Geleganidines AâC, Unusual Monoterpenoid Indole Alkaloids from

Article pubs.acs.org/jnp

Geleganidines A−C, Unusual Monoterpenoid Indole Alkaloids from Gelsemium elegans Wei Zhang,†,‡ Xiao-Jun Huang,† Sheng-Yuan Zhang,† Dong-Mei Zhang,† Ren-Wang Jiang,† Jian-Yang Hu,† Xiao-Qi Zhang,† Lei Wang,*,† and Wen-Cai Ye*,†,‡ †

Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China

‡

S Supporting Information *

ABSTRACT: The first rotameric monoterpenoid indole alkaloids (MIAs), 1a and 1b, and two unusual dimeric MIAs, 2 and 3, with new dimerization patterns, together with their putative biosynthetic intermediates 4−7, were isolated from the roots of Gelsemium elegans. Compounds 2 and 3 represent the first natural aromatic azo- and the first urealinked dimeric MIAs, respectively. Their structures and absolute configurations were elucidated by means of NMR spectroscopy, single-crystal X-ray diffraction, and electronic circular dichroism data analyses. The interconverting mechanism of rotamers 1a and 1b was studied by density functional theory computation. Compounds 2 and 3 showed moderate cytotoxic activity against MCF-7 and PC-12 cells, respectively. In addition, a plausible biosynthesis pathway for the new alkaloids was proposed on the basis of the coexistence of their biosynthetic precursors.

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two new dimerization patterns of MIAs. Herein, we describe the isolation, structural elucidation, and antitumor activities of 1−7. In addition, the interconversion mechanism and equilibrium constant of rotamers 1a and 1b were studied by density functional theory computation. A plausible biogenetic pathway for the new alkaloids (1−3) was also proposed, inspired by the isolation of their putative biosynthetic precursors (4−7).

onoterpenoid indole alkaloids (MIAs) are a class of important natural products constructed from indole and monoterpene moieties.1 MIAs are well-known for their significant antitumor effects, especially the dimeric forms such as vincristine.2 The liana Gelsemium elegans (Loganiaceae), widely distributed in southern China, is a rich source of a class of structurally complex MIAs known as Gelsemium alkaloids. Although it is regarded as a highly toxic plant, G. elegans is used as a folk medicine for the treatment of cancer, furuncle, and carbuncle.3 Previous phytochemical investigations of this plant have led to the isolation of more than 100 Gelsemium alkaloids4−6 that showed antitumor, anti-inflammatory, and analgesic activities.7,8 The complex structures, multiple stereogenic centers, and unique biological effects of Gelsemium alkaloids make them challenging targets for organic chemists.9,10 In a search for structurally unique and biologically interesting natural products,11,12 we reported the isolation of several new alkaloids from G. elegans.13,14 In a continuing investigation, a pair of rotameric Gelsemium alkaloids, 1a and 1b, and two unusual dimeric alkaloids, 2 and 3, together with their putative biosynthetic precursors (4−7) were isolated from the roots of G. elegans. Structurally, geleganidine A (1) has a formyl group at N4 of the humantenine skeleton. Geleganidines B and C (2 and 3) are constructed from two monomeric units that are bridged by azo and carbonyl groups to form aromatic azo- and urea-containing substructures, respectively. Interestingly, 1 is the sole monoterpenoid indole alkaloid that exists as a pair of rotamers (1a and 1b) caused by restricted amide bond rotation. Compound 2 is the first naturally occurring aromatic azo-linked dimeric MIA, and 3 represents the first example of a urea-linked dimeric MIA. Geleganidines B and C (2 and 3) also represent © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The total alkaloid fraction of G. elegans was subjected to column chromatography over silica gel and ODS and preparative HPLC to yield compounds 1−7 (Figure 1). The molecular formula of geleganidine A (1) was determined to be C22H26N2O5 on the basis of its 13C NMR and HRESIMS data (m/z 399.1925 [M + H]+, calcd for C22H27N2O5, 399.1915). Compound 1 exists as a pair of rotamers, as evident from their interchange in solution (Figure 2). The 1H and 13C NMR spectra of 1 displayed two sets of signals in a ratio of approximately 5:4 (1a/1b, Figure 3). The 1H NMR spectrum for the major isomer 1a showed signals caused by three aromatic protons [δH 7.28 (1H, d, J = 8.3 Hz, H-9), 6.62 (1H, dd, J = 8.3, 2.5 Hz, H-10), 6.58 (1H, d, J = 2.5 Hz, H-12)], an N1-methoxy group [δH 3.94 (3H, s)], three oxygenated methylene and methine protons [δH 4.18 (1H, m, H-17α), 4.04 (1H, m, H-17β), 3.62 (1H, m, H-3)], and a formyl proton [δH 8.06 (1H, s, H-22)]. The 13C NMR spectrum of 1a displayed signals similar to those of humantenirine (6),15 except for the changes of the chemical shift of the C-5 and C-21 Received: April 24, 2015

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DOI: 10.1021/acs.jnatprod.5b00351 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Z configuration. To determine the absolute configuration of 1, ECD spectra was calculated using the time-dependent DFT method. The experimental ECD spectrum of 1 showed a positive Cotton effect at 238.8 nm (Δε +5.6) and a negative Cotton effect at 219.0 nm (Δε −38.8), consistent with the calculated ECD spectrum for the stereoisomer with a (3R,5S,7S,15R,16S) absolute configuration (Figure 5). The presence of two sets of NMR signals indicates that the interconversion between 1a and 1b was slow relative to the NMR time scale, which allowed for the accurate determination of the equilibrium constant (K = 0.8) and conformational free energy (ΔGexp = −RT ln K = 0.132 kcal/mol). Quantum chemical calculation was used to rationalize the mechanism and experimental findings using the Gaussian09 package.18 The partial-double-bond character of the formamide C−N bond in 1 arose from conjugation of the N lone pair of electrons with the carbonyl group. Thus, two stable planar conformations (E in 1a and Z in 1b) are feasible formed (Figure 6). The C−N bond lengths changed from 1.366 Å (1a, partial double bond) and 1.359 Å (1b, partial double bond) to 1.432 Å (transition state (TS), single bond), whereas the bond lengths of the carbonyl changed from 1.207 Å (1a) and 1.208 Å (1b) to 1.196 Å (TS), which indicates the conjugation between the N lone pair electrons and the carbonyl group. The computed energy of the transition state of 1b was only 24.60 kcal/mol higher than that of 1a, supporting the feasibility of isomerization between 1a and 1b. In addition, the calculated conformational free energy difference (ΔGcal = 0.121 kcal/mol) between 1a and 1b was consistent with their 5:4 ratio found experimentally in the isomeric equilibrium (Supporting Information). It is noteworthy that geleganidine A (1) is the first Gelsemium alkaloid found to exist as a pair of rotamers. From a biosynthesis point of view, geleganidine A (1) could be an important precursor of geleganidine C (3). Geleganidine B (2) was obtained as orange block crystals from MeOH. The molecular formula of 2 was established as C40H44N4O6 by its 13C NMR and HRESIMS (m/z 677.3338 [M + H]+, calcd for C40H45N4O6, 677.3334). The UV absorption maxima at 209 and 320 nm as well as the IR bands at 1702, 1603, 1497, and 1449 cm−1 imply the presence of a carbonyl and an aromatic ring. The 1H NMR spectrum of 2 shows signals for three aromatic protons [δH 7.41 (1H, d, J = 8.7 Hz, H-9), 6.95 (1H, dd, J = 8.7, 2.8 Hz, H-10), 7.26 (1H, d, J = 2.8 Hz, H-12)], a methoxy group [δH 3.78 (3H, s)], two oxymethylene and an oxymethine proton [δH 4.23 (1H, dd, J = 10.3, 2.0 Hz, H-17α), 3.89 (1H, dd, J = 10.3, 1.3 Hz, H-17β), 4.90 (1H, d, J = 5.0 Hz, H-3), respectively], and an ethylidene unit [δH 5.28 (1H, q, J = 6.9 Hz, H-19) and 1.52 (3H, d, J = 6.9 Hz, H-18)]. The 13C NMR spectrum of 2 exhibits 20 carbon signals, including a benzene ring [δC 159.5 (C-11), 151.3 (C-13), 132.3 (C-8), 128.8 (C-9), 118.1 (C-10), 101.5 (C-12)], an oxygenated methine [δC 80.5 (C-3)], an oxygenated methylene [δC 68.8 (C-17)], a carbonyl [δC 180.5 (C-2)], and an ethylidene unit [δC 118.6 (C-19), 12.8 (C-18)] (Table 2). The spectroscopic data are similar to those of N-demethoxy-11-methoxygelsemamide (4) except for the deshielded C-8 resonance. The NMR features combined with the molecular formula information and the major fragment ion at m/z 340.1779 [C20H24N2O3+] in the Q-TOF-MS/MS of 2 (Supporting Information) indicate that 2 may be a dimer of 4 linked via an azo unit. The 1H−1H COSY spectrum of 2 reveals the presence of the spin systems shown in Figure 7. In the HMBC spectrum, the correlations between H-9 (H-9′) and C-7 (C-7′)/C-11 (C-11′), H-12 (H-12′) and C-8 (C-8′)/C-10 (C-10′), H-3 (H-3′) and C-8 (C-8′)/C-17 (C-17′), H-17 (H-17′)

Figure 1. Structures of compounds 1−7.

Figure 2. HPLC chromatograms of the dynamic interconversion of 1a and 1b at different times.

resonances as well as the presence of the formyl group signal at δC 161.0 (C-22). With the aid of 2D NMR experiments, the 1H and 13C NMR data of 1a were assigned as shown in Table 1. The assignment of the formyl group at N4 was deduced by the HMBC correlations between H-5 and C-22 as well as those between H-21 and C-22. The presence of a formamide group indicates that interconversion between 1a and 1b results from restricted amide bond rotation.16,17 The NOESY spectrum of 1a reveals that the relative configurations of C-3, C-5, C-7, C-15, and C-16 in 1a are identical to those in humantenirine (6).15 Furthermore, the NOE correlation between H-5 and H-22 reveals that the N4−C22 bond in 1a was in the E configuration (Figure 4). Analysis of the 1D and 2D NMR spectroscopic data of minor isomer 1b reveals that the two isomers have the same 2D structure. The main differences between 1a and 1b involved resonances for CH-5, CH2‑6, and CH2-21, which implies that there is a difference in the geometry of the N−C22 bond. The NOESY spectrum of 1b exhibits a cross peak between H-21 and H-22 (Figure 4), indicating that the formyl−N4 amide bond has a B



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Figure 3. 1H NMR spectrum for the mixture of 1a (blue) and 1b (red) in CDCl3 after 24 h.

Table 1. 1H and 13C NMR Data of Compounds 1a and 1b (CDCl3, J in Hz) 1a no. 2 3 5 6

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 N-OMe Ar-OMe

δH

a,c

3.62 4.14 α 2.43 β 1.69

7.28 d (8.3) 6.62 dd (8.3, 2.5) 6.58 d (2.5) α 2.51 d (3.8) β 2.49 m 2.87 m 2.11 d (4.7) α 4.18 β 4.04 1.72 d (6.8) 5.61 dq (13.9, 6.8) α 4.30 β 4.30 8.06 s 3.94 s 3.82 s

(H-14α′)/H-16 (H-16′), and H-15 (H-15′) and H-14β (H-14β′)/ H-17β (H-17β′)/H-19 (H-19′) established the relative configuration of 2, as shown in Figure 8. Crystals suitable for X-ray diffraction were obtained via crystallization from MeOH. The stereostructure of 2 was established by X-ray diffraction analysis as showed in Figure 9. The final refinement of the Cu Kα data resulted in a small Flack parameter of 0.04 (18), allowing the assignment of the absolute configuration of 2. The ECD spectrum of 2 showed the same Cotton effects as those of 11-methoxygelsemamide (5), thus confirming the S configurations of C-7 and C-7′ (Supporting Information).19 Structurally, geleganidine B (2) is an aromatic azo compound. Aromatic azo compounds are high-value molecules because they are widely used in the chemical and medical industries.20−22 The only aromatic azo compound previously obtained from a natural source (Agaricus xanthodermus) was proven to be an artifact.23,24 It is noteworthy that 2 could be detected in the crude methanol extract of G. elegans by LCMS analysis (Supporting Information), confirming the natural occurrence of 2. Thus, geleganidine B (2) represents the first natural aromatic azo compound. Geleganidine C (3) was obtained as an amorphous powder whose molecular formula was deduced by 13C NMR data and the protonated ion at m/z 737.3550 [C42H49N4O8+] in the HRESIMS. The IR spectrum of 3 shows absorptions for an aromatic ring (1504 and 1459 cm−1) and a carbonyl group (1723 and 1631 cm−1). The UV spectrum displays absorption maxima at 209 and 287 nm, characteristic of indolin-2-one chromophores. The 1H NMR spectrum of 3 shows signals for an o-disubstituted benzene ring [δH 7.26 (1H, d, J = 7.2 Hz, H-9′), 7.22 (1H, dd, J = 7.2, 7.2 Hz, H-11′), 7.00 (1H, dd, J = 7.2, 7.2 Hz, H-10′), 6.91 (1H, d, J = 7.2 Hz, H-12′)], a 1,2,4-trisubstituted benzene ring [δH 7.15 (1H, d, J = 9.0 Hz, H-9), 6.50 (1H, dd, J = 9.0, 2.1 Hz, H-10), 6.49 (1H, d, J = 2.1 Hz, H-12)], two ethylidene groups [δH 5.48 (2H, q, J = 6.5 Hz, H-19/19′), 1.64 (6H, d, J = 6.5 Hz, H-18/18′)], two N-methoxy groups [δH 3.93 (3H, s), 3.92 (3H, s)], and an aromatic methoxy group [δH 3.78 (3H, s)]. The 13C NMR and DEPT spectra display 42 carbon signals. Among them, 19 signals are attributable to 2 indolin-2one moieties, and 20 signals could be assigned to two monoterpenoid moieties. In addition, in the 13C NMR spectrum, a urea-type carbonyl at δC 163.6 (C-22) was present. The above

1b δC

b

no.

173.6 73.1 57.2 35.4

2′ 3′ 5′ 6′

54.7 119.9 127.0 108.0

7′ 8′ 9′ 10′

161.0 95.1 140.5 30.1

11′ 12′ 13′ 14′

33.4 37.4 65.9

15′ 16′ 17′

13.5 121.4

18′ 19′

135.5 37.1

20′ 21′

161.0 63.6 55.8

22′ N-OMe′ Ar-OMe′

δH

a,c

3.61 4.97 m α 2.28 β 2.04 dd (15.2, 8.3)

7.22 d (8.2) 6.60 dd (8.3, 2.5) 6.55 d (2.5) α 2.43 d (3.8) β 2.37 m 2.77 m 2.25 m α 4.17 β 4.03 1.68 d (6.8) 5.51 dq (13.9, 6.8) α 4.49 d (4.5) β 4.08 d (4.5) 8.05 s 3.95 s 3.81 s

δ Cb 174.0 72.7 50.8 32.1

55.1 121.0 127.0 107.9 160.7 95.2 140.0 28.5 35.3 38.7 66.3 13.3 123.1 135.0 40.3 161.3 63.7 55.9

a Measured at 500 MHz. bMeasured at 125 MHz. cOverlapped signals are reported without designating multiplicity.

and C-5 (C-5′)/C-15 (C-15′), and H-21 (H-21′) and C-2 (C-2′)/C-5 (C-5′)/C-19 (C-19′) allowed for the assignment of the 2D structure of 2 (Figure 7). In the NOESY spectrum, the correlations between H-3 (H-3′) and H-9 (H-9′)/H-14β (H-14β′), H-5 (H-5′) and H-14α C



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Figure 4. Optimized structures and key NOESY correlations for 1a and 1b.

type molecule moiety (Figure 7). This was confirmed by the IR band for the urea unit at 1631 cm−1 and by the fragment ion peaks at m/z 397.0670 [C22H25N2O5+] and 367.0872 [C21H23N2O4+] in the Q-TOF-MS/MS that originated by fragmentation of the urea C−N bonds (Supporting Information). The relative configuration of 3 was elucidated by the NOESY experiment. The correlations between H-3 and H-14β, H-5 and H-14α/H-16, and H-15 and H-14β/H-17β/H-19 indicate that the relative configurations of C-3, C-5, C-15, and C-16 in 3 are identical to those of humantenirine (6) and rankinidine (7).15 The ECD spectrum of 3 shows negative and positive Cotton effects at 216.2 and 239.2 nm, respectively, suggesting S configurations at C-7 and C-7′.15 An MIA dimer linked via a carbonyl to form a urea-type molecule is reported for the first time. On the basis of the literature and our research results, a plausible biosynthetic pathway to compounds 1−3 is proposed (Scheme 1). The alkaloid humantenirine (6), a major component of Gelsemium plants, originates from strictosidine, which is derived from the Pictet−Spengler reaction between tryptamine and secologanin.25 N4-formylation of 6 would yield 1.26 The nucleophilic attack from the amino group in 7 to the aldehyde group in 1 followed by oxidation could afford 3. In contrast, 11-methoxygelsemamide (5) could be derived from humantenirine (6) by trans-lactamization processes. Reduction of the methoxyamino group in 5,27 may afford amine (4). Oxidative coupling of amine 4,28,29 may afford dimer 2. The inhibitory effects of compounds 1−7 on the viability of MCF-7, HepG2, HepG2/ADM, and PC-12 cells were

Figure 5. Calculated and experimental ECD spectra of 1.

data suggest that 3 could be a dimeric Gelsemium alkaloid in which the two monomeric units are connected via a urea-type carbonyl carbon unit. Comparison of the NMR data of 3 (Table 2) with those of known Gelsemium alkaloids revealed that the signals of 3 may be attributed to humantenirine (6) and rankinidine (7) moieties.15 The HMBC correlations between H-5/H-5′ and C-22 as well as those between H-21/H-21′ and C-22 suggest that the two monomeric units are linked by a carbonyl group to form a ureaD



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Figure 6. Intramolecular hyperconjugation interaction between the natural bond orbitals (NBOs) of the N lone pair of electrons (N4) and π* (C-22O) orbitals as well as the second-order perturbation energies (E(2)) in the chemical structures of 1a [LP(1)N4 → BD*(1)C-22−O (68.38 kcal/mol)] and 1b [LP(1)N4 → BD*(1)C-22−O (69.99 kcal/mol)], calculated at the B3LYP/6-31++g** level in Gaussian09.

Table 2. 1H and 13C NMR Data of Compounds 2 and 3 (CDCl3, J in Hz) 2 δHa,c

no. 2/(2′) 3/(3′) 5/(5′) 6/(6′) 7/(7′) 8/(8′) 9/(9′) 10/(10′) 11/(11′) 12/(12′) 13/(13′) 14/(14′) 15/(15′) 16/(16′) 17/(17′) 18/(18′) 19/(19′) 20/(20′) 21/(21′)

Ar-OMe a

4.90 d (5.0) 3.97 dd (9.0, 5.0) α 2.58 d (11.5) β 1.96 br d (13.3)

7.41 d (8.7) 6.95 dd (8.7, 2.8) 7.26 d (2.8) α 2.41 β 1.96 br d (13.3) 2.42 2.20 m α 4.23 dd (10.3, 2.0) β 3.89 dd (10.3, 1.3) 1.52 d (6.9) 5.28 q (6.9) α 4.73 d (17.1) β 3.48 d (17.1) 3.78 s

3 δ Cb

δHa,c

no.

180.5 80.5 54.6 34.9

2 3 5 6

60.4 132.3 128.8 118.1 159.5 101.5 151.3 32.9

7 8 9 10 11 12 13 14

37.8 37.2 68.8

15 16 17

12.8 118.6 141.5 42.2

18 19 20 21

55.5

22 Ar-OMe N-OMe

3.56 d (6.4) 4.35 m α 2.46 β 1.89 m

7.15 d (9.0) 6.50 dd (9.0, 2.1) 6.49 d (2.1) α 2.45 β 2.39 2.74 m 2.26 m α 4.13 d (10.8) β 4.00 dd (10.8, 5.3) 1.64 d (6.5) 5.48 q (6.5) α 4.17 β 4.17 3.78 s 3.93 s

δ Cb

δHa,c

no.

174.2 72.7 56.8 32.0

2′ 3′ 5′ 6′

55.3 120.2 127.0 107.5 160.3 95.2 140.1 29.3

7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′

33.7 38.1 66.4

15′ 16′ 17′

13.4 121.2 136.7 42.0

18′ 19′ 20′ 21′

163.6 55.7 63.6

N-OMe′

3.60 d (6.5) 4.35 m α 2.46 β 1.89 m

7.26 d (7.2) 7.00 dd (7.2, 7.2) 7.22 dd (7.2, 7.2) 6.91 d (7.2) α 1.24 β 2.39 2.74 m 2.26 m α 4.13 d (10.8) β 4.00 dd (10.8, 5.3) 1.64 d (6.5) 5.48 q (6.5) α 4.10 β 4.10

3.92 s

δ Cb 173.7 72.6 56.1 32.2 54.8 128.5 126.2 123.2 128.4 107.5 138.9 29.4 33.8 38.1 66.4 13.4 121.2 136.7 42.0

63.6

Measured at 400 MHz. bMeasured at 100 MHz. cOverlapped signals are reported without designating multiplicity.

Figure 7. Key 1H−1H COSY and HMBC correlations of 2 and 3.

A pair of rotameric Gelsemium alkaloids (1a and 1b) and two unusual dimeric MIAs (2 and 3), together with their putative biosynthetic intermediates (4−7), were isolated from the roots of G. elegans. Among them, geleganidine A (1) is the first rotameric MIA because of restricted amide bond rotation.

determined by the MTT assay. Compound 3 displayed a growth inhibitory effect against PC-12 cells with an IC50 value of 16.10 ± 1.67 μM. Compound 2 showed moderate activity against MCF-7 cells, with an IC50 value of 38.41 ± 2.68 μM. However, the IC50 values of the other compounds were greater than 50 μM. E



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were carried out on an Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies, CA, USA) and a Waters Q TOF SYNAPT G2 mass spectrometer (Waters MS Technologies, Manchester, U.K.), respectively. NMR spectra were recorded on Bruker AV-400 and AV-500 spectrometers (Bruker, Fällanden, Switzerland). Preparative HPLC was carried out on an Agilent 1260 Chromatograph equipped with a G1311C pump and a G1315D photodiode array detector (Agilent Technologies, CA, USA) with a C18 reversed-phase column (Cosmosil, 10 mm × 250 mm, 5 μm). All solvents used in HPLC were of chromatographic grade (Fisher Scientific, New Jersey, USA). Plant material. The roots of G. elegans were collected from Conghua city, Guangdong province of PR China, in September of 2012. A voucher specimen (no. 2012092801) identified by Prof. Guang-Xiong Zhou (Jinan University) was deposited with the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China. Extraction and Isolation. The air-dried roots of G. elegans (20 kg) were pulverized and extracted with 95% EtOH. The extract (1.9 kg) was suspended in H2O and acidified with 5% HCl to pH 3. The acidic suspension was partitioned with CHCl3 to remove the neutral components. Then, the aqueous layer was basified with 10% aqueous ammonia to pH 9 and extracted with CHCl3 to obtain a total alkaloid fraction. The alkaloid fraction (250 g) was subjected to silica-gel column chromatography using CHCl3/MeOH (100:0 → 0:100) as an eluent to afford 11 fractions (Frs. A−K). Fr. C (35 g) was subjected to silica-gel column chromatography (n-hexane/EtOAc/Et 2NH, 100:0:1 → 0:100:1) to afford nine subfractions (Frs. C1−C9). Fr. C1 (1.8 g) was separated by reversed-phase preparative HPLC (MeOH/H2O/Et2NH, 62:38:0.01) to afford compounds 4 (4.7 mg) and 5 (5.0 mg). Fr. C2 (5.1 g) was purified on Sephadex LH-20 using MeOH as an eluent to yield compounds 6 (15.5 mg) and 7 (23.5 mg). Fr. C3 (2.5 g) was chromatographed on Sephadex LH-20 (CHCl3/MeOH, 1:1) and purified by preparative HPLC using MeOH/H2O (75:25) as the mobile phase to obtain compounds 2 (7.6 mg) and 3 (5.3 mg). Fr. C8 (2.3 g) was subsequently purified by reversed-phase preparative HPLC (MeOH/H2O, 55:45) to yield compounds 1a and 1b (total = 13.1 mg). Geleganidine A (1): amorphous powder; [α]24D = −15 (c = 0.3, MeOH); 1H and 13C NMR data, see Table 1; UV (MeOH) λmax (log ε): 212 (3.77), 280 (2.90) nm; IR (KBr) νmax: 2910, 2900, 2837, 1720, 1655, 1498, 1359, 1217, 1176, 1128, 1073, 1031, 960, 809, 629 cm−1; ECD (MeCN, Δε) λmax 219.0 (−38.8), 238.8 (+5.6); HR-ESI-MS m/z 399.1925 [M + H]+ (calcd for C22H27N2O5, 399.1915).

Figure 8. Key NOESY correlations of 2.

The interconversion mechanism and equilibrium constant of the two rotamers were rationalized by density functional theory computation. Geleganidine B (2) is the first naturally occurring aromatic azo-linked MIA, and geleganidine C (3) is the first dimeric MIA linked by a urea unit. Although more than 120 dimeric MIAs have been reported, none of them was linked via an azo or a urea unit.30−32 Thus, 2 and 3 represent two new dimerization patterns of MIAs. On the basis of the coexistence of compounds 4−7, a plausible biosynthetic pathway of 1−3 was proposed.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained on an X-5 micro melting point apparatus and are uncorrected (Fukai Instrument, Beijing, China). UV spectra were recorded on a Jasco V-550 UV/vis spectrophotometer (Jasco, Tokyo, Japan). IR spectra were determined on a Jasco FT/IR-480 plus Fourier transform infrared spectrometer (Jasco, Tokyo, Japan) using KBr pellets. ECD spectra were obtained on a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan) at room temperature. HR-ESI-MS and Q-TOF-MS/MS

Figure 9. X-ray ORTEP drawing of 2. F



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Scheme 1. Putative Biosynthesis Routes to 1−3

Geleganidine B (2): orange blocks (MeOH); mp 266−267 °C; [α]24D = +10 (c = 0.3, MeOH); 1H and 13C NMR data, see Table 2; UV (MeOH) λmax (log ε) 209 (3.98), 320 (3.46) nm; IR (KBr) νmax 2920, 1702, 1603, 1497, 1449, 1391, 1252, 1105, 1044, 962, 848 cm−1; ECD (MeCN, Δε) λmax 226.2 (+34.1); HR-ESI-MS m/z 677.3338 [M + H]+ (calcd for C40H45N4O6: 677.3334). Geleganidine C (3): amorphous powder; [α]24D = −14 (c = 0.5, MeOH); 1H and 13C NMR data, see Table 2; UV (MeOH) λ max (log ε): 209 (3.65), 287 (3.01) nm; IR (KBr) νmax: 3433, 2925, 2856, 2383, 2347, 1723, 1631, 1504, 1459, 1380, 1218, 1120, 1067, 951, 833, 749 cm−1; ECD (MeCN, Δε) λ max 216.2 (−18.8),

239.2 (+1.9); HR-ESI-MS m/z 737.3550 [M + H] + (calcd for C 42H49N 4O8, 737.3545). Characterization of 4−7. A detailed structural assignment via NMR spectroscopic analyses of 4−7 and their physicochemical data are provided in the Supporting Information. HPLC Analysis of 1a and 1b. Rotamers 1a and 1b isolated by preparative HPLC were analyzed by HPLC at different times (0, 0.5, 1, and 24 h, at room temperature). The HPLC/UV spectra were obtained by an Agilent 1260 instrument equipped with a DAD detector and a Cosmosil RP-18 column (5 μm, 4.6 mm I.D. × 250 mm). The separation was achieved by applying a binary mobile phase consisting of G



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MeOH/H2O (55:45) in isocratic elution mode, (λ= 210 nm; flow rate: 1 mL min−1; Rt for 1a: 11.8 min; Rt for 1b: 13.4 min). X-ray Analysis of Geleganidine B (2): orange blocks, C40H44N4O6, monoclinic, P21, a = 9.9993(2), b = 12.9855(2), c = 13.4171(2) Å, β = 110.184(2), V = 1635.17(5) Å3, Z = 2, dx = 1.379 Mg/m3, F(000) = 724. Data collection was carried out on a SMART CCD using graphite monochromated radiation (λ = 1.54178 Å) under low temperature (nitrogen gas); 4931 unique reflections were collected to θmax = 62.85°, in which 5217 reflections were observed [F2 > 4σ(F2)]. The structures were solved by direct methods (SHELXTL, version 5.1) and refined by full-matrix least-squares on F2. In the structure refinements, nonhydrogen atoms were refined anisotropically. Hydrogen atoms bonded to carbons were placed on the geometrically ideal positions by the ride on method. Hydrogen atoms bonded to oxygen were located by the difference Fourier method and were included in the calculation of structure factors with isotropic temperature factors. The final values were R = 0.0404, RW = 0.1044, S = 1.035, and Flack parameter = 0.04 (18). The crystal data of compound 2 was deposited with the Cambridge Crystallographic Data Centre (CCDC 978571, http://http:// www.ccdc.cam.ac.uk/). Calculation Details. For the ECD calculation of 1, seven conformers were obtained by conformational analysis (Sybyl 8.0 program, MMFF94s molecular mechanic force field, charged with Gasteiger−Hückel). All of them were further optimized at B3LYP/ 6-31+G(d) level using the Gaussian 09 package and were proven to be real minima because no imaginary frequencies were found. The ECD calculation of each single conformer was carried out by means of timedependent DFT (TDDFT) methods at the B3LYP/6-31+G(d) level, as available in Gaussian09. The overall calculated ECD curves were obtained by means of Boltzmann weighting of single ECD spectra. The initial geometries of 1a and 1b were selected from the conformational analysis described above. The optimized structures of 1a, 1b, and TS were calculated in the gas phase at PWPB95-D3(BJ)/def2-QZVPP within the Gaussian09 program. Frequency analyses were carried out to identify the natures (minima or transition state) of the optimized geometries. The relative energies (in kcal/mol) were obtained according to G = E + Gcorr + ΔGsolv. Single-point energies (E) of 1a, 1b, and TS were obtained after optimization at the PWPB95-D3(BJ)/def2-QZVPP level in the ORCA program. Thermochemistry correction energies (Gcorr) were obtained by the optimization and frequency calculation at the M06-2X/6-31G** level in Gaussian09 with the correction scales (0.967 for ZPE, 0.979 for ΔH, and 0.940 for S). The optimized structures of 1a and 1b were also used for NBO property calculation at the B3LYP/6-31++G** level by using the NBO 3.1 program implemented in the Gaussian09 program. Cell Lines and Cell Culture. The human hepatocellular carcinoma cell line HepG2, human breast cancer cell line MCF-7, and rat pheochromocytoma cell line PC-12 were obtained from the American Type Culture Collection. The multidrug-resistant human hepatoma cell line HepG2/ADM was kindly provided by Prof. Kwok-Pui Fung (Chinese University of Hong Kong, Hong Kong). All of the cell lines were cultured in RPMI 1640 medium, supplemented with 10% FBS (v/v) at 37 °C in a humidified atmosphere of 5% CO2 (v/v). Cell Viability Assay. The cytotoxicities were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were cultured in 96-well plates for 24 h. After that, the cells were treated with compounds 1−7 at various concentrations for 72 h. A 30 μL aliquot of MTT solution (5 mg/mL) was added into each well and incubated for another 4 h. Subsequently, the medium was discarded, and 100 μL of DMSO was added to dissolve the produced formazan. The absorbance was measured at 570 nm using a microplate spectrophotometer (Thermo Scientific Multiskan MK3, USA). The concentration giving 50% inhibition (IC50) was determined from the dose−response curves using Prism software and expressed as the mean ± SD.

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structural elucidation of compounds 4−7, and HPLCHR-MS analyses of the methanol extract of the fresh roots of Gelsemium elegans. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00351.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-20-85221559. Fax: +86-20-85221559. *E-mail: [email protected]. Author Contributions

W.Z. and X.-J.H. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of this research was provided by the Program for National Natural Science Foundation of China (no. 81273391), the Ministry of Science and Technology of China (nos. 2013DFM30080, 2013BAI11B05, and 2012ZX09103201056), the Program of Pearl River Young Talents of Science and Technology in Guangzhou, China (2013J2200058), and the High-Performance Computing Platform of Jinan University.

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REFERENCES

(1) Ishikura, M.; Abe, T.; Choshi, T.; Hibino, S. Nat. Prod. Rep. 2013, 30, 694−752. (2) Dudley, A. C.; Shih, S.-C.; Cliffe, A. R.; Hida, K.; Klagsbrun, M. Br. J. Cancer 2008, 99, 118−125. (3) Kitajima, M. J. Nat. Med. 2006, 61, 14−23. (4) Sun, M. X.; Hou, X. L.; Gao, H. H.; Guo, J. S.; Xiao, K. Molecules 2013, 18, 1819−1825. (5) Qu, J.; Fang, L.; Ren, X. D.; Liu, Y. B.; Yu, S. S.; Li, L.; Bao, X. Q.; Zhang, D.; Li, Y.; Ma, S. G. J. Nat. Prod. 2013, 76, 2203−2209. (6) Liang, S.; He, C. Y.; Szabó, L. F.; Feng, Y.; Lin, X.; Wang, Y. Tetrahedron Lett. 2013, 54, 887−890. (7) Kogure, N.; Ishii, N.; Kitajima, M.; Wongseripipatana, S.; Takayama, H. Org. Lett. 2006, 8, 3085−3088. (8) Kitajima, M.; Nakamura, T.; Kogure, N.; Ogawa, M.; Mitsuno, Y.; Ono, K.; Yano, S.; Aimi, N.; Takayama, H. J. Nat. Prod. 2006, 69, 715− 718. (9) Diethelm, S.; Carreira, E. M. J. Am. Chem. Soc. 2013, 135, 8500− 8503. (10) Zhou, X.; Xiao, T.; Iwama, Y.; Qin, Y. Angew. Chem., Int. Ed. 2012, 51, 4909−4912. (11) Wang, C. Q.; Wang, L.; Fan, C. L.; Zhang, D. M.; Huang, X. J.; Jiang, R. W.; Bai, L. L.; Shi, J. M.; Wang, Y.; Ye, W. C. Org. Lett. 2012, 14, 4102−4105. (12) Wu, Z. L.; Zhao, B. X.; Huang, X. J.; Tang, G. Y.; Shi, L.; Jiang, R. W.; Liu, X.; Wang, Y.; Ye, W. C. Angew. Chem., Int. Ed. 2014, 53, 5796− 5799. (13) Ouyang, S.; Wang, L.; Zhang, Q. W.; Wang, G. C.; Wang, Y.; Huang, X. J.; Zhang, X. Q.; Jiang, R. W.; Yao, X. S.; Che, C. T.; Ye, W. C. Tetrahedron 2011, 67, 4807−4813. (14) Wang, L.; Ye, W. C.; Zhang, W.; Zhang, S. Y.; Yin, Z. Q. Heterocycles 2014, 89, 1245−1253. (15) Lin, L. Z.; Cordell, G. A.; Ni, C. Z.; Clardy, J. J. Nat. Prod. 1989, 52, 588−594. (16) Che, Q.; Zhu, T. J.; Keyzers, R. A.; Liu, X. F.; Li, J.; Gu, Q. Q.; Li, D. H. J. Nat. Prod. 2013, 76, 759−763. (17) Patil, A. D.; Freyer, A. J.; Reichwein, R.; Bean, M. F.; Faucette, L.; Johnson, R. K.; haltiwanger, R. C.; Eggleston, D. S. J. Nat. Prod. 1997, 60, 507−510. (18) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.;

ASSOCIATED CONTENT

S Supporting Information *

UV, IR, MS, CD, and NMR spectra for compounds 1−4, calculation details for 1a and 1b, Q-TOF-MS/MS analysis of 2 and 3, H



Article

Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, revision A.02; Gaussian, Inc.: Wallingford CT, 2009. (19) Lin, L. Z.; Cordell, G. A.; Ni, C. Z.; Clardy, J. Tetrahedron Lett. 1989, 30, 1177−1180. (20) Grirrane, A.; Corma, A.; García, H. Science 2008, 322, 1661−1664. (21) Zhang, C.; Jiao, N. Angew. Chem., Int. Ed. 2010, 49, 6174−6177. (22) Zhu, Y. G.; Shi, Y. Org. Lett. 2013, 15, 1942−1945. (23) Gill, M.; Strauch, R. J. Z. Naturforsch. 1984, 39, 1027−1029. (24) Liu, J. K. Chem. Rev. 2005, 105, 2723−2744. (25) Bailey, P. D.; McLay, N. R. Tetrahedron Lett. 1991, 32, 3895− 3898. (26) West, N. P.; Cergol, K. M.; Xue, M.; Randall, E. J.; Britton, W. J.; Payne, R. J. Chem. Commun. 2011, 47, 5166−5168. (27) Kawase, M.; Kitamura, T.; Kikugawa, Y. J. Org. Chem. 1989, 54, 3394−3403. (28) Laha, S.; Luthy, R. G. Environ. Sci. Technol. 1990, 24, 363−373. (29) Zhang, J. M.; Du, Y. M. Huaqiao Daxue Xuebao, Ziran Kexueban 2004, 25, 446−448. (30) Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach, 3rd ed.; John Wiley & Sons, Ltd.: New York, 2009; pp 19, 25. (31) Kitajima, M.; Iwai, M.; Kogure, N.; Kikura-Hanajiri, R.; Goda, Y.; Takayama, H. Tetrahedron 2013, 69, 796−801. (32) Leonard, J. Nat. Prod. Rep. 1999, 16, 319−338.

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