Antiproliferative Activities of Cynaropicrin and Related Compounds against Cancer Stem Cells

Abstract

Glioblastoma (GBM) has a high mortality rate despite the availability of various cancer treatment options. Although cancer stem cells (CSCs) have been associated with poor prognosis and metastasis, and play an important role in the resistance to existing anticancer drugs and radiation; no CSC-targeting drugs are currently approved in clinical practice. Therefore, the development of antiproliferative agents against CSCs is urgently required. In this study, we evaluated the antiproliferative activities of 21 sesquiterpenoids against human GBM U-251 MG CSCs and U-251 MG non-CSCs. Particularly, the guaianolide sesquiterpene lactone cynaropicrin (1) showed strong antiproliferative activity against U-251 MG CSCs (IC₅₀ = 20.4 µM) and U-251 MG non-CSCs (IC₅₀ = 10.9 µM). Accordingly, we synthesized six derivatives of 1 and investigated their structure–activity relationships. Most of the guaianolide sesquiterpene lactones with the α-methylene-γ-butyrolactone moiety showed antiproliferative activities against U-251 MG cells. We conclude that the 5,7,5-ring and the α-methylene-γ-butyrolactone moiety are both important for antiproliferative activities against U-251 MG cells. The results of this study suggest that the α,β-unsaturated carbonyl moiety, which has recently become a research hotspot in drug discovery, is the active center of 1. Therefore, we consider 1 as a potential lead for developing novel drugs targeting CSCs.

Introduction

Glioblastoma (GBM) accounts for approximately 45% of malignant brain tumors with high mortality rates despite the availability of various treatment options, including surgical resection, radiotherapy, and concomitant and adjuvant temozolomide-based chemotherapy.¹⁾ A few cancer stem cells (CSCs), which have been associated with poor prognosis, are present in various cancer types. They are characterized by self-renewal ability, tumorigenic and metastatic potential, and resistance against existing anticancer drugs and radiation. However, no anticancer drugs that target CSCs are currently approved in clinical settings. Therefore, novel drug candidates targeting CSCs must be developed crucially.^2–4) Recent studies have reported sesquiterpenoids to have anti-CSC activities, drawing attention to them as seed compounds for targeting CSCs.^5–7) Therefore, we evaluated the antiproliferative activities of 21 sesquiterpenoids (1–7: guaianolide sesquiterpene lactones⁸⁾; 8–10: germacranolide sesquiterpene lactones and their derivatives⁹⁾; 11,12: eudesmanolide sesquiterpene lactones⁹⁾; and 13–21: cadinene-type sesquiterpenes¹⁰⁾) against human GBM U-251 MG CSCs and U-251 MG non-CSCs (Fig. 1). The guaianolide sesquiterpene lactone cynaropicrin (1) demonstrated strong antiproliferative activity against U-251 MG cells. 1 is a natural compound primarily found in artichoke leaves and reported to show various pharmacological activities, including anti-trypanosomal, anti-spasmodic, anti-inflammatory, and anti-hyperlipidemic activities, probably mediated by inhibiting the nuclear factor-kappaB (NF-κB) pathway which plays a key role in inflammation.^11–16) A recent study has shown that 1 is strongly cytotoxic against human continuous glioblastoma U-87 MG cells.¹⁷⁾ Therefore, we synthesized six derivatives (22–27), evaluated their antiproliferative activities against U-251 MG CSCs and U-251 MG non-CSCs, and determined the structural features that are important for such activities. We discussed the structure–activity relationships (SARs) and the antiproliferative activities of a total of 27 sesquiterpenoids, including six derivatives of 1, against U-251 MG CSCs and U-251 MG non-CSCs.

Fig. 1. Structures of Compounds 1–21

Results and Discussion

Antiproliferative Activities of 21 Sesquiterpenoids against U-251 MG CSCs and U-251 MG Non-CSCs

In this study, we evaluated the antiproliferative activities of 21 sesquiterpenoids against human GBM U-251 MG CSCs and U-251 MG non-CSCs (Tables 1, 2). Adriamycin (ADR) was used as the positive control and CSCs were prepared by sphere formation. The formation of spheres was confirmed from cell morphology.^5,18) Cell viability was assayed using CellTiter-Glo^® 3D (Promega Corp., Madison, WI, U.S.A.). The guaianolide sesquiterpene lactone cynaropicrin (1), dehydrocostus lactone (5), saussureamine B (6), and the germacranolide sesquiterpene lactone saussureamine A (9) demonstrated strong antiproliferative activities with IC₅₀ 20.4, 8.2, 7.9, and 7.7 µM against U-251 MG CSCs and 10.9, 4.0, 7.0, 6.5 µM against U-251 MG non-CSCs respectively. Conversely, most cadinene-type sesquiterpenes were inactive against U-251 MG CSCs and U-251 MG non-CSCs, with only 19 showing antiproliferative activity against U-251 MG CSCs (IC₅₀ = 26.6 µM) and U-251 MG non-CSCs (IC₅₀ = 18.0 µM).

Table 1. Antiproliferative Activities of Compounds 1–21 against U-251 MG CSCs

	Inhibition (%)						IC50 (µM)
Conc. (µM)	0	3.125	6.25	12.5	25	50
1	0.0 ± 2.0	−8.0 ± 3.2	−0.6 ± 2.6	21.0 ± 8.1**	64.2 ± 12.8**	99.1 ± 0.2**	20.4
2	0.0 ± 13.7	1.5 ± 1.6	−2.0 ± 5.1	−2.0 ± 1.9	1.2 ± 2.8	26.1 ± 3.0	>50
3	0.0 ± 3.5	−13.3 ± 12.5	−10.1 ± 6.9	20.4 ± 5.2**	77.9 ± 8.7**	100.3 ± 0.6**	17.8
4	0.0 ± 5.5	−13.7 ± 4.8	−14.5 ± 7.2	−15.9 ± 7.0	−19.0 ± 2.3	−20.7 ± 8.6	>50
5	0.0 ± 3.3	−4.6 ± 3.1	22.0 ± 5.6**	87.7 ± 9.6**	100.2 ± 0.0**	100.2 ± 0.1**	8.2
6	0.0 ± 4.9	6.4 ± 1.2	30.9 ± 6.8**	82.3 ± 3.4**	99.6 ± 0.0**	99.6 ± 0.1**	7.9
7	0.0 ± 1.7	−1.6 ± 4.5	0.3 ± 0.8	5.8 ± 3.7	40.6 ± 7.9**	99.8 ± 0.0**	27.4
8	0.0 ± 3.1	−7.0 ± 0.6	−14.7 ± 15.2	−4.7 ± 2.3	−12.9 ± 5.7**	14.6 ± 8.1**	>50
9	0.0 ± 1.1	−3.3 ± 2.9	35.6 ± 6.6**	81.5 ± 1.4**	98.4 ± 0.6**	99.6 ± 0.1**	7.7
10	0.0 ± 3.2	−3.1 ± 6.2	−10.2 ± 0.7*	−4.4 ± 0.2	−7.4 ± 7.3	10.1 ± 0.6*	>50
11	0.0 ± 3.9	−1.9 ± 6.8	−9.0 ± 8.1	−15.9 ± 5.6**	−5.9 ± 7.1	−7.0 ± 5.1	>50
12	0.0 ± 7.1	−9.1 ± 3.1	−14.1 ± 2.8	−14.4 ± 3.9	−9.0 ± 0.8	1.7 ± 3.4	>50
13	0.0 ± 2.9	−2.8 ± 4.5	−4.4 ± 5.9	−9.7 ± 6.8	−9.8 ± 5.5	5.6 ± 3.7	>50
14	0.0 ± 1.9	−11.2 ± 2.4	−13.3 ± 3.4	−20.2 ± 4.9	−13.6 ± 2.6	−10.5 ± 3.0	>50
15	0.0 ± 2.2	−6.2 ± 5.4	−7.2 ± 6.3*	−14.1 ± 3.4	−6.0 ± 2.7	9.5 ± 3.4	>50
16	0.0 ± 10.9	−1.7 ± 11.5	−3.9 ± 7.2	−2.5 ± 9.1	10.0 ± 8.3	20.8 ± 4.4	>50
17	0.0 ± 1.4	−6.1 ± 0.7	−8.8 ± 7.7	−5.8 ± 0.9	−2.3 ± 1.7	0.2 ± 1.5	>50
18	0.0 ± 2.6	−10.2 ± 2.2*	−16.6 ± 1.0**	−11.6 ± 3.9*	−11.7 ± 2.7*	−9.1 ± 1.8	>50
19	0.0 ± 4.1	4.6 ± 7.5	3.4 ± 6.6	19.5 ± 10.5**	48.3 ± 1.7**	57.7 ± 1.2**	26.6
20	0.0 ± 4.7	2.9 ± 2.4	2.4 ± 2.3	5.1 ± 2.1	4.0 ± 1.4	10.5 ± 2.0*	>50
21	0.0 ± 1.8	−2.7 ± 2.4	−0.9 ± 4.7	3.2 ± 1.6	14.4 ± 8.3**	27.8 ± 5.4**	>50
	Inhibition (%)						IC50 (µM)
Conc. (µM)	Control	0.0125	0.025	0.05	0.1	0.2
Adriamycin	0.0 ± 9.9	24.9 ± 7.8	42.3 ± 10.3**	57.3 ± 3.5**	77.2 ± 4.6**	90.8 ± 1.5**	0.036

Each value represents the mean ± standard deviation (S.D.) (N = 3). The statistical significance of differences was analyzed using Dunnett’s test (* p < 0.05, ** p < 0.01, compared with the control group). Cells were incubated with test samples for 6 d. Adriamycin was used as positive control.

Table 2. Antiproliferative Activities of 1–21 and against U-251 MG Non-CSCs

	Inhibition (%)						IC50 (µM)
Conc. (µM)	Control	3.125	6.25	12.5	25	50
1	0.0 ± 2.3	4.1 ± 3.2	16.0 ± 2.5**	68.8 ± 2.6**	76.0 ± 1.0**	97.9 ± 2.1**	10.9
2	0.0 ± 3.1	6.0 ± 1.0	7.0 ± 7.2	6.8 ± 3.7	17.2 ± 1.3**	58.6 ± 1.8**	44.7
3	0.0 ± 3.7	−5.0 ± 1.9	3.7 ± 1.7	13.5 ± 3.3**	42.4 ± 2.0**	87.4 ± 2.5**	27.7
4	0.0 ± 7.2	1.1 ± 3.1	−4.7 ± 6.1	−0.4 ± 2.1	−1.3 ± 0.7	4.8 ± 0.6	>50
5	0.0 ± 5.4	33.9 ± 6.9**	75.5 ± 3.8**	99.4 ± 0.1**	99.6 ± 0.0**	100.0 ± 0.0**	4.0
6	0.0 ± 7.8	17.4 ± 7.9**	36.9 ± 9.0**	93.3 ± 1.1**	99.4 ± 0.1**	99.8 ± 0.1**	7.0
7	0.0 ± 5.0	6.5 ± 3.9	17.7 ± 8.1**	33.0 ± 3.2**	73.9 ± 5.5**	99.0 ± 0.0**	16.4
8	0.0 ± 0.8	3.6 ± 10.2	5.5 ± 0.7	15.1 ± 11.1	32.0 ± 8.7**	65.9 ± 3.2**	36.0
9	0.0 ± 6.8	17.6 ± 3.5**	46.0 ± 8.1**	84.4 ± 0.7**	98.4 ± 0.1**	99.3 ± 0.1**	6.5
10	0.0 ± 7.6	5.9 ± 10.8	0.1 ± 7.7	11.9 ± 7.3*	25.0 ± 2.0**	96.5 ± 0.9**	34.1
11	0.0 ± 5.6	2.1 ± 3.3	−1.9 ± 5.6	7.4 ± 6.1	16.9 ± 8.3**	17.9 ± 3.4**	>50
12	0.0 ± 11.0	−7.4 ± 5.4	2.2 ± 2.9	14.4 ± 4.6	15.7 ± 8.9	43.4 ± 2.3**	>50
13	0.0 ± 4.8	2.4 ± 5.8	2.8 ± 5.2	8.1 ± 3.1	10.0 ± 4.5	11.8 ± 3.4	>50
14	0.0 ± 3.8	3.1 ± 2.3	7.6 ± 5.3*	2.6 ± 2.4	8.7 ± 4.0**	17.6 ± 2.6**	>50
15	0.0 ± 2.5	2.8 ± 5.5	8.2 ± 4.9	−4.2 ± 7.7	3.2 ± 3.2	11.5 ± 5.7*	>50
16	0.0 ± 2.8	−12.9 ± 1.8	−8.7 ± 12.1	−1.4 ± 9.4	−6.0 ± 7.2	9.9 ± 14.9	>50
17	0.0 ± 9.3	−8.5 ± 21.9	−6.9 ± 4.4	−11.4 ± 5.3	−9.0 ± 6.2	8.7 ± 7.2	>50
18	0.0 ± 9.1	−25.0 ± 3.1	−11.5 ± 3.7	−20.6 ± 8.8	−12.9 ± 5.2	−5.3 ± 4.4	>50
19	0.0 ± 6.6	−0.8 ± 9.9	−1.2 ± 11.9	28.3 ± 12.7**	61.1 ± 5.9**	69.3 ± 1.5**	18.0
20	0.0 ± 3.4	−3.5 ± 6.9	−9.6 ± 3.0	−7.2 ± 4.8	4.5 ± 5.7	19.0 ± 5.1**	>50
21	0.0 ± 9.1	−8.6 ± 5.7	−5.1 ± 8.1	−13.0 ± 11.8*	5.4 ± 7.5	22.9 ± 9.3**	>50
	Inhibition (%)						IC50 (µM)
Conc. (µM)	Control	0.0125	0.025	0.05	0.1	0.2
Adriamycin	0.0 ± 5.5	12.4 ± 8.3	38.0 ± 3.5**	54.3 ± 1.5**	64.3 ± 1.1**	79.9 ± 0.9**	0.045

Each value represents the mean ± S.D. (N = 3). The statistical significance of differences was analyzed using Dunnett’s test (* p < 0.05, ** p < 0.01, compared with the control group). Cells were incubated with test samples for 72 h. Adriamycin was used as positive control.

Synthesis of Cynaropicrin Derivatives and Evaluation of Sesquiterpenoids

In a recent study, 1 was reported to exhibit strong cytotoxicity against U-87 MG cells.¹⁷⁾

Among the four compounds with strong activities, 1 had the largest number of functional groups, including four exo-olefins and two hydroxy groups. To gain further insight into the important structural features in 1; we synthesized six derivatives (22–27) and evaluated their antiproliferative activities against U-251 MG CSCs and U-251 MG non-CSCs (Fig. 2). Methoxylation of 1 was conducted with sodium methoxide (MeONa) in methanol (MeOH) at room temperature (25 °C) for 48 h under nitrogen and yielded 54.0% of 22. Selective oxidation of the hydroxy group at the 3-position of 1 was performed by treatment with phenyliodine (III) bis(trifluoroacetate) (PIFA) and nitroxyl radical catalyst A, sodium hydrogen carbonate (NaHCO₃), in dichloromethane (CH₂Cl₂) at room temperature (25 °C) for 2 h to yield 50.0% of 23. Nitroxyl radical catalyst A is an efficient catalyst for the oxidation of secondary alcohols developed by Hamada et al.¹⁹⁾ Furthermore, 1 was acetylated using acetic anhydride (Ac₂O) with triethylamine (Et₃N) in CH₂Cl₂ at room temperature (25 °C) for 48 h under nitrogen to give 24 (acetoxy group at the 4′-position) and 25 (acetoxy groups at the 3- and 4′-positions) in 34.6 and 2.4% yields, respectively, according to the method reported by Usuki et al.²⁰⁾ Moreover, 1 was epoxidated using m-chloroperoxybenzoic acid (mCPBA) in CH₂Cl₂ at 0 °C for 24 h under nitrogen to yield 24.7% of 26, according to the method reported by Snider and Zhou.²¹⁾ In addition, hydrolysis of 1 was conducted in a 10% potassium hydroxide (KOH) aqueous solution at room temperature (25 °C) to yield 45.0% of 27. Compounds 22–27 were identified based on its ¹H-NMR, ¹³C-NMR, MS, and IR spectra. In addition, two dimensional (2-D) NMR spectra data was obtained except for 27 to determine detailed structure. From the ¹H, ¹³C and 2-D NMR, MS, and IR spectra of 26, the planar structure was determined to be the same as janerin.²²⁾ The orientation of epoxy group of 26 was determined via analysis of their nuclear Overhauser effect spectroscopy (NOESY) spectrum (p. 28 in supplementary-materials). The NOESY cross-peak between H-15a/H-5 indicated that H-15a and H-5 are facing the same direction. This suggested the configuration of an isomer as shown in Fig. 2.

Fig. 2. Synthesis of Compounds 22–27

Further, six synthetic compounds (22–27) were tested for their antiproliferative activities against U-251 MG CSCs and U-251 MG non-CSCs (Tables 3, 4, Fig. 2). CSCs were prepared by sphere formation as previously described.^5,18) Cell viability was assayed using CellTiter-Glo^® 3D (Promega Corp., Madison, WI, U.S.A.), with ADR as the positive control. Compound 23, having a carbonyl group instead of a hydroxy group at the 3-position, showed strong antiproliferative activity against U-251 MG CSCs (IC₅₀ = 8.1 µM) and U-251 MG non-CSCs (IC₅₀ = 3.6 µM).

Table 3. Antiproliferative Activities of Compounds 22–27 and against U-251 MG CSCs

	Inhibition (%)						IC50 (µM)
Conc. (µM)	Control	3.125	6.25	12.5	25	50
22	0.0 ± 0.9	−3.0 ± 5.8	−0.7 ± 6.2	−4.4 ± 7.1	−5.2 ± 4.3	−9.0 ± 3.4	>50
23	0.0 ± 9.9	11.7 ± 5.6	42.7 ± 5.3**	69.4 ± 6.3**	85.9 ± 6.8**	100.0 ± 0.0**	8.1
24	0.0 ± 0.2	5.7 ± 3.0	−1.1 ± 1.9	11.7 ± 2.2**	76.7 ± 5.2**	90.2 ± 1.4**	18.9
25	0.0 ± 7.5	−9.4 ± 3.8	−11.0 ± 3.7	22.0 ± 9.0**	74.8 ± 5.7**	96.9 ± 4.7**	18.0
26	0.0 ± 2.3	2.3 ± 0.9	6.0 ± 1.7	28.0 ± 4.1**	73.2 ± 6.9**	60.2 ± 3.5**	—*1
27	0.0 ± 2.3	−7.8 ± 5.9	−9.2 ± 5.4	−4.6 ± 6.6	−3.1 ± 6.3*	38.8 ± 14.9**	>50
	Inhibition (%)						IC50 (µM)
Conc. (µM)	Control	0.0125	0.025	0.05	0.1	0.2
Adriamycin	0.0 ± 5.8	23.1 ± 5.8**	49.0 ± 1.8**	57.4 ± 1.3**	66.8 ± 1.6**	90.3 ± 1.5**	0.037

Each value represents the mean ± S.D. (N = 3). The statistical significance of differences was analyzed using Dunnett’s test (* p < 0.05, ** p < 0.01, compared with the control group). Cells were incubated with test samples for 6 d. Adriamycin was used as positive control. *¹ Compound 26 did not show concentration-dependent antiproliferative activity.

Table 4. Antiproliferative Activities of Compounds 22–27 and against U-251 MG Non-CSCs

	Inhibition (%)						IC50 (µM)
Conc. (µM)	Control	3.125	6.25	12.5	25	50
22	0.0 ± 13.6	6.9 ± 9.9	3.0 ± 1.7	−5.7 ± 5.4	3.5 ± 2.4	9.4 ± 4.3	>50
23	0.0 ± 15.6	35.7 ± 10.9**	89.2 ± 1.6**	97.3 ± 0.3**	99.9 ± 0.0**	100.0 ± 0.0**	3.6
24	0.0 ± 2.8	1.4 ± 4.2	45.0 ± 6.2**	73.2 ± 7.3**	84.8 ± 1.4**	96.6 ± 1.3**	7.6
25	0.0 ± 11.0	16.1 ± 22.4	43.7 ± 8.7**	76.6 ± 8.2**	82.7 ± 1.6**	99.8 ± 0.1**	7.5
26	0.0 ± 4.1	4.9 ± 6.1	15.5 ± 4.3**	76.3 ± 4.0**	98.4 ± 0.1**	97.9 ± 0.3**	9.5
27	0.0 ± 3.2	1.8 ± 4.1	1.0 ± 3.5	13.6 ± 2.1**	34.0 ± 7.7**	85.1 ± 1.8**	31.0
	Inhibition (%)						IC50 (µM)
Conc. (µM)	Control	0.0125	0.025	0.05	0.1	0.2
Adriamycin	0.0 ± 5.5	12.4 ± 8.3	38.0 ± 3.5**	54.3 ± 1.5**	64.3 ± 1.1**	79.9 ± 0.9**	0.045

Each value represents the mean ± S.D. (N = 3). The statistical significance of differences was analyzed using Dunnett’s test (* p < 0.05, ** p < 0.01, compared with the control group). Cells were incubated with test samples for 72 h. Adriamycin was used as positive control.

Structure–Activity Relationships of Sesquiterpenoids

Although cynaropicrin (1) showed strong antiproliferative activity against U-251 MG CSCs (IC₅₀ = 20.4 µM) and U-251 MG non-CSCs (IC₅₀ = 10.9 µM), 11β,13-dihydro-cynaropicrin (2) obtained by the reduction of the α-methylene-γ-butyrolactone moiety in 1 was inactive. Similar to 2, isoamberboin (4) which lacks an exo-olefin moiety; and 22 which lacks a double bond owing to the methoxylation of 1, were inactive. These results indicate that the α-methylene-γ-butyrolactone moiety is essential for the antiproliferative activities of guaianolide sesquiterpene lactones against U-251 MG cells. In addition, 27 without a side chain at the 8-position exhibited decreased activity, while 23 with a carbonyl group instead of a hydroxy group at the 3-position, showed strong antiproliferative activity against U-251 MG CSCs (IC₅₀ = 8.1 µM) and U-251 MG non-CSCs (IC₅₀ = 3.6 µM). These results suggested that the antiproliferative activities of guaianolide sesquiterpene lactones increase proportionally with the number of the α,β-unsaturated carbonyl moiety which is the activity center of 1. We further aimed to determine the α,β-unsaturated carbonyl moiety in 1, more important for its antiproliferative activity. Compounds 2, 4 and 22 remarkably showed a decrease in activities compared with that of 27, hence; we considered the α-methylene-γ-butyrolactone moiety to be more important for activity than the α,β-unsaturated carbonyl moiety at 8-position.

The α,β-unsaturated carbonyl moiety can typically undergo Michael addition reaction and compounds with this structure are known as Michael acceptors. Michael acceptors usually demonstrate a wide range of physiological activities since they can modify various biomolecules and activate the transcription factor nuclear factor-E2-related factor 2 (Nrf2). Although they are promising leads for the development of novel drugs due to their various physiological activities, they show non-selective reactions and masquerade as true hits with high potential for further development.²³⁾

In addition, Michael acceptors often have a high potential for diverse side effects due to their irreversible binding, which increases their target-residence times. For example, acrylamide, a typical Michael acceptor, has been reported to cause concentration-dependent neurotoxicity in in vitro studies. 1 has been reported that horses ingesting large amounts of Centaurea solstitialis containing 1 caused facial muscle atresia and mastication disorders. 1 was also reported to inhibit smooth muscle contraction in rabbit isolated aortic ring preparations. For these reasons, Michael acceptors tended to be shunned in previous drug discovery efforts.^24–26) Nevertheless, Michael acceptors have gained attention over the past few years as scaffolds for novel drug discovery. Chemists have attempted to use Michael acceptors to design promising compounds with high selectivity. For example, the BTK inhibitor ibrutinib and the EGFR inhibitor afatinib, which contain the α,β-unsaturated carboxamide moiety involved in the Michael addition reaction, are clinically used as anticancer agents.^27–31) Therefore, 1 is suggested to be a potential lead for the development of drugs against CSCs. Although, saussureamine B (6) which lacks the exo-olefin moiety showed strong antiproliferative activity against U-251 MG CSCs (IC₅₀ = 7.9 µM) and U-251 MG non-CSCs (IC₅₀ = 7.0 µM) surprisingly, overall, it is suggested that the combination of a 5,7,5-ring and an α-methylene-γ-butyrolactone moiety is important for antiproliferative activity against U-251 MG cells. Further, the contribution of hydroxy groups and the exo-olefin moiety at the 4-position to the antiproliferative activity was investigated. We observed that 24 with an acetoxy group at the 4′-position, 25 with two acetoxy groups at the 3-position and 4′-position, 26 with an epoxidized moiety at the 4-position, and grosheimin (3) lacking an exo-olefin moiety showed antiproliferative activities similar to those of 1. These results suggest that the hydroxy groups at the 3-position and 4′-position and exo-olefin moiety at the 4-position in 1 do not contribute to its antiproliferative activity (Fig. 3).

Fig. 3. Structure–Activity Relationships of Cynaropicrin (1)

Further, the activities of sesquiterpenes, including costunolide (8) and saussureamine A (9), obtained from Saussurea lappa, were investigated. The results showed that 8 had no significant antiproliferative activity against U-251 MG CSCs (IC₅₀ > 50 µM) or U-251 MG non-CSCs (IC₅₀ = 36.0 µM), although it was previously reported that 8 exhibited strong antiproliferative activity against HL-60 cells which was attributed to the α-methylene-γ-butyrolactone moiety.^32,33) Conversely, 9, which lacks the exo-olefin moiety, showed strong antiproliferative activity against U-251 MG CSCs (IC₅₀ = 7.7 µM) and U-251 MG non-CSCs (IC₅₀ = 6.5 µM). These results indicated that the α-methylene-γ-butyrolactone moiety is not important for the antiproliferative activities of germacranolide sesquiterpene lactones against U-251 MG cells.

Verification of the Activity of Cynaropicrin (1) as a Michael Acceptor

These results suggest that 1 with α,β-unsaturated carbonyl moiety is a Michael acceptor. To obtain further experimental evidence for this hypothesis, we performed the following reaction under conditions similar to the physiological environment.³⁴⁾ In brief, 1 (9.8 mg, 0.028 mmol) was dissolved in dimethyl sulfoxide (DMSO)-d₆ (750 µL) in a microtube. Next, anhydrous dibasic sodium phosphate (3.0 mg) was added to D₂O (450 µL). The phosphate buffered saline (PBS) solution (pH approx. 8) was added to the DMSO stock of 1 and vortexed. Then, ¹H-NMR spectrum of the solution was recorded. Cysteamine hydrochloride (6.3 mg, 0.055 mmol, 2 equivalent (equiv.)) was pre-weighed into a microtube. The solution of 1 was added to the pre-weighed cysteamine hydrochloride and vortexed for 5 min. As a result, the signal of the exo-olefin moiety of the α,β-unsaturated carbonyl moiety disappeared (p. 15 in supplementary-materials). This result makes the hypothesis stronger that the exo-olefin moiety of the α,β-unsaturated carbonyl moiety are involved in the Michael addition reaction and 1 with α,β-unsaturated carbonyl moiety is a Michael acceptor.

Conclusion

In this study, we evaluated the antiproliferative activities of 27 sesquiterpenoids, including six derivatives of 1, against U-251 MG CSCs and U-251 MG non-CSCs. We observed that most guaianolide sesquiterpene lactones with the α-methylene-γ-butyrolactone moiety showed antiproliferative activities against U-251 MG cells. Therefore, we conclude that the 5,7,5-ring and the α-methylene-γ-butyrolactone moiety are both important for antiproliferative activities against U-251 MG cells. In addition, we strongly suggest that 1 with α,β-unsaturated carbonyl moiety is a Michael acceptor. 1 and its derivatives exhibited lower activity compared to adriamycin used as the positive control in this study, but adriamycin is known to be a cardiotoxic agent, whereas 1 has been reported to show preventive effect against coronary heart disease.²⁶⁾ In addition, its small molecular weight makes 1 a seed of oral anticancer drug. Furthermore, 1 improve indigestion, which are typical side effects of existing anticancer drugs.³⁵⁾ Although less active than existing anticancer drugs, these factors lead us to suggest that 1 is an attractive lead for the developing of drugs targeting CSCs with the potential to significantly improve the disadvantages of existing anticancer drugs. In our future studies, we target to further investigate the potential of sesquiterpenoids as leads for the development of novel drugs with activities against U-251 MG CSCs.

Experimental

General Experimental Procedures

The following instruments were used to obtain structural data: Shimadzu FTIR-8100 spectrometer for IR spectra; JEOL JMS-GCMATE mass spectrometer for electron ionization-mass spectra (EIMS) and high resolution (HR)EIMS; JEOL JNM-ECS 400 (400 MHz) and JEOL JNM-ECA 600 (600 MHz) spectrometers for ¹H-NMR spectra; JEOL JNM-ECS 400 (101 MHz) and JEOL JNM-ECA 600 (151 MHz) spectrometer for ¹³C-NMR spectra with tetramethylsilane as the internal standard when CDCl₃ was used as the measurement solvent, but not when DMSO-d₆ was used.

Isolation of Cynaropicrin (1)

Cynaropicrin (1) was isolated from the dried leaves of Cynara scolymus (900 g) stored in our laboratory using the similar method previously reported by us.⁸⁾ Briefly, the methanolic extract was partitioned into an ethyl acetate (EtOAc)–H₂O (1 : 1, v/v) mixture to furnish an EtOAc-soluble fraction and aqueous layer. The EtOAc-soluble fraction was subjected to normal-phase column chromatography [Chloroform (CHCl₃)：MeOH (30 : 1, v/v)] and reversed-phase column chromatography [H₂O：MeOH (80 : 20→50 : 50, v/v)] to give 1 (925 mg, isolation yield from the starting material: 0.1%). 1 was identified based on its ¹H-NMR and ¹³C-NMR spectra data with its reported value.³⁶⁾ In addition, ¹H-NMR, ¹³C-NMR, and 2-D NMR spectra of 1 in a solution [DMSO-d₆: aqueous (D₂O) phosphate buffered saline (5 : 3, v/v)] was obtained to determine detailed structure.

Yellow oil, ¹H-NMR (400 MHz, CDCl₃) δ: 6.32 (s like, 1H, H-3′a), 6.21 (d like, J = 3.0 Hz, 1H, H-13a), 5.95 (s like, 1H, H-3′b), 5.60 (d like, J = 3.0 Hz, 1H, H-13b), 5.48 (s like, 1H, H-15a), 5.35 (s like, 1H, H-15b), 5.15–5.11 (m, 2H, H-8 and H-14a), 4.93 (s like, 1H, H-14b), 4.55 (m, 1H, H-3), 4.37 (s like, 2H, H-4′), 4.24 (dd, J = 10.6, 9.0 Hz, 1H, H-6), 3.19 (m, 1H, H-7), 2.97 (m, 1H, H-1), 2.84 (dd, J = 10.6, 8.8 Hz, 1H, H-5), 2.70 (dd, J = 14.7, 5.2 Hz, 1H, H-9a), 2.38 (dd, J = 14.7, 3.8 Hz, 1H, H-9b), 2.22 (ddd, J = 13.6, 7.3, 6.6 Hz, 1H, H-2a), 1.72 (ddd, J = 13.6, 8.3, 7.6 Hz, 1H, H-2b). ¹³C-NMR (151 MHz, CDCl₃) δ: 169.2 (C-12), 165.4 (C-1′), 152.1 (C-4), 141.7 (C-10), 139.2 (C-2′), 137.3 (C-11), 126.8 (C-3′), 122.8 (C-13), 118.3 (C-14), 113.6 (C-15), 78.5 (C-6), 74.3 (C-8), 73.7 (C-3), 62.2 (C-4′), 51.4 (C-5), 47.5 (C-7), 45.3 (C-1), 39.0 (C-2), 36.9 (C-9). ¹H-NMR [600 MHz, DMSO-d₆: aqueous (D₂O) phosphate buffered saline (5 : 3, v/v)] δ 6.19 (s like, 1H, H-3′a), 6.01 (d like, J = 2.9 Hz, 1H, H-13a), 5.87 (s like, 1H, H-3′b), 5.53 (d like, J = 2.9 Hz, 1H, H-13b), 5.23 (s like, 1H, H-15a), 5.17 (s like, 1H, H-15b), 5.05 (s like, 1H, H-14a), 5.01 (m, 1H, H-8), 4.80 (s like, 1H, H-14b), 4.37 (overlaped, 1H, H-3), 4.26 (dd, J = 10.7, 8.8 Hz, 1H, H-6), 4.14 (s, 2H, H-4′), 3.18 (m, 1H, H-7), 2.88 (m, 1H, H-1), 2.79 (dd, J = 10.7, 9.2 Hz, 1H, H-5), 2.59 (dd, J = 14.7, 5.3 Hz, 1H, H-9a), 2.22 (dd, J = 14.7, 3.7 Hz, 1H, H-9b), 1.95 (m, 1H, H-2a), 1.57 (m, 1H, H-2b). ¹³C-NMR [151 MHz, DMSO-d₆: aqueous (D₂O) phosphate buffered saline (5 : 3, v/v)] δ 171.5 (C-12), 166.9 (C-1′), 153.7 (C-4), 143.4 (C-10), 140.7 (C-2′), 138.7 (C-11), 127.5 (C-3′), 123.6 (C-13), 118.9 (C-14), 112.7 (C-15), 80.0 (C-6), 75.3 (C-8), 73.3 (C-3), 60.9 (C-4′), 51.0 (C-5), 47.6 (C-7), 45.0 (C-1), overlaped (C-2), 37.5 (C-9).

Synthesis of Compounds 22–27

Synthesis of 22: Cynaropicrin (1, 16.2 mg, 0.047 mmol), and MeONa (13.4 mg, 0.248 mmol) were dissolved in MeOH (5.0 mL). The reaction mixture was stirred at room temperature (25 °C) for 48 h under nitrogen. Consequently, the solution was evaporated and the residue was partitioned into EtOAc and water to yield an EtOAc-soluble fraction and aqueous layer. The aqueous layer was further washed four times with EtOAc. After drying the organic layer with anhydrous sodium sulfate (Na₂SO₄), it was evaporated, and the residue was subjected to normal-phase column chromatography [Hexane (Hex) : EtOAc (1 : 1, v/v)] to obtain compound 22 (7.4 mg, 54.0%) which was identified based on its ¹H-NMR, ¹³C-NMR, MS, IR, and 2-D NMR spectra with its reported value.³⁷⁾

Yellow oil, high resolution electrospray ionization (ESI)-MS: Calcd for (C₁₆H₂₂O₅ + Na)⁺: 317.1359. Found: 317.1362. IR (attenuated total reflectance (ATR)): 3400, 2924, 1757, 1640, 1451, 1298, 1262, 1178, 1109, 1069, 1033, 1008 cm⁻¹. ESI-MS: m/z 317 (M + Na)⁺. ¹H-NMR (600 MHz, CDCl₃) δ: 5.40 (dd like, J = 1.9, 1.9 Hz, 1H, H-15a), 5.33 (dd like, J = 1.9, 1.9 Hz, 1H, H-15b), 5.08 (s like, 1H, H-14a), 5.04 (s like, 1H, H-14b), 4.56–4.50 (m, 1H, H-3), 4.14 (dd, J = 10.0, 10.0 Hz, 1H, H-6), 4.00 (dd, J = 9.6, 3.4 Hz, 1H, H-13a), 3.70–3.67 (m, 1H, H-8), 3.49 (dd, J = 9.6, 9.6 Hz 1H, H-13b), 3.46 (s, 3H, OCH₃), 2.96–2.91 (m, 1H, H-1), 2.86–2.80 (m, 2H, H-5 and H-11), 2.69 (dd, J = 13.4, 5.0 Hz, 1H, H-9a), 2.32–2.19 (m, 3H, H-9b and H-7 and H-2a), 1.75 (ddd, J = 15.3, 7.9, 7.4 Hz, 1H, H-2b). ¹³C-NMR (151 MHz, CDCl₃) δ: 174.2 (C-12), 152.8 (C-4), 143.0 (C-10), 116.5 (C-14), 112.8 (C-15), 79.2 (C-6), 73.8 (C-3), 73.2 (C-8), 72.1 (C-13), 59.3 (C-16), 56.1 (C-7), 50.3 (C-5), 48.1 (C-11), 44.4 (C-1), 41.6 (C-9), 38.8 (C-2).

Synthesis of 23: To a mixture of substrate (1, 20.0 mg, 0.058 mmol), catalyst¹⁹⁾ (2.7 mg, 0.012 mmol), and NaHCO₃ (19.4 mg, 0.231 mmol) in CH₂Cl₂ (1.2 mL), PIFA (27.3 mg, 0.064 mmol) was added. The reaction mixture was then stirred at room temperature (25 °C) for 2 h. Further, a saturated aq. NaHCO₃ was added, and the mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo to obtain a residue. The residue was purified by preparative TLC [CHCl₃ : EtOAc (1 : 1, v/v)] to obtain 23 (9.9 mg, 50.0%) which was identified based on its ¹H-NMR, ¹³C-NMR, MS, IR, and 2-D NMR spectra with its reported value.³⁸⁾

Yellow oil, high resolution ESI-MS: Calcd for (C₁₉H₂₀O₆ + Na)⁺: 367.1152. Found: 367.1160. ESI-MS: m/z 367 (M + Na)⁺. IR (ATR): 3450, 2926, 1767, 1720, 1637, 1402, 1295, 1265, 1146, 1055, 1033, 1014 cm⁻¹. ¹H-NMR (600 MHz, CDCl₃) δ: 6.35 (d like, J = 1.0 Hz, 1H, H-3′a), 6.34 (d like, J = 3.4 Hz, 1H, H-13a), 6.30 (dd like, J = 2.6, 0.8 Hz, 1H, H-15a), 6.01 (d like, J = 1.0 Hz, 1H, H-3′b), 5.90 (dd like, J = 2.4, 0.8 Hz, 1H, H-15b), 5.80 (d like, J = 2.9 Hz, 1H, H-13b), 5.14–5.09 (m, 2H, H-8 and H-14a), 4.90 (s like, 1H, H-14b), 4.40 (d, J = 5.9 Hz, 2H, H-4′), 4.11 (dd, J = 10.0, 8.9 Hz, 1H, H-6), 3.43 (m, 1H, H-7), 3.29 (m, 1H, H-5), 3.22 (ddd, J = 8.4, 8.4, 5.2 Hz, 1H, H-1), 2.92 (dd, J = 13.4, 7.6 Hz, 1H, H-9a), 2.64 (dd, J = 18.5, 8.4 Hz, 1H, H-2a), 2.54 (dd, J = 18.5, 5.2 Hz, 1H, H-2b), 2.34 (dd, J = 13.4, 7.6 Hz, 1H, H-9b). ¹³C-NMR (151 MHz, CDCl₃) δ: 203.6 (C-3), 168.9 (C-12), 165.0 (C-1′), 143.5 (C-4), 141.3 (C-10), 139.1 (C-2′), 135.7 (C-11), 126.9 (C-3′), 125.0 (C-13), 123.6 (C-15), 117.9 (C-14), 79.7 (C-6), 74.4 (C-8), 62.3 (C-4′), 49.0 (C-5), 46.6 (C-7), 43.3 (C-1), 41.7 (C-2), 40.4 (C-9).

Synthesis of 24 and 25: Cynaropicrin (1, 30.7 mg, 0.089 mmol), Ac₂O (15.6 µL, 0.166 mmol), and Et₃N (29 µL, 0.21 mmol) were dissolved in CH₂Cl₂ (1.0 mL). The reaction mixture was stirred at room temperature (25 °C) for 50 h under nitrogen. Then, water was added to decompose Ac₂O into acetic acid, and the solution was evaporated. The residue was partitioned into EtOAc and water to yield an EtOAc-soluble fraction and aqueous layer. The aqueous layer was then washed twice with EtOAc. The organic layer was evaporated after drying with Na₂SO₄, and the residue was subjected to normal-phase column chromatography [Hex: EtOAc (2 : 1, v/v)] to obtain 24 (11.9 mg, 34.6%) and 25 (0.9 mg, 2.4%). 24 and 25 were identified based on their ¹H-NMR, ¹³C-NMR, MS, IR, and 2-D NMR spectra with their reported values.²⁰⁾

24: Yellow oil, high resolution ESI-MS: Calcd for (C₂₁H₂₄O₇ + Na)⁺: 411.1414. Found: 411.1409. ESI-MS: m/z 411 (M + Na)⁺. IR (ATR): 3440, 2946, 1764, 1748, 1716, 1640, 1451, 1404, 1367, 1298, 1268, 1229, 1155, 1047, 1014 cm⁻¹. ¹H-NMR (600 MHz, CDCl₃) δ: 6.46 (d like, J = 0.8 Hz, 1H, H-3′a), 6.23 (d like, J = 3.2 Hz, 1H, H-13a), 5.98 (d like, J = 0.8 Hz, 1H, H-3′b), 5.62 (d like, J = 3.2 Hz, 1H, H-13b), 5.51 (s like, 1H, H-15a), 5.37 (s like, 1H, H-15b), 5.17–5.14 (m, 2H, H-8 and H-14a), 4.94 (s like, 1H, H-14b), 4.84 (s, 2H, H-4′), 4.60–4.55 (m, 1H, H-3), 4.26 (m, 1H, H-6), 3.19 (dddd, J = 12.7, 6.1, 3.2, 3.2 Hz, 1H, H-7), 2.99 (ddd, J = 13.6, 9.3, 5.5 Hz, 1H, H-1), 2.86 (dd, J = 10.7, 9.3 Hz, 1H, H-5), 2.71 (dd, J = 14.7, 3.8 Hz, 1H, H-9a), 2.41 (dd, J = 14.7, 3.8 Hz, 1H, H-9b) 2.24 (ddd, J = 13.6, 7.3, 6.8 Hz, 1H, H-2a), 2.11 (s, 3H, H-2″) 1.74 (m, 1H, H-2b) . ¹³C-NMR (151 MHz, CDCl₃) δ: 170.4 (C-1″), 169.0 (C-12), 164.3 (C-1′), 152.2 (C-4), 141.7 (C-10), 137.4 (C-11), 135.1 (C-2′), 129.2 (C-3′), 122.6 (C-13), 118.3 (C-14), 113.7 (C-15), 78.4 (C-6), 74.5 (C-8), 73.8 (C-3), 62.3 (C-4′), 51.4 (C-5), 47.6 (C-7), 45.3 (C-1), 39.1 (C-2), 37.0 (C-9), 20.9 (C-2″).

25: Yellow oil, high resolution ESI-MS: Calcd for (C₂₃H₂₆O₈ + Na)⁺: 453.1520. Found: 453.1535. ESI-MS: m/z 453 (M + Na)⁺. IR (ATR): 2931, 1771, 1735, 1661, 1640, 1451, 1396, 1367, 1240, 1164, 1138, 1069, 1043, 1022 cm⁻¹. ¹H-NMR (600 MHz, CDCl₃) δ: 6.46 (d like, J = 0.8 Hz, 1H, H-3″a), 6.25 (d like, J = 3.1 Hz, 1H, H-13a), 5.98 (d like, J = 0.8 Hz, 1H, H-3″b), 5.63 (d like, J = 3.1 Hz, 1H, H-13b), 5.57 (m,1H, H-3), 5.54 (dd like, J = 2.0, 1.8 Hz, 1H, H-15a), 5.37 (dd like, J = 2.0, 1.8 Hz, 1H, H-15b), 5.18–5.15 (m, 2H, H-8 and H-14a), 4.96 (d like, 1.4 Hz, 1H, H-14b), 4.84 (s, 2H, H-4″), 4.19 (dd, J = 10.7, 9.0 Hz, 1H, H-6), 3.20 (dddd, J = 12.6, 6.2, 3.1, 3.1 Hz, 1H, H-7), 3.03 (ddd, J = 8.7, 8.1, 5.5 Hz, 1H, H-1), 2.86 (dd, J = 10.7, 8.7 Hz, 1H, H-5), 2.69 (dd, J = 14.6, 5.2 Hz, 1H, H-9a), 2.46–2.35 (m, 2H, H-2a and H-9b), 2.11 (s, 3H, H-2′ or H-2‴), 2.10 (s, 3H, H-2′ or H-2‴) 1.80 (ddd, 1H J = 15.9, 8.7, 8.1 Hz, H-2b) . ¹³C-NMR (151 MHz, CDCl₃) δ: 170.8 (C-1′ or C-1‴), 170.3 (C-1′ or C-1‴), 168.9 (C-12), 164.3 (C-1″), 147.1 (C-4), 141.2 (C-10), 137.3 (C-11), 135.1 (C-2″), 129.3 (C-3″), 122.7 (C-13), 118.6 (C-14), 116.3 (C-15), 77.9 (C-6), 74.6 (C-8), 74.4 (C-3), 62.3 (C-4″), 51.7 (C-5), 47.5 (C-7), 45.6 (C-1), 37.1 (C-2), 36.3 (C-9), 21.3 (C-2′ or C-2‴), 20.9 (C-2′ or C-2‴).

Synthesis of 26: Cynaropicrin (1, 63.0 mg, 0.182 mmol) and mCPBA (70.2 mg, 0.407 mmol) were dissolved in CH₂Cl₂ (7.0 mL). The reaction mixture was then stirred at 0 °C for 24 h under nitrogen. The solution was evaporated, and the residue was subjected to normal-phase column chromatography [Hex: EtOAc (2 : 3, v/v)] to obtain compound 26 (16.3 mg, 24.7%) which was identified based on its ¹H-NMR, ¹³C-NMR, MS, IR, and 2-D NMR spectra with its reported value.²²⁾ In addition, NOESY spectrum was obtained to determine the configuration of H-15 and H-5.

Yellow oil, high resolution ESI-MS: Calcd for (C₁₉H₂₂O₇ + Na)⁺: 385.1258. Found: 385.1266. ESI-MS: m/z 385 (M + Na)⁺. IR (ATR): 3420, 2931, 2349, 1761, 1714, 1640, 1451, 1407, 1298, 1267, 1150, 1053, 1029 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 6.33 (d like, J = 1.0 Hz, 1H, H-3′a), 6.23 (d like, J = 3.4 Hz, 1H, H-13a), 5.97 (d like, J = 1.0 Hz,1H, H-3′b), 5.67 (d like, J = 3.0 Hz, 1H, H-13b), 5.20 (s like, 1H, H-14a), 5.09 (ddd, J = 10.2, 5.5, 5.1 Hz, 1H, H-8), 5.04 (s like, 1H, H-14b), 4.39 (s, 2H, H-4′), 4.31 (dd, J = 10.4, 9.4 Hz, 1H, H-6), 4.19 (dd, J = 10.1, 6.3 Hz, 1H, H-3), 3.30 (d like, J = 4.6 Hz, 1H, H-15a), 3.25 (m, 1H, H-7), 3.00 (ddd, J = 11.6, 10.7, 6.8 Hz, 1H, H-1), 2.90 (d like, J = 4.6 Hz, 1H, H-15b), 2.74 (m, 2H, H-9a and H-5), 2.37 (dd, J = 14.0, 5.5 Hz, 1H, H-9b), 2.26 (ddd, J = 13.2, 6.8, 6.3 Hz, 1H, H-2a), 1.76 (ddd, J = 13.2, 11.6, 10.1 Hz, 1H, H-2b) . ¹³C-NMR (101 MHz, CDCl₃) δ: 169.0 (C-12), 165.1 (C-1′), 140.9 (C-10), 139.1 (C-2′), 136.0 (C-11), 126.8 (C-3′), 123.5 (C-13), 118.1 (C-14), 76.0 (C-6), 74.2 (C-8), 69.8 (C-3), 65.3 (C-4), 62.3 (C-4′), 47.6 (C-15), 46.6 (C-7), 45.3 (C-5), 42.7 (C-1), 39.1 (C-2 and C-9).

Synthesis of 27: Cynaropicrin (1, 3.2 mg, 0.009 mmol) was dissolved in 10% aqueous potassium hydroxide (2.0 mL). The reaction mixture was then stirred at room temperature (25 °C) for 18 h. The solution was partitioned into EtOAc and water to obtain an EtOAc-soluble fraction and aqueous layer. The aqueous layer was further washed twice with EtOAc. The organic layer was evaporated after drying with Na₂SO₄, and the residue was subjected to normal-phase column chromatography [Hex: EtOAc (1 : 2, v/v)] to obtain 27 (1.1 mg, 45.0%) which was identified based on its ¹H-NMR, ¹³C-NMR, MS, and IR spectral data with its reported value.³⁶⁾

Colorless oil, high resolution ESI-MS: Calcd for (C₁₅H₁₈O₄ + Na)⁺: 285.1097. Found: 285.1091. ESI-MS: m/z 285 (M + Na)⁺. IR (ATR): 3400, 2924, 1745, 1655, 1640, 1451, 1396, 1338, 1274, 1152, 1069, 1047, 1004 cm⁻¹. ¹H-NMR (400 MHz, CDCl₃) δ: 6.28 (dd like, J = 3.5, 0.8 Hz, 1H, H-13a), 6.15 (dd like, J = 3.1, 0.8 Hz, 1H, H-13b), 5.50 (dd like, J = 1.9, 1.9 Hz, 1H, H-15a), 5.35 (dd like, J = 1.9, 1.9 Hz, 1H, H-15b), 5.14 (s, 1H, H-14a), 4.99 (s like,1H, H-14b), 4.58–4.54 (m, 1H, H-3), 4.17 (dd, J = 10.6, 9.1 Hz, 1H, H-6), 3.98 (m, 1H, H-8), 2.98 (ddd, J = 9.6, 8.4 8.1 Hz, 1H, H-1), 2.85–2.76 (m, 2H, H-5 and H-7), 2.71 (dd, J = 14.1, 5.1 Hz, 1H, H-9a), 2.32–2.21 (m, 2H, H-2a and H-9b), 1.74 (ddd, J = 16.0, 8.4, 8.1 Hz, 1H, H-2b). ¹³C-NMR (101 MHz, CDCl₃) δ: 170.0 (C-12), 152.5 (C-4), 142.8 (C-10), 138.0 (C-11), 123.2 (C-13), 117.2 (C-14), 113.3 (C-15), 78.7 (C-6), 73.8 (C-3), 72.0 (C-8), 51.5 (C-5), 51.1 (C-7), 45.4 (C-1), 41.2 (C-9), 39.4 (C-2).

Cell Culture

The glioblastoma cell line U-251 MG was purchased from the American Type Culture Collection (Manassas, VA, U.S.A.). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/low glucose (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, U.S.A.) and 1% penicillin/streptomycin (PC/SM; Wako).

Cancer Stem Cell Preparation

U-251 MG CSCs were prepared by the sphere formation assay as described previously.^5,18) Briefly, the cells were cultured in DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing 1% PC/SM, 2% B-27 [B-27^® Serum-Free Supplement (50×); Thermo Fisher Scientific], 20 ng/mL epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ, U.S.A.), and 20 ng/mL basic fibroblast growth factor (b-FGF; Peprotech) for 7 d in ultra-low attachment 6-well plates (Corning International, Corning, NY, U.S.A.). The cells were seeded at a density of 1.0 × 10⁵ cells/mL. The formation of spheres was confirmed under an inverted microscope.

CellTiter-Glo 3D^® Cell Viability Assay for CSCs and Non-CSCs

For CSCs: The cells were seeded at a density of 3.0 × 10³ cells/90 µL/well in ultra-low attachment 96-well plates and treated with test compounds (10 µL/well) 24 h after seeding. After 6 d, the cells were transferred to a 96-well white plate (96F Nunclon TM Delta White Microwell SI; Thermo Fisher Scientific). To each well, 100 µL of CellTiter-Glo^® 3D Reagent (Promega, Madison, WI, U.S.A.) was added, mixed by shaking for 5 min at room temperature (25 °C) and incubated for 25 min at 37 °C. Luminescence was measured with a luminometer (GloMax^® Discover System; Promega). For non-CSCs: The cells were seeded at a density of 3.0 × 10³ cells/90 µL/well of 96-well plates and treated with test compounds (10 µL/well) 24 h after seeding. After 3 d, cell viability was evaluated with CellTiter-Glo^® 3D Reagent using a 96-well white plate, as described above.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 8.21 software. One-way ANOVA, followed by Dunnett’s test, was used to analyze differences between treatment groups. Differences were considered significant when * p < 0.05 or ** p < 0.01.

Acknowledgments

This study was funded by JSPS KAKENHI Grant Number 23H02642 (S.N.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

The ¹H-NMR, ¹³C-NMR and 2-D NMR spectral data for 1, a product after Michael addition reaction, and 22–27 are provided as supplementary material.

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