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

Article history: Received August 21, 2016 Received in revised form October 14, 2016 Accepted 15 October 2016 Available online 15 October 2016 Benzyl -L-rhamnopyranoside, prepared by both conventional and microwave assisted glycosidation techniques, was converted into benzyl 2,3-O-isopropylidene-α-Lrhamnopyranoside which after lauroylation followed by removal of isopropylidene group gave the benzyl 4-O-lauroyl-α-L-rhamnopyranoside in good yield. Several derivatives of benzyl 4O-lauroyl-α-L-rhamnopyranoside were prepared and assessed in vitro for their antimicrobial activity against ten human pathogenic bacteria and seven fungi. The structure activity relationship (SAR) study revealed that incorporation of 4-O-lauroyl group in rhamnopyranoside frame work along with 2,3-di-O-acyl group increased the antifungal potentiality of the rhamnopyranosides. © 2017 Growing Science Ltd. All rights reserved.


Introduction
L-Rhamnose, an important member of the monosaccharide series, 1 is widely distributed in nature, it was found in plant gums, plant glycosides and in bacterial polysaccharides. 1,2Some disaccharides having L-rhamnose as the aglycone has been synthesized and are important for the determination of the immunodominant site in antigenic lipopolysaccharides. 3 The aldobiouronic acid 4-O-(8-Dglucopyranosyluronic acid)-L-rhamnose has been isolated from hydrolysates of Acrosiphonia centralis, Ulva lactuca and Klebsiella K9 capsular polysaccharide. 3Also, 5-O--L-rhamnopyranosyl--Larabinofuranose (1, Fig. 1) has been found as the sugar component of sitosterol glycoside and showed rhamnosidase specificity in Aspergillus niger. 4The diacetyl derivative of the natural product kaempferol-3-O-(3',4'-di-O-acetyl-α-L-rhamnopyranoside), also called SL0101 (2), is a highly specific protein kinase (RSK) inhibitor. 5Compound 2 was isolated from Forsteronia refracta, a variety of dogbane found in the South American rainforest.This diacetyl compound 2 was found 12 times more inhibitor of RSK in vitro than that of its non-acetyl analogue 3. Thus, diacyl compound 2 inhibits the growth of cancer cell lines. 5Acylation of the rhamnose moiety in these natural products is necessary for high affinity binding and selectivity.These results should facilitate the development of RSK inhibitors derived from SL0101 as anticancer agents.The branched L-rhamnopyranosides are found abundant in nature. 6][9] Although, regioselectivity is a major challenge as carbohydrates contain several hydroxyl groups of similar reactivity.Small differences in reactivity cannot be utilized for selective protection and modification of hydroxyl group.However, desired protection pattern can be achieved in one or few steps making use of complex reaction sequences. 10For example, organotin reagents, such as tributyltin oxide or dibutyltin oxide [11][12] are often used to accomplish regioselective protection, including acylation [13][14] of hydroxyl group of carbohydrate derivatives.6][17] In this context, our main aim was to establish a method for the synthesis of 4-O-lauroylrhamnopyranoside via protection-deprotection technique.
In recent years, search for new antibacterial agents with novel mode of action represents a major target in chemotherapy 18 as the emergence of multiple antibiotic resistant pathogenic bacteria causing threat to human health worldwide.0] The sugar moieties present in these esters can increase drug water solubility, decrease toxicity, and contribute to the bioactivity of the natural products.4][25][26] Attachment of aryl and acyl group(s) to the sugar molecules enhances the biological activities many times than that of the parent sugar 27,28 .Considering these important observations, we are interested to the introduction of lauroyl group at position C-4 of benzyl -L-rhamnopyranoside (4) instead of acyl group at C-3 position.This may provide important information about positional effects of the acyl group in its role as antimicrobial functionality.

Results and Discussion
Our present research work mainly describes the synthesis of benzyl 4-O-lauroyl--Lrhamnopyranoside (7) with its 2,3-di-O-acyl derivatives (8-10) and antimicrobial evaluation/studies of all the synthesized products.

Synthesis of benzyl 4-O-lauroyl--L-rhamnopyranoside (7)
6][17] Thus, protectiondeprotection method was employed successfully for the 4-O-lauroylation of rhamnopyranoside 4. Initially, benzyl -L-rhamnopyranoside (4) was prepared from L-(+)-rhamnose according to the literature procedure 12,29  To improve the yield of 4, we have applied microwave irradiation to conventional Fischer glycosidation.Thus, microwave irradiation of finely powdered L-rhamnose with little excess dry benzyl alcohol (in a porcelain dish) and Amberlite IR 120 (H + ) ion exchange resin at 160 watts for 90 sec in a domestic microwave oven followed by short silica gel column provided pure benzyl rhamnopyranoside 4 almost in quantitative yield (96%), as a brownish thick liquid.Notable, the achieved in this method yield was very high and the reaction time was shorter (only 90 sec) compare to the conventionaly heated reaction (20 h).Having benzyl -L-rhamnopyranoside (4) in hand, we have protected its cis-vicinal glycol group at position C-2 and C-3 by isopropylidene protecting group.Treatment of 4 with excess 2,2-DMP in the presence of catalytic amount of p-TSA afforded 5, as an oil, in 79% yield.In its IR spectrum, stretching bands at 3450-3300 (br) and 1381 cm 1 belong to hydroxyl group and isopropylidene group, respectively.In the 1 H NMR spectrum, two three-proton singlets at  1.33 and 1.32 ppm confirm the presence of one acetonide group in the molecule.Based on the spectral analysis, the structure of 5 was established as benzyl 2,3-O-isopropylidene--L-rhamnopyranoside.The acetonide protection was formed between cis-vicinal 2,3-diol positions of 4 and Liptak et al. reported the similar type of acetonide formation. 30The monoacetonide 5, having free hydroxyl group at C-4 position, was used in mono-lauroylation in reaction with lauroyl chloride in dry pyridine to afford 6 as a viscouscous oil (Scheme 1).IR spectrum of the compound 6 possessed the carbonyl-stretching band at 1708 cm 1 instead of the C-4 hydroxyl group band at 3450-3300 cm 1 .The proton spectrum was consistent with the structure of compound 6.The presence of lauroyl group was confirmed by the integrating the regions of 1 H NMR spectrum at about 0.87 (3H), 1.21-1.34(16H, overlapping multiple signals) 1.59-1.65 (2H, m), and 2.36 (2H, t) ppm, totaling to 23 proton equivalents.In addition, the downfield shift of H-4 (4.90 ppm) as compared to the precursor compound 5 (4.42-4.48ppm) confirmed the attachment of the lauroyloxy group at C-4 position of the molecule.Thus, the structure of benzyl 2,3-O-isopropylidene-4-O-lauroyl--L-rhamnopyranoside (6) was confirmed.In the subsequent step, removal of the acetonide functionality was achieved by stirring 4-O-lauroate 6 with glacial acetic acid at 40 ºC for 18 h to give a semi-solid 7 (82%).In the IR spectrum of 7, the presence of a new broad band at 3510-3280 cm 1 corresponding to hydroxyl groups witnessed the removal of isopropylidene moiety.This fact was also confirmed by observation of the absence of isopropylidene protons in the 1 H NMR spectrum, while a broad two-proton singlet (exchanged with D2O) at 1.87-2.16ppm in that spectrum corresponds to two hydroxyl groups.Thus, the structure benzyl 4-O-lauroyl--L-rhamnopyranoside (7) was unambiguously assigned.

Scheme 2. Synthesis of compounds 8-10
Similarly, mesylation of 4-O-lauroate 7 gave a compound 9 in 81% yield.Its IR spectrum showed no signal for hydroxyl group and thus indicated the mesylation of the compound.In its 1 H NMR spectrum, two three-proton singlets at 3.15 and 3.12 ppm clearly indicated the attachment of two mesyloxy groups in the molecule.The reasonable downfield shift of H-2 (4.98 ppm) and H-3 (5.05 ppm) protons as compared to that of compound 7 (4.04-4.07ppm) confirmed the attachment of two mesyloxy groups at position C-2 and C-3.The rest of the 1 H NMR spectrum was in complete agreement with the structure assigned as benzyl 2,3-di-O-methanesulfonyl-4-O-lauroyl--L-rhamnopyranoside (9).Finally, dimolar benzoylation of laureate 7 gave a solid benzyl 2,3-di-O-benzoyl-4-O-lauroyl--Lrhamnopyranoside (10) in 87% yield as confirmed a complete analysis of its IR and 1 H NMR spectra.2] Similarly, benzyl -L-rhamnopyranoside (4) was found to exist in regular 1 C4 conformation. 29However, in case of derivatives 5-6, the presence of isopropylidene functionality at C-2 and C-3 positions and/or acyl group(s) increases the steric hindrance in these molecules.Therefore, the conformations of 5-10 were proposed based on the analyses of 1 H NMR spectral data.The coupling constants determined from the 400 MHz 1 H NMR spectra in CDCl3 of 5-10 are shown in Table 1.In case of 7, appearance of a distinct triplet for H-4 at 5.02 (J4,3 = J4,5 = 10.0 Hz) and a doublet of doublet for H-3 at 4.04 (J3,4 = 9.6 and J3,2 = 3.4 Hz) ppm were informative.The large coupling constants (J4,3 = J4,5 = ~10.0Hz) for the H-4 axial proton requires trans-diaxial relationship with H-3 and H-5 protons.This clearly requires H-3 and H-5 protons to be axial.Again, the small coupling constant between H-3 and H-2 protons requires cis axialequatorial relationship.As H-3 is axially oriented, H-2 must be present in equatorial position.These observation confirmed that 4-O-lauroate 7 exists in regular 1 C4 conformation with C-5 substituent (-CH3) equatorially oriented [(5S)].Compound 7 was obtained from monoacetonide 6.Hence, in compound 6, the relative stereochemistry of the substituents at C-2, C-3 is cis and C-3, C-4 is trans (as the same stereochemistry is retained in the product 7 formation).But the 1 H NMR spectrum of rhamnopyranoside 6 contains a doublet of doublet for H-4 at 4.90 ppm (J4,3 = 10.0 and J4,5 = 6.3 Hz).The smaller value of coupling constant between H-4 and H-5 (6.3 Hz) than the expected one (~10.0Hz) could be explained by the presence of a five-membered isopropylidene ring fused to the six-membered rhamnopyranoside ring.This clearly indicated the slight distortion of the pyranose ring from regular 1 C4 conformation.Similar distortion of the pyranose ring from regular 1 C4 conformation was also observed for monoacetonide 5.It could be anticipated from the Table 1 that coupling constants of compounds 8-10 were in good agreement with regular 1 C4 conformation with C-5 substituent (-CH3) equatorially oriented [(5S) configuration].

Antimicrobial studies
In vitro zone of inhibitions of four Gram-positive and six Gram-negative bacteria due to the effect of the rhamnopyranoside derivatives 4-10 are shown in Table 2.The Table 2 indicates that the tested rhamnopyranosides 4-10 were less effective against these Gram-positive and Gram-negative organisms than that of the standard antibiotic kanamycin.Only 2,3-di-O-benzoate 10 exhibited considerable inhibition against these bacterial pathogens.In vitro percentage inhibition results of mycelial growth of seven plant pathogenic fungi due to the effect of rhamnopyranoside derivatives (4-10) are presented in Table 3.All the acylated rhamnopyranosides were found comparatively more active against the tested fungal pathogens than that of bacterial organisms.In case of Aspergillus flavus, diacetate 8 (*66%) and dibenzoate 10 (*62%) showed excellent inhibition, which were comparable to that of standard antifungal antibiotic fluconazole (*62%).

Structure activity relationship (SAR)
It was evident from Table 2 and Table 3 that incorporation of lauroyl group increased the antimicrobial potentiality of rhamnopyranoside 4. Again, the rhamnopyranoside derivatives 4-10 were more active against fungal pathogens than against the bacterial organisms.An important observation was that, compounds 7-10 were found to be more active than compounds 5-6 against the tested pathogens.Compounds 4-7 contain more hydroxyl groups (more hydrophilic) than that of compound 8-10.Compounds 8-10 having fewer or no hydroxyl groups (more hydrophobic) showed much better antimicrobial potentiality than compounds 4-7.The hydrophobicity of compounds is an important parameter for bioactivity such as toxicity or alteration of membrane integrity, and is directly related to membrane permeation. 33Hunt 34 proposed that the antimicrobial activities of alcoholic compounds is directly related to their lipid solubility through the hydrophobic interaction between alkyl chains of alcohols and lipid regions in the membrane.A similar hydrophobic interaction might occur between the acyl chains of glucofuranoses accumulated in the lipid like nature of the bacteria membranes.[35] It was observed from Table 2 and Table 3 that 4-O-lauroyl-2,3-di-O-acetate/mesylate/benzoate (8/9/10) exhibited excellent activity against both bacterial and fungal pathogens which were, in some cases, comparable to that of the standard antibiotic.This led us to conclude that incorporation of 4-Olauroyl group in rhamnopyranoside frame work along with 2,3-di-O-acetyl/mesyl/benzoyl group increased the antimicrobial potentiality of the rhamnopyranoside 4.
microwave oven, MB-3947C, 800 W, 2450 MHz).Chemical shifts were reported in  unit (ppm) with reference to TMS as an internal standard and J values are given in Hz.
(b) Microwave assisted method: Finely powdered L-rhamnose (0.8 g, 4.873 mmol) was taken in a porcelain dish followed by addition of dry benzyl alcohol (1.0 mL) and Amberlite IR 120 (H + ) ion exchange resin (0.8 g).The reaction mixture was mixed with a spatula and covered with a glass plate.The mixture was then placed in a domestic microwave oven (LG microwave oven, MB-3947C, 800 W, 2450 MHz) and irradiated at 160 watts for 1.5 minutes (30 sec×3).Progress of the reaction was monitored every 30 sec intervals by TLC (CHCl3/MeOH = 10/1).The reaction mixture was filtered and the filtrate was evaporated under reduced pressure to leave a thick syrup.The syrup was then passed through a short silica gel column to give pure benzyl rhamnopyranoside (1.19 g, 96%) as brownish thick liquid.The IR and 1 H NMR spectra of this compound were indistinguishable to that of earlier prepared (4) by conventional glycosidation method (literature method).

General procedure for acylation:
To a solution of the benzyl rhamnopyranoside having hydroxyl groups in anhydrous pyridine (1 mL) was added acyl halide at 0 ºC followed by addition of catalytic amount of 4-dimethylaminopyridine (DMAP).The reaction mixture was allowed to attain room temperature and stirring was continued for 10-16 h.A few pieces of ice was added to the reaction mixture to decompose unreacted (excess) acyl halide and extracted with dichloromethane (DCM, 35 mL).The DCM layer was washed successively with 5% hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution and brine.The DCM layer was dried and concentrated under reduced pressure.The residue thus obtained on column chromatography (n-hexane/ethyl acetate) gave the corresponding acylated product.

Antimicrobial screening procedure
Screening of antibacterial activity: For the detection of antibacterial activities, the disc diffusion method 23 was followed.Dimethylformamide (DMF) was used as a solvent for test chemicals and a 2% solution of the compound was used in the investigation.The plates were incubated at 37 °C for 48 h.Proper control was maintained with DMF without chemicals.Mueller-Hinton (agar and broth) medium was used for culture of bacteria.Each experiment was carried out three times.All the results were compared with the standard antibacterial antibiotic kanamycin (50 μg/disc, Taj Pharmaceuticals Ltd., India).

Screening of mycelial growth:
The antifungal activities of the newly synthesized rhamnopyranosides (4-10) were investigated based on food poisoning technique. 25,26Sabouraud (agar and broth, PDA) medium was used for culture of fungi.Linear mycelial growth of fungus was measured after 3~5 days of incubation.The percentage inhibition of radial mycelial growth of the test fungus was
= percentage of inhibition, C = diameter of the fungal colony in control (DMF), T = diameter of the fungal colony in treatment.The results were compared with standard antifungal antibiotic fluconazole (100 μg/mL medium, brand name Omastin, Beximco Pharmaceuticals Ltd., Bangladesh).