Next Article in Journal
Chemical Composition, Antimicrobial and Antioxidant Bioautography Activity of Essential Oil from Leaves of Amazon Plant Clinopodium brownei (Sw.)
Next Article in Special Issue
Novel Tetrahydro-[1,2,4]triazolo[3,4-a]isoquinoline Chalcones Suppress Breast Carcinoma through Cell Cycle Arrests and Apoptosis
Previous Article in Journal
Comprehensive Metabolite Profiling of Berdav Propolis Using LC-MS/MS: Determination of Antioxidant, Anticholinergic, Antiglaucoma, and Antidiabetic Effects
Previous Article in Special Issue
Chloroquine-Based Mitochondrial ATP Inhibitors
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 28
Issue 4
10.3390/molecules28041737
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Synthesis and Biological Evaluation of Sclareolide-Indole Conjugates and Their Derivatives

by
Ying Cheng
1,2,†,
Xilin Lyu
2,†,
Chen Liu
1,2,
Xiancheng Wang
2,3,
Jing Cheng
2,3,
Daizhou Zhang
4,
Xiangjing Meng
4,* and
Yujun Zhao
1,2,3,4,5,*
1
School of Chinese Materia Medica, Nanjing University of Chinese Medicine; Nanjing 210023, China
2
State Key Laboratory of Drug Research and Small-Molecule Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Rd., Shanghai 201203, China
3
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
4
Shandong Provincial Key Laboratory of Biopharmaceuticals, Shandong Academy of Pharmaceutical Sciences, Jinan 250101, China
5
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2023, 28(4), 1737; https://doi.org/10.3390/molecules28041737
Submission received: 27 January 2023 / Revised: 8 February 2023 / Accepted: 10 February 2023 / Published: 11 February 2023
(This article belongs to the Special Issue Natural Compounds and New Analogs in Chemical Synthesis: A Themed Issue in Honor of Professor Dr. Mark Moloney)
Browse Figures
Review Reports Versions Notes

Abstract

:
Sclareolide is a sesquiterpene lactone isolated from various plant sources in tons every year and is commercially used as a flavor ingredient in the cosmetic and food industries. Antitumor and antiviral activities of sclareolide have been previously reported. However, biological studies of sclareolide synthetic analogous are few. In view of these, we developed a robust synthetic method that allows the assembly of 36 novel sclareolide-indole conjugates and their derivatives. The synthetic method was based on TiCl4-promoted nucleophilic substitution of sclareolide-derived hemiacetal 4, while electron-rich aryles including indoles, polyphenol ethers, and pyrazolo [1,5-a]pyridine were good substrates. The stereochemistry of the final products was confirmed by single-crystal X-ray diffraction analysis, while the antiproliferative activities of selected final products were tested in K562 and MV4-11 cancer cell lines. Cytometric flow analysis shows that lead compounds 8k- and 10-induced robust apoptosis in MV4-11 cancer cells, while they exhibited weak impact on cell cycle progression. Taken together, our study suggests that sclareolide could be a good template and substrate for the synthesis of novel antiproliferative compounds.

1. Introduction

Sclareolide (Figure 1) is a sesquiterpene lactone isolated from various plant sources including Salvia sclarea, Salvia yosgadensis, and tobacco leaves, among others [1,2]. It is commercially used as a flavor ingredient in the cosmetic [3,4] and food industries [2,5,6]. In 2016, the consumption volume of sclareolide was up to 100 metric tons in European countries, which represents 82% of its global consumption volume [7]. It has also been reported that sclareolide exhibited robust antitumor activity [8] and anti-viral activity [9]. For example, sclareolide significantly improved the antitumor activities of Gemcitabine, an FDA approved anticancer drug, against pancreatic cancer cells in vitro and in vivo [8]. In addition, sclareolide blocked the Ebola virus fusion process with an inhibitory EC50 value of 8.0 μM and exhibited wide-spectrum activities against additional seven filoviruses [9]. These interesting biological activities of sclareolide have motivated an increasing number of studies in this area.
In natural product synthesis, sclareolide is often used as a starting material for the synthesis of many terpenoids and their derivatives [7,10,11,12,13,14], while a few reports have also documented synthesis of sclareolide analogues and the examination of their biological activities [15,16,17,18,19,20,21]. For instance, González and coauthors reported the synthesis of sclareolide derivatives as well as the evaluation of their cytotoxic, antifungal, and antiviral activities in vitro [18]. Recently, Tran et. al. reported syntheses of 72 analogues of the natural product andrographolide using sclareolide as a key starting material [17]. One lead compound from this series was a potent inhibitor against two low-passage Chikungunya virus isolates from human patients. Despite these recent achievements, to the best of our knowledge, neither sclareolide-indole conjugate nor sclareolide-polyphenol conjugate has been reported, as shown in Figure 1. In view of the interesting biological activities of sclareolide and its derivatives, we aimed to synthesize novel sclareolide derivatives containing various indoles or polyphenol methyl ethers, given their high prevalence in bioactive compounds and drugs [19,22,23,24,25,26,27,28]. As part of our ongoing interest [29] in finding natural product derivatives with potent antitumor activities, we herein report our synthesis and the biological evaluation of a new series of sclareolide-indole conjugates and their derivatives.

2. Results and Discussion

Our synthesis started with NBS-promoted bromination of commercially available sclareolide (1, Scheme 1), while a bromination compound 2 was obtained in a 53% yield. It is very interesting that bromination only took place on β-position of the ester group. In the presence of lithium hydroxide, a bromine atom of 2 was eliminated and a previously known α, β-unsaturated lactone 3 [30,31,32,33] was obtained in an 85% yield, without observation of hydrolysis of the ester group. The stereochemistry of 3 was further verified by single-crystal X-ray diffraction analysis (Figure 2) [34]. Having confirmed the structure of 3, subsequent reduction in the presence of DIBAL-H yielded hemiacetal 4 in 98% yield as a mixture of isomers. It is worth of noting that >1 g of 4 could be obtained in a single run of reaction, which makes it feasible to synthesize its diverse derivatives.
Previously, one of us was involved in studies of Lewis acid-chiral acetal-promoted polyene cyclization [35,36,37] in which oxocarbenium generated from an acetal could be attacked by a weak nucleophile, an olefin [38]. Therefore, we examined whether a hemiacetal 4 could react with an electron-rich indole 5 in the presence of Lewis acid, and the results are summarized in Table 1. TiCl4 was found to be the acid of choice and expected product 6 was obtained in 57% yield (Entry 6), while BF3-OEt2, InBr3, In(OTf)3, and SnCl4 yielded a low yield of or no 6 (entries 1–5). The isolated yield of 6 slightly decreased when 1.5 equiv. of 5 was used (entry 7). Interestingly, the highest yield of 6 in 59% with an isomer ratio of 69:31 was obtained when 0.8 equiv. of TiCl4 was used (entry 9).
With an optimized reaction condition in hand, indoles bearing various substituents were examined to explore substrate scope and the results were summarized in Table 2. Overall, thirty substituted indoles were tested, seven desired products (8b, 8h, 8m, 8v, 8z, 8ab, and 8ad) were obtained in >80% yields, and twenty-six desired products were furnished in >50% yield. In general, the 4, 5, and 6 positions of indole tolerate electron-withdrawing and electron-donating groups as exemplified in 7a to 7y, resulting 8a–8y with a moderate to good yield. In addition, the isomer ratios of 8a–8y were dynamic in a range of 51:49 to 85:15. Surprisingly, 7z bearing a larger pinacolboranyl group (-BPin) at the 4-position provided no desired product, suggesting the steric hindrance was detrimental to the reaction yield. Similarly, indole 7af bearing a large butyl group at the 2-position also yielded the desired product 8af in a relative low yield (35%) with an isomer ratio of 87:13, which suggests that steric hindrance was detrimental for the reaction yield. Nevertheless, 7ab–7ae containing 2-methyl, 2-ethyl, 2-phenyl, and 2-CO2Me groups all furnished the desired products in good yields with isomer ratios around 80:20. The stereochemistry of the major isomer of 8ab was further confirmed by single-crystal X-ray diffraction analysis (Figure 3) [34] and an 1H NMR study of signal crystals. Overall, the sclareolide-indole conjugates 8a–8y and 8ab–8ae were readily obtained in moderate to good yields as a mixture of two isomers, whereas the steric-demanding 8z was an outlier without observation of a desired product.
In view of the robust reactivity of electron-rich indoles in TiCl4 mediated coupling to 4, other electron-rich aromatic rings were further examined and the results are summarized in Scheme 2. Gratifyingly, polyphenol methyl ethers are good substrates and expected conjugation products 9–13 were obtained in good yields with isomer ratios ranging from 66:34 to 87:13.
In addition, pyrazolo [1,5-a]pyridine was a good substrate and N-heterocycle-containing product 14 was obtained in an 82% yield with an isomer ratio of 66:34. Free polyphenol phloroglucinol was also tested but no desired product was obtained, which is in contrast to phloroglucinol trimethyl ether affording 10 in a 62% yield. These results suggest that multiple free OH groups are not tolerated in the reaction condition. Other aromatic substrates, including anisole, veratrole, acetaminophen, and 2-pyridone, were also tested in the optimized reaction condition. Unfortunately, no expected coupling product was obtained. The limitations of the substrate scope were summarized in Table S1 in the supporting information.
Next, we examined the antiproliferative activities of selected sclareolide–indole conjugates and their analogues in K562 and MV4-11 cancer cell lines (Table 3). K562 is a human chronic myelogenous leukemia (CML) cell line and MV4-11 is a human acute myeloid leukemia (AML) cell line, both of which are considered sensitive blood cancer cell lines and are often used as screening cell lines for anticancer agents. [29,39,40] Decitabine, an FDA approved drug for the treatment of blood or bone marrow cancer, was used as a positive control, while sclareolide and 3 were also included as reference compounds. The antiproliferative activities results are summarized in Table 3. Overall, 8c, 8g, 8k, 8m, 8n, 8p, 8q, 8s, 8t, 8w, 8ab, 8ad, and 8ae have inhibitory IC50 values in a range of 12.0 μM to 4.4 μM in K562 cells and in a range of 0.8 μM to 7.9 μM in MV4-11 cells. The best compound of this series was 8k, which inhibited cell growth of K562 and MV4-11 cells with IC50 values of 5.2 ± 0.6 μM and 0.8 ± 0.6 μM, respectively, and 8k is more potent than sclareolide that has IC50 values of 10.8 ± 0.6μM and 4.5 ± 0.3μM, respectively (Figure 4). For sclareolide–polyphenolic conjugates 9, 10, 11, 12, and 13, their antiproliferative activity was very similar to that of sclareolide, while the best compound 10 was about two-fold more potent in K562 and MV4-11 cells. Last but not least, the antiproliferative activities of 14 and sclareolide have minimal differences.
In order to further elucidate the antiproliferative activities of 8k and 10, cytometric flow analysis was performed in MV4-11 to examine cell cycle arrest and apoptosis induction (Figure 5 and Figure 6). MV4-11 cells were treated with 8k or 10 at the indicated concentrations for 48 h before the addition of dyes and subsequent sorting, while Decitabine was used as a positive control. The corresponding cells were counted based on their types, while their numbers were normalized and plotted, as shown in Figure 5 and Figure 6.
The data in Figure 5 shows that both 8k and 10 dose-dependently arrest cell cycle progression at the S phase, while Decitabine has no impact on cell cycle progression at the concentrations tested. The percentage of the S phase for DMSO, all Decitabine-treated groups, and the 0.3μM 8k/10-treated groups is 42%, while that of the 10 μM 8k/10-treated groups is 45.8 ± 1.3% and 45.9 ± 1.9%, respectively, representing a small amount of increase of 3%. These data indicate that 8k and 10 have weak effects on the cell cyle progression of MV4-11 cells up to 10 μM.
The data in Figure 6 show that both 8k and 10 dose-dependently-induced cell apoptosis, while Decitabine was also an effective apoptosis inducer at the concentration tested. In the DMSO-treated group, the percentage of apoptotic cells was 3.89 ± 0.12%, while that in all Decitabine treated groups were in a range of 8.66% to 11.4%, representing a 2~3 fold increase compared with DMSO; 8k and 10 were stronger apoptosis inducers at 30 μM compared with Decitabine, having apoptotic cells at 18.3 ± 0.8% and 12.3 ± 0.3%, respectively. These data suggest that 8k and 10 have strong effects on the apoptotic cell death of MV4-11 cells, which partially explains their antiproliferative phenotype in cell growth inhibition assays.

3. Materials and Methods

3.1. General Chemistry Methods

All reactions were conducted in a round-bottomed flask equipped with a Teflon®-coated magnet stirring bar. Experiments involving moisture and/or air sensitive components were performed under a nitrogen atmosphere. Commercial reagents and anhydrous solvents were used without further purification. The crude reaction products were purified by flash column chromatography packed with silica gel. Proton nuclear magnetic resonance (1H NMR) was performed in Bruker Advance 400 NMR spectrometers (Bruker corp., Billerica, MA, USA). Carbon nuclear magnetic resonance (13C NMR) spectroscopy was performed in Bruker Advance 500 NMR spectrometers (Bruker corp., Billerica, MA, USA). High resolution ESI mass spectrum analysis was performed on Agilent Q-TOF mass spectrometer (G6520, Agilent Technologies Inc., Santa Clara, USA). The analytical UPLC model was Waters Acquity H class (UV detection at 230 nm and 254 nm, Waters Corp., Milford, USA) and the reverse phase column used was the Acquity UPLC® BEH (C18–1.7 µm, 2.1 × 50 mm, Waters Corp., Milford, USA). Further purification of final compounds for biological testing was performed on a preparative HPLC (Waters 2545, Waters Corp., Milford, USA) with a C18 reverse phase column (Waters Corp., Milford, USA). The mobile phase used here was a gradient flow of solvent A (water) and solvent B (CH3CN) at a flow rate of 10 mL/min. All final compounds were purified to ≥95% purity as determined by analytical UPLC analysis. Isomer ratio was determined by integration of corresponding vinyl proton or O-CH peaks in 1H NMR spectrum.

3.2. Procedures for Synthesis of Sclareolide Derivatives

Synthesis of (3aR,5aS,9aS,9bS)-9b-bromo-3a,6,6,9a-tetramethyldecahydronaphtho [2,1-b]furan-2(1H)-one (2): A 250 mL round-bottom flask equipped with a magnetic stirring bar was charged with 1 (5.0 g, 19.97 mmol, 1.0 equiv), N-Bromosuccinimide (8.89 g, 49.92 mmol, 2.5 equiv), dibenzoyl peroxide (483.7 mg, 1.20 mmol, 0.1 equiv), and CCl4 (60 mL). The mixture was stirred at 85 °C for 16 h. The resulting mixture was filtered, and the filtrate was concentrated. The crude was purified by flash column chromatography to afford the title compound 2 as a white solid (3.48 g, 53%). 1H NMR (500 MHz, CDCl3): δ 3.54–3.46 (d, J = 18.0 Hz, 1H), 3.14 (d, J = 18.0 Hz, 1H), 2.25–2.18 (m, 1H), 1.91 (dtd, J = 12.9, 3.3, 1.6 Hz, 1H), 1.75 (s, 3H), 1.71–1.61 (m, 2H), 1.57–1.43 (m, 3H), 1.43–1.32 (m, 2H), 1.29 (s, 3H), 1.19–1.10 (m, 2H), 0.96 (s, 3H), 0.89 (s, 3H). 13C NMR (126 MHz, CDCl3): δ 175.60, 94.65, 88.77, 49.55, 45.47, 43.87, 41.30, 38.50, 36.48, 33.82, 33.52, 26.18, 21.72, 18.88, 18.83, 17.98. HRMS (EI): m/z calculated for C16H2579BrO2 [M]+: 328.1038, Found: 328.1033. Specific Rotation: [α]20 = -46.95° (c = 1.0 g/100 mL, in CHCl3).
Synthesis of (3aR,5aS,9aS)-3a,6,6,9a-tetramethyl-4,5,5a,6,7,8,9,9a-octahydronaphtho [2,1-b]furan-2(3aH)-one (3): Dissolve 2 (3.48 g, 10.58 mmol, 1.0 equiv) in H2O and THF (20/20 mL) followed by addition of LiOH·H2O (3.55 g, 84.62 mmol, 8.0 equiv). The mixture was stirred at ambient temperature for 12 h. When the reaction was completed, the pH was adjusted to be 6 with 1N HCl. The aqueous layer was extracted with ethyl acetate (3 × 50 mL), and the combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residual crude product was purified by flash column chromatography to afford the title compound 3 as a white solid (2.24 g, 85%). 1H NMR (500 MHz, CDCl3): δ 5.51 (s, 1H), 2.29 (dd, J = 8.9, 2.9 Hz, 1H), 1.83–1.68 (m, 3H), 1.63–1.57 (m, 1H), 1.56–1.53 (m, 4H), 1.51–1.50 (m, 1H), 1.49–1.44 (m, 2H), 1.22–1.15 (m, 4H), 0.96–0.93 (m, 1H), 0.90 (d, J = 7.8 Hz, 6H). 13C NMR (126 MHz, CDCl3): δ 197.98, 173.72, 107.09, 84.78, 57.61, 41.49, 40.71, 39.66, 37.09, 34.73, 33.24, 25.22, 22.36, 19.36, 18.07, 17.91. HRMS (EI): m/z calculated for C16H24O2 [M]+: 248.1776, Found: 248.1758. Specific Rotation: [α]20 = −157.13° (c = 1.0 g/100 mL, in CHCl3).
Synthesis of (3aR,5aS,9aS)-3a,6,6,9a-tetramethyl-2,3a,4,5,5a,6,7,8,9,9a-decahydro naphtho [2,1-b]furan-2-ol (4): A 100 mL dry round-bottom flask equipped with a magnetic stirring bar was charged with 3 (1.2 g, 4.83 mmol, 1.0 equiv) and anhydrous CH2Cl2 (40 mL). Subsequently, DIBAL-H (1.0 M in hexane, 8.7 mL, 8.7 mmol, 1.8 equiv.) was added at −78 °C. The resulting solution was allowed to stir from −78 °C to 0 °C for 12 h. The reaction mixture was quenched with water (30 mL) followed by addition of 2N NaOH (10 mL). The aqueous layer was extracted with CH2Cl2 (3 × 40 mL), and the combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford the crude product 4 as a white solid (1.18 g, 98%). The crude product 4 was used for next step without further purification. 1H NMR (500 MHz, CDCl3): δ 5.91 (d, J = 1.5 Hz, 1H), 5.19 (d, J = 1.5 Hz, 1H), 2.10–1.95 (m, 1H), 1.72–1.55 (m, 4H), 1.49–1.44 (m, 4H), 1.40–1.34 (m, 2H), 1.15–1.03 (m, 6H), 0.82 (d, J = 2.0 Hz, 6H). 13C NMR (126 MHz, CDCl3): δ 161.79, 113.90, 102.55, 86.94, 55.52, 42.41, 42.05, 37.86, 37.65, 33.71, 33.47, 28.70, 21.58, 19.98, 18.99, 18.55.

3.3. Procedure for Coupling Reactions of Sclareolide-Indole and Sclareolide-Aromatic Compounds 6, 8a-8y, 8ab-8ae, and 9–14

Synthesis of 3-((3aR,5aS,9aS)-3a,6,6,9a-tetramethyl-2,3a,4,5,5a,6,7,8,9,9a-decahydro naphtha [2,1-b]furan-2-yl)-1H-indole (6): To a 100 mL dry round-bottom flask equipped with a magnetic stirring bar, 4 (0.20 mmol, 1.0 equiv), indole (0.16 mmol, 0.8 equiv), and anhydrous CH2Cl2 (10 mL) were added. The solution was cooled to −78 °C then TiCl4 (1.0 M in CH2Cl2, 0.16 mmol, 0.8 equiv) was added dropwise via a syringe. The reaction mixture was stirred at −78 °C for 2.5 h before quenching with NaHCO3 saturated aqueous solution (15 mL). The aqueous layer was extracted with CH2Cl2 (3 × 30 mL), and the combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography to afford the desired product 6 (white solid, 20.4 mg, 58% yield, Isomer ratio: 69:31). 1H NMR (500 MHz, CDCl3): δ 8.07 (s, 1H), 7.74 (ddd, J = 8.0, 5.6, 1.0 Hz, 1H), 7.35 (dt, J = 8.2, 1.0 Hz, 1H), 7.21–7.16 (m, 2H), 7.14–7.07 (m, 1H), 6.05 (d, J = 1.3 Hz, 0.69H), 6.02 (d, J = 1.3Hz, 0.31H), 5.50(d, J = 1.2Hz, 0.31H), 5.40 (d, J = 1.2 Hz, 0.69H), 2.07 (ddt, J = 12.7, 6.6, 2.8 Hz, 1H), 1.81–1.64 (m, 4H), 1.55–1.38 (m, 7H), 1.34–1.14 (m, 5H), 1.08 (ddd, J = 50.3, 12.2, 2.3 Hz, 1H), 0.94–0.88 (m, 6H). 13C NMR (126 MHz, CDCl3): δ 157.03, 156.15, 136.97, 136.78, 126.21, 126.09, 123.30, 122.81, 121.93, 119.74, 119.61, 119.45, 119.42, 118.09, 117.42, 117.05, 115.77, 111.47, 111.39, 87.67, 87.20, 78.79, 78.30, 56.06, 55.22, 53.55, 42.79, 42.33, 42.28, 42.20, 38.21, 38.18, 38.14, 37.87, 33.83, 33.77, 33.63, 33.54, 28.68, 26.63, 21.63, 21.58, 20.52, 20.33, 20.15, 19.80, 18.74, 18.68. HRMS (ESI): m/z calculated for C24H31NO [M+H]+: 350.2406, Found [M+H]+: 350.2484.
The synthesis of 8a–8y, 8ab–8af, and 9–14 used the same protocol for the synthesis of 6. The procedures of these syntheses and characterization of 8a–8y, 8ab–8af, and 9–14 were summarized in the supporting information.

3.4. Cell Growth Inhibition Assay

K562 and MV4-11 cancer cell lines were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). STR analysis was performed and passed for all cell lines after in-house cell culture. The testing compounds were serially diluted in 96-well plates. Subsequently, the MV4-11 or K562 cells were seeded into wells at appropriate density (8000–25,000 cells/well). Cells in plates were incubated with 5% CO2 at 37 °C for 4 days. Thereafter, Cell Counting Kit-8 (Dojindo) was used to measure cell viability according to manufacturer’s protocols. The absorbance of OD450 were detected by a multimode microplate reader (TECAN SPARK 10M, Tecan Group Ltd., Männedorf, Switzerland). The untreated cells were set as an indicator of 100% cell viability. IC50 values were calculated by nonlinear regression analysis with GraphPad Prism 8 (Version 8.4.3).

3.5. Flow Cytometry Analysis of Cell Cycle Arrest

MV4-11 cells in logarithmic growth phase were seeded into 6-wells plates at a concentration of 1.5 × 106 cells/mL and treated with 8k and 10 at serial diluted concentrations for 48 h. Subsequently, all cells were collected, washed, and fixed in 2 mL 70% ethanol (precooled to −20 °C) at −20 °C for 12 h. The fixed cells were centrifuged, washed, and re-suspended in a cell cycle and apoptosis Kit solution (Beyotime Biotechnology, Code C1052). All samples were incubated at 37 °C in dark for 30 min prior to signal detection (FACSCalibur; BD Biosciences). Data were analyzed with FlowJo software (version 7.6.1) to provide to cell cycle progression status.

3.6. Flow Cytometry Analysis of Apoptosis

The MV4-11 cells in the logarithmic growth phase were seeded into 6-wells plates at a concentration of 1.5 × 106 cells/mL, and treated with 8k and 10 at the concentrations of 0.3, 1, 3, 10, 30, and 60 μM for 48 h, accordingly. After treatment, all the cells were collected and re-suspended cells in buffer at a concentration of 1.0 × 106 cells/mL. One hundred μL of the solution (1.0 × 105 cells) was transferred to a 5 mL EP tube, followed by addition of 5 μL of FITC Annexin V and 5 μL of Propidium Iodide (PI). The mixture of cells was gently vortexed and incubated for 15 min at room temperature in the dark. Four hundred μL of binding buffer was added to each EP tube prior to flow cytometry analysis. Propidium Iodide and AnnexinV staining were performed using FITC Annexin V Apoptosis Detection Kit I from BD Biosciences. Flow cytometry data were acquired on a FACS Calibur (BD).

4. Conclusions

In summary, we have synthesized a class of sclareolide–indole conjugates and their derivatives based on TiCl4-promoted nucleophilic substitution of hemiacetal 4. Electron-rich aryles including indoles, polyphenol ethers, and pyrazolo [1,5-a]pyridine are good substrates for this reaction. The conjugation products were obtained in a moderate to good yield as a mixture of two isomers. The stereochemistry of the final products was confirmed by single-crystal X-ray diffraction analysis, while the antiproliferative activities of selected final products were tested in K562 and MV4-11 cancer cell lines. Cytometric flow analysis shows that lead compounds 8k- and 10-induced robust apoptosis in MV4-11 cancer cells, while they exhibited a weak impact on cell cycle progression. Our study suggests that sclareolide could be a good template and substrate for the synthesis of novel antiproliferative compounds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28041737/s1.

Author Contributions

Conceptualization: Y.Z.; X.M.; methodology, investigation, and visualization: Y.C.; X.L.; C.L.; X.W.; and J.C.; writing—original draft preparation, Y.Z.; X.M.; Y.C.; and X.L.; writing—review and editing, Y.Z.; X.M.; Y.C.; X.L.; C.L.; X.W.; J.C.; and D.Z.; project administration: D.Z.; X.M.; and J.C.; funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China grants (82073682, 81872724, and 82273759), Jinan Innovation Team Project (2021GXRC069), Science and Technology Commission of Shanghai Municipality (22ZR1474500).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds 6, 8a-8y, 8ab-8af, and 9-14 are available from the authors.

References

  1. Kaneko, H. The Aroma of cigar tobacco. Agric. Biol. Chem. 1971, 35, 1461–1462. [Google Scholar]
  2. Frija, L.M.T.; Frade, R.; Afonso, C. Isolation, chemical, and biotransformation routes of labdane-type diterpenes. Chem. Rev. 2011, 111, 4418–4452. [Google Scholar] [CrossRef] [PubMed]
  3. Peres, M.; Huet, F.; Le Bonniec, M.; Guéhenneux, S.; Vié, K.; Misery, L. Evaluation of the effects on cutaneous sensitivity of a face emulsion containing sclareolide in women presenting with sensitive skin through quantitative sensory testing. J. Eur. Acad. Dermatol. Venereol. 2022, 36, e74–e75. [Google Scholar] [CrossRef]
  4. Api, A.M.; Belsito, D.; Botelho, D.; Bruze, M.; Burton, G.A., Jr.; Cancellieri, M.A.; Chon, H.; Dagli, M.L.; Date, M.; Tokura, Y.; et al. RIFM fragrance ingredient safety assessment, sclareolide, CAS Registry Number 564-20-5. Food Chem. Toxicol. 2022, 167, 113379. [Google Scholar] [CrossRef]
  5. Rangan, C.; Barceloux, D.G. Food Additives and Sensitivities. Disease-A-Month 2009, 55, 292–311. [Google Scholar] [CrossRef]
  6. Smith, R.L.; Ford, R.A. 16. GRAS substances. Food Technol. 1993, 47, 104–117. [Google Scholar]
  7. Sclareolide Market Size, Growth, Share: Global Sales Revenue, Emerging Technologies, Key Players Analysis, Development Status, Opportunity Assessment and Industry Expansion Strategies 2028. 2022. Available online: https://www.marketwatch.com/press-release/sclareolide-market-size-growth-share-global-sales-revenue-emerging-technologies-key-players-analysis-development-status-opportunity-assessment-and-industry-expansion-strategies-2028-2022-12-14 (accessed on 11 January 2023).
  8. Chen, S.; Wang, Y.; Zhang, W.-L.; Dong, M.-S.; Zhang, J.-H. Sclareolide enhances gemcitabine-induced cell death through mediating the NICD and Gli1 pathways in gemcitabine-resistant human pancreatic cancer. Mol. Med. Rep. 2017, 15, 1461–1470. [Google Scholar] [CrossRef] [PubMed]
  9. Chen, Q.; Tang, K.; Guo, Y. Discovery of sclareol and sclareolide as filovirus entry inhibitors. J. Asian Nat. Prod. Res. 2019, 22, 464–473. [Google Scholar] [CrossRef]
  10. Czaplyski, W.L.; Na, C.G.; Alexanian, E.J. C–H Xanthylation: A Synthetic Platform for Alkane Functionalization. J. Am. Chem. Soc. 2016, 138, 13854–13857. [Google Scholar] [CrossRef]
  11. Dixon, D.D.; Lockner, J.W.; Zhou, Q.; Baran, P.S. Scalable, divergent synthesis of meroterpenoids via “borono-sclareolide”. J. Am. Chem. Soc. 2012, 134, 8432–8435. [Google Scholar] [CrossRef] [PubMed]
  12. Kumar, C.N.S.S.P.; Chein, R.-J. Synthesis of Labdane Diterpenes Galanal A and B from (+)-Sclareolide. Org. Lett. 2014, 16, 2990–2992. [Google Scholar] [CrossRef]
  13. Kuan, K.K.W.; Pepper, H.P.; Bloch, W.M.; George, J.H. Total Synthesis of (+)-Aureol. Org. Lett. 2012, 14, 4710–4713. [Google Scholar] [CrossRef]
  14. Quinn, R.K.; Könst, Z.A.; Michalak, S.E.; Schmidt, Y.; Szklarski, A.R.; Flores, A.R.; Nam, S.; Horne, D.A.; Vanderwal, C.D.; Alexanian, E.J. Site-Selective Aliphatic C–H Chlorination Using N-Chloroamides Enables a Synthesis of Chlorolissoclimide. J. Am. Chem. Soc. 2016, 138, 696–702. [Google Scholar] [CrossRef]
  15. Duca, G.; Aricu, A.; Kuchkova, K.; Secara, E.; Barba, A.; Dragalin, I.; Ungur, N.; Spengler, G. Synthesis, structural elucidation and biological evaluations of new guanidine-containing terpenoids as anticancer agents. Nat. Prod. Res. 2018, 33, 3052–3056. [Google Scholar] [CrossRef] [PubMed]
  16. Carullo, G.; Saponara, S.; Ahmed, A.; Gorelli, B.; Mazzotta, S.; Trezza, A.; Gianibbi, B.; Campiani, G.; Fusi, F.; Aiello, F.; et al. Novel labdane diterpenes-based synthetic derivatives: Identification of a bifunctional vasodilator that inhibits Ca(V)1.2 and stimulates K(Ca)1.1 channels. Mar. Drugs 2022, 20, 515. [Google Scholar] [CrossRef] [PubMed]
  17. Tran, Q.T.N.; Lee, R.C.H.; Liu, H.J.; Ran, D.; Low, V.Z.L.; To, D.Q.; Chu, J.J.H.; Chai, C.L.L. Discovery and development of labdane-oxindole hybrids as small-molecule inhibitors against chikungunya virus infection. Eur. J. Med. Chem. 2022, 230, 114110. [Google Scholar] [CrossRef] [PubMed]
  18. González, M.A.; Mancebo-Aracil, J.; Tangarife-Castaño, V.; Gómez, L.S.A.; Zapata, B.; Mesa-Arango, A.; Galvis, L.A.B. Synthesis and biological evaluation of (+)-labdadienedial, derivatives and precursors from (+)-sclareolide. Eur. J. Med. Chem. 2010, 45, 4403–4408. [Google Scholar] [CrossRef] [PubMed]
  19. Golonko, A.; Pienkowski, T.; Swislocka, R.; Lazny, R.; Roszko, M.; Lewandowski, W. Another look at phenolic compounds in cancer therapy the effect of polyphenols on ubiquitin-proteasome system. Eur. J. Med. Chem. 2019, 167, 291–311. [Google Scholar] [CrossRef]
  20. Li, D.; Zhang, S.; Song, Z.; Wang, G.; Li, S. Bioactivity-guided mixed synthesis accelerate the serendipity in lead optimization: Discovery of fungicidal homodrimanyl amides. Eur. J. Med. Chem. 2017, 136, 114–121. [Google Scholar] [CrossRef] [PubMed]
  21. Li, D.; Zhang, S.; Song, Z.; Li, W.; Zhu, F.; Zhang, J.; Li, S. Synthesis and bio-inspired optimization of drimenal: Discovery of chiral drimane fused oxazinones as promising antifungal and antibacterial candidates. Eur. J. Med. Chem. 2018, 143, 558–567. [Google Scholar] [CrossRef] [PubMed]
  22. Umer, S.M.; Solangi, M.; Khan, K.M.; Saleem RS, Z. Indole-containing natural products 2019-2022: Isolations, reappraisals, syntheses, and biological activities. Molecules 2022, 27, 7586. [Google Scholar] [CrossRef]
  23. Pacheco, P.A.F.; Santos, M.M.M. Recent Progress in the Development of Indole-Based Compounds Active against Malaria, Trypanosomiasis and Leishmaniasis. Molecules 2022, 27, 319. [Google Scholar] [CrossRef] [PubMed]
  24. Scott, K.A.; Cox, P.B.; Njardarson, J.T. Phenols in Pharmaceuticals: Analysis of a Recurring Motif. J. Med. Chem. 2022, 65, 7044–7072. [Google Scholar] [CrossRef] [PubMed]
  25. Zhu, Y.; Zhao, J.; Luo, L.; Gao, Y.; Bao, H.; Li, P.; Zhang, H. Research progress of indole compounds with potential antidiabetic activity. Eur. J. Med. Chem. 2021, 223, 113665. [Google Scholar] [CrossRef] [PubMed]
  26. Chauhan, M.; Saxena, A.; Saha, B. An insight in anti-malarial potential of indole scaffold: A review. Eur. J. Med. Chem. 2021, 218, 113400. [Google Scholar] [CrossRef] [PubMed]
  27. Dvořák, Z.; Poulíková, K.; Mani, S. Indole scaffolds as a promising class of the aryl hydrocarbon receptor ligands. Eur. J. Med. Chem. 2021, 215, 113231. [Google Scholar] [CrossRef]
  28. Han, Y.; Dong, W.; Guo, Q.; Li, X.; Huang, L. The importance of indole and azaindole scaffold in the development of antitumor agents. Eur. J. Med. Chem. 2020, 203, 112506. [Google Scholar] [CrossRef]
  29. Zhou, X.; Feng, X.; Wang, D.; Chen, D.; Wu, G.; Yan, Z.; Lyu, X.; Wang, H.; Yang, J.-M.; Zhao, Y. Synthesis and bioactivity studies of covalent inhibitors derived from (-)-Chaetominine. J. Mol. Struct. 2021, 1241, 130694. [Google Scholar] [CrossRef]
  30. Huang, D.; Szewczyk, S.M.; Zhang, P.; Newhouse, T.R. Allyl-Nickel Catalysis Enables Carbonyl Dehydrogenation and Oxidative Cycloalkenylation of Ketones. J. Am. Chem. Soc. 2019, 141, 5669–5674. [Google Scholar] [CrossRef]
  31. Chen, Y.; Romaire, J.P.; Newhouse, T.R. Palladium-catalyzed α,β-dehydrogenation of esters and nitriles. J. Am. Chem. Soc. 2015, 137, 5875–5878. [Google Scholar] [CrossRef]
  32. Fehr, C.; Magpantay, I.; Saudan, L.; Sommer, H. trans-Tetrahydrofurans by OH-Assisted Ru-Catalyzed Isomerization of 2-Butene-1,4-diols. Eur. J. Org. Chem. 2010, 2010, 6153–6156. [Google Scholar] [CrossRef]
  33. Cambie, R.; Palmer, B. Chemistry of the Podocarpaceae. LVII. The preparation of some 1,3-Dioxans with ambergris-type odours. Aust. J. Chem. 1981, 34, 1265–1284. [Google Scholar] [CrossRef]
  34. The X-ray structures of 3 and 8ab have been deposited to CCDC (https://www.ccdc.cam.ac.uk/) with access numbers 2238307 and 2236373, respectively.
  35. Zhao, Y.-J.; Chng, S.-S.; Loh, T.-P. Lewis Acid-Promoted Intermolecular Acetal-Initiated Cationic Polyene Cyclizations. J. Am. Chem. Soc. 2006, 129, 492–493. [Google Scholar] [CrossRef]
  36. Zhao, Y.J.; Loh, T.P. Bio-inspired polyene cyclization: Synthesis of tetracyclic terpenoids promoted by steroidal acetal-SnCl4. Chem. Commun. 2008, 12, 1434–1436. [Google Scholar] [CrossRef] [PubMed]
  37. Zhao, Y.-J.; Loh, T.-P. Asymmetric Total Synthesis of Antiochic Acid. Org. Lett. 2008, 10, 2143–2145. [Google Scholar] [CrossRef]
  38. Zhao, Y.J.; Loh, T.P. Bioinspired polyene cyclization promoted by intermolecular chiral acetal-SnCl4 or chiral N-acetal-TiCl4: Investigation of the mechanism and identification of the key intermediates. J. Am. Chem. Soc. 2008, 130, 10024–10029. [Google Scholar] [CrossRef]
  39. Chen, D.; Lu, T.; Yan, Z.; Lu, W.; Zhou, F.; Lyu, X.; Xu, B.; Jiang, H.; Chen, K.; Luo, C.; et al. Discovery, structural insight, and bioactivities of BY27 as a selective inhibitor of the second bromodomains of BET proteins. Eur. J. Med. Chem. 2019, 182, 111633. [Google Scholar] [CrossRef]
  40. Feng, X.; Yan, Z.; Zhou, F.; Lou, J.; Lyu, X.; Ren, X.; Zeng, Z.; Liu, C.; Zhang, S.; Zhu, D.; et al. Discovery of a selective and covalent small-molecule inhibitor of BFL-1 protein that induces robust apoptosis in cancer cells. Eur. J. Med. Chem. 2022, 236, 114327. [Google Scholar] [CrossRef]
Molecules 28 01737 g001 550
Figure 1. Design of sclareolide-indole conjugates and analogues
Figure 1. Design of sclareolide-indole conjugates and analogues
Molecules 28 01737 g001
Molecules 28 01737 sch001 550
Scheme 1. Gram-scale synthesis of sclareolide derivatives.
Scheme 1. Gram-scale synthesis of sclareolide derivatives.
Molecules 28 01737 sch001
Molecules 28 01737 g002 550
Figure 2. Structure of 3 determined by single-crystal X-ray diffraction, CCDC code: 2,238,307 [34].
Figure 2. Structure of 3 determined by single-crystal X-ray diffraction, CCDC code: 2,238,307 [34].
Molecules 28 01737 g002
Molecules 28 01737 g003 550
Figure 3. Structure of the major isomer 8ab determined by single-crystal X-ray diffraction, CCDC code: 2,236,373 [34].
Figure 3. Structure of the major isomer 8ab determined by single-crystal X-ray diffraction, CCDC code: 2,236,373 [34].
Molecules 28 01737 g003
Molecules 28 01737 sch002 550
Scheme 2. Coupling of sclareolide and polyphenol ethers as well as a N-containing heteroaryle.
Scheme 2. Coupling of sclareolide and polyphenol ethers as well as a N-containing heteroaryle.
Molecules 28 01737 sch002
Molecules 28 01737 g004 550
Figure 4. Cell growth inhibition curves of 8k, 10, Decitabine, and Sclareolide in MV4-11 and K-562 cell lines upon 4-day treatment.
Figure 4. Cell growth inhibition curves of 8k, 10, Decitabine, and Sclareolide in MV4-11 and K-562 cell lines upon 4-day treatment.
Molecules 28 01737 g004
Molecules 28 01737 g005 550
Figure 5. Cytometric flow analysis of cell cycle distribution of MV4-11 cells in the presence of 8k (CD105), 10 (CE045), and Decitabine at the indicated concentrations for 48 h. (A). Normalized cell cycle distribution, (B) Cell cycle distribution figures of 8k, while those of 10 and Decitabine can be found in SI Figures S1 and S2. The experiments were repeated twice.
Figure 5. Cytometric flow analysis of cell cycle distribution of MV4-11 cells in the presence of 8k (CD105), 10 (CE045), and Decitabine at the indicated concentrations for 48 h. (A). Normalized cell cycle distribution, (B) Cell cycle distribution figures of 8k, while those of 10 and Decitabine can be found in SI Figures S1 and S2. The experiments were repeated twice.
Molecules 28 01737 g005
Molecules 28 01737 g006 550
Figure 6. Cytometric flow analysis of apoptosis induction of MV4-11 cells in the presence of 8k (CD105), 10 (CE045), and Decitabine at the indicated concentrations for 48 h. (A). Normalized apoptosis induction, (B) Apoptosis figures of 8k, while those of 10 and Decitabine can be found in SI Figures S3 and S4. The experiments were repeated twice.
Figure 6. Cytometric flow analysis of apoptosis induction of MV4-11 cells in the presence of 8k (CD105), 10 (CE045), and Decitabine at the indicated concentrations for 48 h. (A). Normalized apoptosis induction, (B) Apoptosis figures of 8k, while those of 10 and Decitabine can be found in SI Figures S3 and S4. The experiments were repeated twice.
Molecules 28 01737 g006
Table
Table 1. Optimization of sclareolide–indole coupling reaction condition.
Molecules 28 01737 i001
Table 1. Optimization of sclareolide–indole coupling reaction condition.
Molecules 28 01737 i001
EntryLewis AcidEquiv.4 (Equiv.)5 (Equiv.)T (oC)Reaction TimeYield aIsomer Ratio b
1BF3 OEt21.01.01.0−78 °C12nullnull
2InBr31.01.01.0rt12nullnull
3In(OTf)31.01.01.0rt12nullnull
4SnCl41.0 1.0 1.0−78 °C2.533% 69:31
5SnCl41.0 1.0 3.0−78 °C2.527%69:31
6TiCl41.0 1.0 1.0−78 °C2.557% 69:31
7TiCl41.0 1.0 1.5−78 °C2.553%69:31
8TiCl40.91.0 1.0−78 °C2.551%69:31
9TiCl40.81.0 1.0−78 °C2.559%69:31
a. Isolated yield; b. Determined by 1H NMR.
Table
Table 2. Substrate scope of sclareolide–indole coupling reaction.
Molecules 28 01737 i002
Table 2. Substrate scope of sclareolide–indole coupling reaction.
Molecules 28 01737 i002
SMRProductYield aIsomer Ratio bSMRProductYield aIsomer Ratio b
7a4-CO2Me8a52%51:497q6-OMe8q53%59:41
7b5-CO2Me8b92%76:247r6-Me8r67%69:31
7c6-CO2Me8c73%69:317s6-CHO8s77%70:30
7d7-CO2Me8d60%82:187t6-CF38t62%73:27
7e4-NO28e56%68:327u6-NHCbz8u78%69:31
7f5-NO28f67%85:157v6-BPin8v82%70:30
7g6-NO28g55%69:317w6-OH8w48%73:27
7h7-NO28h87%68:327x4-CO2Me, 6-Br8x51%52:48
7i4-Br8i43%62:387y5-BPin8y84%71:29
7j5-Br8j73%76:247z4-BPin8z<1%
7k6-Br8k61%71:297ab2-Me8ab86%78:22
7l7-Br8l68%70:307ac2-Et8ac72%85:15
7m6-CN8m84%67:337ad2-Ph8ad82%82:18
7n6-F8n77%70:307ae2-CO2Me8ae63%67:33
7p6-Cl8p76%70:307af2-tBu8af35%87:13
a. Isolated yield; b. Determined by 1H NMR.
Table
Table 3. Antiproliferative activities of sclareolide derivatives in K562 and MV4-11 cell lines.
Table 3. Antiproliferative activities of sclareolide derivatives in K562 and MV4-11 cell lines.
CmpdK562
(IC50, μM) a		MV4-11
(IC50, μM) a		CmpdK562
(IC50, μM) a		MV4-11
(IC50, μM) a
Decitabine0.112 ± 0.0350.129 ± 0.0358t4.4 ± 0.44.2 ± 0.7
Sclareolide10.8 ± 0.64.5 ± 0.38w10.4 ± 1.45.8 ± 0.7
311.6 ± 2.42.0 ± 1.78ab12.0 ± 1.07.9 ± 1.8
8c9.4 ± 0.25.2 ± 0.68ad9.9 ± 0.16.8 ± 0.4
8g4.5 ± 0.74.5 ± 2.88ae7.0 ± 0.84.6 ± 2.8
8k5.2 ± 0.60.8 ± 0.699.8 ± 0.14.7 ± 2.4
8m5.3 ± 0.24.4 ± 0.4106.8 ± 1.12.3 ± 0.7
8n9.6 ± 0.17.6 ± 0.21110.0 ± 0.13.9 ± 0.1
8p6.1 ± 0.81.7 ± 1.01210.9 ± 1.86.1 ± 1.1
8q6.1 ± 0.91.7 ± 0.71310.7 ± 1.24.6 ± 2.3
8s6.9 ± 0.44.1 ± 2.21410.1 ± 0.83.3 ± 0.2
a The IC50 values were mean of data from at least two independent experiments.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Cheng, Y.; Lyu, X.; Liu, C.; Wang, X.; Cheng, J.; Zhang, D.; Meng, X.; Zhao, Y. Synthesis and Biological Evaluation of Sclareolide-Indole Conjugates and Their Derivatives. Molecules 2023, 28, 1737. https://doi.org/10.3390/molecules28041737

AMA Style

Cheng Y, Lyu X, Liu C, Wang X, Cheng J, Zhang D, Meng X, Zhao Y. Synthesis and Biological Evaluation of Sclareolide-Indole Conjugates and Their Derivatives. Molecules. 2023; 28(4):1737. https://doi.org/10.3390/molecules28041737

Chicago/Turabian Style

Cheng, Ying, Xilin Lyu, Chen Liu, Xiancheng Wang, Jing Cheng, Daizhou Zhang, Xiangjing Meng, and Yujun Zhao. 2023. "Synthesis and Biological Evaluation of Sclareolide-Indole Conjugates and Their Derivatives" Molecules 28, no. 4: 1737. https://doi.org/10.3390/molecules28041737

APA Style

Cheng, Y., Lyu, X., Liu, C., Wang, X., Cheng, J., Zhang, D., Meng, X., & Zhao, Y. (2023). Synthesis and Biological Evaluation of Sclareolide-Indole Conjugates and Their Derivatives. Molecules, 28(4), 1737. https://doi.org/10.3390/molecules28041737

Article Metrics

Back to TopTop