key: cord-0777754-hiwti4fs
authors: Matin, Mohammed M.; Chakraborty, Priyanka; Alam, Muhammad S.; Islam, Mohammad M.; Hanee, Umme
title: Novel mannopyranoside esters as sterol 14α-demethylase inhibitors: Synthesis, PASS predication, molecular docking, and pharmacokinetic studies
date: 2020-08-14
journal: Carbohydrate Research
DOI: 10.1016/j.carres.2020.108130
sha: 39354cbb30662c28675c11daf0e3d49b4bb9aca0
doc_id: 777754
cord_uid: hiwti4fs

Abstract Direct unimolar one-step valeroylation of methyl α-d-mannopyranoside (MDM) furnished mainly 6-O-valeroate. However, similar reaction catalyzed by DMAP resulted 3,6-di-O-valeroate (21%) and 6-O-valeroate (47%) indicating reactivity sequence as 6-OH>3-OH>2-OH, 4-OH. To get potential antimicrobial agents, 6-O-valeroate was converted into four 2,3,4-di-O-acyl esters and 3,6-di-O-valeroate was converted into 2,4-di-O-acetate. Direct tetra-O-valeroylation of MDM gave a mixture of 2,3,4,6-tetra-O-valeroate and 2,3,6-tri-O-valeroate indicating that the C2–OH is more reactive than the equatorial C4–OH. The activity spectra analysis along with in vitro antimicrobial evaluation clearly indicated that these novel MDM esters had better antifungal activities over antibacterial agents. In this connection, molecular docking indicated that these MDM esters acted as competitive inhibitors of sterol 14α-demethylase (CYP51), an essential enzyme for clinical target to cure several infectious diseases. Furthermore, pharmacokinetic studies revealed that these MDM esters may be worth considering as potent candidates for oral and topical administration. Structure activity relationship (SAR) affirmed that saturated valeric chain (C5) in combination with caprylic (C8) chains was more promising CYP51 inhibitor over conventional antifungal antibiotics.

Fatty acid esters of monohydric alcohols such as methanol or ethanol have been reported to be associated with reduced antimicrobial activity of fatty acids [1] . On the other hand, fatty acid esters of the polyhydric glycerol, i.e. monolaurin, have been found to increase their effectiveness as antimicrobial agent [2] . Accordingly, fatty acid esters of sugars with multiple hydroxyl groups constituted an important class of fatty acid derivatives, termed carbohydrate fatty acid (CFA) esters and/or sugar esters (SEs) [3] . These tasteless, and odorless SEs are composed of a hydrophilic carbohydrate moiety, and one or more fatty acids as lipophilic moieties [4] . Therefore, SEs are amphiphilic, nontoxic, biodegradable, nonirritating, and environment friendly [5] . In addition, the good stabilizing, and conditioning properties of CFA esters significantly contributed to their increased inclusion in daily commodities including as low caloric sweeteners, fat replacer, flavorings, and biosurfactants/emulsifiers in foods, food additives, detergents, and in pharmaceutical, biomedical, cosmetic, and oral-care products [6, 7] .

The properties of CFA esters can vary widely from a minute changes in their constituent fatty acid, and sugar moieties and therefore, it is of immense interest to develop CFA esters of diverse physicochemical properties with various biological functions. For examples, several CFA esters have reported to have antitumor, plant growth inhibitory, insecticidal, and miticidal, antibiotic, and antifriction property [8] [9] [10] [11] [12] . Therefore, CFA esters have long been used as versatile intermediates in the syntheses of many natural products due to the presence of multifunctional groups, [13] [14] [15] in addition to their extensive use for design, and development of new drugs [16] .

In recent decades, emergence of multiple drug resistant (MDR) pathogenic strains is a global health concern, a leading cause of nosocomial, and community infections [17] .

Therefore, design and synthesis of new chemotherapeutic agents with novel mode of action is always time-demanding dealing with MDR microbial strains. The structure activity relationship (SAR) of several mannopyranose esters with different side chains concluded that 6-O-myristoyl-D-mannopyranose [( Fig. 1(1 

Staphylococcus aureus ATCC 33591 (MRSA), in addition to its prospective use as environment friendly antimicrobial in food processing, food preservation, and for treating bacterial and fungal diseases in animal, and plants [18] . Very recently, we reported that that incorporation of alkanoyl, and aromatic ester groups on octyl glucopyranoside [ Fig. 1 (2)] as carbohydrate moiety increased antimicrobial potentiality even at very low concentration (10 μgmL −1 ) which may act as competitive inhibitors of lanosterol 14α-demethylase [19] . Synthesis of CFA esters can be achieved by chemical and enzymatic methods, and industrial sugar esters are currently being manufactured by chemical syntheses. However, the two major challenges with CFA ester syntheses are-(i) the presence of several 2° hydroxyl groups of almost similar reactivity, mostly affecting functionalization (esterification) step leading to a mono-, di-, and polyesters [20] , and (ii) the tremendous variation (variety) of carbohydrate structures. Accordingly, various methods have been attempted and reported in the last couple of decades for selective esterification (acylation) [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . All the methods and/or strategies described were successful to some extents, however, suffered from numerous shortcomings, i.e. complex processes of multiple steps, tedious, expensive, low selectivity and extremely low yield [32] [33] . Hence, in most of the cases, direct acylation method maintaining proper reaction conditions is preferred for the monosaccharide-based CFA ester synthesis to reduce the number of steps and increase the final yield(s) [23, 29] .

Most of the antifungal and antiprotozoal drugs are designed through targeting sterol 14α- inhibition [19, 22] similar to azoles, papulacandins etc. In addition, sugar moieties with ester groups as in papulacandins led to higher solubility and reduced hemolytic toxicity [36] .

Hence, one of our major objectives was to get non-azole type MDM based potential CYP51

inhibitor to overcome MDR microbial strains. is δ ~3.75) [37] [38] which clearly demonstrated the incorporation of valeroyloxy group at C-6

position. This observation was furthermore confirmed by the HMBC spectrum ( Figure S8 ). 

In 

To compare biological activities and to examine positional effect of acyl groups we 

Web based PASS (prediction of activity spectra for substances; http://www.pharmaexpert.ru/PASSonline/index.php) was used for the evaluation of antimicrobial activities of the compounds, as reported previously [27] . The PASS prediction data of the mannopyranoside derivatives 4-12 clearly indicated that the MDM esters reported here had reasonable antimicrobial activities (Table 1) . However, the predicted biological activities against bacteria (0.52<Pa<0.58) and fungi (0.62<Pa<0.71) clearly suggested that the MDM esters could be more potent against phytopathogenic fungi as compared to that against bacterial pathogens. The PASS study also revealed that compounds identified from the present study have various potential biological activities, and further studies are needed to validate these predictions. 

The antibacterial activities of the MDM esters were evaluated against one Gram-positive (Staphylococcus aureus), and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria using disc diffusion method [9, 26] . The antibacterial activities were expressed as size of inhibition zone (diameter in mm; J o u r n a l P r e -p r o o f was previously observed for mannose monoester 1 in pyranose form [18] . However, the present MDM ester series (pyranoside form) showed little activity against MRSA probably due to more lipophilicity (presence of multiple acyl chains) and glycosidic nature. Diameter of zone of inhibition in mm (5 μg.dw ⁄ disc) 

The evaluation of in vitro antifungal activities [9] of MDM esters 4-12 against two pathogenic fungi viz. Aspergillus fumigatus ATCC 46645, and Aspergillus niger ATCC 16404 clearly indicated that all the MDM esters had excellent antifungal activities (Table 3 ;

To be more precise, the antifungal activities of MDM esters were very similar and/or even better than that observed with standard antibiotic fluconazole (Table 3) 

To rationalize the excellent in vitro experimental antifungal results of MDM esters 4-12

we conducted their biding affinity with sterol 14α-demethylase (CYP51). Here, we considered XP docking to filter out the false positive ligand by strict scoring function [41] , hydrogen bond, two alkyl bonds, and ten pi-alkyl bonds (Fig. 5a) . On the other hand, compound 8 showed three conventional hydrogen bonds, one carbon hydrogen bond, ten alkyl bonds, and two pi-alkyl bonds (Fig. 5b,6) . (Table S1 ). These observations clearly revealed that novel compound 8 may act as competitive inhibitors of sterol 14α-demethylase (CYP51).

Pharmacokinetic study of the synthetic compounds was conducted using pkCSM [42] , (Table S2) . First, all MDM esters were water soluble, except compound 7 and 11, and all had a good Caco-2 cell line permeability, except the compound 4 and 5 which were very poorly permeable. All CFA esters showed good intestinal absorption rate and skin permeability, the properties of potential candidates as oral and topical drugs. In addition, most of the MDM esters did not show any noticeable interactions with p-glycoprotein, a biological barrier extruding xenobiotics/drugs reducing the bioavailability of the orally taking drugs [43] .

Therefore, all CFA showed overall satisfactory absorption rate.

The volume of distribution (VD) is the theoretical volume that the total dose of a drug would need to be uniformly distributed to give the same concentration as in blood plasma.

The higher the VD is the more of a drug is distributed in tissue rather than plasma. Here, VD of all compounds is low. Thus, our experimental results depicted most these MDM esters as potent antifungal agents via CYP51 inhibition. To this end, we believe that the approaches for new and/or derived non-azole type antimicrobial agents that we report here may pave a way dealing with increased antibiotic resistivity, and may of great interest from global public health viewpoint.

We presented a general and convenient method to synthesize a series of regioselective mannopyranoside based novel CFA esters reasonably in good yields. The applicability of these CFA esters as antimicrobials was tested. Both PASS predication and in vitro evaluation 

To a cooled ( (5) Further elution with chloroform/methanol (12:1) provided slower-moving component (R f = 0.31, chloroform/methanol = 5:1) 4 (2.021 g, 47%) as semi-solid. Its FT-IR, 1 H and 13 C NMR spectra were similar to that of prepared by direct valeroylation in the earlier step.

To a solution of the 4 or 5 (0.1 g) in dry pyridine (1 mL) corresponding acyl halide ( 

Semi-solid; yield 86%; R f = 0.55 (n-hexane/EA = 4:1); FT-IR (KBr) ν max /cm compound were generated and we selected the best conformer with lowest energy.

As the protein target of the molecular docking was Cytochrome P450 sterol 14αdemethylase, its crystal structure was retrieved from the RCSB protein data bank (PDB id:

4UYL; organism: Aspergillus fumigatus). Further, the protein crystal structure 4UYL prepared by using protein preparation wizard of Maestro 11.6 software (Maestro, version J o u r n a l P r e -p r o o f 11.6, Schrödinger, LLC), in which the crystal structure is initially assigned proper hydrogen, charges and bond orders. At neutral pH, all hydrogen bonds in the structure were optimized, deleting unnecessary water. Then the minimization process was run with the OPLS3e force field, considering structural changes of not more than 0.30 Å of RMSD. The active site of the protein was fixed for docking simulation by generating a grid box at the reference ligand (VNI) binding site. Grid generation parameters were kept at default, with a box size of 18Å×18Å×18Å, and the OPLS3e force field utilized for post minimization. The charge cutoff and van der Waals scaling factor were set to 0.25 and 1.00, respectively.

We carried out extra precision (XP) flexible docking using the Glide module of Maestro 11.6 software (Maestro, version 11.6, Schrödinger, LLC), which is more sophisticated than SP/HTVS in scoring function. Here, all ligands were treated flexibly, considering the partial charge and van der Waals factor of 0.15 and 0.80, respectively. Minimization was performed to the docked complex after docking using the OPLS3e force field. The best-docked pose with highest negative Glide score value was recorded for each ligand.

Binding affinity calculations were carried out using the Prime MM-GBSA module of the Maestro 11.6 software (Maestro, version 11.6, Schrödinger, LLC), where greater negative affinity denotes higher stability [46] . The docked Pose viewer file from Glide XP docking was used for calculation, using the sampling minimization protocol used generalized born surface accessible (GBSA) as a continuum model, and OPLS3e force field as molecular mechanics (MM), keeping the protein flexible. For correcting empirical functions of πstacking and H-bond interactions, a dielectric solvent model such as VSGB 2.0 was used. For comparison binding affinity of CFA esters was compared with standard sterol 14αdemethylase (CYP51 ; Table S2 ) inhibitor VNI (the experimental drug for treatment of Chagas disease caused by the fungus Trypanosoma).

In order to predict the pharmacokinetic profile of the derivatives, pkCSM online tool [42] was utilized, where the absorption, distribution, metabolism, excretion and toxicity of each ligand were calculated. Drug-likeness and medicinal chemistry friendliness properties of the derivatives, such as topological polar surface area (TPSA), Ghose violation, bioavailability score and Lipinski's rule of five were predicted by using SwissADME web tool [47] . Before J o u r n a l P r e -p r o o f using these tools, at first, we have drawn all the structures by ChemDraw 18.0 software, and collected InChI key, SMILES and SD file format. InChI key were used to find these compounds in different database and we found our derivatives as not reported in any database yet. SMILES (simplified molecular-input line-entry system) strings were used in pkCSM, and SwissADME online tools while SD file format used in molecular docking. 

Further elution with n-hexane/EA (10:1, v/v) furnished tri-O-valeroate 12 (0.120 g, 26%) as a homogeneous syrup

CDCl 3 ): δ H 5.20 (dd

38 (s, 3H, OCH 3 ), 2.26-2.39 [m, 6H, 3×CH 3 (CH 2 ) 2 CH 2 CO

Anal. Calcd for C 22 H 38 O 9

For antibacterial activities test, the disc diffusion method [9,22] was followed. Dimethylformamide (DMF) was used as a solvent for the test chemicals, and a 2% solution of each compound was used. The plates were incubated at 37 °C for 48 h. The control was DMF without chemicals. Mueller-Hinton (agar and broth) medium was used to culture the bacteria. Each experiment was carried out three times. Tetracycline (antibiotic used for bacterial infections

In vitro antifungal activities were investigated against two human pathogenic fungi viz

Fatty acids and derivatives as antimicrobial agents

Antimicrobial action of isomeric fatty acids on group A Streptococcus

Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida Antarctica

Study of thermal behaviour of sugar esters

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Biological evaluation of some monosaccharide derivatives

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Antibiotic resistance evolution of methicillin resistant Staphylococcus aureus (MRSA) and colloidal silver as the nanoweapon

One-step synthesis of carbohydrate esters as antibacterial and antifungal agents

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Regioselective acylation, alkylation, silylation and glycosylation of monosaccharides

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Synthesis, characterization, ADMET, PASS predication, and antimicrobial study of 6-O-lauroyl mannopyranosides

Synthesis of 6-Ostearoyl-1,2-O-isopropylidene-α-D-gluco-furanose derivatives for antimicrobial evaluation

Synthesis and comparative antimicrobial studies of some acylated D-glucofuranose and Dglucopyranose derivatives

Design and synthesis of benzyl 4-O-lauroyl-α-L-rhamnopyranoside derivatives as antimicrobial agents

Synthesis and antimicrobial study of some methyl 4-O-palmitoyl-α-Lrhamnopyranoside derivatives, Orbital: The Electron

Synthesis, PASS-predication and in vitro antimicrobial activity of benzyl 4-O-benzoyl-α-L-rhamnopyranoside derivatives

Regioselective acylation of a derivative of L-rhamonse using the dibutyltin oxide method

Comparative studies on selective acylation of uridine using the dibutyltin oxide and direct methods, Chittagong Univ

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A critical overview on the effect of microwave irradiation in organic synthesis

Lipase-catalyzed syntheses of sugar esters in non-aqueous media

Substituent effects on the regioselectivity of enzymatic acylation of 6-O-alkylglycopyranosides using Pseudomonas cepacia lipase

Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

Synthesis, spectroscopic characterization, molecular docking, and ADMET studies of mannopyranoside esters as antimicrobial agents

In silico and in vitro screening to identify structurally diverse non-azole CYP51 inhibitors as potent antifungal agent

PASS predication, antiviral, in vitro Antimicrobial, and ADMET studies of rhamnopyranoside esters

Synthesis and antifungal activity of palmitic acid-based neoglycolipids related to papulacandin D

Rapid and efficient synthesis of α(1-2) mannobiosides

Selective benzoylation of methyl α-Dmannopyranoside using the dibutyltin oxide and direct methods

Chemoenzymatic synthesis of GDPazidodeoxymannoses: non-radioactive probes for mannosyltransferase activity

Acetylpyridinium ion intermediate in pyridine-catalyzed hydrolysis and acyl transfer reactions of acetic anhydride. Observation, kinetics, structure-reactivity correlations, and effects of concentrated salt solutions

Highly efficient propionylation and butyralation of cellulose in an ionic liquid catalyzed by 4-dimethylminopyridine

Identification of natural compound inhibitors for multidrug efflux pumps of

Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation

pkCSM: Predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures

Role of P-glycoprotein in pharmacokinetics: clinical implications

The effects of aliphatic alcohols on the biophysical and biochemical correlates of membrane function

Evidence for recycling of cytochrome P450 sterol 14-demethylase from the cis-Golgi compartment to the endoplasmic reticulum (ER) upon saturation of the ER-retention mechanism

The MM/PBSA and MM/GBSA methods to estimate ligandbinding affinities

SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules

We highly acknowledge the financial support from the Ministry of Education, Bangladesh and BANBEIS (grant no. PS 201660, 2016-17).

The authors declare no conflict of interest. All authors have approved the final article.

FT-IR, 1 H & 13 C NMR, 2D COSY, HSQC, HMBC spectra, antimicrobial pictures ( Fig   S1-S68) , MM-GBSA Binding affinity and ADMET evaluation Tables (Table S1 -S2) are available as supplementary data.