Azacyclo-indoles and Phenolics from the Flowers of Juglans regia

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Azacyclo-indoles and Phenolics from the Flowers of Juglans regia Qian Li, An-Jun Deng, Li Li,* Lian-Qiu Wu, Ming Ji, Hai-Jing Zhang, Zhi-Hong Li, Lin Ma, Zhi-Hui Zhang, Xiao-Guang Chen, and Hai-Lin Qin* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Seven new azacyclo-indoles and phenolics and four known alkaloids were isolated from the flowers of Juglans regia. Spectroscopic and chromatographic data revealed that the structures of the new compounds are 5,6,11,12tetrahydropyrrolo[1′,2′:1,2]azepino[4,5-b]indole-3-carbaldehyde (1), (±)-5,6,7,11c-tetrahydro-1H-indolizino[7,8-b]indol3(2H)-one (2), (±)-9-hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-2-carboxamide (3), 5-(ethoxymethyl)-1-(4-hydroxyphenethyl)-1H-pyrrole-2-carbaldehyde (4), (±)-5,8-dihydroxy-4-(1H-indol-3-yl)-3,4-dihydronaphthalen-1(2H)-one (5), (±)-4(6-amino-9H-purin-9-yl)-5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (6), and (±)-4-(6-amino-9H-purin-9-yl)-5-hydroxy3,4-dihydronaphthalen-1(2H)-one (7). The five pairs of enantiomers were resolved, and the absolute configurations of the enantiomers were assigned via electronic circular dichroism data. Compound 1 exhibited significant in vitro growth inhibition against the HCT-116, HepG2, BGC-823, NCI-H1650, and A2780 cancer cell lines, with IC50 values of 2.87, 1.87, 2.28, 2.86, and 0.96 μM, respectively, and low cytotoxicity toward normal IEC-6 cells, with a 79.6% survival rate at a 10 μM concentration.

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RESULTS AND DISCUSSION The air-dried flowers (70 kg) of J. regia were extracted with 95% ethanol under reflux. The 95% ethanol extract (7.5 kg) was subjected to solvent extraction isolation, column chromatography over silica gel and Sephadex LH-20 gel, and preparative HPLC to afford seven new azacyclo-indoles and phenolics (1− 7) and four known alkaloids (8−11). Compound 1 was obtained as a light yellow, amorphous powder. The molecular formula C16H14N2O was established via the protonated molecular ion at m/z 251.1179 in the (+)HRESIMS spectrum, indicating 11 indices of hydrogen deficiency. This molecular formula was compatible with the 1H NMR spectrum of 1 showing 14 proton resonances and the 13C NMR spectrum with 16 carbon signals. The IR spectrum showed absorption bands for an amino group (3275 cm−1), a conjugated formyl group (1642 cm−1), and an aryl group (1538 cm−1). The 14 proton resonances were classified as the signals of an ortho-disubstituted benzene unit resonating at δH 7.34 (1H, br d, J = 8.0 Hz, H-7), 6.93 (1H, ddd, J = 8.0, 7.0, 1.0 Hz, H-8), 7.02 (1H, ddd, J = 8.0, 7.0, 1.0 Hz, H-9), and 7.26 (1H, br d, J = 8.0 Hz, H-10); two protons of a 5-substituted 2formylpyrrole moiety at δH 6.18 (1H, d, J = 4.0 Hz, H-1) and

Juglans regia Linn is a deciduous arbor plant from the Juglandaceae family. It is widely cultivated across China as both ornamental and economic plants, its nut being a popular and delicious food. Extracts of J. regia are used in traditional and folk medicine as an efficacious therapeutic agent to treat various diseases, for example, cancers, urinary system calculi, dermatitis, eczema, dysentery, and chronic bronchitis, among others.1 In previous reports of J. regia, several classes of compounds were isolated, including naphthoquinones from the roots and bark,2−5 naphthols from the roots and fruit,2,5 tetralones from the fruit,6,7 flavones from the stem-bark and whole plant,8−10 diarylheptanoids from the roots and bark,2,11 lignans from the walnut shell oil and whole plant,10,12 terpenes from the fruit and leaves,11,13 and polyphenols from the walnuts.14 However, a literature search indicates that limited chemical studies have been conducted on the flowers of this plant, with only flavones, tetralones, and naphthoquinones having been isolated.15,16 On the basis of the utilization of the flowers of J. regia to treat malignant tumors in several regions of China, a phytochemical investigation of the aqueous ethanol extract of the flowers was performed and resulted in the identification of seven new azacyclo-indoles and phenolics (1−7) and four known alkaloids (8−11). An in vitro evaluation for the growth inhibition of the compounds against several human cancer cell lines and on the viability of the normal intestinal epithelial cell-6 (IEC-6) cell line is also reported. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 29, 2016

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DOI: 10.1021/acs.jnatprod.6b00887 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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between C-2 of the indole moiety and C-2 of the pyrrole unit and an ethylene bridge between C-3 of the indole moiety and N-1 of the pyrrole unit (Figure 1). Thus, the structure of 1 was elucidated as 5,6,11,12-tetrahydropyrrolo[1′,2′:1,2]azepino[4,5b]indole-3-carbaldehyde. Compound 2 was obtained as a light yellow, amorphous powder. The molecular formula was established as C14H14N2O via the protonated molecular ion at m/z 227.1187 in the (+)HRESIMS spectrum, indicating nine indices of hydrogen deficiency. This molecular formula was compatible with the 1H NMR spectrum of 2 showing 14 proton resonances and the 13C NMR spectrum with 14 carbon signals. The IR spectrum showed absorption bands for an amino group (3255 cm−1), an amide carbonyl group (1663 cm−1), and an aryl group (1586, 1494, and 1453 cm−1). The 14 protons were classified as belonging to an ortho-disubstituted benzene moiety at δH 7.30 (1H, br d, J = 8.0 Hz, H-8), 7.04 (1H, br dd, J = 8.0, 7.2 Hz, H9), 6.95 (1H, br dd, J = 7.6, 7.2 Hz, H-10), and 7.38 (1H, br d, J = 7.6 Hz, H-11); a −CH2CH2− group at δH 2.95 (1H, ddd, J = 12.4, 12.4, 4.0 Hz, H2-5a), 4.26 (1H, dd, J = 12.4, 6.0 Hz, H25b), 2.63 (1H, m, H2-6a), and 2.73 (1H, dd, J = 15.2, 4.0 Hz, H2-6b); a −CHCH2CH2− moiety at δH 1.77 (1H, m, H2-1a), 2.54 (1H, m, H2-1b), 2.25 (1H, m, H2-2a), 2.49 (1H, ov, H22b), and 4.89 (1H, m, H-11c); and an amino proton at δH 11.00 (1H, s, H-7). The 14 carbons were classified as four methylene carbons, four aromatic methine carbons, an aliphatic methine carbon, two nitrogenated aromatic tertiary carbons at δC 135.1 (C-6a) and 136.6 (C-7a), two aromatic quaternary carbons at δC 126.9 (C-11a) and 106.4 (C-11b), and a carbonyl carbon. The HMBC cross-peaks from NH-7 to C-6a, 7a, 11a, and 11b; from H-8 to C-10 and 11a; from H-9 to C-7a and 11; from H-10 to C-8 and 11a; from H-11 to C-7a, 9, and 11b; and from H2-6 to C-11b indicated the presence of a 2,3disubstituted indole moiety. The HMBC cross-peaks from H2-1 to C-2, 3, and 11c; from H2-2 to C-1 and 11c; and from H2-5 to C-3 and 11c indicated the presence of a 1,5disubstitued pyrrolidin-2-one moiety. The COSY couplings from CH2-5 to CH2-6; from CH-11c to CH2-1; and from CH21 to CH2-2; along with the aforementioned HMBC cross-peaks from H2-1, H2-5, H2-6, H-11, and H-11c to the relevant carbons, indicated the connection between the indole moiety and the pyrrolidin-2-one moiety via both a −CH2CH2− bridge between C-2 of the indole moiety and N-1 of the pyrrolidin-2one moiety and a carbon−carbon σ-bond between C-3 of the indole moiety and C-5 of the pyrrolidin-2-one unit (Figure 1).

6.93 (1H, d, J = 4.0 Hz, H-2); six methylene protons at δH 4.24 (2H, s, H2-12), 5.01 (2H, t, J = 5.5 Hz, H2-5), and 2.94 (2H, t, J = 5.5 Hz, H2-6); a formyl proton at δH 9.45 (1H, s, H-13); and an amino proton at δH 10.90 (1H, s, H-11). The latter proton was assigned based on its chemical shift, the molecular formula, and the HSQC spectrum, in which no 13C NMR signal was correlated to the 1H NMR signal. The 16 carbon signals were classified as three methylene carbons, six aromatic methine carbons, four nitrogenated aromatic tertiary carbons, two aromatic quaternary carbons, and a formyl carbon. The HMBC cross-peaks from H2-5 to C-6a; from H2-6 to C-6a, 6b, and 11a; from H2-12 to C-6a and 11a; from H-7 to C-6a, 6b, 9, and 10a; from H-8 to C-6b and 10; from H-9 to C-7 and 10a; and from H-10 to C-8 and 6b indicated the presence of a 2,3-disubstituted indole moiety. The HMBC cross-peaks from H-1 to C-2, 3, and 12a; from H-2 to C-1, 3, 12a, and 13; from H2-5 to C-3 and 12a; from H2-12 to C-1 and 12a; and from H13 to C-3 indicated the presence of a 1,2-disubstitued-5formylpyrrole moiety. In addition, the HMBC cross-peaks from H-1 to C-12 and from H2-6 to C-5, along with the aforementioned cross-peaks from H2-5, H2-6, H-7, and H2-12 to the relevant carbons, indicated the connection of the indole moiety and the 5-formylpyrrole unit via a methylene bridge

Figure 1. Key HMBC correlations (H → C) of 1−7 and 1H−1H COSY correlations (bold) of 2−5. B



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Figure 2. Experimental and calculated ECD spectra of 2a and 2b.


Compound 2 had an [α]20D value of −18 (c 0.1, MeOH). The experimental electronic circular dichroism (ECD) spectrum deviated from the common profile, displaying only lowamplitude Cotton effects (CEs) (Figure S16, Supporting Information). Chiral HPLC analysis showed that 2 comprised two peaks of unequal areas (Figure S17, Supporting Information). Thus, 2 was elucidated as a scalemic mixture of (±)-5,6,7,11c-tetrahydro-1H-indolizino[7,8-b]indol-3(2H)one, resulting in weak optical activity and low-amplitude ECD CEs. Resolution of 2 via chiral HPLC afforded two enantiomers, 2a and 2b. Compound 2a had an [α]20D value of +57 (c 0.1, MeOH), and 2b [α]20D −48 (c 0.1, MeOH). The experimental ECD spectrum of 2a displayed CEs at 217 (Δε + 3.16), 245 (Δε +0.15), and 263 (Δε −0.26) nm. The positive CE at 217 nm was compatible with the time-dependent density functional theory (TDDFT)-calculated ECD value for the (11cR) absolute configuration. The experimental ECD spectrum of 2b displayed a mirror-image curve to that of 2a with CEs at 215 (Δε −4.20), 242 (Δε −0.37), and 266 (Δε +0.25) nm. The negative CE at 215 nm was compatible with the TDDFT-calculated ECD value for the (11cS) absolute configuration (Figure 2). Therefore, compounds 2a and 2b were assigned (11cR) and (11cS) absolute configurations, respectively. Compound 3 was obtained as a brown, amorphous powder. The molecular formula was established as C11H12N2O3 via the

protonated molecular ion at m/z 221.0915 in the (+)HRESIMS spectrum, indicating seven indices of hydrogen deficiency. This molecular formula was compatible with the 13C NMR spectrum showing 11 carbon signals. The IR spectrum showed absorption bands for hydroxy and amino groups (3332 and 3206 cm−1), a conjugated carbonyl and an amide carbonyl group (1658 cm−1), and a phenyl group (1587, 1516, and 1468 cm−1). Eleven of the 12 protons of 3 were evident in the 1H NMR spectrum. Eight protons were classified as being from a vic-trisubstituted benzene unit resonating at δH 7.35 (1H, dd, J = 8.0, 1.0 Hz, H-6), 7.26 (1H, dd, J = 8.0, 8.0 Hz, H-7), and 7.06 (1H, dd, J = 8.0, 1.0 Hz, H-8) and a −CHCH2CH2− moiety at δH 5.00 (1H, m, H-2), 2.15 (2H, m, H2-3), 2.49 (1H, ov, H2-4a), and 2.82 (1H, m, H2-4b) based on the chemical shifts, the spin−spin coupling data, and the COSY couplings. Three protons at δH 6.85 (1H, d, J = 6.8 Hz, H-1) and 5.65 (2H, s, NH2) were classified as belonging to exchangeable protons linked with heteroatoms by the HSQC spectrum, in which no 13C NMR signal was correlated with these two 1H NMR signals. The 11 carbons were classified as two methylene carbons, three aromatic methine carbons, an aliphatic methine carbon, a nitrogenated aromatic tertiary carbon at δC 133.1 (C9a), an oxygenated aromatic tertiary carbon at δC 155.8 (C-9), an aromatic quaternary carbon at δC 130.8 (C-5a), a ketocarbonyl carbon at δC 197.8 (C-5), and an amide carbonyl carbon at δC 160.0 (C-10). The three exchangeable protons and C



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6′, and 8′; and from H2-8′ to C-1′ and 7′ indicated the presence of a 4-hydroxyphenethyl moiety. The HMBC cross-peaks from H-3 to C-2, 4, 5, and 7; from H-4 to C-2, 3, 5, and 6; from H2-6 to C-4, 5, and 1″; from H-7 to C-2 and 3; from H2-1″ to C-6 and 2″; and from H3-2″ to C-1″ indicated the presence of a 5(ethoxymethyl)pyrrole-2-carbaldehyde moiety. The HMBC cross-peaks from H2-8′ to C-2 and 5 indicated that the 4hydroxyphenethyl moiety was linked to N-1 of the 5(ethoxymethyl)pyrrole-2-carbaldehyde moiety via C-8′ (Figure 1). Thus, the structure of 4 was defined as 5-(ethoxymethyl)-1(4-hydroxyphenethyl)-1H-pyrrole-2-carbaldehyde. Compound 5 was obtained as a brown, amorphous powder. The molecular formula was established as C18H15NO3 via the protonated molecular ion at m/z 294.1125 in the (+)HRESIMS spectrum, indicating 12 indices of hydrogen deficiency. This molecular formula was compatible with the 1H NMR spectrum with 15 proton resonances and the 13C NMR spectrum with 18 carbon signals. The IR spectrum showed absorption bands for hydroxy and amino groups (3397 cm−1), a conjugated carbonyl group (1640 cm−1), and a phenyl group (1586 and 1468 cm−1). Twelve of the 15 protons were classified as belonging to an aromatic AB spin−spin coupling system resonating at δH 7.11 (1H, d, J = 9.0 Hz, H-6) and 6.75 (1H, d, J = 9.0 Hz, H-7); an ortho-disubstituted benzene unit at δH 7.61 (1H, br d, J = 7.5 Hz, H-4′), 7.01 (1H, dd, J = 7.5, 7.5 Hz, H-5′), 7.09 (1H, dd, J = 7.5, 7.5 Hz, H-6′), and 7.35 (1H, br d, J = 7.5 Hz, H-7′); an aromatic proton only displaying a long-range coupling at δH 6.41 (1H, d, J = 2.0 Hz, H-2′); and a −CHCH2CH2− moiety at δH 2.38 (1H, m, H2-2a), 2.52 (1H, m, H2-2b), 2.31 (2H, m, H23), and 4.85 (1H, br, H-4). Three protons at δH 9.06 (1H, s, OH-5), 10.77 (1H, br s, NH-1′), and 12.03 (1H, s, OH-8) were classified as belonging to exchangeable protons linked with heteroatoms and were confirmed by the HSQC spectrum in which no 13C NMR signal was correlated with these 1H NMR signals. The DEPT NMR spectrum classified the 18 carbons as two methylene carbons; seven aromatic methine carbons; an aliphatic methine carbon; a nitrogenated aromatic tertiary carbon at δC 136.7 (C-7′a); two oxygenated aromatic tertiary carbons at δC 146.1 (C-5) and 154.7 (C-8); four aromatic quaternary carbons at δC 116.5 (C-9), 131.5 (C-10), 115.1 (C3′), and 126.4 (C-3′a); and a keto-carbonyl carbon at δC 205.9 (C-1). The three exchangeable protons were assigned as two phenolic groups and a secondary amino group by the molecular formula and the 13C NMR chemical shifts of the oxygenated and nitrogenated tertiary aromatic carbons. The HMBC crosspeaks from H2-2 to C-1, 3, 4, and 9; from H2-3 to C-1, 2, 4, and 10; from H-4 to C-2, 3, 5, 9, and 10; from OH-5 to C-5 and 10; from H-6 to C-8 and 10; from H-7 to C-5, 8, and 9; and from OH-8 to C-7, 8, and 9 indicated the presence of a 5,8dihydroxy-3,4-dihydronaphthalen-1(2H)-one-4-yl moiety. The HMBC cross-peaks from NH-1′ to C-2′, 3′a, and 7′a; from H2′ to C-3′, 3′a, and 7′a; from H-4′ to C-3′, 3′a, 6′, and 7′a; from H-5′ to C-3′a and 7′; from H-6′ to C-4′, 5′, and 7′a; and from H-7′ to C-3′a and 5′ indicated the presence of a 1Hindole-3-yl moiety. The HMBC correlations from H-4 to C-2′ and 3′a and from H-2′ to C-4 indicated the connection between the 5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one4-yl and 1H-indole-3-yl moieties through a carbon−carbon single bond (Figure 1). Compound 5 had an [α]20D value of −17 (c 0.1, MeOH). The experimental ECD spectrum deviated from the common profile, displaying only low-amplitude CEs (Figure S46, Supporting Information). Chiral HPLC analysis showed that 5 comprised two peaks of unequal areas (Figure

the undected proton were assigned to a phenolic group, an amido NH2, and a secondary NH group based on the molecular formula and the 13C NMR chemical shifts. Along with the COSY couplings, the HMBC cross-peaks from H-2 to C-3, 4, 9a, and 10; from H2-3 to C-2, 4, and 5; from H2-4 to C-2, 3, 5, and 5a; from H-6 to C-5, 5a, 7, and 8; from H-7 to C-8 and 9; from H-8 to C-6, 7, and 9; from NH2 to C-2; and from NH to C-2, 5a, and 10 indicated a 2D structure of 9-hydroxy-5-oxo2,3,4,5-tetrahydro-1H-benzo[b]azepine-2-carboxamide (Figure 1) for 3. Cmpound 3 had an [α]20D value of −13 (c 0.1, MeOH). The experimental ECD spectrum deviated from the common profile, displaying only low-amplitude CEs (Figure S26, Supporting Information). Chiral HPLC analysis showed that 3 comprised two peaks of unequal areas (Figure S27, Supporting Information). Thus, 3 was elucidated as a scalemic mixture of (±)-9-hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-2-carboxamide, resulting in weak optical activity and low-amplitude ECD CEs. Resolution of 3 via chiral HPLC afforded two enantiomers, 3a and 3b. Compound 3a had an [α]20D value of +72 (c 0.1, MeOH), and 3b [α]20D −66 (c 0.1, MeOH). The experimental ECD spectrum of 3a displayed CEs at 221 (Δε +5.45), 260 (Δε −1.19), 306 (Δε +0.31), and 342 (Δε −0.38) nm, compatible with the TDDFT-calculated ECD values for the (2R) absolute configuration. The experimental ECD spectrum of 3b displayed CEs at 221 (Δε −5.82), 261 (Δε +1.28), 303 (Δε −0.35), and 340 (Δε +0.47) nm, compatible with the calculated ECD values for the (2S) absolute configuration (Figure 3). Therefore, compounds 3a and 3b were assigned (2R) and (2S) absolute configurations, respectrively. Compound 4 was obtained as a brown, viscous liquid. The molecular formula C16H19NO3 was established via the protonated molecular ion at m/z 274.1447 in the (+)HRESIMS spectrum, indicating eight indices of hydrogen deficiency. This molecular formula was compatible with the 1H NMR spectrum of 4, showing 19 proton resonances, and the 13C NMR spectrum with 16 carbon signals. The IR spectrum showed absorption bands for a hydroxy group (3227 cm−1), a conjugated formyl group (1658 cm−1), and a phenyl group (1615, 1596, and 1516 cm−1). Eighteen protons were classified as belonging to an AA′BB′ spin system of a para-disubstituted benzene moiety resonating at δH 6.92 (2H, br d, J = 8.0 Hz, H2′,6′) and 6.66 (2H, br d, J = 8.0 Hz, H-3′,5′); two protons of a 5-substituted 2-formylpyrrole moiety at δH 6.98 (1H, d, J = 4.0 Hz, H-3) and 6.20 (1H, d, J = 4.0 Hz, H-4); an oxymethylene group at δH 4.24 (2H, s, H2-6); a −CH2−CH2− group at δH 2.79 (2H, t, J = 7.6 Hz, H2-7′) and 4.33 (2H, t, J = 7.6 Hz, H28′); a formyl proton at δH 9.49 (1H, s, H-7); and an ethoxy group at δH 3.41 (2H, q, J = 7.0 Hz, H2-1″) and 1.10 (3H, t, J = 7.0 Hz, H3-2″). The proton at δH 9.21 (1H, s, 4′-OH) was assigned as being an exchangeable proton linked to a heteroatom, which was confirmed by the HSQC spectrum, in which no 13C NMR signal was correlated with it. The 16 carbons in the 13C NMR spectrum were classified as a methyl carbon; four methylene carbons; six aromatic methine carbons including two pairs of chemically equivalent carbons; two nitrogenated aromatic tertiary carbons at δC 132.3 (C-2) and 139.9 (C-5); an oxygenated aromatic tertiary carbon at δC 156.4 (C-4′), which was evidence of a phenolic group; an aromatic quaternary carbon at δC 128.9 (C-1′); and a formyl carbon at δC 179.8 (C-7). The HMBC cross-peaks from H-2′/ 6′ to C-3′/5′, 4′, 2′/6′, and 7′; from H-3′/5′ to C-1′, 4′, and 3′/5′; from OH-4′ to C-3′, 4′, and 5′; from H2-7′ to C-1′, 2′/ D



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The signals of an aromatic AB spin−spin coupling system resonating at δH 7.14 (1H, d, J = 9.0 Hz, H-6) and 6.93 (1H, d, J = 9.0 Hz, H-7); an aliphatic ABCDX spin−spin coupling system of a −CHCH2CH2− moiety at δH 2.57 (1H, m, H2-2a), 2.74 (1H, ddd, J = 18.0, 14.0, 5.5 Hz, H2-2b), 2.50 (1H, ov, H23a), 2.57 (1H, m, H2-3b), and 6.03 (1H, br, H-4); and two exchangeable protons at δH 9.52 (1H, s, OH-5) and 12.00 (1H, s, OH-8) were highly similar to those of the 5,8-dihydroxy-3,4dihydronaphthalen-1(2H)-one-4-yl moiety in 5. The difference between the spectra of 5 and 6 involved the deshielded shift of H-4 in 6 (Δδ +1.18). The 13C NMR signals of the 5,8dihydroxy-3,4-dihydronaphthalen-1(2H)-one-4-yl moiety also showed high similarity to those of 5 except for the deshielded shift of C-4 in 6 (Δδ +17.8), suggesting the substitution of an electronegative heteroatom at C-4. This identification of the 5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one-4-yl moiety in 6 was confirmed by the HMBC cross-peaks from H2-2 to C-1, 3, 4, and 9; from H2-3 to C-1, 2, and 4; from H-4 to C-2, 3, 5, 9, and 10; from OH-5 to C-5, 6, and 10; from H-6 to C-5, 7, 8, and 10; from H-7 to C-5, 6, 8, and 9; and from OH-8 to C-7, 8, and 9. The remaining 1H and 13C NMR signals of 6 were assigned as those derived from an adenine-9-yl moiety by comparison of the NMR data of 6 and adenine, with the two methines and the primary amino protons resonating at δH 8.15 (1H, s, H-2′), 7.69 (1H, s, H-8′), and 7.23 (2H, s, NH2-6′) in the 1H NMR spectrum and the five carbons resonating at δC

S47, Supporting Information). Thus, 5 was elucidated as a scalemic mixture of (±)-5,8-dihydroxy-4-(1H-indol-3-yl)-3,4dihydronaphthalen-1(2H)-one, resulting in weak optical activity and low-amplitude ECD CEs. Resolution of 5 via chiral HPLC afforded two enantiomers, 5a and 5b. Compound 5a had an [α]20D value of +127 (c 0.02, MeOH), and 5b [α]20D −131 (c 0.02, MeOH). The experimental ECD spectrum of 5a displayed CEs at 222 (Δε −2.13), 258 (Δε +0.95), 281 (Δε −0.60), and 374 (Δε −0.46) nm, compatible with the TDDFT-calculated ECD values for the (4S) absolute configuration. The experimental ECD spectrum of 5b displayed CEs at 222 (Δε +2.70), 257 (Δε −1.61), 281 (Δε +1.03), and 374 (Δε +0.68) nm, compatible with the calculated ECD values for the (4R) absolute configuration (Figure 4). Therefore, compounds 5a and 5b were assigned (4S) and (4R) absolute configurations, respectively. Compound 6 was obtained as light yellow needles. The molecular formula C15H13N5O3 was established via the protonated molecular ion at m/z 312.1087 in the (+)HRESIMS spectrum, indicating 12 indices of hydrogen deficiency. This molecular formula was compatible with the 1H NMR spectrum showing 13 proton resonances and the 13C NMR spectrum with 15 carbon signals. The IR spectrum showed absorption bands for hydroxy and amino groups (3433, 3320, and 3113 cm−1), a conjugated carbonyl and an imino group (1671 and 1643 cm−1), and a phenyl group (1605, 1571, and 1472 cm−1). E



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(1H, dd, J = 8.0, 1.0 Hz, H-6), 7.38 (1H, dd, J = 8.0, 8.0 Hz, H7), and 7.47 (1H, dd, J = 8.0, 1.0 Hz, H-8), with only one phenolic proton detected. The 13C and DEPT NMR spectra showed one more aromatic methine carbon in 7, assigned to the resonance at δC 116.9 (C-8) via the HMBC data (Figure 1). Deshielded shifts of Δδ +10.8 and +17.0 for C-7 and C-9, respectively, were observed in 7 compared with their counterparts in 6. The assignment of 7 as the 8-deoxy analogue of 6, i.e., 4-(6-amino-9H-purin-9-yl)-5-hydroxy-3,4-dihydronaphthalen-1(2H)-one, was confirmed by the HMBC crosspeaks from H2-2 to C-1, 3, 4, and 9; from H2-3 to C-1, 2, 4, and 10; from H-4 to C-2, 3, 4′, 5, 8′, 9, and 10; from H-6 to C-5, 8, and 10; from H-7 to C-5, 6, 8, and 9; from H-8 to C-1, 6, and 10; from H-2′ to C-4′ and 6′; from H-8′ to C-4, 4′, and 5′; and from NH2-6′ to C-5′ (Figure 1). Compound 7 had an [α]20D value of −57 (c 0.1, MeOH). Although the experimental ECD spectrum of 7 displayed CEs at 221 (Δε −0.50), 234 (Δε +0.33), 261 (Δε −1.15), and 309 (Δε +0.54) nm (Figure S65, Supporting Information), chiral HPLC analysis showed that 7 comprised two peaks of unequal areas (Figure S66, Supporting Information). Thus, compound 7 was elucidated as a scalemic mixture. Resolution of 7 via chiral HPLC afforded two enantiomers, 7a and 7b. Compound 7a had an [α]20D value of +160 (c 0.1, MeOH), and 7b [α]20D −155 (c 0.1, MeOH). The experimental ECD spectrum of 7a displayed CEs at 228 (Δε −0.90), 260 (Δε +2.61), 306 (Δε −1.25), and 343 (Δε +0.33) nm, compatible with the TDDFT-calculated ECD values for the (4R) absolute configuration. The experimental ECD spectrum of 7b displayed CEs at 229 (Δε +0.68), 260 (Δε −2.10), 307 (Δε +1.11), and 342 (Δε −0.29) nm, compatible with the calculated ECD values for the (4S) absolute configuration (Figure 6). Moreover, in addition to typical antipodal ECD curves in terms of the aforementioned CEs between 7a and 7b, the two enantiomers showed nearly identical ECD curves to those of 6a and 6b, respectively. Therefore, compounds 7a and 7b were assigned (4R) and (4S) absolute configurations, respectively. The known compounds, 6-bromo-1H-indole-3-carbaldehyde (8),19 N-benzoyl-L-phenylalaninol (9),20 oleracein E (10),21 and dracocephin A (11),22 were identified by comparing their 1 H and 13C NMR and MS data with reported data. This work presents the first isolation and characterization of these compounds from J. regia. The isolated compounds were evaluated for growth inhibition against five human cancer cell lines using the 3-

152.7 (C-2′), 149.9 (C-4′), 119.5 (C-5′), 156.5 (C-6′), and 140.4 (C-8′) in the 13C NMR spectrum.17,18 The deshielded shifts of H-4 and C-4 indicated that the adenine moiety was linked to the 5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one4-yl moiety via a nitrogen−carbon single bond. The HMBC cross-peaks from H-2′ to C-4′ and 6′; from H-8′ to C-4′ and 5′; and from NH2-6′ to C-5′ and 6′ confirmed the structure of the adenine moiety, while the HMBC cross-peaks from H-4 to C-4′ and 8′ confirmed the 2D structure of 6 as 4-(6-amino-9Hpurin-9-yl)-5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (Figure 1). Compound 6 had an [α]20D value of −5 (c 0.1, MeOH). The experimental ECD spectrum deviated from the common profile, displaying only low-amplitude CEs (Figure S55, Supporting Information). Chiral HPLC analysis showed that 6 comprised two peaks of unequal areas (Figure S56, Supporting Information). Thus, 6 was elucidated as a scalemic mixture of (±)-4-(6-amino-9H-purin-9-yl)-5,8-dihydroxy-3,4dihydronaphthalen-1(2H)-one, resulting in weak optical activity and low-amplitude ECD CEs. Resolution of 6 via chiral HPLC afforded two enantiomers, 6a and 6b. Compound 6a had an [α]20D value of +112 (c 0.1, MeOH), and 6b [α]20D −108 (c 0.1, MeOH). The experimental ECD spectrum of 6a displayed CEs at 229 (Δε −0.97), 264 (Δε +2.57), 305 (Δε −0.39), and 346 (Δε +0.24) nm, compatible with the TDDFT-calculated ECD values for the (4R) absolute configuration. The experimental ECD spectrum of 6b displayed CEs at 229 (Δε +1.20), 260 (Δε −3.08), 306 (Δε +1.53), and 343 (Δε −0.50) nm, compatible with the calculated ECD values for the (4S) absolute configuration (Figure 5). Therefore, compounds 6a and 6b were assigned (4R) and (4S) absolute configurations, respectively. Compound 7 was obtained as a light yellow, amorphous powder. The molecular formula C15H13N5O2 was established via the protonated molecular ion at m/z 296.1145 in the (+)HRESIMS spectrum, indicating one less oxygen atom than 6 and 12 indices of hydrogen deficiency. This molecular formula was compatible with the 1H NMR spectrum showing 13 proton resonances and the 13C NMR spectrum with 15 carbon signals. The IR spectrum showed absorption bands for hydroxy and amino groups (3432 and 3137 cm−1), a conjugated carbonyl and an imino group (1674 cm−1), and a phenyl group (1591 and 1507 cm−1). Comparison of the NMR data of 7 and 6 revealed that in the 1H NMR spectrum of 7 the aromatic AB spin−spin coupling system was replaced by an aromatic ABC spin−spin coupling system resonating at δH 7.09 F



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deposited in the Herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, P. R. China. Extraction and Isolation. Air-dried flowers of J. regia (70 kg) were extracted twice (2 and 1 h) under reflux conditions using 95% EtOH (560 and 480 L). After removing the solvent under reduced pressure, a viscous residue (ca. 7.5 kg) was obtained, which was suspended in H2O and defatted in a separatory funnel using petroleum ether until the upper layer became nearly colorless. The aqueous layer was extracted with EtOAc until the EtOAc solution was nearly colorless. The EtOAc extracts were combined and extracted three times using a solution of HCl in H2O (pH = 2). The acidic aqueous extracts were combined, and a 2 N NaOH solution was added dropwise while stirring until a weakly alkaline solution was obtained. This solution was extracted with EtOAc until the solution was nearly colorless. The EtOAc extracts were combined and washed with H2O until the extracting water was neutral. The supernatant EtOAc solution was evaporated under vacuum to near dryness to yield a brown, viscous residue (28 g). The EtOAc solution remaining after extraction with the HCl solution was washed with H2O until the extracting water was neutral. Removing the EtOAc solvent under reduced pressure yielded a brown, sticky, neutral EtOAc residue (450 g). The alkaline EtOAc residue was subjected to silica gel CC and eluted using a 25:1 → 15:1 (v/v) gradient elution of CHCl3/MeOH to yield two subfractions, BFr. 1 and 2. BFr. 2 (2.92 g; eluted with CHCl3/MeOH, 15:1, v/v) was separated on a Sephadex LH-20 column and eluted with MeOH to yield five subfractions, BFr. 2-1 to 2-5. BFr. 2-2 (600 mg) was separated on a Sephadex LH-20 column and eluted with MeOH to yield three subfractions, BFr. 2-2-1 to 2-2-3. BFr. 2-2-3 (32 mg) was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (9:11, v/v) at a flow rate of 6 mL min−1with UV detection at 230 nm] to afford 3 (4 mg; tR = 16 min), which was analyzed using chiral HPLC [mobile phase of n-hexane/2-propanol (1:1, v/v) at a flow rate of 0.6 mL min−1 with a column temperature of 25 °C and UV detection at 230 nm], with two peaks of unequal areas appearing at tR = 23.2 and 37.8 min (Figure S27, Supporting Information). The resolution of 3 (1.6 mg) was performed via the same chiral HPLC procedure to yield 3a (0.4 mg) and 3b (0.8 mg). BFr. 2-4 (650 mg) was separated on a Sephadex LH-20 column and eluted with MeOH to yield a subfraction (40 mg). This subfraction was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (2:3, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford 7 (24 mg; tR = 23 min). Compound 7 was analyzed using chiral HPLC [mobile phase of n-hexane/2-propanol (8:2, v/v) at a flow rate of 0.6 mL min−1 with a column temperature of 25 °C and UV detection at 230 nm], showing two peaks of unequal areas at tR = 10.4 and 11.6 min (Figure S66, Supporting Information). The resolution of 7 (1.9 mg) was performed via chiral HPLC to yield 7a (0.5 mg) and 7b (0.8 mg). BFr. 2-5 (300 mg) was purified by crystallization from MeOH to yield 6 (35 mg) as light yellow needles; 6 was analyzed using chiral HPLC [mobile phase of n-hexane/2-propanol (1:1, v/v) at a flow rate of 0.6 mL min−1 with a column temperature of 25 °C and UV detection at 230 nm], showing two peaks of unequal areas at tR = 8.4 and 10.4 min (Figure S56, Supporting Information). The resolution of 6 (2.4 mg) was performed via chiral HPLC to yield 6a (0.8 mg) and 6b (0.9 mg). The neutral EtOAc residue was subjected to silica gel CC and eluted using a 25:1 → 15:1 (v/v) gradient elution of CHCl3/MeOH to yield two subfractions, Fr. 1 and 2. Fr. 1 (58 g; CHCl3/MeOH, 25:1, v/v) was applied to MCI and eluted with a 30:70 → 50:50 → 70:30 → 85:15 (v/v) gradient elution of EtOH/H2O to yield four subfractions, Fr. 1-1 to 1-4. Fr. 1-1 (15 g; EtOH/H2O, 30:70, v/v) was applied to flash C18 CC and eluted with a 30:70 → 50:50 (v/v) gradient elution of MeOH/H2O to yield two subfractions, Fr. 1-1-1 and 1-1-2. Fr. 1-1-2 (2.5 g; MeOH/H2O, 50:50, v/v) was applied to Sephadex LH-20 CC and eluted with MeOH to yield a subfraction (108 mg). This subfraction was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (3:2, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford a crude eluate at tR = 38 min. Purification of this eluate via preparative RP-HPLC [mobile phase of MeCN/H2O (11:9, v/v) at a flow rate of 6 mL min−1 with UV

(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, as described previously.23 Both Taxol and 1-hydroxy-2-methoxy-12-methyl-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-c]phenanthridin-12-ium chloride (NK109)24 were used as positive controls. All cancerous cells were treated continuously with each sample for 96 h. The results of the means of three replicates were expressed as the concentrations of the compound inhibiting cell growth by 50%, i.e., the IC50 values. Compound 1 exhibited significant growth inhibition against human colorectal (HCT-116), human hepatocellular carcinoma (HepG2), human gastric adenocarcinoma (BGC823), human non-small-cell lung (NCI-H1650), and human ovarian carcinoma (A2780) cancer cell lines with IC50 values of 2.87, 1.87, 2.28, 2.86, and 0.96 μM, respectively. The positive controls exhibited growth inhibition at IC50 values of 3.94, 1.19, 1.84, 1.75, and 0.90 μM for NK109 and 0.0002, 0.0076, 0.0006, >1, and 0.0204 μM for Taxol for the same human cancer cell lines. In contrast, none of the other compounds showed any bioactivity, with IC50 values of more than 10 μM each. Given that the flowers of J. regia are also used as a food in some rural areas in China and considering the tested growth inhibition against HCT-116 cell lines, compound 1 was investigated for its effect on the in vitro viability of the normal IEC-6 cell line using the MTT assay at a concentration of 10 μM with a blank control group. The results showed that compound 1 has low toxicity; it exhibited a weak growth inhibition with a 79.6% survival rate when co-incubated with IEC-6 cells for 48 h. Although not all of the test compounds were found to exhibit growth inhibition against human cancer cell lines, the finding that compound 1 showed selective growth inhibition against the aforementioned human cancer cell lines provided some support for the application of the flowers of J. regia in some folk medicine systems to treat cancers.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined using an XT5B microscopic melting point apparatus (Keyi, Beijing, China). Optical rotations were measured on a PerkinElmer 241 digital polarimeter. UV spectra were recorded on a JASCO V-650 spectrophotometer, and IR spectra were recorded on a Nicolet 5700 spectrometer. ECD experiments were conducted using a JASCO J-815 ECD spectrometer. The 1D and 2D NMR spectra were recorded on either a Varian Mercury-400 NMR spectrometer or a Bruker AV-III-500 NMR spectrometer with tetramethylsilane as an internal standard and DMSO-d6 as a solvent. HRESIMS experiments were conducted using an Agilent 1100 series LC/MSD Trap SL mass spectrometer. The preparative HPLC procedure was conducted on a Shimadzu LC-6AD instrument with an SPD-20A detector and a reversed-phase C18 column (BDS HYPERSIL, ODS-A, 250 × 21.2 mm, particle size 5 μm; Thermo, CA, USA). Chiral HPLC analysis and resolution were conducted on a Shimadzu CTO-10AS VP instrument with an SPD-20A detector and a Chiralpak AD-H column (250 × 4.6 mm, particle size 5 μm) packed with amylose tris(3,5-dimethylphenylcarbarmate) coated on 5 μm silica gel (Daicel, Shanghai, China). Silica gel (200−300 mesh size; Qingdao Marine Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden), and MCI gel (CHP20P, 75−150 μm; Mitsubishi Chemical Industries Ltd., Tokyo, Japan) were used for column chromatography (CC). TLC was conducted using glass plates precoated with silica gel G. Spots were visualized under UV light and by spraying with a 10% H2SO4 solution in 95% EtOH followed by heating. Plant Material. Flowers of J. regia were collected from the Temple of Heaven Park, Beijing, P. R. China, in May 2014. The flowers were identified by one of the authors (L.M.), according to the description for J. regia in the Flora of China.1 A voucher specimen (ID-S-2601) is G



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(2H, s, H2-12), 9.45 (1H, s, H-13); 13C NMR (125 MHz) δ 109.1 (C1), 124.4 (C-2), 131.4 (C-3), 43.5 (C-5), 25.3 (C-6), 108.4 (C-6a), 128.8 (C-6b), 117.8 (C-7), 119.0 (C-8), 121.3 (C-9), 111.1 (C-10), 135.0 (C-10a), 130.3 (C-11a), 25.5 (C-12), 143.6 (C-12a), 179.6 (C13); HRESIMS m/z 251.1179 [M + H]+ (calcd for C16H15N2O, 251.1179). (±)-5,6,7,11c-Tetrahydro-1H-indolizino[7,8-b]indol-3(2H)-one (2): light yellow, amorphous powder; [α]20D −18 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (4.45), 223 (4.51), 282 (3.81) nm; IR (KBr) νmax 3255, 2980, 2729, 1663, 1586, 1494, 1453, 1423, 1312, 1268, 1157, 1005, 906, 837, 749 cm−1; 1H NMR (400 MHz) δ 1.77 (1H, m, H2-1a), 2.54 (1H, m, H2-1b), 2.25 (1H, m, H2-2a), 2.49 (1H, ov, H2-2b), 2.95 (1H, ddd, J = 12.4, 12.4, 4.0 Hz, H2-5a), 4.26 (1H, dd, J = 12.4, 6.0 Hz, H2-5b), 2.63 (1H, m, H2-6a), 2.73 (1H, dd, J = 15.2, 4.0 Hz, H2-6b), 11.00 (1H, s, H-7), 7.30 (1H, br d, J = 8.0 Hz, H-8), 7.04 (1H, br dd, J = 8.0, 7.2 Hz, H-9), 6.95 (1H, br dd, J = 7.6, 7.2 Hz, H-10), 7.38 (1H, br d, J = 7.6 Hz, H-11), 4.89 (1H, m, H-11c); 13C NMR (100 MHz) δ 25.9 (C-1), 31.5 (C-2), 172.8 (C-3), 37.3 (C-5), 21.2 (C-6), 135.1 (C-6a), 136.6 (C-7a) 111.6 (C-8), 121.5 (C-9), 119.1 (C-10), 118.3 (C-11), 126.9 (C-11a), 106.4 (C-11b), 54.1 (C11c); HRESIMS m/z 227.1187 [M + H]+ (calcd for C14H15N2O, 227.1179). (11cR)-5,6,7,11c-Tetrahydro-1H-indolizino[7,8-b]indol-3(2H)-one (2a): light yellow, amorphous powder; [α]20D +57 (c 0.1, MeOH); experimental ECD (MeOH) λmax (Δε) 217 (+3.16), 245 (+0.15), 263 (−0.26) nm. (11cS)-5,6,7,11c-Tetrahydro-1H-indolizino[7,8-b]indol-3(2H)-one (2b): light yellow, amorphous powder; [α]20D −48 (c 0.1, MeOH); experimental ECD (MeOH) λmax (Δε) 215 (−4.20), 242 (−0.37), 266 (+0.25) nm. (±)-9-Hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-2carboxamide (3): brown, amorphous powder; [α]20D −13 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.87), 221 (3.76), 257 (3.32), 316 (3.02) nm; IR (KBr) νmax 3332, 3206, 2922, 2850, 1658, 1587, 1516, 1468, 1291, 1140, 1024, 951, 806 cm−1; 1H NMR (400 MHz) δ 6.85 (1H, d, J = 6.8 Hz, H-1), 5.00 (1H, m, H-2), 2.15 (2H, m, H2-3), 2.49 (1H, ov, H2-4a), 2.82 (1H, m, H2-4b), 7.35 (1H, dd, J = 8.0, 1.0 Hz, H-6), 7.26 (1H, dd, J = 8.0, 8.0 Hz, H-7), 7.06 (1H, dd, J = 8.0, 1.0 Hz, H-8), 5.65 (2H, s, NH2); 13C NMR (100 MHz) δ 41.3 (C2), 28.2 (C-3), 33.3 (C-4), 197.8 (C-5), 130.8 (C-5a), 117.4 (C-6), 129.1 (C-7), 122.0 (C-8), 155.8 (C-9), 133.1 (C-9a), 160.0 (C-10); HRESIMS m/z 221.0915 [M + H]+ (calcd for C11H13N2O3, 221.0921). (2R)-9-Hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-2carboxamide (3a): brown, amorphous powder; [α]20D +72 (c 0.1, MeOH); experimental ECD (MeOH) λmax (Δε) 221 (+5.45), 260 (−1.19), 306 (+0.31), 342 (−0.38) nm. (2S)-9-Hydroxy-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-2carboxamide (3b): brown, amorphous powder; [α]20D −66 (c 0.1, MeOH); experimental ECD (MeOH) λmax (Δε) 221 (−5.82), 261 (+1.28), 303 (−0.35), 340 (+0.47) nm. 5-(Ethoxymethyl)-1-(4-hydroxyphenethyl)-1H-pyrrole-2-carbaldehyde (4): brown, viscous liquid; UV (MeOH) λmax (log ε) 206 (4.00), 223 (3.93), 292 (4.16) nm; IR (KBr) νmax 3227, 2973, 2727, 2252, 2128, 1658, 1615, 1596, 1516, 1453, 1404, 1371, 1351, 1026, 953, 830, 806, 780 cm−1; 1H NMR (400 MHz) δ 6.98 (1H, d, J = 4.0 Hz, H-3), 6.20 (1H, d, J = 4.0 Hz, H-4), 4.24 (2H, s, H2-6), 9.49 (1H, s, H-7), 6.92 (2H, br d, J = 8.0 Hz, H-2′, 6′), 6.66 (2H, br d, J = 8.0 Hz, H-3′, 5′), 2.79 (2H, t, J = 7.6 Hz, H2-7′), 4.33 (2H, t, J = 7.6 Hz, H2-8′), 3.41 (2H, q, J = 7.0 Hz, H2-1″), 1.10 (3H, t, J = 7.0 Hz, H3-2″), 9.21 (1H, s, 4′-OH); 13C NMR (100 MHz) δ 132.3 (C-2), 124.2 (C3), 111.2 (C-4), 139.9 (C-5), 63.3 (C-6), 179.8 (C-7), 128.9 (C-1′), 130.1 (C-2′, 6′), 115.7 (C-3′, 5′), 156.4 (C-4′), 36.7 (C-7′), 47.6 (C8′), 65.4 (C-1″), 15.4 (C-2″); HRESIMS m/z 274.1447 [M + H]+ (calcd for C16H20NO3, 274.1438). (±)-5,8-Dihydroxy-4-(1H-indol-3-yl)-3,4-dihydronaphthalen1(2H)-one (5): brown, amorphous powder; [α]20D −17 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.71), 222 (3.78), 265 (3.17), 373 (2.77) nm; IR (KBr) νmax 3397, 2923, 2852, 1640, 1586, 1468, 1341, 1223, 1170, 1012, 939, 911, 844, 828, 749 cm−1; 1H NMR (500 MHz) δ 2.38 (1H, m, H2-2a), 2.52 (1H, m, H2-2b), 2.31 (2H, m,

detection at 230 nm] afforded 4 (15 mg; tR = 36 min). Fr. 1-2 (6 g; EtOH/H2O, 50:50, v/v) was applied to Sephadex LH-20 CC and eluted with MeOH to yield three subfractions, Fr. 1-2-1 to 1-2-3. Fr. 12-3 (98 mg) was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (11:9, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford 5 (5 mg; tR = 78 min), which was analyzed using chiral HPLC [mobile phase of n-hexane/2-propanol (1:1, v/v) at a flow rate of 0.6 mL min−1 with a column temperature of 25 °C and UV detection at 230 nm], with two peaks of unequal areas appearing at tR = 8.8 and 9.6 min (Figure S47, Supporting Information). The resolution of 5 (0.8 mg) was performed via chiral HPLC to yield 5a (0.2 mg) and 5b (0.2 mg). Fr. 1-4 (12 g; EtOH/ H2O, 85:15, v/v) was applied to flash C18 CC and eluted with a 30:70 → 60:40 (v/v) gradient elution of MeOH/H2O to yield two subfractions, Fr. 1-4-1 and 1-4-2. Fr. 1-4-2 (2 g; MeOH/H2O, 60:40, v/v) was applied to a Sephadex LH-20 column and eluted with MeOH to yield three subfractions, Fr. 1-4-2-1 to 1-4-2-3. Fr. 1-4-2-3 (150 mg) was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (7:3, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford 1 (6 mg; tR = 27 min). Fr. 2 (72 g; CHCl3/MeOH, 15:1, v/v) was subjected to silica gel CC and eluted using a 25:1 → 15:1 gradient elution of CHCl3/MeOH to yield two subfractions, Fr. 2-1 and 2-2. Fr. 2-1 (15 g; CHCl3/ MeOH, 25:1, v/v) was applied to flash C18 CC and eluted with a 25:75 → 35:65 → 45:55 (v/v) gradient elution of MeOH/H2O to yield three subfractions, Fr. 2-1-1 to 2-1-3. Fr. 2-1-2 (3.5 g; MeOH/H2O, 35:65, v/v) was separated on a Sephadex LH-20 column and eluted with MeOH to yield three subfractions, Fr. 2-1-2-1 to 2-1-2-3. Fr. 2-12-3 (80 mg) was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (11:9, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford 8 (10 mg; tR = 18 min). Fr. 2-1-3 (2.5 g; MeOH/H2O, 45:55, v/v) was separated on a Sephadex LH-20 column and eluted with MeOH to yield four subfractions, Fr. 2-1-3-1 to 2-1-3-4. Fr. 2-1-3-1 (180 mg) was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (11:9, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford a crude eluate at tR = 31 min, which was applied to preparative RP-HPLC [mobile phase of MeCN/H2O (2:3, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford 9 (12 mg; tR = 28 min). Fr. 2-1-3-4 (100 mg) was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (11:9, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford 2 (22 mg; tR = 39 min), which was analyzed using chiral HPLC [mobile phase of n-hexane/2-propanol (7:3, v/v) at a flow rate of 0.6 mL min−1 with a column temperature of 25 °C and UV detection at 230 nm], showing two peaks of unequal areas at tR = 6.3 and 7.1 min (Figure S17, Supporting Information). The resolution of 2 (2.0 mg) was performed via chiral HPLC to yield 2a (0.6 mg) and 2b (0.8 mg). Fr. 2-2 (10 g; CHCl3/MeOH, 15:1, v/v) was applied to flash C18 CC and eluted with a 60:40 → 75:25 (v/v) gradient elution of MeOH/H2O to yield two subfractions, Fr. 2-2-1 and 2-2-2. Fr. 2-2-1 (2 g; MeOH/H2O, 60/40, v/v) was applied to a Sephadex LH-20 CC and eluted with MeOH to yield two subfractions, Fr. 2-2-1-1 and 2-21-2. Fr. 2-2-1-2 (65 mg) was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (2:3, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford 10 (15 mg; tR = 15 min). Fr. 2-2-2 (2.2 g; MeOH/H2O, 75:25, v/v) was applied to Sephadex LH20 CC and eluted with MeOH to yield a subfraction (100 mg), which was purified by preparative RP-HPLC [mobile phase of MeOH/H2O (11:9, v/v) at a flow rate of 6 mL min−1 with UV detection at 230 nm] to afford 11 (20 mg; tR = 31 min). 5,6,11,12-Tetrahydropyrrolo[1′,2′:1,2]azepino[4,5-b]indole-3-carbaldehyde (1): light yellow, amorphous powder; UV (MeOH) λmax (log ε) 209 (4.28), 224 (4.35), 293 (4.11) nm; IR (KBr) νmax 3275, 3114, 3078, 2891, 2848, 2738, 1642, 1538, 1488, 1407, 1334, 1259, 1031, 810, 771, 745 cm−1; 1H NMR (500 MHz) δ 6.18 (1H, d, J = 4.0 Hz, H-1), 6.93 (1H, d, J = 4.0 Hz, H-2), 5.01 (2H, t, J = 5.5 Hz, H2-5), 2.94 (2H, t, J = 5.5 Hz, H2-6), 7.34 (1H, br d, J = 8.0 Hz, H-7), 6.93 (1H, ddd, J = 8.0, 7.0, 1.0 Hz, H-8), 7.02 (1H, ddd, J = 8.0, 7.0, 1.0 Hz, H-9), 7.26 (1H, br d, J = 8.0 Hz, H-10), 10.90 (1H, s, H-11), 4.24 H



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H2-3), 4.85 (1H, br, H-4), 9.06 (1H, s, 5-OH), 7.11 (1H, d, J = 9.0 Hz, H-6), 6.75 (1H, d, J = 9.0 Hz, H-7), 12.03 (1H, s, 8-OH), 6.41 (1H, d, J = 2.0 Hz, H-2′), 7.61 (1H, br d, J = 7.5 Hz, H-4′), 7.01 (1H, dd, J = 7.5, 7.5 Hz, H-5′), 7.09 (1H, dd, J = 7.5, 7.5 Hz, H-6′), 7.35 (1H, br d, J = 7.5 Hz, H-7′), 10.77 (1H, br s, 1′-NH); 13C NMR (125 MHz) δ 205.9 (C-1), 33.9 (C-2), 27.4 (C-3), 29.0 (C-4), 146.1 (C-5), 125.0 (C-6), 115.3 (C-7), 154.7 (C-8), 116.5 (C-9), 131.5 (C-10), 123.2 (C2′), 115.1 (C-3′), 126.4 (C-3′a), 118.5 (C-4′), 118.4 (C-5′), 121.1 (C6′), 111.7 (C-7′), 136.7 (C-7′a); HRESIMS m/z 294.1125 [M + H]+ (calcd. for C18H16NO3, 294.1125). (4S)-5,8-Dihydroxy-4-(1H-indol-3-yl)-3,4-dihydronaphthalen1(2H)-one (5a): brown, amorphous powder; [α]20D +127 (c 0.02, MeOH); experimental ECD (MeOH) λmax (Δε) 222 (−2.13), 258 (+0.95), 281 (−0.60), 374 (−0.46) nm. (4R)-5,8-Dihydroxy-4-(1H-indol-3-yl)-3,4-dihydronaphthalen1(2H)-one (5b): brown, amorphous powder; [α]20D −131 (c 0.02, MeOH); experimental ECD (MeOH) λmax (Δε) 222 (+2.70), 257 (−1.61), 281 (+1.03), 374 (+0.68) nm. (±)-4-(6-Amino-9H-purin-9-yl)-5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (6): light yellow needles, mp 207.3−208.3 °C; [α]20D −5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (4.11), 262 (3.88), 369 (3.26) nm; IR (KBr) νmax 3433, 3320, 3113, 1671, 1643, 1605, 1571, 1472, 1340, 1219, 1172, 1006, 940, 914, 844, 777, 646 cm−1; 1H NMR (400 MHz) δ 2.57 (1H, m, H2-2a), 2.74 (1H, ddd, J = 18.0, 14.0, 5.5 Hz, H2-2b), 2.50 (1H, ov, H2-3a), 2.57 (1H, m, H2-3b), 6.03 (1H, br, H-4), 9.52 (1H, s, 5-OH), 7.14 (1H, d, J = 9.0 Hz, H-6), 6.93 (1H, d, J = 9.0 Hz, H-7), 12.00 (1H, s, 8-OH), 8.15 (1H, s, H-2′), 7.69 (1H, s, H-8′), 7.23 (2H, s, NH2); 13C NMR (100 MHz) δ 204.8 (C-1), 33.5 (C-2), 27.5 (C-3), 46.8 (C-4), 147.6 (C-5), 125.9 (C-6), 119.2 (C-7), 155.2 (C-8), 117.1 (C-9), 122.9 (C-10), 152.7 (C-2′), 149.9 (C-4′), 119.5 (C-5′), 156.5 (C-6′), 140.4 (C-8′); HRESIMS m/ z 312.1087 [M + H]+ (calcd for C15H14N5O3, 312.1091). (4R)-4-(6-Amino-9H-purin-9-yl)-5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (6a): light yellow, amorphous powder; [α]20D +112 (c 0.1, MeOH); experimental ECD (MeOH) λmax (Δε) 229 (−0.97), 264 (+2.57), 305 (−0.39), 346 (+0.24) nm. (4S)-4-(6-Amino-9H-purin-9-yl)-5,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (6b): light yellow, amorphous powder; [α]20D −108 (c 0.1, MeOH); experimental ECD (MeOH) λmax (Δε) 229 (+1.20), 260 (−3.08), 306 (+1.53), 343 (−0.50) nm. (±)-4-(6-Amino-9H-purin-9-yl)-5-hydroxy-3,4-dihydronaphthalen-1(2H)-one (7): light yellow, amorphous powder; [α]20D −57(c 0.1, MeOH); UV (MeOH) λmax (log ε) 211 (4.06), 259 (3.83), 315 (3.19) nm; IR (KBr) νmax 3432, 3137, 1674, 1591, 1507, 1469, 1415, 1294, 1180, 945, 915, 878, 811, 743, 724, 647 cm−1; 1H NMR (500 MHz) δ 2.48 (1H, ov, H2-2a), 2.63 (1H, m, H2-2b), 2.48 (1H, ov, H2-3a), 2.52 (1H, m, H2-3b), 6.01 (1H, m, H-4), 10.12 (1H, s, 5-OH), 7.09 (1H, dd, J = 8.0, 1.0 Hz, H-6), 7.38 (1H, dd, J = 8.0, 8.0 Hz, H-7), 7.47 (1H, dd, J = 8.0, 1.0 Hz, H-8), 8.12 (1H, s, H-2′), 7.58 (1H, s, H-8′), 7.21 (2H, br s, NH2); 13C NMR (125 MHz) δ 196.5 (C-1), 33.2 (C-2), 27.6 (C-3), 46.4 (C-4), 155.6 (C-5), 120.5 (C-6), 130.0 (C-7), 116.9 (C-8), 134.0 (C-9), 124.7 (C-10), 152.3 (C-2′), 149.5 (C-4′), 119.1 (C-5′), 156.1 (C-6′), 139.7 (C-8′); HRESIMS m/z 296.1145 [M + H]+ (calcd for C15H14N5O2, 296.1142). (4R)-4-(6-Amino-9H-purin-9-yl)-5-hydroxy-3,4-dihydronaphthalen-1(2H)-one (7a): light yellow, amorphous powder; [α]20D +160 (c 0.1, MeOH); experimental ECD (MeOH) λmax (Δε) 228 (−0.90), 260 (+2.61), 306 (−1.25), 343 (+0.33) nm. (4S)-4-(6-Amino-9H-purin-9-yl)-5-hydroxy-3,4-dihydronaphthalen-1(2H)-one (7b): light yellow, amorphous powder; [α]20D −155 (c 0.1, MeOH); experimental ECD (MeOH) λmax (Δε) 229 (+0.68), 260 (−2.10), 307 (+1.11), 342 (−0.29) nm. Calculation of the ECD Spectra of Compounds 2a, 2b, 3a, 3b, 5a, 5b, 6a, 6b, 7a, and 7b. Conformational Analysis. The quantum-chemical calculations were performed on the assumed R and S configurations of the optically active compounds, 2a, 2b, 3a, 3b, 5a, 5b, 6a, 6b, 7a, and 7b, by the Gaussian 09 package on an IBM cluster machine located at the High Performance Computing Center of Peking Union Medical College. Conformational analysis was initially performed using MOE software together with MMFF94 molecular mechanics methods. MMFF94 structures were reoptimized using ab

initio DFT at the B3LYP/6-31G(d) level. The energy of the stable conformations was calculated and led to the relative energy; this allowed the room-temperature equilibrium populations to be calculated according to the Maxwell−Boltzmann distribution law. ECD Data Calculations. The excitation energy (in nm) and rotatory strength R (velocity form Rvel and length form Rlen in 10−40 erg esu cm per Gauss) between the different states were calculated using TDDFT at the B3LYP/6-31G(d)//B3LYP/6-31G(d) level in a methanol solution. All of the calculations were performed using the Gaussian 09 program package. ECD Simulation. The ECD spectra were simulated by overlapping the Gaussian functions for each transition, and Rvel was used in this work. Conformational analysis was performed, and the theoretically weighted ECD spectra were simulated at the different levels mentioned above. Growth Inhibition Assay against Human Cancer Cell Lines. The growth inhibition of all of the compounds against the HCT-116, HepG2, BGC-823, NCI-H1650, and A2780 cell lines was examined using the published method.23 Measurement of in Vitro Cell Viability on IEC-6 Cells. An in vitro viability assay on IEC-6 cells for compound 1 was performed as described previously, in which growth inhibition was measured by an MTT assay (n = 5) at 0, 24, and 48 h after the coculture of IEC-6 cells and test compound 1.25

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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.6b00887. UV, IR, 1D and 2D NMR, and HRESIMS spectra recorded for each new compound, as well as ECD spectra and chiral HPLC contours for all the scalemic mixtures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-010-63165247. Fax: +86-010-63017757. E-mail: [email protected] (L. Li). *Tel: +86-010-83172503. Fax: +86-010-63017757. E-mail: [email protected] (H.-L. Qin). ORCID

Hai-Lin Qin: 0000-0001-6721-1409 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from NSFC (81361138020), CAMS Initiation Fund for Innovative Medicine (2016-I2M-3-014), and the National Science and Technology Project of China (2012ZX09301002001003). NK109 as the positive control was kindly donated by Dr. QiLin Li at the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.

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REFERENCES

(1) Xi, R. T.; Zhang, Y. P. Juglans Regia Colume in China Fruit Trees Records; China Forestry Press: Beijing, 1996; p 10. (2) Kim, S. H.; Lee, K. S.; Son, J. K.; Je, G. H.; Lee, J. S.; Lee, C. H.; Cheong, C. J. J. Nat. Prod. 1998, 61, 643−645. (3) Hirakawa, K.; Ogiue, E.; Motoyoshiya, J.; Yajima, M. Phytochemistry 1986, 25, 1494−1495. (4) Talapatra, S. K.; Karmacharya, B.; De, S. C.; Talapatra, B. Phytochemistry 1988, 27, 3929−3932. (5) Müller, W. U.; Leistner, E. Phytochemistry 1978, 17, 1739−1742.

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