Absolute Configuration of a New Phenethylcyclohexanetriol and a New Natural Bibenzyl From Amomum celsum

Abstract

Methanol extracts of the rhizomes or fruit of Amomum celsum Lamxay & M.F. Newman (Zingiberaceae) yielded 5 compounds one of which, 1,3S,5S-trihydroxy-1-(4-hydroxyphenylethyl)cyclohexane (2), is new and one, 4-hydroxybibenzyl (1), is reported as a natural product for the first time. In addition, 3 known compounds, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (3), 3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane (4), and 1,4-hydroquinone (5), were also isolated. Their structures were determined by performing extensive analysis of high-resolution electrospray ionization-mass spectrometry and nuclear magnetic resonance spectroscopic data. The absolute configuration of 2 was unambiguously established by X-ray crystallography and Mosher’s method. Cytotoxicity of compounds 1 and 2 against MCF-7 and Hep-G2 human cancer cell lines were tested in the sulforhodamine B cytotoxic assay. A plausible biosynthesis of 1 and 2 in A. celsum was proposed.

The nontimber genus Amomum (Zingiberaceae) was first described by Linnaeus in 1747. Today, some of approximately 170 Amomum species such as A. tsao-ko Crevost & Lemarié, A. villosum Lour., A. krervanh PierreexGagnep., and A. xanthioides Wall. have been recognized as plants of economic importance because of their extensive use as spices and medicinal herbs. The number of phytochemical studies of Amomum species is relatively small including A. pavieanum, ¹ A. krervanh, ² A. koenigii, ³ A. aculeatum, ^4–7 A. xanthioides, ⁸ A. subulatum, ^9–11 A. tsao-ko, ^12–14 A. muricarpum, ^15,16 and A. biflorum. ¹⁷ Amomum celsum has an area of occurrence of 314 km² from Kon Tum province in Vietnam and Attapeu province in Laos. ¹⁸ There has been no phytochemical study of A. celsum. We prepared water-soluble fractions from the rhizomes or fruit of A. celsum in Vietnam and isolated compounds 1– 5 of which 1 was isolated from a natural source for the first time and 2 was new.

MeOH extracts were prepared individually from the dried rhizomes or fresh fruit of A. celsum by extraction at room temperature. Two water-soluble fractions were prepared by successive fractionation of the MeOH extracts with n-hexane, dichloromethane, and ethyl acetate. The water-soluble fractions were chromatographed successively on highly porous Diaion HP-20, RP-18, and silica gel to afford 5 compounds. The structures of the known compounds 3 (5-hydroxy-3,7,3′,4′-tetramethoxyflavone), ¹⁹ 4 (3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane), ²⁰ and 5 (1,4-hydroquinone) ²¹ were determined by comparing their spectroscopic data (MS, ¹H, and ¹³C nuclear magnetic resonance, NMR) with the reported literature values. Compound 1 is a known bibenzyl isolated for the first time from nature and compound 2 is a new phenethyl-1,3,5-trihydroxycyclohexane (Figure 1).

Figure 1

Structures of 1 and 2.

Compound 1 was isolated as a white amorphous powder. Its IR absorption peaks indicated the presence of hydroxy (3368 cm⁻¹) and aromatic (1600, 1514, and 1452 cm⁻¹) groups. A molecular formula of C₁₄H₁₄O was determined for 1 on the basis of negative-ion high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS) (m/z 197.0972, [M − H]⁻). The ¹H NMR spectrum of 1 showed the presence of 2 aromatic systems. A phenyl ring was determined on the basis of 5-proton resonance signals at δ _H7.15 (3H, m), 7.22 (1H, t, J = 7.5 Hz), 7.24 (1H, t, J = 7.5 Hz) and a 4-hydroxyphenyl ring on the basis of 2-proton ortho-coupling signals at δ _H 6.68 (2H, d, J = 8.5 Hz) and 6.97 (2H, d, J = 8.5 Hz). An ethylene (-CH₂-CH₂-) link (δ _H 2.86, m, 2CH₂) was found attached to C-1 and C-1′ of the 2 aromatic rings. On the basis of the ¹H NMR the structure of 1 was proposed to be 4-hydroxybibenzyl. The ¹³C NMR spectrum of 1 showed 14 carbon-13 signals including 2 methylene groups at δ _C 38.3 (CH₂) and 39.5 (CH₂), the phenyl (δ _C 126.8, 129.2, 129.6, and 143.3), and the 4-hydroxyphenyl groups (δ _C 116.0, 130.4, 133.9, and 156.4). The high-field methylene signal at δ _C 38.3 (Δδ _C−1.2 ppm) was assigned to occupy the α-position (C _α ) relative to the 4-hydroxyphenyl group. ²² The lower-field signal at δ _C 39.5 (C _β ) was not influenced by the aromatic electron-donating hydroxy group and was attached to the phenyl group. Thus, the structure of 1 was confirmed to be 4-hydroxy-bibenzyl, as shown in Figure 1. Compound 1 is known ²³ but isolated for the first time from a natural source. The NMR data of 1 were reported herein for the first time.

Compound 2 was isolated as colorless plates and has a specific optical rotation of ${\displaystyle [\alpha {]}_{\mathrm{D}}^{25}}$ −27.9 (c 0.05, CH₃OH). The IR spectrum of 2 showed absorption bands for hydroxy (3335 cm⁻¹) and aromatic (1559, 1513, and 1419 cm⁻¹) groups. A molecular formula of C₁₄H₂₀O₄ was assigned for 2 by positive-ion HR-ESI-MS (m/z 275.1252 [M + Na]⁺). The ¹³C NMR, DEPT, and heteronuclear single quantum correlation (HSQC) spectra of 2 indicated the presence of 4 aromatic methines, 2 aromatic carbons, 2 hydroxymethines, a hydroxy-bearing tertiary carbon, and 5 methylenes. The presence of a 4-hydroxyphenyl ring was indicated by a set of 2-proton resonance signals at δ _H 6.70 (2H, d, J = 8.5 Hz) and 7.02 (2H, d, J = 8.5 Hz) in the ¹H NMR spectrum and 4 carbon-13 signals including 2 doubling signals at δ _C 116.1 (2CH) and 130.2 (2CH), and 134.8 (C) and 156.3 (C-OH). The 2 aromatic proton signals (δ _H 6.70 and 7.02) were correlated in the ¹H-¹H correlation spectroscopy (COSY) spectrum, confirming the 1,4-substitution of the aromatic ring. The signals of the 2 methylene groups were observed at δ _H1.73 (2H, ddd, J = 9.5 Hz, 7.5 Hz, 2.0 Hz) and 2.61 (2H, m), and δ _C 46.9 (CH₂) and 29.5 (CH₂) in the ¹H and ¹³C NMR spectra, respectively; their connection was determined by ¹H-¹H COSY cross peaks (Figure 2) from H₂-7 (δ _H 1.73) to H₂-8 (δ _H 2.61). The signals of both H-7a and H-7b were overlapped and their multiplicities (ddd) were in accordance with their connectivity to the methylene group at δ _H 2.61. Heteronuclear multiple bond correlations (HMBCs) (Figure 2) assembled the 4-hydroxyphenyl moiety to the ethylene group, confirming the presence of a phenethyl group. Thus, HMBC correlations were observed between H-2′/H-6′ (δ _H 7.02) and C-8 (δ _C 29.5), H₂-8 (δ _H 2.61) and C-1′ (δ _C 134.8), and H₂-7 (δ _H 1.73) and C-1′. Owing to hydrogen deficiency, a trihydroxycyclohexane ring was deduced from the carbon-13 resonance signals of an oxygenated tertiary carbon at δ _C 75.6 (C-1), 2 hydroxymethines at δ _C 64.4 (C-5) and 69.1 (C-3), and 3 methylenes at δ _C 41.5 (C-2), 42.5 (C-4), and 46.7 (C-6). In the HSQC spectrum of 2, the 2 hydroxymethines at δ _C 64.4 and 69.1 were correlated with 2 proton signals at 4.23 (1H, m, H-5) and δ _H 4.26 (1H, m, H-3), respectively. The 1,3,5-substitution pattern of the cyclohexane ring was deduced from the ¹H-¹H COSY and HMBC spectra of 2. In the ¹H-¹H COSY spectrum (Figure 2) the proton sequence -(2-CH₂)-(3-CH-OH)-(4-CH₂)-(5-CH–OH)-(6-CH₂)- was established by the correlations between 6-CH₂ (δ _H 1.42/2.08) and H-5 (δ _H 4.23), H-5 and 4-CH₂ (δ _H 1.47/2.15), 4-CH₂ and H-3 (δ _H 4.26), and H-3 and 2-CH₂ (δ _H 1.58/1.87). In the HMBC spectrum H₂-2, H₂-6, and H-3 were correlated with C-1; and H-5 was correlated with C-3. C-1 was connected to the phenethyl moiety through the HMBC correlations between H₂-7 (δ _H 1.73), H₂-8 (δ _H 2.61), H₂-6 (δ _H 1.42/2.08), and H₂-2 (δ _H 1.58/1.87) and C-1 (δ _C 75.6).

Figure 2

¹H-¹H COSY, HMBC, and NOESY correlations of 2.

The mutiplicities and values of coupling constants of the cyclohexyl protons were consistent with the assignments. Judging from the stability of cyclohexane conformations, a chair conformation that is stabilized by an intramolecular hydrogen bond between 2 diaxial hydroxy groups at C-1 and C-3 was chosen. Thus, the values of diaxial coupling constants of H-6_ax at δ _H 1.42 (J = 10.5 Hz) and H-4_ax at δ _H 1.47 (J = 11.5 Hz) indicated that H-5 (δ _H 4.23, m) was also axially orientated. Both axial protons coupled with H-4_eq (δ _H 2.15) and H-6_eq (δ _H 2.08) by the same geminal coupling constant (J = 13.0 Hz). The values of the coupling constants measured for H-2_ax at δ _H 1.58 (J = 3.0 Hz) and H-4_ax at δ _H 1.47 (J = 2.0 Hz) indicated the equatorial orientation of H-3 (δ _H 4.26, m). Long-range W-couplings (⁴ J = 1.7 Hz) were observed between H-2_eq (δ _H 1.87) and H-6_eq and H-4_eq and H-6_eq. The nuclear Overhauser effect spectroscopy (NOESY) experiments (Figure 2) were not selective to rule out ¹H-¹H COSY signals for H-3 and H-5; however, the NOESY correlations between H₂-7 and H-2_ax and H-6_ax confirmed the equatorial orientation of the phenethyl group, thus confirming the axial orientation of the hydroxy group at C-1.

The relative structure assembled by the spectroscopic data was confirmed by the crystallographic analysis and the absolute structure is as shown in Figure 3, where the Flack paramter was 0.02(4). Two independent molecules and water molecules are included in an asymmetric unit, and one water molecule forms a hydrogen bond with 2 independent molecules. The absolute configuration was further confirmed by the modified Mosher’s method ²⁴ as shown in Figure 4. On the basis of the extensive NMR and X-ray analysis, the structure of 2 was determined to be 1,3S,5S-trihydroxy-1-(4-hydroxyphenylethyl)cyclohexane.

Figure 3

X-ray crystallographic structure (H bonding) of 2.

Figure 4

Mosher’s esters of 2. Δδ values are in ppm (δ_S–δ_R ).

Compound 1 showed weak cytotoxicity against MCF-7 (human breast carcinoma) and Hep-G2 (human hepatocellular carcinoma) cell lines (IC₅₀ of 59.01 µg/mL and 76.14 µg/mL, respectively) in the sulforhodamine B (SRB) cytotoxic assay. ²⁵ At the same concentration range with that of 1, compound 2 was not active. A plausible biosynthetic pathway to 1 and 2 from 4-hydroxycinnamoyl coenzyme A is proposed in Figure 5.

Figure 5

Plausible biosynthetic pathway of 1 and 2.

Experimental

General Procedure

Optical rotations were measured on a JASCO P-2200 digital polarimeter. IR spectra were recorded on a Jasco FT-IR 6100 spectrometer. ESI-MS spectra were obtained using a LC-MS 6310 Agilen Ion Trap system. HR-ESI-MS spectra were measured on a Thermo Fischer Scientific LTQ Orbitrap XL mass spectrometer. ¹H, ¹³C NMR, DEPT, ¹H-¹H COSY, HSQC, HMBC, and NOESY spectra were recorded on a Bruker Avance 500 NMR or 600 MHz spectrometers. The diffraction experiment was performed with a Bruker D8VENTURE system (PHOTON-100 CMOS detector, CuKα: λ = 1.54178 Å). All nonhydrogen atoms were refined anisotropically. The hydrogen atoms were refined isotropically on the calculated positions using a riding model except for hydroxy hydrogen. Absorption correction was performed by an empirical method implemented in SADABS. Structure solution and refinement were performed using SHELXT-2014/5 and SHELXL-2018/3. Silica gel (Merck, Germany) and Diaion HP-20 (Mitsubishi, Japan) were used for column chromatography (CC). Merck LiChrolut^® (reversed phase)RP-18 (Merck, Germany) cartridges were used for solid-phase extraction (SPE). Thin-layer chromatography (TLC) was performed on precoated silica gel 60 F₂₅₄ plates (Merck, Germany).

Plant Material

Fresh rhizomes and fruit of A. celsum Lamxay & M.F. Newman were collected in Kon Tum province, Vietnam, in July 2016. The plant material was identified by one of the authors, Dr Nguyen Quoc Binh (Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, Hanoi, Vietnam). A voucher sample (No. EF-12-15) has been deposited at the same Museum.

Extraction and Isolation

The fresh rhizomes and fruit were cleaned and air-dried to remove surface water in the shade. The rhizomes were dried in an oven at 40°C to 50°C then powdered. The fruit (3 kg) was crushed in an electric blender. The dried powdered rhizomes (2 kg) or fruit paste of A. celsum were extracted with MeOH at room temperature 3 times, each time for 7 days. After filtration, the MeOH extracts were combined separately and evaporated under reduced pressure to afford 2 corresponding MeOH extracts which were suspended in distilled water. The suspensions were partitioned between water and n-hexane, CH₂Cl₂, and EtOAc. The water phases were concentrated and chromatographed on a Diaion HP-20 column eluted with gradient mixtures of MeOH-H₂O (20%, 40%, 60%) and MeOH yielding 4 corresponding fractions. From the fruit: The 100% MeOH fraction (89 mg) was separated by silica gel CC with gradient mixtures of n-hexane-EtOAc-HCO₂H (10:3:1, 10:5:1, 10:7:1, 10:10:1) to give 2 fractions. Compound 3 (2 mg) was crystallized as an amorphous white powder (R_f 0.84, silica gel TLC, n-hexane-EtOAc-HCOOH 10:10:1, v:v:v) from fraction 1. Fraction 2 was subjected to Sephadex LH-20 CC with MeOH to give 3 subfractions. Subfraction 2 was purified by silica gel CC with gradient mixtures of CH₂Cl₂-MeOH (30:1, 25:1, 20:1, 15:1, 9:1, 6:1, 3:1, 1:1), RP-18 CC with MeOH-H₂O 1:1, and preparative TLC with CH₂Cl₂-MeOH 9:1 to give 4 (7 mg) as white needles (R _f 0.42, silica gel TLC, CH₂Cl₂-MeOH 9:1, v:v). The 60% MeOH fraction (0.83 g) was separated by silica gel CC with gradient mixtures of CH₂Cl₂-MeOH (25:1, 20:1, 15:1, 9:1, 6:1, 3:1, 1:1) to give 1 (17 mg) as white needles (R _f 0.58, silica gel TLC, CH₂Cl₂-MeOH 9:1, v:v). The 40% MeOH fraction (680 mg) was separated by silica gel CC with gradient mixtures of EtOAc-MeOH (12:1, 9:1, 6:1, 3:1, 1:1) to give 4 fractions. Fraction 2 was crystallized from EtOAc to give 1 (9 mg). The 20% MeOH fraction (1.1 g) was separated by silica gel CC with gradient mixtures of EtOAc-MeOH (15:1, 9:1, 6:1, 3:1, 1:1) to give 5 (13 mg) as white needles (R _f 0.7, silica gel TLC, EtOAc-MeOH 3:1, v:v). From the rhizomes: The 40% MeOH fraction (1.0 g) was purified by RP-18 CC with gradient mixtures of MeOH-H₂O (1:4, 1:3, 1:2, 1:1) to give 2 (9 mg) as white needles (Rf 0.84 (silica gel TLC, EtOAc-MeOH 1:1, v:v). The 60% MeOH fraction (2.0 g) was chromatographed on Sephadex LH-20 with MeOH to obtain 3 fractions. Fraction 2 was purified by repeated SPE on RP-18 with MeOH-H₂O 1:1, Sephadex LH-20 with MeOH, and silica gel CC with gradient mixtures of CH₂Cl₂-MeOH (30:1, 25:1, 20:1, 15:1, 9:1, 6:1, 3:1, 1:1) to give 2 (19 mg).

4-Hydroxybibenzyl (1)

Amorphous powder.

IR (film) ν_max 3368, 1600, 1514, 1452, 1251 cm⁻¹.

¹H NMR (500 MHz, CD₃OD): 2.86 (4H, m, 2H _α , 2H _β ), 6.68 (2H, d, J = 8.5 Hz, H-2, H-6), 6.97 (2H, d, J = 8.5 Hz, H-3, H-5), 7.15 (3H, m), 7.24 (1H, t, J = 7.5 Hz), 7.22 (1H, t, J = 7.5 Hz) (5 H, H-2′, H-3′, H-4′, H-5′, H-6′).

¹³C NMR(125 MHz, CD₃OD): 38.3 (C _α ), 39.5 (C _β ), 116.0 (C-2, C-6), 126.8 (C-4′), 129.2 (C-3′, C-5′), 129.6 (C-2′, C-6′), 130.4 (C-3, C-5), 133.9 (C-1), 143.3 (C-1′), 156.4 (C-4).

Negative-ion HR-ESI-MS: m/z [M–H]^– calcd for C₁₄H₁₃O: 197.0961, found: 197.0972.

1,3S,5S-Trihydroxy-1-(4-Hydroxyphenylethyl)Cyclohexane (2)

Colorless plates.

MP:. 139°C-141°C.

${\displaystyle [\alpha {]}_{\mathrm{D}}^{25}}$ : –27.9 (c 0.05, CH₃OH).

IR (film) ν_max 3335, 1559, 1513, 1419, 1237 cm⁻¹.

¹H NMR (500 MHz, CD₃OD): 1.42 (1H, dd, J = 13.0 Hz, 10.5 Hz, H-6_ax), 1.47 (1H, ddd, J = 13.0 Hz, 11.5 Hz, 2.0 Hz, H-4_ax), 1.58 (1H, dd, J = 14.0 Hz, 3.0 Hz, H-2_ax), 1.73 (2H, ddd, J = 9.5 Hz, 7.5 Hz, 2.0 Hz, 2 H-7), 1.87 (1H, dddd, J = 14.0 Hz, 3.6 Hz, 1.7 Hz, 1.7 Hz, H-2_eq), 2.08 (1H, dddd, J = 13.0 Hz, 2.0 Hz, 2.0 Hz, 1.7 Hz, H-6_eq), 2.15 (1H, br d, J = 13.0 Hz, H-4_eq), 2.61 (2H, m, 2 H-8), 4.23 (1H, m, H-5), 4.26 (1H, m, H-3), 6.70 (2H, d, J = 8.5 Hz, H-3′, H-5′), 7.02 (2H, d, J = 8.5 Hz, H-2′, H-6′).

¹³C NMR (CD₃OD): 29.5 (C-8), 41.5 (C-2), 42.5 (C-4), 46.7 (C-6), 46.9 (C-7), 64.4 (C-5), 69.1 (C-3), 75.6 (C-1), 116.1 (C-3′, C-5′), 130.2 (C-2′, C-6′), 134.8 (C-1′), 156.3 (C-4′).

Positive-ion HR-ESI-MS: m/z [M + Na]⁺ calcd for C₁₄H₂₀O₄Na: 275.1254, found: 275.1252.

Preparation of (S)- and (R)-MTPA Diesters (2a and 2b) From 2

A solution of 2 (0.5 mg) in 0.5 mL of dehydrated CH₂Cl₂ was reacted with (S)-MTPA (30.5 mg) in the presence of EDC (15.4 mg) and 4-DMAP (10.2 mg) and the resulting mixture was occasionally stirred at 25°C for 24 hours. After the addition of 1 mL of each of H₂O and CH₂Cl₂, the solution was washed successively with 5% HCl, NaHCO₃-saturated H₂O and brine. The organic layer was dried over Na₂SO₄ and then evaporated under reduced pressure. The residue was purified by silica gel CC, eluted with CHCl₃ and CHCl₃-MeOH 19:1 to give diester 2a (0.7 mg). Using a similar procedure, diester 2b (0.5 mg) was prepared from 2 (0.5 mg), (R)-MTPA (34.6 mg), EDC (13.2 mg), and 4-DMAP (11.1 mg).

(S)-MTPA Diester (2a)

¹H NMR (600 MHz, CDCl₃): 1.5148 (1H, m, H-2_ax), 1.572 (1H, m, H-6_ax), 1.5580 (1H, m, H-4_ax), 1.7852 (2H, m, 2 H-7), 2.0478 (1H, m, H-2_eq), 2.2868 (1H, br d, J = 13.0 Hz, H-6_eq), 2.3648 (1H, br d, J = 13.0 Hz, H-4_eq), 2.7509 (2H, m, 2 H-8), 3.5486 (3H, s, -OCH₃), 3.6834 (3H, s, -OCH₃), 4.3790 (1H, m, H-3), 5.6981 (1H, m, H-5), 7.04 (2H, d, J = 8.5 Hz, H-3′, H-5′), 7.22 (2H, d, J = 8.5 Hz, H-2′, H-6′), 7.40–7.65 (10H, m, aromatic protons).

Positive-ion HR-ESI-MS: m/z [M + Na]⁺ calcd for C₃₄H₃₄O₈F₆Na: 707.2050, found: 707.2052.

(R)-MTPA Diester (2b)

¹H NMR (600 MHz, CDCl₃): 1.4925 (1H, t, J = 12.0 Hz, H-6_ax), 1.5198 (1H, m, H-2_ax), 1.6581 (1H, m, H-4_ax), 1.7669 (2H, m, 2 H-7), 2.0546 (1H, br d, J = 13.0 Hz, H-2_eq), 2.2378 (1H, br d, J = 13.0 Hz, H-6_eq), 2.4265 (1H, br d, J = 13.0 Hz, H-4_eq), 2.7307 (2H, m, 2 H-8), 3.5513 (3H, s, -OCH₃), 3.6821 (3H, s, -OCH₃), 4.4001 (1H, m, H-3), 5.6988 (1H, m, H-5), 7.031 (2H, d, J = 8.5 Hz, H-3′, H-5′), 7.211 (2H, d, J = 8.5 Hz, H-2′, H-6′), 7.40-7.65 (10H, m, aromatic protons).

Positive-ion HR-ESI-MS: m/z [M + Na]⁺ calcd. for C₃₄H₃₄O₈F₆Na: 707.2050, found: 707.2048.

X-Ray Crystallographic Analysis of 2

C₁₄H₂₂O₅, M _r = 270.31. Monoclinic, space group P2₁, Z = 4, D _calc = 1.321 g cm⁻³, a = 6.1296(14), b = 7.0606(16), c = 31.400(7) Å, β = 90.218(5)°, V = 1359.0(5) ų, 16 182 observed and 5191 independent [I > 2σ(I)] reflections, 381 parameters, final R ₁ = 0.0324, wR ₂ = 0.1012, S = 1.095 [I > 2σ(I)]. Flack parameter: χ = 0.02(4). The largest difference peak and hole were 0.314 and –0.355 eÅ^–3, respectively.

Supplementary X-ray crystallographic data for 2 (CCDC 1859781) can be obtained free of charge via www.ccdc.cam.ac.uk/conts/ retrieving.html (or from the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2 1EZ, UK; fax: [phone]; or [e-mail]).