Oxidized Juncuenin B Analogues with Increased Antiproliferative Activity on Human Adherent Cell Lines: Semisynthesis and Biological Evaluation

Abstract

Phenanthrenes have become the subject of intensive research during the past decades because of their structural diversity and wide range of pharmacological activities. Earlier studies demonstrated that semisynthetic derivatization of these natural compounds could result in more active agents, and oxidative transformations are particularly promising in this regard. In our work, a natural phenanthrene, juncuenin B, was transformed by hypervalent iodine(III) reagents using a diversity-oriented approach. Eleven racemic semisynthetic compounds were produced, the majority containing an alkyl substituted p-quinol ring. Purification of the compounds was carried out by chromatographic techniques, and their structures were elucidated by 1D and 2D NMR spectroscopic methods. Stereoisomers of the bioactive derivatives were separated by chiral-phase HPLC and the absolute configurations of the active compounds, 2,6-dioxo-1,8a-dimethoxy-1,7-dimethyl-8-vinyl-9,10-dihydrophenanthrenes (1a–d), and 8a-ethoxy-1,7-dimethyl-6-oxo-8-vinyl-9,10-dihydrophenanthrene-2-ols (7a,b) were determined by ECD measurements and TDDFT-ECD calculations. The antiproliferative activities of the compounds were tested on different (MCF-7, T47D, HeLa, SiHa, C33A, A2780) human gynecological cancer cell lines. Compounds 1a–d, 4a, 6a, and 7a possessed higher activity than juncuenin B on several tumor cell lines. The structure–activity relationship studies suggested that the p-quinol (2,5-cyclohexadien-4-hydroxy-1-one) moiety has a considerable effect on the antiproliferative properties, and substantial differences could be identified in the activities of the stereoisomers.

Phenanthrenes are a rather uncommon class of aromatic secondary metabolites and have a limited occurrence in nature. They are presumably formed by oxidative coupling of the aromatic rings of stilbene precursors.¹ Hitherto, more than 460 phenanthrenes, among them mono-, di-, and triphenanthrenes, were identified from different plant families, e.g., Orchidaceae, Juncaceae, Combretaceae, and Dioscoreaceae.² A large number of phenanthrenes with different substitution patterns have been reported with various pharmacological activities, such as antimicrobial, anti-inflammatory, cytotoxic, antiproliferative, spasmolytic, antiallergic, and anxiolytic effects.^2,3 The most thoroughly investigated denbinobin, a phenanthrenequinone, showed promising antiproliferative activity.⁴⁻⁶ The mechanism of action of denbinobin involves caspase-dependent and caspase-independent apoptosis of colon cancer (COLO 205) cells.⁴ Moreover, by enhancing the synthesis of reactive oxygen species (ROS), denbinobin induces apoptosis in Jurkat leukemia and human lung adenocarcinoma (A549) cells.^5,7 A structure–activity relationship study showed that the quinone substructure is required for enhancement of mitogen-activated protein kinase (MAPK)-independent ROS production activity.⁵ Another phenanthrenequinone, calanquinone A, differing from denbinobin by the presence of an additional methoxy group, and synthetic phenanthrenequinones also possessed cytotoxic activity on different cell lines (HepG2, Hep3B, Ca9-22, A549, MCF-7, MDA-MB-231, MRC-5). Structure–activity relationship studies showed that intramolecular hydrogen bonding between the C-4 carbonyl and HO-5 in 3-methoxyphenanthrene-1,4-diones may be necessary for the cytotoxicity.⁶

The phenanthrene dehydroeffusol showed activity against two metastatic cancer cell lines, SGC-7901 (human gastric carcinoma) and AGS (human caucasian gastric adenocarcinoma) in a dose-dependent manner (IC₅₀ 35.9 and 32.9 μM for SGC-7901 and AGS cells, respectively). Gastric cancer cell mediated vasculogenic mimicry on SGC-7901 cells were also inhibited by this compound using the tube formation assay.⁸ Dehydroeffusol effectively inhibited the gastric cancer cell invasion and migration without exerting significant acute toxicity.⁹ Juncusol was observed to be cytotoxic on HeLa cells; treatment for 24 h increased the cell population in the G2/M and sub-G1 phases.¹⁰ Juncuenin B possessed significant antiproliferative activity against different human tumor cell lines such as MDA-MB-231 (breast cancer), HeLa (cervical cancer), and A2780 (ovarian cancer) with IC₅₀ values 9.4, 2.9, and 7.3 μM, respectively.¹⁰

The aim of this work was the semisynthetic modification of juncuenin B (1,7-dimethyl-8-vinyl-9,10-dihydrophenanthrene-2,6-diol) to prepare a variety of structurally related compounds with higher antiproliferative activities. The experimental approach was inspired by a 3-fold of reasons. First, the free radical scavenging-related, oxidized metabolites of phenolic compounds represent a chemical space particularly rich in bioactive compounds.¹¹ Second, we have recently demonstrated that the hypervalent iodine(III) reagent [bis(trifluoroacetoxy)iodo]benzene (PIFA) may be efficiently used to obtain product mixtures rich in such oxidized scaffolds.¹² Third, the p-quinol moiety, anticipated from a PIFA-catalyzed oxidation of p-phenols, is the pharmacophore of several potent inhibitors of ATR-dependent signaling,^12,13 a crucial mechanism of DNA damage response that is an emerging antitumor target and a subject of many ongoing clinical studies.¹⁴ Therefore, we followed a diversity-oriented semisynthetic approach using two hypervalent iodine reagents, PIFA, and (diacetoxyiodo)benzene (PIDA) to obtain oxidized derivatives of juncuenin B. Furthermore, various alcohols (MeOH, EtOH, and n-BuOH) were used as solvents to ensure formation of a set of differently substituted derivatives. Eleven chiral oxidation products (1–11), representing a wide structural diversity, were obtained in this way as racemates or stereoisomeric mixtures. Stereoisomers of the bioactive derivatives were resolved by chiral-phase HPLC and absolute configurations were assigned by ECD measurements and TDDFT-ECD calculations. The antiproliferative activity of the racemic compounds and separated optically active stereoisomers was investigated on MCF-7, T47D, HeLa, SiHa, C33A, and A2780 cell lines, which enabled us to establish both structure–activity and stereochemistry–activity relationships. In order to gain insight into their tumor selectivity, the compounds have also been tested on the NIH/3T3 cell line.

Results and Discussion

Eleven racemic and enantiomerically pure chiral semisynthetic derivatives (1–11) (Scheme 1 and Figure 1) have been prepared from juncuenin B, a naturally occurring vinyl-substituted achiral dihydrophenanthrene, in five transformations (I–V) by hypervalent iodine(III) reagents using a diversity-oriented approach. PIFA and PIDA were used as oxidants (PIFA in processes I–IV, PIDA in process V), under different conditions, in MeCN–MeOH (process I), MeOH (II), EtOH (III), n-BuOH (IV, V). PIFA and PIDA are well-known reagents for the oxidation of phenols leading to the formation of quinone-type products. This takes place through an aryloxyiodonium(III) intermediate forming a phenoxenium ion that undergoes a nucleophilic attack,¹⁵ but PIFA and PIDA may also oxidize aromatic compounds via a single-electron transfer mechanism.¹⁶ Following the oxidation, the mixture of products were subjected to solid-phase extraction on silica to remove the remaining oxidizing agent and the oxidation side-products. The purification process was followed by MPLC and HPLC.

Scheme 1. Transformations of Juncuenin B under Different Conditions.

Figure 1.

Structures of the prepared compounds 1–11.

During the reaction processes I–V, racemic mixtures of the compounds were formed, all of them bearing p- or o-quinol rings, substituted with methoxy-, ethoxy-, and n-butoxy groups based on the solvent used. In the case of reaction IV, only compound 10 could be isolated. It did not show structural analogy with the aforementioned compounds; therefore, the reaction was performed with PIDA under the same conditions (process V). This reaction yielded 11, analogous to the compounds derived from 4 and 7. As a result of chromatographic purifications, compounds with one stereogenic center were obtained as racemates (3, 4, 7–11), while compounds with two stereogenic centers were isolated as diastereomeric mixtures of two racemates (1a+1d, 1b+1c, 2a, 2b, 6a, 6b). Separations of stereoisomers were performed using chiral-phase HPLC when the mixtures of stereoisomers or the racemates (1a–d, 4a,b, 7a,b) were found effective in the antiproliferative assay. This was done in order to compare the antiproliferative activity of the pure enantiomers and identify not only 2D structure–activity but also stereochemistry–activity relationships. The separation of the stereoisomers also enabled the characterization and determination of the absolute configurations of the bioactive derivatives by comparison of the experimental and TDDFT-calculated ECD spectra.

The structure determination was carried out by extensive spectroscopic analysis, using 1D and 2D NMR (¹H–¹H COSY, HSQC, and HMBC) spectroscopy and ECD data analysis.

In the NMR spectra of 1a–1d, two sets of signals could be identified that were attributed to two racemic diastereomers 1a+1d and 1b+1c (ratio 1:1). The ¹H NMR spectrum of the 1a–1d mixture showed the presence of a pair of isomeric compounds (1a+1d and 1b+1c) as some signals were duplicated. Since these compounds are derived from juncuenin B, similar signals were found in the ¹H NMR spectrum: signals of two o-coupled aromatic protons (δ_H 6.24, d and 7.23, d/7.25, d), an aromatic proton singlet (δ_H 6.47/6.49), two methyls (δ_H 1.46, s and 2.08, s), a vinylic system at δ_H 6.58, dd, 5.83, dd, and 5.69, dd (H-13, H₂-14), and two methylene groups (δ_H 2.63, m, 1.68, m/1.67, m and δ_H 2.83, m, 2.57, dd). Two methoxy groups (δ_H 3.07, s and 2.92/2.93, s) were also detected in the ¹H NMR spectrum (Table 1/1a+d and 1b+c). The presence of two methylene signals (H₂-9, H₂-10) indicated this isomeric pair to be 9,10-dihydrophenanthrene derivatives. In the JMOD (J-modulated spin–echo experiment) spectrum, 20 carbon signals were displayed (Table 2/1a+d and 1b+c). In the ¹H–¹H COSY spectrum, correlations were observed between δ_H 6.24, d and δ_H 7.23/7.25, d (H-3–H-4), between δ_H 2.63, m, 1.68/1.67, m and δ_H 2.83, m, 2.57, dd (H₂-9–H₂-10), and between δ_H 5.83, brd/5.69, dd and δ_H 6.58, dd (H-13–H₂-14) (Figure 2). The location of the methoxy groups was concluded from the HMBC spectra, as proton signal at δ_H 3.07 (CH₃O-1) showed correlations with δ_C 83.8 (C-1), whereas the signal at δ_H 2.92/2.93 (CH₃O-8a) correlated with δ_C 73.2 (C-8a) (Figure 2). Thus, the C-2 and C-6 hydroxy groups of juncuenin B were oxidized to carbonyl groups (δ_C 202.6 and 185.9, respectively) and methoxy-substituted dienone moieties were formed.

Table 1. ¹H NMR (500 MHz) Data for Compounds 1–11^a.

	δH (J, Hz)
position	1a+d	1b+c	2a	2b	3	4a+b	5	6a	6b	7a+b	8	9	10	11
3	6.24, d (10.2)	6.23, d (10.2)	6.45, d (9.9)	6.52, d (10.2)	6.30, d (10.6)	6.74, d (8.4)	6.40, d (9.9)	6.23, d (10.1)	6.21, d (10.1)	6.72, d (8.4)	6.32, d (10.4)	6.73, d (8.3)	6.21, d (10.6)	6.72, d (8.4)
4	7.23, d (10.2)/7.25, d (10.2)b	7.23, d (10.2)/7.25, d (10.2)b	6.61, d (9.9)	6.89, d (10.2)	9.06, d (10.6)	7.34, d (8.4)	6.66, d (9.9)	7.21, d (10.1)	7.21, d (10.1)	7.3, d (8.4)	8.18, d (10.4)	7.45, d (8.3)	8.71, d (10.6)	7.30, d (8.4)
5	6.47, s/6.49, sb	6.47, s/6.49, sb	6.84, s	6.45, s	–	6.58, s	6.82, s	6.43, s	6.45, s	6.54, s	7.49, s	6.42, s	–	6.55, s
9	1.68, m/1.67, m, 2.63, mb	1.68, m/1.67, m, 2.63, mb	1.92, m, 2.45, m	1.43, m, 2.59, m	8.19, d (8.8)	1.76, ddd (14.4, 11.3, 6.5), 2.69, dd (14.4, 5.7)	1.93, ddd (13.8, 10.0, 4.3), 2.47, m	1.62, m, 2.62, dd (14.0, 5.5)	1.66, m, 2.63, dd (13.9, 5.9)	1.74, ddd (13.9, 11.7, 6.5), 2.68, dd (14.0, 6.0)	8.15, d (8.8)	2.71, mb	7.62, d (8.3)	1.74, ddd (14.1, 11.6, 6.4), 2.68, dd (14.1, 5.8)
10	2.57, dd (20.2, 5.9), 2.83, m	2.50, dd (20.3, 6.0), 2.85, m	2.35, m, 2.83, m	2.67, m, 2.89, m	7.60, d (8.8)	2.75, dd (17.3, 6.5), 3.06, m	2.39, m, 2.87, ddd (13.8, 9.0, 4.3)	2.53, dd (14.4, 5.9), 2.88, ddd (16.5, 10.6, 5.5)	2.51, dd (20.4, 6.0), 2.86, ddd (20.2, 10.7, 6.0)	3.09, m, 2.74, dd (17.3, 6.2)	7.61, d (8.8)	2.84, mb	7.66, d (8.3)	2.73, dd (17.3, 6.4), 3.09, m
11	1.46, s	1.42, s	1.97, s	1.91, s	1.58, s	2.16, s	1.96, s	1.46, s	1.43, s	2.17, s	1.56, s	2.22, s	1.52, s	2.16, s
12	2.08, s	2.08, s	2.02, s	2.00, s	2.42, s	2.09, s	2.01, s	2.07, s	2.07, s	2.09, s	2.42, s	1.49, s	2.04, s	2.08, s
13	6.58, dd (17.8, 12.1)	6.58, dd (17.8, 12.1)	6.50, dd (17.9, 12.0)	6.51, dd (17.9, 11.9)	6.98, dd (17.9, 11.4)	6.63, dd (17.9, 11.9)	6.50, dd (17.8, 11.9)	6.61, dd (17.9, 11.9)	6.61, dd (17.9, 12.0)	6.65, dd (17.9, 11.9)	7.02, dd (17.7, 11.4)	6.67, dd (17.8, 11.8)	6.62, dd (17.9, 11.8)	6.64, dd (17.9, 11.9)
14	5.69, dd (12.1, 1.5), 5.83, dd (17.8, 1.5)	5.69, dd (12.1, 1.5), 5.83, dd (17.8, 1.5)	5.63, dd (12.0, 1.4), 5.78, dd (17.9, 1.4)	5.67, dd (11.9, 1.5), 5.70, dd (17.9, 1.4)	5.38, dd (17.9, 1.7), 5.82, dd (11.4, 1.7)	5,66, dd (11.9, 1.7), 5.85, dd (17.9, 1.7)	5.62, dd (11.9, 1.3), 5.80, dd (17.8, 1.3)	5.68, dd (11.9, 1.3), 5.87, dd (17.9, 1.3)	5.68, dd (12.0, 1.5), 5.89, dd (17.9, 1.5)	5.65, dd (11.9, 1.5), 5.89, dd (17.9, 1.5)	5.41, dd (17.7. 1.6), 5.84, dd (11.4, 1.6)	5.55, dd (11.8, 1.3), 5.87, dd (17.8, 1.3)	5.45, dd (17.9, 1.0), 5.82, d (11.8)	5.65, dd (11.9, 1.7), 5.89, dd (17.9, 1.7)

^aMeasured in CDCl₃.

^bInterchangeable. 1a+d: CH₃O-1: 3.07, s; CH₃O-8a: 2.92, s/2.93, s (interchangeable with 1b); 1b+c: CH₃O-1: 3.12, s; CH₃O-8a: 2.92, s/2.93,s (interchangeable with 1a); 2a: CH₃O-4a: 3.04, s; CH₃O-8a: 2.72, s; 2b: CH₃O-4a: 3.10, s; CH₃O-8a: 3.14, s; 3: CH₃O-1: 3.08, s; CH₃O-5: 3.79, s; 4a+b: CH₃O-8a: 2.85, s; 5: C₂H₅O-4a (interchangeable with 8a): 2.76 dq (7.0, 14.1), 2.96, dq (7.0, 14.1) (CH₂), 0.80, t (7.0) (CH₃); C₂H₅O-8a (interchangeable with 4a): 3.08 dq (7.0, 14.1), 3.29, dq (7.0, 14.1) (CH₂), 1.11, t (7.0) (CH₃); 6a: C₂H₅O-1 (interchangeable with 8a): 2.93, m, 3.36 dq (14.3, 7.1) (CH₂), 1.21, t (7.0) (CH₃); C₂H₅O-8a (interchangeable with 1): 2.91, m, 3.19 dq (14.3, 7.1) (CH₂), 1.01, t (7.0) (CH₃); 6b: C₂H₅O-1:3.10, dq (14.2, 7.0), 3.34, dq (14.3, 7.1) (CH₂), 1.23, t (7.0) (CH₃); C₂H₅O-8a: 2.93, dq (14.2, 7.0), 3.17, dq (14.3, 7.1) (CH₂), 0.97, t (7.0) (CH₃); 7a+b: C₂H₅O-8a: 2.92, m, 3.11, m (CH₂), 0.86, t (7.0) (CH₃); 8: C₂H₅O-1: 3.00 dq (14.2, 7.0), 3.28 dq (14.2, 7.0) (CH₂), 1.20, t (7.0) (CH₃); 9: C₂H₅O-7: 3.13, dq (13.9, 7.1), 3.26, dq (14.0, 7.1) (CH₂), 1.17, t (7.0) (CH₃); 10: CH₃-(CH₂)₃-O-1: 3.22, m, 2.92, m (1′-CH₂), 1.58, m (2′-CH₂), 1.28–1.40, m (3′-CH₂), 0.87, t (7.4) (4′-CH₃); CH₃-(CH₂)₃-O-5: 3.14 m, 3.54, m (1″-CH₂), 1.47, m (2″-CH₂), 1.28–1.40, m (3″-CH₂), 0. 81, t (7.5)/0.83, t (7.5) (4″-CH₃) (interchangeable with 4‴-CH₃); 3.10, m, 3.45, m (1‴-CH₂), 1.47, m (2‴-CH₂), 1.28–1.40, m (3‴-CH₂), 0. 81, t (7.5)/0.83, t (7.5) (4‴-CH₃) (interchangeable with 4″-CH₃); 11: CH₃-(CH₂)₃-O-8a: 2.83, m, 3.07, m (1′-CH₂), 1.13–1.23, m (2′-CH₂), 0.86–1.03, m (3′-CH₂), 0.62, t (7.4) (4′-CH₃).

Table 2. ¹³C NMR (125 MHz) Data for Compounds 1–11^a.

	δc, type
position	1a+d	1b+c	2a	2b	3	4a+b	5	6a	6b	7a+b	8	9	10	11
1	83.8, C	83.1, C	133.0, C	132.8, C	83.5, C	122.0, C	132.5, C	83.2, C	82.4, C	121.8, C	82.5, C	121.9, C	81.9, C	121.8, C
1a	157.2, C	157.0, C	150.9, C	154.4, C	145.1, C	133.9, C	152.0, C	157.9, C	157.5, C	139.1, C	144.2, C	141.0, C	147.3, C	139.2, C
2	202.6, C	202.4, C	185.4, C	184.7, C	202.4, C	155.8, CH	185.3, C	202.7, C	202.5, C	155.5, C	202.9, C	156.3, C	201.4, C	155.5, C
3	125.9, CH	125.9, CH	131.9, CH	131.8, CH	124.1, CH	113.8, CH	131.2, CH	125.8, CH	125.8, CH	113.7, CH	124.6, CH	114.2, CH	125.1, CH	113.6, CH
4	139.7, CH	139.7, CH	144.8, CH	146.5, CH	143. 6, CH	124.6, CH	145.6, CH	139.6, CH	139.6, CH	124.5, CH	139.8, CH	125.7, CH	142.7, CH	124.6, CH
4a	125.4, C	125.4, C	73.1, C	77.3, C	123.7, C	125.2, C	73.0, C	125.0, C	125.0, C	125.5, C	123.7, C	126.0, C	131.0, C	125.5, C
5	123.1, CH	123.1, CH	131.0, CH	128.8, CH	139.9, C	122.2, CH	130.6, CH	122.6, CH	122.6, CH	121.8, CH	103.3, CH	117.3, CH	94.6, C	121.7, CH
5a	151.9, C	151.9, C	159.6, C	156.8, C	122.9, C	156.7, C	160.5, C	152.7, C	152.9, C	157.7, C	130.7, C	150.6, C	136.1, C	157.8, C
6	185.9, C	185.9, C	186.2, C	186.1, C	147.8, C	186.5, C	186.3, C	185.9, C	186.0, C	186.6, C	154.0, C	202.6, C	194.4, C	186.7, C
7	135.8, C	135.8, C	134.1, C	133.9, C	125.2, C	135.7, C	133.8, C	135.2, C	135.3, C	135.1, C	124.4, C	82.1, C	130.4, C	135.1, C
8	149.6, C	149.6, C	150.2, C	153.6, C	134.1, C	149.6, C	150.7, C	150.2, C	150.4, C	150.6, C	138.2, C	145.9, C	147.9, C	150.5, C
8a	73.2, C	73.2, C	75.1, C	76.7, C	127.9, C	73.8, C	74.5, C	72.7, C	72.7, C	73.4, C	127.3, C	131.6, C	132.6, C	73.2, C
9	33.1/34.0, CH2b	33.1/34.0, CH2b	34.3, CH2	41.1, CH2	129.7, CH	34.5, CH2	34.1, CH2	33.2, CH2	34.2, CH2	34.8, CH2	129.0, CH	25.8/26.6, CH2b	129.0, CH	34.8, CH2
10	21.8, CH2	22.4, CH2	22.8, CH2	22.7, CH2	121.3, CH	23.9, CH2	22.9, CH2	21.9, CH2	22.5, CH2	23.9, CH2	121.0, CH	25.8/26.6, CH2b	127.0, CH	23.9, CH2
11	28.0, CH3	26.6, CH3	10.7, CH3	10.9, CH3	31.1, CH3	11.2, CH3	10.7, CH3	27.2, CH3	26.7, CH3	11.2, CH3	30.9, CH3	11.3, CH3	31.3, CH3	11.2, CH3
12	12.3, CH3	12.3, CH3	12.0, CH3	12.3, CH3	13.9, CH3	12.3, CH3	12.0, CH3	12.2, CH3	12.3, CH3	12.3, CH3	13.6, CH3	28.2, CH3	13.0, CH3	12.3, CH3
13	131.7, CH	131.7, CH	131.6, CH	132.0, CH	133.9, CH	132.1, CH	131.8, CH	131.9, CH	131.8, CH	132.2, CH	134.0, CH	131.2, CH	132.9, CH	132.2, CH
14	123.9, CH2	123.9, CH2	124.0, CH2	124.0, CH2	122.5, CH2	123.3, CH2	123.8, CH2	123.7, CH2	123.8, CH2	123.3, CH2	122.4, CH2	121.7, CH2	123.2, CH2	123.3, CH2

^aMeasured in CDCl₃.

^bInterchangeable. 1a+d: CH₃O-1: 51.7; CH₃O-8a: 54.7; 1b+c: CH₃O-1: 51.7; CH₃O-8a: 54.5; 2a: CH₃O-4a: 51.1; CH₃O-8a: 52.5; 2b: CH₃O-4a: 51.6; CH₃O-8a: 52.9; 7: CH₃O-1: 54.4; CH₃O-5: 61.1; 4a+b: CH₃O-8a: 51.6; 5: C₂H₅O-4a (interchangeable with 8a): 60.2, CH₂, 14.7, CH₃; C₂H₅O-8a (interchangeable with 4a): 59.0, CH₂, 15.8, CH₃; 6a: C₂H₅O-1 (interchangeable with 8a): 62.5, CH₂, 15.7, CH₃; C₂H₅O-8a (interchangeable with 1): 59.6, CH₂, 15.5, CH₃; 6b: C₂H₅O-1: 62.2, CH₂, 15.8, CH₃; C₂H₅O-8a: 59.6, CH₂, 15.6, CH₃; 7a+b: C₂H₅O-8a: 59.5, CH₂, 15.4 CH₃; 8: C₂H₅O-1: 62.2, CH₂, 15.8, CH₃; 9: C₂H₅O-7: 61.6, CH₂, 15.6, CH₃; 10: CH₃-(CH₂)₃-O-7: 66.3 (1′-CH₂), 32.4 (2′-CH₂), 19.2/19.3/19.4 (3′-CH₂) (interchangeable with 3″-CH₂ and 3‴-CH₂), 14.1 (4′-CH₃); CH₃-(CH₂)₃-O-5: 63.3 (1″-CH₂), 31.8/31.9 (2″-CH₂) (interchangeable with 2‴-CH₂), 19.2/19.3/19.4 (3″-CH₂) (interchangeable with 3′-CH₂ and 3‴-CH₂), 13.8/13.9 (4″-CH₂) (interchangeable with 4‴-CH₃); 63.6 (1‴-CH₂), 31.8/31.9 (2‴-CH₂) (interchangeable with 2″-CH₂), 19.2/19.3/19.4 (3‴-CH₂) (interchangeable with 3′-CH₂ and 3″-CH₂), 13.8/13.9 (4‴-CH₂) (interchangeable with 4″-CH₃); 11: CH₃-(CH₂)₃-O-8a: 63.5 (1′-CH₂), 31.9 (2′-CH₂), 19.1 (3′-CH₂), 13.7 (4′-CH₃).

Figure 2.

Diagnostic COSY (−) and HMBC (H → C) correlations of compounds 1a–d.

All of the above evidence confirmed the 2D structures of the 1a–1d isomers (Figure 2).

The NMR spectra of the mixture of 1b+1c were similar to those of 1a+1d, only small differences were observed for the CH₃O-1, C-10, and C-11 resonances of 1a+1d and 1b+1c, which suggested that these compounds are structurally related to each other. Some ¹H and ¹³C NMR signals were duplicated in the spectra of 1b+1c, revealing the presence of a diastereomeric pair as for 1a+1d.

After NMR studies the stereoisomers 1a–1d were separated on a Lux amylose-1 chiral-phase column [t_R = 13.6 min (1a), 17.4 min (1b), 21.6 min (1c), and 23.6 min (1d)]. Experimental ECD spectra indicated mirror-image relationships between 1a/1d, and 1b/1c, respectively, verifying their enantiomeric relationships (Figure 3).

Figure 3.

(a) Experimental ECD spectra of 1a–1d in MeCN and (b) experimental UV spectra of 1a/1d and 1b/1c in MeCN.

In order to elucidate the absolute configurations of 1a–d, the solution TDDFT-ECD method was applied to (1R,8aR)-1 and (1R,8aS)-1.¹⁷ The initial Merck Molecular Force Field (MMFF) conformational search resulted in nine conformers for each in a 21 kJ/mol energy window, respectively. These geometries were reoptimized at the ωB97X/TZVP PCM/MeCN level¹⁸ resulting in three conformers for both stereoisomers (Figures S8 and S9, Supporting Information). ECD spectra were computed at various levels and compared to the experimental spectra of 1a–1d. There were only minor differences in the experimental ECD spectra of 1a versus 1b and 1c versus 1d, and the computed ECDs are mainly influenced by one of the two chirality centers. Boltzmann-averaged ECD data of both (1R,8aR)-1 and (1R,8aS)-1 gave acceptable to good agreement with the experimental data of 1a and 1b with the C-1 stereogenic center definitively influencing the ECD profile (Figure 4). Thus, the absolute configuration of this stereocenter could be determined as (1R) for the first- and second-eluted stereoisomers 1a and 1b and (1S) for the third- and fourth-eluting 1c and 1d. The shape of the B3LYP/TZVP PCM/MeCN and PBE0/TZVP PCM/MeCN ECD spectra of (1R,8aR)-1 reproduced the experimental ECD curve of 1a better, while those of (1R,8aS)-1 were similar to the experimental spectrum of 1b (Figure 5). The better agreements allowed tentative assignment (8aR) for 1a and (8aS) for 1b. DFT-NMR calculations of the B3LYP/6-31+G(d,p) conformers of (1R,8aR)-1 and (1R,8aS)-1 at the mPW1PW91/6-311+G(2d,p) level¹⁹ could not differentiate the epimers due to the small chemical shift differences of the experimental values.²⁰

Figure 4.

Experimental ECD spectrum of 1a in MeCN (black line) compared with the calculated PBE0/TZVP PCM/MeCN spectrum of (1R,8aR)-1 (purple line). Level of DFT optimization: ωB97X/TZVP PCM/MeCN. Bars represent the rotational strengths of conformer A.

Figure 5.

Experimental ECD spectrum of 1b in MeCN (black line) compared with the calculated B3LYP/TZVP PCM/MeCN spectrum of (1R,8aS)-1 (red line). Level of DFT optimization: ωB97X/TZVP PCM/MeCN. Bars represent the rotational strengths of conformer A.

The ¹H NMR spectrum of compound 2a showed signals characteristic of juncuenin B analogues: signals of two o-coupled aromatic protons (δ_H 6.45, d and 6.61, d), one aromatic proton singlet (δ_H 6.84), two methyl singlets (δ_H 1.97 and 2.02), a vinyl group at δ_H 6.50, dd, 5.78, dd and 5.63, dd, two methylene groups (δ_H 1.92, m, 2.45, m, and δ_H 2.35, m, 2.83, m), and two methoxy singlets (δ_H 2.72 and 3.04), respectively (Table 1). In the JMOD spectrum, signals of 20 carbon atoms were detected (Table 2). In the ¹H–¹H COSY spectrum, correlations between δ_H 6.45, d and 6.61, d (H-3–H-4), between δ_H 2.35, m, 2.83, m, and δ_H 1.92, m, 2.45, m (H-9–H-10), and between δ_H 6.50, dd, and 5.78, dd, 5.63, dd (H-13–H₂-14) were observed. The position of the methyl groups were determined based on the HMBC correlations of the methyl protons at δ_H 1.97 and 2.02 with C-1 (δ_C 133.0) and C-7 (δ_C 134.1), respectively. Methoxy groups attached at C-4a and C-8a were positioned by means of the HMBC interactions between δ_H 3.04 (CH₃O-4a) and δ_C 73.1 (C-4a), and between δ_H 2.72 (CH₃O-8a) and δ_C 75.1 (C-8a). The vinylic system was connected to C-8 via cross peaks between δ_H 5.63, dd, 5.78, dd and δ_C 150.2.

All NMR characteristics of compound 2b were similar to compound 2a, indicating that they are diastereomers. The main differences between 2b and 2a were found between the ¹³C NMR chemical shifts of C-4a and 8a and adjacent carbons, suggesting that 2b and 2a differ in the configurations of C-4a and 8a.

In the case of racemic compound 3, the signals of four o-coupled aromatic protons [(δ_H 6.30, d and 9.06, d (H-3–H-4), δ_H 8.19, d and 7.60, d (H-9–H-10)], two methyls (δ_H 1.58, s and 2.42, s), a vinyl group (δ_H 6.98, dd, 5.82, dd and 5.38, dd, H-13 and H₂-14), and two methoxy groups (δ_H 3.08, s, and 3.79, s) were detected in the ¹H NMR data (Table 1). The differences between juncuenin B and compound 3 involved the presence of an aromatic ring B, the oxidation of the phenolic group at C-2 to a carbonyl moiety (δ_C 202.4), and the addition of methoxy groups at C-1 and C-5.

The ¹H NMR spectrum of the racemic mixture of 4a and 4b differ from juncuenin B in the presence of a methoxy group (δ_H 2.85) (Table 1) and the carbonyl group at ring C. In the JMOD spectrum 19 carbon signals were observed (Table 2). The methoxy group was located at C-8a, as the aforementioned CH₃O proton signal showed HMBC correlation with the carbon signal resonating at δ_C 73.8. Following the NMR studies the enantiomers 4a and 4b were separated on a Lux amylose-1 chiral-phase HPLC column [t_R = 5.5 min (4a) and 10.3 min (4b)].

The 1D NMR data of compound 5 were found to be similar to those of 2a and 2b, except for the presence of two ethoxy groups in the molecule [δ_H 2.96, dq/3.29, dq, 2.76 dq/3.08, dq (CH₂) and 0.80, t/1.11, t (CH₃); δ_C 59.0/60.2 (CH₂), 14.7/15.8 (CH₃)]. Relevant HMBC interactions suggested the side chains to be attached to the phenanthrene skeleton at C-4a and C-8a (Tables 1 and 2).

Compounds 6a and 6b were obtained as racemic diastereomers. The difference between compounds 1a–d and compounds 6a and 6b involved the replacement of the C-1 and C-8a methoxy groups with ethoxy substituents (Tables 1 and 2).

Compounds 7a and 7b differ from compounds 4a and 4b in the presence of an ethoxy group [δ_H 2.92, m, 3.11, m (CH₂), 0.86, t (CH₃); δ_C 59.5 (CH₂), 15.4 (CH₃)] at C-8a instead of a methoxy group (Tables 1 and 2). Compounds 7a and 7b, similar to 1a–d and 4–6, also contain a carbonyl moiety at C-6 (δ_C 186.6). Following the NMR analysis, the enantiomers 7a and 7b were separated by HPLC on a Lux amylose-1 chiral-phase column [t_R = 4.7 min (7a) and 9.1 min (7b)] and mirror-image ECD spectra were recorded (Figure 6).

Figure 6.

Experimental ECD spectra of 7a and 7b in MeCN.

For the configurational assignments of 7a and 7b, the above-mentioned TDDFT-ECD protocol was applied to (R)-7 and also on the truncated methoxy model compound (R)-4.

DFT reoptimization of the initial six MMFF conformers of both (R)-4 and (R)-7 yielded six conformers above 1% population indicating that the ethoxy group does not have considerable freedom (Figure S64, Supporting Information). The Boltzmann-weighted ECD spectra of both sets of conformers reproduced well the experimental ECD spectrum of the first-eluting enantiomer 7a (Figure 7 and Figure S37, Supporting Information). Consequently, the absolute configurations of 7a and 4a, which were separated under the same conditions, could be elucidated as (R) and that of the second-eluting 7b and 4b as (S).

Figure 7.

Experimental ECD spectrum of 7a in MeCN (black line) compared with the calculated CAM-B3LYP/TZVP PCM/MeCN spectrum of (R)-7a (olive line). Level of DFT optimization: ωB97X/TZVP PCM/MeCN. Bars represent the rotational strengths of conformer A.

Interestingly, racemic compound 8 is the only one in which ring C is unchanged compared to the starting material, juncuenin B. An ethoxy group [δ_H 3.00, dq, 3.28, dq (CH₂), 1.20, t (CH₃); δ_C 62.2 (CH₂), 15.8 (CH₃)] is attached at C-1, and the phenolic group at C-2 of juncuenin B was oxidized to a carbonyl moiety (δ_C 202.9). Moreover, 8 is an unsaturated compound containing a Δ^9,10 double bond (δ_H 8.15, d and 7.61, d, H-9–H-10).

Compound 9 is a unique derivative because an ethoxy group [δ_H 3.13, dq, 3.26, dq (CH₂), 1.17, t (CH₃); δ_C 61.6 (CH₂), 15.6 (CH₃)] is attached to the phenanthrene skeleton at C-7, as suggested by spectroscopic data evidence. The HMBC correlation of H₃-12 (δ_H 1.49) with the oxygen-bearing C-7 at δ_C 82.1, as well as the relevant cross peaks H-5/C-7, H-13/C-7, H-5/C-8a, and H-13/C-8a indicated the presence of a Δ^8,8a double bond instead of a Δ^7,8 olefinic bond, to support the above conclusion.

In the case of compound 10, it was apparent from the 1D NMR spectra that three n-butoxy groups are connected to the phenanthrene skeleton. Both the phenolic substituents of juncuenin B were oxidized to carbonyl groups, and a Δ^9,10 double bond was also formed. The location of the methyl, vinyl, and n-butoxy groups were determined by their diagnostic HMBC correlations. Three-bond heteronuclear interactions of C-1 (δ_C 81.9) with the oxymethylene protons at δ_H 2.92, m, and of the deshielded C-5 (δ_C 94.6) with two additional oxymethylenes at δ_H 3.14, m, and 3.54, m confirmed the positions of the n-butoxy groups at C-1 and C-5, respectively.

The ¹H NMR spectrum of the racemic compound 11 displayed signals attributable to an n-butoxy moiety [δ_H 2.83, m, 3.07, m (CH₂), 1.13–1.23, m (CH₂) 0.86–1.03, m (CH₂), and 0.62, t (CH₃); δ_C 63.5 (CH₂), 31.9 (CH₂), 19.1 (CH₂), and 13.7 (CH₃)]. Since the oxidative transformation was performed in a reaction medium containing n-BuOH as solvent, and the aromatic A-ring and HO-2 remained intact during the reaction, it was anticipated that the n-butoxy chain is situated at C-8a. This presumption was corroborated by a strong HMBC cross peak between the oxymethylene protons and C-8a.

The antiproliferative assays of the products were carried out by a standard MTT method on human breast (MCF-7, T47D), cervical (HeLa, SiHa, C33A), and ovarian (A2780) cancer cells, and NIH/3T3 mouse embryonic fibroblast cell lines. In the first step, the diastereomeric and racemic mixtures 1–11 and the starting compound, juncuenin B, were tested to obtain information about their antiproliferative activity. Among the tested samples, compounds 1a–d, 4a+4b, 6a, 7a+7b, and 11 exhibited significant tumor cell proliferation inhibitory activities with IC₅₀ values close to that of juncuenin B (Table 3, and Tables S2 and S3, Supporting Information). The IC₅₀ value of juncuenin B on MCF-7 cell line (11.7 μM) was determined for the first time. Similar to the structural points of view, it is evident that compounds without a p-quinol ring (3, 8, 9, and 10) and compounds with two p-quinol moieties (2a, 2b, 5) did not exert antiproliferative activity. Compounds substituted at C-4a with an alkoxy group were also inactive. Compounds 1a–d (IC₅₀s 11.6 μM on MCF-7, 15.0 μM on T47D, 15.6 μM on HeLa, 20.0 μM on SiHa, 6.7 μM on C33A, and 7.7 μM on A2780, respectively), 4a+4b (IC₅₀s 6.5 μM on MCF-7, 1.9 μM on HeLa, 7.0 μM on C33A, and 9.1 μM on A2780, respectively), and 7a+7b (IC₅₀s 6.2 μM on MCF-7, 16.9 μM on T47D, 5.4 μM on HeLa, 6.1 μM on C33A and 5.9 μM on A2780, respectively) with the highest activity were selected for further studies.

Table 3. IC₅₀ Values (μM, Mean ± SEM) of the Diastereomeric and/or Racemic Mixtures^a.

	calculated IC50 values (μM ± SEM)
compound	MCF-7	HeLa	C33A	A2780	NIH/3T3
juncuenin B	–	2.9 ± 0.5b	–	7.3 ± 1.3b	>30
1a–1d	–	–	6.7 ± 0.5	7.7 ± 1.0	17.7 ± 0.7
4a+4b	6.5 ± 1.1	1.9 ± 0.4	7.0 ± 1.8	9.1 ± 0.5	14.9 ± 3.7
7a+7b	6.2 ± 0.2	5.4 ± 0.1	6.1 ± 0.4	5.9 ± 1.1	>30
cisplatin	5.8	–	1.8	1.3b	2.7

^aCompounds 2a, 2b, 3, 5, 6b, 8, 9, and 10 were found to be inactive or moderately active (Table S1, Supporting Information). Determinations were performed using an MTT assay by treating the cells with compounds (0.1–30 μM) for 72 h. Data are based on two independent experiments.

^bIC₅₀ value from literature.¹⁰

In the second step, the enantiomerically pure compounds (1a, 1b, 1c, 1d, 4a, 4b, and 7b) were evaluated for their antiproliferative activity against the same cell lines (Table 4). Among these compounds, the highest activities were recorded for 1c, 4a, and 7a, reaching or exceeding that of the positive control cisplatin against HeLa and/or T47D cell lines. (R)-7a was found to be the most promising compound with substantial antiproliferative effects against all tested cell lines except for SiHa, but it was inactive against the nontumoral NIH/3T3 cells.

Table 4. IC₅₀ Values of the Enantiopure Compounds 1a–1d, 4a, 4b, 7a, and 7b^a.

	Calculated IC50 values (μM ± SEM)
compound	MCF-7	T47D	HeLa	C33A	A2780	NIH/3T3
(1R,8aS)-1b	–	–	–	–	9.0 ± 1.0	>30
(1S,8aR)-1c	–	–	–	5.3 ± 0.9	7.7 ± 0.6	21.6 ± 0.4
(8aR)-4a	6.2 ± 1.6	–	0.9 ± 0.4	5.3 ± 0.6	8.7 ± 0.8	25.8 ± 4.6
(8aR)-7a	–	8.8 ± 1.1	3.7 ± 0.9	4.9 ± 0.3	2.8 ± 0.3	>30
cisplatin	5.8	9.8b	–b	1.8	1.3b	2.7

^aDeterminations were performed using an MTT assay by treating the cells with compounds (0.1–30 μM) for 72 h. Data are based on two independent experiments.

^bIC₅₀ value from literature.¹⁰

Compounds 4a, 4b, 7a, 7b, and 11 contain a nonoxidized A ring and a C-ring comprising a p-quinol alkyl ether moiety. Moreover, 4a and 4b are substituted with a methoxy-, 7a, and 7b with an ethoxy-, and 11 with an n-butoxy group at C-8a, respectively. In the case of these compounds, the effect of the length of the ether chain at C-8a is undefined: on the T47D, HeLa, C33A, and A2780 cell lines, compound 7a with an ethoxy group, showed the highest inhibition, while its enantiomer (7b) had no activity. On the HeLa cell line, the antiproliferative activity was inversely proportional to the chain length of the alkoxy groups (IC₅₀ values 11 > 7a > 4a). Compounds 4a and 7a showed an antiproliferative effect on HeLa, C33A, MCF-7, and A2780 cells, which was comparable to or stronger than that of juncuenin B, while their stereoisomers 4b and 7b had no relevant influence on the cell proliferation, indicating that (8aR) is the preferred absolute configuration of these compounds. Regarding the pure enantiomers 1a–d, the (1S,8aR)-configuration (1c) was the most beneficial for the antiproliferative effect. The activities of (1S,8aR)-1c, (R)-4a, and (R)-7a suggest the preference of the (R) configuration for the antiproliferative activity. Since 1a is less active than 1c, the (1S) configuration seems to be also essential in the case of quinoidal compounds.

It is worth mentioning that several of the semisynthetic compounds demonstrated an altered cell line specificity as compared to their parent compound, juncuenin B. For example, while juncuenin B exerted approximately 2.5-fold stronger activity on MCF-7 than on T47D cells, a similar rate of selectivity in the opposite direction was observed for the enantiopure compound 7a. While both of these cell lines are estrogen receptor α positive and therefore estrogen-dependent, they show critical differences in their bioenergetic properties,²¹ as well as in apoptotic events including the activation of different caspases and mitochondrial changes.²² This selectivity inversion suggests that compounds obtained from juncuenin B were not only more potent than the starting material, but also these compounds likely act on different biochemical targets. Further studies are necessary for the clarification of these targets, but chemical analogies from our previous work suggest the involvement of ATR-dependent signaling at least to some extent.^12,13

In conclusion, oxidation of juncuenin B with hypervalent iodine reagents under different conditions resulted in the identification of phenanthrene derivatives substituted with hydroxy, methyl, methoxy, vinyl, ethoxy and n-butoxy groups. The majority of the components contain a substituted p-quinol ring. All compounds are reported here for the first time. Several compounds showed promising antiproliferative activities, and based on their structural analogy, structure–activity relationships were deduced. No significant activities were observed either for compounds without a substituted p-quinol ring (3, 8, 9, and 11), or for those containing two such rings (2a, 2b, and 5). However, derivatives containing one substituted p-quinol ring showed promising antiproliferative activities against different human cancer cell lines, and this was observed for compounds with an o-alkoxy-quinone moiety (1a–d, 6a) and for those with an intact A ring (4a, 7a, and 11). Clarifying the mechanism of action for these compounds requires further studies.

In the current work, similar structural elements were introduced in juncuenin B as present in denbinobin and calanquinone. The aromatic ring oxidations of juncuenin B afforded quinols which were found to be bioactive to the same degree as the lead compounds denbinobin and calanquinone. The starting compound juncuenin B can be readily isolated in a good yield (0.043% w/w) from Juncus inflexus root. There is a strong rationale for further development of oxidized derivatives of juncuenin B in the future.

Experimental Section

General Experimental Procedures

NMR spectra were recorded in methanol-d₄ and CDCl₃ on a Bruker Avance DRX 500 spectrometer at 500 MHz (¹H) and 125 MHz (¹³C). The signals of the deuterated solvents were used as references. The chemical shift values (δ) are given in ppm and coupling constants in Hz. 2D experiments were performed with standard Bruker software. In the ¹H–¹H COSY, HSQC, and HMBC experiments, gradient-enhanced versions were applied. The HRMS data were acquired on a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer equipped with ESI ion source in positive ionization mode. The data were acquired and processed with MassLynx software. ECD and UV spectra were recorded on a J-810 spectropolarimeter.

In the reaction processes [bis(trifluoroacetoxy)iodo]benzene (PIFA) and (diacetoxyiodo)benzene (PIDA) were used as oxidizing agents (Sigma-Aldrich, Stockholm, Sweden). To reaction progress and separation steps were monitored by TLC on Kieselgel 60F₂₅₄ silica plates obtained from Merck (Merck, Darmstadt, Germany), and examined under UV light at 254 and 366 nm. Solid phase extraction (SPE) was carried out on silica gel (Kieselgel 60 GF₂₅₄, 15 μm, Merck). MPLC was performed with a Combi Flash Rf⁺ Lumen instrument (Teledyne Isco). All solvents used for MPLC were of at least analytical grade (VWR Ltd., Szeged, Hungary). Both the reaction mixtures and processed fractions were investigated by HPLC on a Jasco instrument equipped with an MD-2010 Plus PDA detector (Jasco Analytical Instruments, Japan) in a detection range of 210–400 nm, with an Agilent Eclipse XDB C₈ column (4.6 × 150 mm, 5 μm) (Agilent Technologies, Inc., USA). MeCN–H₂O solvent system was used as eluent. The final purification steps were carried out by using HPLC with an Agilent Eclipse XDB C₈ (9.4 × 250 mm, 5 μm) (Agilent Technologies, Inc., USA) equipment, and MeCN–H₂O mixture as the mobile phase. Enantiomers were separated by using a Lux amylose-1 column (250 × 21.2 mm) (Phenomenex, USA), using a cyclohexane–isopropanol solvent system as mobile phase. The HPLC system for semipreparative separations comprised of a Waters 600 controller, Waters 600 pump, and Waters 2998 photodiode array detector. The data were acquired and processed with the Empower software.

The antiproliferative activity and tumor selectivity of the compounds were measured by standard MTT assays. Tumor cell lines MCF-7, T47D, HeLa, A2780, and noncancerous NIH/3T3 were provided by European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK); cell lines SiHa and C33A by LGC Standards GmbH (Wesel, Germany). All chemicals and materials used for the antiproliferative assays were purchased from Sigma Aldrich Ltd. (Budapest, Hungary).

Juncuenin B

Juncuenin B was isolated previously from the MeOH extract of Juncus inflexus roots by chromatographic methods (VLC on silica gel, and gel chromatography on Sephadex LH-20). J. inflexus is a good source of this compound as approximately 1.5 g was isolated (0.043% w/w) in crystalline form from 3.5 kg dried plant material.²³

Synthesis and Purification Process

In reaction I, 50 mg juncuenin B was dissolved in MeCN–MeOH (9:1) at a concentration of 1 mg/mL, and stirred with 2 equiv of PIFA (161.2 mg) for 30 min at 0 °C. After drying under nitrogen gas, SPE was applied to absorb the remaining oxidizing agent and the possibly decomposed compounds. Thereafter, the reaction mixture was fractionated by MPLC with n-hexane–EtOAc gradient solvent system to obtain five fractions (I/1–5). From fraction I/1 compounds 1a–d (1.00 mg, 2% each) were isolated by RP-HPLC using MeCN–H₂O (1:1) as eluent (3 mL/min flow rate). Fraction I/2 was purified by RP-HPLC, MeCN–H₂O (3:2) (3 mL/min flow rate) was applied as eluent to gain compounds 2a (0.88 mg) and 2b (0.75 mg).

For process reaction II, 100 mg juncuenin B was dissolved in anhydrous MeOH at a concentration of 1 mg/mL, and stirred with 2 equiv of PIFA (322.32 mg) for 30 min at room temperature. After evaporating the solvent under reduced pressure, SPE was applied to absorb the remaining oxidizing agents and the possibly decomposed compounds. The mixture was fractionated by MPLC with n-hexane–EtOAc gradient solvent system to obtain four fractions. Fraction II/1, after being chromatographed by RP-HPLC using MeCN–H₂O (1:1) (flow rate 3 mL/min) as mobile phase, compounds 1a–d (2.00 mg) and 5 (3.8 mg) were obtained. Fractions II/2 and II/3 were separated under the same conditions as II/1. From fraction II/2 1a–d (1.00 mg) and 3 (1.8 mg); from II/3 compounds 1a–d (3.00 mg) and 4a,b (2.00 mg) were isolated.

In the case of reaction III, 100 mg juncuenin B was dissolved in anhydrous EtOH at a concentration of 1 mg/mL, and stirred with 2 equiv of PIFA (322.32 mg) for 30 min at room temperature. After evaporating the solvent under reduced pressure, SPE was applied to remove the remaining oxidizing agents and decomposed compounds. The mixture was fractionated by MPLC with a gradient solvent system of n-hexane–EtOAc to obtain 10 fractions (III/1–10). Fraction III/1 afforded compound 5 (1.6 mg) after purification by HPLC with MeCN–H₂O (1:1) as mobile phase (flow rate 3 mL/min). Fraction III/2 was rechromatographed by RP-HPLC, using an MeCN–H₂O gradient system (flow rate 3 mL/min) as eluent to yield compounds 6a (3.0 mg) and 6b (1.85 mg). Fraction III/4 was found to be pure and gave compounds 7a,b (13.0 mg). Fraction III/6 was subjected to HPLC using MeCN–H₂O (1:1) (flow rate 3 mL/min) as mobile phase and afforded compounds 7a,b (4.4 mg, 4.4%) and 8 (2.1 mg). Fraction III/7 was purified by RP-HPLC with MeCN–H₂O (1:1) as eluent to give compound 9 (1.80 mg).

In reaction IV, 50 mg juncuenin B was dissolved in n-BuOH at a concentration of 1 mg/mL, and stirred with 2 equiv of PIFA (161,15 mg) for 30 min at room temperature. After evaporating the solvent under reduced pressure, prepurification with SPE was performed and the resulting mixture was fractionated by MPLC with an n-hexane–EtOAc gradient system to afford six fractions (IV/1–6). From fraction IV/1 compound 10 (1.80 mg) was isolated.

For reaction V, 100 mg juncuenin B was dissolved in n-BuOH at a concentration of 1 mg/mL, and stirred with 2 equiv of PIDA (242 mg) for 30 min at room temperature. The solvent was evaporated under reduced pressure, and then SPE was used to absorb the remaining oxidizing agents and other impurities. The mixture was fractionated by MPLC with an n-hexane–EtOAc gradient solvent system to afford four fractions (V/1–4). Fraction V/2 was subjected to HPLC, eluting with MeCN–H₂O (3:2) (flow rate 3 mL/min) as mobile phase to give compound 11 (5.5 mg).

Separation of Enantiomers

Racemic mixtures, which had high antiproliferative activity, were separated on a Lux amylose-1 chiral-phase column in order to compare the antiproliferative activity of the pure enantiomers. For the separation of compounds 1a–d, cyclohexane–isopropanol (95:5) and for compounds 4a+b and 7a+b cyclohexane–isopropanol (8:2) solvent systems (15 mL/min) were used as eluent.

(1R,8aR)-1a

Pale yellow solid; [α]_D: −17 (c 0.1, CHCl₃); HRESIMS m/z 327.1596 [M + H]⁺ (calcd for C₂₀H₂₃O₄, 327.1592); UV (MeCN) λ_max (log ε) 295sh (3.53), 220sh (3.95) nm; ECD {CH₃CN, λ_max [nm] (Δε)}, c 5.97 × 10^–4 M: 382 (−0.52), 341 (+2.05), 304sh (−0.67), 236 (−5.31), 207sh (−0.59); ¹H and ¹³C NMR data, see Tables 1 and 2; HPLC: Lux amylose-1 chiral-phase column, cyclohexane–isopropanol (95:5) solvent system, flow rate 15 mL/min, t_R = 13.6 min.

(1S,8aR)-1b

[α]_D: −26 (c 0.1, CHCl₃); UV (MeCN) λ_max (log ε) 294sh (3.57), 221sh (4.01) nm; ECD {CH₃CN, λ_max [nm] (Δε)}, c 4.90 × 10^–4 M: 393 (−0.74), 338sh (+0.80), 319 (+1.24), 246 (−4.01), 222sh (−2.92), 196 (+4.29); ¹H and ¹³C NMR data, see Tables 1 and 2. HPLC: Lux amylose-1 chiral-phase column, cyclohexane–isopropanol (95:5) solvent system, flow rate 15 mL/min, t_R = 17.4 min.

(1S,8aS)-1c

[α]_D: +26 (c 0.1, CHCl₃); UV (MeCN) λ_max (log ε) 294sh (3.57), 221sh (4.01) nm; ECD {CH₃CN, λ_max [nm] (Δε)}, c 5.31 × 10^–4 M: 390 (+0.49), 336sh (−0.81), 317 (−1.14), 253sh (+2.87), 237 (+3.81), 218sh (+2.27), 194 (−3.41); ¹H and ¹³C NMR data, see Tables 1 and 2. HPLC: Lux amylose-1 chiral-phase column, cyclohexane–isopropanol (95:5) solvent system, flow rate 15 mL/min, t_R = 21.6 min.

(1R,8aS)-1d

[α]_D: +23 (c, 0.1, CHCl₃); UV (MeCN) λ_max (log ε) 295sh (3.53), 220sh (3.95) nm; ECD {CH₃CN, λ_max [nm] (Δε)}, c 5.36 × 10^–4 M: 384 (+0.39), 338 (−1.52), 303sh (+0.43), 236 (+3.79), 209sh (+0.49); ¹H and ¹³C NMR data, see Tables 1 and 2. HPLC: Lux amylose-1 chiral-phase column, cyclohexane–isopropanol (95:5) solvent system, flow rate 15 mL/min, t_R = 23.6 min.

Ent-2a

Pale yellow solid; HRESIMS m/z 327.1598 [M + H]⁺ (calcd for C₂₀H₂₃O₄, 327.1591); ¹H and ¹³C NMR data, see Tables 1 and 2.

Ent-2b

Pale yellow solid; HRESIMS see ent-2a; ¹H and ¹³C NMR data, see Tables 1 and 2.

Rac-3

White solid, HRESIMS m/z 325.1435 [M + H]⁺ (calcd for C₂₀H₂₁O₄, 325.1434); ¹H and ¹³C NMR data, see Tables 1 and 2.

(8aR)-4a + (8aS)-4b

Pale yellow solid; HRESIMS m/z 297.1488 [M + H]⁺ (calcd for C₁₉H₂₁O₃ 297.1485); ¹H and ¹³C NMR data, see Tables 1 and 2; HPLC: Lux amylose-1 chiral column, cyclohexane–isopropanol (8:2) solvent system, flow rate 50 mL/min, t_R = 5.5 min (4a) and 10.3 min (4b).

(4aR,S;8aR,S)-5

Pale yellow solid; HRESIMS m/z 355.1913 [M + H]⁺ (calcd for C₂₂H₂₇O₄ 355.1904; ¹H and ¹³C NMR data, see Tables 1 and 2.

Ent-6a

White solid; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 355.1903 [M + H]⁺ (calcd for C₂₂H₂₇O₄ 355.1904).

Ent-6b

White solid; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS see ent-6a.

(8aR)-7a

Pale yellow solid; [α]_D: −124 (c, 0.1, CHCl₃); HRESIMS m/z 355.1915 (calcd for C₂₀H₂₃O₃ 355.1909); ECD {CH₃CN, λ_max [nm] (Δε)}, c 5.07 × 10^–4 M: 378 (−1.01), 333sh (−0.23), 302sh (+0.19), 271 (+1.52), 250 (−1.13), 232 (+0.28), 218 (−0.84); ¹H and ¹³C NMR data, see Tables 1 and 2; HPLC: Lux amylose-1 chiral-phase column, cyclohexane–isopropanol (8:2) solvent system, flow rate 15 mL/min, t_R = 4.7 min.

(8aS)-7b

Pale yellow solid; [α]_D: +148 (c, 0.1, CHCl₃); HRESIMS see (8aR)-7a; ECD {CH₃CN, λ_max [nm] (Δε)}, c 4.91 × 10^–4 M: 379 (+2.48), 334sh (+0.55), 271 (−3.54), 250 (+2.89), 233 (−0.81), 217 (+1.94); ¹H and ¹³C NMR data, see Tables 1 and 2. HPLC: Lux amylose-1 chiral-phase column, cyclohexane–isopropanol (8:2) solvent system, flow rate 15 mL/min, t_R = 9.1 min.

Rac-8

White solid; HRESIMS m/z 309.1491 [M + H]⁺ (calcd for C₂₀H₂₁O₃ 309.1485); ¹H and ¹³C NMR data, see Tables 1 and 2.

Rac-9

White solid; HRESIMS m/z 311.1646 [M + H]⁺ (calcd for C₂₀H₂₃O₃ 311.1642); ¹H and ¹³C NMR data, see Tables 1 and 2.

Rac-10

Light brown solid; HRESIMS m/z 481.2948 [M + H]⁺ (calcd for C₃₀H₄₁O₅ 481.2949); ¹H and ¹³C NMR data, see Tables 1 and 2.

Rac-11

Light brown solid; HRESIMS m/z 339.1959 [M + H]⁺ (calcd for C₂₂H₂₇O₃ 339.1955); ¹H and ¹³C NMR data, see Tables 1 and 2.

Computational Methods

Mixed torsional/low-mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the MMFF with an implicit solvent model for CHCl₃ applying a 21 kJ/mol energy window.²⁴ Geometry reoptimizations of the resultant conformers (ωB97X/TZVP with PCM solvent model for MeCN) and TDDFT ECD calculations were performed with Gaussian 09.²⁵ For ECD using various functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0) and the TZVP basis set with the same solvent model as in the preceding DFT optimization step. ECD spectra were generated as the sum of Gaussians with 3000 cm^–1 half-height widths, using dipole-velocity computed rotational strengths.²⁶ Boltzmann distributions were estimated from the ωB97X energies. The MOLEKEL program was used for visualization of the results.²⁷

Antiproliferative (MTT) Assay

Growth-inhibition properties of racemates and pure enantiomer compounds were determined by standard MTT assays on six human malignant gynecological cell lines (MCF-7, T47D, HeLa, SiHa, C33A, A2780). To investigate tumor selectivity, the MTT assay was repeated on mouse embryonic fibroblast cells (NIH/3T3) under the same conditions. All cell lines were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 1% penicillin–streptomycin–amphotericin B mixture, and were stored in humidified air containing 5% CO₂ at 37 °C. All cell types were seeded into 96–well plates at a density of 5000 with the exception of C33a, which was seeded at a density of 10 000 and treated by increasing concentrations (0.1–30 μM) of the compounds for 72 h under cell culturing conditions. After the incubation, 5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution was added to samples for 4 h and precipitated blue formazan crystals were dissolved in DMSO. Absorbance values of the samples were measured at 545 nm using a microplate reader (Stat Fax-2100, Awareness Technologies Inc., Palm City, FL, USA) and untreated cells were used as control. Normalized sigmoidal dose–response curves were fitted to the determined data and the IC₅₀ values were calculated by GraphPad Prism 5.01 (GraphPad Software, San Diego, CA, USA). Cisplatin (Ebewe Pharma GmbH, Unterach, Austria) was used as a reference agent in the same concentration range.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00499.

• 1D and 2D NMR spectra of compounds 1–11 and ECD spectra of compounds 1, 4, and 7 (PDF)

Author Contributions

^∥ Csaba Bús and Ágnes Kulmány are co-first authors.

The authors declare no competing financial interest.