α-Glucosidase inhibitory derivatives of protocetraric acid

Abstract Lichen-derived depsidones have been a successful source for alpha-glucosidase inhibitory agents with numerous advantages. In this article, derivatives of protocetraric acids were designed and synthesised. Diels-Alder reaction, esterification, and Friedel-Crafts alkylation of protocetraric acid with different reagents under Lewis acid were performed. Eleven products were prepared, including 10 new compounds and parmosidone A. Among them, compounds 2–4 and 6 had the novel skeletons. The newly synthetic products were evaluated for alpha-glucosidase inhibition. Among tested compounds, 9 showed the strongest activity, with an IC50 value of 5.9 µM. The molecular docking model indicated the consistency between in vitro and in silico data of alpha-glucosidase inhibition. Graphical Abstract


Introduction
Lichens are complex symbiotes of fungi and algae that produce a range of bioactive metabolites (M€ uller 2001(M€ uller , Huneck 2001. Among them, depsidone, an unique scaffold, possesses diverse biological properties, such as UV blockers Yoshimura 1996, Russo et al. 2008), antioxidant (Ismed et al. 2017, Chomcheon et al. 2009), antifungal (Millot et al. 2017), inhibitor of malignant cancer cells, antiviral, antimicrobial, and enzyme inhibitor (Rankovi c 2015, Nishanth et al. 2015, Boustie and Grube 2005, Boustie et al. 2011, Huneck 1999, Micheletti et al. 2009, Honda et al. 2010, Nakazawa et al. 1962, Pittayakhajonwut et al. 2006. Lichen-derived depsidones have also been reported to have promising a-glucosidase inhibitory properties (Ngoupayo et al. 2008, Karunaratne et al. 2014, Duong et al. 2020, Devi et al. 2020, Pham et al. 2022. In 2020, Devi and co-workers reported strong alpha-glucosidase inhibition of salazinic acid derivatives isolated from Parmotrema indicum with IC 50 values in the range 2.2-82.7 mM. In the same year, three natural derivatives of protocetraric acid (1) were isolated and they exhibited alpha-glucosidase inhibition with IC 50 values: 10.7, 11.4, and 17.6 mM (Duong et al. 2020). More recently, Pham and her colleagues synthesised eight new derivatives of salazinic acid and provided their complete in vitro and in silico data of alpha-glucosidase inhibition (Pham et al. 2022). These results indicated that the transformation of 3-CHO and 3 0 -CH 2 OH groups of salazinic acid would significantly enhance the activity. Parmosidone K, a natural derivative of parmosidone A (5) was reported as a potent inhibitor against alpha-glucosidase with an IC 50 value of 3.12 mM (Nguyen et al. 2022). Protocetraric acid (1), a depsidone having a benzyl alcohol moiety, were derived from the lichen-genera Usnea, Parmotrema, Cladonia, and Sticta (Culberson 1966, B ezivin et al. 2004, Nguyen et al. 2018, Duong et al. 2015, Verma et al. 2011). Sala and Sargent first reported its synthesis via a 15-step process starting with methyl b-orsellinate (Sala and Sargent 1981). Protocetraric acid (1) is a significant broad-spectrum antimicrobial against medically-significant human pathogens (Nishanth et al. 2015). Several protocetraric acid derivatives have been synthesised, yielding phenylhydrazone, thiosermicarbazone, and benzimidazole derivatives from the addition of the aldehyde moiety (Klosa 1952, Keogh 1977, Asahina 1933. Other ester derivatives have been prepared by attacking the hydroxymethylene group. Data on these synthetic derivatives are by far scarce, and few reports have addressed their biological properties. Therefore, in this study, we investigated the synthesis of novel depsidones using various reactions of 1 under Lewis acid. Synthetic compounds were evaluated for alpha-glucosidase inhibition. Molecular docking studies were performed to clarify the molecular understanding to alpha-glucosidase inhibition.

Results and discussion
Diels-Alder cycloaddition of 1 and several chalcones was conducted in the search for novel a-glucosidase inhibitory products. Among the chalcones, the occurrence of the electron-donating group in the C-ring, a methoxy group, was expected to yield products 2 and 3 by application of the inverse Diels-Alder cycloaddition via an ortho-quinone methide intermediate (Figure 1). It is known that the two ortho benzyl alcohol and hydroxyl groups of the B-ring enable the formation of ortho-quinone methide (Water and Pettus 2002, Lumb et al. 2008, Yoshida et al. 2011. This intermediate was reacted with chalcone to form products (Scheme S1). The chemical structures of compounds 2-3 were clarified by analysis of the 1 D and 2 D NMR data. The NMR data on 2 closely matched those of 1, indicating that they shared the same A-and B-rings. The upfield chemical shift of H 2 -8 0 (d H 3.16 and 3.12) and the presence of nine novel aromatic protons in the d H range 8.00-6.70 indicated that the reaction occurred at C-8 0 . In addition, the absence of the two trans olefinic protons of chalcone and the presence of two aliphatic methines (d H 5.14, 1H, d, 9.0, H-11 0 and 4.40 1H, m, H-10 0 ) confirmed that Diels-Alder cycloaddition had completed. This was further supported by HMBC correlations of H 2 -8 0 and H-10 0 to C-11 0 (d C 78.7) and C-1", of H-11 0 to C-2"'/6"' (d C 128.1), and by the mass spectroscopic data. Furthermore, the trans pseudo-diaxial orientations of H-10 0 and H-11 0 were defined by a large 3 J 10'-11 0 coupling constant of 9.0 Hz (Boyer et al. 2006).
In contrast, when the chalcones lacked the methoxy group in the C-ring (entry 4, Table S1), none of the expected products were obtained. Instead, self-conversion of 1 occurred and produced a novel product (4) and parmosidone A (5). The NMR data on 4 and 1 were similar, except for the two structural differences. The first was the absence of carbon signals at d C 170.2 (Brandão et al. 2013) and the presence of an additional aromatic proton (d H 6.55), indicating the decarboxylation of 1 0 -COOH. The second was a structural change from the 7-member ring of 1 to the 8-member lactone of 4. This has also been reported for penicillide compounds (Komai et al. 2006, Zhao et al. 2015. It was reflected in a downfield chemical shift of H 2 -7 0 (d H 5.13 in 4 compared with d H 4.60 in 1) (Brandão et al. 2013). To confirm this facile conversion, treatment 1 with AlCl 3 was repeated under the same conditions, affording 4 and parmosidone A (5) (Figure 1). This is the well-known Smile rearrangement (Snape 2008). The formation of these products indicated that 1 and parmosidone A could be interchanged via their respective intermediates I and II (Scheme S2). This mechanism was proposed for the biosynthesis of parmosidones (Duong et al. 2015). The formation of 4 could be explained by intra- When chalcones are replaced with a,b-unsaturated carboxylic acid, such as 4-methoxycinnamic acid, both Diels-Alder product and esterified products were formed. Esterification of 1 and 4-methoxycinnamic acid produced a cycloaddition product (6) and the two esters 7-8. Although the NMR data on 6 and 5 were similar, the absence of the E-double bond indicated that 6 had a different scaffold. The NMR results for 6 were also very similar to those for 2-3, with the exception of the replacement of the carboxylic acid group on the D-ring. In the reaction, two esters were formed: ester 7 from PA and ester 8 from 5. This further confirmed the conversion of 1 to 5 in the reaction.
Molecular docking studies were conducted for compounds 1-3, 9, 10, and 12. For estimating the binding affinity and binding pose of a ligand to a protein, AutoDock 4 and AutoDock Vina have been widely used. The latter has been reported to be up to 77% more accurate and precise than the former in successful docking of over 800 protein-ligand complexes, according to the benchmark reported by Nguyen et al. (2020). In this study, AutoDock 4 was used to assess the binding affinity of the targeted complexes. Table S3 shows the experimental and docked free energy of binding while Figure S42 illustrates their correlation. All the tested compounds were found to have stronger a-glucosidase inhibitory activity than that of acarbosean approved anti-diabetic drug (Table S3). In addition, a good correlation (R ¼ 0.81) was found between IC 50 experiments and docking values ( Figures S42 and S43), thus confirming the effectiveness of the in silico screening for the activity against a-glucosidase of the synthesised compounds. Interactions between the protein and ligands were analysed using MOE 2015.10. The complexes of the protein and ligands 10 and 11 formed five Hbonds with a-glucosidase binding sites, giving their binding affinities significantly lower than the one of ligands 1 and 9 which had only four H-bonds. Some key residues must be mentioned such as Phe 163, His 203, Asn 258, and Arg 411. They are present in most of the H-bonding interactions of the ligands studied ( Fig S43) and these results are in agreement with previously published observations (Barmak et al. 2019, Eawsakul et al. 2021).

General experimental procedures
NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer using TMS as internal reference and the solvent residual signals of DMSO-d 6 (d H 2.50, d C 39.5) and acetone-d 6 (d H 2.05, d C 29.4). HR-ESI-MS data were recorded on a Bruker microTOF-Q II apparatus. Open column chromatography was carried out on silica gel 60 (40-63 mm) (Merck, Darmstadt, Germany). All solvents (Chemsol, Vietnam) were distilled before use. The progress of all reactions was monitored by TLC on silica gel 60 F254 plates (Merck, Darmstadt, Germany). Spots were visualised under UV by dissolving vanilin in H 2 SO 4 followed by heating. Analytical and preparative TLC were performed on precoated 0.20 mm thick plates of silica gel 60 F254 (Merck, Darmstadt, Germany). Protocetraric acid was isolated from the lichen Usnea baileyi (Nguyen et al. 2018).
3.2. General procedure for the preparation of Diels-Alder products 2, 3, and 6 A solution of protocetraric acid (1, 50.0 mg, 0.134 mmol) and the corresponding chalcone (2.68 mmol) was stirred in dimethylformamide at room temperature. A solution of AlCl 3 (2.75 mg, 0.0206 mmol) in DMF was added and the mixture was stirred at 90 C for 3 h. The reaction was periodically monitored using TLC. The mixture was partitioned between ethyl acetate and water (50 mL each). Organic layers were pooled, washed with brine, and dried over anhydrous Na 2 SO 4 . The residue was purified by normal phase silica gel column chromatography, eluted with n-hexane-ethyl acetateacetone-acetic acid (3.5:10:4:2.4:0.8, v/v/v/v), yielding products 2, 3, and 6 (Scheme 1).

General procedure for the preparation of 4 and parmosidone A (5)
AlCl 3 (5.5 mg, 0.041 mmol) was added to a solution of protocetraric acid (1, 50.0 mg, 0.134 mmol) in DMF solvent (2 mL). The mixture was stirred at 90 C for 3 h. The reaction was periodically monitored using TLC. The mixture was partitioned between ethyl acetate and water (50 mL each). Organic layers were pooled, washed with brine, and dried over anhydrous Na 2 SO 4 . The residue was purified by normal phase silica gel column chromatography, eluted with chloroform-ethyl acetate-acetone-acetic acid (100: 40: 24: 8, v/v/v/v), yielding product 4 and parmosidone A (5) (Scheme 1).

General procedure for synthesis of esterification analogues 7-12
A solution of protocetraric acid (1, 20.0 mg, 0.0534 mmol) and 4-methoxylcinnamic acid (437.9 mg, 2.46 mmol) in DMF was stirred at room temperature. A solution of AlCl 3 (2.1 mg, 0.0164 mmol) in DMF was added and the mixture was stirred at 90 C for 3 h. The reaction was periodically monitored using TLC. The mixture was partitioned between ethyl acetate and water (50 mL each). Organic layers were pooled, washed with brine, and dried over anhydrous Na 2 SO 4 . The residue was purified by normal phase silica gel column chromatography, eluted with n-hexane-ethyl acetateacetone-acetic acid (10:1:0.2:0.2, v/v/v/v), yielding 7 and 8. Using benzoic acid, a-methylcinnamic acid, cinnamic acid, and 4-methylcinnamic acid as reagents, the reactions were conducted as previously described, providing products 9, 10, 11, and 12, respectively (Scheme 1).
(2S,3S)-8-formyl-9-hydroxy-3-(4-methoxyphenyl)-6,11-dimethyl-12-oxo-2,3-dihydro-1H,12H-benzo (E)-4-formyl-3,7-dihydroxy-1, 9-dimethyl-11-oxo-6-(((3-(p-tolyl)  3.5. General procedure for in silico molecular docking The crystal structure of alpha-glucosidase in complex with maltotriose was retrieved from the Protein Data Bank (PDB) (Berman et al. 2000, Auiewiriyanukul et al. 2018. The structure of the alpha-glucosidase protein was then obtained by the removal of maltotriose. This allowed the binding site of the inhibitors to be determined. The three-dimensional structures of the inhibitors were generated via chemicalize.com. All the parameters employed for investigating the alpha-glucosidase protein and inhibitors were extracted using AutoDockTools 1.5.6 (ADT) (Morris et al. 2009). The receptor and ligands were assigned polar hydrogen bonds and charges using the Gasteiger-Marsili method (Gasteiger and Marsili 1980). A grid box of 2.4 Â 2.4 Â 2.4 nm was created for preparation of a grid map, using AutoGrid. A genetic algorithm (GA) was used to search for the conformational position of the ligand binding to the protein with the lowest binding energy. The GA executed 250 runs in the long option of the maximum number of evals ). The population size was 300 and the maximum number of generations was set at 27000 . AutoDock version 4.2 was used to simulate the molecular docking of those protein-ligand complexes that showed IC 50 < 100 mM. The best predicted free energy of binding and root-mean-square deviation (RMSD) identified the 250 configurations with the most favourable energy of binding. The interactions between protein-ligand were analysed using Molecular Operating Environment software version 2015.10 (MOE 2015.10) (Stierand and Rarey 2010).

a-Glucosidase inhibition assay
The a-glucosidase inhibition assay was performed using a slight modification of a published method (Tran et al. 2021).

Conclusions
Eleven products (2-12) were successfully synthesised from protocetraric acid using Diels-Alder cycloaddition, Smile rearrangement, esterification, and Friedel-Crafts alkylation under Lewis acid. Compounds 2-4, and 6 were shown to have novel scaffolds. Conversion between protocetraric acid (1) and parmosidone A (5) was confirmed. All synthesised compounds were evaluated for a-glucosidase inhibition. Products 1-3, 9, 10, and 12 showed good activity against a-glucosidase with IC50 values in the range of 5.9-50.7 mM, being stronger than both a positive control (acarbose, IC 50 330.3 mM) and the original compound (1, IC 50 81.6 mM). Compound 9 showed the strongest activity, with an IC 50 value of 5.9 mM. An in silico molecular docking study provided the understanding of mechanism of alpha-glucosidase inhibition of bioactive compounds.