NMR Assignments of Rotameric Aporphine Alkaloids from Liriodendron tulipifera

Abstract

Liriodendron tulipifera, belonging to the family Magnoliaceae, is commonly called tulip tree. Four N-acetylated aporphine alkaloids, N-acetylnornuciferine (1), N-acetylanonaine (2), N-acetyl-3-methoxynornuciferine (3), and N-acetyl-3-methoxynornantenine (4) were isolated from the roots of L. tulipifera. Although the purity of each compound (1 - 4) was determined to be 97, 96, 99, and 98%, respectively, the ¹H and ¹³C NMR spectroscopic data of the aporphine alkaloids 1 - 4 displayed all signals in duplicate, indicating the presence of two rotamers due to restricted rotation of N-COCH₃ functionality in solution status. The absolute configurations of 1 - 4 w ere established by measuring specific rotation and comparison with the reported data. This is the first report on the ¹H and ¹³C NMR assignments of N-acetyl-3-methoxynornuciferine (3) and N-acetyl-3-methoxynornantenine (4). This study provides advanced NMR spectroscopic data for the structure determination of rotameric aporphine alkaloids.

Introduction

Aporphine alkaloids possess a tetracyclic skeleton. In the family Magnoliaceae, they are found in the genera of Liriodendron, Elmerrillia, Magnolia, Michelia, Talauma, and Tsoongiodendron.¹ Many studies showed that naturally occurring aporphine alkaloids exhibited diverse range of therapeutic potential, such as antibacterial, antiviral, antioxidant, and anticancer activities.^1,2

Liriodendron is a genus in the subfamily Liriodendroidae (family Magnoliaceae) that encompasses only two species, L. tulipifera and L. chinense. Liriodendron tulipifera, a hardwood, is commonly called tuliptree or yellow-poplar. This plant, indigenous to the Northern United States, is cultivated for ornamental tree in Korea. The bark of L. tulipifera was utilized by the Native Americans as a febrifuge to treat the intermittent fevers associated with malaria.³ The aporphine alkaloids, particularly liriodenine, anonaine, and oxoglaucine are considered as the major bioactive constituents of L. tulipifera based on their antiarrhythmic,⁴ anticancer,⁵ antiplasmodial³ properties. N-Acetylated aporphine alkaloids such as tuliferoline exist in two isomeric forms due to rotation of N-COCH₃ functionality. In the ¹H and ¹³C NMR spectroscopic data of these aporphine alkaloids, the spectra displayed all signals in duplicate, indicating the presence of two rotamers, which occur due to restricted rotation of the N-acetyl group. Four rotameric aporphine alkaloids (1 - 4) were afforded from the roots of L. tulipifera. To date, there are a few reports on the structure determination of N-acetyl aporphine alkaloids.^6-9 However, the NMR spectroscopic data assignments on each E- and Z-rotamer of N-acetyl aporphine alkaloids are limited.⁶ The structures were elucidated by NMR and MS spectroscopic data interpretation. Absolute stereochemistry was identified by specific rotation measurement. This report details the isolation and structure elucidation of compounds 1 - 4.

Experimental

General experimental procedures - Specific rotations were measured on a JASCO DIP-370 polarimeter (Tokyo, Japan). UV spectra were recorded on a Shimadazu SPDM20A PDA detector. NMR spectroscopic data were recorded on a Bruker DMX 600 ( ¹H-600 MHz, ¹³C-150 MHz) spectrometers equipped with a 5 mm direct detection BBFO probe. All NMR experiments were performed at 294 K, using CDCl ₃ as the solvent. Chemical shifts were given on the δ scale and referenced by CDCl ₃ as an internal standard (δ_H = 7.26, δ_C = 77.2). Coupling constants (J) are in Hz. Data processing was carried out with MestReNova v6.0.2 program. Precoated TLC silica gel 60 F₂₅₄ and RP-18 F₂₅₄ plates from Merck was used for analytical purposes followed by detection under UV light at 254 and 365 nm wavelengths or after spraying with 10% sulfuric acid and dragendorff reagents. VLC was implemented on Merck silica gel (70 - 230 mesh), and MPLC (Biotage Isolera^TM) was carried out utilizing silica (KP-Sil, Biotage, Biotage, Uppsala, Sewden) and C18 (KP-C18-HS, Biotage, Uppsala, Sewden) cartridges. Preparative HPLC was accomplished on a Gilson system (PLC 2020) with a flow rate of 5.0 mL/min using a Phenomenex kinetex biphenyl column (250 × 21.2 mm, 5 μm), a Hector C₁₈ column (250 × 21.2 mm, 5 μm), or Hector phenyl column (250 × 21.2 mm, 5 μm).

Plant materials - The roots of Liriodendron tulipifera were collected from Chungnam National University in August 2016. A voucher specimen (CNU201608) has been deposited at the Laboratory of Pharmacognosy of College of Pharmacy, Chungnam National University, Daejeon, Korea.

Extraction and isolation - The EtOH extract (239.3 g) of L. tulipifera roots was subjected to silica gel VLC and eluted with a stepwise gradient of n-hexane-EtOAc (8:2, 5:5, 3:7, 1:9; each step 6 L) and CHCl₃ -MeOH (9:1, 7:3, 5:5, 2:8; each step 6 L; final washing with 10 L of 100% MeOH) to produce seven fractions (LT1 ~ LT7). Fraction LT4 (20.7 g) was separated by MPLC (Biotage SNAP cartridge, KP-C18-HS, 400 g) using a step gradient mixures of MeOH-H₂O (5:5, 7:3, 9:1, 10:0, MeCN 100%) to yield 1 (300.4 mg) and six subfractions (LT4-1 ~ LT4-6). LT4-5 (5.5 g) was separated by MPLC (Biotage SNAP cartridge, KP-C18-HS, 400 g) with a stepwise gradient elution using MeOH-H₂O (7:3, 8:2, 9:1, 10:0) to give seven fractions (LT4-5-1 ~ LT4-5-7). Fr. LT4-5-5 (461.0 mg) was repeatedly subjected to HPLC (Hector C₁₈ column) with a gradient mixtures of MeOH-H₂O (80:20 → 90:10) and to yield three fractions (LT4-5-5-2 ~ LT4-5-5-4). LT4-5-5-3 (41.0 mg) and LT4-5-5-4 (207.1 mg) were subjected to HPLC [Hector phenyl column, MeOH-H₂O (80:20 → 90:10)] purification to obtain 4 (30.5 mg) and 3 (63.4 mg). Compound 2 (6.1 mg) was isolated from LT4-5-6 (907.4 mg) by HPLC (Phenomenex biphenyl column) eluting with isocratic solvent of MeOH-H₂O (90:10).

N-Acetylnornuciferine (1) - yellow needles; [α]_D²² -311.6 (c=0.55, CHCl₃); ¹H and ¹³C NMR spectroscopic data, see Tables 1 and 2; ESIMS, m/z 324 [M+H]⁺ , 346 [M+ Na]⁺

Table 1. ¹H NMR assignments for 1 - 4 in CDCl₃ (600 MHz)

Table 2. ¹³C NMR assignments for 1 - 4 in CDCl₃ (150 MHz)

*Because the chemical shifts are very similar, the signals may change.

N-Acetylanonaine (2) - yellow amorphous solid; [α]_D²² -322.0 (c=0.05, CH₂Cl₂); ¹H and ¹³C NMR spectroscopic data, see Tables 1 and 2; ESIMS, m/z 308 [M+H]⁺ , 330 [M+Na]⁺

N-Acetyl-3-methoxynornuciferine (3) - yellowish amorphous powder; [α]_D²² -333.5 (c=0.2, CHCl₃); ¹H and ¹³C NMR spectroscopic data, see Tables 1 and 2; ESIMS, m/z 354 [M+H]⁺ , 376 [M+Na]⁺

N-Acetyl-3-methoxynornantenine (4) - yellowish amorphous powder; [α]_D²² +429.0 (c=0.1, CHCl₃); ¹H and ¹³C NMR spectroscopic data, see Tables 1 and 2; ESIMS, m/z 398 [M+H]⁺ , 420 [M+Na]⁺

Result and Discussion

Compounds 1 - 4 were determined as N-acetylated aporphine alkaloids (Fig. 1). HPLC analysis on compounds 1 - 4 revealed the purity should be 97, 96, 99, and 98%, respectively. However, the ¹H and ¹³C NMR spectroscopic data of 1 - 4 displayed all signals in dupli-cate, indicating the presence of two rotamers, Z- and E-isomers, due to rotation about N-COCH₃ functionality in solution status (Fig. 2). The ¹H and ¹³C NMR signals at C-4, C-5, C-6a, C-7, and N-acetyl group were distinguished because of the shielding effect of N-acetyl group (Fig. 2), which enabled us to determine the structures of Z- and Eforms, respectively. The absolute configurations of rotameric aporphine alkaloids 1 - 4 were established by measuring specific rotation.

Fig. 1. Structures of compounds 1 - 4.

Fig. 2. Z- and E-rotamers of compound 1.

The ¹H NMR spectroscopic data of 1 was complex, because of the resonances of the two rotational isomers arising from restricted rotation about the N-acetyl group. Two separate signals in the ratio of 1.3/1 (1Z/1E) were estimated based on the integration of aromatic signals. The ¹H NMR spectroscopic data of 1Z displayed signals of two benzene protons at δ 8.42 (d, J = 7.7 Hz, H-11) and 6.66 (s, H-3), while these two benzene protons revealed in 1E at δ 8.47 (d, J = 7.7 Hz, H-11) and 6.70 (s, H-3) in the same spectrum. Other aromatic protons at δ 7.38 - 7.24 for H-8, H-9, and H-10 were observed. In addition, the ¹H NMR spectrum revealed two methoxy groups and one N-acetyl moiety at δ 3.90, 3.67, and 2.22 for 1Z as well as at δ 3.90, 3.67, and 2.17 for 1E (Table 1). The presences of the two methoxy and an N-acetyl groups were further confirmed by the ¹³C NMR spectroscopic data, which showed characteristic signals at δ 60.1, 56.1, 169.2, and 22.7 for 1Z as well as at 60.1, 56.1, 169.9, and 21.7 for 1E. The resonances of one methine proton at δ 5.09/4.56 (dd, J = 13.4 and 2.9 Hz, H-6a of 1Z/br, J = 13.5 Hz, H-6a of 1E) and three methylenes at δ 4.96 (d, J = 11.6 Hz, H₂ -5α of 1E), 4.00 (d, J = 12.6 Hz, H₂ -5α of 1Z), 3.31 (t, J = 12.6 Hz, H₂ -5β of 1Z), 3.10 (t, J = 13.5 Hz, H₂ -7α of 1E), 2.89 (t, J = 12.1 Hz, H 2 -4α of 1Z), 2.83 (m, H₂ -4α of 1E), 2.76 (m, overlap, H₂ -7β of 1E and 1Z, H₂ -5β of 1E), and 2.68 (m, H₂ -4β of 1E and 1Z) were observed (Table 2). Each structure of 1E and 1Z was confirmed by ¹³C NMR, HSQC, and HMBC spectra. The absolute configuration at C-6a was assigned as R based on the specific rotation value [α]_D²² -311.6 (c=0.55, CHCl₃) in com- parison with the reported data.⁶ The structure of 1 was determined as N-acetylnornuciferine, whose NMR spec- troscopic data was displayed in Tables 1 and 2.

The ¹H NMR spectrum of 2 closely resembled those of 1. However, the two methoxy groups at δ 3.90 and 3.67 of 1 were replaced by one dioxymethylene at δ 6.10 (s, -OCH₂O- of 2E) and 5.98 (s, -OCH₂O- of 2Z). Two separate peaks for 2Z and 2E were observed with relative intensities of 1.5/1. The specific rotation of 2 was levorotatory ([α]_D¹⁸ -322.0). By comparison with the literature data, ⁷ 2 was identified as N-acetylanonaine.

The ¹H NMR spectroscopic data for 3 was similar to those of 1 except for the absence of one benzene proton and the presence of one additional methoxy group at δ 3.96 (s, 3-OCH₃ of 3Z/E). The ¹H and ¹³C NMR spectra showed 3Z and 3E with integrated intensities of 1.5/1. The structure of compound 3 was determined as N-acetyl-3-methoxynornuciferine by comparison of specific rotation value with that in the literature.⁸

Compound 4 was also identified as an N-acetylated aporphine alkaloid based on the NMR spectroscopic data. The differences in 4, compared with 3, was the absence of two benzene protons and the presence of one oxymethylene protons at δ 5.97 (m, -OCH₂O- of 4Z/E). The ratio of two rotamers, 4Z and 4E, was 2/1 based on ¹H NMR spectrum. The S configuration at C-6a was deduced by the positive specific rotation value ([α]_D²² +429.0). 9 Based on the above evidence, the structure of compound 4 was assigned as N-acetyl-3-methoxynornantenine.

Although N-acetyl aporphine alkaloids have often been found in natural products, the NMR assignments for E-and Z-rotamers are limited. The ¹H and ¹³C NMR spectroscopic data on N-acetyl-3-methoxynornuciferine (3) and N-acetyl-3-methoxynornantenine (4) were assigned for the first time in this study. Our results provide advanced NMR spectroscopic data for the structure determination of rotameric aporphine alkaloids.