Trogopterins A–C: Three new neolignans from feces of Trogopterus xanthipes

Summary Seven compounds, including three neolignans 1–3, a norlignan 4, and three diterpenoids 5–7, were isolated from the feces of Trogopterus xanthipes. Structures of these compounds were identified by 1D and 2D NMR as well as MS. The absolute configurations of compounds 1, 2, and 4 were determined by comparing CD spectra and optical rotations. Among the isolated compounds, 1–3 were novel and subsequently named trogopterins A, B, and C, respectively. Likewise, compound 4 was isolated from nature for the first time. Cytotoxic activities of compounds 1–4 were evaluated. Compounds 1–3 exhibited moderate cytotoxic activities against HL-60 cells with IC50 values of 34.77–45.68 μM.


Introduction
Chemical studies of natural products including ones derived from plants and microorganisms have led to the isolation of numerous novel metabolites with biological activities [1,2]. As a continuation of these investigations, feces from Trogopterus were selected since organic extracts from this material were found to exhibit potent cytotoxic activities in our preliminary study. Trogopterus feces, called "Wulingzhi", are dried excre-ments of Trogopterus (T.) xanthipes Milne-Edwars (Petauristidae) that are known as complex-toothed flying squirrels to eat branches, leaves, and fruits of pine trees [3]. Trogopterus feces have been reported to promote blood circulation and resolve stasis. Thus, this material has been used as traditional medicine for treating amenorrhea, dysmenorrheal, menstrual pain, and retained lochia due to stasis [4]. Recent studies have indicated that Trogopterus feces mainly consist of terpenoids [5][6][7][8][9], phenolic acids, sterols, aliphatics, fatty acids, and flavonoids [10,11]. They have been reported to possess various pharmacological properties such as controlling of antithrombin levels, inhibition of platelet aggregation, cytotoxic activity, immunity enhancement, and anti-inflammatory activities. Isolation of compounds from the methanol extract of Trogopterus feces was presented before by our group [12]. In the present investigation, chemical evaluation of fecal extracts led to the isolation of seven compounds including three new neolignans named trogopterins A-C (1)(2)(3). Here we describe the isolation, structure, and cytotoxic activities of compounds 1-3.
This compound was also presumed to have three rings in its structure based on the unsaturation degree since it contained two aromatic moieties and an ester group. 1 H, 1 H COSY spectrum of compound 1 revealed the presence of two isolated proton spin systems of CH-CH-CH 2 corresponding to C7′-C8′-C9′ and CH 2 -CH 2 corresponding to C7-C8. In addition, coupling constants as well as the 1 H, 1 H COSY data indicated the presence of a 1,3,4-trisubstituted benzene moiety at δ 6.82 (br d, J = 8.4 Hz, H-6′), 6.74 (d, J = 8.4 Hz, H-5′), and 6.95 (br s, H-2′). The extension of the spin systems and attachments of functional groups were confirmed by HMBC correlations ( Figure 2).
HMBC correlations of a methoxy proton at δ H 3.63 and H 2 -7 at δ H 2.78 (t, J = 7.8 Hz) with the ester carbonyl carbon at δ C 175.3 indicated the presence of a methyl propanoate. HMBC correlations of H-2-H-7 with C-1 at δ C 135.2, C-2 at δ C 116.5, and C-6 at δ C 116.9 demonstrated that the methyl propanoate was attached to the C-1 position of one phenylpropanoid moiety in the lignan skeleton. Together with HMBC correlations, two singlet aromatic protons for H-2 and H-6 indicated that the first phenylpropanoid moiety had a 1,3,4,5-tetrasubstituted benzene ring, which was attached to the C-1 position of the methyl propanoate. H-7' at δ H 5.47 (d, J = 6.0 Hz) of the CH-CH-CH 2 spin system had HMBC correlations with C-1', C-2', and C-6' of the 1,3,4-trisubstituted benzene ring, indicating that the CH-CH-CH 2 spin system was linked to C-1' of the 1,3,4-trisubstituted benzene ring. In addition, HMBC correlations of H-8' with C-3 and C-4 suggested that C-8' was linked to the C-3 position of the first phenylpropanoid moiety. HMBC correlations of H-7' with C-4 allowed linkage of the oxygenated sp 3 methine carbon C-7' to the oxygenated sp 2 carbon C-4 through oxygen to form a dihydrofuran ring. The presence of a dihydrofuran moiety in compound 1 was also demonstrated by chemical shifts of C-4 (δ 146.9) and C-7' (δ 88.8) together with the unsaturation requirement. Thus, compound 1 was proved to be a neolignan containing a dihydrobenzofuran skeleton. HMBC correlations of the methoxy proton at δ H 3.80 with C-4' (δ 149.1) showed that the methoxy group was attached to C-4' of the 1,3,4-trisubstituted benzene ring, and a hydroxy group was presumed to be attached to C-3' based on the chemical shifts. Ultimately, the chemical structure of compound 1 was identified as shown in Figure 1 and named trogopterin A. Detailed 1 H and 13 C NMR data are presented in Table 1 and Table 2.
The relative stereochemistry of compound 1 was established by interpreting the NOESY data. A strong NOESY correlation  between H-7' and H-9' indicated that the 1,3,4-trisubstututed benzene ring and hydroxymethylene group were on opposite faces of the molecule. The absolute configurations of C-7' and C-8' in compound 1 were established by comparing the circular dichroism (CD) data to those of previously reported neoligans containing a dihydrobenzofuran skeleton [17]. The CD of compound 1 showed positive cotton effects at 255 and 327 nm along with a negative cotton effect at 234 nm. These features were very similar to those of neolignans [17], indicating that C-7' and C-8' in compound 1 have S and R configurations, respectively. Thus, compound 1 was determined to be methyl 3-((2S,3R)-7-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydrobenzofuran-5-yl)propanoate.
Compound 2 was isolated as a light brown oil. The molecular formula of compound 2 was determined to be C 19 Figure 2). HMBC correlations of the methine proton H′-8 in the CH 2 -CH-CH 2 spin system with C-2 at δ C 120.2, C-3 at δ C 146.4, C-4 at δ C 114.8, and C-1′ at δ C 143.7 suggested that C-8′ of the second phenylpropanoid was attached to the C-3 position of the first phenylpropanoid moiety. In addition, HMBC correlation of H-7′ with C-2′ together with the coupling constant of H-2′ (1.8 Hz) supported that the second phenylpropanoid moiety has a 1,3-disubstituted benzene ring. Based on these findings, compound 2 was identified as methyl (S)-methyl 3-(3-hydroxy-5-(1-hydroxy-3-(3-hydroxyphenyl)propan-2-yl)phenyl)propanoate and named trogopterin B. Compound 2 was different in that it did not contain a methoxy group at the C-4′ position nor an ether linkage between C-4 and C-7′ to form a dihydrofuran ring. The absolute configuration of compound 2 was determined by comparing its optical rotation with those of (R)-2,3-diphenyl-1-propanol and (S)-2,3-diphenyl-1-propanol [18,19]. Compound 2 had a positive optical rotation value of [a] D 25 +20.2, which was similar to that of (S)-2,3diphenyl-1-propanol (+107°). Thus, C-8′ was determined to have S configuration.
The relative stereochemistry of compound 3 was deduced based on NOESY data and comparison with interproton distances calculated by MM2 with ChemDraw. A strong NOESY correla-tion between H-7′ and H-8′ indicated that the phenyl group at C-7′ and the hydroxymethylene group at C-8′ are on the same face of the molecule (Figure 3). Additionally, a strong NOESY correlation between H-8 and H-8′ indicated that the hydroxymethylene group at C-8 and the hydroxymethylene group at C-8′ are on the same face of the molecule (Figure 3). A structure model of this compound was created using MM2 with ChemDraw 3.0 ( Figure 3). The resulting calculated interproton distances were in close agreement with the NOESY data (Table 3). A careful modeling study considering all the possible conformers of compound 3 exhibited that interproton distances between H-7, H-8, and H-8′ are within 3 Å when two hydroxymethylene groups and a hydroxyphenyl group in compound 3 are on the same face of the molecule. Thus, the structure of compound 3 was determined to be ((1RS,2RS,3RS)-6-hydroxy-2-(3-hydroxyphenyl)-1,2,3,4-tetrahydronaphthalene-1,3diyl)dimethanol and named trogopterin C.
The in vitro cytotoxic activities of compounds 1-4 against HL-60 (human leukemia), HeLa (human cervical carcinoma), and MCF-7 (human breast cancer) cells were evaluated using an MTT assay. As shown in Table 4

Conclusion
In summary, two novel neoligans (trogopterins A and B) and a new phenolic compound (trogopterin C) were isolated from the crude methanol extract of Trogopterus feces for the first time.
The absolute configurations of compounds 1, 2, and 4 were determined by comparing CD spectra and optical rotations. The levels of cytotoxicity against three tumor cell lines (HL-60, HeLa, and MCF-7) were evaluated. Compounds 1-3 had moderate cytotoxic effects against HL-60 cells even though the activities were not significant. Thus, Trogopterus feces could be a potential source of lignans with cytotoxic activity.

Experimental General Methods
Optical rotations were measured with a Jasco P1000 digital polarimeter. UV spectra were recorded by a Hewlett Packard 8453 UV-vis spectrometer. CD spectra were obtained with a Jasco J-715 circular dichroism spectrophotometer. 1D and 2D NMR were performed on a VNS 600 MHz spectrometer operating at 600 MHz for protons and 150 MHz for carbon. Chemical shifts are expressed in ppm and referenced relative to the residual solvent signals. Mass spectra were recorded with a Micromass LCT mass spectrometer, and the lock mass calibration was applied to accurately measure masses. Semi-preparative HPLC was performed with an Agilent system consisting of a vacuum degasser, quaternary pump, diode array detector (DAD), and Luna 5u C 18 (2)

Extraction and isolation
The air-dried Trogopterus feces (1 kg) were subjected to extraction three times (3 h per cycle) with refluxing methanol. The solvent was evaporated under reduced pressure to recover the methanolic extracts (98 g) that were partitioned successively between H 2 O and CHCl 3 and EtOAc. The EtOAc extract (6.5 g) was subjected to silica gel CC (5 × 60 cm column) using a gradient of methylene chloride (MC) and acetonitrile (ACN) as eluents to acquire 27 fractions (Fr.

Assessment of cytotoxicity
Different types of cancer cells (HL-60, HeLa, and MCF-7) were maintained in RPMI 1640 medium supplemented with L-glutamine, 10% fetal bovine serum (FBS), and 2% penicillin-streptomycin. The cells were cultured at 37 °C in a 5% CO 2 incubator. Cytotoxic activity was measured using a modified MTT assay [20]. Viable cells were seeded with the growth medium (100 µL) in 96-well microtiter plates (1 × 10 4 cells per well) and incubated at 37 °C in a 5% CO 2 incubator. The test sample was dissolved in DMSO for the final sample concentrations to be adjusted from 5.0 to 150 µM by diluting with the growth medium. Each sample was prepared in triplicate. The final DMSO concentration was adjusted to be below 0.1%. After standing for 24 h, 10 µL of the test sample was added to each well. The same volume of DMSO alone was added to the control wells. The medium was removed after 48 h of incubation with the test samples, and 10 µL of MTT were then added to the each well (final concentration, 5 mg/mL). After an additional 4 h of incubation, the resulting formazan crystals were dissolved in 150 mL of DMSO and the optical density (O.D.) was measured at 570 nm. The IC 50 value was defined as the concentration of sample that reduced absorbance by 50% relative to the vehicle-treated control. Adriamycin was used as a positive control.

Supporting Information
Supporting Information File 1 NMR and MS spectra of compounds.