A new 5H-purin-6-amine from the leaves of Sedum sarmentosum

Abstract

Phytochemical constituents were isolated from Sedum sarmentosum leaves using open column chromatography and medium-pressure liquid chromatography. Their structures were identified as 2,4-pyrimidinedione (1), N-methylhydroxylamine (2), 5H-purin-6-amine (3), uridine (4), l-tyrosine (5), and l-prolyl-l-tyrosine (6) using mass spectrometry and ¹H- and ¹³C-nuclear magnetic resonance spectroscopic analysis. Among them, compound 3 (5H-purin-6-amine) was isolated for the first time from a natural source.

Introduction

Sedum sarmentosum Bunge is a perennial herb distributed across Asia, Europe, and North America [1]. It is commonly known as Dolnamul in Korea. Fresh leaves of S. sarmentosum have been used in salads as an alternative to pepper because of their pungent taste. Moreover, traditionally, it has been used as a hepatoprotective medicinal plant in Asian countries [2]. Few studies have focused on the biological activities of S. sarmentosum [1]. S. sarmentosum shows unique angiotensin-converting enzyme inhibitory activity. Some flavonoids also show this activity [3]. Kim et al. [4] suggested that S. sarmentosum contains estrogens to promote a better life in menopausal women [4].

The objective of this study was to find new bioactive compounds from the leaves of S. sarmentosum. Therefore, our investigation was designed for the systematic isolation and identification of these valuable phytochemicals.

Materials and methods

Plant materials

Fresh S. sarmentosum leaves were cultivated at Juksan-myeon, Anseong, Republic of Korea.

Apparatus and chemicals

Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker Avance 500 NMR (Rheinstetten, Germany) spectrometer. Mass spectrometry (MS) was performed using a JEOL JMS-600 W (Tokyo, Japan) mass spectrometer. Optical rotations were measured on a Jasco P-2000 digital polarimeter (Tokyo, Japan). Medium-pressure liquid chromatographic separation was carried out on the Biotage (Uppsala, Sweden) system.

Extraction, fractionation, and isolation

The fresh S. sarmentosum leaves were dried for 3 days, and the powdered S. sarmentosum leaves (1.3 kg) were subjected to extraction with EtOH (8 L × 10) under reflux at 65–75 °C. The filtrate was concentrated until dry in vacuo to afford a dark green EtOH extract (286.1 g). The EtOH extract (250.5 g) was suspended in H₂O and then partitioned successively using n-hexane (57.8 g), CH₂Cl₂ (5.5 g), EtOAc (6.9 g), and n-BuOH (76.5 g). Among the fractions, a portion of the EtOAc fraction (5 g) was subjected to medium-pressure liquid chromatography (MPLC) with a gradient elution of CHCl₃–MeOH (1:0 → 5:5) to yield 21 (EF1 to EF21) sub-fractions. EF9 sub-fraction was recrystallized under CHCl₃–MeOH to yield compound 1. A portion of the n-BuOH fraction (60 g) was subjected to MPLC on silica gel using a gradient elution of CHCl₃–MeOH (100:0 → 50:50) to obtain ten (BF1 to BF10) fractions. BF2 fraction was recrystallized under CHCl₃–MeOH to yield compound 2. BF6 fraction was repeatedly chromatographed on a Sephadex LH-20 column (St. Louis, MO, USA) to obtain seven (BF6-1 to BF6-7) fractions, and BF6-3 fraction was recrystallized to yield compound 3. BF8 and BF9 fractions were repeatedly subjected to MPLC and eluted with CHCl₃–MeOH (80:20 → 50:50) to yield five (BF8-1 to BF8-5) and six (BF9-1 to BF9-6) fractions, respectively. BF8-3 fraction was separated on a Sephadex LH-20 column to obtain seven fractions (BF8-3-1 to BF8-3-7) and isolated compound 4. BF9-4 fraction was repeatedly chromatographed on a Sephadex LH-20 column to obtain five (BF9-4-1 to BF9-4-5) fractions, and BF9-4-5 fraction was recrystallized to yield compound 5. BF9-5 fraction was separated on a Sephadex LH-20 column and isolated compound 6.

Compound 1

EI-MS: m/z (rel. int.) 112 [M]⁺ (100), 85 (0.9), 69 (45), 57 (3.8); ¹H-NMR (DMSO-d ₆ , 500 MHz): δ 5.44 (1H, d, J = 7.6 Hz, H-5), 7.38 (1H, d, J = 7.6 Hz, H-6), 10.81 (NH), 11.00 (NH); ¹³C-NMR (DMSO-d ₆ , 125 MHz): δ 100.1 (C-5), 142.0 (C-6), 151.3 (C-2), 164.4 (C-4).

Compound 2

¹H-NMR (DMSO-d ₆ , 500 MHz): δ 3.16 (3H, d, J = 5.5 Hz, –CH₃), 4.08 (1H, q, J = 5.0 Hz, –NH), 8.31 (1H, s, –OH); ¹³C-NMR (DMSO-d ₆ , 125 MHz): δ 48.5 (–NCH₃).

Compound 3

EI-MS: m/z (rel. int.) 135 [M]⁺ (100), 119 (1.4), 108 (25.3), 92 (1.1), 81 (9.7), 66 (4.4), 54 (6.1); ¹H- and ¹³C-NMR (DMSO-d ₆ , 500 MHz): see Table 1.

Table 1 ¹H- and ¹³C-NMR spectral data for compound 3 (DMSO, 500 MHz)

Compound 4

EI-MS: m/z (rel. int.) 154 (10.0), 151 (21.0), 112 (100), 95 (17.4), 83 (8.7), 69 (23.2); ¹H-NMR (DMSO-d ₆ , 500 MHz): δ 3.58 (1H, dd, J = 2.5, 12.0 Hz, H-5′), 3.70 (1H, dd, J = 2.5, 12.0 Hz, H-5′), 3.88 (1H, m, H-4′), 3.95 (1H, t, J = 5.5 Hz, H-3′), 4.02 (1H, t, J = 5.5 Hz, H-2′), 5.71 (1H, d, J = 3.5 Hz, H-4), 5.99 (1H, d, J = 8.0 Hz, H-1′), 8.21 (1H, d, J = 8.0 Hz, H-3); ¹³C-NMR (DMSO-d ₆ , 125 MHz): δ 60.1 (C-5′), 69.0 (C-3′), 74.1 (C-2′), 84.5 (C-4′), 89.4 (C-1′), 93.8 (C-4), 143.1 (C-3), 159.1 (C-2), 168.1 (C-5).

Compound 5

[α] ²⁷_D -6.5 (c 0.4, DMSO); EI-MS: m/z (rel. int.) 181 [M]⁺ (6.5), 136 (3.2), 119 (1.5), 107 (100), 91 (8.2), 77 (7.7), 68 (2.5); ¹H-NMR (DMSO-d ₆ , 500 MHz): δ 3.01 (2H, br d, CH₂), 4.09 (1H, br s, CH), 6.66 (2H, d, J = 8.0 Hz, H-2, H-6), 7.03 (2H, d, J = 8.0 Hz, H-3, H-5). ¹³C-NMR (DMSO-d ₆ , 125 MHz): δ 48.6 (C-7), 55.9 (C-8), 127.2 (C-1), 115.1 (C-3, C-5), 130.2 (C-2, C-6), 155.5 (C-4), 177.2 (C=O).

Compound 6

[α] ²⁷_D -60 (c 0.075, MeOH); EI-MS: m/z (rel. int.) 181 (9.7), 151 (8.3), 140 (14.5), 112 (25.4), 107 (100), 91 (5.3), 77 (6.8); ¹H-NMR (DMSO-d ₆ , 500 MHz): δ 3.02 (2H, br d, CH₂), 3.95 (1H, br s, CH), 7.04 (2H, d, J = 8.5 Hz, H-2, H-6), 8.04 (2H, d, J = 8.5 Hz, H-3, H-5); ¹³C-NMR (DMSO-d ₆ , 125 MHz): δ 48.6 (C-7), 53.4 (C-8), 60.3 (C-13), 69.1 (C-12), 74.1 (C-14), 84.3 (C-11), 115.4 (C-3, C-5), 124.6 (C-1), 130.5 (C-2, C-6), 156.6 (C-4), 170.5 (C-10), 175.9 (C-9).

Results and discussion

A chromatographic separation of the MeOH extract obtained six compounds (Fig. 1). The known compounds, 2,4-pyrimidinedione (1) [5], N-methylhydroxylamine (2) [6], uridine (4) [5], l-tyrosine (5) [7], and l-prolyl-l-tyrosine (6) [8], were identified by comparison with spectroscopic data from the studies.

Fig. 1

Structures of compounds 1–6

Compound 3 was obtained as a white amorphous powder. A molecular ion peak was measured at m/z 135 [M]⁺ in the EI-MS, which corresponds to a molecular formula of C₅H₅N₅ by HREI-MS (m/z 135.0546 [M]^+, calcd. for 135.0545). ¹H-NMR spectra of 3 showed a methine proton at δ 3.16 and the presence of two aromatic protons at δ 8.45 and 8.46. The ¹³C-NMR spectrum indicated 5 carbon resonances. The ¹³C-NMR spectrum showed a methine carbon at δ 48.5 and two olefin quaternary and two olefin methane carbons at δ 142.7, 146.1, 149.3 and 151.4. The structure of 3 was similar to that of adenine [9]. Thus, the structure of 3 was assigned as 5H-purin-6-amine via spectroscopic analysis.

Six compounds were isolated for the first time from S. sarmentosum leaves. According to previous studies, compound 3 (5H-purin-6-amine) was isolated for the first time from a natural source.