Lasianosides F–I: A New Iridoid and Three New Bis-Iridoid Glycosides from the Leaves of Lasianthus verticillatus (Lour.) Merr.

A series of iridoid glycosides were isolated from the leaves of Lasianthus verticillatus (Lour.) Merr., belonging to family Rubiaceae. A new iridoid glycoside, lasianoside F (1), and three new bis-iridoid glycosides, lasianosides G–I (2–4), together with four known compounds (5–8) were isolated. The structures were established by spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, HMBC, and NOESY) in combination with HR-ESI-MS and CD spectra.


Introduction
Rubiaceae is the fourth-largest angiosperm family, comprising approximately 660 genera and 11,500 species and classified into 42 tribes [1]. Rubiaceae has a long history of investigation on the distribution of iridoid glycoside through its species. These investigations were started by isolation of asperuloside from six plants belonging to the family Rubiaceae, as a characteristic iridoid for this genus [2]. The classification of the occurrence of iridoid glucoside in Rubiaceae subfamilies was initiated by Kooiman 1969 [3]. Later, this classification was approved by investigation of 35 selected Rubiaceae plants by TLC, GC, and GC-MS; the result revealed that asperuloside and deacetylasperulosidic acid occur in most plants of the Rubioideae subfamily, especially in Lasianthus species [4]. Previous phytochemical studies on some Lasianthus species revealed the presence of iridoids, iridoid glycosides, anthraquinones, and terpenes [5][6][7][8][9][10]. In our previous study, we isolated a bis-iridoid glycoside from L. wallichii for the first time [11] and five undescribed iridane type glycosides, lasianosides A-E, from L. verticillatus [12]. These results indicated that the genus Lasianthus is a promising rich source in secondary metabolites; however, only limited numbers of Lasianthus species have been investigated until now. To continue research of this genus, we performed further phytochemical investigation of the leaves of L. verticillatus. As a result, a new iridoid glycoside, lasianoside F (1), and three new bis-iridoid isolated in this study. The chemical structures were determined by spectroscopic ( Figures S1-S41) and chemical analyses, as shown in Figure 1.

Chemical Structure of Compound 1
Compound (1) was obtained as a colorless amorphous powder with a specific optical rotation of [α] 22 D − 65.5. The molecular formula was deduced to be C21H28O11 from HR-ESI-MS (m/z 479.1521 [M + Na] + , calcd for C21H28O11Na, 479.1524), which suggested eight degrees of unsaturation. The UV spectrum showed absorption maxima at 234 nm, indicating the presence of an enone system, and IR absorption bands at 3406, 1733, 1658, and 1634 cm −1 that corresponded to hydroxy, carbonyl, and olefinic groups. The 1 H-NMR spectrum of 1 (Table 1) showed one oxymethylene at δH 4.69 and 4.81 ppm, two olefinic protons; one at δH 7.32 ppm assigned to conjugated enol ether and the other at δH 5.75 ppm, two methines at δH 3.70 and 3.31 ppm, two oxymethines at δH 5.59 and δH 5.98 ppm, one anomeric proton at δH 4.70, together with signals of isovaleroyl unit (one methylene at δH 2.27ppm,

Chemical Structure of Compound 2
Compound (2) was isolated as a colorless amorphous powder with a specific optical rotation of [α] 22 D − 55.0. Its molecular formula C36H44O22, from its HR-ESI-MS (m/z 851.2214 [M + Na] + (calcd for C36H44O22Na 851.2216), indicating 15 degrees of unsaturation. The UV spectra of 2 exhibited absorption maxima at 236 nm, characteristic of an enol ether system. Similarly, IR spectra displayed absorption bands corresponding to hydroxy, carbonyl, and olefinic groups at 3309, 1736, 1541 cm −1 , respectively. Duplication of the signals in both 1 H and 13 C-NMR spectra ( Table 2 and (Table 2). Furthermore, two sp 2 methine proton signals at δH 7.15 (1H, d, J = 1.9 Hz) and 7.70 (1H, d, J = 1.1 Hz), which are characteristic for C-3 protons confirmed the presence of two iridoid moieties having an enol ether function. Consistent with these observations, the 13

Chemical Structure of Compound 2
Compound (2) was isolated as a colorless amorphous powder with a specific optical rotation of [α] 22 D − 55.0. Its molecular formula C36H44O22, from its HR-ESI-MS (m/z 851.2214 [M + Na] + (calcd for C36H44O22Na 851.2216), indicating 15 degrees of unsaturation. The UV spectra of 2 exhibited absorption maxima at 236 nm, characteristic of an enol ether system. Similarly, IR spectra displayed absorption bands corresponding to hydroxy, carbonyl, and olefinic groups at 3309, 1736, 1541 cm −1 , respectively. Duplication of the signals in both 1 H and 13 C-NMR spectra ( Table 2 and (Table 2). Furthermore, two sp 2 methine proton signals at δH 7.15 (1H, d, J = 1.9 Hz) and 7.70 (1H, d, J = 1.1 Hz), which are characteristic for C-3 protons confirmed the presence of two iridoid moieties having an enol ether function. Consistent with these observations, the 13

Chemical Structure of Compound 2
Compound (2) (Table 2). Furthermore, two sp 2 methine proton signals at δ H 7.15 (1H, d, J = 1.9 Hz) and 7.70 (1H, d, J = 1.1 Hz), which are characteristic for C-3 protons confirmed the presence of two iridoid moieties having an enol ether function. Consistent with these observations, the 13 C-NMR spectrum showed 36 signals comprising four carbonyl carbons (δc 167. 6, 172.2, 172.2 and 172.7), eight olefinic carbons (δc 106. 3, 107.7, 129.1, 131.6, 143.9, 146.5, 150.1 and 156.3), six oxygenated carbons (four methines δc 75. 2, 86.1, 94.1 and 101.8; two methylenes δc 61.9 and 63.8), four sp 3 methine carbons (δc 37. 6, 42.9, 45.1 and 45.9) together with two anomeric carbons (δc 98.7, 100.9) and oxygenated carbons arising in the region of δc 62.7-78.6 belonging to two glucose moieties (Table 3). Thus, the two partial structures of 2 were referred to as units "A" and "B" and determined to be asperuloside (5) and asperulosidic acid [5,13], respectively. The 1D and 2D-NMR data of compound 2 were very similar to those of the bis-iridoid glucoside (8) that reported in [11]. The only evident difference was observed in the glycosyl part of unit "A", i.e., the lower field shifted H-2 at δ H 4.80, and upper field shifted H 2 -6 , δ H 3.69 and 3.94 ppm, indicating that 2 was a positional isomer of 8, and the attachment site between "A" and "B" units was deduced to be at C-2 of unit "A" via an ester linkage. This assumption was further verified by a correlation from H-2 (δ H 4.80) of unit "A" to C-11 (δc 167.6) of unit "B" in the HMBC spectrum ( Figure 2). Moreover, acid hydrolysis of 2 gave D-glucose, which was identified by HPLC analysis with a chiral detector, while β-anomeric configurations were established from the coupling constant of anomeric protons, 8.2 and 7.8 Hz. The relative and absolute configurations of aglycone parts of 2 were determined to be identical to 5 by comparison of their chemical shift values, coupling constants, NOESY experiment (Figure 3), and CD data. Therefore, the structure of 2 was characterized as shown in Figure 1, named lasianoside G.   (11.9, 1.9) 3.63 dd (11.9, 5.8) 3.87 dd (11.9, 1.7) m: multiplet or overlapped signals.  (Tables 2 and 3) showed that the structure of 3 was similar to that of 8. The significant change occurred in glucose moiety of unit "A", i.e., the chemical shift of H-3 moved to downfield at δ H 5.08 ppm, and the chemical shift of H 2 -6 moved to upfield at δ H 3.74 and 3.95 ppm, which suggested that the position of esterification between unit "A" and "B" was changed from H-6 to H-3 . This suggestion was supported by a correlation between the H-3 (δ H 5.08) of unit "A" and C-11(δc 168.6) of unit "B" in HMBC spectrum ( Figure 2). The structure of this compound was verified by further analysis of 2D-NMR data, including COSY, HSQC, and HMBC spectra. The relative and absolute configurations of aglycone parts of 3 were identical to those of 2 by comparison of their chemical shift values, coupling constants, NOESY experiment (Figure 3), and CD analysis. From these data, the structure of 3 was characterized as shown in Figure 1, designated as lasianoside H.

Chemical Structure of Compound 4
Compound (4)  . The 13 C-NMR data showed signals resembling those of 8, except the presence of two sp 3 methines, C-4 at δc 44.4 and C-3 at 97.4 ppm in unit "B" of 4, instead of resonances of two olefinic carbons at the same position of 8, in addition to lower field shift of C-11 and C-6 to δc 176.9 and 87.9 ppm, respectively (Table 4). This change coincided with the disappearance of an enol ether proton signal and the appearance of methine proton at δ H 3.36 ppm together with oxymethine proton at δ H 5.27 ppm that correspond to H-4 and H-3 of unit "B", respectively ( Table 4). The above data suggested the absence of a double bond between C-3 and C-4 and the presence of γ-lactone ring in the aglycone part of unit "B". The occurrence of γ-lactone was confirmed by HMBC correlation from H-6 (δ H 5.41) to C-11 (δc 176.9) (Figure 2). A detailed analysis of NMR data (COSY, HSQC, and HMBC) suggested two partial structures in 4, i.e., asperuloside (5) [13] and 3,4-dihydro-3-oxy asperuloside [15]. The attachment between "A" and "B" units was found to be between C-6 of unit "A" and C-3 of unit "B" via O-linkage due to a long-range correlation between H 2 -6 of unit "A" (δ H 3.95 and 4.18) and C-3 of unit "B" (δc 97.4) in the HMBC spectrum ( Figure 2). HPLC analysis after acid hydrolysis of 4 revealed that the glycosyl units were D-configurations. The relative and absolute configurations of unit "A" were the same as 5 by comparison of NOESY, chemical shifts, and coupling constants. On the other hand, the stereochemistry of part "B" was achieved by NOESY analysis, particularly for those of chiral centers H-4, H-5, H-6, and H-9. In the NOESY spectrum, the correlations between H-5/H-4, H-6, and H-9, indicated β-orientation of H-4, H-5, H-6, and H-9 ( Figure 3). The stereochemistry of C-3 in unit "B" was also determined as Figure 1, because of the chemical shift similarity with 3,4-dihydro-3-methoxy asperuloside [15], coupling constants, and the absence of NOE correlation between H-3/H-4,5,9. The CD spectrum showed essentially the same cotton effect as asperuloside (5). Base on the above findings, the structure of 4 was assigned as shown in Figure 1, named lasianoside I.  (11.7, 1.4) 4.18 dd (11.7, 5.0) 62. 8 3.67 dd (11.7, 3.6) 3.88 br d (11.7) m: multiplet or overlapped signals.

Plant Material
Leaves of L. verticillatus were collected in 2000 from Iriomote Island, Okinawa Prefecture, Japan. A voucher specimen of the plant was deposited in the herbarium of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Hiroshima University (IR0009-LT).