A new polyphenolic isoprenylated acetophenone dimer from the stem bark of Acronychia pedunculata (L.) Miq

Abstract A new acetophenone dimer, 5′-prenylacrovestone (1), together with nineteen known compounds (2–20), were isolated from the stem bark of Acronychia pedunculata (L.) Miq. Their structures were identified by thorough analysis of spectroscopic (IR, 1D and 2D NMR) and mass spectrometric data. The isolated compounds were tested against the bacterial pathogens MRSA, B. cereus, S. aureus and E. coli. Compound 3 demonstrated exceptionally potent antibacterial activity against each of the four strains (MIC values of 1 µg/mL). Graphical Abstract


Results and discussion
Compound 1 was isolated as a yellow viscous liquid. Its molecular formula was determined as C 37 H 50 O 8 on the basis of HRESITOFMS (m/z 621.3441 [M À H] À , calcd 621.3433). Its IR spectrum showed the presence of hydroxy groups (3260 cm À1 ). The 1 H NMR spectrum (Supplementary material, Table S1) showed three olefinic proton signals at d H 5.21 (H-2 0 , 1H, m), 5.19 (H-2 0000 , 1H, m) and 5.04 (H-6 0 , 1H, brt, 5.8 Hz) ppm. A deshielded, benzylic methine proton stood out at d H 4.75 (H-1 00 , 1H, br) ppm. A methoxy group at d H 3.71 (MeO-2 000 , 3H, s) ppm and two acetyl resonances at d H 2.71 (MeCO-1 000 , 3H, s) and 2.67 (MeCO-1, 3H, s) ppm were also detected. The 1 H NMR spectrum also displayed resonance peaks belonging to two deshielded methylene protons at d H 3.42 (H-1 0 , 2H, d, 6.8 Hz) and 3.29 (H-1 0000 , 2H, brs) ppm, and of six methylene protons at d H 2.05-2.20 (H-2 00 , H-4 0 , H-5 0 , 6H, brm). In addition, five deshielded methyl signals were found at d H 1.82 (H-10 0 , 3H, s), 1.76 (H-4 0000 , 3H, s), 1.69 (H-5 0000 , 3H, s), 1.67 (H-8 0 , 3H, s), and 1.59 (H-9 0 , 3H, s) ppm. Eventually, signals of a methine proton at d H 1.40 (H-3 00 , 1H, m) ppm, and of two methyl groups at d H 0.87 (H-4 00 , H-5 00 , 6H, brs) ppm were observed. The 13 C NMR spectroscopic data (Supplementary material, Table S1) revealed 37 signals. The low field region indicated the presence of two ketone carbons at d C 204.5 and 204.3 ppm, twelve aromatic carbons at d C 162. 8, 161.2, 161.0, 160.3, 158.5, 158.5, 116.9, 113.2, 109.3, 108.3, 106.4 and 104.7 ppm, as well as three pairs of olefinic carbons at d C 140.8, 132.3, 131.9, 123.6, 123.2 and 121.7 ppm. In the high field region, the 13 C NMR spectrum displayed signals of five methylene carbons at d C 39. 8, 39.5, 26.4, 23.3 and 22.3 ppm, two methine carbons at d C 28.7 and 27.2 ppm, seven methyl groups at d C 25.9, 25.8, 22.8, 22.6, 18.1, 17.9 and 16.4 ppm, a methoxy carbon at d C 62.7 ppm, as well as two acetyl groups at d C 32.9 and 30.8 ppm. Such spectroscopic data, including signal broadening from rotational limitations, were reminiscent of prenylated acetophenone dimers, such as acrovestone (2) (Supplementary material, Table S2), which are chemotaxonomic markers of the Acronychia genus. The detection of three sets of olefinic signals and five allylic methyl groups, however, indicated the presence of an extra prenyl unit in compound 1, forming a 3,7-dimethyl-2,6-octadienyl unit on one of the two aromatic rings of the acetophenone dimer. This assessment was confirmed by analysis of 2D NMR spectra, which showed similar correlations to those of acrovestone (2). The COSY spectrum (Supplementary material, Figure S8) showed correlations between H-2 0000 and H-1 0000 , H-1 0 and H-2 0 , as well as H-5 0 with H-6 0 , leading to the identification of each pair of olefinic/allylic methylene signals. The double bond geometry of the octadienyl unit was determined as (E), as a NOESY correlation between H-2 0 and H-4 0 was observed. Moreover, the correlations between H-6 0 and H-8 0 , and H-2 0000 with H-5 0000 were used to define the relative orientations of the methyl groups. A NOESY correlation between the methoxy group and the acetyl group at position 1 000 confirmed its location at C-2 000 . The position of the 3,7-dimethyl-2,6-octadienyl unit was determined through analysis of HMBC correlations. H-2 0 correlated with the methyl carbon at d C 16.4 (C-10 0 ) ppm, and with two methylene carbons at d C 22.3 and 39.8 ppm. These positions were therefore assigned as C-1 0 and C-4 0 , respectively. The methylene protons H-1 0 correlated to two phenolic carbons (C-2 and C-4, at d C 158.5 and 161.0 ppm, respectively) and one quaternary aromatic carbon at d C 106.4 ppm (C-3). It is important to mention that, in a similar fashion to acrovestone (2), the NMR spectra of compound 1 displayed signal broadening due to rotational limitations. As a result, clear multiplicities, expected for prenyl units, for example, were sometimes not observed in the 1 H NMR spectrum. In addition, many carbon resonances appeared as broad peaks, while several HMBC correlations were not observed, including those of the exchangeable phenolic protons (appearing as broad signals).
The absolute configuration of compound 1 was also investigated. Although C-1 00 is a chiral centre, its circular dichroism (CD) spectrum was nearly flat in the 250-500 nm region, indicating the presence of a racemate. This result is consistent with absolute configuration studies reported for related isoprenylated acetophenone dimers (Kouloura et al. 2012;Tanjung et al. 2018;Miyake et al. 2019).
Several isolated compounds were tested in search of antibacterial activities, using a broth microdilution method. The results are shown in Table S3 (Supplementary material). The antibacterial activities of compounds 2, 4 and 5 were previously discussed on the same strains . Compound 3 demonstrated exceptionally potent antibacterial activity against the four bacterial strains tested, with MIC values of 1 mg/mL against each strain. Compound 3 surpassed the control drugs chloramphenicol and kanamycin against the four strains tested. It also outperformed vancomycin against MRSA and B. subtilis but performed equally well against S. aureus and B. cereus. Compound 3 also outperformed gentamicin against three strains (MRSA, B. cereus and B. subtilis). Gentamicin proved more potent (MIC ¼ 0.25 mg/mL) against S. aureus. Remarkably, compound 3 was much more potent than acrovestone (2), a closely related compound. They differ structurally by the presence of an extra alcohol at C-2 0 and double bond migration to a terminal position in compound 3. The second prenylated chain seems to play a key role in the observed antibacterial activities. We reported a similar conclusion for acropyrone (4). The latter showed strong antibacterial activity against MRSA, with a MIC value of 8 mg/mL, but was inactive against the other strains, despite being closely related to acrovestone (2). Indeed, the presence of a pyran ring starting at C-1 0 dramatically altered the antibacterial activities. Interestingly, compound 1 (5 0 -prenylacrovestone) proved inactive against the four strains tested. Elongation of the chain at C-1 0 of acrovestone (2) seems deleterious to its antibacterial properties. Compound 9 demonstrated strong antibacterial activity against S. aureus with a MIC value of 8 mg/mL, which was as high as that of chloramphenicol. Compounds 10, 12, 13, 18, 19 and 20 showed moderate activities against S. aureus (MIC values of 16-32 mg/mL) while compound 17 showed weak activity (MIC ¼ 64 mg/ mL) against this strain. Compound 18 showed strong activity against B. cereus, with a MIC value of 8 mg/mL. Compound 20 showed moderate activity against B. cereus, with a MIC value of 16 mg/mL, while compound 13 showed weak activity (MIC ¼ 64 mg/mL) against this strain. Eventually, compound 18 showed weak activity against B. subtilis, with a MIC value of 64 mg/mL.

General experimental procedures
Optical rotations were measured with a JASCODIP-1000 digital polarimeter (JASCO Inc., Japan). UV spectra were recorded using a JASCO J-810 apparatus. Melting points were determined using an Electrothermal IA9200 digital melting point apparatus (Bibby Scientific Limited, Staffordshire, UK). IR analyses were performed using a Bruker Tenser 27 spectrophotometer (Bruker, Germany). NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer (Varian Inc., U.S.A.) or on a Bruker Avance 400 NMR spectrometer (Bruker, Germany), using CDCl 3 and CD 3 OD as solvents. The residual peaks of these solvents were used as internal references. The HRESITOFMS were carried out on a Bruker micrOTOF mass spectrometer (Brucker, Germany). Column chromatography was carried out on MERCK silica gel 60 (230-400 mesh) (Merck, Darmstadt, Germany). Thin-layer chromatography was carried out with precoated MERCK silica gel 60 PF254 (Merck, Darmstadt, Germany); the spots were visualized under UV light (254 and 365 nm) and further stained by spraying p-anisaldehyde and then heated until charred. Unless otherwise noted, all chemicals were obtained from commercially available sources and were used without further purification.

Plant material
The stem bark of A. pedunculata was collected in Pla Pak District, Nakhon phanom Province, Thailand, in August 2018. The plant material was identified by Prof. Pranom Chantaranothai, Department of Biology, Khon Kaen University, Thailand, where a voucher specimen (R. Lekphrom017) was deposited.

Extraction and isolation
The stem bark of A. pedunculata (4.2 kg) was ground to powder and then extracted at room temperature with hexanes and EtOAc three times each (3 Â 3 L). Removal of solvents under reduced pressure gave the crude hexane (23.0 g) and EtOAc (31.0 g) extracts.

Antibacterial assay
The minimum inhibitory concentrations (MICs) were determined by the dilution method as described in the M07-A9 (CLSI 2014

Conclusions
Phytochemical investigation of the stem bark of A. pedunculata led to the isolation of twenty compounds. One of them is a new isoprenylated acetophenone dimer, 5 0 -prenylacrovestone (1). Compounds 8, 9 and 10 are reported for the first time in the Rutaceae family. Compounds 12, 13, 14 and 20 are reported from the genus Acronychia for the first time. Compound 3 showed exceptionally potent antibacterial activity against the bacterial pathogens MRSA, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis.