A Bis-benzopyrroloisoquinoline Alkaloid Incorporating a Cyclobutane

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A Bis-benzopyrroloisoquinoline Alkaloid Incorporating a Cyclobutane Core and a Chlorophenanthroindolizidine Alkaloid with Cytotoxic Activity from Ficus f istulosa var. tengerensis Amjad Ayad Qatran Al-Khdhairawi,† Premanand Krishnan,† Chun-Wai Mai,‡ Felicia Fei-Lei Chung,⊥ Chee-Onn Leong,‡,⊥ Kien-Thai Yong,# Kam-Weng Chong,§ Yun-Yee Low,§ Toh-Seok Kam,§ and Kuan-Hon Lim*,† †

School of Pharmacy, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia School of Pharmacy, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia ⊥ Center for Cancer and Stem Cell Research, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia # Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia § Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia ‡

S Supporting Information *

ABSTRACT: Tengerensine (1), isolated as a racemate and constituted from a pair of bis-benzopyrroloisoquinoline enantiomers, and tengechlorenine (2), purified as a scalemic mixture and constituted from a pair of chlorinated phenanthroindolizidine enantiomers, were isolated from the leaves of Ficus f istulosa var. tengerensis, along with three other known alkaloids. The structures of 1 and 2 were determined by spectroscopic data interpretation and X-ray diffraction analysis. The enantiomers of 1 were separated by chiral-phase HPLC, and the absolute configurations of (+)-1 and (−)-1 were established via experimental and calculated ECD data. Compound 1 is notable in being a rare unsymmetrical cyclobutane adduct and is the first example of a dimeric benzopyrroloisoquinoline alkaloid, while compound 2 represents the first naturally occurring halogenated phenanthroindolizidine alkaloid. Compound (+)-1 displayed a selective in vitro cytotoxic effect against MDA-MB-468 cells (IC50 7.4 μM), while compound 2 showed pronounced in vitro cytotoxic activity against all three breast cancer cell lines tested (MDA-MB-468, MDA-MB-231, and MCF7; IC50 values of 0.038−0.91 μM).

T

both classes of indolizidine alkaloids may share a common biosynthetic origin. The genus Ficus is represented by about 100 species in Peninsular Malaysia and approximately 725 species worldwide.8 Previous alkaloidal investigations of Ficus species have been limited to three species, namely, F. septica, F. hispida, and F. f istulosa.1,9−11 Plants of the genus Ficus are therefore considerably underinvestigated and as such represent potential sources of biologically useful alkaloids. Ficus tengerensis was categorized as a variety under the species Ficus f istulosa, i.e., F. f istulosa var. tengerensis (Miq.) Kuntze, in 1891. However, Berg and Corner reclassified F. f istulosa var. tengerensis as a synonym

he benzopyrroloisoquinoline or naphthoindolizidine alkaloids are rare in Nature and are represented by only three members, namely, (−)-fistulosine ((−)-3), vincetene (4), and 2,3-dimethoxy-6-(3-oxobutyl)-7,9,10,11,11a,12hexahydrobenzo[f ]pyrrolo[1,2-b]isoquinoline (5), which were previously isolated from Ficus f istulosa (Moraceae), Cynanchum vincetoxicum, and Cynanchum komarovii (Asclepiadaceae), respectively.1−3 In contrast to the closely related phenanthroindolizidine alkaloids, which have attracted considerable attention due to their potent cytotoxic properties, benzopyrroloisoquinoline alkaloids were reported previously to possess no apparent cytotoxic activity.4 It is also notable that the distribution of both phenanthroindolizidine and benzopyrroloisoquinoline alkaloids is limited mainly to plants of the families Asclepiadaceae and Moraceae,5−7 which suggests that © 2017 American Chemical Society and American Society of Pharmacognosy

Received: June 10, 2017 Published: September 19, 2017 2734

DOI: 10.1021/acs.jnatprod.7b00500 J. Nat. Prod. 2017, 80, 2734−2740

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of F. f istulosa in 2005,8 only for this to be revised in 2011 by Berg, who reinstated F. f istulosa var. tengerensis as a distinct variety of F. fistulosa.12 The search for alkaloid-containing Ficus species in Malaysia has led our group to investigate the alkaloid content of the leaves of F. fistulosa var. tengerensis (Miq.) Kuntze. Herein, we report the structures and biological activity of the bis-benzopyrroloisoquinoline alkaloid, tengerensine (1), and the halogenated phenanthroindolizidine alkaloid, tengechlorenine (2), along with the isolation of three known compounds, namely, (±)-fistulosine ((±)-3),1 (+)-antofine,6 and (−)-seco-antofine,6 which were identified by comparison of their physical and spectroscopic data with literature values.

(δ 206.27 and 207.82), six methyls (of which four were methoxy groups), 10 methylenes (of which four were Nmethylenes, δ 53.70−55.51), six aliphatic methines (of which two were N-methines, δ 60.05 × 2C), and 20 aromatic carbons (including four that were oxygenated, δ 148.59−149.36). The presence of six oxygen atoms in 1 as revealed by the HRESIMS data, coupled with the fact that there were four oxygenated aromatic carbons and two ketone carbonyl resonances, indicated that the four methoxy groups are attached to the four oxygenated aromatic carbons. A detailed inspection of the 13 C NMR data suggested 1 to be a dimeric compound with a large number of resonances observed appearing in pairs. Comparison of the 1H and 13C NMR data of 1 with those of fistulosine (3) revealed 1 to be a bis-benzopyrroloisoquinoline alkaloid. In fact, 1 and 3 were found to possess closely comparable benzopyrroloisoquinoline moieties with methoxy substitution at positions C-2 and C-3.1,3 The presence of the two benzopyrroloisoquinoline moieties in 1 was also consistent with the HMBC data (Figure 1). The HMBC correlations of 1 (Figure 1) revealed the attachment of the two acetyl groups to two adjacent carbons (C-14 and C-14′) of a cyclobutane ring on the basis of the correlations from H-16 to C-14; H-16′ to C14′; H-14 to C-15′; and H-14′ to C-15. On the other hand, the remaining adjacent carbons (C-13 and C-13′) of this cyclobutane ring were shown to be attached to C-6 and C-6′ of the two benzopyrroloisoquinoline moieties as a result of the correlations from H-14 to C-6; H-5 to C-13; H-14′ to C-6′; and H-5′ to C-13′. The structure of 1 was thus deduced to be an unsymmetrical cyclobutane adduct arising from the union of two seemingly identical benzopyrroloisoquinoline moieties, each possessing a 3-buten-2-one side chain at position C-6. The relative configuration of the cyclobutane ring at compound 1 was determined from the NOESY data (Figure 2). The indolizidine units of the benzopyrroloisoquinoline moieties were found to be oriented toward one another on the basis of the NOEs observed for H-14′/H-5, H-14′/H-5′, and H-7α/H-7′α. The H-14′/H-5 and H-14′/H-5′ NOEs also required both the benzopyrroloisoquinoline moieties and H-14′ be oriented on the same face of the cyclobutane ring, i.e., with H-13 and H-13′ cis to each other, and H-13′ and H-14′ trans to one another. Additionally, the NOEs observed for H-16′/H-13′ and H-16′/H-14 indicated that the acetyl group at C-14′, and H-13′ and H-14 are oriented on the same face of the cyclobutane ring, i.e., H-14 and H-14′ are trans to each other. Taken together, it was concluded that the cyclobutane ring in 1 is cis,trans,trans-configured, i.e., H-13, H-14, and H-13′ are oriented on the same face, while H-14′ is on the opposite face of the cyclobutane ring. Since 1 developed suitable crystals from CH2Cl2−MeOH, an X-ray diffraction analysis was carried out (Figure 3), which confirmed the structure proposed based on analysis of the spectroscopic data. The X-ray data revealed a centrosymmetric P1̅ space group, which supported 1 as being a racemic crystal.14 This is consistent with the observation that 1 was optically inactive. Chiral resolution of 1 was performed using chiralphase HPLC, which afforded (+)-1 and (−)-1 in a ratio of approximately 1:1 (Figure S3, Supporting Information). As expected, the electronic circular dichroism (ECD) curves for both (+)-1 and (−)-1 showed opposite Cotton effects (Figure 4). Finally, comparison of the experimental and calculated ECD spectra of the enantiomers (Figure 4) allowed the absolute configurations of (+)-1 and (−)-1 to be established as

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RESULTS AND DISCUSSION Tengerensine (1) was obtained initially as a light yellowish oil and subsequently crystallized from CH2Cl2−MeOH as light yellowish block crystals, mp >190 °C (dec), [α]25D 0 (CHCl3, c 0.03). The IR spectrum showed a carbonyl absorption band at 1705 cm−1, while the UV spectrum showed characteristic naphthalene maxima at 239, 280, and 332 nm (log ε 4.62, 4.13, and 3.89, respectively).3 The ESIMS showed a [M + H]+ peak at m/z 703, and HRESIMS measurements were used to establish the molecular formula as C44H50N2O6. The 1H NMR data of compound 1 (Table 1) exhibited the presence of six aromatic singlets (δ 6.55−7.51), indicating the presence of six isolated aromatic hydrogens. Six methyl singlets were also apparent, of which four were due to methoxy groups observed at δ 3.76, 3.91, 3.94, and 3.98. The remaining two methyl singlets (δ 1.65 and 2.31) were attributed to two acetyl groups on the basis of the HSQC and HMBC data. It is notable that the acetyl group at δ 1.65 was shielded substantially compared to the other one (δ 2.31). In addition, there were four distinct methine triplets at δ 4.02, 4.37, 4.54, and 4.84 (J = 9.5 Hz), which, on the basis of the COSY and HSQC data, were deduced to be due to four contiguous methine hydrogens, indicating the presence of a tetrasubstituted cyclobutane partial structure.13 The 13C NMR data of 1 (Table 1) showed the presence of 39 discrete carbon resonances, of which five were overlapping resonances accounting for two carbons each. In agreement with the HRESIMS data, the total number of carbons in 1 was therefore determined as 44, comprising two ketone carbonyls 2735

DOI: 10.1021/acs.jnatprod.7b00500 J. Nat. Prod. 2017, 80, 2734−2740

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Table 1. 1H and 13C NMR Spectroscopic Data of 1 and 2 in CDCl3 tengerensine (1) H/C 1 2 3 4 4a 5 6 6a 7α 7β 9α 9β 10 11β 11α 11a (α) 12β 12α 12a 12b 13 14 15 16 2-OMe 3-OMe a

δHa mult. (J in Hz)

δCb

6.96 s

101.51 149.36 148.59 106.98 128.98d 124.84 129.26 126.94d 55.51e

6.55 s 6.94 s

3.47 m 4.37 d (13.7) 2.33 m 3.43 m 1.82 mc 1.92 mc 1.70 m 2.19 m 2.16 m 2.74 m 3.16 d (15)

4.54 t (9.5) 4.02 t (9.5) 1.65 s 3.94 s 3.76 s

600 MHz. b150 MHz.

c−h

55.51e 21.55 31.14 60.05 33.51 128.67d 126.53 39.42 50.60 206.27 28.43 55.69f 55.75f

H/C 1′ 2′ 3′ 4′ 4a′ 5′ 6′ 6a′ 7′α 7′β 9′α 9′β 10′ 11′β 11′α 11a′ (α) 12′β 12′α 12a′ 12b′ 13′ 14′ 15′ 16′ 2′-OMe 3′-OMe

tengechlorenine (2) δHa mult. (J in Hz)

δ Cb

H/C

δHa mult. (J in Hz)

δ Cb

6.96 s

101.85 149.19 148.79 106.73 129.67d 123.02 130.87 127.38d 53.70

1 2 3 4 4a 4b 5 6 7 8 8a 8b 9α 9β 11α 11β 12α 12β 13α 13β 13a (β) 14α 14β 14a 14b 2-OMe 3-OMe 6-OMe

7.32 s

103.45 148.96 146.73 109.83 123.16 126.31g 118.34 153.88 111.30 121.91 126.38g 127.78 54.20

7.04 s 7.51 s

2.75 d (14.5) 4.16 d (14.5) 2.16 m 3.34 m 1.95 mc 2.04 mc 1.58 m 2.05 m 1.83 m 2.64 m 3.04 d (15)

4.37 t (9.5) 4.84 t (9.5) 2.31 s 3.91 s 3.98 s

55.16e 21.55 31.14 60.05 33.14 128.87d 126.63 40.88 47.78 207.82 29.05 55.75f 55.81f

9.39 s

7.27 d (9) 7.79 d (9)

3.66 d (14.8) 4.62 d (14.8) 2.44 m 3.45 td (8.5, 1.7) 1.92 m 2.02 m 1.77 m 2.23 m 2.47 m 2.90 dd (15.6, 10.5) 3.29 dd (15.7, 2.4)

4.06 s 4.08 s 4.05 s

55.08 21.63 31.27 60.04 33.96 127.12 128.84 55.80h 56.03h 57.00

Signals interchangeable.

Figure 1. Selected HMBC correlations of 1.

Figure 2. Selected NOESY correlations of 1.

11aS,13S,14S,11a′R,13′R,14′S and 11aR,13R,14R,11a′S,13′S,14′R, respectively. The X-ray crystal structure of 1 also revealed an intriguing structural feature of the molecule whereby the configurations of the two indolizidine stereocenters at C-11a and C-11a′ are opposite. In other words, if the cyclobutane part of the structure is disregarded, the resultant monomeric halves are mirror images of each other. The X-ray crystal structure also revealed that the acetyl group at C-14 is in the vicinity of an aromatic shielding zone, thus providing an explanation for the notably shielded acetyl signal (δ 1.65, Me-16) observed in the 1 H NMR spectrum. The structure of tengerensine (1) represents a rare instance of an unsymmetrical cyclobutane

adduct15 and the first example of a dimeric benzopyrroloisoquinoline alkaloid. Tengechlorenine (2) was obtained initially as a light yellowish oil and subsequently crystallized from CH2Cl2 as light yellowish plates, mp 190−195 °C, [α]25D +11 (CHCl3, c 0.08). The IR spectrum showed the presence of Bohlmann bands at 2834 and 2795 cm−1, indicating the presence of a trans-indolizidine ring junction, while the UV spectrum showed absorption maxima at 223, 269, 280, and 320 nm (log ε 3.60, 3.87, 3.77, and 3.20, respectively). The ESIMS showed two [M + H]+ peaks at m/z 398 and 400 with an intensity ratio of about 3:1, suggesting the presence of a Cl atom. HRESIMS measurements were consistent with the molecular formulas 2736

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singlets at δ 4.05, 4.06, and 4.08 in the 1H NMR spectrum indicated that the three oxygenated aromatic carbons are sites of methoxylation in the molecule of 2. The COSY and HSQC data revealed the presence of an NCH2CH2CH2CHCH2 fragment corresponding to the N−C11−C-12−C-13−C-13a−C-14−Ar partial structure, thus establishing an unsubstituted indolizidine unit in 2. Attachment of the indolizidine unit to the phenanthrene moiety was facilitated by the HMBC data (Figure 5). Three-bond correlations from H-9 to C-8a and C-14a, and from H-14 to C-8b supported the linkage of C-9 to C-8b and C-14 to C-14a, respectively. The substitution pattern of the phenanthrene moiety was determined on the basis of the NOESY data (Figure 5). NOESY correlations from H-14α and H-14β to the aromatic singlet at δ 7.32 enabled this singlet signal to be assigned to H1. This in turn inferred that the aromatic singlet at δ 9.39 was due to H-4. Similarly, NOESY correlations from H-9α and H9β to the aromatic doublet at δ 7.79 confirmed the assignment of the doublet signal to H-8 and the other aromatic doublet at δ 7.27 to H-7. The phenanthrene moiety was therefore deduced to be substituted at C-2, C-3, C-5, and C-6, with C-2, C-3, and C-6 being the sites of methoxylation on the basis of the NOEs observed for H-1/OMe-2, H-4/OMe-3, and H-7/OMe-6. Finally, the remaining Cl atom was determined to be substituted at C-5, which was consistent with the observation that H-4 was substantially deshielded (δ 9.39) due to its spatial proximity to the lone pair electrons of Cl resulting in a paramagnetic anisotropic deshielding effect. The structure proposed for 2 was consistent with the HMBC and NOESY data (Figure 5). Since suitable crystals were obtained, the structure of 2 was confirmed by X-ray diffraction analysis (Figure 6).16 As in the case with 1, the X-ray data revealed the crystals of 2 to possess a centrosymmetric P1̅ space group (racemic crystals).14 However, in addition to showing optical activity with a specific rotation value of +11, positive Cotton curves were observed in the ECD spectrum of 2. Compound 2 was therefore deduced to be a scalemic mixture with the enantiomer in excess being dextrorotatory and showing positive Cotton effects. This

Figure 3. X-ray crystal structure of 1.

associated with both the pseudomolecular ion peaks as C23H2435ClNO3 and C23H2437ClNO3, respectively. The 13C NMR data of 2 (Table 2) showed the presence of 23 carbons, in agreement with the HRESIMS data. The presence of 14 aromatic carbon resonances suggested 2 to be a phenanthroindolizidine alkaloid, while the resonances at δ 146.73, 148.96, and 153.88 indicated the presence of three oxygenated aromatic carbons. The 1H NMR data of 2 (Table 2) showed the presence of a 1,2,4,5-tetrasubstituted benzene ring from the two aromatic singlets observed at δ 7.32 and 9.39, while the pair of AB doublets (J = 9 Hz) at δ 7.27 and 7.79 supported the occurrence of a 1,2,3,4-tetrasubstituted benzene ring. Another pair of AB doublets (J = 14.8 Hz) observed at δ 3.66 and 4.62 were attributable to the isolated benzylic aminomethylene group (H2-9). The presence of three methoxy

Figure 4. Experimental and calculated ECD spectra of (+)-1 and (−)-1. 2737

DOI: 10.1021/acs.jnatprod.7b00500 J. Nat. Prod. 2017, 80, 2734−2740

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Table 2. Cytotoxic Effects of (±)-1, (+)-1, (−)-1, 2, (±)-3, and Paclitaxel IC50 ± SD (μM)

a

compound

MDA-MB-468a

MDA-MB-231a

MCF7a

MCF10Ab

(±)-1 (+)-1 (−)-1 2 (±)-3 paclitaxel

>10 7.4 ± 2.1 >10 0.038 ± 0.01 >10 0.0061 ± 0.001

>10 >10 >10 0.48 ± 0.05 >10 0.059 ± 0.012

>10 >10 >10 0.91 ± 0.06 >10 0.0088 ± 0.003

>10 >10 >10 10.7 ± 3.7 >10 0.0058 ± 0.002

MDA-MB-468, MDA-MB-231, and MCF7: human breast adenocarcinomas. bMCF10A: nontumorigenic human breast epithelial cells.

(MCF10A). Compounds (±)-1 and (−)-1 did not show any appreciable cytotoxic activity against all the breast cancer cell lines tested, while (+)-1 displayed a selective cytotoxic effect against MDA-MD-468 cells (Table 2). On the other hand, compound 2 displayed a pronounced cytotoxic effect against all three breast cancer cell lines tested, while it was moderately cytotoxic toward the nontumorigenic cell line. Consistent with the previous report,4 the monomeric benzopyrroloisoquinoline alkaloid (±)-fistulosine ((±)-3), which was purified as a racemic mixture in the present study, was found to be ineffective against all the cell lines tested.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on an Electrothermal IA9100 digital melting point apparatus and are uncorrected. Optical rotations were determined on a JASCO P-1020 automatic digital polarimeter. UV spectra were obtained on a Shimadzu UV-3101PC spectrophotometer. IR spectra were recorded on a PerkinElmer Spectrum 400 FT-IR/FT-FIR spectrometer. ECD spectra were obtained on a J-815 circular dichroism spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 using tetramethylsilane as internal standard on a Bruker 600 MHz NMR spectrometer. HRESIMS were obtained on a JEOL Accu TOF-DART mass spectrometer. HPLC was performed on a Waters liquid chromatograph with a Waters 600 controller and a Waters 2489 tunable absorbance detector, using Chiralpak AS-H (4.6 × 150 mm) and Chiralpak IA (4.6 × 150 mm) columns. Plant Material. Plant material (Ficus f istulosa var. tengerensis) was collected in December 2014 from Berembun Forest Reserve, Negeri Sembilan, Malaysia, and was identified by K. T. Yong (Institute of Biological Sciences, University of Malaya, Malaysia). Herbarium voucher specimens (KLU49073, KLU49074, KLU49075, and KLU49076) are deposited at the Herbarium, University of Malaya. Extraction and Isolation. The dried leaves (15 kg) of F. f istulosa var. tengerensis were extracted with 95% EtOH. The concentrated ethanolic extract was added into 2% tartaric acid solution with vigorous stirring. The acidic solution was then filtered through kieselguhr to remove the insoluble nonalkaloidal substances. The pH of the acidic filtrate was then adjusted to about 10 by addition of concentrated NH3 solution. The liberated alkaloids were exhaustively extracted with EtOAc, washed with water, dried over anhydrous Na2SO4, and concentrated to afford a crude basic alkaloid mixture (10.5 g). The basic alkaloid mixture was initially fractionated by silica gel column chromatography using CHCl3 with increasing proportions of MeOH, followed by rechromatography of the appropriate semipurified fractions using preparative radial chromatography (Chromatotron). Solvent systems used for preparative radial chromatography were Et2O−hexane (2:1, NH3-saturated), Et2O− MeOH (20:1, NH3-saturated), THF−hexane (2:1, NH3-saturated), THF−hexane−MeOH (2:1:1, NH3-saturated), CHCl3−MeOH (20:1, NH3-saturated), and CHCl3−MeOH (10:1, NH3-saturated). The yields of the alkaloids from the crude alkaloid mixture were as follows: 1 (20 mg), 2 (12 mg), (±)-fistulosine ((±)-3) (18 mg),1 (+)-antofine (0.8 mg),6 and (−)-seco-antofine (18 mg).6

Figure 5. COSY, HMBC, and NOESY correlations of 2.

Figure 6. X-ray crystal structure of 2. Only one orientation of the C13a−N moiety is shown for clarity.

deduction was further supported by chiral-phase HPLC analysis of 2, which showed two peaks in a ratio of approximately 1:1.2 in the chromatogram (Figure S8, Supporting Information). The absolute configuration of (+)-2 (the enantiomer in excess) was determined to be C-13aS since its ECD spectrum displayed a positive Cotton effect at 290 nm.17−19 Tengechlorenine (2) represents the first naturally occurring halogenated phenanthroindolizidine alkaloid and a rare instance of a halogenated terrestrial plant alkaloid.20 Compounds (±)-1, (+)-1, (−)-1, 2, and (±)-3 were evaluated for in vitro cytotoxic activity using three human breast cancer cell lines (MDA-MB-468, MDA-MB-231, and MCF7) and a nontumorigenic human breast epithelial cell line 2738

DOI: 10.1021/acs.jnatprod.7b00500 J. Nat. Prod. 2017, 80, 2734−2740

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(±)-Tengerensine (1): light yellowish block crystals; mp >190 °C (dec); [α]25D 0 (c 0.03, CHCl3); UV (EtOH) λmax (log ε) 239 (4.62), 280 (4.13), 332 (3.89) nm; IR (dry film) νmax 1705, 2787, 2856 cm−1; 1 H NMR and 13C NMR data, see Table 1; HRESIMS m/z 703.3752 [M + H]+ (calcd for C44H50N2O6 + H, 703.3747). Chiral-Phase HPLC Separation and ECD Data of (+)-1 and (−)-1. Separation was carried out using a Chiralpak AS-H column (4.6 × 150 mm) packed with amylose tris[(S)-α-methylbenzylcarbamate] coated on 5 μm silica gel at ambient temperature, and fractions were collected manually. (±)-Tengerensine (1) (2.3 mg) was dissolved in EtOH (0.75 mL) and resolved using the eluting solvent hexane−EtOH− Et2NH, 85:15:0.1, at a flow rate of 1.0 mL/min (150 injections, 5.0 μL each). Compound (+)-1: tR 10 min 58 s; 0.9 mg; colorless oil; [α]25D +62 (c 0.02, CHCl3); ECD (MeOH) (Δε) 233 (−20.0), 249 (+13.4), 262 (−3.6), 295 (+6.8) nm. Compound (−)-1: tR 18 min 32 s; 1.2 mg; colorless oil; [α]25D −58 (c 0.06, CHCl3); ECD (MeOH) (Δε) 234 (+16.3), 249 (−13.2), 263 (+2.1), 295 (−6.2) nm. Tengechlorenine (2): light yellowish plates; mp 192−195 °C; [α]25D +11 (c 0.08, CHCl3); UV (EtOH) λmax (log ε) 223 (3.60), 269 (3.87), 280 (3.77), 320 (3.20) nm; IR (dry film) νmax 754, 2795, 2834 cm−1; ECD (CH3CN) (Δε) 224 (+0.58), 290 (+0.55) nm; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 398.1512 [M + H]+ (calcd for C23H2435ClNO3 + H, 398.1523) and 400.1507 [M + H]+ (calcd for C23H2437ClNO3 + H, 400.1494). Chiral-Phase HPLC Analysis of Tengechlorenine (2). Chiral HPLC analysis was carried out using a Chiralpak IA column (4.6 × 150 mm) packed with amylose tris(3,5-dimethylphenylcarbamate) immobilized on 5 μm silica gel at ambient temperature. Tengechlorenine (2) (0.4 mg) was dissolved in EtOH (50 μL) and resolved using the eluting solvent hexane−EtOH−Et2NH, 90:10:0.1, at a flow rate of 1.0 mL/ min to give two peaks at tR 17 min 54 s and tR 24 min 38 s, in a ratio of approximately 1:1.2. The lack of material precluded enantiomeric separation. X-ray Crystallographic Analysis of (±)-Tengerensine (1) and (±)-Tengechlorenine (2). X-ray diffraction analysis was carried out on a Rigaku Oxford (formerly Agilent Technologies) SuperNova Dual diffractometer with Cu Kα radiation (λ = 1.541 84 Å) at 167 K or rt. The structures were solved by direct methods (SHELXS-2014) and refined with full-matrix least-squares on F2 (SHELXL-2014). All nonhydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Crystallographic data for compounds 1 and 2 have been deposited at the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44 (0)1223-336033, or e-mail: [email protected]. uk). Crystallographic data of (±)-tengerensine (1): light yellowish block crystals, C44H50N2O6, Mr 702.86, triclinic, space group P1,̅ a = 12.709(2) Å, b = 12.8666(12) Å, c = 14.9876(16) Å, α = 112.962(10)°, β = 111.821(16)°, γ = 90.977(11)°, V = 2057.0(5) Å3, Z = 2, Dcalc = 1.135 mg/mm3, crystal size 0.40 × 0.15 × 0.02 mm3, F(000) = 752, T = 293 K. The final R1 value is 0.0856 (wR2 = 0.2399) for 4506 reflections [I > 2σ(I)]. CCDC number: 1517078. Crystallographic data of (±)-tengechlorenine (2): light yellowish plates, C23H24N1O3Cl, Mr 397.88, triclinic, space group P1̅, a = 8.7014(6) Å, b = 10.2583(7) Å, c = 11.1747(8) Å, α = 99.612(6)°, β = 106.611(6)°, γ = 93.943(6)°, V = 935.23(12) Å3, Z = 2, Dcalc = 1.413 mg/mm3, crystal size 0.1 × 0.1× 0.01 mm3, F(000) = 420, T = 167(2) K. The final R1 value is 0.0555 (wR2 = 0.1383) for 3723 reflections [I > 2σ(I)]. CCDC mumber: 1551311. Computational Analysis. The conformations of (11aS,13S,14S,11a′R,13′R,14′S)-tengerensine (1) were obtained by Spartan’14 software21 using the MMFF94 force field. Conformers occurring within a 5 kcal/mol energy window from the global minimum were then imported into the Gaussian 09 software22 for DFT-level geometry optimization and frequency calculation using the B3LYP functional with the basis set 6-31G(d). TDDFT ECD calculations were performed at the B3LYP/6-311++G(d,p) level with the optimized conformers using a PCM solvation model for

MeOH. The ECD curve for each optimized conformer was weighted by a Boltzmann distribution after UV correction, and the overall ECD curves were produced by SpecDis, version 1.64, software.23 Optical rotation calculations at the wavelength of 589.3 nm were performed with the optimized conformers at the B3LYP/6-311++G(d,p) computational level using a PCM solvation model for CHCl3. Cell Lines and Cell Culture. A small panel of human breast cancer cells (MDA-MB-468, MDA-MB-231, and MCF7) and human nontumorigenic breast epithelial cells (MCF10A) was purchased from the American Type Culture Collection. All cancer cells were maintained in RPMI 1640 medium with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), while MCF10A cells were cultured with 5% horse serum, 20 ng/mL epidermal growth factor, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 100 IU/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained in an incubator at 37 °C and 5% carbon dioxide. Luminescent Cell Viability Assay. Cell viability of cells after treatment with (±)-1, (+)-1, (−)-1, 2, (±)-3, and paclitaxel were determined using a CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, WI, USA). All compounds were prepared in 100 mM DMSO as a stock solution and diluted to various concentrations (1.5 to 100 μM) using sterile phosphate buffer solution. Paclitaxel was tested at concentrations of 0.01−10 μM as a positive control in this assay. The cancer and noncancer cells were seeded in 384-well opaque plates for 24 h at a density of 1000 cells/well followed by treatment with (±)-1, (+)-1, (−)-1, 2, (±)-3, and paclitaxel for 72 h. Cells treated with 0.1% DMSO were used as negative controls. Luminescence reading was measured using a SpectraMax M3 MultiMode microplate reader (Radnor, PA, USA). The inhibitory concentration of 50% cell viability (IC50) was determined based on the luminescent reading of treated cells and cells treated with the negative control.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00500. 1 H and 13C NMR, chiral HPLC chromatograms, and Xray data for compounds 1 and 2; Cartesian coordinates for (+)-1 (11aS,13S,14S,11a′R,13′R,14′S) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +603-89248208. Fax: +603-89248018. E-mail: KuanHon. [email protected]. ORCID

Toh-Seok Kam: 0000-0002-4910-6434 Kuan-Hon Lim: 0000-0003-1462-3324 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to MOHE Malaysia (FRGS/2/2013/ SG01/UNIM/02/1 and HIR-005) and International Medical University for financial support. The authors thank Professor J. F. F. Weber (Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus) for help with the circular dichroism measurements, and Dr. H. Khaledi (Department of Chemistry, University of Washington, Seattle, WA, USA) for further refining the X-ray data of compound 2. 2739

DOI: 10.1021/acs.jnatprod.7b00500 J. Nat. Prod. 2017, 80, 2734−2740

Journal of Natural Products

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Article

REFERENCES

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