Syntheses and Glycosidase Inhibitory Activities, and in Silico Docking Studies of Pericosine E Analogs Methoxy-Substituted at C6.

Inspired by the significant -glucosidase inhibitory activities of (+)- and (-)-pericosine E, we herein designed and synthesized 16 analogs of these marine natural products bearing a methoxy group instead of a chlorine atom at C6. Four of these compounds exhibited moderate -glucosidase inhibitory activities, which were weaker than those of the corresponding chlorine-containing species. The four compounds could be prepared by coupling reactions utilizing the (-)-pericosine B moiety. An additional in silico docking simulation suggested that the reason of reduced activity of the C6-methoxylated analogs might be an absence of hydrogen bonding between a methoxy group with the surrounding amino acid residues in the active site in -glucosidase.


Syntheses of Pericosine E Analogs Methoxy-Substituted at C6
All possible isomers derived from pericosine B or pericosine C were considered. As mentioned in our previous paper, the acceptor of the coupling reaction was limited to trans-epoxide 11 enantiomers [10,11]. (+)-6, (−)-7, (−)-8, and (−)-9 were prepared following a previously reported strategy (Scheme 1). Both enantiomers of 10 and 15, which are synthetic precursors of pericosines C and B respectively, could be used as donor molecules in the Lewis-acid-catalyzed coupling reaction [12,13].
Considering the above, we herein aimed to design and synthesize pericosine E analogs bearing a methoxy group at C6 instead of a chlorine atom and elucidate the corresponding structure-activity relationships. The design of these targets was inspired by the possible existence of the hitherto unknown naturally occurring pericosine E-type O-linked carbadisaccharides comprising known pericosine B or C units with various combinations of chiralities, as the parent pericosine E is thought to be biologically synthesized from pericosines A and B. Moreover, we aimed to determine whether the replacement of the chlorine atom at C6 with a methoxy group influences enzyme inhibitory activity by electronic effect or size of the substituent. In our previous work, the characteristic structural pattern of pericosine E analogs was denoted as (donor, acceptor)-type for a better understanding of the puzzling stereochemistry with eight chiral centers in each compound. For example, (−)-4 was denoted as (−pA, +pB)-type, as it can be constructed from (−)-pericosine A and (+)-pericosine B moieties. A similar notation was used in the present paper. Inspection of naturally occurring possibilities resulted in the design and synthesis of novel carbadisaccharides 6, 7, 8, and 9, with (+pC, −pB)-, (+pB, −pB)-, (−pC, −pB)-, and (−pB, −pB)-type structures, respectively. The corresponding enantiomers were also synthesized.
Herein, we report the syntheses of sixteen pericosine E analogs bearing a methoxy group at C6 instead of a chlorine atom and evaluate their glycosidase inhibitory activities. In addition, results of the docking simulation on active compounds and α-glucosidase is described.

Syntheses of Pericosine E Analogs Methoxy-Substituted at C6
All possible isomers derived from pericosine B or pericosine C were considered. As mentioned in our previous paper, the acceptor of the coupling reaction was limited to trans-epoxide 11 enantiomers [10,11]. (+)-6, (−)-7, (−)-8, and (−)-9 were prepared following a previously reported strategy (Scheme 1). Both enantiomers of 10 and 15, which are synthetic precursors of pericosines C and B respectively, could be used as donor molecules in the Lewis-acid-catalyzed coupling reaction [12,13].
The above results imply that original pericosine E analogs containing a chlorine atom at C6 are more potent α-glucosidase inhibitors than their methoxy analogues. Notably, compounds featuring (−)-11 as an acceptor exhibited a certain anti-α-glucosidase activity, except for (−)-18. These findings provided useful information for the design of new molecules with better activities.

Docking Simulation
A molecular docking study using the Dock function [18] of Molecular Operating Environment (MOE) version 2018.0101 (Chemical Computing Group Inc., Quebec, Canada) was conducted for better understanding of the inhibitory mechanisms (Figures 2 and 3). The interactions between α-glucosidase (Protein Data Bank (PDB) code 3A4A) [19] with compounds (−)-5 and (−)-24 as well as glucose were investigated using the MOE-Dock. glucosidase (Protein Data Bank (PDB) code 3A4A) [19] with compounds (−)-5 and (−)-24 as well as glucose were investigated using the MOE-Dock.    As a result, the peripheral amino acids important for glucose binding were observed to be involved in the binding of compounds (−)-5 and (−)-24. It was shown that the chlorine atom of compound (−)-5 could form hydrogen bonds with the surrounding amino acid residues such as Val216, Glu277, Gln279, and Phe303. No such binding was observed for glucose and the methoxy group of the compound (−)-24. Furthermore, the configurations of compound (−)-5 and compound (−)-24 were quite different, and the surrounding amino acids that bind α-glucosidase were also As a result, the peripheral amino acids important for glucose binding were observed to be involved in the binding of compounds (−)-5 and (−)-24. It was shown that the chlorine atom of compound (−)-5 could form hydrogen bonds with the surrounding amino acid residues such as Val216, Glu277, Gln279, and Phe303. No such binding was observed for glucose and the methoxy group of the compound (−)-24. Furthermore, the configurations of compound (−)-5 and compound (−)-24 were quite different, and the surrounding amino acids that bind α-glucosidase were also different.
Tang and co-workers identified 12 amino acid residues, including Val 216, Glu 277, Gln 279, and Phe 303, that interact with the α-glucosidase inhibitors salvianolic acid A and salvianolic acid C. The chlorine-containing compound (−)-5 might inhibit α-glucosidase in a similar binding manner to those inhibitors [20].
Comparisons of affinity scores between different substrates are known to be useful in assessing agonist/antagonist activity [21]. The binding affinity scores of compounds (−)-5, (−)-24, and glucose were −6.8, −4.7, and −8.2 kcal/mol, respectively. Glucose, which is included as a partial residue of natural substrates, exhibited the highest affinity among the three docked compounds. Also, the affinities of compounds (−)-5 and (−)-24 reflected the inhibitory activities, 1.2 × 10 −5 and 1.7 × 10 −3 , respectively. These results indicate that the docking studies were achieved with a certain accuracy. On the other hand, the threshold value of the hydrogen bond strength depicted in Figure 2 was set to −1.0 kcal/mol for glucose and −0.1 kcal/mol for derivatives. Compound (−)-5 is considered to contribute to stronger binding affinity by interacting with more peripheral amino acids than compound (−)-24. However, the accuracy of the docking simulation is limited. It is considered necessary to perform molecular dynamics (MD) simulation to obtain more detailed knowledge.
α-glucosidase 3A4A is one of the crystal structures representative of α-glucosidase and is generally used for the study of this enzyme [22][23][24][25]. On the other hand, α-glucosidase, β-glucosidase, α-mannosidase, α-galactosidase, and β-galactosidase used in this study showed different inhibition profiles against various pericosine derivatives. These results suggest that the steric configurations of amino acids at the ligand-binding site among these enzymes are significantly different. The relationship among the amino acid sequences of the ligand-binding sites and the inhibitory activities will be rigorously evaluated in a subsequent study.

Conclusions
We designed and synthesized 16 pericosine E analogs bearing a methoxy group at C6 and tested their inhibitory activities against five glycosidases. Among the 16 compounds, four exhibited lower α-glucosidase inhibitory activities than the corresponding chlorine-containing species. Thus, the chlorine atom at C6 in pericosine E analogs was concluded to play an important role in determining α-glucosidase inhibitory activity. The identified active compounds generally featured the (−)-pB moiety as an acceptor. Contrarily, some compounds mainly comprising the (+)-pB moiety showed β-galactosidase inhibitory activity, with (−)-24 acting as a dual inhibitor. Docking simulation suggested that (−)-24 synthesized in this study binds α-glucosidase in different manner to more potent (−)-5, whose chlorine atom forms hydrogen bonds with the surrounding amino acid residues.

Docking Simulation
The docking analysis was carried out using α-glucosidase protein (PDB code 3A4A) using the Dock induced-fit function in Molecular Operating Environment (MOE) version 2018.0101 (Chemical Computing Group Inc., Quebec, Canada) to better understand the inhibitory mechanisms. [18,19] The calculation of the binding affinity scoring function was performed with the amber 10:eht force field, triangle matcher as placement, and GBVI/WSA dG as the binding affinity scoring function [26]. In the protein preparation with respect to charged residues in the binding site, the Protonate three dimensional (3D) option in MOE was used to determine the ionization states and add hydrogens to the system [27]. The function of the Protonate 3D allows to assign ionization states and position hydrogens in a macromolecular structure given its 3D coordinates from the crystal structure. Hydrogen atoms are required for all atom molecular mechanics, dynamics, or electrostatic calculations. The addition of hydrogen atoms to a macromolecule is a non-trivial task; generally, one must determine the rotamers of -SH -OH -CH 3 and -NH 3 groups in cysteine (CYS), serine (SER), tyrosine (TYR), threonine(THR), methionine (MET), and lysine (LYS), the ionization states of acids and bases in arginine (ARG), aspartic acid (ASP), glutamic acid (GLU), LYS, histidine (HIS), the tautomers of imidazoles (HIS) and carboxylic acids (ASP, GLU), the protonation state of metal-ligand atoms CYS, HIS, ASP, GLU, etc., and the ionization state of metals.