Terpecurcumins AâI from the Rhizomes of Curcuma longa

Article pubs.acs.org/jnp

Terpecurcumins A−I from the Rhizomes of Curcuma longa: Absolute Configuration and Cytotoxic Activity Xionghao Lin,† Shuai Ji,† Rui Li,† Yinhui Dong,‡ Xue Qiao,† Hongbo Hu,*,‡ Wenzhi Yang,† Dean Guo,† Pengfei Tu,† and Min Ye*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, People's Republic of China ‡ College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghua East Road, Beijing 100083, People's Republic of China S Supporting Information *

ABSTRACT: Terpecurcumins A−I (1−9), together with three known analogues (10−12), were isolated from the rhizomes of Curcuma longa (turmeric). They were derived from the hybridization of curcuminoids and bisabolanes. The structures and absolute configurations of 1−9 were elucidated on the basis of extensive spectroscopic data analysis, including NMR and electronic circular dichroism spectra. The configuration of 10 was further confirmed by X-ray crystallography. A plausible biogenetic relationship for 1−12 is proposed. Compounds 4, 6, 7, 10, and 11 showed higher cytotoxic activities (IC50, 10.3−19.4 μM) than curcumin (IC50, 31.3−49.2 μM) against human cancer cell lines (A549, HepG2, and MDA-MB-231).

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RESULTS AND DISCUSSION In our preliminary analysis of chemical constituents in the EtOH extract of turmeric, HPLC-DAD-ESI-MSn analysis indicated the presence of a series of bisabolane−curcuminoid hybrids. They showed similar UV absorption to curcuminoids (λmax around 425 nm), but remarkably larger molecular weights ([M − H]− m/z 601, 587, 571, and 541). Their electrospray ionization tandem mass spectra also showed fragments similar to curcuminoids (data not shown).7 Guided by LC/MS analysis, the fraction containing these target compounds was first enriched by MCI and silica gel column chromatography, then subjected to repeated C18 and Sephadex LH-20 column chromatography, and finally purified by semipreparative HPLC to afford terpecurcumins A−I (1−9) and three known analogues (10−12) (Figure 1). Terpecurcumin A (1) was obtained as an orange, amorphous powder. The HRESIMS spectrum gave an [M + H]+ ion at m/z 589.3160 (calcd 589.3165), indicating the molecular formula of C36H44O7. By analyzing 1D and 2D NMR spectra of 1, the proton and carbon signals (Tables 1 and 2) were assigned to moieties A and B, as illustrated in Figure 2. Moiety A showed signals for two aromatic ABX systems [δH 7.35 (1H, d, 1.2 Hz), 7.05 (1H, d, 7.8 Hz), 7.23 (1H, d, 7.8 Hz), 7.34 (1H, d, 1.2 Hz), 6.89 (1H, d, 7.8 Hz), and 7.19 (1H, dd, 7.8, 1.2 Hz)], two pairs of trans-olefinic protons [δH 6.75 (1H, d, 15.6 Hz), 7.62

urmeric, the rhizomes of Curcuma longa L. (Zingiberaceae), is a popular herbal medicine worldwide. Pharmacological studies have proved significant chemoprevention effects of turmeric against various cancers.1 Curcuminoids, mainly curcumin, demethoxycurcumin, and bisdemethoxycurcumin, together with bisabolane sesquiterpenoids including arturmerone, α-turmerone, and β-turmerone are considered to be the anticancer constituents of turmeric.2,3 Particularly, curcumin is a new antineoplastic agent, which is currently in phase II clinical trials for the treatment of colorectal and pancreatic cancer.4 A total of 27 curcuminoids and 38 sesquiterpenoids have been isolated from turmeric, and some of them show even more potent cytotoxic activities than curcumin.5 Recently, four hybrids conjugating the curcumin skeleton with a bisabolane-type sesquiterpene were isolated from turmeric, although the absolute configurations and biological significance were not defined.6 These reports encouraged us to explore the structural diversity of these hybrids and to elucidate their absolute configuration and bioactivity. In this paper, we report nine new [terpecurcumins A−I (1−9)] and three known (10−12) hybrids from turmeric. Their structures were identified on the basis of extensive spectroscopic analysis, including NMR spectroscopy and electronic circular dichroism (ECD) spectra. The configuration of compound 10 was further confirmed by X-ray crystallography. Cytotoxic activities of the new compounds against A549, HepG2, and MDA-MB-231 human cancer cells are reported. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 14, 2012

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dx.doi.org/10.1021/np300551g | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Terpecurcumins A−I (1−9) and three known analogues (10−12) from Curcuma longa.

Table 1. 1H NMR Data for Compounds 1−7 in Acetone-d6 Recorded at 600 MHz (δ in ppm, J in Hz) no. 1 3 4 6 7 9 10 11 3′ 4′ 6′ 9′ 10′ 11′ 1″ 2″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 12″ 13″ 14″ 15″ a

1

2

3

4

5

6

6.00, 6.75, 7.62, 7.35,

s d (15.6) d (15.6) d (1.2)

6.01, 6.74, 7.62, 7.35,

s m m s

6.00, 6.75, 7.61, 7.34,

s d (16.2) d (16.2) s

6.01, 6.74, 7.61, 7.33,

s d (16.2) d (16.2) s

6.01, 6.78, 7.61, 7.35,

s d (15.6) d (15.6) s

5.99, 6.76, 7.61, 7.33,

s d (15.6) d (15.6) s

7.05, 7.23, 3.90, 6.72, 7.62, 7.34, 6.89, 7.19, 3.92, 4.75, 5.55, 4.00, 1.80, 1.55, 2.25, 1.99, 1.38, 1.29, 2.03, 5.15, 1.66, 1.60, 0.82, 1.79,

d (7.8) d (7.8) s d (15.6) d (15.6) d (1.2) d (7.8) dd (7.8, 1.2) s d (9.6) s s ma mb m m m m m t (7.2) s s d (7.2) s

7.09, 7.23, 3.88, 6.74, 7.62, 7.35, 6.89, 7.19, 3.93, 3.95, 5.70, 4.69, 1.95, 1.37, 1.85, 2.09, 1.18,

m m s m m s d (7.8) d (7.8) s m s s ma mb m m m

7.12, 7.24, 3.88, 6.72, 7.61, 7.34, 6.89, 7.19, 3.92, 5.64, 5.64, 4.48, 1.90,

d (7.8) d (7.8) s d (16.2) d (16.2) s d (7.8) d (7.8) s s s d (3.0) m

7.10, 7.23, 3.87, 6.74, 7.61, 7.33, 6.89, 7.18, 3.92, 5.67, 5.70, 4.44, 1.92,

d (7.8) d (7.8) s d (16.2) d (16.2) s d (7.8) d (7.8) s d (2.4) d (2.4) s m

2.35, 2.18, 2.46, 2.20,

m m m m

m t (7.2) s s d (7.2) s

m m m m m t (7.2) s s d (7.2) s

6.09, 1.82, 2.06, 0.86, 1.36,

s s s d (7.2) s

d (7.8) d (7.8) s d (15.6) d (15.6) s d (7.8) d (7.8) s dd (10.2, 3.0) d (10.2) d (4.2) ma mb m m m m m t (7.2) s s d (7.2) s

7.13, 7.33, 3.87, 6.71, 7.61, 7.17, 6.88, 7.18, 3.91, 5.71, 5.76, 4.00, 1.96, 1.70, 2.32, 2.13, 2.38, 2.13,

1.95, 5.06, 1.60, 1.48, 0.78, 1.77,

2.38, 1.60, 1.33, 1.17, 1.94, 5.06, 1.63, 1.52, 0.86, 1.37,

7.12, 7.19, 3.89, 6.72, 7.62, 7.35, 6.89, 7.17, 3.93, 5.70, 5.75, 4.02, 1.96, 1.70, 2.34, 1.54, 1.37, 1.14, 1.96, 5.08, 1.68, 1.58, 0.79, 1.41,

6.05, 1.84, 2.07, 0.78, 1.40,

7

d (8.4) d (8.4) s d (15.6) d (15.6) s d (8.4) d (8.4) s dd (10.8, 1.2) dd (10.8, 2.4) d (6.6) ma mb m m m m

5.99, 6.71, 7.61, 7.58, 7.07, 7.07, 7.58,

s d d d d d d

6.71, 7.61, 7.33, 6.88, 7.18, 3.91, 5.79, 5.74, 4.04, 1.96, 1.75, 2.18, 2.05, 2.43, 2.19,

d (15.0) d (15.0) s d (7.8) d (7.8) s dd (10.2, 2.4) dd (10.8, 0.6) d (3.6) ma mb m m m m

s s s d (7.2) s

6.09, 1.85, 2.08, 0.84, 1.45,

s s s d (6.4) s

(15.0) (15.0) (7.8) (7.8) (7.8) (7.8)

H-5″ was at the lower field. bH-5″ was at the higher field.

(1H, d, 15.6 Hz), 6.72 (1H, d, 15.6 Hz), and 7.62 (1H, d, 15.6 Hz)], an enolic proton [δH 6.00 (1H, s)], and two methoxy

groups [δH 3.90 (3H, s) and 3.92 (3H, s)]. These signals were consistent with those of curcumin. Moiety B was determined to B



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Table 2. 13C NMR Data for Compounds 1−7 in Acetone-d6 Recorded at 150 MHz (δ in ppm) no.

1

1 2 3 4 5 6 7 8 9 10 11 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″

101.7, 184.1, 122.9, 141.0, 129.1, 112.2, 151.5, 151.0, 115.7, 123.4, 56.3, 184.7, 122.3, 141.5, 128.1, 111.5, 148.8, 150.0, 116.2, 123.8, 56.3, 76.7, 124.8, 139.8, 67.6, 30.7, 38.3, 31.1, 35.8, 26.6, 125.6, 131.5, 25.8, 17.7, 15.1, 20.8,

2 CH C CH CH C CH C C CH CH CH3 C CH CH C CH C C CH CH CH3 CH CH C CH CH2 CH CH CH2 CH2 CH C CH3 CH3 CH3 CH3

101.7, 184.0, 123.1, 140.9, 129.6, 112.1, 152.0, 151.2, 116.2, 123.2, 56.2, 184.8, 122.3, 141.5, 128.1, 111.5, 148.7, 150.0, 116.9, 123.8, 56.3, 68.9, 134.2, 133.3, 76.2, 26.1, 40.7, 30.7, 35.9, 26.6, 125.6, 131.3, 25.8, 17.5, 14.6, 20.7,


101.7, 184.1, 123.0, 140.9, 129.5, 112.5, 151.7, 151.1, 116.1, 123.3, 56.4, 184.8, 122.3, 141.5, 128.1, 111.5, 148.7, 150.0, 116.7, 123.8, 56.3, 133.1, 133.1, 69.5, 81.8, 24.4, 36.3, 36.7, 34.9, 26.5, 125.4, 131.5, 25.8, 17.6, 16.4, 25.8,


101.7, 184.0, 123.1, 140.9, 129.5, 112.5, 151.8, 151.1, 116.2, 123.3, 56.4, 184.8, 122.3, 141.5, 128.1, 111.5, 148.7, 150.0, 117.0, 123.8, 56.3, 132.1, 133.5, 69.4, 81.8, 25.2, 36.7, 33.5, 49.0, 200.3, 124.8, 154.6, 27.3, 20.4, 16.9, 25.3,

5 CH C CH CH C CH C C CH CH CH3 C CH CH C CH C C CH CH CH3 CH CH C CH CH2 CH CH CH2 C CH C CH3 CH3 CH3 CH3

101.8, 185.2, 123.8, 140.7, 131.7, 112.3, 154.6, 150.1, 125.4, 123.9, 56.2, 183.6, 122.3, 141.7, 128.1, 111.5, 147.8, 148.8, 116.2, 122.2, 56.3, 135.1, 130.9, 82.8, 71.1, 28.5, 37.4, 37.0, 35.0, 26.6, 125.5, 131.5, 25.8, 17.7, 16.7, 22.4,


101.8, 183.5, 123.8, 140.6, 131.7, 111.5, 154.6, 147.9, 125.3, 112.3, 56.3, 185.1, 122.2, 141.7, 128.0, 122.1, 148.7, 150.1, 116.2, 123.9, 56.2, 134.3, 131.3, 82.6, 71.1, 29.0, 37.5, 33.6, 49.1, 200.2, 124.8, 154.5, 27.4, 20.4, 17.2, 22.5,

7 CH C CH CH C CH C C CH CH CH3 C CH CH C CH C C CH CH CH3 CH CH C CH CH2 CH CH CH2 C CH C CH3 CH3 CH3 CH3

101.8, 183.5, 123.3, 140.4, 130.1, 129.9, 123.4, 158.8, 123.4, 129.9,

CH C CH CH C CH CH C CH CH

185.1, 122.2, 141.6, 128.1, 111.4, 148.7, 150.1, 116.2, 123.9, 56.3, 134.9, 130.9, 80.8, 71.1, 28.9, 37.3, 33.5, 49.2, 200.2, 124.8, 154.5, 27.4, 20.4, 17.1, 22.8,

C CH CH C CH C C CH CH CH3 CH CH C CH CH2 CH CH CH2 C CH C CH3 CH3 CH3 CH3

17.7, and 15.1, three methylenes at δC 35.8, 30.7, and 26.6, six methines at δC 125.6, 124.8, 76.7, 67.6, 38.3, and 31.1, and two quaternary carbons at δC 139.8 and 131.5. An oxygenated cyclohexene ring with a methyl substituent was established by HMBC correlations of H-1″/C-3″, H-2″/C-4″ and C-6″, and CH3-15″/C-2″, C-3″, and C-4″, as well as 1H−1H COSY correlations of H-4″/H-5″/H-6″/H-1″/H-2″. Furthermore, the HMBC correlations of CH3-12″/C-10″, C-11″, and C-13″, H8″/C-9″, C-10″, and C-14″, and CH3-14″/C-7″ and C-8″ revealed the presence of a (CH3)2CCH−CH2−CH2− CH(CH3)− moiety, which was further confirmed by the 1 H−1H COSY correlations of H-10″/H-9″/H-8″/H-7″/H-14″. This side chain was connected to C-6″ of the cyclohexene ring, as evidenced by the HMBC correlations of CH3-14″/C-6″, H1″/C-7″, and H-7″/C-5″, as well as the 1H−1H COSY correlation between H-6″ and H-7″(Figure 2). Moiety B represented a new bisabolane structure and was named bisabolane A in this context. Li and co-workers isolated a similar compound possessing a C-9″ carbonyl group from C. longa.5 The linkage of moieties A and B involved a C−O−C bond between C-8 and C-1″, as evidenced by the HMBC correlations of H-1″/C-8 (Figure 2) and NOE enhancement of

Figure 2. Key HMBC, 1H−1H COSY correlations, and NOE enhancements for 1.

be a bisabolane-type sesquiterpene unit. The DEPT experiment revealed that moiety B included four methyls at δC 25.8, 20.8, C



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H-1″/H-9. The above analyses established the planar structure of compound 1. Compound 1 is a new bisabolane−curcumin hybrid. Recently, three analogues (10−12) of 1 were isolated from C. longa, although the relative and absolute configurations of these compounds were not defined.6 These three compounds were also obtained in the present study. They exhibited the same NOE enhancements as observed for compound 1 (Figure 2). The NOE enhancements of H-1″/H-5″b and H-4″/H-5″b revealed that H-1″ and H-4″ were located on the same side of the cyclohexene ring. The large trans-diaxial coupling constant (J1″−6″ = 9.6 Hz) and the absence of NOE enhancement suggested a trans relationship between H-1″ and H-6″. Although these curcuminoid hybrids were difficult to crystallize, we obtained single crystals of compound 10 from MeOH, which further confirmed their relative configurations (Figure 3).

Figure 4. Comparison of the experimental ECD spectrum of 10 in MeCN (blue) with calculated ECD spectra for (1″R,4″S,6″R,7″S)-10 (red) and (1″S,4″R,6″S,7″R)-10 (green) after a UV correction of 20 nm.

(1″R,4″S,6″R,7″S)-10, but not (1″S,4″R,6″S,7″R)-10, agreed well with the recorded spectrum for 10. Molecular orbital (MO) analysis of conformers 10b and 10c (Figure S6) at the B3LYP/6-31G* level with the IEFPCM model in MeCN facilitated understanding of the ECD spectrum of 10 (Figure 5). The first calculated peak at 206 nm could be assigned to the

Figure 3. X-ray crystal structure and crystal cell diagram of 10 (crystallized in MeOH). Note: A different carbon numbering system is used for the structural data deposited at CCDC. Figure 5. Important molecular orbitals involved in the key transitions in ECD spectra of conformers 10b and 10c in MeCN with the IEFPCM model at the B3LYP/6-31G* level.

Single-crystal X-ray analysis of compound 10 was conducted by using anomalous scattering of Cu Kα radiation. The data indicated that 10 was a long-chain flexible molecule and that the crystals belonged to the P21 space group. However, the quality of crystals was insufficient to determine the absolute configuration. The absolute configuration of compound 10 was determined by comparing the experimental and calculated ECD spectra.8 According to the established relative configuration, compound 10 should be one of the two enantiomers (1″R,4″S,6″R,7″S)-10 or (1″S,4″R,6″S,7″R)-10. On the basis of literature reports, the curcumin unit of 10 may undergo keto−enol tautomerism and exists entirely in the enolic form.9 This was different from the crystallographic analysis (keto form), but was highly consistent with the results of NMR experiments (H-1, 1H, 6.00, s) and molecular dynamics (MD) simulation of 10, which indicated that the enol form of 10 had a lower average energy (about 6.73 kcal/mol) than the keto form (Figure S3). Thus, the enol form of 10 was selected as an original structure for the optimization and conformational analysis in ECD calculation. As illustrated in Figure 4, the calculated ECD spectrum for

experimentally observed positive Cotton effect at 203 nm. The transitions from MO155 to MO164 including n (lone pair orbital of O) and π (bonding CC and CO) to π* (antibonding CC and CO) transitions may contribute to this absorption band. The second calculated peak was located at 436 nm, which could be assigned to the experimental positive Cotton effect at around 427 nm. The n → π* and π → π* transitions from MO161 to MO162 in the curcumin unit may play a dominant role. On the basis of the above evidence, the absolute configuration of 10 was established as (1″R,4″S,6″R,7″S)-10. Compounds 1, 11, and 12 share the same relative configurations as 10. In addition, they exhibit ECD spectra similar to 10, showing positive Cotton effects at around 205 and 420 nm (Figure 6). Therefore, the absolute configurations of compounds 1, 11, and 12 were also established as 1″R, 4″S, 6″R, 7″S. The configuration of C-7″ in all bisabolanes previously reported from turmeric was also assigned as S on D



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and H-6″. This was consistent with bisacurone A but was different from bisacurones B and C. The absolute configurations of bisacurones A−C were determined by chemical conversions.10 Therefore, the absolute configuration of 3 was proposed as 3″R, 4″S, 6″R, 7″S based on biogenetic considerations. In order to verify this, the ECD spectra of 3 and its enantiomer were predicted by calculation. The calculated ECD spectrum of (3″R,4″S,6″R,7″S)-3 showed two positive Cotton effects at 202 and 413 nm, which were consistent with the experimental spectrum (two positive Cotton effects at around 200 and 420 nm) (Figure 7). MO Figure 6. Experimental ECD spectra for compounds 1, 2, and 10−12 in MeCN.

the basis of chemical conversions and single-crystal X-ray crystallography.5,10 This may be because bisabolanes isolated from turmeric share a common biosynthetic origin.11 They are derived from α-turmerone and zingiberene (C-7″, S) due to oxidative rearrangement, respectively. Zingiberene could be oxidized to α-turmerone by an enzyme system.12 Therefore, C7″ of the bisabolane unit in terpecurcumins also possesses the S-configuration. Compound 2 has the same molecular formula as 1, as deduced from the HRESIMS spectrum (m/z 589.3162, [M + H]+, calcd 589.3165). The UV, IR, and NMR spectra of 2 indicated that it also possessed the curcumin and bisabolane A units. The only difference was the linkage position between curcumin and bisabolane A. The HMBC correlation of H-4″/ C-8 in compound 2 suggested these two units were connected by C-8−O−C-4″ rather than C-8−O−C-1″. This deduction was confirmed by the upfield shift of C-1″ (ΔδC −7.8) and the downfield shift of C-4″ (ΔδC +8.6) (Table 2), as well as by the NOE enhancement between H-4″ and H-9. The relative configuration of 2 was defined by the NOE experiment. The NOE enhancements of H-1″/H-5″b and H-4″/H-5″b indicated that H-1″ and H-4″ were located on the same side of the cyclohexene ring. The absence of NOE enhancement suggested a trans relationship between H-1″ and H-6″. The ECD spectrum of 2 showed similar positive Cotton effects to 1 at ca. 205 and 400 nm (Figure 6). Therefore, the absolute configuration of 2 was established as 1″R, 4″S, 6″R, 7″S, and it was named terpecurcumin B. Terpecurcumin C (3) has the molecular formula C36H44O7 by HRESIMS analysis (m/z 589.3161, [M + H]+, calcd 589.3165). The UV, IR, and NMR spectra of 3 were similar to those of 2, indicating that their structures are closely related. By analyzing the NMR data of 3, we found that changes mainly occurred in the cyclohexene part of the molecule. The chemical shifts of δC 133.1 (C-1″), 133.1 (C-2″), 69.5 (C-3″), and 81.8 (C-4″) for 3, different from δC 68.9 (C-1″), 134.2 (C-2″), 133.3 (C-3″), and 76.2 (C-4″) for 2, indicated that the Δ2″,3″ double bond in 2 has moved to Δ1″,2″ in 3 (Table 2). Correspondingly, the hydroxy group also moved from 1″ to 3″. These were confirmed by the HMBC correlations of H-1″/C3″ and C5″, H-2″/C-6″, H-4″/C-2″ and C6″, and CH3-15″/C2″ and C4″. Thus, the planar structure of the bisabolane unit was established, which was consistent with the known turmeric compound 4,5-dihydroxybisabola-2,10-diene.5 According to the NOE experiments, enhancement of H-4″/CH3-15″ indicated that H-4″ and CH3-15″ were located on the same side of the cyclohexene ring. H-6″ should be located on the other side, as no NOE enhancement was observed between H-4″ or CH3-15″

Figure 7. Comparison of the experimental ECD spectrum of 3 in MeCN (blue) with calculated ECD spectra for (3″R,4″S,6″R,7″S)-3 (red) and (3″S,4″R,6″S,7″R)-3 (green) after a UV correction of 29 nm.

analysis of conformers 3d and 3h (Figure S7) provided information to understand the ECD spectrum (Figure 8). The

Figure 8. Important molecular orbitals involved in the key transitions in ECD spectra of conformers 3d and 3h in MeCN with the IEFPCM model at the B3LYP/6-31G* level.

positive Cotton effect at around 200 nm may be caused by the electronic transitions from MO145 to MO159 involving n → π*, σ → π*, and π → π* transitions, while the transitions in the curcumin unit from MO158 to MO159 involving n → π* and π → π* transitions may contribute to the absorption band at ca. 420 nm. In addition, the peak at 262 nm in the calculated spectrum may originate from the n → π* and π → π* transitions from MO152 to MO159. All these results were consistent with our experimental data. Thus, the absolute E



Article

was replaced by an A2B2 system in 7 (Table 1). Collectively, this information suggested that 7 had a unit of demethoxycurcumin rather than curcumin, which was also supported by NMR data of the known compound 11. The remaining proton and carbon signals were consistent with those of the bisacurone unit in 6. This was further confirmed by HMBC and 1H−1H COSY correlations. However, the linkage position (C-8 or C8′) of the bisacurone unit was difficult to assign via HMBC correlation, as C-8, C-8′, and C-3″ were all quaternary carbons. However, weak enhancements between CH3-15″ and H-7 or H-9 were observed in the NOE experiment, which suggested that the bisacurone unit was connected to C-8. The NOE enhancement of CH3-15″/H-4″ and no enhancement between CH3-15″ or H-4″ and H-6″ suggested 7 had the same relative configuration as 6. Moreover, their similar ECD spectra permitted definition of the absolute configuration of 7 as 3″R, 4″S, 6″R, 7″S (Figure S4). The HRESIMS of 8 gave an [M + H]+ ion at m/z 589.3162 (calcd 589.3165), indicating its molecular formula to be C36H44O7. The 13C NMR and DEPT spectra (Table 4) of 8 were similar to bisabocurcumin recently isolated from turmeric, which has a new bisabolane curcuminoid skeleton.6 The main differences were the chemical shifts of C-8″ at δC 35.2, C-9″ at δC 25.5, and C-11″ at δC 130.4 for 8, in contrast to δC 50.2, 200.4, and 154.1 for bisabocurcumin, respectively. These differences indicated that C-9″ in 8 was a methylene instead of a carbonyl group, which was confirmed by the triplet of H10″ (Table 3) and HMBC correlations of H-9″/C-8″ and C11″ and H-8″/C-9″, as well as the presence of 1H−1H COSY cross-peaks of H-8″/H-9″/H-10″. In addition, compound 8 was an isomer of 1. The main difference between 8 and 1 was the linkage between curcumin and bisabolane A (C-9−C1″ for 8 and C-8−O−C1″ for 1). This was further confirmed by the HMBC correlations of H-1″/C-10, H-2″/C-9, and H-10/C-1″ and NOE enhancement of H-6″/H-10. Thus, compound 8 represents the second new compound isolated from turmeric possessing a bisabolane curcuminoid skeleton, although the absolute configuration of bisabocurcumin has not been determined yet. The NOE enhancements of H-1″/H-5″b, H4″/H-5″b, and H-6″/H-5″a as well as J1″‑6″ values (about 10.2 Hz) revealed that H-1″ and H-4″ should be located on the same side of the cyclohexene ring, whereas H-1″ and H-6″ had a trans relationship. Thus, the relative configuration of bisabocurcumin in the previous report needs to be revised.6 The absolute configuration of 8 was deduced by comparing the experimental and calculated ECD spectra for the enantiomers (1″R,4″S,6″R, 7″S)-8 and (1″S,4″R,6″S,7″R)-8. The calculated ECD spectrum for (1″R,4″S,6″R,7″S)-8 was consistent with the experimental spectrum. The peaks at 207 and 436 nm in the calculated spectrum corresponded to the positive Cotton effects at 204 and 410 nm in the experimental ECD spectrum, respectively (Figure 9). The electronic excitations from MO145 and MO147 to MO159 involving n → π*, σ → π*, and π → π* transitions contributed dominantly to the positive Cotton effect at around 207 nm, as shown in Figure 10. The electronic transitions from MO158 to MO159 in the curcumin unit involving n → π* and π → π* transitions played a key role in the other positive Cotton effects at ca. 410 nm. Thus, the absolute configuration of 8 was established as 1″R, 4″S, 6″R, 7″S, and it was named terpecurcumin H. Terpecurcumin I (9) has the molecular formula C36H42O7, as established by HRESIMS spectrum ([M + H]+ m/z 587.3014, calcd 587.3009), one oxygen atom less than the previously

configuration of 3 was unambiguously established as 3″R, 4″S, 6″R, 7″S, and it was named terpecurcumin C. The HRESIMS spectrum of terpecurcumin D (4) exhibited a quasi-molecular ion at m/z 603.2965 ([M + H]+, calcd for 603.2952), consistent with the molecular formula C36H42O8. The NMR spectroscopic data for 4 (Tables 1 and 2) were similar to those of terpecurcumin C (3), except that C-9″ in 4 was a carbonyl group (δC, 200.3) instead of a methylene group (δC 26.5 for 3). This was supported by the singlet of H-10″ (triplet for 3), the downfield shifts of C-8″ (ΔδC +14.1) and C11″ (ΔδC +23.1) (Tables 1 and 2), and the HMBC correlations of H-8″/C-9″and H-10″/C-9″. In addition, the absence of 1 H−1H COSY cross-peaks of H-8″/H-9″/H-10″ in 4 also indicated the carbonyl group was at C-9″. This information suggested that compound 4 was derived from curcumin and bisacurone.10 The NOE enhancement of H-4″/CH3-15″ indicated that they were located on the same side of the cyclohexene ring. The absence of NOE enhancement between H-4″ or CH3-15″ and H-6″ suggested H-6″ was located on the other side. Compound 4 should have the same absolute configuration as 3 due to their similar planar structure, relative configuration, and ECD spectra (Figure S4). Therefore, the structure of 4 was established as shown in Figure 1. Compound 5 has the same molecular formula as 3 according to its HRESIMS spectrum ([M + H]+ m/z 589.3161, calcd 589.3165). The NMR spectra of 5 (Tables 1 and 2) also exhibited characteristic signals for curcumin and 4,5-dihydroxybisabola-2,10-diene units. The main difference between 5 and 3 was the linkage position between curcumin and 4,5dihydroxybisabola-2,10-diene (C-8−O−C4″ for 3 and C-8− O−C3″ for 5).5 This difference could not be identified by HMBC correlation, as both C-8 and C-3″ in 5 were quaternary carbons. However, by comparing the 13C NMR spectra of 5 with those of 3 (Table 2), it was clear that signals for C-3″ (Δδ, +13.3) and C-4″ (Δδ, −10.7) shifted remarkably for 5. The NOE enhancement of CH3-15″/H-4″ and no enhancement between CH3-15″ or H-4″ and H-6″ indicated that CH3-15″ and H-4″ were located on the same side of the cyclohexene ring, while H-6″ was located on the other side. The ECD spectrum of 5 showed similar positive Cotton effects at 200 and 409 nm with 3 (Figure S4), indicating they had the same absolute configurations. Thus, the structure of 5 was established as terpecurcumin E. The HRESIMS spectrum ([M + Na]+ m/z 625.2778, calcd 625.2777) of compound 6 established its molecular formula as C36H42O8. The NMR data were similar to those of 5 except for the presence of a new carbonyl resonance (δC, 200.2) (Table 2), indicating that compound 6 is an oxygenated analogue of 5. The carbonyl group was assigned to C-9″ by the HMBC correlations of H-8″/C-9″ and H-10″/C-9″ and the absence of 1 H−1 H COSY cross-peaks of H-8″/H-9″/H-10″. This information suggested that compound 6 was derived from curcumin and bisacurone. Compared with those of 5, the same NOE enhancement (the correlation of CH3-15″/H-4″ and no enhancement between CH3-15″ or H-4″ and H-6″) and similar ECD spectra (Figure S4) indicated compound 6 also had the configuration 3″R, 4″S, 6″R, 7″S. Thus, its structure was established, and the compound was named terpecurcumin F. According to its HRESIMS spectrum ([M + Na]+ m/z 595.2669, calcd 595.2672), terpecurcumin G (7) was deduced to have a molecular formula of C35H40O7, CH2O less than that of 6. The 1H NMR spectrum of 7 displayed only one methoxy signal (δH 3.91). One of the two aromatic ABX systems in 6 F



Article

Table 3. 1H NMR Data for Compounds 8−12 in Acetone-d6 Recorded at 600 MHz (δ in ppm, J in Hz) no. 1 3 4 6

8 6.01, s 6.69, d (15.6) 7.56, d (15.6) 7.21, s

9 5.98, s 6.66, d (15.6) 7.56, d (15.6) 7.20, s

10

11

6.00, s 6.76, d (15.6) 7.62, d (15.6) 7.35, s

6.00, s 6.76, d (15.6) 7.62, d (15.6) 7.36, s

7.04, d (7.8) 7.23, d (7.8) 3.90, s 6.72, d (15.6) 7.62, d (15.6) 7.35, s

7.05, d (7.8) 7.23, d (7.8) 3.90, s 6.68, d (15.6) 7.62, d (15.6) 7.57, d (8.4) 6.91, d (8.4) 6.91, d (8.4) 7.57, d (8.4)

7 9 10

7.10, s

6.99, s

11 3′

3.93, s 6.65, d (15.6) 7.59, d (15.6) 7.32, s

3.93, s 6.69, d (15.6) 7.58, d (15.6) 7.32, s

6.87, d (7.8) 7.17, d (7.8) 3.91, s 3.78, d (10.2) 5.28, s 4.01, s

6.87, d (7.8) 7.15, d (7.8) 3.91, s 3.83, d (9.6) 5.17, s 2.06, m 1.79, m 1.44, ma 1.27, mb 1.59, m 2.01, m 2.32, m

4′ 6′ 7′ 9′ 10′ 11′ 1″ 2″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 12″ 13″ 14″ 15″ a

1.83, ma 1.54, mb 2.10, m 1.41, m 1.28, m 1.17, m 1.87, m 1.83, m 4.90, t (7.2) 1.47, s 1.51, s 0.84, d (7.2) 1.80, s

5.89, s 1.73, s 2.02,s 0.86, d (6.6) 1.69, s

6.89, d (7.8) 7.18, d (7.8) 3.92, s 4.76, d (9.6) 5.53, s 4.01, s 1.87, 1.61, 2.15, 2.52, 2.50, 2.35,

ma mb m m m m

6.14, s 1.85, s 2.06, s 0.84, d (6.6) 1.78, s

4.76, d (9.0) 5.53, s 4.01, s 1.86, 1.60, 2.15, 2.52, 2.50, 2.35,

ma mb m m m m

6.14, s 1.85, s 2.06, s 0.85, d (6.6) 1.78, s

absolute configuration of compound 9 was assigned as 1″R, 6″R, 7″S. According to our HPLC-DAD-ESIMSn analysis of rhizomes and roots of Curcuma plants (C. longa, C. phaeocaulis, C. wenyujin, and C. kwangsiensis), terpecurcumins exist only in C. longa (data not shown). Biosynthetically, terpecurcumins are derived from the novel hybridization of curcuminoids and bisabolanes by “C−C” or “C−O−C” bonds at various linking positions. The bisabolane units are derived from α-turmerone or zingiberene due to oxidative rearrangements. Both αturmerone and zingiberene had been reported from turmeric, and the latter could be oxidized to produce the former by an enzyme system. 12 A plausible biogenetic pathway for compounds 1−12 is proposed in Scheme 1. The cytotoxicity of compounds 1−12 against three human cancer cell lines, namely, human alveolar adenocarcinoma cell line (A549), human hepatocellular liver carcinoma cell line (HepG2), and human breast cancer cell line (MDA-MB-231), was evaluated by crystal violet staining using Taxol as the positive control. Compounds 4, 6, 7, 10, and 11 exhibited moderate cytotoxicities against all the tested cell lines, and their IC50 values (10.3−19.4 μM) were remarkably lower than curcumin (31.3−49.2 μM). Compounds 1, 2, 3, 5, 8, and 9 showed weak or no cytotoxicities, with IC50 values above 22.7 μM (Table 5). These results appeared to indicate that “C−O− C” linkage, α,β-unsaturated carbonyl substructures formed by oxygenation of C-9″, and a methoxy group at the curcuminoid unit may contribute to the cytotoxic activities. Interestingly, previous studies reported that these functional groups were apt to bind or interact with biological molecules.13

12 6.00, s 6.71, d (15.6) 7.63, d (15.6) 7.64, d (8.4) 7.01, d (8.4) 7.01, d (8.4) 7.64, d (8.4) 6.67, d (15.6) 7.60, d (15.6) 7.56, d (8.4) 6.90, d (8.4) 6.90, d (8.4) 7.56, d (8.4) 4.77, d (9.6) 5.52 s 4.01, s 1.83, 1.61, 2.13, 2.47, 2.45, 2.34,

■

ma mb m m m m

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. UV spectra were measured on a Cary 300 Bio UV−visible spectrophotometer. IR spectra were recorded as KBr disks on a Nicolet NEXUS-470 FT-IR spectrometer. ECD spectra were recorded on a JASCO J-810 CD spectrometer. NMR spectra were obtained at 600 MHz for 1H and 150 MHz for 13C, respectively, on an Inova 600 MHz spectrometer in acetone-d6, with TMS as reference. HRESIMS data were performed on a Bruker APEX IV FT-MS spectrometer. TLC was carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., China). Spots were visualized under UV light (365 nm). Column chromatography (CC) was performed using silica gel (200−300 mesh, Qingdao Marine Chemical Inc., China), ODS C18 (DAISO Company, Japan), and Sephadex LH-20 (GE Healthcare BioScience AB, USA). Semipreparative HPLC was performed on an Agilent 1200 liquid chromatograph with a YMC Pack ODS-A column (250 mm × 10 mm, i.d. 5 μm, YMC Co. Ltd., Japan). Plant Material. Dried rhizomes of Curcuma longa L. were purchased from Pengzhou City, Sichuan Province, China, in December 2009. The plant was identified by the authors. A voucher specimen (No. JH200912) was deposited at the School of Pharmaceutical Sciences, Peking University, Beijing, China. Extraction and Isolation. The drug materials (30 kg) were powdered and then extracted with 95% EtOH(aq) at 80 °C. After condensation in vacuo, the crude extract (1.2 kg) was fractionated by an MCI gel column eluted with MeOH/H2O (50−90%, v/v) to afford three fractions. The third fraction (100 g) was chromatographed over silica gel (2.3 kg, 200−300 mesh) using CHCl3/MeOH (1:0 to 2:1) as eluent to produce seven fractions (A−G) based on TLC analysis. Fractions E (20 g) and F (25 g) were then chromatographed, respectively, over silica gel (1.0 kg, 200−300 mesh) using petroleum ether/EtOAc (1:0 to 2:1) as eluent to produce fractions EA−EG and FA−FJ. Fractions EE (5.18 g), EF (3.15 g), and FG (7.0 g) were fractionated by an ODS C18 column eluted with MeOH/H2O (50−

6.13, s 1.84, s 2.06, s 0.82, d (7.2) 1.77, s

H-5″ was at the lower field. bH-5″ was at the higher field.

reported bisabocurcumin. The NMR spectra for 9 (Tables 3 and 4) were similar to those of bisabocurcumin, except for the absence of the H-4″ signal around δH 3.90 and the upfield shifts of C-4″ (ΔδC, −35.7) and C-5″ (Δδ, −9.1) in 9. According to the DEPT spectrum of 9, C-4″ was a methylene instead of a methine. This information indicated that compound 9 was a C4″ reduction product of bisabocurcumin. This was further confirmed by the HMBC correlations of CH3-15″/C-2″ and C4″ as well as the 1H−1H COSY correlations of H-4″/H-5″/H6″/H-1″/H-2″. The relative configuration of 9 was established by the NOE enhancements of H-1″/H-5″b and H-6″/ H-5″a, indicating H-1″ and H-6″ had a trans relationship. Compound 9 possesses similar planar structure, relative configuration, and ECD spectrum to those of 8 (Figure S5). Therefore, the G



Article

Table 4. 13C NMR Data for Compounds 8−12 in Acetone-d6 Recorded at 150 MHz (δ in ppm) no.

8

1 2 3 4 5 6 7 8 9 10 11 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″

100.5, 183.4, 121.5, 141.1, 127.3, 107.1, 147.1, 147.5, 131.4, 124.5, 56.4, 183.8, 121.1, 140.4, 126.6, 110.6, 147.9, 149.1, 115.3, 123.3, 56.3, 38.0, 128.9, 135.3, 67.2, 31.3, 37.4, 31.4, 35.2, 25.5, 124.6, 130.4, 24.8, 16.8, 14.4, 20.5,

9 CH C CH CH C CH C C C CH CH3 C CH CH C CH C C CH CH CH3 CH CH C CH CH2 CH CH CH2 CH2 CH C CH3 CH3 CH3 CH3

101.3, 184.8, 122.1, 141.9, 127.4, 108.3, 148.3, 147.8, 132.7, 123.8, 56.4, 184.4, 122.3, 141.2, 128.2, 111.5, 148.8, 149.9, 116.2, 123.7, 56.3, 38.4, 126.2, 134.6, 30.8, 22.9, 44.4, 30.6, 51.0, 200.4, 124.4, 154.3, 27.4, 20.4, 15.3, 23.6,

10 CH C CH CH C CH C C C CH CH3 C CH CH C CH C C CH CH CH3 CH CH C CH2 CH2 CH CH CH2 C CH C CH3 CH3 CH3 CH3

101.7, 184.1, 122.9, 141.0, 129.2, 112.1, 151.6, 150.7, 115.8, 123.4, 56.3, 184.8, 122.3, 141.5, 128.1, 111.5, 148.8, 150.1, 116.2, 123.8, 56.3, 76.7, 124.8, 139.9, 67.5, 31.7, 39.5, 29.4, 50.5, 200.2, 124.8, 154.4, 27.4, 20.4, 15.5, 20.7,

CH C CH CH C CH C C CH CH CH3 C CH CH C CH C C CH CH CH3 CH CH C CH CH2 CH CH CH2 C CH C CH3 CH3 CH3 CH3

11

12

101.8, 184.1, 122.9, 140.9, 129.3, 112.1, 151.6, 150.7, 115.8, 123.4, 56.3, 184.8, 122.0, 141.2, 127.6, 130.9, 116.8, 160.5, 116.8, 130.9,

CH C CH CH C CH C C CH CH CH3 C CH CH C CH CH C CH CH

101.8, 184.2, 122.7, 140.6, 128.6, 130.8, 116.7, 160.9, 116.7, 130.8,

CH C CH CH C CH CH C CH CH

184.8, 122.0, 141.2, 127.7, 130.9, 116.7, 160.5, 116.7, 130.9,

C CH CH C CH CH C CH CH

76.7, 124.4, 139.9, 67.5, 31.7, 39.5, 28.9, 50.5, 200.2, 124.8, 154.4, 27.4, 20.4, 15.5, 20.7,

CH CH C CH CH2 CH CH CH2 C CH C CH3 CH3 CH3 CH3

75.7, 124.1, 140.2, 67.4, 31.6, 39.2, 28.9, 50.2, 200.1, 124.9, 154.4, 27.4, 20.4, 15.4, 20.7,

CH CH C CH CH2 CH CH CH2 C CH C CH3 CH3 CH3 CH3

Figure 10. Important molecular orbitals involved in the key transitions in the ECD spectrum of conformer 8g in MeCN with the IEFPCM model at the B3LYP/6-31G* level. Figure 9. Comparison of the experimental ECD spectrum of 8 in MeCN (blue) with calculated ECD spectra for (1″R,4″S,6″R,7″S)-8 (red) and (1″S,4″R,6″S,7″R)-8 (green) after a UV correction of 15 nm.

semipreparative HPLC (80% MeCN−H2O) to give 1 (23 mg), 2 (12 mg), 3 (11 mg), and 5 (8 mg). Fraction FGE (425 mg) was separated repeatedly by semipreparative HPLC (62% MeCN−H2O) to give 4 (13 mg), 6 (15 mg), 7 (9 mg), 10 (25 mg), 11 (13 mg), and 12 (8 mg). Terpecurcumin A (1): orange, amorphous powder; [α]25D +2.9 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 420 (4.74) nm; ECD (MeCN) λmax (Δε) 200 (+5.09), 439 (+0.51) nm; IR (KBr) νmax 3413, 2917, 1755, 1626, 1584, 1509, 1451, 1258, 1129, 1031, 967 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS m/z 589.3160 [M + H]+ (calcd for C36H45O7, 589.3165).

90%, v/v) to obtain fractions EEA−EED, EFA−EFC, and FGA−FGF. Fractions EEA (879 mg), EFB (965 mg), and FGE (1.2 g) were subjected to column chromatography over Sephadex LH-20 by eluting with MeOH to afford fractions EEAA−EEAG, EFBA−EFBH, and FGEA−FGEF. Fraction EEAA (60 mg) was chromatographed by semipreparative HPLC (70% MeCN−H2O) to give 8 (15 mg) and 9 (3 mg). Fraction EFBF (365 mg) was purified repeatedly by H



Article

Scheme 1. Plausible Biogenetic Pathway for Compounds 1−12

2925, 1755, 1626, 1587, 1509, 1452, 1266, 1129, 1030, 968 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS m/z 589.3161 [M + H]+ (calcd for C36H45O7, 589.3165). Terpecurcumin D (4): orange, amorphous powder; [α]25D +2.5 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 418 (4.65) nm; ECD (MeCN) λmax (Δε) 200 (+3.61), 406 (+1.66) nm; IR (KBr) νmax 3429, 2952, 1724, 1678, 1625, 1508, 1446, 1265, 1131, 1030, 970 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS m/z 603.2965 [M + H]+ (calcd for C36H43O8, 603.2952). Terpecurcumin E (5): orange, amorphous powder; [α]25D +4.1 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 418 (4.76) nm; ECD (MeCN) λmax (Δε) 207 (+4.73), 409 (+1.24) nm; IR (KBr) νmax 3427, 2924, 1751, 1626, 1588, 1509, 1453, 1267, 1126, 1031, 968 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS m/z 589.3161 [M + H]+ (calcd for C36H45O7, 589.3165). Terpecurcumin F (6): orange, amorphous powder; [α]25D +3.8 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 414 (4.61) nm; ECD (MeCN) λmax (Δε) 208 (+5.05), 404 (+1.17) nm; IR (KBr) νmax 3483, 2952, 2923, 1719, 1679, 1624, 1582, 1509, 1454, 1265, 1130, 1032, 972, 771 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS m/z 625.2778 [M + Na]+ (calcd for C36H42O8Na, 625.2777). Terpecurcumin G (7): orange, amorphous powder; [α]25D +1.9 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 414 (4.71) nm; ECD (MeCN) λmax (Δε) 202 (+3.89), 407 (+0.44) nm; IR (KBr) νmax 3524, 2956, 2924, 1679, 1626, 1582, 1509, 1453, 1382, 1278, 1133, 970 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS m/z 595.2669 [M+ Na]+ (calcd for C35H40O7Na, 595.2672). Terpecurcumin H (8): orange, amorphous powder; [α]25D +1.4 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 420 (4.69) nm; ECD (MeCN) λmax (Δε) 204 (+4.12), 404 (+0.38) nm; IR (KBr) νmax 3504, 2956, 2924, 2853, 1717, 1623, 1591, 1512, 1455, 1428, 1292, 1268, 1133, 1074, 1031, 970, 759 cm−1; 1H and 13C NMR data, see Tables 3

Table 5. Cytotoxicity of Compounds 1−12 against Human Cancer Cell Lines A549, HepG2, and MDA-MB-231 with IC50 Values (μM) cell lines compound

A549

HepG2

MDA-MB-231

1 2 3 4 5 6 7 8 9 10 11 12 curcumin demethoxycurcumin bisdemethoxycurcumin Taxol

30.6 61.9 35.0 14.3 41.4 15.2 14.5 84.3 41.1 16.1 16.6 27.4 49.2 38.1 38.1 0.056

25.0 64.1 29.5 18.3 35.9 15.7 17.1 54.6 50.3 15.4 13.0 22.7 31.3 58.1 62.9 0.054

27.3 74.9 24.6 15.9 84.1 19.4 15.9 43.7 69.5 10.6 10.3 30.8 37.8 44.3 38.5 0.052

Terpecurcumin B (2): orange, amorphous powder; [α]25D +2.6 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 418 (4.77) nm; ECD (MeCN) λmax (Δε) 205 (+2.78), 419 (+0.37) nm; IR (KBr) νmax 3432, 2923, 1757, 1626, 1589, 1508, 1450, 1258, 1126, 1031, 972 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS m/z 589.3162 [M + H]+ (calcd for C36H45O7, 589.3165). Terpecurcumin C (3): orange, amorphous powder; [α]25D +3.5 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 419 (4.75) nm; ECD (MeCN) λmax (Δε) 200 (+2.49), 420 (+0.43) nm; IR (KBr) νmax 3423, I



Article

and 4; positive HRESIMS m/z 589.3161 [M + H]+ (calcd for C36H45O7, 589.3165). Terpecurcumin I (9): orange, amorphous powder; [α]25D +2.4 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 412 (4.24) nm; ECD (MeCN) λmax (Δε) 207 (+4.02) nm; IR (KBr) νmax 3736, 3371, 2925, 1715, 1677, 1607, 1457, 1287, 1216, 1031, 771, 410 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive HRESIMS m/z 587.3014 [M + H]+ (calcd for C36H43O7, 587.3009). Bisabolocurcumin ether (10): red crystals; mp 100−102 °C; [α]25D +3.5 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 419 (4.79) nm; ECD (MeCN) λmax (Δε) 203 (+3.64) 427 (+0.42) nm; IR (KBr) νmax 3747, 3175, 1717, 1667, 1592, 1478, 1402, 1001, 716 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive HRESIMS m/z 625.2776 [M + Na]+ (calcd for C36H42O8Na, 625.2777). Demethoxybisabolocurcumin ether (11): orange, amorphous powder; [α]25D +3.1 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 417 (4.78) nm; ECD (MeCN) λmax nm (Δε) 202 (+3.82), 420 (+0.56); IR (KBr) νmax 3695, 3528, 3170, 1655, 1579, 1516, 1402, 1074, 670 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive HRESIMS m/z 595.2673 [M + Na]+ (calcd for C35H40O7 Na, 595.2672). Didemethoxybisabolocurcumin ether (12): orange, amorphous powder; [α]25D +4.1 (c 0.01, MeCN); UV (MeCN) λmax (log ε) 409 (4.35) nm; ECD (MeCN) λmax nm (Δε) 200 (+3.89), 409 (+0.28); IR (KBr) νmax 3738, 3394, 2922, 1677, 1624, 1599, 1509, 1442, 1245, 1168, 978, 772 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive HRESIMS m/z 565.2567 [M + Na]+ (calcd for C34H38O6 Na, 565.2566). X-ray Crystal Structure Analysis. Red crystals of 10 were obtained from CH3OH. The data were collected on a Rigaku MicroMax 002+ diffractometer with Cu Kα radition by using the ω and κ scan technique to a maximum 2θ value of 117.84°. The crystal structure was solved by direct methods by using SHELXS-97, and all non-hydrogen atoms were refined anisotropically using the leastsquares method. All hydrogen atoms were positioned by geometric calculations. Crystallographic data for the structure of 10 have been deposited with the Cambridge Crystallographic Data Center as supplementary publication CCDC 875533. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44(0) (1223) 336 033; e-mail: [email protected]]. Bisabolocurcumin ether (10): C36H42O8, M = 602.27, monoclinic, space group P21, a = 9.336(5) Å, b = 9.701(5) Å, c = 19.187(11) Å, β = 100.4(2)°, V = 1709.0(16) Å3, Z = 2, Dcalcd = 1.234 g/cm−3, 3778 reflections independent, 3129 reflections observed (|F|2 ≥ 2σ|F|2), R1 = 0.0637, wR2 = 0.1494, S = 1.097. Computational Details. Before the ECD calculation, the molecular dynamics simulations of the keto and enolic forms of 10 were undertaken in the canonical (NVT) ensemble at 298 K by using SYBYL-X 1.114 with the MMFF94 force field. The 1000 fs MD simulation was carried out after the equilibrium stage of about 1000 fs. Reaching the equilibrium state is characterized by the potential energy gradually decreasing to fall into a narrow range (e.g., within 10%) when we started to collect the trajectories with a snapshot every 5 fs. Finally, we adopted the trajectories of the 1000 fs run in the statistical analysis. A preliminary conformational analysis was also undertaken by SYBYL-X 1.1 using the random search method with the MMFF94 force field. The conformers were successively optimized by using the semiempirical method at the AM1 level and the density functional theory (DFT) method at the B3LYP/6-31G* level. The stable conformers with populations greater than or equal to 1% and without imaginary frequencies were submitted to ECD calculation by the TDDFT [B3LYP/6-31G*] method. Considering solvent effects on the calculated ECD spectra, we took the IEFPCM model in MeCN. The 30 lowest electronic transitions were calculated, and the rotational strength of each electronic excitation was given both in dipole velocity and dipole length forms. ECD spectra of different conformers were simulated using SpecDis15 with a half-bandwidth of 0.4 eV, and the final ECD spectra were obtained according to the Boltzmann weighting of each conformer. The calculated ECD spectra were

compared with the experimental data after a UV correction. The ECD spectra of 3, 8, and 10 were calculated similarly. All calculations have been performed with the Gaussian 09 program package.16 Cytotoxicity Assay. All cell lines used in this study, including A549, MDA-MB-231, and HepG2, originated from the American Type Culture Collection. MDA-MB-231 and HepG2 cells were grown in DMEM supplemented with 10% fetal bovine serum without antibiotics. A549 was grown in DMEM/F12K medium supplemented with 10 μg/mL insulin, 100 ng/mL cholera toxin, 5 mg/mL hydrocortisol, and 20 ng/mL recombinant human epidermal growth factor. After plating 24−48 h, when cells were 50% to 60% confluent, the medium was changed before starting the treatment with terpecurcumins. For the evaluation of overall inhibitory effects of terpecurcumins on cell number, the cells were treated with terpecurcumins for 72 h. After treatment, the culture medium was removed and the cells were fixed in 1% glutaraldehyde solution in PBS for 15 min. The fixed cells were stained with a 0.02% aqueous solution of crystal violet for 30 min. After washing with PBS, the stained cells were solubilized with 70% EtOH(aq). The absorbance at 570 nm with the reference filter 405 nm was evaluated using a microplate reader (Thermo, USA).

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

X-ray crystal structure and crystal cell diagram of 10; CIF files, tables of atomic coordinates and equivalent isotropic displacement parameters for the oxygen and carbon atoms, bond lengths, and bond angles for 10. ECD spectra calculation details of 3, 8, and 10. Copies of 1D and 2D NMR, IR, HRESIMS, and ECD spectra for compounds 1−12. This may be accessed free of charge via the Internet at http://pubs.acs.org.

Corresponding Author

*Tel: +86-10-8280-1516. Fax: +86-10-8280-2024. E-mail: [email protected] (M.Y.); [email protected] (H.B.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Program for New Century Excellent Talents in University from Chinese Ministry of Education (No. BMU20110269). We wish to thank Prof. Y. Lv and N. B. Gong (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) for their kind help with crystal structure analysis.

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REFERENCES

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