ATR) technique and are reported in cm-1. HRESIMS analysis was determined using a Bruker Daltonics microTOF spectrometer. Optical rotations were measured on a JASCO P-1020 polarimeter. All glassware was heat-dried prior to use. TLC were visualized using UV light (254 and 366 nm) and Godin’s reagent.

The rhizomes of wild-type K. elegans used in this study were from the rural area in Sai Yok District, Kanchanaburi Province, Thailand and were collected by the local people who live in the area under their permission. The plant was authenticated by a taxonomist, Professor Dr. Wongsatit Chuakul of the Faculty of Pharmacy, Mahidol University, Thailand. The voucher specimen number BKF 192,348 (Thongnest No. 2) was deposited at the Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand. All procedures for plant collection were performed in accordance with relevant institutional, national, and international guidelines and legislation.

All solvents were distilled from commercial grade solvents except where indicated otherwise. Dichlromethane (CH2Cl2) was further purified by pressure filtration through activated alumina for reaction set up. Spectral grade solvents were used for spectroscopic measurements. Thin layer chromatography was performed on Merck precoated silica gel 60 F254 plates. Silica gel 60 (Silicycle, 230–400 mesh) was used for flash column chromatography, and Silica gel 60 PF254 (Merck) was used for preparative thin layer chromatography. Silica gel precoated aluminum plates (F254, 0.25 mm) were used for TLC detection. Muse® Annexin V & Dead Cell Kit was purchased from Luminex (Austin, TX, USA). Doxorubicin, Etoposide, and shikonin were obtained from Sigma-Aldrich (St. Louis, MO, USA). FAK specific inhibitor (1,2,4,5-benzenetetraamine tetrahydrochloride or FAK inhibitor 14) was purchased from Tocris Bioscience (Bristol, U.K.). Protease/phosphatase inhibitor cocktail and all antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). SuperSignal™ ECL substrates were purchased from Thermo Scientific (Rockford, IL, USA).

MDA-MB-231 (triple-negative breast cancer), T-47D (hormone-dependent breast cancer), HepG2 (hepatocellular carcinoma), HL-60 (acute promyelocytic leukemia), MOLT-3 (T-cell acute lymphoblastic leukemia), A549 (lung adenocarcinoma), H69AR (multidrug-resistant small cell lung carcinoma), HeLa (cervical carcinoma), and MRC-5 (normal embryonic lung fibroblast) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HuCCA-1 (cholangiocarcinoma derived from a Thai patient) was obtained from Laboratory of Immunology, Chulabhorn Research Institute, Thailand.

The rhizomes of K. elegans (25 kg) were extracted with dichloromethane (2 × 70 L) at room temperature. After evaporating the solvent, a crude extract (420.6 g) was obtained. The crude dichloromethane extract (420.6 g) was chromatographed over silica gel column, using a step gradient system with hexane-CH2Cl2 (100:0 to 0:100) and CH2Cl2-MeOH (100:0 to 0:100) to yield eighteen fractions (F1-F18) after TLC detection. One of the ACP rich fractions (Fraction F7, 38.5 g) was purified by silica column chromatography eluted with n-hexane-CH2Cl2 (95:5 to 0:100) and CH2Cl2-MeOH (100:0 to 90:10) to give pure ACP as white amorphous (34.5 g). The structure of ACP obtained from the plant extract was identified based on 1H and 13C NMR spectral data compared with the previous data16. Several other ACP-rich fractions will be used for future isolation in due course.

To a solution of ACP (25 mg, 0.08 mmol, 1.0 equiv.) in CH2Cl2 (0.8 mL, 0.1 M) was added HOBt (16.5 mg, 0.12 mmol, 1.5 equiv) and EDCl (23.4 mg, 0.12 mmol, 1.5 equiv.). The mixture was stirred at room temperature for 30 min and then concentrated in vacuo. The residue was dissolved in THF (0.8 mL, 0.1 M) and cooled to 0 °C, and NaBH4 (9.2 mg, 0.24 mmol, 3.0 equiv.) was added into the mixture. Then, H2O (2 µL, 0.12 mmol, 1.5 equiv.) was added into a solution. The resulting mixture was stirred at 0 °C for 30 min, and then quenched with MeOH (1 mL). The mixture was dried under vacuum and redissolved in EtOAc. The organic layer was wash with 10% citric acid, brine, and dried over Na2SO4, and concentrated under vacuum. The product was purified by column chromatography eluting with EtOAc:hexane (10:90) to provide 6 mg (25%) of anticopalol (2)16 as a colorless oil. \({[\mathrm{\alpha }]}_{\mathrm{D}}^{27}\) =  + 30.3 (c 0.95, CHCl3); FTIR (neat) Ѵmax: 3356, 2923, 2847, 1662, 1642, 1457, 1442, 1387, 996, 887 cm‒1; 1H NMR (600 MHz, CDCl3) δH 4.83 (1H, d, J = 1.3 Hz, H-17), 4.51 (1H, d, J = 1.0 Hz, H-17), 4.18 (1H, dd, J = 6.9, 1.2 Hz, H-14), 4.18 (1H, d, J = 6.9 Hz, H-15), 2.40 (1H, ddd, J = 12.8, 4.2, 2.5 Hz, H-7), 2.16 (2H, ddd, J = 14.0, 9.8, 4.0 Hz, H2-12), 1.98 (1H, td, J = 12.9, 5.0 Hz, H-7), 1.76 (1H, m, H-1), 1.71 (1H, m, H-6), 1.67 (3H, s, H3-16), 1.61 (1H, m, H-11), 1.57 (1H, m, H-9), 1.56 (1H, m, H-2), 1.49 (1H, m, H-2), 1.43 (1H, m, H-11), 1.39 (1H, m, H-3), 1.32 (1H, tdd, J = 12.9, 12.9, 4.3 Hz, H-6), 1.18 (1H, td, J = 12.8, 4.9 Hz, H-3), 1.10 (1H, dd, J = 12.5, 2.7 Hz, H-5), 0.90 (3H, s, H3-19), 0.80 (3H, s, H3-18), 0.67 (3H, s, H3-20); 13C NMR (150 MHz, CDCl3) δ 148.6 (C-8, s), 140.6 (C-13, s), 123.1 (C-14, d), 106.2 (C-17, t), 59.4 (C-15, t), 56.4 (C-9, d), 55.6 (C-5, d), 42.2 (C-3, t), 39.7 (C-10, s), 39.1 (C-1, t), 38.5 (C-12, t), 38.4 (C-7, t), 33.7 (C-19, q), 33.6 (C-4, s), 24.5 (C-6, t), 21.9 (C-11, t), 21.7 (C-19, q), 19.4 (C-2, t), 19.3 (C-16, q), 14.5 (C-20, q); ESI-TOF MS: calcd for C20H34NaO, m/z 313.2502 [M + Na]+ found 340.2502.

Anticopalol (2) (5.5 mg, 0.018 mmol, 1.0 equiv.) was dissolved in CH2Cl2 (0.18 mL, 0.1 M). Then, Dess-Martin periodinane (9.6 mg, 0.022 mmol, 1.2 equiv.) was slowly added into a mixture. The solution was kept stirring at room temperature for 3 h. The reaction was worked up with NH4Cl (aq.). The mixture was extracted with EtOAc, brine, and dried over Na2SO4.The crude extract was concentrated under vacuum and purified by column chromatography eluting with EtOAc:hexane (20:80) to give 6.6 mg (100%) of the aldehyde 3 as a yellow oil. \({[\mathrm{\alpha }]}_{\mathrm{D}}^{27}\) =  + 10.5 3 (c 0.48, CHCl3); FTIR (neat) Ѵmax: 2925, 2865, 1694, 1642, 1459, 1441, 1387, 1260, 1094, 1018, 887, 802 cm‒1; 1H NMR (400 MHz, CDCl3) δH 9.99 (1H, d, J = 8.0 Hz, H-15), 5.87 (1H, d, J = 8.0 Hz, H-14), 4.84 (1H, brs, H-17), 4.46 (1H, brs, H-17), 2.39 (1H, m, H-7), 2.32 (1H, m, H-12), 2.15 (3H, s, H3-16), 2.04 (1H, m, H-12), 1.94 (1H, m, H-7), 1.74 (1H, m, H-11), 1.74 (1H, m, H-6), 1.65 (1H, m, H-1), 1.62 (1H, m, H-9), 1.50 (1H, m, H-11), 1.45 (2H, m, H2-2), 1.33 (1H, m, H-3), 1.25 (1H, m, H-6), 1.16 (1H, m, H-3), 1.10 (1H, m, H-5), 0.85 (3H, s, H3-19), 0.78 (3H, s, H3-18), 0.68 (3H, s, H3-20)); 13C NMR (100 MHz, CDCl3) δC 191.3 (C-15, d), 164.9 (C-13, s), 148.1 (C-8, s), 127.1 (C-14, d), 106.3 (C-17, t), 56.1 (C-9, d), 55.5 (C-5, d), 42.0 (C-3, t), 39.7 (C-12, t), 39.5 (C-10, s), 39.0 (C-1, t), 38.2 (C-7, t), 33.5 (C-4, s), 33.5 (C-19, q), 24.4 (C-6, t), 21.7 (C-18, q), 21.2 (C-11, t), 19.3 (C-2, t), 17.6 (C-16, q), 14.4 (C-20, q); ESI MS: calcd for C20H32NaO, m/z 311.2345 [M + Na]+ found 311.2352.

A mixture of ACP (25 mg, 1.0 equiv.), 1-hydroxybenzotriazole hydrate (HOBt) (1.5 equiv.), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI) (1.5 equiv.), amine derivative (1.5 equiv.) in CH2Cl2 (0.1 M) was stirred at 0 °C for 5 min, and then N,N-diisopropylethylamine (DIPEA) (3 equiv.) was slowly added into the mixture. The reaction mixture was stirred at 0 °C for additional 15 min and further stirred at room temperature for 24 h. Then, the reaction was quenched with aqueous 1 N hydrochloric acid solution and extracted with EtOAc (× 3). The organic layers were combined, washed with brine, and dried with MgSO4 (s) to obtain a crude fraction, which was further purified by flash silica gel column chromatography, eluted with either EtOAc: hexane or CH2Cl2:hexane to provide the amide derivatives of ACP 4a–4r.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of CH2Cl2:MeOH (99:1) as an eluent to provide 4a (17.2 mg, 67%) as yellow oil. \({[\mathrm{\alpha }]}_{D}^{25}\) +44.0 (c 1.69 CHCl3); FTIR (neat) Ѵmax: 3298, 3078, 2927, 2866, 2843, 1660, 1630, 1548, 1458, 1442, 1410, 1387, 1365, 1262, 1181, 886, 863, 737, 673 cm‒1; 1H NMR (400 MHz, CDCl3) δH 5.49 (1H, br s, H-14), 4.81 (1H, br s, H2-17), 4.47 (1H, br s, H2-17), 2.81 (3H, d, J = 5.0 Hz, H3-1ʹ), 2.36 (1H, ddd, J = 13.0, 4.0, 2.0 Hz, H2-7), 2.21 (1H, ddd, J = 14.0, 9.0, 4.0 Hz, H2-12), 2.12 (3H, d, J = 2.0 Hz, H3-16), 1.96 (1H, m, H2-7), 1.87 (1H, m, H-12), 1.73 (1H, m, H2-1), 1.73 (1H, m, H2-6), 1.65 (1H, m, H2-11), 1.60 (1H, m, H2-2), 1.57 (1H, m, H-9), 1.53 (1H, m, H2-2), 1.47 (1H, m, H2-11), 1.43 (1H, m, H2-3), 1.30 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.17 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.05 (1H, dd, J = 13.0, 3.0 Hz, H-5), 0.96 (1H, td, J = 13.0, 4.0 Hz, H2-1), 0.85 (3H, s, H3-18), 0.78 (3H, s, H3-19), 0.66 (3H, s, H3-20); 13C NMR (100 MHz, CDCl3) δC 168.0 (C-15, s), 154.8 (C-13, s), 148.5 (C-8, s), 117.6 (C-14, d), 106.3 (C-17, t), 56.1 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 39.7 (C-10, s), 39.5 (C-12, t), 39.0 (C-1, t), 38.3 (C-7, t), 33.6 (C-18, q), 33.6 (C-4, s), 26.0 (C-1ʹ, q), 24.4 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, t), 19.4 (C-2, t), 18.2 (C-16, q), 14.5 (C-20, q); ESI-TOF MS: calcd for C21H35NNaO, m/z 340.2610 [M + Na]+ found 340.2610.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (5:95) as an eluent to provide 4b (21.4 mg, 78%) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{25}\) +38.3 (c 1.98, CHCl3); FTIR (neat) Ѵmax: 3293, 3078, 2926, 2866, 2844, 1660, 1630, 1536, 1459, 1442, 1388, 1365, 1256, 1178, 988, 915, 887 cm‒1. 1H NMR (400 MHz, CDCl3) δ 5.86 (1H, m, H-2ʹ), 5.53 (1H, dd, J = 2.4, 1.2 Hz, H-14), 5.20 (1H, m, H2-3ʹ), 5.13 (1H, m, H2-3ʹ), 4.83 (1H, d, J = 1.6 Hz, H2-17), 4.49 (1H, d , J = 1.2 Hz, H2-17), 3.92 (2H, tt, J = 6.0, 1.6 Hz., H2-1ʹ), 2.3389 (1H, ddd, J = 13.0, 4.4, 2.4 Hz, H2-7), 2.24 (1H, ddd, J = 12.0, 10.0, 4.4 Hz, H2-12), 2.16 (3H, d, J = 1.2 Hz, H3-16), 1.97 (1H, m, H2-7), 1.91 (1H, m, H2-12), 1.75 (1H, m, H2-1), 1.72 (1H, m, H2-6), 1.67 (1H, m, H2-11), 1.58 (1H, m, H2-2), 1.55 (1H, m, H-9), 1.50 (1H, m, H2-2), 1.46 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.32 (1H, dd, J = 13.0, 4.4 Hz, H2-6), 1.17 (1H, td, J = 13.4, 4.2 Hz, H2-3), 1.08 (1H, dd, J = 12.0, 2.7 Hz, H-5), 1.01 (1H, td, J = 13.0, 4.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H,s, H3-20). 13C NMR (100 MHz, CDCl3) δ 166.9 (C-15, s), 155.6 (C-13, s), 148.5 (C-8, s) 134.6 (C-2ʹ, d), 117.5 (C-14, d), 116.3 (C-3ʹ, t), 106.3 (C-17, t), 56.1 (C-9, d), 55.5 (C-5, d), 42.1 (C-1ʹ, t), 41.6 (C-3, t), 39.7 (C-12, t), 39.6 (C-10, s), 39.0 (C-1, t), 38.3 (C-7, t), 33.6 (C-4, s), 33.6 (C-18, q), 24.5 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, t), 19.4 (C-2, t), 18.4 (C-16, q), 14.5 (C-20, q)); ESI-TOF MS: calcd for C23H37NNaO, m/z 366.2767 [M + Na]+, found 366.2766.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (5:95) as an eluent to provide 4c (21.1 mg, quantitative yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{25}\) +32.9 (c 1.69, CHCl3); FTIR (neat) Ѵmax: 3309, 2927, 2847, 1662, 1637, 1527, 1459, 1442, 1387, 1366, 1253, 1176, 1115, 886 cm‒1. 1H NMR (400 MHz, CDCl3) δ 5.59 (1H, br s, NH), 5.51 (1H, br d, J = 0.8 Hz, H-14), 4.83 (1H, d, J = 1.6 Hz, H2-17), 4.48 (1H, d, J = 1.2 Hz, H2-17), 4.10 (2H, dd, J = 5.6, 2.8 Hz, H2-1ʹ), 2.38 (1H, ddd, J = 13.0, 4.4, 2.4 Hz, H2-7), 2.26 (1H, dd, J = 10.0, 4.0 Hz, H2-12), 2.23 (1H, t, J = 2.8 Hz, H-3ʹ), 2.16 (3H, d, J = 1.2 Hz, H3-16), 1.97 (1H, dd, J = 13.0, 5.0 Hz, H2-7), 1.91 (1H, m, H2-12), 1.75 (1H, m, H2-1), 1.71 (1H, m, H2-6), 1.64 (1H, m, H2-11), 1.58 (1H, m, H2-2), 1.55 (1H, m, H-9), 1.49 (1H, m, H2-2), 1.45 (1H, m, H-11), 1.39 (1H, m, H2-3), 1.32 (1H, dd, J = 13.0, 4.4 Hz, H2-6), 1.17 (1H, td, J = 13.0, 4.4 Hz, H2-3), 1.08 (1H, dd, J = 13.0, 2.8 Hz, H-5), 1.00 (1H, td, J = 13.0, 4.4 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20). 13C NMR (100 MHz, CDCl3) δ 166.6 (C-15, s), 156.9 (C-13, s), 148.4 (C-8, s), 116.8 (C-14, d), 106.3 (C-17, t), 79.9 (C-2ʹ, s), 71.4 (C-3ʹ, d), 56.0 (C-9, d), 55.4 (C-5, d), 42.1 (C-3, t), 39.66 (C-10, s), 39.65 (C-12, t), 39.0 (C-1, t), 38.3 (C-7, t), 33.59 (C-18, q), 33.57 (C-4, s), 28.9 (C-1ʹ, t), 24.4 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, t), 19.4 (C-2, t), 18.4 (C-16, q), 14.5 (C-20, q). ESI-TOF MS: calcd for C23H36NO, m/z 342.2791 [M + H]+, found 342.2794.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (10:90) as an eluent to provide 4d (26.9 mg, 80% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{26}\) +28.2 (c 2.51, CHCl3); FTIR (neat) Ѵmax: 3293, 3078, 2924, 2851, 1659, 1628, 1542, 1459, 1441, 1387, 1365, 1259, 887 cm‒1. 1H NMR (400 MHz, CDCl3) δ 5.5 (1H, d, J = 1.2 Hz, H-14), 5.39 (1H, br s, NH), 4.83 (1H, d, J = 1.5 Hz, H2-17), 4.49 (1H, d, J = 1.2 Hz, H2-17), 3.27 (2H, m, H2-1ʹ), 2.38 (1H, ddd, J = 12.0, 4.4, 2.4 Hz, H2-7), 2.23 (1H, ddd, J = 12.0, 10.0, 4.0 Hz, H2-12), 2.14 (3H, d, J = 1.2 Hz, H3-16), 1.97 (1H, m, H2-7), 1.90 (1H, m, H2-12), 1.76 (1H, m, H2-1), 1.72 (1H, m, H2-6), 1.65 (1H, m, H2-11), 1.58 (1H, m, H2-2), 1.56 (1H, m, H-9), 1.53‒1.47 (2H, m, H-2'), 1.49 (2H, m, H2-2/H2-2ʹ), 1.45 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.33 (1H, m, H2-6), 1.32‒1.25 (10H, m, H-3ʹ/H-4ʹ/ H-5ʹ/H-6ʹ/H-7ʹ), 1.17 (1H, td, J = 13.0, 4.0 Hz, H-3), 1.08 (1H, dd, J = 13.0, 4.0 Hz, H-5), 1.01 (1H, td, J = 13.0, 4.0 Hz, H-1), 0.88 (3H, m, H3-8ʹ), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20). 13C NMR (100 MHz, CDCl3) δ 167.2 (C-15, s), 154.7 (C-13, s), 148.5 (C-8, s), 117.9 (C-14, d), 106.3 (C-17, t), 56.1 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 39.7 (C-10, s), 39.6 (C-12, t), 39.2 (C-1ʹ, t), 39.0 (C-1, t), 38.3 (C-7, t), 33.6 (C-18, q), 33.58 (C-4, s), 31.8 (C-6ʹ, t), 29.8 (C-2ʹ, t), 29.3 (C-4ʹ, t), 29.2 (C-5ʹ, t), 27.0 (C-3ʹ, t), 24.5 (C-6, t), 22.6 (C-7ʹ, t), 21.7 (C-19, q), 21.5 (C-11), 19.3 (C-2), 18.3 (C-16, q), 14.5 (C-20, q), 14.1 (C-8ʹ, q). ESI-TOF MS: calcd for C28H49NNaO, m/z 438.3706 [M + Na]+, found 438.3695.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (5:95) as an eluent to provide 4e (16.7 mg, 54% yield) as a yellow oil.\({[\mathrm{\alpha }]}_{D}^{25}\) +35.2 (c 0.69, CHCl3); FTIR (neat) Ѵmax: 3308, 2927, 2845, 1661, 1641, 1598, 1541, 1499, 1440, 1387, 1309, 1252, 1152, 1079, 888, 752, 691 cm‒1. 1H NMR (400 MHz, CDCl3) δ 7.55 (1H, d, J = 7.6 Hz, H-2ʹ), 7.32 (3H, m, H-3ʹ/H-4ʹ/H-5ʹ), 7.08 (1H, br t, J = 7.6 Hz, H-6ʹ), 5.66 (1H, d, J = 1.0 Hz, H-14), 4.82 (1H, d, J = 1.1 Hz, H2-17), 4.52 (1H, br s, H2-17), 2.40 (1H, ddd, J = 13.0, 4.4, 2.4 Hz, H2-7), 2.27 (1H, ddd, J = 13.0, 10.4, 1.2 Hz, H2-12), 2.22 (3H, d, J = 1.2 Hz, H3-16), 1.98 (1H, m, H2-7), 1.95 (1H, m, H2-12), 1.76 (1H, m, H2-1), 1.74 (1H, m, H2-6), 1.71 (1H, m, H2-11), 1.61 (1H, m, H2-2), 1.58 (1H, m, H-9), 1.53 (1H, m, H2-2), 1.48 (1H, m, H2-11), 1.40 (1H, m, H2-3), 1.33 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.18 (1H, td, J = 13.0, 4.4 Hz, H2-3), 1.10 (1H, dd, J = 12.4, 3.0 Hz, H-5), 1.02 (1H, td, J = 13.0, 4.0 Hz, H2-1), 0.88 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.70 (3H, s, H3-20). 13C NMR (100 MHz, CDCl3) δC 165.0 (C-15, s), 157.9 (C-13, s), 148.5 (C-8, s), 138.3 (C-1ʹ, s), 129.0 (C-4ʹ, d), 124.0 (C-3ʹ/C-5ʹ, d), 119.7 (C-2ʹ/C-6ʹ, d), 117.9 (C-14, d), 106.4 (C-17), 56.1 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 39.9 (C-12, t), 39.7 (C-10, s), 39.1 (C-1, t), 38.4 (C-7, t), 33.6 (C-18, q), 33.6 (C-4, s), 24.5 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, t), 19.4 (C-2, t), 18.6 (C-16, q), 14.5 (C-20, q); ESI MS: Calcd for C26H37NNaO, m/z 402.2767 [M + Na]+, found 402.2779.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (10:90) as an eluent to provide 4f (30.7 mg, 97% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{25}\) +31.0 (c 0.70, CHCl3); FTIR (neat) Ѵmax: 3295, 2925, 2843, 1657, 1631, 1536, 1454, 1387, 1365, 1255, 1175, 887, 697 cm‒1. 1H NMR (400 MHz, CDCl3) δ 7.31‒7.27 (5H, m, H-2ʹ/H-3ʹ/H-4ʹ/H-5ʹ/H-6ʹ), 5.63 (1H, br s, NH), 5.53 (1H, d, J = 1.1 Hz, H-14), 4.83 (1H, d, J = 1.4 Hz, H2-17), 4.48 (1H, s, H2-17), 4.47 (2H, m, H2-7ʹ), 2.36 (1H, ddd, J = 13.0, 4.4, 2.4 Hz, H2-7), 2.24 (1H, ddd, J = 13.0, 10.4, 1.2 Hz, H2-12), 2.18 (3H, d, J = 1.1 Hz, H3-16), 1.96 (1H, m, H2-7), 1.89 (1H, m, H2-12), 1.75 (1H, m, H2-1), 1.71 (1H, m, H2-6), 1.65 (1H, m, H2-11), 1.59 (1H, m, H2-2), 1.56 (1H, m, H-9), 1.49 (1H, m, H2-2), 1.44 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.31 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.17 (1H, td, J = 13.0, 4.4 Hz, H2-3), 1.08 (1H, dd, J = 12.4, 3.0 Hz, H-5), 1.00 (1H, td, J = 13.0, 4.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.67 (3H, s, H3-20). 13C NMR (100 MHz, CDCl3) δ 167.9 (C-15, s), 156.9 (C-13, s), 149.4 (C-8, s), 139.5 (C-1ʹ, s), 129.5 (C-3ʹ/C-5ʹ, d), 128.7 (C-2ʹ/C-6ʹ, d), 128.2 (C-4ʹ, d), 118.2 (C-14, d), 107.0 (C-17, t), 56.5 (C-9, d), 55.8 (C-5, d),43.6 (C-7ʹ, t), 42.4 (C-3, t), 39.99 (C-10, s), 39.90 (C-12, t), 39.3 (C-1, t), 38.6 (C-7, t), 33.8 (C-4, s), 33.8 (C-18, q), 24.6 (C-6, t), 21.9 (C-19, q), 21.7 (C-11, t), 19.5 (C-2, t), 18.6 (C-16, q), 14.6 (C-20, q). ESI-TOF MS: calcd for C27H39NNaO, m/z 416.2924 [M + Na]+, found 416.2940.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (20:80) as an eluent to provide 4g (29.1 mg, 91% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{27}\) +33.3 (c 0.71, CHCl3); FTIR (neat) Ѵmax: 3312, 3077, 2925, 2866, 2843, 1661, 1641, 1599, 1571, 1524, 1459, 1437, 1387, 1365, 1234, 1153, 887, 750, 692 cm‒1. 1H NMR (400 MHz, CDCl3) δ 8.54 (1H, ddd, J = 4.0, 1.6, 0.4 Hz, H-6ʹ), 7.67 (1H, td, J = 8.0, 2.0 Hz, H-4ʹ), 7.30 (1H, d, J = 8.0 Hz, H-3ʹ), 7.20 (1H, ddd, J = 4.0, 1.6, 0.8 Hz, H-5ʹ), 6.60 (1H, br s, NH), 5.65 (1H, br d, J = 1.3 Hz, H-14), 4.84, d, J = 1.6 Hz, H2-17), 4.61 (1H, br s, H2-7ʹ), 4.60 (1H, br s, H2-7ʹ), 4.50 (1H, d, J = 1.2 Hz, H2-17), 2.38 (1H, ddd, J = 13.0, 4.4, 2.4 Hz, H2-7), 2.26 (1H, ddd, J = 16.0, 10.4, 1.2 Hz, H2-12), 2.17 (3H, d, J = 1.2 Hz, H3-16), 1.98 (1H, dd, J = 13.0, 2.4 Hz, H2-7), 1.91 (1H, m, H2-12), 1.74 (1H, m, H2-1), 1.71 (1H, m, H2-6), 1.66 (1H, m, H2-11), 1.58 (1H, m, H2-2), 1.56 (1H, m, H-9), 1.50 (1H, m, H2-2), 1.46 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.32 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.17 (1H, td, J = 13.0, 4.4 Hz, H2-3), 1.09 (1H, dd, J = 12.4, 3.0 Hz, H-5), 1.01 (1H, td, J = 13.0, 5.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20). 13C NMR (100 MHz, CDCl3) δ 167.2 (C-15, s), 156.8 (C-2ʹ, s), 155.6 (C-13, s), 149.0 (C-6ʹ, d), 148.5 (C-8, s), 136.8 (C-4ʹ, d), 122.31 (C-3ʹ, d) 122.24 (C-5ʹ, d), 117.6 (C-14, d), 106.3 (C-17, t), 56.1 (C-9, d), 55.5 (C-5), 44.3 (C-7ʹ, d), 42.1 (C-3, t), 39.7 (C-10, s), 39.7 (C-12, t), 39.1 (C-1, t), 38.3 (C-7, t), 33.6 (C-4, s), 33.6 (C-18, q), 24.5 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, t), 19.4 (C-2, t), 18.4 (C-16, q), 14.5 (C-20, q). ESI-TOF MS: calcd for C26H38N2NaO, m/z 417.2876 [M + Na]+, found 417.2893.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (20:80) as an eluent to provide 4h (33 mg, 91% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{25}\) +26.2 (c 2.21, CHCl3); FTIR (neat) Ѵmax: 3299, 2928, 2845, 1689, 1641, 1521, 1457, 1440, 1387, 1365, 1254, 1230, 887, 740 cm‒1. 1H NMR (400 MHz, CDCl3) δ 8.30 (1H, br s, NH-tryptamine), 7.61 (1H, d, J = 8.0 Hz, H-7ʹ tryptamine), 7.37 (1H, d, J = 8.0 Hz, H-4ʹ tryptamine), 7.21 (1H, t, J = 7.0 Hz, H-5ʹ tryptamine), 7.11 (1H, t, J = 7.0 Hz, H-6ʹ tryptamine), 7.03 (1H, d, J = 2.0 Hz, H-2ʹ tryptamine), 5.49 (1H, br s, NH), 5.43 (1H, br s, H-14), 4.81 (1H, d, J = 1.2 Hz, H2-17), 4.47 (1H, br s, H2-17), 3.63 (2H, ddd, J = 7.0, 5.0, 2.0 Hz, H2-α tryptamine), 2.99 (2H, t, J = 7.0 Hz, H2-β tryptamine), 2.37 (1H, ddd, J = 13.0, 4.0, 3.0 Hz, H2-7), 2.20 (1H, ddd, J = 13.0, 10.0, 4.0 Hz, H2-12), 2.14 (3H, d, J = 0.8 Hz, H3-16), 1.95 (1H, m, H2-7), 1.86 (1H, m, H2-12), 1.74 (1H, m, H2-1), 1.70 (1H, m, H2-6), 1.63 (2H, m, H2-11), 1.57 (1H, m, H2-2), 1.54 (1H, m, H-9), 1.48 (1H, m, H2-2), 1.43 (1H, m, H2-11), 1.38 (1H, m, H2-3), 1.31 (1H, dd, J = 13.0, 4.4 Hz, H2-6), 1.16 (1H, td, J = 12.0, 4.1 Hz, H2-3), 1.07 (1H, dd, J = 12.0, 2.4 Hz, H-5), 0.99 (1H, td, J = 12.0, 4.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.67 (3H, s, H3-20); 13C NMR (100 MHz, CDCl3) δ 167.2 (C-15, s), 155.0 (C-13, s), 148.6 (C-8, s), 136.4 (C-7ʹa, s), 127.4 (C-3ʹa, s), 122.1 (C-2ʹ, d), 122.1 (C-6ʹ, d), 119.4 (C-5ʹ, d), 118.9 (C-7ʹ, d), 117.8 (C-14, d), 113.1 (C-3ʹ, s), 111.3 (C-4', d), 106.3 (C-17, t), 56.1 (C-9, d), 55.4 (C-5, d), 42.1 (C-3, t), 39.7 (C-10, s), 39.6 (C-12, t), 39.4 (C-α, d), 39.0 (C-1, t), 38.3 (C-7, t), 33.59 (C-18, q), 33.57 (C-4, s), 25.5 (C-β, t), 24.2 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, d), 19.4 (C-2), 18.3 (C-16, q), 14.4 (C-20, q); ESI-TOF MS: calcd for C30H42N2NaO, m/z 469.3189 [M + Na]+, found 469.3197.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (20:80) as an eluent to provide 4i (19 mg, 71% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{27}\) +37.5 (c 1.54, CHCl3); FTIR (neat) Ѵmax: 3176, 2931, 2843, 1638, 1507, 1459, 1440, 1387, 1366, 1255, 1201, 1076, 1048, 887, 788 cm‒1. 1H NMR (400 MHz, CDCl3) δ 5.50 (1H, br s, H-14), 4.84 (1H, d, J = 1.2 Hz, H2-17), 4.49 (1H, s, H2-17), 3.77 (3H, s, OCH3), 2.38 (1H, ddd, J = 12.0, 4.4, 2.4 Hz, H2-7), 2.27 (1H, ddd, J = 13.0, 10.0, 4.4 Hz, H2-12), 2.17 (3H, d, J = 1.1 Hz, H3-16), 1.98 (1H, m, H2-7), 1.94 (1H, m, H2-12), 1.75 (1H, m, H2-1), 1.71 (1H, m, H2-6), 1.65 (1H, m, H2-11), 1.59 (1H, m, H2-2), 1.56 (1H, m, H-9), 1.49 (1H, m, H-2), 1.48 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.32 (1H, dd, J = 13.0, 4.2 Hz, H2-6), 1.17 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.09 (1H, dd, J = 12.0, 2.4 Hz, H-5), 1.01 (1H, td, J = 12.8, 4.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s,H3-19), 0.68 (3H, s, H3-20); 13C NMR (100 MHz, CDCl3) δ 171.0 (C-15, s), 148.4 (C-13/C-8, s), 113.0 (C-14, d), 106.3 (C-17, t), 64.7 (OCH3, q), 56.1 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 39.9 (C-10, s), 39.7 (C-12, t), 39.1 (C-1, t), 38.3 (C-7, t), 33.6 (C-18, s), 33.59 (C-4, s), 24.4 (C-6, t), 21.7 (C-19, s), 21.5 (C-11, t), 19.4 (C-2, t), 18.8 (C-16, s), 14.5 (C-20, s); ESI-TOF MS: calcd for C21H35NNaO2, m/z 356.2560 [M + Na]+, found 356.2565.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (40:80) as an eluent to provide 4j (4.7 mg, 20% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{26}\) +33.5 (c 0.55, CHCl3); FTIR (neat) Ѵmax: 3212, 2925, 2843, 1641, 1459, 1442, 1387, 1366, 1087, 1031, 887, 861, 738 cm‒1; 1H NMR (300 MHz, CDCl3) δ 5.45 (1H, br s, H-14), 4.84 (1H, d, J = 1.3 Hz, H2-17), 4.47 (1H, br s, H2-17), 2.38 (1H, ddd, J = 13.0, 4.0, 2.0 Hz, H2-7), 2.27 (1H, ddd, J = 14.0, 10.0, 5.0 Hz, H2-12), 2.18 (3H, br s, H3-16), 1.97 (1H, m, H-7), 1.93 (1H, m, H2-12), 1.75 (1H, m, H2-1), 1.71 (1H, m, H2-6), 1.65 (1H, m, H2-11), 1.58 (1H, m, H2-2), 1.55 (1H, m, H-9), 1.50 (1H, m, H2-2), 1.46 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.32 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.17 (1H, td, J = 13.3, 4.0 Hz, H2-3), 1.08 (1H, dd, J = 10.0, 3.0 Hz, H-5), 1.00 (1H, td, J = 12.0, 4.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20); 13C NMR (75 MHz, CDCl3) δ 166.5 (C-15, s), 158.7 (C-13, s), 148.3 (C-8, s), 112.3 (C-14, d), 106.4 (C-17, t), 56.0 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 39.72 (C-12, t), 39.70 (C-10, s), 39.1 (C-1, t), 38.3 (C-7, t), 33.6 (C-18, q), 33.6 (C, C-4, s), 24.4 (C-6, t), 22.6 (C-19, q), 21.7 (C-11, t), 19.3 (C-16, q), 18.9 (C-2, t), 14.5 (C-20, q); ESI-TOF MS: calcd for C20H34NO2, m/z 320.2584 [M + H]+, found 320.2583.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (20:80) as an eluent to provide 4k (9.8 mg, 40% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{27}\) +35.5 (c 1.16, CHCl3); FTIR (neat) Ѵmax: 3240, 2924, 2865, 2844, 1655, 1624, 1542, 1458, 1440, 1386, 1365, 1289, 1194, 1006, 887, 851, 685 cm‒1; 1H NMR (400 MHz, CDCl3) δ 5.48 (1H, s, H-14), 4.84 (1H, d, J = 1.3 Hz, H2-17), 4.47 (1H, br s, H2-17), 2.38 (1H, ddd, J = 13.0, 4.0, 2.0 Hz, H2-7), 2.27 (1H, ddd, J = 14.0, 10.0, 5.0 Hz, H2-12), 2.18 (3H, br s, H3-16), 1.97 (1H, m, H-7), 1.93 (1H, m, H2-12), 1.75 (1H, m, H2-1), 1.71 (1H, m, H2-6), 1.65 (1H, m, H2-11), 1.58 (1H, m, H2-2), 1.55 (1H, m, H-9), 1.50 (1H, m, H2-2), 1.46 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.32 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.17 (1H, td, J = 13.3, 4.0 Hz, H2-3), 1.08 (1H, dd, J = 10.0, 3.0 Hz, H-5), 1.00 (1H, td, J = 12.0, 4.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20);13C NMR (100 MHz, CDCl3) δ 163.0 (C-15, s), 157.1 (C-13, s), 148.4 (C-8, s), 114.6 (C-14, d), 106.3 (C-17, t), 56.0 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 39.67 (C-10, s), 39.6 (C-12, t), 39.0 (C-1, t), 38.3 (C-7, t), 33.59 (C-18, q), 33.58 (C-4, s), 21.7 (C-19, q), 21.6 (C-11, t), 19.4 (C-16, q), 18.6 (C-2, t), 14.5 (C-20, q); ESI-TOF MS: calcd for C20H34N2O, m/z 341.2563 [M + H]+, found 341.2553.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (10:90) as an eluent to provide 4l (22.9 mg, 74% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{25}\) +11.8 (c 1.06 CHCl3); FTIR (neat) Ѵmax: 3197, 2925, 2849, 1661, 1642, 1456, 1441, 1387, 1365, 1257, 1204, 1113, 1037, 1064, 1021, 951, 895, 875, 817 cm‒1. 1H NMR (400 MHz, CDCl3) δ 5.49 (1H, br s, H-14), 4. 94 (1H, br s, H-1ʹ), 4.82 (1H, s, H2-17), 4.47 (1H, s, H2-17), 3.95 (1H, t, J = 9.0 Hz, H2-5ʹ), 3.63 (1H, m, H2-5ʹ), 2.37 (1H, ddd, J = 13.0, 4.0, 2.4 Hz, H2-7), 2.25 (1H, ddd, J = 14.0, 10.1, 4 Hz, H2-12), 2.15 (3H, d, J = 1.0 Hz, H3-16), 1.97 (1H, m, H2-7), 1.93 (1H, m, H2-12), 1.81 (2H, m, H2-2ʹ/H2-3ʹ), 1.74 (1H, m, H2-1), 1.71 (1H, m, H2-6), 1.65 (1H, m, H2-11), 1.63 (1H, m, H2-4ʹ), 1.58–1.53 (2H, m, H2-3ʹ/H2-4ʹ), 1.58 (1H, m, H2-2), 1.54 (1H, m, H-9), 1.48 (1H, m, H2-2), 1.46 (1H, m, H2-11), 1.38 (1H, m, H2-3), 1.30 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.16 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.07 (1H, dt, J = 12.0, 2.0 Hz, H-5), 0.99 (1H, br t, J = 12.0 Hz, H2-1), 0.86 (3H, s, H3-18), 0.79 (3H, s, H3-19), 0.67 (3H, s, H3-20); 13C NMR (100 MHz, CDCl3) δ 158.0 (C-15, s), 158.0 (C-13, s), 148.4 (C-8, s), 148.4 (C-13, s), 113.2 (C-14, d), 106.3 (C-17, t), 102.6 (C-1ʹ, d), 62.6 (C-5ʹ, t), 56.0 (C-9, d), 55.4 (C-5, d), 42.0 (C-3, t), 39.8 (C-10, s), 39.6 (C-12, t), 39.0 (C-1, t), 38.3 (C-7, t), 33.6 (C-18, q), 33.6 (C-4, s), 28.1 (C-2ʹ, t), 25.0 (C-4ʹ, t), 24.4 (C-6, t), 21.7 (C-19, q), 21.4 (C-11, t), 19.3 (C-2, t), 18.67 (C-3ʹ, t), 18.66 (C-16, q), 14.4 (C-20, q). ESI-TOF MS: calcd for C25H41NNaO3, m/z 426.2979 [M + Na]+, found 426.2969.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of CH2Cl2:MeOH (98:2) as an eluent to provide 4m (22.6 mg, 85% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{27}\) +31.9 (c 2.11, CHCl3); FTIR (neat) Ѵmax: 2925, 2848, 1651, 1629, 1458, 1442, 1388, 1371, 1265, 1131, 1056, 887, 852 cm‒1. 1H NMR (300 MHz, CDCl3) δ 5.76 (1H, d, J = 0.8 Hz, H-14), 4.84 (1H, d, J = 1.6 Hz, H2-17), 4.50 (1H, d, J = 1.2 Hz, H2-17), 3.02 (3H, s, H3-1ʹʹ), 2.98 (3H, s, H3-1ʹ), 2.39 (1H, ddd, J = 13.0 4.4, 2.4 Hz, H2-7), 2.24 (1H, ddd, J = 13.0, 10.0, 4.0 Hz, H2-12), 1.97 (1H, m, H2-7), 1.93 (1H, m, H2-12), 1.90 (3H, d, J = 1.2 Hz, H3-16), 1.75 (1H, m, H2-1), 1.71 (1H, m, H2-7), 1.65 (1H, m, H2-11), 1.58 (1H, m, H-9), 1.50 1.58 (1H, m, H-9), 1.55 (1H, m, H2-2), 1.50 (1H, m, H2-2), 1.48 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.33 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.17 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.08 (1H, dd, J = 12.0, 3.0 Hz, H-5), 1.01 (1H, td, J = 13.0, 4.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20). 13C NMR (75 MHz, CDCl3) δ 168.8 (C-15, s), 149.9 (C-13, s), 148.5 (C-8, s), 117.4 (C-14, d), 106.3 (C-17, t), 56.1 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 39.7 (C-10, s), 39.1 (C-1, t), 38.6 (C-12, t), 38.3 (C-7, t), 37.8 (C-1ʹʹ, q), 34.7 (C-1ʹ, q), 33.61 (C-18, q), 33.58 (C-4, s), 24.4 (C-6, t), 21.7 (C-19, q), 21.4 (C-11, t), 19.4 (C-2, t), 18.5 (C-16, q), 14.5 (C-20, q); ESI-TOF MS: calcd for C22H38NO, m/z 322.2948 [M + H]+, found 332.2949.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of CH2Cl2:MeOH (99:1) as an eluent to provide 4n (20.3 mg, 68% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{27}\) +24.9 (c 2.08, CHCl3); FTIR (neat) Ѵmax: 3077, 2932, 2848, 1626, 1440, 1381, 1255, 1228, 1137, 1124, 1021, 886, 850 cm‒1. 1H NMR (400 MHz, CDCl3) δ 5.72 (1H, d, J = 0.8 Hz, H-14), 4.84 (1H, d, J = 1.6 Hz, H2-17), 4.51 (1H, br s, H2-17), 3.59 (1H, m, H2-a), 3.45 (1H, m, H2-a), 2.39 (1H, ddd, J = 13.0, 4.0, 2.4 Hz, H2-7), 2.22 (1H, ddd, J = 14.0, 9.5, 4.3 Hz, H2-7), 1.94 (2H, m, H2-12), 1.83 (3H, br s, H3-16), 1.76 (1H, m, H2-1), 1.71 (1H, m, H2-c), 1.65 (1H, m, H2-11), 1.63 (1H, m, H2-6), 1.61 (1H, m, H2-b), 1.58 (2H, m, H2-c/H-9), 1.55 (1H, m, H2-2), 1.53 (1H, m, H2-b), 1.52 (1H, m, H2-b), 1.49 (1H, m, H2-2), 1.47 (2H, m, H2-11), 1.39 (1H, br d, J = 13.0 Hz, H2-3), 1.32 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.17 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.07 (1H, dd, J = 13.0, 2.4 Hz, H-5), 1.00 (1H, td, J = 13.0, 4.0 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.69 (3H, s, H3-20); 13C NMR (100 MHz, CDCl3) δ 167.5 (C-15, s), 148.4 (C-8, s), 147.9 (C-13, s), 118.1 (C-14, d), 106.4 (C-17, t), 56.1 (C-9, d), 55.6 (C-5, d), 47.4 (C-a of piperidine, t), 42.21 (C-a of piperidine, t), 42.16 (C-3, t), 39.7 (C-10, s), 39.1 (C-1, t), 38.4 (C-12, t), 38.2 (C-7, t), 33.64 (C-18, q), 33.58 (C-4, s), 26.7 (C-b of piperidine, t), 25.7 (C-b of piperidine, t), 24.7 (C-c of piperidine, t), 24.5 (C-6, t), 21.7 (C-19, q), 21.3 (C-11, t), 19.4 (C-2, t), 18.5 (C-16, q), 14.5 (C-20, q); ESI-TOF MS: calcd for C25H41NNaO, m/z 394.3080 [M + Na]+, found 394.3070.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of CH2Cl2:MeOH (99:1) as an eluent to provide 4o (29.4 mg, quantitative yield) as a colorless oil. \({[\mathrm{\alpha }]}_{D}^{27}\) +29.6 (c 2.61, CHCl3); FTIR (neat) Ѵmax: 2933, 2844, 1656, 1634, 1458, 1441, 1387, 1366, 1319, 1175, 1003, 887 cm‒1; 1H NMR (300 MHz, CDCl3) δ 6.08 (1H, s, H-14), 4.86 (1H, br s, H2-17), 4.53 (1H, br s, H2-17), 3.68 (3H, s, OCH3), 3.20 (3H, s, H3-1ʹ), 2.40 (1H, ddd, J = 12.0, 4.0, 3.0 Hz, H2-7), 2.30 (1H, ddd, J = 14.0, 9.0, 4.0 Hz, H2-12), 2.13 (3H, d, J = 1.1 Hz, H3-16), 2.06–1.91 (2H, m, H2-7/H2-12), 1.78 (1H, m, H2-1), 1.74 (1H, m, H2-11), 1.65 (1H, m, H2-2), 1.56 (1H, m, H-9), 1.50 (1H, m, H2-2), 1.47 (1H, m, H2-11), 1.40 (1H, m, H2-3), 1.33 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.20 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.10 (1H, dd, J = 12.0, 2.0 Hz, H-5), 1.01 (1H, td, J = 13.0, 4.0 Hz, H2-1), 0.88 (3H, s, H3-18), 0.81 (3H, s, H3-19), 0.70 (3H, s, H3-20); 13C NMR (75 MHz, CDCl3) δ 168.3 (C-15, s), 157.2 (C-13, s), 148.4 (C-8, s), 113.7 (C-14, d), 106.3 (C-17, t), 61.4 (OCH3, q), 56.0 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 40.0 (C-12, t), 39.7 (C-10, s), 39.0 (C-1, t), 38.3 (C-7, t), 33.59 (C-18, q), 33.57 (C-4, s), 32.0 (C-1ʹ, q), 24.5 (C-6, t), 21.7 (C-19, q), 21.6 (C-11, t), 19.4 (C-2, t), 18.7 (C-16, q), 14.5 (C-20, q); ESI-TOF MS: calcd for C22H37NNaO2, m/z 370.2717 [M + Na]+, found 370.2741.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of CH2Cl2:MeOH (99:1) as an eluent to provide 4p (27.5 mg, 51% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{27}\) +38.8 (c 0.42, CHCl3); FTIR (neat) Ѵmax:3170, 3078, 2926, 2843, 1764, 1687, 1643, 1597, 1440, 1387, 1201, 1110, 965, 886, 742 cm‒1. 1H NMR (400 MHz, CDCl3) δ 5.73 (1H, br s, NH), 5.68 (1H, d, J = 1.0 Hz, H-14), 4.85 (1H, d, J = 1.4 Hz, H2-17), 4.50 (1H, s, H2-17), 3.35 (3H, s, H3-1ʹ), 2.39 (1H, ddd, J = 13.0, 4.4, 2.4 Hz, H2-7), 2.28 (H, ddd, J = 12.0, 10.0, 4.0 Hz, H2-12), 2.04 (3H, s, H3-16), 1.98 (1H, m, H2-12), 1.94 (1H, m, H2-7), 1.75 (1H, m, H2-1), 1.73 (1H, m, H2-6), 1.66 (1H, m, H2-11), 1.60 (1H, m, H2-2). 1.56 (1H, m, H-9), 1.50 (H, m, H2-2), 1.47 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.33 (1H, dd, J = 13.0, 4.0 Hz, H2-6), 1.18 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.09 (1H, dd, J = 12.0, 4.0 Hz, H-5), 1.01 (1H, td, J = 13.0, 4 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20); 13C NMR (100 MHz, CDCl3) δ 163.5 (C-15, s), 148.4 (C-13/C-8, s), 114.9 (C-14, d), 106.4 (C-17, t), 56.1 (C-9, d), 55.5 (C-5, d), 42.1 (C-3, t), 40.0 (C-10, s), 39.7 (C-12, t), 39.1 (C-1, t), 38.3 (C-7, t), 36.3 (C-1ʹ, q), 33.6 (C-18, q), 33.59 (C-4, s), 24.5 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, t), 19.4 (C-2, t), 18.7 (C-16, q), 14.5 (C-20, q). ESI-TOF MS: calcd for C21H35NNaO2, m/z 356.2560 [M–H]+, found 356.2564.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of EtOAc:hexane (10:90) as an eluent to provide 4q (22.3 mg, 74% yield) as a yellow oil. \({[\mathrm{\alpha }]}_{D}^{26}\) + 29.0 (c 0.88, CHCl3); FTIR (neat) Ѵmax: 3314, 2928, 2865, 2844, 1754, 1661, 1638, 1638, 1532, 1438, 1366, 1205, 1175, 887 cm‒1; 1H NMR (400 MHz, CDCl3) δ 5.86 (1H, br s, NH), 5.59 (1H, br s, H-14), 4.84 (1H, d, J = 1.4 Hz, H2-17), 4.49 (1H, d, J = 1.0 Hz, H2-17), 4.10 (2H, d, J = 5.2 Hz, H2-2ʹ), 3.77 (3H, s, OCH3), 2.39 (1H, ddd, J = 12.0, 4.4, 2.4 Hz, H2-7), 2.26 (1H, ddd, J = 12.0, 10.0, 4.0 Hz, H2-12), 2.15 (3H, d, J = 0.8 Hz, H3-16), 1.98 (1H, m, H2-7), 1.92 (1H, m, H2-12), 1.76 (1H, m, H2-1), 1.72 (1H, m, H2-6), 1.65 (1H, m, H2-6), 1.5589 (1H, m, H2-2), 1.56 (1H, m, H-9), 1.50 (1H, m, H2-2), 1.46 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.32 (1H, dd, J = 13.0, 4.4 Hz, H2-6), 1.17 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.09 (1H, dd, J = 13.0, 3.0 Hz, H-5), 1.01 (1H, td, J = 13.0, 4.4 Hz, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20); 13C NMR (100 MHz, CDCl3) δ 170.8 (C-1ʹ, s), 167.0 (C-15, s), 156.8 (C-13, s), 148.4 (C-8, s), 116.9 (C-14, d), 106.3 (C-17, t), 56.1 (C-9, d), 55.4 (C-5, d), 52.3 (OCH3, q), 42.1 (C-3, t), 41.0 (C-2ʹ, t), 39.7 (C-12, t), 39.68 (C-10, s), 39.1 (C-1, t), 38.3 (C-7, t), 33.6 (C-18, q), 33.6 (C-4, s), 24.5 (C-6, t), 21.7 (C-19, q), 21,5 (C-11, t), 19.4 (C-2, t), 18.5 (C-16, q), 14.5 (C-20, q); ESI-TOF MS: calcd for C23H37NNaO3, m/z 398.2666 [M + Na]+, found 398.2660.

The crude residue was pre-absorbed on silica gel and purified by flash silica gel column chromatography using a mixture of CH2Cl2:MeOH (95:5) as an eluent to provide 4r (22.4 mg, 72% yield) as a colourless oil.\({[\mathrm{\alpha }]}_{D}^{27}\) +30.5 (c 1.41 CHCl3); FTIR (neat) Ѵmax: 3313, 2928, 2844, 1750, 1661, 1638, 1524, 1460, 1443, 1373, 1263, 1195, 1178, 1025, 886, 863, 736 cm‒1; 1H NMR (300 MHz, CDCl3) δH 5.94 (H, br s, NH), 5.60 (H, d, J = 1.0 Hz, H-14), 4.85 (1H, d, J = 1.2 Hz, H2-17), 4.50 (1H, s, H2-17), 4.23 (2H, q, J = 7.0 Hz, H2-3ʹ), 4.08 (2H, d, J = 5.0 Hz, H2-2ʹ), 2.39 (1H, ddd, J = 13.0, 4.0, 2.0 Hz, H2-7), 2.26 (1H, ddd, J = 13.0, 10.0, 4.0 Hz, H2-12), 2.15 (3H, d, J = 1.0 Hz, H3-16), 1.98 (1H, m, H2-7), 1.94 (1H, m, H2-12), 1.78 (1H, m, H2-1), 1.72 (1H, m, H2-6), 1.68 (1H, m, H2-11), 1.61 (1H, m, H2-2), 1.56 (1H, m, H-9), 1.50 (1H, m, H2-2), 1.48 (1H, m, H2-11), 1.40 (1H, m, H2-3), 1.33 (1H, m, H-6), 1.30 (3H, t, J = 7.0 Hz, H3-4ʹ), 1.21 (1H, td, J = 13.0, 4.0 Hz, H2-3), 1.10 (1H, dd, J = 12.0, 3.0 Hz, H-5), 0.99 (1H, td, J = 13.0, 4.0 Hz, H2-1), 0.88 (3H, s, H3-18), 0.81 (3H, s, H3-19), 0.69 (3H, s, H3-20); 13C NMR (75 MHz, CDCl3) δC 170.3 (C-1ʹ, s), 167.0 (C-15, s), 156.5 (C-13, s), 148.4 (C-8, s), 117.0 (C-14, d), 106.3 (C-17, t), 61.4 (C-3ʹ, t), 56.1 (C-9, d), 55.4 (C-5, d), 42.1 (C-3, t), 41.2 (C-2ʹ, t), 39.68 (C-12, t), 39.65 (C-10, s), 39.0 (C-1, t), 38.3 (C-7, t), 33.5 (C-18, q), 33.5 (C-4, s), 24.4 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, t), 19.3 (C-2, t), 18.4 (C-16, q), 14.4 (C-20, q), 14.1 (C-4ʹ, q); ESI-TOF MS: calcd for C24H39NNaO3, m/z 412.2822 [M + Na]+, found 412.2836.

A mixture of 4r (16.5 mg, 0.04 mmol, 1.0 equiv.) and lithium hydroxide monohydrate (2.1 mg, 0.5 mmol, 1.2 equiv.) were dissolved in THF:MeOH:H2O (0.4 mL, 0.1 M). The reaction was allowed to stir at room temperature for 4 h, and then MeOH and THF were removed under vacuo. The crude was diluted with H2O and extracted with EtOAc, and the organic layer was discarded. The aqueous layer was acidified to ~ pH 3 with 1N hydrochloric acid solution, and then extracted with EtOAc. The organic layers were combined, washed with brine, dried over Mg2SO4 (s), and dried under vacuo to afford 4s (11.7 mg, quantitative yield) as a colourless oil. The target compound was obtained as a yellow oil in quantitative yield. \({[\mathrm{\alpha }]}_{D}^{25}\) + 20.7 (c 0.83 CHCl3); FTIR (neat) Ѵmax: 3336, 2926, 2867, 1715, 1662, 1541, 1457, 1441, 1388, 1366, 1261, 1218, 1087, 888, 733 cm‒1; 1H NMR (400 MHz, CDCl3) δ 6.11 (1H, br s, NH), 5.61 (1H, br s, H-14), 4.84 (1H, br s, H2-17), 4.48 (1H, br s, H2-17), 4.10 (2H, br s, H2-2ʹ), 2.39 (1H, br d, J = 12.0 Hz, H2-7), 2.25 (1H, br d, J = 12.0 Hz, H2-12), 2.15 (3H, s, H3-16), 1.97 (1H, m, H2-7), 1.91 (1H, m, H2-12), 1.75 (1H, m, H2-1), 1.71 (1H, m, H2-6), 1.65 (1H, m, H2-11), 1.58 (1H, m, H2-2), 1.56 (1H, m, H-9), 1.50 (1H, m, H2-2), 1.45 (1H, m, H2-11), 1.39 (1H, m, H2-3), 1.32 (1H, m, H2-6), 1.17 (1H, m, H2-3), 1.08 (1H, br d, J = 12.0 Hz, H-5), 1.00 (1H, m, H2-1), 0.87 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.68 (3H, s, H3-20); 13C NMR (100 MHz, CDCl3) δ 172.9 (C-1ʹ, s), 167.9 (C-15, s), 158.0 (C-13, s), 148.4 (C-8, s), 116.5 (C-14, d), 106.3 (C-17, t), 56.1 (C-9, d), 55.4 (C-5, d), 42.1 (C-3, t), 41.1 (C-2ʹ, t), 39.8 (C-10, s), 39.7 (C-12, t), 39.0 (C-1, t), 38.3 (C-7, t), 33.6 (C-18, q), 33.6 (C, C-4, s), 24.4 (C-6, t), 21.7 (C-19, q), 21.5 (C-11, t), 19.4 (C-2, t), 18.6 (C-16, q), 14.5 (C-20, q); ESI-TOF MS: calcd for C22H36NO3, m/z 362.2690 [M + H]+, found 362.2681.

The cells were seeded into 96-well plates (100 µL for adherent cells and 75 µL for suspended cells) at a density of 5000–20,000 cells per well, depending on their growth rates. Adherent and suspended cells were then allowed to grow at 37 °C with 95% humidity and 5% CO2 for 24 h and 30 min, respectively. The cytotoxicity assay was initiated by adding an equal volume of cell culture medium containing tested compound at predetermined concentrations. Following 48 h of exposure, cell viability was determined using MTT assay for adherent cells51,52or XTT assay for suspeended cells53. Briefly, for adherent cells, 100 µL of the MTT reagent was added to each well, and the microtiter plates were further incubated for 2.5–4 h. The medium was subsequently replaced with 100 µL of DMSO to dissolve the purple formazan before the absorbance at 550 nm was measured using a microplate reader (a SpectraMax Plus 384) with a reference wavelength of 650 nm. For suspended cells, 75 µL of the XTT reagent was added to each well, and the cells were further incubated for 4 h. Afterwards, the absorbance of orange formazan at 492 nm was measured with a reference wavelength of 690 nm using a microplate reader. The IC50 value was finally calculated from the dose–response curve as the concentration that inhibits the cell growth by 50% in comparison with the negative control following 48 h of exposure to each tested compound.

Annexin V and 7-AAD double staining was used to distinguish apoptotic and non-apoptotic cell death modes. Briefly, MDA-MB-231 cells were seeded into 6-well culture plates at 1 × 106 cells/well and left in a CO2 incubator for 24 h. Subsequently, the cells were treated with tested compounds for 24–48 h. DMSO was used as a vehicle and its final concentration was kept at 0.2% (v/v) throughout the study. The treated cells were harvested by trypsinization and subjected to annexin V/7-AAD double staining using Muse® Annexin V & Dead Cell Kit as previously described54. The stained cells were analyzed by flow cytometric technique using Muse® Cell Analyzer. Population of annexin V-positive cells was defined as early apoptotic cells, and the population positive for both annexin V and 7-AAD was defined as late apoptotic cells. The sequential events of increasing early and late apoptotic cell populations over time is a characteristic of apoptotic cell death.

MDA-MB-231 cells in 6-well culture plates (1 × 106 cells/well) were treated with tested compounds for 24 h. Then the cells were harvested and western blot analysis was used to detect alteration of targeted proteins in the treated cells, as previously described55. Briefly, the cells in 6-well culture plates were scraped in RIPA cell lysis buffer supplemented with protease/phosphatase inhibitor cocktail before being lysed by sonication. Cell lysates were centrifuged at 12,000×g for 5 min, at 4 °C, to remove cell debris. Total proteins (20 μg) in the cell lysates were separated by SDS-PAGE and blotted to PVDF membranes. The membranes were blocked with 3% (w/v) BSA and incubated overnight at 4 °C with primary antibodies specific to the following proteins: EGFR, phospho-EGFR (Y845), FAK, phospho-FAK (Y397), Akt, phospho-Akt (S473), ERK, and phospho-ERK (T202/Y204). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Bands of specific proteins were detected by using SuperSignal™ ECL substrates and visualized on X-ray films.

iGEMDOCK v2.1 software56 with accurate docking setting was employed to perform molecular docking to predict the possible binding between 4l and FAK. The molecular structure of 4l was obtained at the B3LYP/6-31G* level using Gaussian09. The protein structures of FERM domain (PDB:2AL6) and kinase domain (PDB ID: 2JKK) of FAK were obtained from Protein Data Bank (http://www.rcsb.org/). The BIOVIA Discovery Studio Visualizer57 was used to analyze and image the docking results.

Drug-likeness properties and Lipinski’s rule of five were studied by using SwissADME website services58,59.

ACP is a labdane diterpenoid that could be obtained in a high quantity from K. elegans. With its attractive structure to use as a starting point for optimization into novel natural product-based bioactive agents, a series of 21 ACP derivatives were synthesized and evaluated for their in vitro cytotoxic activity against a panel of cancer cell lines. ACP and most of its derivatives showed moderate activity. Interestingly, compound 4l demonstrated notable cytotoxic activity against the triple-negative breast cancer MDA-MB-231 cell line. Further mechanistic study revealed FAK as a potential target of compound 4l in MDA-MB-231 breast cancer cells, and FAK inhibition appeared to be the mechanism underlying non-apoptotic cell death induction in 4l-treated cells. An in-silico study suggests that 4l could potentially inhibit FAK activation by binding to the ATP binding pocket of FAK kinase domain, resulting in suppression of Tyr397 autophosphorylation of FAK. To our knowledge, this is the first report demontrating FAK inhibitory activity of ACP derivative. The data obtained from this work is important for further development of natural product-based bioactive agents for treatment of the triple-negative breast cancer based on the ACP scaffold.

The data generated and/or analyzed during the current study are available in the Supplementary Information. These included the un-cropped films of western blot results, and the 1H, 13CNMR, and MS spectra of compounds 1–3 and 4a–4s.

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This research work was supported in part by the Thailand Science Research and Innovation (TSRI), Chulabhorn Research Institute (Grant Nos. 36824/4274394, 36824/4274396, and 36827/4274407) and Center of Excellence on Environmental Health and Toxicology (EHT), OPS, Ministry of Higher Education, Science, Research and Innovation. P.C. was supported by the Royal Golden Jubilee Ph.D. Program, the National Research Council of Thailand (NRCT5-RGJ63023-176), and the Chulabhorn Graduate Scholarship Commemorating the 84th Birthday Anniversary of His Majesty King Bhumibol Adulyadej the Great. The authors also would like to thank Professor Dr. Wongsatit Chuakul from Mahidol University for identification of the plant, and Pakamas Intachote, Suchada Sengsai, and Busakorn Saimanee from Chulabhorn Research Institute for cytotoxicity evaluations.

These authors contributed equally: Pornsuda Chawengrum and Natthaorn Luepongpatthana.

Chemical Biology Program, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok, Thailand

Pornsuda Chawengrum, Prasat Kittakoop & Somsak Ruchirawat

Applied Biological Sciences Program, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok, Thailand

Natthaorn Luepongpatthana & Jisnuson Svasti

Laboratory of Natural Products, Chulabhorn Research Institute, Bangkok, Thailand

Sanit Thongnest, Jutatip Boonsombat, Patcharin Kongwaen, Prasat Kittakoop & Somsak Ruchirawat

Center of Excellence on Environmental Health and Toxicology (EHT), Office of the Permanent Secretary (OPS), Ministry of Higher Education, Science, Research and Innovation (MHESI), Bangkok, Thailand

Sanit Thongnest, Jutatip Boonsombat, Kriengsak Lirdprapamongkol, Prasat Kittakoop & Somsak Ruchirawat

Department of Chemistry, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand

Jitnapa Sirirak

Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand

Kriengsak Lirdprapamongkol, Siriporn Keeratichamroen, Phreeranat Montatip & Jisnuson Svasti

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Conceptualization, J.B., K.L.; data curation, S.T., J.B., J.S.; K.L., S.K. formal analysis, S.T., J.B., J.S., K.L., S.K.; funding acquisition, J.B., K.L., P.Kittakoop; investigation, P.C., N.L., J.S., J.B., P.K., P.M.; methodology, S.T., J.B., J.S., K.L., S.K.; project administration, J.B., K.L.; resources, S.R., J.Svasti; supervision, S.R., J.Svasti; validation, S.T., J.B., J.S., K.L., S.K.; writing-original draft preparation, J.B., K.L.; writing-review and editing, S.T., J.B., J.S., K.L., S.K., P.Kittakoop, J.Svasti, S.R.; All authors read and approved the final manuscript.

Correspondence to Jutatip Boonsombat or Kriengsak Lirdprapamongkol.

The authors declare no competing interests.

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Chawengrum, P., Luepongpatthana, N., Thongnest, S. et al. The amide derivative of anticopalic acid induces non-apoptotic cell death in triple-negative breast cancer cells by inhibiting FAK activation. Sci Rep 13, 13456 (2023). https://doi.org/10.1038/s41598-023-40669-6

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Received: 25 March 2023

Accepted: 16 August 2023

Published: 18 August 2023

DOI: https://doi.org/10.1038/s41598-023-40669-6

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