Two new ent-kaurane-type diterpene diastereomers isolated from Coffea canephora

Abstract Phytochemical investigation of the trunks of Coffea canephora yielded two new ent-kaurane diterpene diastereomers, which have been named coffecanepholide A, ent-3β,16β,17-trihydroxykauran-18-al (1) and coffecanepholide B, ent-3β,16β,17-trihydroxykauran-19-al (2). Structural elucidation and configurational assignment were deduced from extensive spectroscopic NMR/HRESIMS analysis and by comparison with the spectral data of the literature relevant structures. The isolated compounds were assayed for in vitro inhibitory activities against α-glucosidase. Structure 2 showed the α-glucosidase inhibitory activity with an IC50 value of 294.7 ± 0.9 μM, while compound 1 exhibited inactivity. In addition, the docking results revealed that structure 2 can form more interactions with amino acid residues at the active site of α-glucosidase, which gave a more negative binding energy (−9.56 kcal/mol) compared with 1 (−8.60 kcal/mol). This observation might be responsible for a better activity of 2 against α-glucosidase. Graphical Abstract


Introduction
Robusta coffee, Coffea canephora (C. canephora) is one of five Coffea species popularly planted in Vietnam (Pham 2000). The numerous chemical compositions of coffee beans have been reported. Diterpene derivatives were to be widely found from Coffea genus and were structurally classified into eight groups: oxidized diterpenoids, rearranged diterpenes, tetrahydrofuran-type diterpenes, lysis-type diterpenes, c-lactonetype diterpenes, D 4,18 -type diterpenes, degraded-type diterpenes, and Villanova-type diterpenes (Hu et al. 2019). Studies on diterpenoids from coffee have been received much attention from researchers. Shu et al has phytochemically investigated and reported the identification of the ent-kaurane diterpenoid glucosides from the acetone extract of roasted coffee beans of Coffea arabica var. yunnanensis (Shu et al. 2014). Investigations on the isolation by Wang et al, which led to the elucidation of the entkaurane c-lactone-type diterpene fatty acid esters from green beans of Coffea arabica (C. arabica). From a biological point of view, the structure-activity relationship (SAR) studies revealed that a hemiketal group at C-3, substituent groups at C-1, and types of fatty acids displayed induction effect for platelet aggregation in vitro (Wang et al. 2018). The N-containing diterpenoids and lactam-type diterpene fatty acid esters isolated from roasted beans of C. arabica and showed moderate inhibitory effects on a-glucosidase (Hu et al. 2020). Furthermore, the ent-kaurane diterpene derivatives belonging to six among eight typical diterpene groups of Coffea genus were also identified from roasted beans of C. arabica. The SAR analysis revealed that diterpenes possessing a C-15/C-16 double bond exhibited stronger a-glucosidase inhibitory activities than the others (Hu et al. 2021).
As part of our ongoing research on phytochemistry and exploring a-glucosidase inhibitory agents from C. canephora, we now focus on the isolation and elucidation of two ent-kaurane-skeleton diterpene diastereomers and their a-glucosidase inhibitory activity evaluation.
The stereochemistry of compound 1 was established by the analysis of coupling constants, chemical shifts, and NOESY experiment (Figure S10, S11 and S30). The NOESY spectrum showed the cross-peaks H-5/H-9 and H-9/H-15b in accordance with the usual stereochemistry of the ent-kaurane skeleton (Martin et al. 1997). The NOESY correlations between H-17/H-15b, H-15b/H-9, H-9/H-5 are clear clues to confirm the b-orientation of 17-oxymethylene group. In other words, the 16a-hydroxy stereochemistry at C-16 was completely determined. The assignment was secured by comparison with the following literature data. The configuration at C-16 of 16a-OH stereoisomer can be distinguished from 16b-OH diastereomer by the differences in the 13 C-NMR chemical shifts of C-16 and C-17. The C-16 and C-17 signals in 16a-OH isomer were located at d C 81.0-85.0 and d C 66.0-68.0 ppm, respectively (Piacente et al. 1994;Wu et al. 1996;Yang et al. 2002;Shu et al. 2014). While the signals of C-16 and C-17 in 16b-OH isomer were observed at d C 78.0-80.0 and d C 69.0-70.0 ppm, respectively (Wu et al. 1996;Yang et al. 2002;Wu 2006;Lee et al. 2020). Herein, in compound 1, the C-16 and C-17 signals were found to resonance at d C 82.7 and d C 66.8 ppm, respectively. Therefore, the relative configuration at C-16 in 1 can be determined as 16a-OH. In addition, it was reported that the diagnostic carbon signal C-13 is a key point to distinguish 16a-OH stereoisomer (C-13, d C 45-46 ppm) from 16b-OH diastereomer (C-13, d C 40-42 ppm) (Wu 2006). Practically, the chemical shift d C 46.4 (C-13) confirmed the 16a-hydroxyl at C-16 of compound 1. The large coupling constant of H-3 (dd, J ¼ 10.5/ 5.5) and the NOE cross peak between H-3 and H-5 suggested an equatorial orientation for the hydroxyl group at C-3. The observation of NOE correlation between H-19 and H-20 was in agreement with the a-axial position of methyl group at C-4. Therefore, the stereochemistry of the formyl group was assigned as b-equatorial orientation. On the basis of the above analysis, compound 1 was elucidated as an enantiomer of a kaurane-skeleton diterpene and named as ent-3b,16b,17-trihydroxykauran-18-al ( Figure 1).
Compound 2 was obtained as white amorphous powder. It had the molecular formula C 20 H 32 O 4 , as evidenced by the HRESIMS (Figure S12) with the pseudo-molecular ion peak for [M þ Na] þ at m/z 359.2201 (calcd for [C 20 H 32 O 4 þNa] þ , 359.2199, D À 0.2 mu). Firstly, the 1 D and 2 D NMR spectra of 2 have been recorded in acetoned 6 . However, a comparison of the overall NMR data revealed significant similarities between 2 and 1, the 1 H and 13 C NMR spectra were secondly measured in methanold 4 for further comparison. When compared with 1, some differences in chemical shifts (Table S1) and NOE correlations at neighbour atoms around C-4 were observed in 2. The difference between 1 and 2 was the chemical shifts of the aldehyde and methyl groups attached at C-4. In the 1 H NMR spectrum of 2 (CD 3 OD), the formyl proton d H 9.97 (s, H-19) was shifted to the downfield, while the value of d H 9.26 (s, H-18) was observed in 1. In the 13 C NMR (CD 3 OD) spectrum, the resonance of the angular methyl group attached C-4 in 2 was downfield shifted (d C 21.1, C-18) with respect to the spectrum of 1 (d C 9.1, C-19). These spectroscopic data of 2 were partly similar to those of ent-16b,17-dihydroxykauran-19-oic acid (Wu et al. 1996) and 16a,17-dihydroxy-entkauran-19-al (Piacente et al. 1994). The observations were attributed to the anisotropic effect of the ent-kaurane A-ring and helpful to assign the stereochemistry on C-4 (Wu 2006). Furthermore, the additional difference was also observed for the chemical shift of the carbon neighbouring the C-4 position. The C-5 signal appeared at d C 48.5 in 1 vs d C 57.8 in 2. The shifted upfield in 1 was attributed to the c-gauche effect of the 18b-CHO group, resulting in a shielding effect on the C-5, which described in the literature. (Wu et al. 1996;Yang et al. 2002;Wu 2006). Indeed, these data suggested that 2 possessed an a-oriented formyl group instead of being b-oriented as in 1. This was further supported by no observable NOE correlation ( Figures S22, S23 and S30) between H-18 and H-20, and the strong NOE correlations between H-19/H-20 and H- 18/H-3 in 2. These lines of evidence established the structure ent-3b,16b,17-trihydroxykauran-19-al for 2 (Figure 1).
The in vitro bio-guided assay against a-glucosidase was carried out on ethyl acetate (EtOAc) extract. The result was described as IC 50 (mg/mL) value. The EtOAc fraction exhibited poor inhibitory activity. We focus on both biological and chemical investigations on C. canephora trunks. Therefore, after the pure compounds were successfully isolated, we further investigated the inhibitory activity against a-glucosidase of compounds 1, 2 (Table S2). For pure compounds, the unit of the IC 50 was converted from mg/mL to mM. Acarbose was used as a positive control (IC 50 ¼ 209.7 ± 0.3 mM). Structure 1 gave no inhibition effect, while compound 2 showed better activity with an IC 50 value of 294.7 ± 0.9 lM.
In order to provide the structural insights of the a-glucosidase pocket, molecular docking was performed to reveal interactions of (1, 2) and amino acid residues of enzyme. The binding energies of stable a-glucosidase-ligand complexes and number of closest amino acid residues surrounding the active sites of the enzyme were determined for the isolated compounds ( Figure S31 and Table S2). It can be assumed that the better inhibitory activity of 2 is mainly attributed to its binding energy, i.e. more negative docking score, DS (2, DS ¼ À9.56 kcal/mol compared with 1, DS ¼ À8.60 kcal/mol). Compound 2 can establish eight interactions with Lys506, Phe476, Asp568, Ala234, Arg552, Ile233, Asp232 and Tyr243, while the Tyr243-ligand interaction was found to be absent in 1. Structure 1 differs from 2 only at the absolute configuration at C-4. This orientation causes no hydrogen bond between the aldehyde group in 1 and Lys506 residue. Although compound 2 gave less hydrogen bonds than 1, it can form a more stable a-glucosidase-ligand complex by forming more interactions at the active sites, which is responsible for the higher activity against a-glucosidase.

General experimental procedures
NMR spectra were recorded on a Br€ uker Avance III spectrometer, 500 MHz for 1 H NMR and 125 MHz for 13 C NMR (Br€ uker BioSpin, Switzerland). HRESIMS spectra were acquired using 1100 Series LC-MSD-Trap-SL mass spectrometer (Agilent Technologies, USA). Optical rotation was performed on a polarimeter P8000 (A. KR € USS Optronic, Germany). The optical absorbances for a-glucosidase inhibitory experiments were measured with a BioTek ELX800 microplate reader (BIOTEK, USA).

Plant material
The trunk of C. canephora was collected in Lam Dong province, Vietnam, in August 2018. Plant material was identified as Coffea canephora Pierre ex A.Froehner by Dr. Van Son DANG from the Institute of Tropical Biology, Vietnam Academy Science and Technology, Vietnam. A voucher specimen was deposited at the Department of Chemical Technology, Ho Chi Minh City University of Technology and Education, Vietnam with the UTE-A001 code.

Extraction and isolation
From the dried powdered trunk (30 kg) of C. canephora, the procedure of the preparation of n-hexane extract (H, 300 g) and ethyl acetate extract (EtOAc, 180 g) from the trunk of C. canephora was described in our previous work (Hoang et al. 2021).

a-Glucosidase inhibitory assay
The procedure for the inhibitory activity of a-glucosidase of the EtOAc extract and the isolated compounds 1, 2 was carried out following the reported literature (Hoang et al. 2021, Apostolidis et al. 2007) with acarbose as a positive control.

Molecular docking
Molecular docking calculations were carried out by using the Molecular Operating Environment (MOE 2008.10) software. The 3 D-crystal structure of a-glucosidase was retrieved from the protein data bank (PDB: 3W37) (Tagami et al. 2013). Prior to docking, enzyme was prepared by LigX suite of MOE following 5 steps: verify ligand, choose ligand, protonate, tether and minimize and delete unbound waters. Acarbose, a co-crystallized ligand with a-glucosidase receptor was selected as a reference ligand. The 3 D-structures of (1, 2) were drawn, then minimized energy by MOE. The scoring function, calculating the binding energies of ligand poses with enzyme pocket was set to 'London dG'. For calculation, the top 10 docking poses were retained for each ligand and then refined by the force field method. Redocking gave a Root Mean Square Deviation (RMSD) value less than 2 Å (RMSD ¼ 1.13 Å), which indicated the reliability of the docking protocol (Plewczynski et al. 2011).

Conclusion
Coffecanepholide A, ent-3b,16b,17-trihydroxykauran-18-al (1) and coffecanepholide B, ent-3b,16b,17-trihydroxykauran-19-al (2) were isolated as two new ent-kaurane diterpenes from the ethyl acetate extract of the trunk of C. canephora. The structures 1, 2 were determined from spectroscopic data. Diastereomeric set of coffecanepholides differed in configuration at C-4. Biologically, compound 2 was shown to possess the inhibitory activity against a-glucosidase (IC 50 ¼ 294.7 ± 0.9 lM), while compound 1 gave no effects. The simulation results showed that compound 2 containing four hydrogen donor/acceptor centers and ent-kaurane diterpene skeleton created efficient interactions with amino acid residues surrounding the binding sites. The formation of the favorable enzyme-ligand complex with lower binding energy can be responsible for the inhibitory activity against a-glucosidase of this structure.