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Article

Synthesis and Antiproliferative Activity of Novel Dehydroabietic Acid-Chalcone Hybrids

by
Sophia Grigoropoulou
1,
Dimitra Manou
2,
Antonia I. Antoniou
1,
Artemis Tsirogianni
1,
Carlo Siciliano
3,
Achilleas D. Theocharis
2,* and
Constantinos M. Athanassopoulos
1,*
1
Synthetic Organic Chemistry Laboratory, Department of Chemistry, University of Patras, GR-26504 Patras, Greece
2
Biochemistry, Biochemical Analysis & Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, GR-26504 Patras, Greece
3
Department of Pharmacy, Health and Nutritional Sciences, Edificio Polifunzionale, I-87036 Arcavacata di Rende, CS, Italy
*
Authors to whom correspondence should be addressed.
Molecules 2022, 27(11), 3623; https://doi.org/10.3390/molecules27113623
Submission received: 27 April 2022 / Revised: 26 May 2022 / Accepted: 31 May 2022 / Published: 5 June 2022
(This article belongs to the Special Issue Discovery, Synthesis and Evaluation of Bioactive Compounds)
Browse Figures
Versions Notes

Abstract

:
Dehydroabietic Acid (DHA, 1) derivatives are known for their antiproliferative properties, among others. In the context of this work, DHA was initially modified to two key intermediates bearing a C18 methyl ester, a phenol moiety at C12, and an acetyl or formyl group at C13 position. These derivatives allowed us to synthesize a series of DHA-chalcone hybrids, suitable for structure–activity relationship studies (SARS), following their condensation with a variety of aryl-aldehydes and methyl ketones. The antiproliferative evaluation of the synthesized DHA-chalcone hybrids against three breast cancer cell lines (the estrogen-dependent MCF-7 and the estrogen-independent MDA-MB-231 and Hs578T) showed that eight derivatives (33, 35, 37, 38, 39, 41, 43, 44) exhibit low micromolar activity levels (IC50 2.21–11.5 μΜ/MCF-7). For instance, some of them showed better activity compared to the commercial anticancer drug 5-FU against MCF-7 cells (33, 41, 43, 44) and against MDA-MB231 (33 and 41). Hybrid 38 is a promising lead compound for the treatment of MCF-7 breast cancer, exhibiting comparable activity to 5-FU and being 12.9 times less toxic (SI = 22.7). Thus, our findings suggest that DHA-chalcone hybrids are drug candidates worth pursuing for further development in the search for novel breast cancer therapies.

1. Introduction

Cancer is the second leading cause of deaths worldwide, after cardiovascular diseases [1]. One of the main reasons for the failure of the conventionally used chemotherapeutic agents to treat cancer is the development of resistance and the lack of selectivity against normal cells, which is also accompanied by high cytotoxicity and undesired side effects. Thus, there is a considerable need for the design and development of new, more effective molecules displaying high selectivity for cancer cells. Over the last decades, natural products have played a crucial role in the field of drug discovery, and they became the major source of new anticancer drug candidates [2,3,4,5]. Among them, a special place is taken by dehydroabietic acid (DHA, 1; Figure 1), a natural diterpenic resin acid, and its derivatives. They have shown a plethora of biological activities, such as antioxidant [6], anti-microbial [7], anti-inflammatory [8], anti-proliferative [9], and anti-cancer [6,10,11,12].
In Figure 1, some representative DHA analogues are depicted, featuring functional and structural modifications in one or more positions of the parent molecule. These compounds are active against several cancer cell lines, with their corresponding IC50 values ranging between 0.08 and 83.3 μM. The most studied modifications of the DHA skeleton concern the carboxyl group at C(18) and involve its transformation to esters (2, 7, 12–16) and amides (5, 6), as well as its reduction to the aldehyde 3 or alcohol 4. Comparing the antitumor effectiveness against HeLa and Jurkat cell lines of 2, 3 and 4, the latter showed to be the most active [13]. In another study, DHA amides with natural amino acids were evaluated against HL60, A549, AZS21, and SK-BR-3 cancer cell lines, and the L-tyrosine derivative 5 proved to be the most potent with IC50 values ranging from 2.3 to 8.1 μM [14]. On the other hand, replacement of the amino acid moiety at C(18) amides with a dipeptide and oxidation of C-7 resulted in a lead compound 6 [15], which exhibited higher cytotoxicity against HeLa, NCl-H460, and MGC-803 human cancer cell lines in the range of 7.7–24.3 μM compared to the positive control 5-fluorouracil (5-FU), a well-established anticancer drug (IC50 = 30.4–36.5 μM).
Li et al. reported a series of DHA derivatives functionalized with an acylhydrazone moiety at C(18) [16]. Among them, compound 7 showed the most potent activity against CNE-2, HepG2, HeLa, and BeL-7402 cancer cells, while it exhibited low cytotoxicity against normal HL-7702 human liver cells. It is also worth noting that the urea and acylthiourea derivatives 8–9 and 10, respectively, showed enhanced cytotoxic activity against SMMC7721 cells [17,18]. A series of reduced DHA triazolyl derivatives, suitable for structure–activity relationship studies (SARS), has been prepared by Pertino et al. using click chemistry strategies [19]. Among them, derivative 11 showed the highest activity against three human cancer cell lines, MRC-5, AGS, and SK-MES-1.
Modifications at positions C(11) and C(12) led to other potential anticancer agents, such as the oxime 12 [20], with cytotoxic activity against Aspc-1 pancreatic cancer cells, and catechol 13 [21] against MCF-7 breast cancer cells. Functionalization of the C(13) and C(14) positions has also been reported. Miao et al. prepared a library of 2-aryl-benzimidazole derivatives with compound 14 [22] to be the most potent against SMMC-7721, MDA-MB-231, HeLa, and CT-26 cancer cell lines (IC50 = 0.08–0.42 μΜ). Moreover, a series of quinoxaline derivatives was synthesized by Gu et al. [23] and evaluated for cytotoxic activity against three cancer cell lines (MCF-7, SMMC-7721, and HeLa), two of which (15 and 16) are considered as lead compounds with IC50 values between 0.7 and 2.4 μΜ.
Main cancer therapy limitations, such as the therapeutical efficacy and the drug resistance, can be dealt with the development of hybrid molecules bearing more than one pharmacophore, thus allowing them to act simultaneously on multiple targets [24]. In this field, the chalcone moiety has proven to be a useful template for the development of novel anticancer agents. Thus, various conjugates and hybrids of natural products bearing a chalcone moiety have been synthesized and evaluated for their anticancer activity. Artemisinin derivatives conjugated with chalcone moieties, e.g., compounds 17–19 [25,26] (Figure 2), as well as dimeric artemisinin derivatives containing substituted chalcones as a linker, such as compound 20, displayed enhanced and selective cytotoxicity against human cancer cell lines, compared to dihydroartemisinin [26]. Moreover, coumarin and quinoline scaffolds have played an important role in anticancer drug development for the treatment of breast, colon, lung, and stomach cancers [27,28]. Thus, hybridization of the above natural products with chalcone provided new potent anticancer candidates, e.g., compounds 21–25 [29,30,31,32,33] (Figure 2), possessing bioactivities that have been found to be comparable to those of commercially available anticancer drugs, such as combrestatin [24], topotecan [26], and paclitaxel [28].
In connection to our previous studies where we used dehydroabietic acid as a chiral template to prepare either anticancer [34] or antimicrobial agents [35] and considering the potential of a chalcone pharmacophore, we thought it of interest to synthesize a series of hybrid DHA-chalcone molecules (suitable for structure–activity relationship studies) and to evaluate them for their antiproliferative activity against three breast cancer cell lines (MCF-7, MDA-MB-231, and Hs578T). The synthetic methodology is described below and involves two key intermediates derived from DHA, the DHA C13 ketone 28 and the DHA C13 aldehyde 30.

2. Results and Discussion

2.1. Synthesis of the DHA-Chalcone Hybrids 31–53

The proposed DHA-chalcone hybrids (Scheme 1) were synthesized from the readily available DHA (1), which was easily transformed to the C13 acetyl 28 and formyl 30 derivatives the based on experimental procedures described previously [34]. Cross-aldol condensation of 27 or 28 with arylaldehydes in the presence of barium hydroxide octahydrate gave the corresponding hybrid compounds 31–47, where the DHA’s aromatic ring coincides with the chalcone “A” ring [36].
On the other hand, ipso-formylation of 26 by treatment with dichloromethyl methyl ether/AlCl3 at −35 °C provided the 12-methoxy aldehyde 29 [37], which upon further treatment with excess AlCl3 under reflux in DCM gave the desired 12-hydroxy aldehyde 30. Condensation of 29 or 30 with suitable aryl methyl ketones mediated by barium hydroxide octahydrate afforded the corresponding hybrid compounds 48–53, where the DHA’s aromatic ring coincides with the chalcone “B” ring.

2.2. Biological Evaluation

The antiproliferative activity of the synthesized DHA-chalcone hybrids 31–53 was evaluated using three characteristic breast carcinoma cell lines, MDA-MB-231, MCF-7, and Hs578T. For the sake of comparison, 5-FU, a widely used drug in clinical practice, was selected as positive control.
Regarding the different characteristics of the used breast cancer cell lines, we can notice that MDA-MB-231 and Hs578T are triple negative. Nevertheless, MDA-MB-231 is (ERβ)-positive and both cell lines exhibit high metastatic and aggressive potential, whereas MCF-7 cells are estrogen receptor alpha (Erα)-positive and exhibit low metastatic potential and low aggressiveness. The estimated IC50 values of these compounds are provided in Table 1.
In order to calculate the selectivity index of the most potent compounds (33, 35, 38, 39, 41, 43, 44, 49, and 51), their cytotoxicity was evaluated against normal primary fibroblasts (FB) and the corresponding results (FB-IC50 and Sis) are presented again in Table 1 and Figure 3.
A general comment regarding the activity of the hybrid chalcones is that the presence of a phenol at the C12 position of DHA, as well as the incorporation of the aromatic “A” ring of the chalcone to the skeleton of DHA (compounds 31–47), represent two essential structural elements for the potency of the hybrids. Moreover, we notice an enhanced cytotoxicity of our compounds against MCF-7 cells, in comparison to the other two cancer cell lines, MDA-MB231 and Hs578T.
According to Figure 3 and Table 1, is evident that compound 33 is the most potent hybrid for all three cancer cell lines and shows a slightly better activity, in comparison to the control drug 5-FU (2.4–3.3 times more active). Regarding its selectivity indices (SIs), over the three tested breast cancer lines, compound 33 seems to be 2.2–3 times less toxic than 5-FU.
The IC50 values presented in Figure 4A suggest that the compounds 51 and 53 exhibit a comparable 5-FU activity against MDA-MB-231 cells, whereas compounds 33 and 41 a better one. Regarding the MCF-7 evaluation results (Figure 4B), we notice that compound 33, together with compounds 41, 43, and 44, showed higher activity than 5-FU.
Accordingly, the bar graph for the Hs578T cell line shows that 5-FU is more effective than all the synthesized compounds with the exception of 33 (Figure 4C).
Finally, in terms of selectivity, it is apparent that the less toxic compounds are 35, 38, 39, 43, 44, 49, and 51 (Figure 4D). It is worth mentioning that compound 38 (IC50 = 7.06 μM) combines a comparable to 5-FU (IC50 = 5.37 μM) activity for MCF-7 with a very low toxicity (IC50 = 160.65 μΜ, SI = 22.75), being 12.9 times less toxic than 5-FU against normal fibroblasts.

2.3. Structure-Activity Relationships

Structurally, the hybrid chalcones can be classified in two series, Series A, where the DHA’s aromatic ring coincides with the chalcone “A” ring (compounds 31–47), and Series B, where the DHA’s aromatic ring coincides with chalcone “B” ring (compounds 48–53).
Regarding the structural elements of the Series A hybrids, which affect their activity, we notice the following:
The introduction of a heteroatom into the aromatic system of ring B remarkably improved their cytotoxicity. More specifically, the pyrrole (43) and thiophene (44) derivatives are highly cytotoxic against the MCF-7 cancer cell line. However, a fused heterocyclic form for ring B, such as an indole (46) or benzothiophene (47), as well as the presence of a bromo substituent on the thiophene heterocycle of 45, led to less active compounds or to complete loss of activity.
Among pyridyl type derivatives 32–35, the 3-pyridyl derivative 33 showed to be the most active compound against all the cancer cell lines (IC50 = 2.21–5.89 μM) and had similar cytotoxicity with 5-FU (Figure 3, Table 1).
On the other hand, the quinoline derivatives 38–41 showed moderate activity, except hybrid 41, which was active against all cancer cell lines, with IC50 = 7.12 μΜ (ΜDA-MB-231), 3.99 μΜ (MCF-7), and 10.31 μΜ (Hs578T), however it showed high toxicity against normal fibroblasts (IC50 = 7.96 μΜ).
Regarding Series B hybrids (48–53), we observe that all of them presented low to moderate activity (over 17 μΜ).

3. Materials and Methods

3.1. General Methods

1H NMR spectra were obtained at 600.13 MHz and 13C NMR spectra at 150.90 MHz on a Bruker AVANCEIII HD spectrometer. Chemical shifts (δ) are indicated in parts per million (ppm) downfield from TMS and coupling constants (J) are reported in hertz. ESI mass spectra were recorded at 30 V, on a Micromass-Platform LC spectrometer using MeOH as solvent. HR mass spectra were performed using an ESI-LTQ-ORBITRAP XL unit (Thermo Scientific, Bremen, Germany). The Orbitrap Unit was operated in positive mode, with a spay voltage of 3.2 kV, while the sheath gas flow rate and auxiliary gas flow rate were adjusted to 12 and 2 arbitrary units, respectively. The capillary voltage and the tube lens voltage were set to 10 and 110 V, respectively. The scan ranged from m/z 150 up to 2000. All solvents were dried and/or purified according to standard procedures prior to use. Anhydrous Na2SO4 was used for drying solutions, and the solvents were then routinely removed at ca. 40 °C under reduced pressure using a rotary vacuum evaporator. All reagents employed in the present work were commercially available and used without further purification. When required, reactions were carried out under dry argon atmosphere in preflamed glassware. Flash column chromatography (FCC) was performed on Merck silica gel 60 (230–400 mesh) and analytical thin layer chromatography (TLC) was performed on Merck silica gel 60F254 (0.2 mm) precoated on aluminum foil. Spots on the TLC plates were visualized with UV light at 254 nm using ninhydrin solution or para-anisaldehyde solution (Supplementary Material).

3.2. Experimental Procedures

General Procedure for the Synthesis of the DHA-Chalcone Hybrids

To a stirred solution of 27 or 28 or 29 or 30 (1 eq) and carboxaldehyde or aromatic methyl ketone (1 eq) in absolute EtOH was added portion of Ba(OH)2·8H2O (1 eq) every 10 min. The reaction mixture was stirred at room temperature for 24–48 h and the progress of the reaction was monitored by TLC. In case of non-completion of the reaction, a 10% excess of Ba(OH)2·8H2O was added. After completion of the reaction, the mixture was diluted with AcOEt and the organic layer was washed sequentially with cold 5% aqueous citric acid, H2O, and brine. After being dried over anhydrous Na2SO4, the organic extracts were filtered and evaporated to dryness under reduced pressure. The residue was subjected to FCC, using system PhMe/AcOEt, to give the pure product as yellow oil in a yield range of 62–90%.
(1R,4aS)-methyl 7-((E)-3-(2-hydroxyphenyl)-3-oxoprop-1-en-1-yl)-6-methoxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (31): 84%; MS (ESI, 30eV): m/z 471.21 [M + Na], 449.25 [M + H]; 1H NMR (CDCl3) δ 13.00 (s, 1H), 8.16 (d, J = 15.5 Hz, 1H), 7.93 (dd, J = 8.1, 1.7 Hz, 1H), 7.74 (d, J = 15.6 Hz, 1H), 7.49–7.45 (m, 1H), 7.01 (dd, J = 8.3, 1.2 Hz, 1H), 6.95–6.91 (m, 1H), 6.82 (s, 1H), 3.91 (s, 3H), 3.69 (s, 3H), 2.91–2.85 (m, 2H), 2.30 (d, J = 11.7 Hz, 1H), 2.23 (dd, J = 12.5, 2.2 Hz, 1H), 1.88–1.72 (m, 5H), 1.70–1.65 (m, 1H), 1.48–1.43 (m, 1H), 1.30 (s, 3H), 1.25 (s, 3H); 13C NMR (CDCl3) δ 194.5, 179.0, 163.7, 157.6, 154.4, 141.4, 136.1, 130.5, 129.8, 127.6, 121.5, 120.4, 120.1, 118.8, 118.6, 107.2, 55.8, 52.2, 47.8, 44.7, 38.1, 36.7, 29.1, 24.9, 21.7, 18.7, 16.7.
(1R,4aS)-methyl 6-methoxy-1,4a-dimethyl-7-((E)-3-(pyridin-3-yl)acryloyl)-1,2,3,4,4a, 9,10,10a-octahydrophenanthrene-1-carboxylate (32): 76%; MS (ESI, 30eV): m/z 456.12 [M + Na], 434.09 [M + H]; 1H NMR (CDCl3) δ 8.81 (s, 1H), 8.60–8.57 (m, 1H), 7.89–7.86 (m, 1H), 7.62 (d, J = 15.9 Hz, 1H), 7.52 (d, J = 15.9 Hz, 1H), 7.38 (s, 1H), 7.34–7.31 (m, 1H), 6.86 (s, 1H), 3.88 (s, 3H), 3.68 (s, 3H), 2.89–2.84 (m, 2H), 2.31 (d, J = 11.5 Hz, 1H), 2.22 (dd, J = 12.5, 2.3 Hz, 1H), 1.83–1.73 (m, 5H), 1.47–1.41 (m, 2H), 1.29 (s, 3H), 1.25 (s, 3H); 13C NMR (CDCl3) δ 191.3, 178.8, 156.8, 155.5, 150.5, 149.7, 138.2, 134.7, 131.4, 131.2, 129.0, 127.8, 126.4, 123.7, 107.6, 55.8, 52.0, 47.6, 44.5, 38.0, 37.9, 36.5, 28.8, 24.8, 21.5, 18.5, 16.6.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-(pyridin-3-yl)acryloyl)-1,2,3,4,4a, 9,10,10a-octahydrophenanthrene-1-carboxylate (33): 74%; MS (ESI, 30eV): m/z 442.21 [M + Na]; 1H NMR (CDCl3) δ 12.30 (s, 1H), 8.90 (s, 1H), 8.65 (d, J = 4.9 Hz, 1H), 8.01 (dt, J = 7.9, 4.0 Hz, 1H), 7.86 (d, J = 15.6 Hz, 1H), 7.69 (d, J = 15.6 Hz, 1H), 7.55 (s, 1H), 7.42 (dd, J = 8.0, 4.7 Hz, 1H), 6.92 (s, 1H), 3.68 (s, 3H), 2.95–2.86 (m, 2H), 2.28 (d, J = 12.8 Hz, 1H), 2.21 (dd, J = 12.5, 2.4 Hz, 1H), 1.88–1.64 (m, 5H), 1.56–1.43 (m, 2H), 1.29 (s, 3H), 1.23 (s, 3H); 13C NMR (CDCl3) δ 192.5, 179.0, 161.7, 160.0, 150.8, 149.6, 140.8, 135.5, 131.0, 130.0, 126.2, 124.1, 122.8, 118.1, 114.0, 52.2, 47.8, 38.2, 37.7, 36.7, 29.2, 24.7, 21.7, 18.6, 16.8; HR-ESI: m/z 420.2164; [M + H+] for the compound C26H29NO4 requires 420.2169.
(E)-1-((4bS,8R)-3-hydroxy-8-methoxy-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl)-3-(pyridin-2-yl)prop-2-en-1-one (34): 86%; MS (ESI, 30eV): m/z 442.39 [M + Na], 420.46 [M + H]; 1H NMR (CDCl3) δ 12.31 (s, 1H), 8.76 (d, J = 5.1 Hz, 1H), 8.47 (br s, 1H), 7.91 (s, 1H), 7.83 (s, 1H), 7.80 (d, J = 15.2 Hz, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.44 (s, 1H), 6.91 (s, 1H), 3.68 (s, 3H), 2.99–2.85 (m, 2H), 2.35 (t, J = 7.6 Hz, 1H), 2.27 (d, J = 11.6 Hz, 1H), 2.21 (dd, J = 12.5, 2.5 Hz, 1H), 1.87–1.64 (m, 5H), 1.48–1.43 (m, 1H), 1.29 (s, 3H), 1.22 (s, 3H); 13C NMR (CDCl3) δ 192.9, 178.8, 161.5, 159.9, 153.4, 151.0, 130.8, 126.5, 126.3, 124.9, 118.2, 113.5, 52.0, 47.6, 44.2, 38.1, 37.6, 36.5, 28.9, 24.5, 21.5, 18.4, 16.6.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-(pyridin-4-yl)acryloyl)-1,2,3,4,4a, 9,10,10a-octahydrophenanthrene-1-carboxylate (35): 85%; MS (ESI, 30eV): m/z 442.34 [M + Na], 420.18 [M + H]; 1H NMR (CDCl3) δ 12.22 (s, 1H), 8.71 (d, J = 6.1 Hz, 2H), 7.76 (d, J = 3.8 Hz, 2H), 7.53 (s, 1H), 7.50 (d, J = 6.1 Hz, 2H), 6.93 (s, 1H), 3.69 (s, 3H), 2.93–2.87 (m, 2H), 2.28 (d, J = 12.8 Hz, 1H), 2.21 (dd, J = 12.5, 2.6 Hz, 1H), 1.88–1.64 (m, 5H), 1.54–1.43 (m, 2H), 1.29 (s, 3H), 1.23 (s, 3H); 13C NMR (CDCl3) δ 178.6, 177.2, 160.5, 157.8, 154.7, 150.6, 133.9, 129.0, 128.2, 125.7, 122.4, 120.3, 113.2, 52.1, 47.5, 44.1, 38.2, 38.0, 36.5, 31.1, 29.1, 25.0, 21.2, 18.4, 16.6.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-(2,4,5-trifluorophenyl)acryloyl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (36): 89%; MS (ESI, 30eV): m/z 495.23 [M + Na], 473.44 [M + H]; 1H NMR (CDCl3) δ 12.30 (s, 1H), 7.86 (d, J = 15.7 Hz, 1H), 7.62 (d, J = 15.7 Hz, 1H), 7.53–7.46 (m, 2H), 7.06–7.00 (m, 1H), 6.92 (s, 1H), 3.69 (s, 3H), 2.94–2.87 (m, 2H), 2.27 (d, J = 12.8 Hz, 1H), 2.21 (dd, J = 12.5, 2.4 Hz, 1H), 1.88–1.73 (m, 4H), 1.69–1.66 (m, 1H), 1.55–1.45 (m, 2H), 1.29 (s, 3H), 1.23 (s, 3H); 13C NMR (CDCl3) δ 192.6, 179.0, 161.7, 160.0, 135.4, 130.0, 128.1, 126.2, 123.7, 118.2, 113.9, 106.5, 52.2, 47.8, 44.3, 38.2, 37.7, 36.7, 29.2, 24.7, 21.7, 18.6, 16.8.
(1R,4aS)-6-hydroxy-1,4a-dimethyl-7-((E)-3-(naphthalen-2-yl)acryloyl)-1,2,3,4,4a,9,10, 10a-octahydrophenanthrene-1-carboxylic acid (37): 76%; MS (ESI, 30eV): m/z 507.33 [M + K], 469.36 [M + H]; 1H NMR (CDCl3) δ 12.52 (s, 1H), 8.06 (d, J = 12.8 Hz, 1H), 7.92–7.81 (m, 5H), 7.73–7.32 (d, J = 15.5 Hz, 1H), 7.62 (s, 1H), 7.59–7.52 (m, 2H), 6.93 (s, 1H), 3.69 (s, 3H), 2.96–2.90 (m, 2H), 2.29 (d, J = 11.6 Hz, 1H), 2.23 (dd, J = 12.5, 2.3 Hz, 1H), 1.90–1.65 (m, 5H), 1.53–1.45 (m, 2H), 1.30 (s, 3H), 1.24 (s, 3H); 13C NMR (CDCl3) δ 193.2, 179.0, 161.6, 159.5, 145.2, 134.6, 133.5, 132.4, 131.1, 130.0, 129.0, 128.9, 128.0, 127.7, 127.00, 126.0, 123.9, 120.6, 118.5, 113.9, 52.2, 47.8, 44.4, 38.2, 37.8, 36.7, 29.2, 24.7, 21.8, 18.6, 16.8.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-(quinolin-2-yl)acryloyl)-1,2,3,4,4a, 9,10,10a-octahydrophenanthrene-1-carboxylate (38): 71%; MS (ESI, 30eV): m/z 492.11 [M + Na], 470.38 [M + H]; 1H NMR (CDCl3) δ 12.30 (s, 1H), 8.99 (d, J = 4.4 Hz, 1H), 8.60 (d, J = 15.4 Hz, 1H), 8.21 (d, J = 14.3 Hz, 1H), 8.18 (d, J = 9.7 Hz, 1H), 7.81–7.76 (m, 2H), 7.69–7.64 (m, 2H), 7.57 (s, 1H), 6.95 (s, 1H), 3.68 (s, 3H), 2.95–2.87 (m, 2H), 2.30–2.26 (m, 1H), 2.21 (dd, J = 12.5, 2.6 Hz, 1H), 1.88–1.66 (m, 5H), 1.60–1.51 (m, 1H), 1.50–1.45 (m, 1H), 1.29 (s, 3H), 1.24 (s, 3H); 13C NMR (CDCl3) δ 193.4, 179.0, 161.5, 159.4, 138.4, 138.2, 129.9, 129.8, 129.2, 128.4, 127.3, 125.9, 125.5, 120.2, 118.4, 113.8, 52.2, 47.8, 44.4, 38.2, 37.8, 36.7, 29.2, 24.7, 21.8, 18.6, 16.8; HR-ESI: m/z 470.2305; [M + H+] for the compound C30H31NO4 requires 470.2326.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-(quinolin-3-yl)acryloyl)-1,2,3,4,4a, 9,10,10a-octahydrophenanthrene-1-carboxylate (39): 62%; MS (ESI, 30eV): m/z 492.29 [M + Na], 470.29 [M + H]; 1H NMR (CDCl3) δ 12.33 (s, 1H), 8.48 (s, 1H), 8.33 (s, 1H), 8.29–8.14 (m, 1H), 8.03 (d, J = 15.7 Hz, 1H), 7.96 (d, J = 8.7 Hz, 1H), 7.91 (d, J = 6.8 Hz, 1H), 7.85–7.81 (m, 1H), 7.70–7.64 (m, 1H), 7.62 (s, 1H), 6.94 (s, 1H), 3.69 (s, 3H), 2.63–2.58 (m, 2H), 2.98–2.90 (m, 2H), 2.37–2.32 (m, 2H), 2.29 (d, J = 12.4 Hz, 1H), 2.23 (dd, J = 12.5, 2.4 Hz, 1H), 1.70–1.47 (m, 3H), 1.30 (s, 3H), 1.24 (s, 3H); 13C NMR (CDCl3) δ 194.8, 178.8, 161.6, 159.9, 150.0, 148.2, 131.8, 131.3, 129.9, 129.8, 129.2, 127.9, 127.8, 126.2, 124.5, 120.8, 118.0, 113.8, 52.0, 47.6, 44.2, 38.1, 37.6, 36.6, 29.6, 24.5, 21.6, 18.4, 16.6.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-(quinolin-4-yl)acryloyl)-1,2,3,4,4a, 9,10,10a-octahydrophenanthrene-1-carboxylate (40): 68%; MS (ESI, 30eV): m/z 508.37 [M + K], 497.47 [M + Na], 470.25 [M + H]; 1H NMR (CDCl3) δ 12.28 (s, 1H), 8.60 (d, J = 15.4 Hz, 1H),8.24–8.18 (m, 2H), 7.82–7.75 (m, 2H), 7.70–7.63 (m, 3H), 7.57 (s, 1H), 6.95 (s, 1H), 3.68 (s, 3H), 2.94–2.86 (m, 2H), 2.29 (d, J = 12.6 Hz, 1H), 2.22 (dd, J = 12.6, 2.4 Hz, 1H), 1.94–1.63 (m, 5H), 1.56–1.40 (m, 2H), 1.29 (s, 3H), 1.24 (s, 3H); 13C NMR (CDCl3) δ 192.4, 179.0, 161.8, 160.3, 159.4, 150.1, 140.8, 139.2, 131.2, 130.2, 127.7, 126.9, 126.4, 126.0, 123.6, 118.4, 114.0, 113.9, 52.2, 47.8, 44.3, 38.3, 37.7, 36.7, 29.1, 24.7, 21.7, 18.6, 16.8; HR-ESI: m/z 470.2304; [M + H+] for the compound C30H31NO4 requires 470.2326.
(1R,4aS)-methyl 7-((E)-3-(2-chloroquinolin-4-yl)acryloyl)-6-hydroxy-1,4a-dimethyl-1,2,3,4, 4a,9,10,10a-octahydrophenanthrene-1-carboxylate (41): 67%; MS (ESI, 30eV): m/z 526.40 [M + Na], 504.23 [M + H]; 1H NMR (CDCl3) δ 12.30 (s, 1H), 8.52 (s, 1H), 8.29 (d, J = 15.5 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.82–7.78 (m, 1H), 7.71 (d, J = 15.5 Hz, 1H), 7.64–7.61 (m, 1H), 7.59 (s, 1H), 6.95 (s, 1H), 3.69 (s, 3H), 2.95–2.91 (m, 2H), 2.29 (d, J = 12.6 Hz, 1H), 2.22 (dd, J = 12.6, 2.4 Hz, 1H), 1.90–1.67 (m, 6H), 1.51–1.49 (m, 1H), 1.60 (s, 3H), 1.24 (s, 3H); 13C NMR (CDCl3) δ 192.4, 179.0, 161.8, 160.0, 157.4, 148.2, 139.6, 136.6, 131.9, 130.1, 128.7, 128.2, 127.9, 127.2, 126.2, 125.0, 118.0, 114.0, 52.2, 47.8, 44.3, 38.3, 37.7, 36.7, 29.2, 24.7, 21.8, 18.6, 16.8; HR-ESI: m/z 504.1908; [M + H+] for the compound C25H29NO4 requires 504.1936.
(E)-3-(furan-2-yl)-1-((4bS,8R)-3-hydroxy-8-methoxy-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl)prop-2-en-1-one (42): 90%; MS (ESI, 30eV): m/z 431.36 [M + Na]; 1H NMR (CDCl3) δ 12.53 (s, 1H), 7.65 (d, J = 15.2 Hz, 1H), 7.56 (s, 2H), 7.51 (d, J = 15.2 Hz, 1H), 6.90 (s, 1H), 6.75 (d, J = 3.5 Hz, 1H), 6.55–6.52 (m, 1H), 3.68 (s, 3H), 2.92–2.88 (m, 2H), 2.27 (d, J = 11.1 Hz, 1H), 2.20 (dd, J = 12.5, 2.5 Hz, 1H), 1.88–1.61 (m, 6H), 1.55–1.44 (m, 1H), 1.29 (s, 3H), 1.23 (s, 3H); 13C NMR (CDCl3) δ 192.8, 179.0, 161.5, 159.4, 151.8, 145.4, 130.8, 130.0, 125.9, 118.4, 118.0, 117.0, 113.7, 113.0, 52.2, 47.8, 44.4, 38.2, 37.8, 36.7, 29.2, 24.7, 21.8, 18.6, 16.8.
(1R,4aS)-methyl 7-((E)-3-(1H-pyrrol-3-yl)acryloyl)-6-hydroxy-1,4a-dimethyl-1,2,3,4, 4a,9,10,10a-octahydrophenanthrene-1-carboxylate (43): 72%; MS (ESI, 30eV): m/z 446.24 [M + K], 430.19 [M + Na], 408.23 [M + H]; 1H NMR (CDCl3) δ 12.71 (s, 1H), 8.76 (s, 1H), 7.79 (d, J = 15.4 Hz, 1H), 7.50 (s, 1H), 7.03–6.99 (m, 1H), 6.89 (s, 1H), 6.77–6.74 (m, 1H), 3.69 (s, 3H), 2.91–2.86 (m, 2H), 2.26 (d, J = 11.5 Hz, 1H), 2.21 (dd, J = 12.4, 2.3 Hz, 1H), 1.88–1.63 (m, 5H), 1.50–1.43 (m, 2H), 1.29 (s, 3H), 1.22 (s, 3H); 13C NMR (CDCl3) δ 192.6, 179.0, 161.4, 158.8, 138.0, 134.4, 129.2, 128.4, 125.5, 123.6, 118.5, 116.2, 113.8, 111.9, 52.2, 47.8, 44.4, 38.1, 37.8, 36.7, 29.2, 24.7, 21.8, 18.6, 16.8; HR-ESI: m/z 408.2152; [M + H+] for the compound C25H29NO4 requires 408.2169.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-(thiophen-3-yl)acryloyl)-1,2,3,4, 4a,9,10,10a-octahydrophenanthrene-1-carboxylate (44): 83%; MS (ESI, 30eV): m/z 463.40 [M + K], 447.09 [M + Na], 425.38 [M + H]; 1H NMR (CDCl3) δ 12.49 (s, 1H), 7.88 (d, J = 15.4 Hz, 1H), 7.66–7.63 (m, 1H), 7.53 (s, 1H), 7.47–7.45 (m, 1H), 7.43 (d, J = 15.4 Hz, 1H), 7.40–7.38 (m, 1H), 6.91 (s, 1H), 3.68 (s, 3H), 2.94–2.87 (m, 2H), 2.28 (d, J = 11.9 Hz, 1H), 2.21 (dd, J = 12.5, 2.4 Hz, 1H), 1.89–1.65 (m, 5H), 1.55–1.45 (m, 2H), 1.29 (s, 3H), 1.23 (s, 3H); 13C NMR (CDCl3) δ 193.2, 178.9, 161.4, 159.2, 138.3, 138.1, 129.8, 129.6, 127.1, 125.7, 125.3, 120.0, 118.2, 113.7, 52.0, 47.6, 44.2, 38.0, 37.6, 36.6, 29.0, 24.5, 21.6, 18.4, 16.6; HR-ESI: m/z 425.1764; [M + H+] for the compound C25H28SO4 requires 425.1781.
(1R,4aS)-methyl 7-((E)-3-(5-bromothiophen-2-yl)acryloyl)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (45): 67%; MS (ESI, 30eV): m/z 541.47 [M + K], 525.40 [M + Na], 503.33 [M + H]; 1H NMR (CDCl3) δ 12.41 (s, 1H), 7.87 (d, J = 15.2 Hz, 1H), 7.47 (s, 1H), 7.28 (d, J = 15.2 Hz, 1H), 7.13 (d, J = 3.9 Hz, 1H), 7.08 (d, J = 3.9 Hz, 1H), 6.90 (s, 1H), 3.69 (s, 3H), 2.93–2.86 (m, 2H), 2.27 (d, J = 11.3 Hz, 1H), 2.21 (dd, J = 12.5, 2.4 Hz, 1H), 1.88–1.71 (m, 4H), 1.69–1.64 (m, 1H), 1.54–1.45 (m, 2H), 1.29 (s, 3H), 1.22 (s, 3H); 13C NMR (CDCl3) δ 192.1, 178.9, 161.4, 159.4, 141.8, 136.3, 123.6, 131.4, 129.6, 125.9, 119.4, 118.0, 116.9, 113.7, 52.0, 47.6, 44.2, 38.0, 37.6, 36.5, 29.0, 24.5, 21.6, 18.4, 16.6.
(1R,4aS)-methyl 7-((E)-3-(1H-indol-3-yl)acryloyl)-6-hydroxy-1,4a-dimethyl-1,2,3,4, 4a,9,10,10a-octahydrophenanthrene-1-carboxylate (46): 62%; MS (ESI, 30eV): m/z 496.12 [M + K], 480.18 [M + Na], 458.26 [M + H]; 1H NMR (CDCl3) δ 12.87 (s, 1H), 8.82 (s, 1H), 8.17 (d, J = 15.3 Hz, 1H), 8.04–7.99 (m, 1H), 7.66–7.58 (s, 3H), 7.47–7.44 (m, 1H), 7.34–7.31 (m,2H), 6.92 (s, 1H), 3.70 (s, 3H), 2.98–2.88 (m, 2H), 2.28 (d, J = 11.8 Hz, 1H), 2.23 (dd, J = 12.6, 2.5 Hz, 1H), 1.90–1.65 (m, 5H), 1.57–1.45 (m, 2H), 1.31 (s, 3H), 1.24 (s, 3H); 13C NMR (CDCl3) δ 193.3, 179.0, 161.3, 158.5, 139.0, 137.3, 130.6, 129.7, 129.0, 128.2, 123.7, 121.9, 120.6, 118.5, 115.8, 114.6, 113.5, 112.1, 52.0, 47.7, 44.3, 37.9, 37.7, 36.6, 29.1, 24.5, 21.6, 18.5, 16.6.
(1R,4aS)-methyl 7-((E)-3-(benzo[b]thiophen-3-yl)acryloyl)-6-hydroxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (47): 72%; MS (ESI, 30eV): m/z 497.26 [M + Na], 475.48 [M + H]; 1H NMR (CDCl3) δ 12.81 (s, 1H), 8.58 (s, 1H), 8.18 (d, J = 15.4 Hz, 1H), 8.04–7.99 (m, 1H), 7.67–7.65 (m, 1H), 7.62 (d, J = 19.3 Hz, 1H), 7.48–7.45 (m, 1H), 7.36–7.32 (m, 2H), 6.91 (s, 1H), 3.69 (s, 3H), 2.98–2.92 (m, 2H), 2.29 (d, J = 11.6 Hz, 1H), 2.24 (dd, J = 12.5, 2.5 Hz, 1H), 1.91–1.82 (m, 1H), 1.79–1.72 (m, 3H), 1.68–1.66 (m, 1H), 1.57–1.51 (m, 2H), 1.30 (s, 3H), 1.24 (s, 3H); 13C NMR (CDCl3) δ 193.4, 179.1, 161.5, 158.7, 139.0, 138.0, 137.4, 130.5, 129.8, 129.2, 127.1, 125.6, 125.5, 123.9, 122.9, 120.8, 118.6, 113.7, 52.2, 47.8, 44.5, 38.1, 37.8, 36.7, 29.3, 24.7, 21.8, 18.6, 16.8; HR-ESI: m/z 475.1925; [M + H+] for the compound C25H29SO4 requires 475.1938.
Tert-butyl 4-(4-((E)-3-((4bS,8R)-3-methoxy-8-(methoxycarbonyl)-4b,8-dimethyl-4b,5, 6,7,8,8a,9,10-octahydrophenanthren-2-yl)acryloyl)phenyl)piperazine-1-carboxylate (48): 88%; MS (ESI, 30eV): m/z 625.37 [M + Na], 603.61 [M + H]; 1H NMR (CDCl3) δ 8.03–7.99 (m, 2H), 7.90 (d, J = 9.1 Hz, 1H), 7.58 (d, J = 15.8 Hz, 1H), 7.28 (s, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.80 (s, 1H), 3.88 (s, 3H), 3.68 (s, 3H), 3.63 (t, J = 5.3 Hz, 2H), 3.40–3.31 (m, 5H), 2.89–2.84 (m, 2H), 2.29 (d, J = 12.4 Hz, 1H), 2.23 (dd, J = 12.5, 2.2 Hz, 1H), 1.87–1.72 (m, 5H), 1.68–1.65 (m, 1H), 1.57–1.52 (m, 1H), 1.49 (s, 9H), 1.46–1.41 (m, 1H), 1.29 (s, 3H), 1.24 (s, 3H); 13C NMR (CDCl3) δ 196.6, 189.1, 179.1, 157.3, 154.7, 153.5, 139.6, 130.8, 130.6, 130.0, 127.5, 125.4, 122.1, 115.2, 114.5, 107.2, 80.5, 55.8, 52.1, 47.8, 44.8, 38.1, 38.0, 36.7, 29.1, 28.5, 26.3, 25.0, 21.8, 21.6, 18.7, 16.7.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-oxo-3-(pyridin-2-yl)prop-1-en-1-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (49): 74%; 1H NMR (CDCl3) δ 10.65 (s, 1H), 9.79 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.34–7.29 (m, 1H), 7.28–7.26 (m, 2H), 7.21 (s, 1H), 6.87 (s, 1H), 3.68 (s, 3H), 2.90–2.85 (m, 2H), 2.26 (d, J = 12.9 Hz, 1H), 2.19 (dd, J = 12.5, 2.4 Hz, 1H), 1.86–1.64 (m, 5H), 1.53–1.43 (m, 2H), 1.28 (s, 3H), 1.22 (s, 3H); 13C NMR (CDCl3) δ 195.9, 182.9, 182.0, 164.6, 15.9, 159.4, 137.5, 136.6, 134.0, 127.9, 127.0, 119.0, 112.9, 92.0, 52.0, 47.6, 44.1, 38.2, 37.6, 36.5, 28.7, 24.5, 21.4, 18.4, 16.6.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (50): 65%; MS (ESI, 30eV): m/z 442.47 [M + Na]; 1H NMR (CDCl3) δ 9.22 (d, J = 46.1 Hz, 1H), 8.79 (br s, 1H), 8.37–8.24 (m, 1H), 8.00 (d, J = 15.7 Hz, 1H), 7.75 (d, J = 15.7 Hz, 1H), 7.58 (s, 1H), 7.51–7.47 (m, 1H), 7.46–7.42 (m, 1H),. 6.87 (s, 1H), 3.68 (s, 3H), 2.87–2.81 (m, 2H), 2.26 (d, J = 12.5 Hz, 1H), 2.21 (dd, J = 12.5, 2.4 Hz, 1H), 1.80–1.64 (m, 5H), 1.54–1.41 (m, 2H), 1.28 (s, 3H), 1.25 (s, 3H); 13C NMR (CDCl3) δ 195.9, 159.9, 159.4, 137.3, 136.6, 134.0, 128.6, 127.9, 126.4, 117.0, 112.9, 52.0, 47.6, 44.1, 38.2, 37.6, 36.5, 28.7, 24.5, 21.6, 18.4, 16.6.
(1R,4aS)-methyl 6-hydroxy-1,4a-dimethyl-7-((E)-3-oxo-3-(pyrazin-2-yl)prop-1-en-1-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (51): 69%; MS (ESI, 30eV): m/z 459.56 [M + K], 421.44 [M + H]; 1H NMR (CDCl3) δ 9.25 (s, 1H), 8.78 (d, J = 2.4 Hz, 1H), 8.75 & 8.71 (2d, J = 2.6 & 2.5 Hz, 1H), 8.67–8.46 (m, 1H), 7.56 (s, 1H), 7.17 (d, J = 7.3 Hz, 2H), 6.87 (s, 1H), 3.68 (s, 3H), 2.87–2.80 (m, 2H), 2.25 (d, J = 12.3 Hz, 1H), 2.19 (dd, J = 12.5, 2.7 Hz, 1H), 1.84–1.61 (m, 7H), 1.28 (s, 3H), 1.21 (s, 3H); 13C NMR (CDCl3) δ 178.9, 159.9, 159.1, 147.9, 143.6, 142.9, 142.5, 137.9, 130.9, 129.0, 128.2, 126.4, 125.3, 112.8, 52.0, 47.6, 44.2, 37.9, 37.6, 36.5, 28.7, 24.6, 21.4, 18.4, 16.6; HR-ESI: m/z 421.2117; [M + H+] for the compound C25H29N2O4 requires 421.2122.
(1R,4aS)-methyl 7-((E)-3-(benzofuran-2-yl)acryloyl)-6-hydroxy-1,4a-dimethyl-1,2,3, 4,4a,9,10, 10a-octahydrophenanthrene-1-carboxylate (52): 74%; MS (ESI, 30eV): m/z 481.21 [M + Na], 459.18 [M + H]; 1H NMR (CDCl3) δ 7.53 (dd, J = 7.6, 3.5 Hz, 1H), 7.45 (dd, J = 8.1, 2.8 Hz, 1H), 7.38–7.35 (m, 1H), 7.29 (dd, J = 8.2, 4.0 Hz, 1H), 7.22–7.19 (m, 1H), 7.10 (t, J = 7.1 Hz, 1H), 7.05 (t, J = 7.1 Hz, 1H), 6.68 (s, 1H), 6.23 (s, 1H), 3.67 (s, 3H), 3.63 (s, 1H), 2.80–2.73 (m, 1H), 2.56 (dd, J = 6.5, 2.4 Hz, 1H), 2.54 (dd, J = 6.5, 2.4 Hz, 1H), 1.69–1.63 (m, 2H), 1.51–1.44 (m, 2H), 1.41–1.35 (m, 1H), 1.25–1.18 (m, 2H), 1.11 (s, 3H), 1.10 (s, 3H); 13C NMR (CDCl3) δ 196.5, 179.0, 161.4, 155.7, 154.6, 152.2, 149.5, 128.8, 128.7, 128.4, 127.9, 126.4, 123.3, 122.8, 122.7, 112.2, 111.1, 103.1, 75.5, 51.9, 47.5, 44.4, 37.4, 36.7, 36.3, 21.6, 21.4, 18.3, 16.4.
(1R,4aS)-methyl 7-((E)-3-(5-bromothiophen-2-yl)-3-oxoprop-1-en-1-yl)-6-hydroxy-1,4a-dimthyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (53): 73%; MS (ESI, 30eV): m/z 541.28 [M + K], 525.98 [M + Na], 503.12 [M + H]; 1H NMR (CDCl3) δ 8.02 (d, J = 15.5 Hz, 1H), 7.58 (d, J = 4.0 Hz, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.12 (d, J = 4.0 Hz, 1H), 6.74 (s, 1H), 5.89 (s, 1H), 3.68 (s, 3H), 2.87–2.78 (m, 2H), 2.22–2.16 (m, 2H), 1.85–1.63 (m, 5H), 1.52–1.46 (m, 1H), 1.45–1.40 (m, 1H), 1.27 (s, 3H), 1.20 (s, 3H); 13C NMR (CDCl3) δ 181.8, 179.0, 153.8, 147.4, 140.4, 131.7, 131.3, 130.4, 129.0, 128.2, 122.4, 120.7, 119.6, 112.1, 52.0, 47.6, 44.4, 37.8, 37.5, 36.5, 28.8, 24.7, 21.6, 18.5, 16.5.

3.3. Biological Assays

MCF-7 (ER-positive breast cancer cells, epithelial, low metastatic), MDA-MB-231, and Hs578T (triple negative breast cancer cells, mesenchymal, high metastatic) were obtained from American Type Culture Collection (ATCC) and cultured with DMEM high glucose/10% Fetal Bovine Serum (FBS) (supplied by Biosera). Primary skin normal fibroblasts were kindly provided by Prof. Emer. A. Aletras, Laboratory of Biochemistry, University of Patras, Greece and cultured with DMEM low glucose/10% FBS (supplied by Biosera). Cancer cells were seeded in 24 well plates (30,000 MCF-7 cells/well, 30.000 MDA-MB-231 cells/well, 25,000 Hs578T cells/well) and fibroblasts in 12 well plates (40,000 cells/well) and incubated in proper medium completed with 10% FBS for 48 h. Compounds were diluted in DMSO and working dilutions were prepared ranging from 5 to 200 μΜ for cancer cells and 1 to 100 μM for fibroblasts. All cells were incubated with working dilutions for 48 h. Afterwards, cells were detached by trypsinization, centrifuged at 3000 rpm for 3 min, and counted using a hemocytometer. Antiproliferation values were then calculated relatively to cancer or normal cells treated only with DMSO. The DMSO concentration equivalents were tested for their toxicity in all cells and no statistical significance at the cellular viability was observed at the used concentrations. IC50 values were calculated using GraphPad Prism 5 (Graph Pad Software).

4. Conclusions

In conclusion, from the total of 23 synthetic DHA-chalcone hybrids tested, two compounds, 33 and 41, exhibited high activity levels in all three breast cancer cell lines, namely the estrogen-dependent MCF-7 and the estrogen-independent MDA-MB-231 and Hs578T. Four DHA-chalcone hybrids (38, 43, 44, and 47) present selective strong antiproliferative activity only against MCF-7 breast cancer cells. Finally, one compound (51) displays a selective moderate anticancer activity against the triple negative cell lines MDA-MB-231 and Hs578T. Compound 38 appears to be a promising drug lead for the treatment of hormone dependent breast cancer cases, which account for the majority of breast cancers, taking into consideration it’s comparable activity with the control drug 5-FU on MCF-7 breast cancer cells and its very high selectivity index (SI = 22.7), which makes it 12.9 times less toxic than 5-FU. Further studies aiming to reveal new hybrid lead compounds are currently in progress, focusing on the structural elements of the chalcone’s ring B (taking into consideration the so far SARS) and the replacement of the C18 ester moiety with various amides and carbamates.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27113623/s1. Figure S1–S54: Copies of 1H and 13C NMR spectra of compounds.

Author Contributions

Conceptualization, C.M.A.; methodology, C.M.A., A.D.T., A.I.A., D.M., S.G. and A.T.; NMR and MS analysis, C.M.A., C.S. and A.I.A.; investigation, D.M., S.G. and A.T.; writing—original draft preparation, C.M.A., A.D.T., C.S., A.I.A. and D.M.; writing—review and editing, C.M.A., A.D.T., C.S., A.I.A. and D.M.; supervision, C.M.A. and A.D.T.; project administration, C.M.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank the University of Patras for financial support, as well as the Instrumental Analysis Laboratory (IAL, School of Natural Sciences, University of Patras) for recording the NMR spectra and Alexios Aletras, Dept. of Chemistry, University of Patras, for kindly providing us the primary normal fibroblasts. In addition, we wish to thank the Unit of Environmental, Organic and Biochemical high-resolution analysis-Orbitrap-LC-MS of the University of Ioannina for providing access to their facilities and Vasiliki Boti for performing the HRMS analyses.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

References

  1. Wang, H.; Naghavi, M.; Allen, C.; Barber, R.M.; Bhutta, Z.A.; Carter, A.; Casey, D.C.; Charlson, F.J.; Chen, A.Z.; Coates, M.M.; et al. Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990–2015: A systematic analysis for the Global Burden of Disease Study 2015. Lancet 2016, 388, 1459–1544. [Google Scholar] [CrossRef] [Green Version]
  2. Newman, D.J.; Cragg, G.M.; Snader, K.M. Natural products as sources of new drugs over the period 1981–2002. J. Nat. Prod. 2003, 66, 1022–1037. [Google Scholar] [CrossRef] [PubMed]
  3. Lee, K.-H. Current Developments in the Discovery and Design of New Drug Candidates from Plant Natural Product Leads. J. Nat. Prod. 2004, 67, 273–283. [Google Scholar] [CrossRef] [PubMed]
  4. You, X.; Zhu, D.; Lu, W.; Sun, Y.; Qiao, S.; Luo, B.; Du, Y.; Pi, R.; Hu, Y.; Huang, P.; et al. Design, synthesis and biological evaluation of N-arylsulfonyl carbazoles as novel anticancer agents. RSC Adv. 2018, 8, 17183–17190. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Lee, M.M.-L.; Chan, B.D.; Wong, W.-Y.; Leung, T.-W.; Qu, Z.; Huang, J.; Zhu, L.; Lee, C.-S.; Chen, S.; Tai, W.C.-S. Synthesis and Evaluation of Novel Anticancer Compounds Derived from the Natural Product Brevilin A. ACS Omega 2020, 5, 14586–14596. [Google Scholar] [CrossRef] [PubMed]
  6. Gazim, Z.C.; Rodrigues, F.; Amorin, A.C.L.; de Rezende, C.M.; Soković, M.; Tešević, V.; Vučković, I.; Krstić, G.; Cortez, L.E.R.; Colauto, N.B.; et al. New natural diterpene-type abietane from Tetradenia riparia essential oil with cytotoxic and antioxidant activities. Molecules 2014, 19, 514–524. [Google Scholar] [CrossRef] [Green Version]
  7. Neto, Í.; Faustino, C.; Rijo, P. Antimicrobial abietane diterpenoids against resistant bacteria and biofilms. In the Battle against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs; Formatex Research Center: Badajoz, Spain, 2015; pp. 15–26. [Google Scholar]
  8. Johnson, J.J. Carnosol: A promising anti-cancer and anti-inflammatory agent. Cancer Lett. 2011, 305, 1–7. [Google Scholar] [CrossRef] [Green Version]
  9. Burmistrova, O.; Simões, M.F.; Rijo, P.; Quintana, J.; Bermejo, J.; Estévez, F. Antiproliferative Activity of Abietane Diterpenoids against Human Tumor Cells. J. Nat. Prod. 2013, 76, 1413–1423. [Google Scholar] [CrossRef] [Green Version]
  10. Islam, M.T. Diterpenes and Their Derivatives as Potential Anticancer Agents. Phytother. Res. 2017, 31, 691–712. [Google Scholar] [CrossRef]
  11. Yang, N.-Y.; Liu, L.; Tao, W.-W.; Duan, J.-A.; Tian, L.-J. Diterpenoids from Pinus massoniana resin and their cytotoxicity against A431 and A549 cells. Phytochemistry 2010, 71, 1528–1533. [Google Scholar] [CrossRef]
  12. Fronza, M.; Murillo, R.; Ślusarczyk, S.; Adams, M.; Hamburger, M.; Heinzmann, B.; Laufer, S.; Merfort, I. In vitro cytotoxic activity of abietane diterpenes from Peltodon longipes as well as Salvia miltiorrhiza and Salvia sahendica. Bioorg. Med. Chem. 2011, 19, 4876–4881. [Google Scholar] [CrossRef] [PubMed]
  13. González, M.A.; Pérez-Guaita, D.; Correa-Royero, J.; Zapata, B.; Agudelo, L.; Mesa-Arango, A.; Betancur-Galvis, L. Synthesis and biological evaluation of dehydroabietic acid derivatives. Eur. J. Med. Chem. 2010, 45, 811–816. [Google Scholar] [CrossRef] [PubMed]
  14. Ukiya, M.; Kawaguchi, T.; Ishii, K.; Ogihara, E.; Tachi, Y.; Kurita, M.; Ezaki, Y.; Fukatsu, M.; Kushi, Y.; Akihisa, T. Cytotoxic Activities of Amino Acid-Conjugate Derivatives of Abietane-Type Diterpenoids against Human Cancer Cell Lines. Chem. Biodivers. 2013, 10, 1260–1268. [Google Scholar] [CrossRef] [PubMed]
  15. Huang, X.-C.; Wang, M.; Wang, H.-S.; Chen, Z.-F.; Zhang, Y.; Pan, Y.-M. Synthesis and antitumor activities of novel dipeptide derivatives derived from dehydroabietic acid. Bioorg. Med. Chem. Lett. 2014, 24, 1511–1518. [Google Scholar] [CrossRef]
  16. Li, F.-Y.; Wang, X.; Duan, W.-G.; Lin, G.-S. Synthesis and In Vitro Anticancer Activity of Novel Dehydroabietic Acid-Based Acylhydrazones. Molecules 2017, 22, 1087. [Google Scholar] [CrossRef] [Green Version]
  17. Rao, X.; Song, Z.; He, L.; Jia, W. Synthesis, structure analysis and cytotoxicity studies of novel unsymmetrically n,n’-substituted ureas from dehydroabietic Acid. Chem. Pharm. Bull. 2008, 56, 1575–1578. [Google Scholar] [CrossRef] [Green Version]
  18. Rao, X.-P.; Wu, Y.; Song, Z.-Q.; Shang, S.-B.; Wang, Z.-D. Synthesis and antitumor activities of unsymmetrically disubstituted acylthioureas fused with hydrophenanthrene structure. Med. Chem. Res. 2011, 20, 333–338. [Google Scholar] [CrossRef]
  19. Pertino, M.W.; Verdugo, V.; Theoduloz, C.; Schmeda-Hirschmann, G. Synthesis and antiproliferative activity of some novel triazole derivatives from dehydroabietic acid. Molecules 2014, 19, 2523–2535. [Google Scholar] [CrossRef] [Green Version]
  20. Kolsi, L.E.; Leal, A.S.; Yli-Kauhaluoma, J.; Liby, K.T.; Moreira, V.M. Dehydroabietic oximes halt pancreatic cancer cell growth in the G1 phase through induction of p27 and downregulation of cyclin D1. Sci. Rep. 2018, 8, 15923. [Google Scholar] [CrossRef]
  21. Gigante, B.; Santos, C.; Silva, A.M.; Curto, M.J.M.; Nascimento, M.S.J.; Pinto, E.; Pedro, M.; Cerqueira, F.; Pinto, M.M.; Duarte, M.P.; et al. Catechols from abietic acid: Synthesis and evaluation as bioactive compounds. Bioorg. Med. Chem. 2003, 11, 1631–1638. [Google Scholar] [CrossRef]
  22. Miao, T.-T.; Tao, X.-B.; Li, D.-D.; Chen, H.; Jin, X.-Y.; Geng, Y.; Wang, S.-F.; Gu, W. Synthesis and biological evaluation of 2-aryl-benzimidazole derivatives of dehydroabietic acid as novel tubulin polymerization inhibitors. RSC Adv. 2018, 8, 17511–17526. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Gu, W.; Wang, S.; Jin, X.; Zhang, Y.; Hua, D.; Miao, T.; Tao, X.; Wang, S. Synthesis and Evaluation of New Quinoxaline Derivatives of Dehydroabietic Acid as Potential Antitumor Agents. Molecules 2017, 22, 1154. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Shalini; Kumar, V. Have molecular hybrids delivered effective anti-cancer treatments and what should future drug discovery focus on? Expert Opin. Drug Discov. 2021, 16, 335–363. [Google Scholar] [CrossRef] [PubMed]
  25. Xie, L.; Zhai, X.; Ren, L.; Meng, H.; Liu, C.; Zhu, W.; Zhao, Y. Design, Synthesis and Antitumor Activity of Novel Artemisinin Derivatives Using Hybrid Approach. Chem. Pharm. Bull. 2011, 59, 984–990. [Google Scholar] [CrossRef] [Green Version]
  26. Gaur, R.; Pathania, A.S.; Malik, F.A.; Bhakuni, R.S.; Verma, R.K. Synthesis of a series of novel dihydroartemisinin monomers and dimers containing chalcone as a linker and their anticancer activity. Eur. J. Med. Chem. 2016, 122, 232–246. [Google Scholar] [CrossRef]
  27. Palmieri, C.; Januszewski, A.; Stanway, S.; Coombes, R.C. Irosustat: A first-generation steroid sulfatase inhibitor in breast cancer. Expert Rev. Anticancer Ther. 2011, 11, 179–183. [Google Scholar] [CrossRef]
  28. Afzal, O.; Kumar, S.; Haider, M.R.; Ali, M.R.; Kumar, R.; Jaggi, M.; Bawa, S. A review on anticancer potential of bioactive heterocycle quinoline. Eur. J. Med. Chem. 2015, 97, 871–910. [Google Scholar] [CrossRef]
  29. Elshemy, H.A.H.; Zaki, M.A. Design and synthesis of new coumarin hybrids and insight into their mode of antiproliferative action. Bioorg. Med. Chem. 2017, 25, 1066–1075. [Google Scholar] [CrossRef]
  30. Shin, S.Y.; Yoon, H.; Ahn, S.; Kim, D.-W.; Kim, S.H.; Koh, D.; Lee, Y.H.; Lim, Y. Chromenylchalcones showing cytotoxicity on human colon cancer cell lines and in silico docking with aurora kinases. Bioorg. Med. Chem. 2013, 21, 4250–4258. [Google Scholar] [CrossRef]
  31. Tseng, C.-H.; Chen, Y.-L.; Hsu, C.-Y.; Chen, T.-C.; Cheng, C.-M.; Tso, H.-C.; Lu, Y.-J.; Tzeng, C.-C. Synthesis and antiproliferative evaluation of 3-phenylquinolinylchalcone derivatives against non-small cell lung cancers and breast cancers. Eur. J. Med. Chem. 2013, 59, 274–282. [Google Scholar] [CrossRef]
  32. Li, W.; Xu, F.; Shuai, W.; Sun, H.; Yao, H.; Ma, C.; Xu, S.; Yao, H.; Zhu, Z.; Yang, D.-H.; et al. Discovery of Novel Quinoline–Chalcone Derivatives as Potent Antitumor Agents with Microtubule Polymerization Inhibitory Activity. J. Med. Chem. 2019, 62, 993–1013. [Google Scholar] [CrossRef] [PubMed]
  33. Lindamulage, I.K.; Vu, H.-Y.; Karthikeyan, C.; Knockleby, J.; Lee, Y.-F.; Trivedi, P.; Lee, H. Novel quinolone chalcones targeting colchicine-binding pocket kill multidrug-resistant cancer cells by inhibiting tubulin activity and MRP1 function. Sci. Rep. 2017, 7, 10298. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Antoniou, A.; Chatzopoulou, M.; Bantzi, M.; Athanassopoulos, C.M.; Giannis, A.; Pitsinos, E.N. Identification of Gli-mediated transcription inhibitors through synthesis and evaluation of taepeenin D analogues. Med. Chem. Comm. 2016, 7, 2328–2331. [Google Scholar] [CrossRef]
  35. Chaban, M.F.; I Antoniou, A.; Karagianni, C.; Toumpa, D.; Joray, M.B.; Bocco, J.L.; Sola, C.; M Athanassopoulos, C.; Carpinella, M.C. Synthesis and structure-activity relationships of novel abietane diterpenoids with activity against Staphylococcus aureus. Future Med. Chem. 2019, 11, 3109–3124. [Google Scholar] [CrossRef]
  36. Mori, M.; Tottone, L.; Quaglio, D.; Zhdanovskaya, N.; Ingallina, C.; Fusto, M.; Ghirga, F.; Peruzzi, G.; Crestoni, M.E.; Simeoni, F.; et al. Identification of a novel chalcone derivative that inhibits Notch signaling in T-cell acute lymphoblastic leukemia. Sci. Rep. 2017, 7, 2213. [Google Scholar] [CrossRef]
  37. Gutierrez, P.; Altarejos, J.; Linares-Palomino, P.J.; Chahboun, R.; Alvarez-Manzaneda, E. Synthesis of cassane-type diterpenes from abietane compounds: The first synthesis of taepeenin F. Org. Chem. Front. 2018, 5, 2537–2541. [Google Scholar] [CrossRef]
Molecules 27 03623 g001 550
Figure 1. Structures of DHA (1) and its analogues (2–16) with anticancer activity.
Figure 1. Structures of DHA (1) and its analogues (2–16) with anticancer activity.
Molecules 27 03623 g001
Molecules 27 03623 g002 550
Figure 2. Structures of artemisinin-, coumarin- and quinoline-chalcone hybrids 17–25 with anticancer activity.
Figure 2. Structures of artemisinin-, coumarin- and quinoline-chalcone hybrids 17–25 with anticancer activity.
Molecules 27 03623 g002
Molecules 27 03623 sch001 550
Scheme 1. Synthesis of the DHA-chalcone hybrids 31–53; Reagents and conditions: (a) CH3COCl, AlCl3, DCM, 0 °C to rt, 7 h, 93% (b) AlCl3, DCM, reflux, 2 h, 97%; (c) CH3OCHCl2, AlCl3, DCM, −35 °C, 1 h, 85%; (d) aryl methyl ketone or arylaldehyde, Ba(OH)2 · 8H2O, EtOH, rt, 24 h, 62–90%.
Scheme 1. Synthesis of the DHA-chalcone hybrids 31–53; Reagents and conditions: (a) CH3COCl, AlCl3, DCM, 0 °C to rt, 7 h, 93% (b) AlCl3, DCM, reflux, 2 h, 97%; (c) CH3OCHCl2, AlCl3, DCM, −35 °C, 1 h, 85%; (d) aryl methyl ketone or arylaldehyde, Ba(OH)2 · 8H2O, EtOH, rt, 24 h, 62–90%.
Molecules 27 03623 sch001
Molecules 27 03623 g003 550
Figure 3. Structures of the most potent compounds and their IC50 values against all tested cell lines including the positive control (5-FU).
Figure 3. Structures of the most potent compounds and their IC50 values against all tested cell lines including the positive control (5-FU).
Molecules 27 03623 g003
Molecules 27 03623 g004 550
Figure 4. IC50 values of the most potent compounds and 5-FU (positive control) against each cancer cell line; (A) MDA-MB-231; (B) MCF-7; (C) Hs578T; and (D) against normal primary FB.
Figure 4. IC50 values of the most potent compounds and 5-FU (positive control) against each cancer cell line; (A) MDA-MB-231; (B) MCF-7; (C) Hs578T; and (D) against normal primary FB.
Molecules 27 03623 g004
Table
Table 1. Antiproliferation activity of synthesized compounds 32–54 in the MDA-MB-231, MCF-7 and Hs578T breast cancer cell lines as well as against normal human fibroblast cell line. Selectivity index values are also provided for each IC50 value.
Table 1. Antiproliferation activity of synthesized compounds 32–54 in the MDA-MB-231, MCF-7 and Hs578T breast cancer cell lines as well as against normal human fibroblast cell line. Selectivity index values are also provided for each IC50 value.
CompoundIC50 (μM) Selectivity Index for
MDA-MB-231MCF-7Hs578TFibroblastsMDA-MB-231MCF-7Hs578T
31>7523.90 ± 4.47>75n/an/an/an/a
32>75>75>75n/an/an/an/a
335.89 ± 0.772.21 ± 0.402.48 ± 0.138.68 ± 0.881.473.933.50
34>7521.36 ± 0.6028.09 ± 7.76n/an/an/an/a
3537.05 ± 1.829.90 ± 0.058.69 ± 0.5533.83 ± 6.630.913.423.89
36>75>75>75n/an/an/an/a
37>7511.39 ± 1.55>75n/an/an/an/a
3873.00 ± 2.317.06 ± 1.71>75160.65 ± 13.082.2022.75-
39>7511.50 ± 1.0215.71 ± 2.7432.88 ± 2.27-2.862.09
40>75>75>7592.11 ± 5.47---
417.12 ± 0.203.99 ± 0.1510.31 ± 0.257.96 ± 0.101.121.990.77
42>7537.01 ± 6.8943.33 ± 4.32n/an/an/an/a
4329.20 ± 3.264.29 ± 0.2916.64 ± 2.8333.05 ± 6.101.137.701.99
4425.21 ± 0.843.45 ± 0.3621.48 ± 1.2044.03 ± 1.841.7512.762.05
45>7529.94 ± 5.26>75n/an/an/an/a
46>75>75>75n/an/an/an/a
4740.37 ± 0.6712.52 ± 8.43>75n/an/an/an/a
48>7525.62 ± 0.0923.15 ± 2.50n/an/an/an/a
4922.43 ± 0.8145.93 ± 1.5217.07 ± 2.1323.81 ± 1.071.060.521.39
50>75>75>75192.25 ± 51.41---
5118.01 ± 2.78>7523.11 ± 0.0420.47 ± 2.461.14-0.88
52>75>7560.21 ± 6.94n/an/an/an/a
5317.12 ± 0.4141.44 ± 1.7428.44 ± 1.97n/an/an/an/a
28115.44 ± 40.11206.95 ± 7.28135.95 ± 2.47n/an/an/an/a
5-FU19.17 ± 0.395.37 ± 1.167.38 ± 2.499.43 ± 0.800.491.761.28
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Grigoropoulou, S.; Manou, D.; Antoniou, A.I.; Tsirogianni, A.; Siciliano, C.; Theocharis, A.D.; Athanassopoulos, C.M. Synthesis and Antiproliferative Activity of Novel Dehydroabietic Acid-Chalcone Hybrids. Molecules 2022, 27, 3623. https://doi.org/10.3390/molecules27113623

AMA Style

Grigoropoulou S, Manou D, Antoniou AI, Tsirogianni A, Siciliano C, Theocharis AD, Athanassopoulos CM. Synthesis and Antiproliferative Activity of Novel Dehydroabietic Acid-Chalcone Hybrids. Molecules. 2022; 27(11):3623. https://doi.org/10.3390/molecules27113623

Chicago/Turabian Style

Grigoropoulou, Sophia, Dimitra Manou, Antonia I. Antoniou, Artemis Tsirogianni, Carlo Siciliano, Achilleas D. Theocharis, and Constantinos M. Athanassopoulos. 2022. "Synthesis and Antiproliferative Activity of Novel Dehydroabietic Acid-Chalcone Hybrids" Molecules 27, no. 11: 3623. https://doi.org/10.3390/molecules27113623

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