Dihydroisocoumarins and Dihydroisoflavones from the Rhizomes of Dioscorea collettii with Cytotoxic Activity and Structural Revision of 2,2′-Oxybis(1,4-di-tert-butylbenzene)

Abstract

The investigation of the constituents of the rhizomes of Dioscorea collettii afforded one new dihydroisocoumarin, named (−)-montroumarin (1a), along with five known compounds—montroumarin (1b), 1,1′-oxybis(2,4-di-tert-butylbenzene) (2), (3R)-3′-O-methylviolanone (3a), (3S)-3′-O-methylviolanone (3b), and (RS)-sativanone (4). Their structures were elucidated using extensive spectroscopic methods. To the best of our knowledge, compound 1a is a new enantiomer of compound 1b. The NMR data of compound 2 had been reported but its structure was erroneous. The structure of compound 2 was revised on the basis of a reinterpretation of its NMR data (1D and 2D) and the assignment of the ¹H and ¹³C NMR data was given rightly for the first time. Compounds 3a–4, three dihydroisoflavones, were reported from the Dioscoreaceae family for the first time. The cytotoxic activities of all the compounds were tested against the NCI-H460 cell line. Two dihydroisocoumarins, compounds 1a and 1b, displayed moderate cytotoxic activities, while the other compounds showed no cytotoxicity.

1. Introduction

Dioscorea collettii HK. f. (Dioscoreaceae) is a perennial herbaceous plant of the Dioscorea genus that is widely distributed in Southwest China, Myanmar, and India. The rhizomes of D. collettii have been used clinically in combination with its sister species D. collettii HK. f. var. hypoglauca in the treatment of gouty arthritis, hyperuricemia, hyperlipidemia, inflammation, and tumors in China as a traditional medicine [1,2,3,4,5]. Previous studies have revealed that the major active constituents of D. collettii are steroidal saponins [6,7]. An unusual tricyclic diarylheptanoid derivative with thirty-four compounds has been reported on the basis of the previous work of our group [8,9]. Pharmacological studies have demonstrated that steroidal saponins of the Dioscorea genus have significant tumor-suppressive activities in human cancer cells [10,11]. In our previous study, tsaokoarylone, a diarylheptanoid obtained from D. collettii, also showed strong cytotoxic activity against the NCI-H460 cell line [8]. However, to date, the chemical constituents of D. collettii and whether its other types of compounds have significant cytotoxic activities have remained unclear.

As part of our continuous search for structurally unique and biologically valuable natural products from the Dioscorea genus [8,9,12,13], this study aimed to investigate the phytochemical constituents and cytotoxic activity of D. collettii. In the present study, one new dihydroisocoumarin, named (−)-montroumarin (1a), together with five known compounds were obtained from the rhizomes of D. collettii (Figure 1). Compound 1a is the enantiomer of the known compound montroumarin (1b). Compound 2 was first discovered as a natural product from the marine mollusk Onchidium struma [14]. The originally proposed structure of compound 2 (2′) (Figure 1) possessed a phenyl ether skeleton bearing four tert-butyl substituents located in the aromatic rings at the positions C-1, C-4, C-1′, and C-4′. However, during our structure elucidation of compound 2, a closer inspection of its NMR data indicated that the originally proposed structure of compound 2 might not be correct. Herein, details of the isolation and structure elucidation of these compounds, the structural revision of compound 2, and their cytotoxic activities against the NCI-H460 cell line are described.

2. Results and Discussion

2.1. Structural Elucidation of the Compounds

Compound 1 (1a/1b) was obtained as a colorless solid. Its molecular formula was elucidated to be C₁₅H₁₂O₄ by HR-ESI-MS at m/z 279.0626 for [M + Na]⁺ (calculated. for C₁₅H₁₂O₄Na, 279.0633), with ten degrees of unsaturation. The ¹³C NMR data (

Table 1. ¹H-NMR (600 MHz), ¹³C NMR (150 MHz) data, and HMBC correlations of compound 1 in CD₃OD.

No.	δH, mult (J in Hz)	δC	HMBC (H→C)
1		171.5, C
3	5.56, dd (12.0, 3.0)	81.8, CH	C-1, C-4, C-4a, C-1′, C-2′, C-6′
4	3.21, dd (16.3, 12.2) 3.06, dd (16.4, 3.2)	36.0, CH2	C-3, C-4a, C-5, C-8a, C-1′
4a		143.4, C
5	6.26, br s	108.0, CH	C-4, C-6, C-7, C-8a
6		166.4, C
7	6.24, d (1.8)	102.4, CH	C-5, C-6, C-8, C-8a
8		165.7, C
8a		101.7, C
1′		140.1, C
2′, 6′	7.48, d (7.5)	127.3, CH	C-3, C-3′, C-4′, C-5′
3′, 5′	7.41, t (7.5)	129.7, CH	C-1′, C-3′, C-5′
4′	7.36, t (7.0)	129.7, CH	C-2′, C-6′

) in combination with analysis of the HSQC spectrum for 1 showed the presence of one carbonyl carbon, nine olefinic or aromatic carbons, one oxymethine, and one methylene. In its ¹H NMR spectrum, the five aromatic proton signals between δ_H 7.48 and 7.36 (δ_H 7.48 (2H, d, J = 7.5 Hz), 7.39 (2H, t, J = 7.5 Hz), and 7.36 (1H, t, J = 7.0 Hz)) indicated the presence of one mono-substituted benzene ring. Two meta-coupled aromatic signals at δ_H 6.26 (1H, br s) and 6.24 (1H, J = 1.8 Hz) suggested that 1 possessed one 1,2,3,5-tetrasubstituted aromatic ring. Additionally, one oxymethine proton at δ_H 5.56 (1H, dd, J = 12.0, 3.0 Hz) and one methylene group at δ_H 3.21 (1H, dd, J = 16.3, 12.2 Hz) and 3.06 (1H, dd, J = 16.4, 3.2 Hz) were observed in the ¹H NMR spectrum, perfectly matching the above ¹³C NMR data. In addition to the two aromatic rings and one carbonyl, the remaining 1 degree of unsaturation suggested the existence of a one ring structure. All ¹H and ¹³C chemical shifts for 1 were essentially identical to those observed for montroumarin [15]. Based on the interpretation of the HMBC and ¹H-¹H COSY spectra (Figure 2) as well as a comparison with the literature data, the planar structure of 1 was characterized as 6, 8-dihydroxy-3-phenyl-3, 4-dihydroisocoumarin.

Due to having a single C-3 stereogenic center, 1 might exist in two potential configurations, (3R)-1 and (3S)-1, and only the 3S-form (montroumarin) has been isolated previously [15]. Subsequent chiral HPLC separation on 1 afforded compounds 1a and 1b, a pair of enantiomers, in a ratio of 15:85 (Supplementary materials Figure S8), showing that 1 is a scalemic mixture. The opposite optical rotations and mirror-like electronic circular dichroism (ECD) spectra confirmed their enantiomeric relationship (Figure 3). The absolute configuration of 1a and 1b was determined by comparing their ECD spectra and optical rotations with those of montroumarin [15] and other similar dihydroisocoumarin derivatives [16]. The absolute configuration of (+)-1 (1b) was established as S on the basis of the strong positive Cotton effect (CE) at 233 nm in its ECD spectrum (Figure 3), which is consistent with that of montroumarin. However, (−)-1 (1a) showed a strong negative CE at 233 nm; thus, its absolute configuration was deduced to be R. A comparison of the optical rotations of 1a and 1b with that of montroumarin also supported the above conclusions. Thus, the structure of compound 1a was defined and named as (−)-montroumarin, and 1b was identified as the known compound montroumarin (see Figure 1).

Compound 2 was purified as a pale-yellow oil. The ¹H NMR spectrum (

Table 2. ¹H-NMR (600 MHz), ¹³C NMR (150 MHz) data, and HMBC correlations of compound 2 in CDCl₃.

No.	δH, mult (J in Hz)	δC	HMBC (H→C)
1 (1′)		147.8 (147.8), C
2 (2′)		138.6 (138.7), C
3 (3′)	7.36, t (2.5)	124.6, CH	C-7, C-11, C-5, C-1 (C-7′, C-11′, C-5′, C-1′)
4 (4′)		147.2, C
5 (5′)	7.13, dd (8.6, 2.5)	124.1, CH	C-11, C-6, C-3, C-1 (C-11′, C-6′, C-3′, C-1′)
6 (6′)	7.54, d (8.6)	119.3, CH	C-2, C-4 (C-2′, C-4′)
7 (7′)		35.0, C
8/9/10 (8′/9′/10′)	1.33, s	30.4, CH3	C-7, C-2 (C-7′, C-2′)
11 (11′)		34.7, C
12/13/14 (12′/13′/14′)	1.28, s	31.6, CH3	C-11, C-4 (C-11′, C-4′)

) of 2 in CDCl₃ showed signals attributable to an ABX-type aromatic ring at δ_H 7.54 (1H, d, J = 8.6 Hz, H-6),7.36 (1H, t, J = 2.5 Hz, H-3),and 7.13 (1H, dd, J = 8.6, 2.5 Hz, H-5), which indicated the presence of a 1,2,4-trisubstituted benzene ring [17,18]. In addition, two sets of non-equivalent signals each containing three methyl groups at δ_H 1.33 (9H, s, H-8/9/10) and 1.28 (9H, s, H-12/13/14) were also observed. The ¹³C NMR and HSQC spectra displayed 12 carbon resonances, which were classified as eight olefinic or aromatic carbons (δ_C 147.8, 147.8, 147.2, 138.7, 138.6, 124.6, 124.1, and 119.3), two quaternary carbons (δ_C 35.0 and 34.7), and two methyl carbons (δ_C 31.6 and 30.4). The ¹H and ¹³C NMR data in CDCl₃ (Figure S15) for compound 2 were highly consistent with the experimental data for 2, 2′-oxybis (1, 4-di-tert-butylbenzene) (2′), suggesting that 2 was the same substance isolated by Sun B-N and coworkers [14]. The molecular formula of compound 2′ is C₂₈H₄₂O and the structure of 2′ was originally identified as a phenyl ether derivative bearing four tert-butyl substituents located in the aromatic rings at the positions C-1, C-4, C-1′, and C-4′ (Figure 1). We subsequently performed 2D NMR experiments on 2. Careful examination of the HMBC spectrum (Figure 2) of 2 revealed a strong HMBC correlation from H-5 (δ_H 7.13) to C-1 (δ_C 147.8), which is apparently inconsistent with structure 2′ (from H-2 to C-5 of 2′) but consistent with 2. This key HMBC correlation indicated that the structure elucidation of 2′ is clearly incorrect. The ¹³C NMR data (δ_C 147.8, 147.8, 138.7, and 138.6) and the molecular formula of 2′ indicated that 2 was a dimer. In the HMBC spectrum, the correlation from the three equivalent methyl groups signals at δ_H 1.33 (9H, H-8/9/10) to the quaternary carbon signal at δ_C 35.0 (C-7) suggested a direct connection between C-8/9/10 (δ_C 30.4) and C-7, forming one tert-butyl substituent, which was further confirmed by the HMBC correlations from H-8 (δ_H 1.33, 9H) to C-9/10 (δ_C 30.4). This tert-butyl substituent attached to C-2 (δ_C 138.6) of the aromatic ring was clarified by the correlation from H-8/9/10 to C-2 in the HMBC spectrum. Similar, the HMBC correlations from H-12/13/14 (δ_H 1.28, 9H) to C-11 (δ_C 34.7) and C-4 (δ_C 147.2) suggested a connection between C-12/13/14 (δ_C 31.6) and C-11, forming another tert-butyl substituent attached to C-4 of the aromatic ring. The ¹H-¹H COSY correlation (Figure 2) of H-5/H-6 (δ_H 7.54) and the HMBC correlations of H-5/C-11 and H-6/C-4 elucidated the connectivity of C-4/C-5/C-6. The linkage between C-2 and C-3 was verified by the HMBC correlation from H-3 (δ_H 7.36) to C-7. The HMBC correlations from H-5 to C-3 (δ_C 124.6) and H-3 to C-5 (δ_C 124.1), together with the C-3 and C-5 located at the meta-position of the aromatic ring, indicated the connectivity of C-2/C-3/C-4/C-5/C-6. The established linkage of C-2/C-3/C-4/C-5/C-6 as well as the HMBC correlations from H-3 and H-5 to C-1 (δ_C 147.8) constructed the aromatic ring. Considering the downfield chemical shift of C-1, and the molecular formula required for 2, C-1 and C-1′ should be linked to the remaining one oxygen atom to form a dimer. The structure of 2, shown in Figure 1, was identified as 1,1′-oxybis(2,4-di-tert-butylbenzene).

This explained well the strong HMBC correlation from H-5 (δ_H 7.13) to C-1 (δ_C 147.8), which is apparently inconsistent with structure 2′. In addition, all the HMBC correlations reported for 2′ [14], including the weak ⁴J_C,H long-range HMBC correlation from H-6 (δ_H 7.54) to C-7 (δ_C 35.0) [19,20], were also consistent with structure 2. Moreover, compound 2 might be a naturally occurring dimer of 2,4-Di-tert-butylphenol (2,4-DTBP), which is a common secondary metabolite produced by various groups of organisms [21]. Therefore, we compared the chemical shifts of compounds 2, 2′, and 2,4-DTBP and found that the structure elucidation of 2 was more reasonable than that of 2′ [22,23] (Figure S15). When running a ¹³C spectrum prediction for compounds 2,4-DTBP and 2′, it is trivial to see that the ¹³C-data of 2,4-DTBP are consistent with structure 2, whereas in the case of 2′ there is a massive inconsistency. By searching SciFinder, we found that the structure of 2 was not reported in any literature or patents but only had one commercial source. This was the first report of its detailed structure elucidation based on 1D and 2D NMR spectroscopy data.

Compound 3 (3a/3b) was obtained as a colorless needle. The results of a comparison between the ¹H, ¹³C NMR data of 3 and those reported by Guimarães et al. [24] suggested that the planar structure of 3 was 3′-O-methylviolanone. Compound 3 was optically inactive, suggesting that it was a racemic mixture. Both of the two configurations (3R)-3 and (3S)-3 have been reported previously [25,26], but the ECD spectra of (3R/3S)-3 and the specific rotation of (3R)-3 have remained undefined. Subsequent chiral resolution of 3 (Figure S18) afforded a pair of enantiomers, 3a and 3b, and they displayed almost mirror-image ECD curves that showed opposite CEs at 193, 210, 230, 272, and 325 nm. The respective absolute configurations of (−)-3 and (+)-3 (3a and 3b) were defined as (3R) and (3S) via a comparison of their experimental and calculated ECD spectra (Figure 3).

Compound 4 was also obtained as a colorless needle. A comparison of the ¹H and ¹³C NMR data of 4 with those of compound 3 showed that their structures are closely related, suggesting that 4 is also a dihydroisoflavone analogue. Through a comparison of its NMR data with those reported [27], the planar structure of 4 was identified as sativanone. Similarly to compound 3, 4 was also optically inactive, suggesting that it was a racemate. Both of the two configurations (3R)-4 and (3S)-4, including their ECD spectra and the specific rotations, have been reported [28,29]. Thus, compound 4 was identified as (RS)-sativanone.

2.2. Cytotoxic Activity against NCI-H460 Cell Line

All the isolated compounds 1–4 were further evaluated for their cytotoxicity against the human lung cancer NCI-H460 cell line (

Table 3. The IC₅₀ value of compounds on NCI-H460 cells (μM).

Sample	IC50 (μM)	Sample	IC50 (μM)
Compound 1a	33.37	Compound 1b	32.06
Compound 2	>100	Compound 3a	57.83
Compound 3b	59.34	Compound 4	64.01
Cisplatin	1.27

). The results indicated that compounds 1a and 1b, two dihydroisocoumarins, possess moderate cytotoxic activities, with IC₅₀ values of 33.37 and 32.06 μM, respectively. Compounds 2–4 showed no activity, with an IC₅₀ > 50 μM.

3. Materials and Methods

3.1. General Information

One-dimensional- and two-dimensional-NMR spectra were obtained from a Bruker Avance III 600 MHz spectrometer (Bruker BioSpin Inc., Zurich, Switzerland). Optical rotations were measured with an MCP 200 Modular Circular Polarimeter (Anton Paar, Graz, Austria). HRESIMS spectrum was obtained from a MicrOTOF-Q II ESI mass spectrometer (Bruker, Bremen, Germany). UV spectrum was obtained from a Hitachi U-3900 UV-visible spectrophotometer (Hitachi High-Tech Science Corporation, Tokyo, Japan). ECD spectra were recorded by a MOS-500 Circular Dichroism spectropolarimeter (Bio-Logic, Grenoble, France). Semi-preparative HPLC was performed on an LC-6A (Shimadzu, Kyoto, Japan) equipped with preparative YMC Pack ODS-A column (250 × 20 mm, 5 μm, YMC, Kyoto, Japan). A Daicel Chiralpak AD-H (250 × 4.6 mm, 5 μm, Daicel, Tokyo, Japan) was used for the chiral separations. Column chromatography (CC) was carried out over silica gel (100–200 and 200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and ODS RP-C18 (40–63 μm, YMC, Kyoto, Japan). Thin Layer Chromatography (TLC) was performed on pre-coated silica gel GF-254 (Qingdao Marine Chemical Factory, Qingdao, China).

3.2. Materials

The rhizomes of D. collettii were collected from Mount Emei, Leshan City, Sichuan province, China, and authenticated by Prof. Wenyuan Gao (School of Pharmaceutical Science and Technology, Tianjin University). A voucher specimen (ID: 245020328) was deposited in the School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China.

3.3. Extraction and Isolation

The air-dried rhizomes of D. collettii (16.2 kg) were extracted three times with 90% aqueous ethanol and three times with 60% aqueous ethanol under reflux (30 L, each for 2 h). After removal of the solvent under reduced pressure, the residue was combined and suspended in water to a final volume of 10 L, and then sequentially partitioned with petroleum ether (PE, 60–90 °C), ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH).

The EtOAc extract (305.0 g) was subjected to silica gel column chromatography (CC) eluted with CH₂Cl₂-MeOH gradient (10:0 to 0:10, v/v) to afford 19 fractions (A–S). Fraction G was purified by Sephadex LH-20 CC eluted with CH₂Cl₂-MeOH (1:1) to yield 5 fractions (G1–G5). Fraction G2 was exposed to silica gel CC eluted with PE-EtOAc gradient (1:0 to 7:3, v/v) followed by Sephadex LH-20 CC eluted with CH₂Cl₂-MeOH (1:1), and finally purified by semi-preparative HPLC (YMC C18, 250 × 20 mm, 5 μm, 10 mL/min) eluted with 65% aqueous MeOH to yield compound 1 (15 mg, t_R = 8 min). Fraction G4 was isolated by Sephadex LH-20 CC eluted with CH₂Cl₂-MeOH (1:1) to yield 51 subfractions (Fr.1–Fr.51). Fr.28–Fr.30 were submitted to silica gel CC eluted with PE-EtOAc (75:25, v/v) and then purified by Sephadex LH-20 CC eluted with CH₂Cl₂-MeOH (1:1) to obtain compound 3 (16 mg). Fr.31–Fr.39 was applied on silica gel CC eluted with PE-EtOAc (8:2, v/v) to obtain compound 4 (7 mg). Fraction L was chromatographed over silica gel CC with PE-EtOAc solvent system with increasing polarity to afford 3 fractions (L1–L3). Fraction L1 was isolated by Sephadex LH-20 CC eluted with CH₂Cl₂-MeOH (1:1) to obtain compound 2 (9 mg).

Compound 1 was separated using chiral-phase HPLC (AD-H column, n-hexane/ethanol, 70:30, flow rate: 1.0 mL/min) to afford 1b (t_R = 10.0 min) and 1a (t_R = 11.9 min). Compound 3 was further resolved using chiral-phase HPLC (AD-H column, n-hexane/ethanol, 85:15, flow rate: 1.0 mL/min) to obtain 3b (t_R = 8.2 min) and 3a (t_R = 9.2 min).

3.3.1. (−)-Montroumarin (1a)

Colorless solid; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ − 93.65 (c 0.20, MeOH); UV (MeOH) λmax 195, 217, 271, 303 nm; ¹H-NMR (600 MHz, CD₃OD) and ¹³C-NMR (150 MHz, CD₃OD) spectroscopic data, see Table 1; HR ESI-TOF MS m/z 279.0626 [M + Na]⁺ (calcd. for C₁₅H₁₂O₄Na 279.0633).

3.3.2. Montroumarin (1b)

Colorless solid; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ + 71.72 (c 0.20, MeOH); ¹H-NMR (600 MHz, CD₃OD) and ¹³C-NMR (150 MHz, CD₃OD) spectroscopic data, see Table 1; HR ESI-TOF MS data, see compound 1a.

3.3.3. 1,1′-Oxybis(2,4-di-tert-butylbenzene) (2)

Pale-yellow oil; ¹H-NMR (600 MHz, CDCl₃) and ¹³C-NMR (150 MHz, CDCl₃) spectroscopic data, see Table 2; HR ESI-TOF MS m/z 395.3373 [M + H]⁺ (calcd. for C₂₈H₄₃O 395.3314).

3.3.4. (3R)-3′-O-Methylviolanone (3a)

Colorless needle; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ − 39.17 (c 0.20, MeOH); ¹H-NMR (600 MHz, C₅D₅N) δ: 4.79 (1H, t, J = 11.3 Hz, H-2ax), 4.44 (1H, dd, J = 11.7 Hz, 5.4 Hz, H-3ax), 4.60 (1H, dd, J = 10.8 Hz, 5.5 Hz, H-2eq), 8.23 (1H, d, J = 8.6 Hz, H-5), 6.93 (1H, dd, J = 8.6 Hz, 2.2 Hz, H-6), 6.84 (1H, d, J = 2.2 Hz, H-8), 6.71 (1H, d, J = 8.5 Hz, H-5′), 7.03 (1H, d, J = 8.5 Hz, H-6′), 3.84 (1H, s, 2′-OMe), 3.92 (1H, s, 3′-OMe), 3.72 (1H, s, 4′-OMe). ¹³C-NMR (150 MHz, C₅D₅N) δ: 72.1 (C-2), 49.1 (C-3), 191.6 (C-4), 130.4 (C-5), 112.1 (C-6), 166.5 (C-7), 104.2 (C-8), 164.9 (C-8a), 115.6 (C-4a), 123.1 (C-1′), 153.1 (C-2′), 143.4 (C-3′), 154.5 (C-4′), 108.7 (C-5′),125.4 (C-6′), 61.3 (C-2′-OMe), 60.9 (C-4′-OMe), 56.4 (C-3′-OMe); HR ESI-TOF MS m/z 353.0998 [M + Na]⁺ (calcd. for C₁₈H₁₈O₆Na 353.1001).

3.3.5. (3S)-3′-O-Methylviolanone (3b)

Colorless needle; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ + 28.33 (c 0.20, MeOH); ¹H-NMR (600 MHz, C₅D₅N), ¹³C-NMR (600 MHz, C₅D₅N) spectroscopic data and HR ESI-TOF MS data, see compound 3a.

3.3.6. (RS)-Sativanone (4)

Colorless solid; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ − 0.06 (c 0.38, MeOH); ¹H-NMR (600 MHz, C₅D₅N) δ: 4.76 (1H, t, J = 11.1 Hz, H-2ax), 4.50 (1H, dd, J = 11.3 Hz, 5.2 Hz, H-3ax), 4.59 (1H, dd, J = 10.8 Hz, 5.3 Hz, H-2eq), 8.25 (1H, d, J = 8.6 Hz, H-5), 6.92 (1H, dd, J = 8.6 Hz, 2.1 Hz, H-6), 6.84 (1H, d, J = 2.1 Hz, H-8), 6.67 (1H, d, J = 2.0 Hz, H-3′), 6.58 (1H, dd, J = 8.3 Hz, 2.0 Hz, H-5′), 7.24 (1H, d, J = 8.3 Hz, H-6′), 3.63 (1H, s, 2′-OMe), 3.69 (1H, s, 4′-OMe). ¹³C-NMR (150 MHz, C₅D₅N) δ: 71.8 (C-2), 48.2 (C-3), 191.6 (C-4), 130.4 (C-5), 112.0 (C-6), 166.5 (C-7), 104.0 (C-8), 164.9 (C-8a), 115.7 (C-4a), 117.5 (C-1′), 159.4 (C-2′), 99.8 (C-3′), 161.4 (C-4′), 105.7 (C-5′), 131.7 (C-6′), 55.9 (C-2′-OMe), 55.6 (C-4′-OMe); HR ESI-TOF MS m/z 301.1006 [M + H]⁺ (calcd. for C₁₇H₁₇O₅ 301.1076).

3.4. Cytotoxicity Assays

Compounds 1–4 were evaluated for their cytotoxic activities by the MTT method using NCI-H460 cell line. The NCI-H460 cells were seeded at a density of 1 × 104/well in a complete growth medium in 96-well plates. The cells were incubated with the test compounds for 24 h before the MTT assay. Then, a fresh solution of MTT (0.5 mg/mL) was added to each single well of the 96-well plate. The plate was incubated in a CO₂ incubator for another 4 h. Finally, the cells were dissolved with 100 μL of DMSO and then analyzed in a multiwall plate reader with a wavelength of 570 nm.

4. Conclusions

The chemical investigation of D. collettii led to the isolation of six compounds (1–4), including one new dihydroisocoumarin, (−)-montroumari (1a). The structure of 2,2′-oxybis(1,4-di-tert-butylbenzene) (2′) was revised to be 1,1′-oxybis(2,4-di-tert-butylbenzene) (2), assisted by a careful re-examination of the structural elucidation process. All the compounds 1–4 were isolated from D. collettii for the first time. The isolation of compounds 3a, 3b, and 4 from the Dioscorea species has not been reported yet [30,31]. Notably, to the best of our knowledge, dihydroisoflavones have not been described from any other species in the Dioscoreaceae family. Our discovery of these dihydroisoflavones (3a, 3b, and 4) enriches the structural diversity of the Dioscoreaceae family. Dihydroisocoumarins (1a–1b) exhibited moderate cytotoxic activities against the NCI-H460 cell line, with IC₅₀ values ranging from 32.06 to 33.37 μM, whereas the other compounds, including the three dihydroisoflavones (3a–4), did not show any activities at the tested concentrations (IC₅₀ > 50 μM). Mounting evidence has revealed that steroidal saponins exert strong cytotoxic activities against human cancer cells, which means they could be considered as promising cytotoxic agents against human cancer cells. Dihydroisocoumarins and diarylheptanoids showed moderate cytotoxic activities against the NCI-H460 cell line. However, more selectivity studies are needed to determine whether these two types of compounds in D. collettii have cytotoxic activities against human cancer cells.