New acylphloroglucinols from a crude acetone extract of Eucalyptus camaldulensis Dehnh. leaf

Abstract Acylphloroglucinols are well-known Eucalyptus secondary metabolites which exhibit a variety of structures and bioactivities. The investigation of a crude acetone extract of Eucalyptus camaldulensis leaves led to the isolation of two new acylphloroglucinols, eucalypcamals O and P (1 and 2) together with seven phloroglucinols (3–9), and a benzene derivative (10). Their chemical structures were elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy. The absolute configurations of compounds 1 and 2 were established by comparison of experimental and calculated electronic circular dichroism (ECD) data. In the putative biosynthetic pathway, eucalypcamals O and P should be derived from hetero-Diels-Alder reaction between grandinol and trans-isoeugenol. Graphical Abstract


Introduction
Acylphloroglucinols are secondary metabolites that have been shown to exert a variety of bioactivities.In various studies, they have exhibited cytotoxicity (Wang et al. 2012), anti-inflammatory (Gu et al. 2020), and acetylcholinesterase inhibitory activities (Qin et al. 2018a).The Eucalyptus plants, belonging to the family Myrtaceae, are the important natural sourced of acylphloroglucinol metabolites with different type of acyl groups as isovaleryl (Kokumai et al. 1991), isobutyryl (Qin et al. 2018a), and 2-methylbutanoyl (Cheng and Snyder 1991).Eucalyptus species have also yielded acyphloroglucinols coupled with terpenes (Shang et al. 2019), b-triketones (Wang et al. 2014) or other phloroglucinol (Qin et al. 2018a) moieties providing more complex natural products with multiple bioactivities.
Eucalyptus camaldulensis (Myrtaceae) is known to contain bioactive compounds that display antibacterial (Cimanga et al. 2002;Salem et al. 2015;Nasr et al. 2019), antifungal (Su et al. 2006, Elansary et al. 2017), analgesic, anti-inflammatory effects (Silva et al. 2003), antioxidative and antiradical activities (Siramon and Ohtani 2007).In our previous study of a non-polar extract of the leaves of E. camaldulensis, we reported a series of new acylphloroglucinol-meroterpenoids that displayed cytotoxicity and antimicrobial activity (Daus et al. 2022).In the course of our continued investigation, we isolated two new acylphlorglucinols, eucalypcamals O and P (1 and 2) together with eight known compounds from a polar extract of E. camaldulensis leaves.Herein, we described the structure characterisation and putative biosynthetic pathway of the new compounds.In addition, the purified known compounds were evaluated for cytotoxicity and antimicrobial activity.
Compound (1) was obtained as a pale yellowish gum.The HRESIMS of 1 revealed an m/z at 415.1751  ) implied that methine carbon C-8 0 was substituted by a methyl group (Supplementary material, Figure S2).The complete structure of 1 was finalised by an HMBC experiment, HMBC correlations of 1 0 -OH/H-3 0 to C-1 0 (d C 146.4) and 2 0 -OCH 3 /H-6 0 to C-2 0 (d C 147.0) placed the hydroxy and methoxy groups at C-1 0 and C-2 0 , respectively.The 1,2,4-trisubtituted aromatic ring was connected to C-7 0 which was confirmed by the HMBC correlations of H-3 0 to C-7 0 (d C 86.5) and H-7 0 to C-3 0 (d C 109.6), C-4 0 (d C 130.1), and C-5 0 (d C 121.2).The relative stereochemistry of 1 was determined by the magnitude of the coupling constant (J ¼ 10.0 Hz) between H-7 0 and H-8 0 which indicated a trans relationship between these two protons (Satoh et al. 1992;Singh et al. 1997).The trans relationship was supported by NOESY correlations of H-7 0 to H 3 -9 0 and H-7a which confirmed the b-orientation of H-7a, H 3 -9 and H-7 0 , and a-orientation of H-7b, H-8 0 and phenyl ring.The electronic circular dichroism (ECD) spectrum displayed a positive Cotton effect at 241 (De þ 8.80) nm and negative Cotton effects at 212 (De À 21.35), and 270 (De À 8.23) nm.The absolute configuration of 1 as 7 0 S,8 0 S was determined by the comparison of calculated and experimental ECD data (Supplementary material, Figure S3).Thus, 1 was a new phloroglucinol and was given the name eucalypcamal O.
Eucalypcamal P (2) was obtained as a pale yellowish gum.Compound 2 possessed the same molecular formula C 23 H 26 O 7 as that of 1 which was established by HRESIMS The similar UV, IR and NMR data (Supplementary material, Table S1) of 1 and 2 demonstrated that they were structural isomers; the only difference being the alternation of isovaleryl and formyl , respectively (Supplementary material, Figure S2).Compound 2 showed the same NOESY correlations as 1, confirming the b-orientation of H-7a, H 3 -9 0 and H-7 0 and the a-orientation of the phenyl ring.The comparison of calculated and experimental data and calculated ECD spectra confirmed the absolute configuration of 2 to be 7 0 S,8 0 S (Supplementary material, Figure S3).Compound 2 was thus a new phloroglucinol and given the name eucalypcamal P. Acylphloroglucinols in Eucalyptus species are produced via different biosynthetic pathways.As a result, these compounds exhibit a certain structural diversity.In this study, the plausible biosynthetic pathway of eucalypcamals O and P (1-2) were proposed.The key o-quinone methines (i and ii) are generated from grandinol (9) (Singh et al. 1997) and couple with trans-isoeugenol, the essential oil of Eucalyptus plants (Rencoret et al. 2011), via the hetero-Diels-Alder reaction (Tran and Cramer 2014).The reaction produces the new O-C-7 0 and C-7-C-8 0 bonds that complete the structure of compounds 1 and 2 (Supplementary material, Figure S4).
Compounds 3-10 were evaluated for cytotoxicity against human cancer cell lines including the colorectal cancer cells LoVo and SW48, the cervical cancer cell HeLa, and the bone cancer cell SW1353.However, all the compounds exhibited weak cytotoxicity against these cancer cell lines with IC 50 values >100 lg/mL.Compounds 3-10 were further tested for antimicrobial activity against both Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli) bacterial strains and a fungus (Candida albicans).All were inactive against the tested microbes.

Plant material
The leaves of E. camadulensis Dehnh.were collected from Nong Song Hong village, Huai Haeng subdistrict, Kaeng Khoi district, Saraburi Province, Thailand in April 2017 and identified by Dr. Phattaravee Prommanut.A voucher specimen (BK No. 070170) was deposited in the Bangkok Herbarium, Bangkok, Thailand.

ECD calculations
All configurations of compounds 1 and 2 in this work were optimised using the density functional theory (DFT) approach at the B3LYP/6-31G(d, p) level of theory.The excited states were calculated by using TD-DFT at the B3LYP functional with the 6-311þþG(d, p) basis.The geometry optimisation and TD-DFT calculations were both performed using a polarisable continuum salvation model (PCM) with methanol.The rotary strengths of 80 excited states were calculated.All calculations were performed using the Gaussian09 program package with a fitting parameter of r ¼ 0.3 eV and were processed with the SpecDis 1.64 program (Frisch et al. 2010).

Cytotoxicity assay
Cell proliferation or cell viability of the cell lines was measured by MTT colorimetric assay.Human cancer cell lines including colorectal cancer cells (LoVo and SW48), cervical cancer cell (HeLa), and bone cancer (SW1353) were obtained from the American Type Culture Collection.This study was carried out in accordance with a previously reported procedure (Daus et al. 2022).

Antimicrobial assay
This study was carried out in accordance with a previously reported procedure (Daus et al. 2022).

Conclusions
This investigation of a polar extract (acetone) of the leaves of E. camaldulensis yielded two new acylphloroglucinols together with seven known phloroglucinols and a known benzene derivative.The structure of the new compounds was likely derived from the hetero-Diels-Alder reaction involving the o-quinone methine of grandinol and transisoeugenol.However, the known compounds (3-10) exhibited neither cytotoxicity nor antimicrobial activity.

Figure 1 .
Figure 1.Structure of compounds 1 and 2 from the leaves of E. camaldulensis.