Taburnaemines A–I, Cytotoxic Vobasinyl-Iboga-Type Bisindole Alkaloids from Tabernaemontana corymbosa

Abstract

Nineteen vobasinyl-ibogan-type bisindole alkaloids, including nine new compounds, taburnaemines A–I (1–9), were isolated from the twigs and leaves of Tabernaemontana corymbosa. The structures and absolute configurations of the new alkaloids were determined by a combination of MS, NMR, and ECD analyses. Alkaloids 1–5 contain a rare 1,3-oxazinane moiety in the vobasinyl unit, while 6 has an uncommon 1,3-oxazolidine moiety in the iboga unit. The absolute configurations of alkaloid 1 and the known alkaloid tabernaecorymbosine A (10) were confirmed by single-crystal X-ray diffraction analysis. All of the bisindole alkaloids, except 2 and 16′-decarbomethoxytabernaecorymbosine A (14), showed antiproliferative activity (IC₅₀ 2.6–9.8 μM) against several human cancer cell lines, including A-549, MDA-MB-231, MCF-7, KB, and P-glycoprotein-overexpressing multidrug-resistant KB cells. The preliminary structure–activity relationship correlations are also discussed.

Graphical Abstract

Monoterpene indole alkaloids are a major group of plant alkaloids, found mainly in the families Apocynaceae, Loganiaceae, and Rubiaceae.¹ More than 3000 compounds of this type, including the anticancer drug vincristine and its derivatives, have been isolated and characterized.² These natural products are notable due to their structural complexity and diverse pharmacological activities.³ Plants of the genus Tabernaemontana are rich in monoterpene indole alkaloids, especially dimeric representatives. Tabercorymine A,⁴ alasmontamine A,⁵ and ervatensine B⁶ are recently reported examples with interesting structural frameworks and significant potential anticancer activity. During an ongoing investigation of these structurally unique and bioactive compounds,^4,7 we isolated 19 vobasinyl-ibogan-type bisindole alkaloids, including nine new compounds, taburnaemines A–I (1–9), from the twigs and leaves of Tabernaemontana corymbosa Roxb. (Apocynaceae). Alkaloids 1–6 are vobasinyl-iboga-type bisindole alkaloids characterized by 1,3-oxazinane and 1,3-oxazolidine moieties in the vobasinyl and iboga units, respectively. The 10 known bisindole alkaloids were identified as tabernaecorymbosine A (10),⁸ tabercorine B (11),^7d tabernaricatine C (12),⁹ tabernaricatine D (13),⁹ 16′-decarbomethoxytabernaecorymbosine A (14),⁸ tabernaecorymbosine B (15),⁸ tabernaricatine E (16),⁹ ervachinine C (17),^7b 16′-decarbomethoxydihydrovocamine (18),¹⁰ and conodurine (19).¹¹ Herein are discussed the isolation, structure elucidation, and antiproliferative activity against several human cancer cell lines of these isolated monoterpene indole alkaloids.

RESULTS AND DISCUSSION

Taburnaemine A (1) was obtained as colorless orthorhombic crystals (acetone) with ${[\alpha ]}_{\text{D}}^{20}$ −15.0 (c 0.25, MeOH). Its molecular formula, C₄₇H₅₆N₄O₈, with 22 degrees of unsaturation was established by HRESIMS (m/z 803.4022 [M − H]⁻, calcd 803.4022) and ¹³C NMR spectroscopic data (Table 2). Its UV spectrum showed the characteristic absorptions of an indole chromophore at 222 and 285 nm.¹² The IR absorptions at 3446 and 1717 cm⁻¹ implied the presence of amino or hydroxy group and ester carbonyl functions, respectively. The ¹³C NMR and DEPT spectroscopic data of 1 showed 47 carbon signals including seven methyls, nine methylenes, 15 methines, and 16 quaternary carbons, which indicated that 1 is a vobasinyl-ibogan-type bisindole alkaloid. Direct comparison of its NMR data with those of tabernaricatine A⁹ suggested 1 to be a 2-oxopropyl derivative of tabernaricatine A. From the HMBC correlations of H-21′ (δ_H 3.33, s) and H-22′b (δ_H 2.34, dd, J = 16.5, 8.5 Hz) to C-3′ (δ_C 54.8) the additional 2-oxopropyl group [C-23′ (δ_C 208.8), C-22′ (δ_C 46.7), and C-24′ (δ_C 31.0)] could be located at C-3′. Thus, the planar structure of 1 (taburnaemine A) was assigned and further verified by a combination analysis of the HSQC, ¹H–¹H COSY, and HMBC spectra (Figure 2).

Table 2.

¹³C NMR Data (δ) for Compounds 1–5

position	1a	2b	3c	4d	5c
2	137.2	137.2	138.7	137.4	137.8
3	35.3	34.5	36.6	36.6	36.0
5	61.8	62.7	62.8	62.2	61.7
6	30.1	30.4	31.0	29.5	26.4
7	108.7	108.7	109.4	109.1	109.7
8	128.9	129.0	129.8	129.4	130.3
9	118.2	118.1	118.8	117.7	118.9
10	119.7	119.5	119.8	119.2	119.6
11	122.4	122.3	122.6	121.8	122.5
12	110.2	110.0	111.1	110.1	110.8
13	135.0	135.9	137.3	135.7	137.9
14	32.3	31.8	33.6	33.6	36.5
15	42.1	36.3	43.4	41.9	40.3
16	50.0	48.8	50.8	49.9	47.6
17	72.6	73.7	73.0	72.7	77.0
18	17.1	13.6	17.5	17.2	11.6
19	59.4	57.5	59.1	59.3	113.6
20	64.8	63.8	65.2	64.7	142.7
21	89.7	89.8	90.2	89.6	50.3
22					88.8
N-Me	40.7	41.2	40.7	40.8
CO2Me	171.7	171.5	172.1	171.3	174.0
	50.5	50.9	53.1	50.4	50.5
2’	136.1	136.2	134.1	190.5	136.8
3’	54.8	54.5	175.2	48.7	62.2
5’	51.3	51.3	42.6	49.0	52.7
6’	22.0	22.0	21.1	33.9	22.3
7’	108.9	108.9	108.6	88.2	109.7
8’	124.3	124.5	124.4	134.5	125.4
9’	117.0	117.2	117.4	120.5	117.5
10’	104.5	104.7	105.9	132.2	106.3
11’	152.1	152.0	153.1	156.6	152.9
12’	114.2	114.3	115.6	104.0	116.2
13’	135.9	135.1	135.9	150.8	136.1
14’	30.6	30.5	38.8	27.0	28.8
15’	26.7	26.8	31.7	31.9	27.2
16’	54.2	54.3	55.7	49.9	55.1
17’	35.6	35.9	34.4	35.1	37.0
18’	11.6	11.6	11.5	11.5	11.8
19’	26.7	26.7	28.2	26.5	27.5
20’	38.2	38.2	35.8	37.5	38.8
21’	58.5	58.8	56.2	58.0	58.2
22’	46.7	46.7			62.0
23’	208.8	208.8
24’	31.0	31.0
CO2Me’	174.5	174.7	172.1	173.4	174.9
	52.3	52.5	50.6	53.2	52.7
OMe-11’	56.4	56.7	56.9	55.8	57.5

^a125 MHz, CDCl₃.

^b200 MHz, CDCl₃.

^c200 MHz, acetone-d₆.

^d150 MHz, CDCl₃.

Figure 2.

¹H–¹H COSY (A: bold lines), selected HMBC (A: →), and ROESY (B: ↔) correlations of 1.

The ROESY spectrum showed that 1 has the same relative configuration as tabernaricatine A, and the β-orientation of H-3′ was established by the ROESY correlation of H-3′ (δ_H 2.96) with H-17′b (δ_H 0.64, br d, J = 14.0 Hz). The absolute configuration of (3R,5S,15R,16S,19S,20R,21S,3′R,14′R,16′R,20′S,21′S)-1 was finally established by X-ray diffraction analysis with a Flack parameter of 0.05(4) (Figure 3).¹³

Figure 3.

Single-crystal X-ray structure of 1 drawn with 30% probability displacement ellipsoids (oxygen atoms are red, nitrogen atoms are blue, hydrogen atoms are cyan).

Taburnaemine B (2) and 1 gave the same molecular formula, as established by HRESIMS (m/z 805.4171 [M + H]⁺; calcd for C₄₇H₅₆N₄O₈, 805.4164) and ¹³C NMR spectroscopic data (Table 2). Comparison of the ¹H and ¹³C NMR data (Tables 1 and 2) of 2 and 1 suggested that both compounds are similar and have the same basic skeleton. The upfield shifts of C-15 (δ_C 36.3, Δδ_C −5.8) and C-18 (13.6, Δδ_C −3.5) observed in 2 suggested a γ-steric compression effect,^7d with the two compounds being epimeric at the C-19 and C-20 positions involving the oxirane ring. In 2, an α-orientation was assigned to the oxirane ring, and the complete structure of taburnaemine B (2) was assigned as shown in Figure 1.

Table 1.

¹H NMR Data (δ) for Compounds 1–5

position	1a	2b	3c	4d	5c
3	4.96 br d (12.0)	5.07 br d (12.0)	4.91 br d (12.0)	4.76 br d (11.4)	5.33 br d (13.6, 3.2)
5	3.53 dd (9.5, 6.0)	3.61 t (8.0)	3.50 dd (9.6, 6.4)	3.49 dd (10.2, 6.6)	4.14 t (9.6)
6a	4.02 dd (15.5, 9.5)	4.00 dd (15.2, 8.0)	4.17 dd (15.2, 9.6)	3.85 dd (15.0, 8.4)	3.75 dd (15.2, 11.2)
6b	3.20 dd (15.5, 6.0)	3.37 dd (15.2, 8.0)	3.25 dd (15.2, 6.4)	3.25 dd (15.0, 6.6)	3.58 dd (15.2, 7.2)
9	7.70 d (7.5)	7.69 d (8.0)	7.77 d (7.2)	7.53 dd (7.2, 1.2)	7.77 d (8.0)
10	7.14 dt (7.5, 2.0)	7.13 td (8.0, 1.6)	7.11 td (7.2, 1.6)	7.07 dd (7.2, 1.2)	7.10 t (8.0)
11	7.08 td (7.5, 2.0)	7.08 td (8.0, 1.6)	7.09 td (7.2, 1.6)	7.08 dd (7.2, 1.2)	7.06 t (8.0)
12	7.07 td (7.5, 2.0)	7.06 t (8.0)	7.15 dd (7.2, 1.6)	7.10 dd (7.2, 1.2)	7.13 d (8.0)
14a	3.08e	2.96 m	3.04 m	3.08 br d (15.0)	2.61 (dt, 16.0, 12.8)
14b	2.27 dt (15.0, 3.0)	1.89 m	2.34 m	2.21 br d (15.0)	1.94 ddd (16.0, 7.2, 3.2)
15	2.98e	2.97 t (7.2)	2.82 dd (15.2, 11.2)	2.89	3.81 dd (10.4, 7.2)
17a	3.89 d (8.5)	3.82 (d, 8.8)	3.74 d (8.8)	3.85 d (8.4)	3.59 br d (14.4)
17b	3.78 d (8.5)	3.68 d (8.8)	3.70 d (8.8)	3.76 d (8.4)	3.59 br d (14.4)
18	1.43 d (5.5)	1.34 d (4.8)	1.40 d (5.6)	1.45 d (5.4)	1.62 d (7.2)
19	3.05 q (5.5)	3.04 q (4.8)	3.03 q (5.6)	3.09 q (5.4)	5.10 q (7.2)
21a	3.57 s	3.50 s	3.63 s	3.61 s	4.26 br d (16.0)
21b					3.21 br d (16.0)
22a					4.66 d (9.6)
22b					4.57 d (9.6)
NH			9.88 s		9.72 s
N-Me	2.67 s	2.73 s	2.66 s	2.70 s
CO2Me	2.34 s	2.39 br s	2.34 s	2.27 s	2.47 s
3’a	2.96e	3.02e		2.72e	2.48 m
3’b				2.72e
5’a	3.13 ddd (14.5, 9.0, 6.5)	3.12 m	4.21 dd (12.0, 4.0)	3.39 t (13.8)	3.15 ddd (16.8, 13.6, 7.2)
5’b	3.07e	3.12 m	3.05 m	2.89	3.15 ddd (16.8, 13.6, 7.2)
6’a	2.98e	3.00 ddd (16.0, 9.6, 5.2)	3.03e	1.87 br d (12.0)	2.95 dt (16.8, 6.4)
6’b	2.83 dt (16.5, 5.5)	2.82 dt (16.0, 5.2)	3.03e	1.82 br d (12.0)	2.83e
9’	7.23 d (9.0)	7.22 d (8.8)	7.31 d (8.8)	6.85 s	7.23 d (8.0)
10’	6.80 d (9.0)	6.79 d (8.8)	6.88 d (8.8)		6.86 d (8.0)
11’
12’				7.11 s
14’	1.23 m	1.26 m	1.85 m	1.89 m	1.53 br s
15’a	1.43 m	1.30 m	1.73 ddd (12.8, 9.6, 3.2)	1.75 m	1.46 m
15’b	1.02 m	1.04 m	1.06 ddt (12.8, 6.4, 2.4)	1.07 m	1.30 m
17’a	1.67 br d (14.0)	1.79 br d (13.6)	0.54 br d (13.6)	2.73 br d (13.2)	1.85 br d (13.6)
17’b	0.64 br d (14.0)	0.74 br d (13.6)	1.55 br d (13.6)	2.39 br d (13.2)	0.64 br d (13.6)
18’	0.78 t (7.0)	0.79 t (7.2)	0.90 t (8.0)	0.83 t (7.2)	0.82 t (7.2)
19’a	1.30 m	1.45 m	1.43 m	1.41 m	1.27 m
19’b	1.01 m	1.30 m	1.26 m	1.41 m	1.12 m
20’	1.03 m	1.05 m	1.60 m	1.35 m	1.11 m
21’	3.33 br s	3.33 br s	4.33 br s	3.73 s	3.42 s
22’a	2.53 dd (16.5, 5.5)	2.56 dd (16.0, 4.0)			3.46 br d (10.4)
22’b	2.34 dd (16.5, 8.5)	2.36 dd (16.0, 8.8)			3.25 br d (10.4)
24’	2.03 s	2.03 s
CO2Me’	3.67 s	3.71 s	3.77 s	3.64 s	3.70 s
OMe-11’	3.92 s	3.93 s	3.96 s	3.94 s	3.96 s

^a500 MHz, CDCl₃.

^b800 MHz, CDCl₃.

^c800 MHz, acetone-d₆.

^d600 MHz, CDCl₃.

^eOverlapped, without designating multiplicity.

Figure 1.

Chemical structures of 1–10.

Taburnaemine C (3) was obtained as a white, amorphous powder with ${[\alpha ]}_{\text{D}}^{20}$ −33.3 (c 0.11, MeOH). HRESIMS data (m/z 761.3556 [M − H]⁻, calcd 761.3558) established its molecular formula as C₄₄H₅₀N₄O₈. The ¹³C NMR and DEPT spectroscopic data (Table 2) of 3 showed 44 carbon signals, including six methyls, eight methylenes, 14 methines, and 16 quaternary carbons. Detailed analysis of its NMR data indicated that 3 is analogous structurally to tabernaricatine A,⁹ except for the presence of an ester/amide carbonyl (δ_C 175.2) in the iboga unit of 3. The key HMBC correlations of protons H-5′a (δ_H 4.21, dd, J = 12.0, 4.0 Hz) and H-21′ (δ_H 4.33, br s) with the oxo carbon (δ_C 175.2) led to the assignment of the carbonyl group at C-3′. Detailed analysis of the 2D NMR spectra (HSQC, ¹H–¹H COSY, HMBC, and ROESY) confirmed the remaining molecular units to be the same as those of tabernaricatine A. The structure of alkaloid 3 was thereby characterized as shown in Figure 1.

Taburnaemine D (4) was obtained as a white, amorphous powder, and its HRESIMS signal at m/z 765.3785 ([M + H]⁺; calcd 765.3850) established the molecular formula C₄₄H₅₂N₄O₈ with 21 degrees of unsaturation. Detailed analysis of the NMR data (Tables 1 and 2) indicated that 4 is also a vobasinyl-ibogan bisindole alkaloid, similar to tabernaricatine A.⁹ The major difference was the presence of an iboga indolenine moiety (unit B) rather than the common iboga indole unit in 4, which was supported by the key carbon resonances for C-7′ (δ_C 88.2) and C-2′ (δ_C 190.5). Moreover, the presence of two singlets at δ_H 6.85 and 7.11 in the aromatic ring of the iboga unit indicated that the monomeric units in 4 are linked between C-3 and C-10′. This assignment was supported by the key HMBC correlations of H-3 (δ_H 4.76, br d, J = 11.4 Hz) to C-9′ (δ_C 120.5) and C-10′ (δ_C 132.2) and of H-9′ (δ_H 6.85) and H-12′ (δ_H 7.11) to C-11′ (δ_C 156.6). Furthermore, analysis of the 2D NMR spectra (HSQC, ¹H–¹H COSY, HMBC, and ROESY) verified the above structural conclusions. The relative configuration of 4 was identical to that of tabernaricatine A, and the α-orientation of OH-7′ was established by the ROESY correlation of H-21′ (δ_H 3.80, s) with OH-7′ (δ_H 5.49, br s) (Figure S35, Supporting Information).

Taburnaemine E (5) was obtained as a white, amorphous powder and gave a molecular formula of C₄₅H₅₄N₄O₇, as established by HRESIMS (m/z 763.3993 [M + H]⁺, calcd 763.4065). Its UV absorption maxima at 237, 261, and 284 nm were assigned as characteristic indole chromophores,¹² while the IR spectrum indicated the presence of amino or hydroxy (3430 cm⁻¹) and ester carbonyl (1725 cm⁻¹) functional groups. The ¹³C NMR and DEPT spectra show 45 carbon signals, classified as five methyls, 11 methylenes, 14 methines, and 15 quaternary carbons. Detailed analysis of the NMR data (Tables 1 and 2) indicated that the structures of tabernaricatine B⁹ and 5 are closely comparable with the exception of an added hydroxymethyl group (δ_H 3.46, 3.25, br d, J = 10.4 Hz; δ_C 62.0) and a nitrogenated methine (δ_H 2.48, m; δ_C 62.2) in 5. These two groups were assigned as C-22′ and C-3′, respectively, based on the key ¹H–¹H COSY correlations of H-3′ (δ_H 2.48) with H-22′ (δ_H 3.46, 3.25), as well as the HMBC correlations of H-21′ (δ_H 3.42, s) to C-3′ (δ_C 62.2) and of H-3′ to C-5′ (δ_C 52.7). The remaining structural units were identical with those of tabernaricatine B and were verified from the 2D NMR spectra (HSQC, ¹H–¹H COSY, and HMBC) (Figure 4). The ROESY correlation of H-3′ with H-17′b (δ_H 0.64, br d, J = 13.6 Hz) established the α-orientation of the 3′-hydroxymethyl group.

Figure 4.

¹H–¹H COSY (A: bold lines), selected HMBC (A: →), and ROESY (B: ↔) correlations of 5.

Taburnaemine F (6) was assigned the molecular formula C₄₄H₅₂N₄O₇ with 21 degrees of unsaturation, as established by HRESIMS ([M + H]⁺ at m/z 749.3920, calcd 749.3836). Based on the 1D (¹H and ¹³C) and 2D (¹H–¹H-COSY, HSQC, and HMBC) NMR data, the overall structure of 6 was found to be closely related to that of tabernaecorymbosine A,⁸ with both vobasinyl and iboga units. However, a striking difference was the presence of two oxygenated methines based on carbon resonances at δ_C 69.5 (δ_H 5.02, d, J = 6.0 Hz) and 93.9 (δ_H 4.75, d, J = 4.2 Hz)]. A ¹H–¹H COSY correlation between H-6′ (δ_H 5.02, d, J = 6.0 Hz) and H-5′a (δ_H 3.36, br d, 12.0 Hz) and a HMBC correlation between H-5′a and C-3′ (δ_C 93.9) supported the placement of the two oxygenated methines at C-6′ and C-3′, respectively. Although HMBC correlations were not observed between H-3′ (δ_H 4.75, d, J = 4.2 Hz) and C-6′ or between H-6′ and C-3′, the presence of an oxygen atom linking C-3′ and C-6′ was deduced from the higher molecular weight (14 mass units) and more degrees of unsaturation (one degree) in 6 compared with those of tabernaecorymbosine A. Thus, the structure of 6 was completely elucidated and characterized by a 1,3-oxazolidine moiety incorporating C-3′, N, C-5′, C-6′, and O in the iboga unit (Figure 5A). The ROESY correlation of H-21′ with H-5′ a suggested that CH₂-5 is α-oriented and, thus, H-3′ and H-6′ also have α-orientations (Figure 5B).

Figure 5.

¹H–¹H COSY (A: bold lines), selected HMBC (A: →), and ROESY (B: ↔) correlations of 6.

Taburnaemine G (7) was obtained as a white, amorphous powder, ${[\alpha ]}_{\text{D}}^{20}$ −62.5 (c 0.22, MeOH), and its HRESIMS signal at m/z 765.4222 ([M + H]⁺, calcd 765.4222) established the molecular formula C₄₅H₅₆N₄O₇ with 20 degrees of unsaturation. Comparison of the NMR data of 8 with those of tabernaecorymbosine A⁸ showed that both alkaloids are closely related, but differ in the presence of an additional hydroxymethyl (δ_H 3.45, dd, J = 11.0, 3.5 Hz; 3.23, dd, J = 11.0, 8.0 Hz; δ_C 61.9) in 7. The key HMBC correlation from H-21′ (δ_H 3.41, s) to C-3′ (δ_C 62.1) as well as the ¹H–¹H COSY correlation of H-3′ (δ_H 2.48, dd, J = 8.0, 3.5 Hz) with H-22′ (δ_H 3.45, 3.23) indicated that the hydroxymethyl is located at C-3′, which was identical with the iboga unit in 5. Therefore, 7 (taburnaemine G) was elucidated as shown based on analysis of 2D NMR (HSQC, ¹H–¹H COSY, HMBC, and ROESY) data.

Taburnaemine H (8) was obtained as a white, amorphous powder, and its HRESIMS signal at m/z 705.4013 ([M + H]⁺, calcd 705.4010) established the molecular formula C₄₃H₅₂N₄O₅. The ¹³C NMR and DEPT spectra of 8 (Table 4) suggested that 8 possesses 43 carbons, including five methyls, 10 methylenes, 14 methines, and 14 quaternary carbons. Alkaloid 8 could be readily identified as the 11′-demethoxy derivative of ervachinine C by comparison of their NMR data.^7b The observation of a ¹H–¹H COSY correlation between H-11′ (δ_H 6.86, d, J = 8.0 Hz) and H-12′ (δ_H 7.15, d, J = 8.0 Hz), in addition to the key HMBC correlations of H-9′ (δ_H 7.28, s) and H-11′ with C-3 (δ_C 46.2), verified the above conclusion. Thus, the structure of 8 (taburnaemine H) was fully elucidated.

Table 4.

¹³C NMR Data (δ) for Compounds 6–9

position	6a	7b	8c	9d
2	138.0	137.7	139.7	137.3
3	35.8	36.1	46.2	36.2
5	61.2	61.2	61.2	60.2
6	18.5	18.4	17.9	17.3
7	110.1	110.2	110.0	110.2
8	130.6	130.7	131.2	130.0
9	118.8	118.8	118.2	117.7
10	119.3	119.3	118.8	121.9
11	122.4	122.2	121.6	119.0
12	110.7	110.6	110.6	109.9
13	137.7	137.7	137.6	136.1
14	35.5	35.1	40.1	35.6
15	35.9	35.8	36.0	35.7
16	53.6	53.6	55.9	52.1
17	70.2	70.2	70.3	70.6
18	12.2	12.2	12.2	12.2
19	119.3	119.3	119.2	120.2
20	138.6	138.5	139.0	136.7
21	52.4	52.5	52.5	52.0
N-Me	42.5	42.5	42.4	42.1
CO2Me	174.6	173.5	173.5	173.9
	53.0	49.8	49.6	50.2
2’	138.3	136.2	138.9	190.4
3’	93.9	62.1	53.3	48.8
5’	60.9	52.8	54.3	49.0
6’	69.5	22.4	22.5	34.0
7’	117.9	109.6	110.4	88.2
8’	123.4	125.4	129.6	134.5
9’	116.5	117.4	117.6	120.1
10’	107.5	106.3	135.7	132.3
11’	152.9	152.8	122.3	156.9
12’	116.9	116.4	111.6	104.1
13’	134.0	136.8	138.2	150.9
14’	30.8	28.7	28.3	27.0
15’	30.4	27.5	32.9	32.0
16’	54.3	55.1	53.5	58.7
17’	31.6	37.0	36.8	35.1
18’	11.8	11.9	11.9	11.5
19’	27.2	27.1	27.8	26.6
20’	39.0	38.8	39.4	37.5
21’	60.7	58.3	57.8	58.1
22’		61.9
CO2Me’	173.3	174.9	175.1	173.4
	49.8	52.8	52.6	53.2
OMe-11’	57.6	54.7		56.0

^a150 MHz, acetone-d₆.

^b125 MHz, acetone-d₆.

^c200 MHz, acetone-d₆.

^d125 MHz, CDCl₃.

Taburnaemine I (9) was obtained as a white, amorphous powder, ${[\alpha ]}_{\text{D}}^{20}$ −23.0 (c 0.20, MeOH). The HRESIMS peak at m/z 751.4061 [M + H]⁺ (calcd 751.4065) established its molecular formula as C₄₄H₅₄N₄O₇. The IR absorptions at 3445 and 1722 cm⁻¹ suggested the presence of NH/OH and ester carbonyl functions, respectively. Comparison of NMR data of compound 9 with those of ervachinine C indicated that these two bisindole alkaloids share the same carbon skeleton.^7b The major difference was the replacement of the common iboga indole unit by an iboga indolenine unit in 9. HMBC correlations of H-5′b (δ_H 2.89, dd, J = 15.0, 3.0 Hz) and H-6′a (δ_H 1.90, br d, J = 13.0 Hz) with C-7′ (δ_C 88.2), as well as H-6′a with C-2′ (δ_C 190.4), verified the presence of the iboga indolenine unit. The 2D NMR spectra (HSQC, ¹H–¹H COSY, HMBC, and ROESY) confirmed that the remaining portion of the molecule was identical to tabernaricatine C (Figure 6A). The α-orientation of OH-7′ was established by the ROESY correlation of H-21′ (δ_H 3.78, s) with OH-7′ (δ_H 5.50, br s) (Figures 6B and S82, Supporting Information), and the structure of alkaloid 9 was thereby characterized as shown in Figure 1.

Figure 6.

¹H–¹H COSY (A: bold lines), selected HMBC (A: →), and ROESY (B: ↔) correlations of 9.

The absolute configurations of compounds 2–9 were identical to that of 1, as evidenced by similar curves in the ECD spectra (Figures S86 and S87, Supporting Information). Furthermore, the absolute configuration of (3R,5S,15S,16S,14′R,16′R,20′S,21′S)-10 was established initially on the basis of its similar ECD curve (Figures S86 and S87, Supporting Information), which was confirmed by X-ray diffraction analysis with a Flack parameter of 0.04(3) (Figure 7).¹³ Establishment of the absolute configurations of 1–10 provided solid evidence for those known vobasinyl-iboga-type alkaloids with unassigned absolute configurations.

Figure 7.

Single-crystal X-ray structure of 10 drawn with 30% probability displacement ellipsoids (oxygen atoms are red, nitrogen atoms are blue, hydrogen atoms are cyan).

All isolates were evaluated for antiproliferative activity against five human cancer cell lines, A-549, MDA-MB-231, KB, KB-VIN, and MCF-7, using the sulforhodamine B (SRB) method with vincristine (VIN) and paclitaxel (PXL) as positive controls.¹⁴ All of the compounds, except 2 and 14, exhibited antiproliferative activity (IC₅₀ 2.6–8.3 μM) against multidrug-resistant KB subline KB-VIN (Table 5). Based on the activities determined for 1 and 2, the absolute configurations of the 19,20-oxirane ring and 16′-carbomethoxy group appear to be critical factors contributing to the antiproliferative effects of such alkaloids.

Table 5.

Antiproliferative Activities of 1–19 (IC₅₀ in μM) in Vitro

	A-549	MDA-MB-231	KB	KB-VIN	MCF-7
1	8.6 ± 0.1	7.7 ± 0.1	7.4 ± 0.3	6.6 ± 0.1	>10
2	>10	>10	>10	>10	>10
3	5.9 ± 0.0	8.2 ± 0.1	5.7 ± 0.2	5.8 ± 0.2	8.3 ± 0.3
4	4.3 ± 0.2	4.5 ± 0.2	4.7 ± 0.0	4.8 ± 0.0	4.6 ± 0.1
5	4.7 ± 0.0	5.1 ± 0.1	4.8 ± 0.4	4.9 ± 0.3	6.0 ± 0.1
6	5.3 ± 0.0	4.8 ± 0.0	4.8 ± 0.1	4.7 ± 0.1	5.6 ± 0.2
7	4.3 ± 0.1	4.5 ± 0.2	4.5 ± 0.2	4.9 ± 0.0	4.9 ± 0.0
8	9.8 ± 0.0	5.2 ± 0.4	6.7 ± 0.4	8.3 ± 0.4	>10
9	3.1 ± 4.4	2.8 ± 3.9	2.7 ± 3.8	2.6 ± 3.6	3.0 ± 4.3
10	4.5 ± 0.1	4.8 ± 0.1	4.2 ± 0.2	4.1 ± 0.0	5.3 ± 0.1
11	4.9 ± 0.0	8.2 ± 0.2	4.5 ± 0.1	4.7 ± 0.2	6.5 ± 0.0
12	4.3 ± 0.2	4.5 ± 0.0	4.3 ± 0.2	4.4 ± 0.3	4.8 ± 0.0
13	3.5 ± 0.1	4.8 ± 0.1	4.3 ± 0.1	4.7 ± 0.1	4.8 ± 0.1
14	>10	>10	>10	>10	>10
15	4.2 ± 0.1	4.3 ± 0.1	4.1 ± 0.1	4.3 ± 0.1	4.9 ± 0.1
16	4.4 ± 0.0	3.2 ± 0.1	4.6 ± 0.1	4.4 ± 0.0	4.4 ± 0.1
17	5.6 ± 0.1	4.8 ± 0.1	5.0 ± 0.1	5.1 ± 0.0	6.1 ± 0.6
18	4.3 ± 0.0	4.5 ± 0.1	4.8 ± 0.1	4.4 ± 0.1	4.7 ± 0.3
19	4.4 ± 0.0	5.2 ± 0.0	5.0 ± 0.2	4.1 ± 0.2	5.6 ± 0.0
VIN (nM)a	23.0 ± 2.4	32.0 ± 0.5	4.4 ± 0.1	2480 ± 28.2	7.3 ± 0.2
PXL (nM)b	4.5 ± 0.9	7.0 ± 0.9	3.6 ± 1.1	2360 ± 59.5	8.8 ± 1.0

^aVIN = vinblastine.

^bPXL = paclitaxel. VIN and PXL were positive controls.

EXPERIMENTAL SECTION

General Experimental Procedures.

Melting points were measured using a Yuhua X-4 digital microdisplay melting point apparatus. Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. ECD spectra were recorded with an Applied Photophysics Chirascan spectrometer. IR spectra were recorded on a Tenor 27 spectrophotometer as KBr pellets. NMR spectra were performed on Bruker AV-500, 600, and 800 instruments with tetramethylsilane as the internal standard. Mass spectra were measured on VG Auto Spec-3000 or API-Qstar-Pulsar instruments. X-ray data were collected using a Bruker APEX DUO instrument. Semipreparative HPLC was performed on a Waters X-Bridge (5 μm; 10 mm × 150 mm) C₁₈ reversed-phase column. Column chromatography (CC) was performed on silica gel (100–200, 200–300, and 300–400 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), Sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), and MCI gel 20P (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). The visualizing reagent was bismuth potassium iodide.

Plant Material.

The twigs and leaves of T. corymbosa were collected in December 2014 from Menglun County of Yunnan Province, People’s Republic of China, and were identified by Mr. Shun-Cheng Zhang, Xishuangbanna Tropical Plant Garden. A voucher specimen (no. 20141228) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Science (CAS).

Extraction and Isolation.

The dried twigs and leaves of T. corymbosa (20 kg) were extracted with CH₃OH, and the pH of the crude extract was adjusted to 2–3 with saturated HCl. The acidic mixture was then extracted three times with petroleum ether and ethyl acetate, respectively. The aqueous phase was basified to pH 9–10 with saturated sodium hydroxide and then extracted with CH₂Cl₂ to obtain the crude alkaloids present. The crude alkaloids (108 g) were subjected to silica gel CC (100–200 mesh; petroleum ether/acetone, 1:0 → 0:1), yielding five major fractions (A–E). Fraction A (17.9 g) was subjected to a series of silica gel CC eluting with petroleum ether/acetone (20:1–10:1, v/v) and then purified by Sephadex LH-20 (acetone) to give compounds 10 (65 mg), 17 (86 mg), and 19 (45 mg). Fraction B (32 g) was further purified by reversed-phase chromatography on a C₁₈ column (MeOH/H₂O, 30:70 → 100:0, v/v) to give three subfractions (BI–III). Subfraction BI (2.3 g) was chromatographed by silica gel CC (petroleum ether/acetone/Et₂NH, 15:1:0.1, v/v) to afford 9 (190 mg), 11 (12 mg), and 13 (9 mg). Subfraction BII (3.2 g) was further separated using a Sephadex LH-20 column (MeOH), followed by semipreparative HPLC using a Waters X-Bridge C₁₈ (10 × 150 mm, 5 μm) column with CH₃CN/H₂O (73:27, 0.1% v/v diethylamine), to give 1 (4.0 mg, t_R = 45.6 min), 2 (1.8 mg, t_R = 46.9 min), 4 (6 mg, t_R = 31.5 min), 5 (9.8 mg, t_R = 28.8 min), and 16 (22 mg, t_R = 43.2 min). Fraction C (26 g) was subjected to silica gel CC eluting with petroleum ether/acetone (10:1–0:1, v/v) to give three subfractions (CI–III). Subfraction CI (2.6 g) was separated using a Sephadex LH-20 column (acetone) to give 6 (1.6 mg). Subfraction CII was separated by semipreparative HPLC using a Waters X-Bridge C₁₈ (10 × 150 mm, 5 μm) column with CH₃CN/H₂O (57:43, 0.1% v/v diethylamine) to afford 3 (6 mg, t_R = 48.6 min), 7 (1.5 mg, t_R = 36.1 min), and 8 (3.0 mg, t_R = 42.0 min). Subfraction CIII (900 mg) was separated using a Sephadex LH-20 column (acetone) followed by semipreparative HPLC using a Waters X-Bridge C₁₈ (10 × 150 mm, 5 μm) column with CH₃CN/H₂O (63:37, 0.1% v/v diethylamine) to afford 12 (4.6 mg, t_R = 53.2 min) and 18 (7 mg, t_R = 59.6 min). Silica gel CC (petroleum ether/acetone/Et₂NH, 8:1:0.1, v/v) of fraction D (22 g) gave three subfractions (I–III). Subfraction DII (1.3 g) was separated using a Sephadex LH-20 column (MeOH) followed by silica gel CC eluting with petroleum ether/acetone (10:1–0:1, v/v) to afford 14 (60 mg) and 15 (44 mg).

Taburnaemine A (1): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ −15.0 (c 0.25, MeOH); UV (MeOH) λ_max (log ε) 226 (3.80), 283 (3.11) nm; ECD (0.000 14 M, MeOH) λ_max (Δε) 222 (−98.9), 237 (+97.6) nm; IR (KBr) ν_max 3387, 1728, 1619 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 803 [M − H]⁻; HRESIMS m/z 803.4022 [M − H]⁻ (calcd for C₄₇H₅₆N₄O₈, 803.4022).

Taburnaemine B (2): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ −26.7 (c 0.14, MeOH); UV (MeOH) λ_max (log ε) 226 (3.78), 283 (3.06) nm; ECD (0.000 13 M, MeOH) λ_max (Δε) 222 (−87.4), 238 (+77.6) nm; IR (KBr) ν_max 3384, 1726, 1619 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 805 [M + H]⁺; HRESIMS m/z 805.4171 [M + H]⁺ (calcd for C₄₇H₅₆N₄O₈, 805.4164).

Taburnaemine C (3): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ −33.3 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 222 (3.69), 286 (3.02) nm; ECD (0.000 11 M, MeOH) λ_max (Δε) 221 (−102.3), 238 (+80.7) nm; IR (KBr) ν_max 3431, 1729, 1630 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 761 [M − H]⁻; HRESIMS m/z 761.3556 [M − H]⁻ (calcd for C₄₄H₅₀N₄O₈, 761.3558).

Taburnaemine D (4): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ +7.4 (c 0.18, MeOH); UV (MeOH) λ_max (log ε) 210 (3.66), 237 (3.87), 261 (3.27), 284 (3.27) nm; ECD (0.000 13 M, MeOH) λ_max (Δε) 222 (−104.7), 242 (+78.9) nm; IR (KBr) ν_max 3430, 2854, 1725, 1627 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 765 [M + H]⁺; HRESIMS m/z 765.3785 [M + H]⁺ (calcd for C₄₄H₅₂N₄O₈, 765.3850).

Taburnaemine E (5): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ −96.7 (c 0.09, MeOH); UV (MeOH) λ_max (log ε) 223 (3.84), 294 (3.13) nm; ECD (0.00009 M, MeOH) λ_max (Δε) 223 (−161.8), 238 (+102.9) nm; IR (KBr) ν_max 3431, 1724, 1627 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 763 [M + H]⁺; HRESIMS m/z 763.3993 [M + H]⁺ (calcd for C₄₅H₅₄N₄O₇, 763.4065).

Taburnaemine F (6): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ −7.1 (c 0.17, MeOH); UV (MeOH) λ_max (log ε) 205 (3.77), 224 (3.84), 383 (3.15) nm; ECD (0.00012 M, MeOH) λ_max (Δε) 211 (−26.8), 223 (−110.6), 238 (+90.9) nm; IR (KBr) ν_max 3431, 1729, 1630 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 3 and 4, respectively; ESIMS m/z 749 [M + H]⁺; HRESIMS m/z 749.3920 [M + H]⁺ (calcd for C₄₄H₅₂N₄O₇, 749.3836).

Table 3.

¹H NMR Data (δ) for Compounds 6–9

position	6a	7b	8c	9d	position	6a	7b	8c	9d
3	5.41 br d (12.6)	5.34 dd (13.0, 3.0)	4.66 br d (13.6)	5.10 br d (12.5)	5’a	3.36 br d (12.0)	3.71 m	3.36 dd (13.6, 7.2)	3.39 t (12.0)
5	3.84 t (9.0)	3.84 t (10.0)	3.80 t (8.8)	3.89 t (9.0)	5’b	3.27 dd (12.0, 4.8)	3.15 m	3.08 dd (13.6, 6.4)	2.89 dd (15.0, 3.0)
6a	3.56 dd (14.4, 9.6)	3.54 br d (8.0)	3.65 dd (12.8, 8.0)	3.52 dd (15.0, 9.5)	6’a	5.02 d (6.0)	2.94 dd (16.0, 6.0)	3.11 dd (9.6, 6.4)	1.90 br d (13.0)
6b	3.43 dd (14.4, 8.4)	3.49 br d (8.0)	3.30e	3.24 dd (15.0, 8.5)	6’b		2.82 dd (16.0, 6.0)	2.91 dd (9.6, 7.2)	1.81 br d (13.0)
9	7.64 d (7.2)	7.68 d (7.5)	7.55 d (7.2)	7.52 d (7.0)	9’	7.35 d (8.4)	7.21 d (8.5)	7.28 s	6.89 s
10	7.02 t (7.2)	7.03 td (7.5, 2.0)	6.96 t (7.2)	7.07 t (7.0)	10’	6.90 d (8.4)	6.84 d (8.5)
11	7.01 t (7.2)	7.01 td (7.5, 2.0)	6.96 t (7.2)	7.06 t (7.0)	11’			6.86 d (8.0)
12	7.08 d (7.2)	7.09 d (7.5)	7.10 d (7.2)	7.08 d (7.0)	12’			7.15 d (8.0)	7.11 s
14a	2.76 m	2.70 dd (13.5, 13.0)	2.83 m	2.66 dd (15.0, 8.0)	14’	1.94 m	1.51 m	1.86e	1.89
14b	1.85 m	1.87 br d (13.5)	1.94 m	1.91 br d (15.0)	15’a	1.71 m	1.26 m	1.73 m	1.75 m
15	3.70 dd (10.8, 7.8)	3.70 dd (11.5, 7.0)	3.67 br d (12.0)	3.50 t (15.0)	15’b	0.92 m	1.10 m	1.09 ddt (12.8, 6.4, 2.4)	1.06 dd (12.5, 7.0)
17a	3.81 d (12.6)	3.83 d (11.0)	3.82 d (10.4)	3.69 d (13.0)	17’a	1.92 br d (15.0)	1.87 m	2.71 dt (13.6, 2.4)	2.73 br d (13.5)
17b	3.63 d (12.6)	3.64 d (11.0)	3.63 d (10.4)	3.69 d (13.0)	17’b	1.50 br d (15.0)	0.64 br d (13.5)	1.86e	2.39 br d (13.5)
18	1.62 d (6.6)	1.62 d (7.0)	1.64 dd (6.4, 1.6)	1.66 d (7.0)	18’	0.79 t (7.2)	0.80 t (7.0)	0.87 t (8.0)	0.83 t (7.0)
19	5.33 q (6.6)	5.32 q (7.0)	5.35 q (6.4)	5.41 q (7.0)	19’a	1.46 m	1.27 m	1.56 m	1.40 m
21a	3.61 d (13.2)	3.56 d (13.5)	3.66 d (13.6)	3.64 d (13.5)	19’b	1.27 m	1.45 m	1.41 m	1.40 m
21b	2.92 d (13.2)	2.90 d (13.5)	2.91 d (13.6)	2.98 d (13.5)	20’	1.07 m	1.09 m	1.37 m	1.34 m
NH		9.60 br s	9.23 br s	7.69 br s	21’	3.34 d (10.8)	3.41 s	3.54 br s	3.72 s
N-Me	2.55 s	2.57 br s	2.54 s	2.57 s	22’a		3.45 dd (11.0, 3.5)
CO2Me	2.27 s	2.33 s	2.30 s	2.37 s	22’b		3.23 dd (11.0, 8.0)
3’a	4.75 d (4.2)	2.48 dd (8.0, 3.5)	2.93 dt (8.0, 3.2)	2.69 br d (18.5)	NH’		7.61 br s	9.38 br s
3’b			2.83 br d (8.0)	2.69 br d (18.5)	CO2Me’	3.79 s	3.72 s	3.65 s	3.65 s
					OMe-11’	3.96 s	3.95 s		3.98 s

^a600 MHz, acetone-d₆.

^b500 MHz, acetone-d₆.

^c800 MHz, acetone-d₆.

^d500 MHz, CDCl₃.

^eOverlapped, without designating multiplicity.

Taburnaemine G (7): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ −62.5 (c 0.22, MeOH); UV (MeOH) λ_max (log ε) 225 (3.84), 286 (3.11) nm; ECD (0.000 08 M, MeOH) λ_max (Δε) 223 (−161.9), and 239 (+ 87.3) nm; IR (KBr) ν_max 3431, 1729, 1630 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 3 and 4, respectively; ESIMS m/z 765 [M + H]⁺; HRESIMS m/z 765.4222 [M + H]⁺ (calcd for C₄₅H₅₆N₄O₇, 765.4222).

Taburnaemine H (8): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ −99.2 (c 0.15, MeOH); UV (MeOH) λ_max (log ε) 231 (3.74), 289 (3.16) nm; ECD (0.000 17 M, MeOH) λ_max (Δε) 225 (−40.6), 243 (22.07) nm; IR (KBr) ν_max 3429, 1723, 1638 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 3 and 4, respectively; ESIMS m/z 705 [M + H]⁺; HRESIMS m/z 705.4013 [M + H]⁺ (calcd for C₄₃H₅₂N₄O₅, 705.4010).

Taburnaemine I (9): white, amorphous powder; ${[\alpha ]}_{\text{D}}^{20}$ −23.0 (c 0.20, MeOH); UV (MeOH) λ_max (log ε) 211 (3.08), 237 (3.38), 262 (2.62), 286 (2.75) nm; ECD (0.000 37 M, MeOH) λ_max (Δε) 229 (−30.6), 246 (+18.3) nm; IR (KBr) ν_max 3428, 1728, 1625 cm⁻¹; ¹H NMR and ¹³C NMR data, see Tables 3 and 4, respectively; ESIMS m/z 751 [M + H]⁺; HRESIMS m/z 751.4061 [M + H]⁺ (calcd for C₄₄H₅₄N₄O₇, 751.4065).

X-ray Crystal Structure Analysis of Compounds 1 and 10.

Colorless crystals of 1 and 10 were recrystallized from acetone and MeOH, respectively. Intensity data were collected on a Bruker Apex Duo diffractometer equipped with an Apex II CCD using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT software. The structure was solved by direct methods using SHELXL-97. Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The H atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON.

Crystal Data of 1. C₄₇H₅₆N₄O₈, M = 804.95, a = 9.4439(2) Å, b = 18.2011(3) Å, c = 24.5795(4) Å, V = 4224.95(13) ų, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.699 mm⁻¹, 26 373 reflections measured, 7659 independent reflections (R_int = 0.0289). The final R₁ values were 0.0315 (I > 2σ(I)). The final wR(F²) values were 0.0818 (I > 2σ(I)). The final R₁ values were 0.0321 (all data), and the final wR(F²) values were 0.0824 (all data). The goodness of fit on F² was 1.044. Flack parameter = 0.05(4). Crystallographic data (excluding structure factor tables) for compound 1 have been deposited with the Cambridge Crystallographic Data Center as supplementary publication (deposit number CCDC 1579166).

Crystal Data of 10. C₄₄H₅₄N₄O₆·CH₄O, M = 766.95, a = 11.9273(2) Å, b = 15.9773(3) Å, c = 20.8434(4) Å, V = 3972.04(13) ų, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.695 mm⁻¹, 24 297 reflections measured, 7217 independent reflections (R_int = 0.0282). The final R₁ values were 0.0317 (I > 2σ(I)). The final wR(F²) values were 0.0811 (I > 2σ(I)), and the final R₁ values were 0.0318 (all data). The final wR(F²) values were 0.0811 (all data). The goodness of fit on F² was 1.088. Flack parameter = 0.04(3). Crystallographic data (excluding structure factor tables) for compound 10 were deposited with the Cambridge Crystallographic Data Center as supplementary publication (deposit number CCDC 1579167). Copies of the data can be obtained free of charge by application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44 (0) (1223) 336 033; e-mail: deposit@ccdc.cam.ac.uk].

Antiproliferative Activity Assay.

The antiproliferative activity of the compounds was determined by an SRB assay, as described previously.¹⁴ Briefly, cell suspensions were seeded on 96-well microtiter plates at a density of 4000–12 000 cells per well and treated with the test compounds. After a 72 h culture with the compounds, the cells were fixed in 10% trichloroacetic acid and then stained with 0.04% SRB. The absorbance at 515 nm of protein-bound dye solubilized with 10 mM Tris base was measured using a microplate reader (ELx800, BioTek) operated by Gen5 software (BioTek). Then, IC₅₀ data were calculated statistically (MS Excel) from at least three independent experiments performed with duplication. The following human tumor cell lines were used in this study: A549 (lung carcinoma), MDA-MB-231 (triple-negative breast cancer), KB (originally isolated from epidermoid carcinoma of the nasopharynx), KB-VIN (vincristine-resistant KB subline showing MDR phenotype by overexpressing P-gp), and MCF-7 (estrogen receptor-positive and HER2-negative breast cancer). The four cell lines A549, MDA-MB-231, KB, and MCF-7 were obtained from the Lineberger Comprehensive Cancer Center (UNC-CH) or from ATCC (Manassas, VA, USA). The KB-VIN cell line was a generous gift from Prof. Y.-C. Cheng of Yale University. The cells were cultured in RPMI-1640 medium supplemented with 2 mM l-glutamine and 25 mM HEPES (Corning), containing 10% fetal bovine serum (Corning), 100 μg/mL streptomycin, and 100 IU penicillin (Corning). KB-VIN stock cells were maintained in the presence of 100 nM vincristine. Vincristine and paclitaxel were used as experimental controls.