CHIRALITY 26:825–828 (2014)

Determination of the Absolute Configuration of Two Pairs of C-8 C-9′ Linked Neolignan Enantiomers JING-MEI BAO,1 ZHONG-BIN CHENG,1 JUN-SHENG ZHANG,1 JIAN-YONG ZHU,1 GUI-HUA TANG,1 LI-SHE GAN,2 AND SHENG YIN1* 1 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China 2 College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China

ABSTRACT Two pairs of new neolignan enantiomers, (±)-torreyayunan A (1a/1b) and (±)-torreyayunan B (2a/2b), featuring a rare C-8 C-9′ linked skeleton, were isolated from leaves and twigs of Torreya yunnanensis. Their absolute configuration involving two chiral centers was determined by combined spectral and Density Functional Theory (DFT) calculation. This is the first report of the absolute configuration of this group of neolignans. Chirality 26:825–828, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: Torreya yunnanensis; neolignan enantiomers; C-8 configuration; ECD; DFT INTRODUCTION

Lignans, widely distributed in the plant kingdom, are a class of secondary metabolites produced by oxidative dimerization of two phenylpropanoid units. Although their molecular backbone consists only of two phenylpropane (C6 C3) units, lignans exhibit an enormous structural diversity originating from various linkage patterns of these phenylpropane units. There is a growing interest in lignans and their synthetic derivatives due to their applications in various pharmacological effects.1 Neolignans with C-8 C-9′ linked skeleton are very rare in nature. So far only eighteen natural neolignans of this skeleton have been reported and all of them came from the genus Alpinia and Morina.2–4 As the C-8 C-9′ linked lignans contain two chiral centers (C-7 and C-8), it may give rise to four stereoisomers, which can be grouped into two pairs of enantiomers. Although the relative configuration of C-7 and C-8 could be clarified by analysis of the 1 H 1H coupling constant of H-7 and H-8 in its 7,8-acetonide derivatives,2,4 the absolute configuration (AC) could not be determined by other conventional approaches. For example, Mosher’s method has limitations in the determination of the AC of C-7 (bearing a secondary hydroxyl) in this class of neolignans, as the ΔδS R values of the (S/R)-MTPA-esters would be affected not only by the phenyl ring of the MTPA but also by the aromatic ring adjacent to C-7.5 The method of comparison of the circular dichroism (CD) spectra with those of the known analogs is also inapplicable, as the different substituent patterns of the benzene rings may complicate the interpretation of the Cotton effects and even reverse the CD curves, leading to a dubious conclusion.6 Moreover, this class of neolignans was usually obtained as oil or powder, which hampers the application of single-crystal X-ray diffraction analysis. Thus, the ACs of this group of neolignans have never been assigned. Compared with conventional approaches to determine the ACs, the comparison of computational and experimental spectroscopic data, such as electronic circular dichroism (ECD), vibrational circular dichroism (VCD), optical rotation (OR), nuclear magnetic resonance (NMR), and ultraviolet (UV) can provide a streamlined approach that, at a minimum, significantly reduces the number of possible stereoisomers.7 As a consequence, the computational chiroptical spectroscopy is widely used by organic chemists in determining ACs. In © 2014 Wiley Periodicals, Inc.

C-9′ linked skeleton; absolute

recent years, a number of useful theoretical methods, such as the configuration interaction (CI) method, Density Functional Theory (DFT), Time-Dependent Density Functional Theory (TDDFT), and time-dependent Hartree-Fock (TDHF), have been established for computed spectra.8 Among them, DFT and TDDFT, which have proven to be reliable and feasible approaches, are perhaps the most widely used ab initio methodologies in the field of chiroptical properties prediction, particularly for the prediction of ECD spectra. Many successful applications of CD spectra prediction by TDDFT are being reported in the literature.7 During our search for discovering structurally interesting metabolites from medicinal plants, two pairs of new neolignan enantiomers featuring a rare C-8 C-9′ linked skeleton were isolated from the leaves and twigs of Torreya yunnanensis. Herein, we report the determination of the absolute configuration of these two pairs of enantiomers using a combined theoretical and experimental method. This is the first report on the absolute configuration of this group of neolignans. MATERIALS AND METHODS General Optical rotations were measured on a Rudolph Autopol I automatic polarimeter, and CD spectra were obtained on an Applied Photophysics Chirascan spectrometer. UV spectra were recorded on a Shimadzu UV2450 spectrophotometer. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. Electrospray ionization, mass spectrometry (ESIMS) was measured on a Finnigan LCQ Deca instrument, and high-resolution ESIMS (HRESIMS) was performed on a Waters Micromass Q-TOF. A Shimadzu LC-20 AT equipped with a SPD-M20A PDA detector was used for high-performance liquid chromatography (HPLC). A chiral column (Phenomenex Lux, cellulose-2, 250 × 10 mm, 5 μm) was used for semipreparative HPLC separation. Silica gel Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: 81102339. Contract grant sponsor: Guangdong Natural Science Foundation; Contract grant number: S2011040002429. *Correspondence to: Sheng Yin, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China. E-mail: [email protected] Received for publication 1 July 2014; Accepted 8 September 2014 DOI: 10.1002/chir.22395 Published online 29 October 2014 in Wiley Online Library (wileyonlinelibrary.com).

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(300–400 mesh, Qingdao Haiyang Chemical), C18 reversed-phase silica gel (12 nm, S-50 μm, YMC), Sephadex LH-20 gel (Amersham Biosciences), and MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries) were used for column chromatography. All solvents used were of analytical grade (Guangzhou Chemical Reagents).

Plant Material Leaves and twigs of T. yunnanensis were collected in October 2012 in the Yunnan Province, P.R. China, and were authenticated by Prof. You-Kai Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (accession number: FM201210) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University.

( 0.65), 278 (+0.71); UV, IR, NMR, MS, and HRESIMS data are the same with those of 1a. 20

( )-Torreyayunan B (2a). Light yellow oil; [α] D 28.8 (c 0.19, 4 CH2Cl2); UV (MeOH) λmax (log ε) 288 (3.32) nm; ECD (c 2.62 × 10 M, CH3CN) λmax (Δε) 202 ( 1.44), 223 (+0.47), 264 (+0.28); IR (KBr) νmax 1 3473, 2942, 2838, 1588, 1469, 1242, 1106, 1029, 800, 729, and 589 cm ; 1 13 + H and C NMR data, see Table 1; positive ESIMS m/z 411.2 [M + Na] ; HRESIMS m/z 387.1810 [M H] (calcd. for C22H27O6, 387.1808). 20

(+)-Torreyayunan B (2b). Light yellow oil; [α] D +28.5 (c 0.20, 4 CH2Cl2); ECD (c 2.80 × 10 M, CH3CN) λmax (Δε) 201 (+1.95), 222 ( 0.85), 262 ( 0.17); UV, IR, NMR, MS, and HRESIMS data are the same with those of 2a.

Extraction and Isolation The air-dried powder of the leaves and twigs of T. yunnanensis (600 g) was extracted with 95% EtOH (3 × 5 L) at room temperature to give 59 g of crude extract. The extract was suspended in H2O (0.5 L) and successively partitioned with petroleum ether (PE, 3 × 1 L), EtOAc (3 × 1 L), and n-BuOH (3 × 1 L). The EtOAc extract (17 g) was subjected to MCI gel column chromatography (CC) eluted with MeOH/H2O gradient (3:7 → 10:0) to afford five fractions (I V). Fraction IV (5.3 g) was chromatographed over reversed-phase C18 (RP-C18) silica gel CC eluted with MeOH/H2O (4:6 → 10:0) to afford five fractions (IVa IVe). Fraction IVd was subjected to Sephadex LH-20 eluted with CHCl3/MeOH, 1:1, to give three fractions (IVd1 IVd3). Fraction IVd2 was applied to silica gel CC (CH2Cl2/MeOH, 100:1 → 20:1) to afford five fractions (IVd2a IVd2e). Fraction IVd2b was purified on Sephadex LH-20 (MeOH), followed by HPLC equipped with a chiral column (MeOH/H2O, 80:20, 3 mL/min) to give 2b (6.5 mg, tR = 21.0 min) and 2a (6.1 mg, tR = 34.3 min). Fraction IVd2c was further purified by HPLC equipped with a chiral column (MeOH/H2O, 80:20, 3 mL/min) to give 1b (3.3 mg, tR = 17.7 min) and 1a (2.5 mg, tR = 28.5 min). 20

(+)-Torreyayunan A (1a). Light yellow oil; [α] D +16.4 (c 0.07, CH2Cl2); 4 UV (MeOH) λmax (log ε) 289 (3.34) nm; ECD (c 1.11 × 10 M, CH3CN) λmax (Δε) 209 (+4.81), 256 (+0.77), 281 ( 1.25); IR (KBr) νmax 2929, 2842, 1587, 1 1 13 1467, 1242, 1107, 1025, 1107, 1025, 774, and 724 cm ; H and C NMR data, + see Table 1; positive ESIMS m/z 799.4 [2 M + Na] ; HRESIMS m/z 387.1803 [M H] (calcd. for C22H27O6, 387.1808). 20

( )-Torreyayunan A (1b). Light yellow oil; [α] D 14.6 (c 0.11, 4 CH2Cl2); ECD (c 1.49 × 10 M, CH3CN) λmax (Δε) 212 ( 4.57), 256

TABLE 1.

1

H and 13C NMR data of 1 2 in Acetone-d6 (400 and 100 MHz, respectively) 1a/1b

position 1 2/6 3/5 4 7 8 9 1′ 2′/6′ 3′/5′ 4′ 7′ 8′ 9′ 2/6-OCH3 2′/6′-OCH3

2a/2b

δH (mult, J in Hz)

δC

6.68, d, 8.4, 1H 7.21, t, 8.4, 1H 5.17, t, 10, 1H

120.2 159.3 105.4 129.5 70.0

2.30, m, 1H 3.91, m, 2H

6.59, 7.09, 6.47, 6.47, 1.94, 2.09, 3.85, 3.79,

d, 8.4, 1H t, 8.4, 1H m, 1H m, 1H m, 1H m, 1H s, 3H s, 3H

Chirality DOI 10.1002/chir

47.4 64.1 115.9 159.1 105.0 128.1 122.2 134.2 34.5 56.2 56.1

δH (mult, J in Hz)

6.70, d, 8.4, 1H 7.22, t, 8.4, 1H 5.11, dd, 11.4, 8.6, 1H 2.17, m, 1H 3.41, m, 2H

6.62, 7.11, 6.67, 6.67, 2.50, 2.65, 3.86, 3.82,

d, 8.4, 1H t, 8.4, 1H m, 1H m, 1H m, 1H m,1H s, 3H s, 3H

δC 120.3 159.0 105.5 129.3 69.0 48.6 62.7 116.2 159.2 105.0 128.1 122.5 134.5 33.3 56.3 56.1

COMPUTATIONAL SECTION

In general, conformational analyses were carried out via Monte Carlo searching using molecular mechanism with MMFF94 force field in the Spartan 08 software package.9 Subsequently, the resulting conformers with relative energy within 2.0 kcal/mol were reoptimized using DFT at the B3LYP/6-31 + G(d) level in gas phase by the Gaussian 09 program.10 The B3LYP/6-31 + G(d) harmonic vibrational frequencies were also calculated to confirm their stability. The energies, oscillator strengths, and rotational strengths (velocity) were calculated using the TDDFT methodology at the B3LYP/6-311++G(2d,2p) level in vacuum. The ECD spectra were simulated by the overlapping Gaussian function.11 To get the final spectra, the simulated spectra of the lowest energy conformers for each structure were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (ΔG). Theoretical ECD spectra were obtained by directly inverting the ECD spectra of the corresponding enantiomers. RESULTS AND DISCUSSION

The air-dried powder of the leaves and twigs of T. yunnanensis was extracted with 95% EtOH at room temperature to give a crude extract, which was suspended in H2O and successively partitioned with petroleum ether, EtOAc, and n-BuOH. Various column chromatographic separations of the EtOAc extract afforded two pairs of new neolignan enantiomers, 1a/1b and 2a/2b. Torreyayunan A (1), a light yellow oil, had the molecular formula C22H28O6, as established by HRESIMS and 13C NMR data. Its IR absorption bands at 1587 and 1467 cm 1 indicated the presence of a benzene ring. Detailed analyses of the 1H NMR revealed the presence of four methoxy groups [δH 3.79 (3H × 2, s) and 3.85 (3H × 2, s)], a methylene [δH 1.94 (1H, m), 2.09 (1H, m)] and an oxymethylene [δH 3.91 (2H, m)], a methine [δH 2.30 (1H, m)] and an oxymethine [δH 5.17 (1H, t, J = 10 Hz)], two olefinic protons [δH 6.47 (2H, m)], and two symmetrically 1,2,3-trisubstituted benzene ring [δH 6.59 (2H, d, J = 8.4 Hz) and 7.09 (1H, t, J = 8.4 Hz); 6.68(2H, d, J = 8.4 Hz) and 7.21 (1H, t, J = 8.4 Hz)]. The 13C NMR spectrum, in combination with DEPT experiments, showed 22 carbon resonances attributable to two benzene rings, a double bond, two sp3 methylene (one bearing O-atom), two methines (one bearing Oatom), and four methoxy groups. The aforementioned data implied that 1 possessed a rare 8-9′ linked neolignan skeleton, similar to that of galanganol A.2 2D NMR analyses allowed the structure of 1 to be postulated as depicted in Figure 1. In particular, the 1H 1H COSY spectrum first established a spin system of C7′-C8′-C9′-C8(C7)-C9. The HMBC correlations from

C-8

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H7 to C-1, C-2, and C-6 established the connectivity between C-7 and ring A. Ring B was connected to C-7′ as apparent from the HMBC correlations of H-7′/C-1′, C-2′ and C-6′. The locations of methoxy groups on rings A and B were assigned by HMBC correlations from the methoxy protons at δH 3.85 (3H × 2) to C-2/C-6 (δC 159.3) and from methoxy protons at δH 3.79 (3H × 2) to C2′/C-6′ (δC 159.1). The trans configuration of Δ7′ in 1 was determined to be the same as that of conchigeranal B by comparing their 13C chemical shifts regarding C-7′ and C-8′ (δC 122.2 and 134.2, respectively, in 1, δC 123.8 and 132.9, respectively, in conchigeranal B).3 Therefore, the structure of 1 was elucidated as shown in Figure 2 and was given the trivial name torreyayunan A. Torreyayunan B (2), displayed a molecular ion at m/z 387.1810 [M H]+, consistent with the same molecular formula as that of 1. The 1H and 13C NMR data of 2 were very similar to those of 1, with the differences being raised from the chiral centers C-7 and C-8, indicating 2 was a diastereoisomer of 1. Detailed interpretation of its 2D NMR data further confirmed that 2 shared the same gross structure of 1. Thus, 2 was given the trivial name torreyayunan B. It is worth noting that compounds 1 and 2 were primarily obtained with the specific rotations being almost zero and gave flat lines in their CD spectra, indicating a racemic nature. Subsequent chiral resolution performed on a chiral column afforded the anticipated enantiomers, respectively, which were opposite in terms of CD curves (Figs. 4 and 5) and specific rotations (1a: +16.4; 1b: 14.6; 2a: 28.8; 2b: +28.5). As the coupling constants between H-7 and H-8 in both 1 and 2 were quite similar (around 10 Hz), which hampered the determination of their relative configuration by analysis of the coupling constants, the stereochemistry of the two pairs of enantiomers was determined by theoretical TDDFT calculations and compared with the corresponding experimental ECD spectra, which directly generated the information of the ACs of 1a/1b and 2a/2b. Firstly, in order to avoid large amounts of lowest energy conformers caused by the flexible chain, the 2,6dimethoxystyryl groups in 1 and 2, which are not directly

connected to the asymmetric carbons and have very little influence on the ECD spectrum, were cut off to give a model compound, M (Fig. 3). Secondly, the theoretical ECD spectra of two possible isomers of M, (7R,8R)-M and (7S,8R)-M, were calculated using the procedures described in the Computational Section. Theoretical ECD spectra of (7S,8S)-M and (7R,8S)-M were obtained by directly inverting the ECD spectra of the corresponding enantiomers.

Fig. 3. Model compounds used in ECD spectra calculations.

Fig. 4. Experimental ECD spectra (200–400 nm) of 1a (black dash line) and 1b (black solid line), and B3LYP/6-311++G(2d,2p)//B3LYP/6-31 + G(d) calculated ECD spectra for (7S,8S)-M (red dash line) and (7R,8R)-M (red solid line).

1

Fig. 1. Selected H

1

H COSY (

) and HMBC (→) correlations of 1.

Fig. 2. Structures of compounds 1a/1b and 2a/2b isolated from Torreya yunnanensis.

Fig. 5. Experimental ECD spectra (200–400 nm) of 2a (black solid line) and 2b (black dash line), and B3LYP/6-311++G(2d,2p)//B3LYP/6-31 + G(d) calculated ECD spectra for (7S,8R)-M (red solid line) and (7R,8S)-M (red dash line). Chirality DOI 10.1002/chir

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The experimental ECD spectrum of 1b (Fig. 4) showed first positive and second negative Cotton effects at 278 nm and 212 nm, respectively, which matched the calculated ECD curve for (7R,8R)-M exactly, indicating 1b possessed a 7R,8R configuration. Accordingly, 1a was assigned as 7S,8S. For 2a and 2b, the calculated ECD spectra of the model compounds (7S,8R)-M and (7R,8S)-M were also in good agreement with the experimental ones (Fig. 5), indicating that the ACs of 2a and 2b were 7S,8R and 7R,8S, respectively. Thus, the above calculations allowed the determination of the ACs of these two pairs of enantiomers. CONCLUSION

Two pairs of new neolignan enantiomers, (±)-torreyayunan A (1a/1b) and (±)-torreyayunan B (2a/2b), featuring a rare C-8 C-9′ linked skeleton, were isolated from leaves and twigs of Torreya yunnanensis. Their gross structures were established on the basis of extensive spectroscopic analysis. The ACs of 1a (7S,8S), 1b (7R,8R), 2a (7S,8R), and 2b (7R,8S) were determined by comparison of their calculated CD spectra with the experimental results. The ECD spectra were good enough to assign the ACs of the two pairs of new neolignan enantiomers in comparison with the experimental data. This is the first report on the absolute configuration of this group of neolignans. ACKNOWLEDGMENTS

The authors thank the National Natural Science Foundation of China (No. 81102339) and Guangdong Natural Science Foundation (No. S2011040002429) for providing financial support for this work. SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article at the publisher’s web-site.

Chirality DOI 10.1002/chir

LITERATURE CITED 1. Muhammad S, Hyoung JK, Muhammad SA, Yong SL. An update on bioactive plant lignans. Nat Prod Rep 2005;22:696–716. 2. Toshio M, Shin A, Hisashi M, Shinya K, Osamu M, Masayuki Y. Inhibitors of nitric oxide production from the rhizomes of Alpinia galanga: structures of new 8–9′ linked neolignans and sesquineolignan. Chem Pharm Bull 2005;53:625–630. 3. Xu JJ, Zeng GZ, Yang SC, Shen Y, Tan NH. 8-9′ linked neolignans with cytotoxicity from Alpinia conchigera. Fitoterapia 2013;91:82–86. 4. Su BN, Takaishi Y, Kusumi T, Morinols A-L. Twelve novel sesquineolignans and neolignans with a new carbon skeleton from Marina chinensis. Tetrahedron 1999;55:14571–14586. 5. José MS, Emilio Q, Ricardo R. The assignment of absolute configuration by NMR. Chem Rev 2004;104:17–117. 6. Antus S, Kurtán T, Juhász L, Kiss L, Hollósi M, Májer ZS. Chiroptical properties of 2,3-dihydrobenzo[b]furan and chromane chromophores in naturally occurring O-Heterocycles. Chirality 2001;13:493–506. 7. Wu RB, Cheng ZB, Han QH, Lin TT, Zhou JW, Tang GH, Yin S. Determination of the absolute stereochemistry of two new aristophyllene sesquiterpenes: a combined theoretical and experimental investigation. Chirality 2014;26:189–193. 8. Hirata S, Head-Gordon M, Bartlett RJ. Configuration interaction singles, time-dependent Hartree–Fock, and time-dependent density functional theory for the electronic excited states of extended systems. J Chem Phys 1999;111:10774–10786. 9. Spartan 04, Wavefunction Inc., Irvine, CA, 92612 USA, 2004. 10. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery JJA, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas Ö, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ. Gaussian 09, Rev. C 01. Wallingford, CT: Gaussian; 2009. 11. Stephens PJ, Harada N. ECD cotton effect approximated by the Gaussian curve and other methods. Chirality 2010;22:229–233.

Determination of the absolute configuration of two pairs of C-8 - C-9' linked neolignan enantiomers.

Two pairs of new neolignan enantiomers, (±)-torreyayunan A (1a/1b) and (±)-torreyayunan B (2a/2b), featuring a rare C-8 - C-9' linked skeleton, were i...
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