Five new 2-(2-phenylethyl)chromone derivatives and three new sesquiterpenoids from the heartwood of Aquilaria sinensis,an aromatic medicine in China

Lu Zhang, Ping Yi, Hui Yan, Xiao-Nian Li, Meng-Yuan Xia, Jun Yang, Ji-Feng Luo, Yue-Qiu He and Yue-Hu Wang*

Abstract Five new 2-(2-phenylethyl)chromone derivatives, (5S,6R,7R,8S,7′R)-7′-hydroxyagarotetrol (1), (5S,6R,7R,8S,7′S)-7′-hydroxyagarotetrol (2), (6S,7S,8R)-2-[2-(4-methoxyphenyl)ethyl]-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone (3),(6S,7S,8R)-2-(2-phenylethyl)-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone (4), (5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)-7-methoxychromonyl-6-oxy]chromone (5), three new sesquiterpenoids,(4S,5S,7S,8S,10S,13R)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (6), (4S,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (7), and (4R,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (8), along with 14 known compounds were isolated from the resinous heartwood of Aquilaria sinensis (Thymelaeaceae).The chemical structures of these new compounds were elucidated by 1D and 2D NMR and MS data, single-crystal X-ray diffraction analysis,and electronic circular dichroism (ECD) calculations.The neuroprotective activities of these isolates were evaluated using an in vitro model of rat adrenal pheochromocytoma (PC12) cell injury induced by corticosterone.At concentrations from 5 to 40 μM, compounds 4 and 6, agarotetrol (9), and 6-hydroxy-2-(2-phenylethyl)chromone (17) showed significant protective activities against corticosterone-induced PC12 cell injury (P ＜ 0.001).

Keywords: Thymelaeaceae, Aquilaria sinensis, Sesquiterpenoids, 2-(2-Phenylethyl)chromones, Neuroprotective

1 Introduction

Chen-xiang (Aquilariae Lignum Resinatum), resinous heartwoods of the Thymelaeaceous plantAquilaria sinensis(Lour.) Spreng., is one of the most well-known aromatic medicines in China [1, 2].More than 240 compounds, mainly sesquiterpenoids, diterpenoids,steroids, benzyl acetones, chromones, phenolic acids,and aliphatic compounds, have been found in chenxiang.Some compounds showed antibacterial, anticancer, acetylcholinesterase inhibitory, and other pharmacological activities [3].Aromatic plants are thought to be a source of chemical constituents with neuroprotective effects [4].In our continuing efforts to search for neuroprotective compounds from chenxiang [5, 6], five new 2-(2-phenylethyl)chromone derivatives (1-5, Fig.1) and three new sesquiterpenoids(6-8, Fig.1), along with 14 known compounds (9-22,Additional file 1: Fig.S1), were isolated.In the present paper, structural elucidation of these new compounds and bioassay results for the neuroprotective activity of these isolates are reported.

Fig.1 Chemical structures of compounds 1-8 from Aquilaria sinensis

2 Results and discussion

2.1 Structure elucidation

Compound 1 was obtained as colorless needles (MeOH).Based on the HRESIMS atm/z357.0942 [M + Na]+(calcd for C17H18NaO7, 357.0950) and13C NMR data(Table 1), its molecular formula was deduced to be C17H18O7with nine indices of hydrogen deficiency.Its IR spectrum indicated the presence of hydroxy groups(3406 cm-1), anα,β-unsaturated carbonyl (1658 cm-1),and a monosubstituted phenyl ring (1601, 1448, and 701 cm-1).According to the1H NMR data of compound 1 (Table 1), a trisubstituted double bond [δH6.19 (1H,s)] and a monosubstituted phenyl ring [δH7.26-7.41(5H, m)] were deduced.Its NMR data (Table 1) were very similar to those of agarotetrol (9) [7, 8], a common 2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone found inAquilariaplants.The main difference was that signals for a methylene in agarotetrol were replaced by signals[δH5.10 (dd,J= 8.0, 5.8 Hz);δC72.4] for an oxygenated methine in compound 1.

Table 1 1H (500 MHz) and 13C (126 MHz) NMR data of 1 and 2 in methanol-d4 (δ in ppm, J in Hz)

Based on the1H-1H COSY correlations of 1 (Fig.2),fragments of C-5 to C-8, C-2′ to C-6′, and C-7′ to C-8′were deduced.HMBC correlations (Fig.2) fromδH7.41(H-2′, H-6′) toδC72.3 (C-7′) and fromδH2.97 (H-8′β)toδC144.8 (C-1′) indicated the presence of a 2-hydroxy-2-phenylethyl fragment in 1.HMBC correlations fromδH2.97 (H-8′β) toδC115.5 (C-3), fromδH6.19 (H-3) toδC44.4 (C-8′), and fromδH5.11 (H-7′) toδC169.1 (C-2)implied that the 2-hydroxy-2-phenylethyl fragment was located at C-2.HMBC correlations fromδH4.75 (H-5) toδC181.9 (C-4) andδC165.4 (C-8a), fromδH4.56 (H-8)toδC121.8 (C-4a), and fromδH6.19 (H-3) toδC121.8(C-4a), a planar structure 2-(2-phenylethyl)-5,6,7,8,7′-pe ntahydroxy-5,6,7,8-tetrahydrochromone was deduced.

Fig.2 Key 2D NMR correlations of compounds 1-8

It is difficult to determine the relative configuration of 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromones by ROESY correlations, while the coupling constants of H-5 to H-8 are helpful.J5,6(4.0 Hz),J6,7(2.4 Hz), andJ7,8(7.5 Hz) values of compound 1 were close to those of 5α,6β,7β,8α-tetrahydroxy-5,6,7,8-tetrahydrochromone analogs [9], implying the 5α,6β,7β,8α-tetrahydroxy configuration in 1.The relative configuration of H-7′ was also determined by comparing its coupling constant with that of known compounds.In isomers (5S,6R,7S,8R,7′R)-7′-hydroxyisoagarotetrol (15) [10] and (5S,6R,7S,8R,7′S)-7′-hydroxyisoagarotetrol (16) [10], the main differences in1H NMR spectra wereJ7′,8′values.BecauseJ7′,8′α(8.0 Hz)andJ7′,8′β(5.8 Hz) values of compound 1 were close to those of the 7′α-OH isomer (15;J7′,8′α= 8.0 Hz andJ7′,8′β= 5.5 Hz), 7′-OH of 1 was deduced to beα-oriented.The absolute configuration of 1 was determined to be(5S,6R,7R,8S,7′R)-7′-hydroxyagarotetrol (Fig.3) by singlecrystal X-ray diffraction using graphite monochromated CuKαradiation with a Flack parameter of 0.11 (10).

The molecular formula of 2 was deduced to be the same as that of compound 1, C17H18O7, by13C NMR (Table 1) and HRESIMS atm/z357.0945[M + Na]+(calcd for C17H18NaO7, 357.0950).Detailed comparison of its NMR data with those of (5S,6R,7S,8R,7′R)-7′-hydroxyisoagarotetrol (1),(5S,6R,7S,8R,7′R)-7′-hydroxyisoagarotetrol (15), and(5S,6R,7S,8R,7′S)-7′-hydroxyisoagarotetrol (16) [10],compound 2 was elucidated to be 7′-epimer of 1, which was confirmed by 2D NMR correlations of 2 (Fig.2).BecauseJ7′,8′α(5.4 Hz) andJ7′,8′β(8.0 Hz) values of compound 2 were close to those of the 7′β-OH isomer (16;J7′,8′α= 5.0 Hz andJ7′,8′β= 8.5 Hz) [10], the 7′-OH of 2 was deduced to beβ-oriented.Thus, compound 2 was elucidated to be (5S,6R,7S,8R,7′S)-7′-hydroxyisoagarotetrol.

Compound 3 had the molecular formula C18H20O6based on13C NMR data (Table 2) and the positive ion atm/z355.1150 [M + Na]+(calcd for C18H20NaO6,355.1158) in the HRESIMS.The1H and13C NMR spectra showed resonances for onep-disubstituted phenyl ring [δH7.12 (2H, br d,J= 8.6 Hz) and 6.82 (2H, br d,J= 8.6 Hz);δC159.8, 133.2, 130.4 × 2, and 115.0 × 2],one trisubstituted 4H-pyran-4-one [δH6.07 (s);δC182.0,171.2, 163.0, 120.9, and 113.1], one methoxy group [δH3.75 (3H, s);δC55.6], threesp3methylenes (δC36.6,33.0, and 26.5), and three oxygenated methine [δH4.50(d,J= 5.0 Hz), 4.11 (ddd,J= 7.4, 5.1, 2.2 Hz), and 3.90(dd,J= 5.0, 2.2 Hz);δC75.0, 71.2, and 67.3], implying that it might also be a 5,6,7,8-tetrahydrochromone with the substituted mode of three hydroxy groups at ring B rather than the usual mode of four hydroxy groups at ring B.Four fragments, C-5 to C-8, C-2′ to C-3′,C-5′ to C-6′, and C-7′ to C-8′, were deduced by1H-1H COSY correlations (Fig.2).Its planar structure was deduced to be 2-[2-(4-methoxyphenyl)ethyl]-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone by key HMBC correlations (Fig.2) from H-3 to C-4a and C-8′, from H-5 to C-4 and C-8a, from H-6 and H-8 to C-4a, from H-7 to C-8a, from H-2′ and H-6′ to C-7′, H-3′ and H-5′ to C-1′,4′-OMe to C-4′, H2-7′ to C-2, C-2′, and C-6′, and H2-8′to C-1′ and C-3.Finally, the absolute configuration of 3 was determined to be (6S,7S,8R)-2-[2-(4-methoxyphenyl)ethyl]-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone(Fig.3) by single-crystal X-ray diffraction using graphite monochromated CuKαradiation with a Flack parameter of 0.09 (4).

Fig.3 X-ray crystallographic structures of 1 and 3

Compound 4 was assigned the molecular formula C17H18O5based on13C NMR data (Table 2) and the positive ion atm/z325.1046 [M + Na]+(calcd for C17H18NaO5, 325.1052) in the HRESIMS.By extensively comparing the NMR data (Table 2) of compounds 3 and 4, signals for one monosubstituted phenyl ring were found in 4 rather than the disubstituted phenyl ring in 3, and signals for a methoxy group disappeared in 4.Otherwise, the NMR data of these two compounds were very close to each other, implying that compound 4 is the 4′-demethoxy derivative of 3, which was confirmed by the1H-1H COSY and HMBC correlations of 4 (Fig.2).The chemical shifts and coupling constants of H-6 [δH4.11 (ddd,J= 7.5, 5.0, 2.1 Hz)],H-7 [δH3.90 (dd,J= 5.0, 2.1 Hz)], and H-8 [δH4.50 (d,J= 5.0 Hz)] of compound 4 were very close to those of H-6 [δH4.11 (ddd,J= 7.4, 5.1, 2.2 Hz)], H-7 [δH3.90(dd,J= 5.0, 2.2 Hz)], and H-8 [δH4.50 (d,J= 5.0 Hz)]of compound 3, implying 6α-OH, 7α-OH, and 8β-OH configurations in 4.The absolute configuration of 4 wassuggested to be (6S,7S,8R)-2-(2-phenylethyl)-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone in view of its ECD spectrum similar to that of compound 3 (Fig.4).

Table 2 1H and 13C NMR data of 3 and 4 in methanol-d4 (δ in ppm, J in Hz)

Fig.4 ECD spectra of compounds 3-5 and 20

A molecular formula, C35H32O9, was assigned to compound 5 by positive HRESIMS with an ion atm/z619.1939 [M + Na]+(calcd C35H32NaO9, 619.1944) and13C NMR data (Table 3).According to its NMR data(Table 3), signals for two sets of phenylethyl moieties (δC141.3, 140.9, 129.2 × 2, 129.6 × 2, 129.5 × 4, 127.5, 127.4,37.0, 36.2, 34.1, and 33.4), one 5,6,7,8-tretrahydroxy-5,6,7,8-tetrahydrochromone moiety [δH6.13 (s), 5.45(dd,J= 7.3, 1.3 Hz), 4.75 (dd,J= 6.9, 1.3 Hz), 4.00 (dd,J= 9.6, 7.3 Hz), and 3.83 (dd,J= 9.6, 6.9 Hz);δC182.0,171.2, 160.7, 122.5, 114.4, 79.5, 74.9, 73.6, and 70.3],one trisubstituted chromone [δH7.90 (s), 7.22 (s), and 6.11 (s);δC179.7, 170.9, 157.3, 155.0, 148.8, 117.4, 110.4,110.1, and 101.7], and one methoxy group [δH3.98 (s);δC57.2], were observed, which implied that this compound might be a dimer of a 2-(2-phenylethyl)chromone and a 5,6,7,8-tretrahydroxy-5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone.In unit A of 5 (Fig.2), three1H-1H COSY fragments, H-5/H-6/H-7/H-8, H-2′/H-3′/H-4′/H-5′/C-6′,and H2-7′/H2-8′, along with HMBC correlations from H-3 to C-4a and C-8′, from H-5 and H-7 to C-8a, from H-2′ and H-6′ to C-7′, from H2-7′ to C-2, C-2′, and C-6′,and from H2-8′ to C-1′ and C-3, were observed, which implied the presence of a 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone moiety in 5.In unit B of 5 (Fig.2), two1H-1H COSY fragments, H-2′/H-3′/H-4′/H-5′/C-6′ and H2-7′/H2-8′, along with HMBC correlations from H-3 to C-4a and C-8′, from H-5 to C-4 and C-8a, from 7-OMe to C-7, from H-8 to C-4a and C-6, from H-2′ and H-6′ to C-7′, from H2-7′ to C-2, C-2′,and C-6′, and from H2-8′ to C-1′ and C-3, were observed,which implied the presence of a 6-hydroxy-7-methoxy-2-(2-phenylethyl)chromone moiety in 5.Units A and B were connected through an ether bond by the HMBC correlation from H-8 of unit A to C-6 of unit B (Fig.2).The relative configuration of unit A was deduced to bethe same as that of a structural analog (5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)chromonyl-6-oxy]chromone (20)[11] by comparing the coupling constants of H-5 to H-8 (J5,6= 6.9 Hz,J6.7= 9.6 Hz, andJ7.8= 7.3 Hz) in 5 with those (J5,6= 7.0 Hz,J6.7= 9.8 Hz, andJ7.8= 7.5 Hz)of H-5 to H-8 in the known analog [11].The absolute configuration of 5 was suggested to be (5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)-7-methoxychromonyl-6-oxy]chromone because the ECD spectrum of 5 was similar to that of(5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)chromonyl-6-oxy]chromone (20) (Fig.4).

Table 3 1H (600 MHz) and 13C (151 MHz) NMR data of 5 in methanol-d4 (δ in ppm, J in Hz)

Compound 6 was assigned the molecular formula C15H20O4, as determined by13C NMR data (Table 4)and the positive ion atm/z287.1255 [M + Na]+(calcd for C15H20NaO4, 287.1259) in the HRESIMS.The1H and13C NMR data (Table 4) indicated the presence of oneα,β-unsaturated ketone [δH5.91 (br s);δC214.1,188.3, and 130.4], one exocyclic double bond [δH5.55(br s) and 5.36 (br s);δC156.3 and 112.3], two methyl groups [δH1.29 (s) and 1.14 (d,J= 7.4 Hz);δC25.2 and 15.4], one methylene, four methines including two oxygenated groups [δH4.12 (t,J= 2.2 Hz) and 3.88(dd,J= 10.7, 5.3 Hz);δC75.4 and 71.8], and two quaternary carbon atoms (δC77.3 and 42.8).Its NMR data were similar to those of (4R,5S,7R,8S,10S,13R)-8,13-dihydroxyrotunda-1,11-dien-3-one with a rare tricyclic rotundane skeleton [5].According to the1H-1H COSY correlations (Fig.2), two fragments, C-15-C-4-C-5-C-6 and C-8-C-9, were deduced.According to HMBC correlations (Fig.2) from H-2 to C-4 and C-5, from H3-15 to C-3, C-4, and C-5, from H2-6 to C-1, C-8, and C-11,from H2-12 to C-7 and C-13, and from H3-14 to C-1,C-9, C-10, and C-13, the planar structure of 6 was elucidated to be 7,8,13-trihydroxyrotunda-1,11-dien-3-one.The ROESY correlations (Fig.2) of H3-15/H-5 and H-5/H-9αindicated that these protons should be cofacial; the ROESY correlations of H-9β/H-13 and H-8/H-13 indicated that these protons should also be cofacial.Thus, the relative configuration of 6 was determined, as shown in Fig.2.By comparing its experimental and calculated ECD spectra (Fig.5), the structure of compound 6 was finally elucidated to be (4S,5S,7S,8S,1 0S,13R)-7,8,13-trihydroxyrotunda-1,11-dien-3-one.

Fig.5 Experimental and calculated ECD spectra of compounds 6-8

Compound 7 has the molecular formula C15H20O4according to its13C NMR data (Table 4) and HRESIMS at 287.1253 [M + Na]+(calcd for C15H20NaO4,287.1259).Its13C NMR data exhibited 15 signals for oneα,β-unsaturated ketone functionality (δC214.1,191.5, and 128.5), one exocyclic double bond (δC156.3 and 116.5), two quaternary carbon atoms (δC77.1 and 43.7), one methine, four methylenes including two oxygenated groups (δC78.5 and 71.7), and two methyl groups (δC24.5 and 15.9).The NMR data of compounds6 and 7 were very close to each other, and both of these compounds have the same molecular formula, which implied that compound 7 might also be a rotundanetype sesquiterpenoid.

Table 4 1H and 13C NMR data of 6-8 in methanol-d4 (δ in ppm, J in Hz)

The1H-1H COSY fragments H3-15/H-4/H-5/H2-6 and H-8/H2-9 were determined from the1H-1H COSY correlations of 7 (Fig.2).Based on the HMBC correlations(Fig.2) from H-2 to C-4 and C-5, from H3-15 to C-3, C-4,and C-5, from H2-6 to C-1, C-8, and C-11, from H2-12 to C-7 and C-13, and from H3-14 to C-1, C-9, C-10, and C-13, the planar structure of 7 was elucidated to be the same as that of compound 6, namely, 7,8,13-trihydroxyrotunda-1,11-dien-3-one.BecauseJ8,9α(6.0 Hz) andJ8,9β(10.5 Hz) values in the1H NMR data of compound 7 were similar to those (J8,9α= 5.3 Hz andJ8,9β= 10.8 Hz)of compound 6, H-8 in compound 7 was elucidated to beβ-oriented.Correlations of H3-15/H-5, H-5/H-9α,and H-2/H-13 were observed in the ROESY spectrum(Fig.2), indicating that compound 7 is a C-13 epimer of compound 6.Finally, the absolute configuration of 7 was elucidated to be (4S,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one based on the ECD calculations(Fig.5).

Compound 8 was assigned the molecular formula C15H20O4, the same as that of 6 and 7, by13C NMR data(Table 4) and the ion peak atm/z264.1359 [M]+(calcd for C15H20O4, 264.1362) in the HREIMS.The1H and13C NMR data (Table 4) indicated that this compound might also be a rotundane-type sesquiterpenoid with oneα,βunsaturated ketone functionality (δC214.5, 191.7, and 128.5), one exocyclic double bond (δC156.3 and 116.5),two quaternary carbon atoms (δC77.4 and 43.6), one methine, four methylenes including two oxygenated groups (δC78.6 and 71.8), and two methyl groups (δC24.5 and 10.5).Based on its1H-1H COSY and HMBC correlations (Fig.2), the planar structure of 8, namely, 7,8,13-trihydroxyrotunda-1,11-dien-3-one, was deduced to be the same as that of compounds 6 and 7.The H-4α, H-5α,H-8β, and H-13βconfigurations were determined by the key ROESY correlations of H3-15/H-6α, H3-15/H-6β,H-5/H-9α, and H-2/H-13 (Fig.2) and by comparingJvalues in its1H NMR spectrum with those of compounds 6 and 7.Finally, the absolute configuration of 8 was elucidated to be (4R,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one, a C-4 epimer of 7, based on the ECD calculations (Fig.5).

Table 5 The effects of compounds at a single concentration on PC12 cell injury induced by corticosteronea

NMR data of C-5 to C-8 in agarotetrol (9) were not correctly assigned before [7, 8], which were revised by 2D NMR correlations (Additional file 1: Fig.S2).NMR data of 4′-methoxyagarotetrol (11) in DMSO-d6[12],2′-hydroxyagarotetrol (13) in DMSO-d6[13], (5S,6R,7R,8S)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)chromonyl-6-oxy]chromone (19) in DMSO-d6[14], and (-)-aquisinenone G (21) in CDCl3[15]have been reported in the literature.Their NMR data in methanol-d4are presented in this paper.Other known compounds, isoagarotetrol (10) [8], 4′-methoxyisoagarotetrol(12) [16], (5S,6R,7S,8R,7′R)-7′-hydroxyisoagarotetrol(15) [10], (5S,6R,7S,8R,7′S)-7′-hydroxyisoagarotetrol(16) [10], (5S,6S,7S,8R)-8-chloro-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydrochromone (14) [5, 17],6-hydroxy-2-(2-phenylethyl)chromone (17) [18], 2,2′-di-(2-phenylethyl)-8,6′-dihydroxy-5,5′-bichromone (18) [19],(5R,6R,7R,8S)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)chromonyl-6-oxy]chromone(20) [11], and syringin (22) [20], were identified by comparing their spectroscopic data with those in literature.

Rat adrenal pheochromocytoma (PC12) cell injury induced by corticosterone is an in vitro model for screening neuroprotective and antidepressant compounds [6].All isolates except for compound 3 were evaluated for their protective activities against corticosterone-induced damage in PC12 cells.After testing these compounds at a single concentration of 20 μM (Table 5), several compounds were selected for testing at gradient concentrations of 2.5, 5, 10, 20, and 40 μM.Among them,(6S,7S,8R)-2-(2-phenylethyl)-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone (4), (4S,5S,7S,8S,10S,13R)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (6), agarotetrol (9), and 6-hydroxy-2-(2-phenylethyl)chromone (17) showed the most protective activities against corticosterone-induced PC12 cell injury at concentrations from 5 to 40 μM(P＜ 0.001) (Table 6).Among the chromone derivatives(1-5 and 9-21), the types and positions of substituent groups seem to have effects on the activity, although no obvious patterns of structure-activity relationships (SAR)were observed.Nevertheless, a hydroxy substituent at C-2′ or C-7′ would reduce the activity by comparing the bioassay data of agarotetrol (9) and their derivatives (1, 2,11, and 13) (Tables 5 and 6).

Table 6 The effects of compounds at gradient concentrations on PC12 cell injury induced by corticosteronea

3 Experimental section

3.1 General experimental procedures

The material and instruments used for isolating compounds and measuring spectroscopic data are provided in the Additional file 1.

3.2 Plant material

The plant material was purchased from the Flagship Store of Jiabaohua Pharmacy, Zhuhai, Guangdong, China(order number: 172979790097330735) in June 2018, produced by Guangdong Huiqun Chinese Traditional Medicine Co., Ltd., Shantou, Guangdong, China (lot number:20171101), and identified as the resinous heartwood ofAquilaria sinensis(Lour.) Spreng.by Professor Shu-De Yang at Yunnan University of Traditional Chinese Medicine, China.The voucher specimen (No.GD171101) was kept in the Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences.

3.3 Extraction and isolation

The dried resinous heartwood ofA.sinensis(2.9 kg)was ultrasonically extracted with 90% EtOH (10 L × 5)at 60 °C for half an hour each time.The crude extract(459.3 g) was suspended in 1 L of water, followed byextraction with petroleum ether (1 L × 5), EtOAc (1 L × 5), andn-BuOH (1 L × 5).After removing the solvent, the petroleum ether-soluble fraction (0.7 g), EtOAcsoluble fraction (374.8 g), andn-BuOH-soluble fraction(55.9 g) were obtained.

Then-BuOH-soluble fraction (55.9 g) was separated by a silica gel column with EtOAc/MeOH (100:0 → 0:1,v/v) as the eluent to yield five further fractions (Fr.1 to Fr.5).Fr.1 (786.8 mg) was subjected to a reversed-phase(RP) C18silica gel column (MeOH/H2O, 5% → 100%) to yield 10 further fractions (Fr.1-1 to Fr.1-10).Compound 17 (2.1 mg) is obtained from Fr.1-6 by recrystallization(MeOH).

Fr.2 (16.7 g) was purified by an RP C18column(MeOH/H2O, 5% → 100%) to yield 13 further fractions (Fr.2-1 to Fr.2-13).Fr.2-6 was recrystallized from MeOH to yield 9 (1.1 g).Fr.2-3 (456.6 mg) was applied to a silica gel column via elution by CH2Cl2/MeOH(50:1 → 1:1) to yield five further fractions (Fr.2-3-1 to Fr.2-3-5).Fr.2-3-2 (114.7 mg) was purified by semipreparative HPLC (Welch Ultimate AQ-C18,φ7.8 × 250 mm;MeOH/H2O, 20:80,v= 2 mL/min) to yield 6 (10.6 mg,tR= 15.072 min) and 7 (11.9 mg,tR= 22.831 min).Fr.2-3-5 (58.3 mg) was further purified by semipreparative HPLC (Welch Ultimate AQ-C18,φ7.8 × 250 mm;MeOH/H2O, 40:60,v= 2 mL/min) to yield 1 (14.1 mg,tR= 9.282 min) and 2 (7.1 mg,tR= 10.161 min).Fr.2-3-4(41.3 mg) was separated by a silica gel column (CH2Cl2/MeOH, 30:1 → 1:1) and was then purified by semipreparative HPLC (Welch Ultimate AQ-C18,φ7.8 × 250 mm;MeCN/H2O, 10:90,v= 2 mL/min) to yield 15 (1.5 mg,tR= 22.158 min).Fr.2-8 (524.3 mg) was separated by a silica gel column (CH2Cl2/MeOH, 50:1) to yield 11 further fractions (Fr.2-8-1 to Fr.2-8-11).Fr.2-8-6 was recrystallized from MeOH to yield 12 (204.0 mg).Fr.2-8-9 (51.3 mg) was purified by semipreparative HPLC(Welch Ultimate AQ-C18,φ7.8 × 250 mm; MeOH/H2O,30:70,v= 2 mL/min) to yield 4 (4.2 mg,tR= 37.011 min)and 3 (2.0 mg,tR= 41.898 min).Fr.2-8-10 (49.3 mg)was applied to a silica gel column via elution by CH2Cl2/MeOH (30:1 → 1:1) and was further purified by semipreparative HPLC (Welch Ultimate AQ-C18,φ7.8 × 250 mm; MeOH/H2O, 33:67,v= 2 mL/min) to yield 11 (1.4 mg,tR= 21.190 min).Fr.2-9 (489.4 mg) was applied to a silica gel column and eluted with CH2Cl2/MeOH (100:1 → 1:1) to yield five further fractions (Fr.2-9-1 to Fr.2-9-5).Fr.2-9-2 (118.8 mg) was separated by Sephadex LH-20 (MeOH) and was further purified by semipreparative HPLC (YMC-Pack ODS-A,φ10 × 250 mm; MeOH/H2O, 40:60,v= 2 mL/min) to yield 14 (2.9 mg,tR= 14.865 min).Fr 2-9-4 (46.9 mg) was purified by Sephadex LH-20 (MeOH) to yield 10 (4.0 mg).Fr 2-12 (599.0 mg) was subjected to a silica gel column via elution by CH2Cl2/MeOH (100:1 → 1:1) to yield five further fractions (Fr.2-12-1 to Fr.2-12-5).Fr.2-12-1 was recrystallized from MeOH to yield 18 (1.2 mg).Fr.2-12-2(164.1 mg) was applied to a silica gel column (CH2Cl2/MeOH, 100:1 → 1:1) to yield three further fractions (Fr.2-12-2-1 to Fr.2-12-2-3).Fr.2-12-2-1 (80.0 mg) was purified by semipreparative HPLC (YMC-Pack ODS-A,φ10 × 250 mm; MeCN/H2O, 48:52,v= 2 mL/min) to yield 21 (4.0 mg,tR= 27.886 min).Fr.2-12-2-3 (60.9 mg)was purified by semipreparative HPLC (Welch Ultimate AQ-C18,φ7.8 × 250 mm; MeOH/H2O, 65:35,v= 2 mL/min) to yield 5 (2.3 mg,tR= 26.957 min).Fr.2-12-4(29.0 mg) was separated by Sephadex LH-20 gel column chromatography (MeOH) and then by semipreparative HPLC (Welch Ultimate AQ-C18,φ7.8 × 250 mm;MeOH/H2O, 65:35,v= 2 mL/min) to yield 19 (3.7 mg,tR= 25.515 min) and 20 (3.9 mg,tR= 30.107 min).

Fr.3 (6.0 g) was separated by an RP C18silica gel column (MeOH/H2O, 5% → 100%) to yield 11 further fractions (Fr.3-1 to Fr.3-11).Fr.3-3 (158.0 mg) was separated by a silica gel column (CH2Cl2/MeOH, 50:1)and then purified by semipreparative HPLC (Welch Ultimate AQ-C18,φ7.8 × 250 mm; MeOH/H2O, 30:70,v= 2 mL/min) to yield 8 (0.8 mg,tR= 10.757 min).Fr.3-4(195.5 mg) was separated by a silica gel column (CH2Cl2/MeOH, 50:1) and was then purified by semipreparative HPLC (YMC-Pack ODS-A,φ10 × 250 mm; MeOH/H2O,35:65,v= 2 mL/min) to yield 22 (7.1 mg,tR= 11.198 min)and 16 (0.9 mg,tR= 15.727 min).Fr.3-5 (39.0 mg) was purified by semipreparative HPLC (YMC-Pack ODS-A,φ10 × 250 mm; MeCN/H2O, 20:80,v= 2 mL/min) to yield 13 (7.5 mg,tR= 31.475 min).

3.4 Spectroscopic data of compounds 1-9, 11, 13,and 19-21

3.4.1 (5S,6R,7R,8S,7′P)-7′-hydroxyagarotetrol (1)

Colorless needle crystal (MeOH);- 29.1 (c= 0.13,MeOH); UV (MeOH)λmax(logε) 252 (4.03), 207 (4.17)nm; ECD (c0.013, MeOH)λmax(Δε) 298 (+ 0.68), 261(- 0.14), 245 (+ 0.13), 222 (- 1.02), 212 (+ 1.35), 194(+ 3.92) nm; IRvmax(KBr) 3406, 1658, 1601, 1448, 1089,1057, 1039, 1018, 701 cm-1;1H NMR and13C NMR data see Table 1; ESIMS (positive)m/z357 [M + Na]+,691 [2 M + Na]+; HRESIMS (positive)m/z357.0942[M + Na]+(calcd for C17H18NaO7, 357.0950).

Crystal data of compound 1: C17H18O7·2(H2O),M= 370.34,a= 5.5739(3) ?,b= 8.0353(5) ?,c= 19.5345(12) ?,α= 90°,β= 97.596(2)°,γ= 90°,V= 867.23(9) ?3,T= 100(2) K, space groupP1211,Z= 2,μ(Cu Kα) = 0.987 mm-1, 7517 reflections measured, 2687 independent reflections (Rint= 0.0345).The finalR1values were 0.0287 [I＞ 2σ(I)].The finalwR(F2) values were 0.0916 [I＞ 2σ(I)].The finalR1values were 0.0292 (all data).The finalwR(F2) values were 0.0929 (all data).The goodness of fit onF2was 0.837.Flack parameter = 0.11(10).The crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number CCDC 2,118,605).Copies of the data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk.

3.4.2 (5S,6R,7R,8S,7′Σ)-7′-hydroxyagarotetrol (2)

Light yellow powder; [28.3 (c= 0.35, MeOH);UV (MeOH)λmax(logε) 252 (3.85), 207 (4.00) nm; ECD(c0.018, MeOH)λmax(Δε) 301 (+ 0.14), 254 (- 0.93),223 (+ 1.69) nm; IRvmax(KBr) 3423, 1658, 1601, 1447,1384, 1044, 703 cm-1;1H NMR and13C NMR data see Table 1; ESIMS (positive)m/z357 [M + Na]+;HRESIMS (positive)m/z357.0945 [M + Na]+(calcd for C17H18NaO7, 357.0950).

3.4.3 (6S,7S,8R)-2-[2-(4-methoxyphenyl)ethyl]-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone (3)

Colorless plate crystal (MeOH);+ 7.4 (c= 0.23,MeOH); UV (MeOH)λmax(logε) 435 (1.81), 365 (2.05),254 (3.88), 220 (3.99), 201(4.04) nm; ECD (c0.0099,MeOH)λmax(Δε) 221 (- 1.43) nm;1H NMR and13C NMR data see Table 2; ESIMS (positive)m/z302[M + Na]+, 627 [2 M + Na]+; HRESIMS (positive)m/z355.1150 [M + Na]+(calcd for C18H20NaO6, 355.1158).

Crystal data of compound 3: C18H20O6,M= 332.34,a= 4.9601(2) ?,b= 7.0550(2) ?,c= 23.2769(7)?,α= 90°,β= 93.5950(10)°,γ= 90°,V= 812.94(5)?3,T= 100.(2) K, space groupP1211,Z= 2,μ(Cu Kα) = 0.850 mm-1, 12,448 reflections measured, 3180 independent reflections (Rint= 0.0352).The finalR1values were 0.0271 [I＞ 2σ(I)].The finalwR(F2) values were 0.0704 [I＞ 2σ(I)].The finalR1values were 0.0277 (all data).The finalwR(F2) values were 0.0710(all data).The goodness of fit onF2was 1.031.Flack parameter = 0.09(4).The crystallographic data for the structure of 3 have been deposited in the Cambridge Crystallographic Data Centre (deposition number CCDC 2118606).Copies of the data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk.

3.4.4 (6S,7S,8R)-2-(2-phenylethyl)-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone (4)

3.4.5 (5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8- [2-(2-phenylethyl)-7-methoxychromonyl-6-oxy]chromone (5)

3.4.6 (4S,5S,7S,8S,10S,13R)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (6)

3.4.7 (4S,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (7)

3.4.8 (4R,5S,7S,8S,10S,13S)-7,8,13-trihydroxyrotunda-1,11-dien-3-one (8)

3.4.9 Agarotetrol (9)

3.4.10 4′-Methoxyagarotetrol (11)

3.4.11 2′-Hydroxyagarotetrol (13)

3.4.12 (5S,6R,7R,8S)-2-(2-Phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8- [2-(2-phenylethyl)chromonyl-6-oxy]chromone (19)

3.4.13 (5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydro-8-[2-(2-phenylethyl)chromonyl-6-oxy]chromone (20)

3.4.14 (-)-Aquisinenone G (21)

3.5 Computational methods

Theoretical calculations of ECD spectra for compounds 6-8 were performed with the Gaussian 16 program package [21].The preliminary conformational distribution search was performed by Tripos sybyl- × 2 software [22].Selected conformers with distributions higher than 1% were further optimized by the DFT method at the B3LYP/6-311 g (d) level in the Gaussian 16 program package.The ECD of the conformer of selected conformers was then calculated by the TDDFT method at the CAM-B3LYP/tzvp levels with the PCM model in methanol solution.The overall calculated ECD curves were weighted by Boltzmann distribution.The calculated ECD spectra were produced by SpecDis 1.71 [23].Detailed calculated parameters are provided in the Additional file 1.

3.6 Corticosterone-induced damage in PC12 cellular assay

The method for bioassay testing was carried out according to our previously published papers [5, 6].

4 Conclusion

Five new 2-(2-phenylethyl)chromone derivatives,three new sesquiterpenoids, and 14 known compounds were isolated from the resinous heartwood ofAquilaria sinensis.The neuroprotective activities of these isolates were evaluated using an in vitro model of PC12 cell injury induced by corticosterone. (6S,7S,8R)-2-(2-Phenylethyl)-6,7,8-trihydroxy-5,6,7,8-tetrahydrochromone (4), (4S,5S,7S,8S,10S,13R)-7,8,13-trihydroxyrotunda-1,11-dien-3-one(6), agarotetrol (9), and 6-hydroxy-2-(2-phenylethyl)chromone (17) showed the most protective activities against corticosterone-induced PC12 cell injury at concentrations from 5 to 40 μM (P＜ 0.001).

Supplementary Information

The online version contains supplementary material available at https:// doi.org/ 10.1007/ s13659- 022- 00326-3.

Additional file 1.Chemical structures of known compounds (9-22), key 2D NMR correlations of agarotetrol (9), general experimental procedures,computational methods for ECD of compounds 6-8, and NMR, HRMS, and ECD spectra of compounds 1-8.

Acknowledgements

This study was supported by Beijing Sino-Science Aquilaria Technology Co.,Ltd., Beijing, China (no.KET202101).

Authors’ contributions

All authors read and approved the final manuscript.

Declarations

Competing interests

The authors declare that there are no conflicts of interest associated with this work.