Regioselective Synthesis, Characterization, and Antimicrobial Activities of Some New Monosaccharide Derivatives

A regioselective acylation series of methyl α-D-glucopyranoside (1), methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-mannopyranoside (1A), and methyl 4,6-O-benzylidene-2-O-(3,5-dinitrobenzoyl)-α-D-mannopyranoside (1B) has been carried out by the direct acylation method and afforded the 2,6-di-O-glucopyranoside and 2 or 3-O-mannopyranoside derivatives in an excellent yield. In order to obtain newer products, the 2,6-di-O-glucopyranoside derivative was further transformed to a series of 3,4-di-O-acyl derivatives containing a wide variety of functionalities in a single molecular framework. The structures of the newly synthesized compounds were elucidated on the basis of IR, 1H-NMR, 13C-NMR, 13C-DEPT spectral data, and elemental analysis. These synthesized derivatives were screened for in vitro antimicrobial activities against ten human pathogenic and five phytopathogenic microorganisms. A number of test compounds showed remarkable antimicrobial activity comparable to, and in some cases even higher than, the standard antibiotics employed. It was observed that methyl 3,4-di-O-(3-chlorobenzoyl)-2,6-di-O-hexanoyl-α-D-glucopyranoside (8) exhibited a varied range of MIC from 12.5 μg/disc to 25 μg/disc by the disk diffusion method and 1000 μg/mL to 1250 μg/mL by the broth macrodilution method.


Introduction
Carbohydrate chemistry is now found in the field of organic synthesis, protein and nucleic acid chemistry, enzymology, antibiotics, immunology, and biotechnology. Carbohydrates also play an important role in our industrial development and many industries are based on the utilization of carbohydrates. With the development of modern and sophisticated techniques, the isolation of various natural products from plants and other sources become easier. Of the carbohydrates isolated from natural sources, acyl-and alkyl-glycoses or glycoside derivatives are important and some of these have effective biological activity [1][2][3]. Selective acylation is considered by carbohydrate chemists as one of the most useful and versatile methods for the preparation of the hydroxyl groups [4,5]. Various selective acylation methods have so been developed and employed successfully in carbohydrate chemistry [6,7]. Of these, the direct method has been considered by the carbohydrate chemists as one of the most effective for selective acylation of carbohydrates. Selective acylation is also important because of its usefulness for the synthesis of biologically active carbohydrates [8][9][10] and nucleosides [11,12]. From the literature survey, it was revealed that a large number of biologically active compounds possess aromatic and heteroaromatic nuclei [13][14][15]. The benzene and substituted benzene nuclei play an important role as the common denominator for various biological activities [16,17]. Nitrogen (N)-and sulphur (S)-containing substitution products also showed marked antimicrobial activities [18,19]. As a continuation of a research project on the biological evaluation of carbohydrate derivatives and guided by some encouraging results obtained in this field [20][21][22], we deliberately synthesized some acylated derivatives of D-glucopyranoside (Scheme 1) and D-mannopyranoside (Scheme 2) containing a variety of substituents in a single molecular framework. We also evaluated the antibacterial and antifungal activities of the synthesized compounds using various bacterial and fungal strains and the results are reported here for the first time.
We then used a number of partially substituted monosaccharide derivatives, synthesized earlier in this laboratory, for acylation with a number of acylating agents in order to attain newer test chemicals for antimicrobial evaluation studies. Thus, pentanoylation of methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-mannopyranoside (1A) with pentanoyl chloride in pyridine gave compound 10 in 90% yield which is very similar to [23]. The IR spectrum of this compound indicated an absorption band at 1718 cm −1 corresponding to carbonyl stretching. In its 1 H-NMR spectrum, three two-proton multiplets at δ 2.41, 1.59, 1.32, and a three-proton multiplet at δ 0.86 corresponded to the presence of one pentanoyl group in the molecule. The introduction of the pentanoyl group at position 2 was demonstrated by a downfield shift of H-2 to δ 5.48. The 13 C-NMR spectrum of compound 10 also showed the presence of one pentanoyl group by displaying the following characteristic peaks: δ 172.52 {CH 3 (CH 2 ) 3 CO-}, δ 33.85, 26.98,22.11 {CH 3 (CH 2 ) 3 CO-}, and δ 13.62 {CH 3 (CH 2 ) 3 CO-}. The benzylidene derivative (1A) was then derivatized by using hexanoyl chloride in pyridine followed by the usual work-up and purification procedures. The hexanoyl derivative (11) was isolated in 92% yield as syrup.  (10)(11)(12)(13)(14).
The next substituted monosaccharide we used was methyl 4,6-O-benzylidene-2-O-(3,5dinitrobenzoyl)-α-D-mannopyranoside (1B) and employed pentanoyl chloride, hexanoyl chloride, and 4-methoxybenzoyl chloride as the acylating agents. Thus, the benzylidene derivative (1B) upon treatment with pentanoyl chloride in pyridine, followed by the usual work-up and purification, afforded the pentanoyl derivative (12)  Hexanoylation of the benzylidene derivative (1B) with hexanoyl chloride in pyridine provided compound 13 in 82% yield as needles, m.p. 137-138°C. The IR spectrum of this compound showed the following characteristic peak: 1712 cm −1 for -CO stretching. The introduction of one hexanoyl group was established by observing the following characteristic peaks in its 1 H-NMR spectrum: δ 2.26 (2H, m), δ 1.49 (2H, t, J= 7.1 Hz), δ 1.11 (4H, m), and δ 0.67 (3H, t, J= 6. 7 Hz). Also, we observed the downfield shift of H-3 to δ 5.79 (as t, J = 9. 4 Hz) showing the introduction of the hexanoyl group at position 3. Its 13 C-NMR spectrum also showed the presence of one hexanoyl group by displaying the following characteristic peaks: δ 172.80 {CH 3 (CH 2 ) 4 CO-}, δ 34. 19, 31.02, 24.66, 22.14 {CH 3 (CH 2 ) 4 CO-}, and δ 13.68 {CH 3 (CH 2 ) 4 CO-}. The benzylidene derivative (1B) was then allowed to react with 4-methoxybenzoyl chloride in pyridine and after the usual work-up and chromatographic purification, we obtained compound 14 in 85% yield as syrup. The IR spectrum of compound 14 showed an absorption band at 1716 cm −1 (-CO stretching), thereby suggesting the presence of a carbonyl group in the molecule. In its 1 H-NMR spectrum, the characteristic two-proton doublets at δ 8.08 and δ 6.97 (J = 8.8 Hz in each case) and a three-proton singlet at δ 3.88 were due to one 4-methoxybenzoyl group in the molecule. Also, the deshielding of H-3 to δ 6.04 (as t, J = 9.5 Hz) indicated the formation of the 3-substitution product. The 13 C-NMR spectrum also displayed the introduction of one 4-methoxybenzoyl group. Thus, the regioselective acylation of D-glucopyranoside (Scheme-1) and D-mannopyranoside (Scheme-2) by applying the direct acylation method was unique in that the reaction provided a single monosubstitution product in reasonably high yields.

Anibacterial Activity
The antibacterial evaluation results of the test compounds and the standard antibiotic, ampicillin, against Gram-positive and Gram-negative bacteria are listed in Table 1, Table  2

Fig. 1.
Zone of inhibition against B. cereus (A) and S. typhi (B) by the compounds 6-9. From the minimum inhibition concentration (MIC) experimental results, it was observed that chemical 8 exhibited a varied range of values from 12.5 µg/disc to 25 µg/disc and 1000 µg/mL to 1250 µg/mL by the disk diffusion (Table 3) and broth macrodilution methods, (Table 4) respectively. The lowest MIC (12.5 µg/disc) was recorded against B. cereus, B. megaterium, S. aureus, S. Typhi, and INABAET (Vibrio) by the disk diffusion method and the lowest MIC (1000 µg/mL) was recorded against B. cereus, B. megaterium, S. Aureus, and INABAET (Vibrio) by the broth macrodilution method. The MIC is indicative of the usefulness of these compounds as antimicrobial drugs, but some other experiments must be carried out before these can be used as effective drugs. As chemical 8 exhibited remarkable inhibitory activity against ten pathogenic bacteria, the efficacy of the chemical cannot be ignored. This chemical along with others, which showed promising inhibitory activity against particular bacterial strains, should be subjected to further experiments to evaluate their efficacy and this will be the subject of our future research works.

Tab. 3.
MIC test of compound 8 against ten human pathogenic bacteria by disk diffusion method.

Antifungal Activity
The results obtained from the present investigation of the antifungal studies as mentioned in Table 5 and Figure 2 clearly demonstrated that compound 8 showed the highest inhibition against all of these fungal strains. Here the percent inhibition of compound 8 is higher than the standard antibiotic, Nystatin in all the cases. The rest of the compounds was moderate or less sensitive towards the five tested fungal phytopathogens. The antifungal activities of our test compounds are in accordance with the results we observed before [24][25]. From the MIC test results reported in

Statistical Analysis
The standard deviation value is expressed in terms of ±SD. On the basis of the calculated value by using the ANOVA method, it has been observed that the differences below the 0.0001 level (p ≤ 0.0001) were considered as statistically significant.

Materials and Methods
The 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded for solutions in deuteriochloroform (CDCl 3 ) using tetramethylsilane (TMS) as internal standard with a Bruker DPX-400 spectrometer at the Bangladesh Council of Scientific and Industrial Research (BCSIR) Laboratories, Dhaka, Bangladesh. Evaporations were carried out under reduced pressure using a VV-1 type vacuum rotary evaporator (Germany) with a bath temperature below 40°C. Melting points were determined on an electro-thermal melting point apparatus (England) and are uncorrected. All reagents used were commercially available (Aldrich) and were used as received, unless otherwise specified. Column chromatography was performed with silica gel G 60 .

Synthesis of methyl 2,6-di-O-hexanoyl-α-D-glucopyranoside (2)
A solution of methyl α-D-glucopyranoside (1) (5 g, 25.75 mmol) in dry pyridine (60 mL) was cooled to −5°C whereupon hexanoyl chloride (3.9 mL, 28.97 mmol) was added to it. The mixture was stirred at the same temperature for 4 hours and then stirred overnight at room temperature. The progress of the reaction was monitored by TLC, which indicated the formation of two products, the slower-moving component being the major one. A few pieces of ice were added to the flask and then the product mixture was extracted with chloroform (3×10 mL). The combined chloroform layer was washed successively with dilute hydrochloric acid (10%), saturated aqueous sodium hydrogen carbonate (NaHCO 3 ) solution, and distilled water. The chloroform layer was dried (MgSO 4 ), filtered, and the filtrate was concentrated under reduced pressure to leave a syrup. The syrup was passed through a silica gel column and eluted with methanol-chloroform (1:20

General Procedure of the Synthesis of Compounds (3-9)
A stirred and cooled (0°C) solution of the 3,4-diol (2) 1:4) showed the complete conversion of reactant into a single product. Excess reagent was destroyed by the addition of a few pieces of ice and the reaction mixture was extracted with chloroform (3×10 mL). The combined organic extract was washed successively with dilute hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution, and water. The organic layer was dried (MgSO 4 ), filtered, and the filtrate was evaporated off. The resulting syrupy residue was passed through silica gel column chromatography and eluted with ethyl acetate-n-hexane to afford compounds 3,4-di-Oacetyl derivative (3), 4, 5, 6, 7, 8, and 9, respectively.

General Procedure for the Synthesis of Compounds 10 and 11
A cooled (0°C) and stirred solution of methyl 3-O-benzoyl-4,6-O-benzylidene-α-Dmannopyranoside (1A) [26] (100 mg, 0.26 mmol) in anhydrous pyridine (3 mL) was allowed to react with pentanoyl chloride (0.07 mL, 0.56 mmol) and stirring was continued for 8 hours. TLC examination indicated the formation of a faster-moving product. A few pieces of ice were added to the flask with constant shaking and the mixture was extracted three times with chloroform. The combined chloroform extract was washed successively with dilute hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution, and distilled water. The organic layer was dried (Na 2 SO 4 ), filtered, and concentrated. Purification of the resulting syrupy residue was effected by silica gel column chromatography (ethyl acetate-hexane as eluant) to furnish the title compound (10). A similar reaction and purification procedure was applied to prepare compound 11.

General Procedure for the Synthesis of Compounds (12-14)
An ice-cooled solution of methyl 4,6-O-benzylidene-2-O-(3,5-dinitrobenzoyl)-α-D-mannopyranoside (1B) [27] (100 mg, 0.21 mmol) in dry pyridine (6 mL) was treated with pentanoyl chloride (0.052 mL, 0.42 mmol) and the solution was stirred at this temperature for 4 hours and then at room temperature for 12 hours. TLC examination indicated the full conversion of the starting material into a single product. Excess reagent was destroyed by the addition of a few pieces of ice and the reaction mixture was processed as usual. The resulting syrup was purified by column chromatography (with ethyl acetate-hexane as eluant) to afford the pentanoyl derivative (12). Recrystalization from ethyl acetate-hexane gave compound 12. A similar reaction and purification was used to isolate compounds 13, 14, and 15.

Antibacterial Activity Assay
The in vitro antibacterial activities of the synthesized compounds were detected by the disk diffusion method [28] with little modification [29]. Sterilized paper discs of 4 mm in diameter and Petri dishes of 150 mm in diameter were used throughout the experiment. The autoclaved Mueller-Hinton agar medium, cooled to 45°C, was poured into sterilized Petri dishes to a depth of 3 to 4 mm and after solidification of the agar medium; the plates were transferred to an incubator at 37°C for 15 to 20 minutes to dry off the moisture that developed on the agar surface. The plates were inoculated with the standard bacterial suspensions (as McFarland 0.5 standard) followed by the spread plate method and allowed to dry for 3 to 5 minutes. Dried and sterilized filter paper discs were treated separately with 50 µg dry weight/disc from 2% solution (in CHCl 3 ) of each test chemical using a micropipette, dried in air under aseptic condition, and were placed at equidistance in a circle on the seeded plate. A control plate was also maintained in each case without any test chemical. These plates were kept for 4-6 hours at low temperature (4-6°C) and the test chemicals diffused from the discs to the surrounding media by this time. The plates were then incubated at 35±2°C for 24 hours to allow maximum growth of the organisms. The antibacterial activity of the test agent was determined by measuring the mean diameter of the zones of inhibition in millimeters. Each experiment was repeated thrice. The standard antibiotic, ampicillin (BEXIMCO Pharm Bangladesh Ltd), was used as a positive control and compared with tested compounds under identical conditions. The MICs of the tested compounds were determined by the disk diffusion [28] method and broth macrodilution [30] method.

Antifungal Activity Assay
The in vitro antifungal activity of the acylated chemicals was done by the Poisons Food technique [31] with some modifications [29]. Two percent solution of the test chemical (in CHCl 3 ) was mixed with sterilized melted Saburaud agar medium to obtain the desired concentration (2%) and this was poured into sterilized Petri dishes. At the center of each plate, 5 days-old fungal mycelial block (4 mm in diameter) was inoculated and incubated at 27°C. A control set was also maintained in each experiment. The 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 calculated as follows: Where, I = Percentage of inhibition, C = Diameter of the fungal colony in the control (CHCl 3 ), T = Diameter of the fungal colony in the treatment. All the results were compared with the standard antifungal antibiotic Nystatin (100 µg/mL medium, BEXIMCO Pharm Bangladesh Ltd.).

Conclusion
In this paper we have explored the synthesis, characterization, and antibacterial screening studies of some acylated monosaccharide derivatives obtained from the direct acylation method. This method demonstrates a very simple and efficient method for the synthesis. Methyl 2,6-di-O-hexanoyl-3,4-di-O-methanesulphonyl-α-D-glucopyranoside (4) and methyl 3,4-di-O-(4-chlorobenzoyl)-2,6-di-O-hexanoyl-α-D-glucopyranoside (7) were found to be encouraging in terms of high selectivity and excellent yields as 95% and 93%, respectively. Thus, a good number of test compounds reported herein exhibited promising antibacterial activity. Methyl 3,4-di-O-(3-chlorobenzoyl)-2,6-di-O-hexanoyl-α-D-glucopyranoside (8) exhibited the highest antibacterial and antifungal activities against all of the tested microorganisms. So, this compound may be targeted for future studies for its usage as a broad-spectrum antibiotic. This piece of work, in our opinion, has created an opportunity for further work with these test compounds, ultimately leading to the development of new pesticides/medicines for human disease control with fewer environmental hazards.