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Synthesis and antimicrobial study of some methyl 4-O-palmitoyl-[alpha]-L-rhamnopyranoside derivatives.

1. INTRODUCTION

L-Rhamnose is an important member of the monosaccharide series [1]. Rhamnolipids comprise one of the most important classes of biosurfactants and exhibit diverse biological functions [2]. For the above reasons, the total synthesis of rhamnolipids has attracted considerable attention [3]. Several oligosaccharides have been isolated from plants which contain L-rhamnose with aglycon moiety at C-4 position [4] e.g. Kaempferol 3,7-di-O-[alpha]-L rhamnopyranoside (1) was isolated from Consolida armeniaca. Recently, Gohar et al. [5] isolated new 3 0- acyl-[alpha]-L-rhamnopyranoside flavonol glycoside named ceratoside for the first time from Ceratonia siliqua L. seeds. Ceratoside showed significant antioxidant activity comparable to that of most common antioxidant ascorbic acid and could be used as a potential source for natural antioxidants. In the last few decades, syntheses of several oligosaccharides containing rhamnose have been accomplished from natural products by hydrolysis [6]. Tsai et al. [7] reported the synthesis and crystallographic structure of phenyl 2,3,4-tri-O-acetyl 1- thio-[alpha]-L-rhamnopyranoside which acts as a glycosyl donor.

Acylation of monosaccharide derivatives is of growing importance in the field of synthetic carbohydrate chemistry because of its usefulness for providing newer derivatives of biological importance [8]. For example, the human pathogens M. tuberculosis and M. avium produce sulfated glycolipids and recently it was shown that the core region of glycopeptidolipids could be selectively sulfated at position C-2 of 3,4-di-O-methyl-L-rhamnose [9]. Due to the role of rhamnose as mediator of cell-cell and host-pathogen interactions, Liptak et al. [10] have reported the synthesis of 4-Osulfonic acid (3) of rhamnopyranoside 2.

Our previous projects on selective acylation and antimicrobial evaluation of rhamnopyranosides revealed that selective decanoylation of methyl [alpha]-L-rhamnopyranoside (2) using dibutyltin oxide method gave methyl 3-O-decanoyl-[alpha]-L-rhamnopyranoside (4) which exhibited higher antimicrobial activity than that of the standard antibiotic [11]. We have been interested to extend studies by the introduction of acyl group (i.e. palmitoyl group as aglycon moiety) at position C-4 instead of position C-3 of methyl [alpha]-L rhamnopyranoside (2). This may provide important information about positional effects of the acyl group in its role as antimicrobial functionality.

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2. MATERIAL AND METHODS

General experimental procedure

Melting points (mp) were determined on an electrothermal melting point apparatus and are uncorrected. Evaporations were performed under diminished pressure on a Buchi rotary evaporator. IR spectra were recorded on a FTIR spectrophotometer (Shimadzu, IR Prestige-21) using KBr and CHCL technique. Thin layer chromatography (TLC) was performed on Kieselgel G[F.sub.254] and visualization was accomplished by spraying the plates with 1% [H.sub.2]S[O.sub.4] followed by heating the plates at 150-200 [degrees]C until coloration took place. Column chromatography was carried out with silica gel (100-200 mesh). [sup.1]H (400 MHz) and [sup.13]C (100 MHz) NMR spectra were recorded using CD[Cl.sub.3] as a solvent. Chemical shifts were reported in [delta] unit (ppm) with reference to TMS as an internal standard and J values are given in Hz. The assignments of the signals were confirmed by decoupling and DEPT experiments. All reagents used were commercially available (Aldrich) and were used as received unless otherwise specified.

Synthesis

Methyl [alpha]-L-rhamnopyranoside (2): The title compound (2) was prepared from L-rhamnose (Merck) and anhydrous methanol with Amberlite IR 120 ([H.sup.+]) ion exchange resin in 82% yield as a crystalline solid, mp 109-110 [degrees]C (Lit. mp 108-109 [degrees]C) [12].

Methyl 2,3-O-isopropylidene-[alpha]-L-rhamnopyranoside (5): To a solution of methyl [alpha]-L-rhamnopyranoside (2) (2.0 g, 11.235 mmol) in 2,2-Dimethoxypropane (DMP, 30 mL) was added p-toluenesulfonic acid (p-TSA, catalytic) at room temperature with stirring. Stirring was continued for 2 h when excess DMP was removed under reduced pressure, water (5 mL) was added to the reaction mixture, extracted with dichloromethane (3*15 mL) and concentrated successively. The residue thus obtained on chromatography with n-hexane/ethyl acetate (9.3/0.7) afforded (5) (2.403 g, 98%) as a colorless thick liquid.

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[R.sub.f] = 0.51 (n-hexane/ethyl acetate = 9.3/0.7). IR (CH[Cl.sub.3]): 3420-3300 (br) (OH), 1370 [cm.sup.-1] [C[(C[H.sub.3]).sub.2]]. [sup.1]H NMR (400 MHz, CD[Cl.sub.3]) : [delta] 4.80 (1H, s, H-1), 4.07 (1H, d, J = 5.8 Hz, H-2), 4.01 (1H, dd [apparent t], J = 6.9 and 5.8 Hz, H-3), 3.50-3.60 (1H, m, H-5), 3.33 (3H, s, O-C[H.sub.3]), 3.28-3.33 (1H, m, H-4), 3.09-3.14 (1H, br s, exchange with [D.sub.2]O, OH), 1.47 (3H, s, C[H.sub.3]), 1.30 (3H, s, C[H.sub.3]), 1.24 (3H, d, J = 6.2 Hz, 6-C[H.sub.3]). [sup.13]C NMR (100 MHz, CD[Cl.sub.3]): 5 109.4 [C[(C[H.sub.3]).sup.2]], 98.0 (C-1), 78.4 (C-4), 75.7 (C-3), 74.3 (C-2), 65.6 (C-5), 54.8 (O-C[H.sub.3]), 27.9, 26.0 [C[(C[H.sub.3]).sub.2]], 17.4 (C-6).

Methyl 2,3-O-isopropylidene-4-O-palmitoyl-[alpha]-L-rhamnopyranoside (6): Palmitoyl chloride (3.48 g, 15.945 mmol) was added to a stirred solution of the monoacetonide 5 (2.3 g, 10.54 mmol) in anhydrous pyridine (8 mL) at 0 [degrees]C followed by addition of 4-dimethylaminopyridine (DMAP, catalytic). The reaction mixture was stirred at room temperature for 12 h, treated with cold water (2 mL) and extracted with chloroform (3x10 mL). Usual work-up and chromatography (elution with n-hexane/ethyl acetate = 18/1) afforded the title compound 6 (4.62 g, 96%) as a pale-yellow needles, mp 40-41 [degrees]C.

[R.sub.f] = 0.5 (n-hexane/ethyl acetate = 9.3/0.7). IR (KBr): 1730 (CO), 1372 [cm.sup.-1] [C[(C[H.sub.3]).sub.2]] [sup.1]H NMR (400 MHz, CD[Cl.sub.3]): 5 4.88 (1H, s, H-1), 4.85 (1H, dd, J = 10.0 and 7.0 Hz, H-4), 4.10-4.15 (2H, m, H-2 and H-3), 3.67-3.71 (1H, m, H-5), 3.37 (3H, s, O-C[H.sub.3]), 2.28-2.37 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.13] C[H.sub.2] CO], 1.58-1.63 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.55 (3H, s, C[H.sub.3]), 1.33 (3H, s, C[H.sub.3]), 1.19-1.29 [24H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.15 (3H, d, J = 6.3 Hz, 6-C[H.sub.3]), 0.89 [3H, t, J = 6.6 Hz, C[H.sub.3][(C[H.sub.2]).sub.14]CO]. [sup.13]C NMR (100 MHz, CD[CI.sub.3]): [delta] 173.0 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 109.8 [C[(C[H.sub.3]).sub.2]], 98.1 (C-1), 76.0 (C-4), 75.6 (C-3), 74.2 (C-2), 64.0 (C-5), 54.0 (O-C[H.sub.3]), 34.4, 32.0, 29.7, 29.6, 29.4, 29.3 (x5), 29.2, 29.1, 24.9, 22.7 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 27.7, 26.4 [C[(C[H.sub.3]).sub.2]], 17.0 (C-6), 14.1 [C[H.sub.3][(C[H.sub.2]).sub.14]CO].

Methyl 4-O-palmitoyl-[alpha]-L-rhamnopyranoside (7): 4-O-Palmitoate 6 (4.5 g, 9.854 mmol) was gently dissolved in glacial acetic acid (25 mL) at room temperature. The solution was slowly warmed to 40 [degrees]C and stirred at this temperature for 18 h. After completion of the reaction, acetic acid was evaporated in vacuum and co-evaporated with toluene (3x3 mL) to remove traces of acetic acid. The residue thus obtained on chromatography with n-hexane/ethyl acetate (2/1) afforded 7 (3.57 g, 87%) as yellow solid, mp 53-54 [degrees]C.

[R.sub.f] = 0.45 (n-hexane/ethyl acetate = 1/1). IR (KBr): 3480-3400 (br) (OH), 1735 [cm.sup.-1] (CO). [sup.1]H NMR (400 MHz, CD[Cl.sub.3]): 5 4.80 (1H, t, J = 9.6 Hz, H-4), 4.68 (1H, s, H-1), 3.90 (1H, d, J = 3.3 Hz, H-2), 3.81 (1H, dd, J = 9.6 and 3.3 Hz, H-3), 3.70-3.78 (1H, m, H-5), 3.35 (3H, s, O-C[H.sub.3]), 2.93-3.22 (2H, br s, exchange with [D.sub.2]O, 2 x OH), 2.33 [2H, t, J = 7.3 Hz, C[H.sub.3][(C[H.sub.2]).sub.13]C[H.sub.2]CO], 1.50-1.63 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.20-1.33 [24H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.18 (3H, d, J = 6.3 Hz, 6-C[H.sub.3]), 0.86 [3H, t, J = 6.5 Hz, CHs[(C[H.sub.2]).sub.14]CO]. [sup.13]C NMR (100 MHz, CD[Cl.sub.3]): [delta] 174.9 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 100.5 (C-1), 75.1 (C-4), 70.9, 70.2 (C-2/C-3), 65.5 (C-5), 55.0 (O-C[H.sub.3]), 34.5, 31.9, 29.7 (x4), 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 24.9, 22.6 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 17.4 (C-6), 14.1 [C[H.sub.3][(C[H.sub.2]).sub.14]CO].

General procedure for 2,3-di-O-acylation of palmitoate 7: To a solution of the 2,3-dihydroxy compound 7 (0.4 g) in anhydrous pyridine (1 mL) was added 2.2 molar equivalent acyl halide at 0 [degrees]C followed by addition of catalytic amount of 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) acylating agent and extracted with dichloromethane (DCM) (3 x 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 2,3 -di-O-acyl-4-O-palmitoate.

Methyl 2,3-di-O-acetyl-4-O-palmitoyl-[alpha]-L rhamnopyranoside (8): Obtained as a semi-solid, 98%.

[R.sub.f] = 0.51 (n-hexane/ethyl acetate = 8.5/1.5). [sup.1]H NMR (400 MHz, CD[Cl.sub.3]): [delta] 5.26 (1H, dd, J = 10.1 and 3.3 Hz, H-3), 5.21 (1H, d, J = 3.3 Hz, H-2), 5.06 (1H, t, J = 9.9 Hz, H-4), 4.60 (1H, s, H-1), 3.78-3.87 (1H, m, H-5), 3.36 (3H, s, O-C[H.sub.3]), 2.25 [2H, t, J = 7.4 Hz, C[H.sub.3][(C[H.sub.2]).sub.13]C[H.sub.2]CO], 2.12 (3H, s, COC[H.sub.3]), 1.95 (3H, s, COC[H.sub.3]), 1.51-1.60 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.20-1.29 [24H, br s, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.20 (3H, d, J = 6.5 Hz, 6-C[H.sub.3]), 0.86 [3H, t, J = 6.6 Hz, C[H.sub.3][(C[H.sub.2]).sub.14]CO]. [sup.13]C NMR (100 MHz, CD[Cl.sub.3]): [delta] 172.8 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 170.1, 169.9 (C[H.sub.3]CO), 98.5 (C-1), 70.7, 69.8, 69.1 (C-2/C-3/C-4), 66.2 (C-5), 55.1 (O-C[H.sub.3]), 34.3, 31.9, 29.7 (x5), 29.4, 29.3 (x2), 29.2, 29.1, 25.0, 22.7 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 20.9, 20.6 (C[H.sub.3]CO), 17.4 (C-6), 14.0 [C[H.sub.3][(C[H.sub.2]).sub.14]CO].

Methyl 2,3-di-O-methanesulfonyl-4-O palmitoyl-[alpha]-L-rhamnopyranoside (9): Appeared as needles, mp 57-58 [degrees]C, 92%.

[R.sub.f] = 0.50 (n-hexane/ethyl acetate = 8.8/1.2). [sup.1]H NMR (400 MHz, CD[Cl.sub.3]): [delta] 5.06 (1H, t, J = 9.8 Hz, H-4), 5.03 (1H, dd, J = 9.8 and 2.6 Hz, H-3), 4.98 (1H, d, J = 2.6 Hz, H-2), 4.83 (1H, s, H-1), 3.78-3.88 (1H, m, H-5), 3.39 (3H, s, O-C[H.sub.3]), 3.15 (3H, s, S[O.sub.2]C[H.sub.3]), 3.04 (3H, s, S[O.sub.2]C[H.sub.3]), 2.33 [2H, t, J = 7.4 Hz, C[H.sub.3][(C[H.sub.2]).sub.13]C[H.sub.2]CO], 1.56-1.66 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.15-1.39 [27H, br s, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO and 6-C[H.sub.3]], 0.85 [3H, t, J = 6.7 Hz, C[H.sub.3][(C[H.sub.2]).sub.14]CO]. [sup.13]C NMR (100 MHz, CD[Cl.sub.3]): [delta] 172.5 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 98.5 (C-1), 78.0, 74.5, 69.6 (C-2/C-3/C-4), 66.5 (C-5), 55.4 (O-C[H.sub.3]), 38.7 (S[O.sub.2]C[H.sub.3]), 38.6 (S[O.sub.2]C[H.sub.3]), 34.1, 31.9, 30.2, 29.7 (x4), 29.6, 29.4, 29.3, 29.2, 29.1, 24.7, 22.7 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 17.3 (C-6), 14.1 [C[H.sub.3][(C[H.sub.2]).sub.14]CO].

Methyl 2,3-di-O-pivaloyl-4-O-palmitoyl-a-Lrhamnopyranoside (10): Isolated as a thick syrup, 88%.

[R.sub.f] = 0.51 (n-hexane/ethyl acetate = (9.2/0.8). [sup.1]H NMR (400 MHz, CDQ3): [delta] 5.27 (1H, dd, J = 10.1 and 3.0 Hz, H-3), 5.17 (1H, d, J = 3.0 Hz, H-2), 5.11 (1H, t, J = 9.9 Hz, H-4), 4.56 (1H, s, H-1), 3.82-3.88 (1H, m, H-5), 3.36 (3H, s, O-C[H.sub.3]), 2.18-2.27 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.51-1.58 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.19-1.32 [33H, br s, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO and C[(C[H.sub.3]).sub.3]], 1.19 (3H, d, J = 6.3 Hz, 6-C[H.sub.3]), 1.08 [9H, s, C[(C[H.sub.3]).sub.3]], 0.85 [3H, t, J = 6.6 Hz, C[H.sub.3][(C[H.sub.2]).sub.14]CO]. [sup.13]C NMR (100 MHz, CD[Cl.sub.3]): [delta] 177.1 [2xC0C[(C[H.sub.3]).sub.3]], 172.6 [C[H.sub.3](C[H.sub.2])i4CO], 98.5 (C-1), 70.9, 69.7, 69.2 (C-2/C3/C-4), 66.4 (C-5), 55.0 (O-C[H.sub.3]), 38.9, 38.7 [2xC[(C[H.sub.3]).sub.3]], 27.1 (x3) [C[(C[H.sub.3]).sub.3]], 27.0 (x3) [C[(C[H.sub.3]).sub.3]], 34.2, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 27.1 (x2), 27.0 (x2), 24.8, 22.7 [C[H.sub.3][(C[H.sub.2]).sub.14]CO], 17.5 (C-6), 14.0 [C[H.sub.3][(C[H.sub.2]).sub.8]CO].

Methyl 2,3-di-O-octanoyl-4-O-palmitoyl-[alpha]-L-rhamnopyranoside (11) : Colorless liquid, 90%.

[R.sub.f] = 0. 52(w-hexane/ethyl acetate = 9/1). [sup.1]H NMR (400 MHz, CD[Cl.sub.3]): 5 5.25 (1H, dd, J = 10.0 and 3.1 Hz, H-3), 5.21 (1H, d, J = 3.1 Hz, H-2), 5.07 (1H, t, J = 10.0 Hz, H-4), 4.58 (1H, s, H-1), 3.78-3.87 (1H, m, H-5), 3.37 (3H, s, O-C[H.sub.3]), 2.18-2.35 [6H, br m, C[H.sub.3][(C[H.sub.2]).sub.13]C[H.sub.2]CO and 2 C[H.sub.3][(C[H.sub.2]).sub.5]C[H.sub.2]C0], 1.43-1.61 1.6H, [6H, br m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]C0 and 2xC[H.sub.3][(C[H.sub.2]).sub.4]C[H.sub.2]C[H.sub.2]C0], 1.20-1.32 [40H, br m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]C0 and 2 x C[H.sub.3][(C[H.sub.2]).sub.4]C[H.sub.2]C[H.sub.2]C0], 1.18 (3H, d, J = 6.4 Hz, 6-C[H.sub.3]), 0.81-0.88 [9H, m, C[H.sub.3][(C[H.sub.2]).sub.14]C0 and 2 x C[H.sub.3][(C[H.sub.2]).sub.6]C0].

Methyl 2,3-di-O-decanoyl-4-O-palmitoyl-[alpha]-L-rhamnopyranoside (12) : Thick oil, 89%.

[R.sub.f] = 0.50 (w-hexane/ethyl acetate = 9/1). [sup.1]H NMR (400 MHz, CD[Cl.sub.3]): [delta] 5.27 (1H, dd, J = 10.0 and 3.3 Hz, H-3), 5.23 (1H, d, J = 3.3 Hz, H-2), 5.06 (1H, t, J = 10.0 Hz, H-4), 4.59 (1H, s, H-1), 3.79-3.86 (1H, m, H-5), 3.36 (3H, s, O-C[H.sub.3]), 2.37 [2H, t, J = 7.3 Hz, C[H.sub.3][(C[H.sub.2]).sub.13] C[H.sub.2]C0], 2.21-2.30 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.7]C[H.sub.2]C0], 2.12-2.18 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.7]C[H.sub.2]C0], 1.45-1.66 [6H, br m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]C0 and 2 x C[H.sub.3][(C[H.sub.2]).sub.6]C[H.sub.2]C[H.sub.2]C0], 1.21-1.31 [48H, br m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]C0 and 2 x C[H.sub.3][(C[H.sub.2]).sub.6]C[H.sub.2]C[H.sub.2]C0], 1.20 (3H, d, J = 6.3 Hz, 6C[H.sub.3]), 0.86 [9H, t, J = 6.3 Hz, C[H.sub.3][(C[H.sub.2]).sub.14]C0 and 2 x C[H.sub.3][(C[H.sub.2]).sub.8]C0].

Methyl 2,3-di-O-(2-chlorobenzoyl)-4-Opalmitoyl-[alpha]-L-rhamnopyranoside (13): Thick oil, 83%.

[R.sub.f] = 0.45 (w-hexane/ethyl acetate = 8.8/1.2). [sup.1]H NMR (400 MHz, CD[Cl.sub.3]): [delta] 7.85 (1H, d, J = 8.0 Hz, Ar-H), 7.75 (1H, d, J = 8.0 Hz, Ar-H), 7.29-7.42 (5H, br m, Ar-H), 7.19-7.25 (1H, m, Ar-H), 5.69 (1H, dd, J = 10.0 and 3.2 Hz, H-3), 5.63 (1H, d, J = 3.2 Hz, H-2), 5.38 (1H, t, J = 9.9 Hz, H-4), 4.85 (1H, s, H-1), 3.934.02 (1H, m, H-5), 3.45 (3H, s, O-C[H.sub.3]), 2.25 [2H, t, J = 7.4 Hz, C[H.sub.3](C[H.sub.2])1bC[H.sub.2]C0], 1.40-1.52 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]C0], 1.27 (3H, d, J = 6.7 Hz, 6 C[H.sub.3]), 1.11-1.22 [24H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]C0], 0.86 [3H, t, J = 6.5 Hz, C[H.sub.3][(C[H.sub.2]).sub.14]C0]. [sup.13]C NMR (100 MHz, CDQ3): 5 172.9 [C[H.sub.3][(C[H.sub.2]).sub.14]C0], 164.6, 164.2 (2-Cl.[C.sub.6][H.sub.4]CO), 134.0, 133.3, 132.9, 132.8, 131.7, 131.3, 131.1, 130.9, 129.1, 128.8, 126.6, 126.5 (Ar-C), 98.4 (C-1), 71.0, 70.9, 70.2 (C-2/C-3/C-4), 66.5 (C-5), 55.3 (O-C[H.sub.3]), 34.2, 31.9, 29.7 (x5), 29.6, 29.4, 29.3, 29.2, 29.0, 24.8, 22.7 [C[H.sub.3][(C[H.sub.2]).sub.14]C0], 17.5 (C-6), 14.1 [C[H.sub.3][(C[H.sub.2]).sub.14]C0].

Methyl 2,3-di-O-(4-chlorobenzoyl)-4-Opalmitoyl-[alpha]-L-rhamnopyranoside (14) : Oil, 85%.

[R.sub.f] = 0.47 (n-hexane/ethyl acetate = 9/1). [sup.1]H NMR (400 MHz, CDCls): [delta] 8.05 (2H, d, J = 8.0 Hz, Ar-H), 7.99 (2H, d, J = 8.0 Hz, Ar-H), 7.49 (2H, d, J = 7.8 Hz, Ar-H), 7.42 (2H, d, J = 7.8 Hz, Ar-H), 5.44 (1H, d, J = 2.9 Hz, H-2), 5.37 (1H, dd, J = 9.6 and 2.8 Hz, H-3), 5.18 (1H, t, J = 9.6 Hz, H-4), 4.75 (1H, s, H-1), 3.89-3.95 (1H, m, H-5), 3.44 (3H, s, O-C[H.sub.3]), 2.242.30 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.13]C[H.sub.2]CO], 1.55-1.62 [2H, m, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.21-1.33 [24H, br s, C[H.sub.3][(C[H.sub.2]).sub.12]C[H.sub.2]C[H.sub.2]CO], 1.20 [3H, d, J = 6.4 Hz, 6C[H.sub.3]], 0.86 [3H, t, J = 6.3 Hz, C[H.sub.3][(C[H.sub.2]).sub.14]C0].

Antimicrobial evaluation

Evaluation of chemicals against bacteria: Four Gram-positive bacteria viz Bacillus cereus BTCC 19, Bacillus megaterium BTCC 18, Bacillus subtilis BTCC 17 and Staphylococcus aureus ATCC 6538 and six Gram-negative bacteria viz. Escherichia coli ATCC 25922, INABAET (vibrio) AE 14748, Pseudomonas species, Salmonella paratyphi AE 14613, Salmonella typhi AE 14612 and Shigella dysenteriae AE 14369 were used to study in vitro antibacterial activities of the synthesized rhamnopyranoside derivatives (2, 5-14). For the detection of antibacterial activities, the disc diffusion method [13] was followed. Chloroform (CHCF) was used as a solvent for test chemicals and a 2% solution of the compound was used in the investigation. Proper control was maintained with chloroform without chemicals. Mueller-Hinton (agar and broth) medium was used for culture of bacteria. All the results were compared with the standard antibacterial antibiotic ampicillin (50 pg/disc, Beximco Pharmaceuticals Ltd., Bangladesh).

Evaluation of chemicals against fungi: The antifungal activities of the synthesized palmitoylated derivatives (2, 5-14) were investigated against four plant pathogenic fungi viz. Alternaria alternata (Fr) Kedissler, Curvularia lunata (Wakker Boedijin), Fusarium equiseti (Corda) Sacc. and Macrophomina phaseolina (Maubl) Ashby. The investigation was based on food poisoning technique [14]. Sabouraud (agar and broth) medium was used for culture of fungi. The results were compared with standard antifungal antibiotic nystatin (100 [micro]g/mL medium, Beximco Pharmaceuticals Ltd., Bangladesh).

3. RESULTS AND DISCUSSION

Synthesis of 4-O-palmitoylrhamnopyranoside 7

The present study mainly describes the selective 4-O-palmitoylation of methyl [alpha]-L-rhamnopyranoside (2). Dibutyltin oxide method in this regard was found to be unsuccessful and furnished the 3-O-acyl derivatives only [11]. Thus, blocking-deblocking technique was employed successfully for the 4-O-palmitoylation of rhamnopyranoside 2. Initially, methyl [alpha]-L- rhamnopyranoside (2) was prepared from L-(+)-rhamnose according to the literature procedure [12] (Scheme 1). Reaction of 2 with excess DMP in the presence of p-TSA (cat.) afforded a product in 98% yield as a colourless thick liquid. In its IR spectrum, stretching bands at 3420-3300 (br) and 1370 [cm.sup.-1] were due to hydroxyl and isopropylidene groups, respectively. In the 'H NMR spectrum, two threeproton singlets at S 1.47 and 1.30 and in the [sup.13]C NMR spectrum, three carbon signals at [delta] 109.4 (CMe2), 27.9 and 26.0 (C[Me.sub.2]) were indicative of the presence of one acetonide group in the molecule. The acetonide protection was formed between cis-vicinal 2,3-diol positions of 2 and the similar formation was observed by Liptak et al. [10].

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The monoacetonide 5, having free hydroxyl group at C-4 position, on palmitoylation with palmitoyl chloride in pyridine afforded a compound as needles (Scheme 1). IR spectrum of the compound showed the presence of a carbonyl-stretching band at 1730 [cm.sup.-1] and absence of hydroxyl stretching band. In the [sup.1]H NMR spectrum, two two-proton multiplets at [delta] 2.28-2.37 and 1.58-1.63, a twenty four-proton multiplet at [delta] 1.19-1.29 and a three-proton triplet at [delta] 0.89 were indicative of the presence of one palmitoyloxy group in the molecule. In addition, the downfield shift of H-4 (~ [delta] 4.85) as compared to the precursor compound 5 ([delta] 3.28-3.33 ppm) confirmed the attachment of the palmitoyloxy group at position C-4. The rest of the [sup.1]H and [sup.13]C NMR spectra were in complete accord with the structure assigned as methyl 2,3-0-isopropylidene-4-0-palmitoyl-a-L rhamnopyranoside (6).

In the subsequent step, removal of the isopropylidene functionality was conducted by stirring 4-O-palmitoate 6 with glacial acetic acid at 40 [degrees]C for 18 h to give a yellow solid. In its IR spectrum, the presence of a new broad band at 3480-3400 [cm.sup.-1] corresponding to hydroxyl region was indicative for the removal of isopropylidene moiety in the molecule. This fact was also confirmed by observing the absence of methyl protons and carbons corresponding to isopropylidene group in its both [sup.1]H and [sup.13]C NMR spectra. In the [sup.1]H NMR spectrum, a broad two-proton singlet (exchanged with D2O) at 5 2.93-3.22 was due to the presence of two hydroxyl groups. Thus, the structure of the compound was assigned as methyl 4-O-palmitoyl-a- L-rhamnopyranoside (7).

Synthesis of 2,3-di-O-acyl derivatives of palmitoate 7

To confirm the structure of palmitoate 7 as well as to get newer derivatives of biological importance seven 2,3-di-O-acyl derivatives (8-14) containing various groups (e.g. acetyl, methanesulfonyl, pivaloyl, octanoyl, decanoyl, 2chlorobenzoyl and 4-chlorobenzoyl, as shown in Scheme 1, were prepared. Thus, treatment of diol 7 with acetic anhydride in pyridine provided a compound in 98% yield. In its [sup.1]H NMR spectrum, two three-proton singlets at [delta] 2.12 and 1.95 corresponding to two methyl groups and in the [sup.13]C NMR spectrum two carbonyl signals at 170.1 and 169.9 clearly indicated the attachment of two acetyloxy groups in the molecule. Thus, the structure was assigned as methyl 2,3-di-O-acetyl-4-O palmitoyl-[alpha]-L-rhamnopyranoside (8).

In the next step, mesylation of 7 with methanesulfonyl chloride in pyridine gave needles in 92% yield. In its [sup.1]H NMR spectrum, two three-proton singlets at 5 3.15 and 3.04 clearly indicated the attachment of two mesyloxy groups in the molecule. The reasonable down field shift of H-2 ([delta] 4.98) and H-3 ([delta] 5.03) protons as compared to that of compound 7 confirmed the attachment of two mesyloxy groups at position C-2 and C-3. The rest of the [sup.1]H NMR spectrum and [sup.13]C NMR spectrum were in complete agreement with the structure accorded as methyl 2,3-di-O-methanesulfonyl-4-O-palmitoyl-[alpha]-L- rhamnopyranoside (9).

Similarly, 4-O-palmitoate 7 was converted to 2,3-di-O-pivaloate (10), 2,3-di-O-octanoate (11), 2,3-di-O-decanoate (12), 2,3-di-O-(2-chlorobenzoate) (13) and 2,3-di-O-(4-chlorobenzoate) (14) in reasonably high yields (Scheme 1). These compounds (10-14) were characterized by spectroscopic analysis and comparing the structure with that of diacetate 8.

Conformational study of the L-rhamnopyranosides (2, 5-14)

Methyl [alpha]-L-rhamnopyranoside (2) is well known to exist in 1C4 conformation [15]. However, in case of compounds 5-14, the presence of acyl group(s) and/or isopropylidene functionality at 2,3 position increases the bulk in the molecule. Therefore, it was thought to derive the conformations of 5-14 using [sup.1]H NMR spectral data. The coupling constants determined from the 400 MHz [sup.1]H NMR spectra in CD[Cl.sub.3] of 5-14 are shown in Table 1.

In case of 7, appearance of a distinct triplet for H-4 at [delta] 4.80 ([J.sub.4,3] = [J.sub.4,5] = 9.6 Hz) and a doublet of doublet for H-3 at [delta] 3.81 ([J.sub.3,4] = 9.6 and [J.sub.3,2] = 3.3 Hz) were informative. The large coupling constants ([J.sub.4,3] = [J.sub.4,5] = 9.6 Hz) for the H-4 axial proton requires transdiaxial 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 axial-equatorial relationship. As H-3 is axially oriented, H-2 must be present in equatorial position. These observation confirmed that 4-O-palmitoate 7 exist in [sup.1]C4 conformation with C-5 substituent (-C[H.sub.3]) equatorially oriented [(5S) configuration].

In one step earlier compound 6, the relative stereochemistry of the substituents at C-2, C-3 is cis and C-3, C-4 is trans and the same stereochemistry is retained in the product 7 formation. But rhamnopyranoside 6 showed a doublet of doublet for H-4 at [delta] 4.85 ([J.sub.4,3] = 10.0 and [J.sub.4,5] = 7.0 Hz). The smaller coupling constant between H-4 and H-5 ([J.sub.4,5] = 7.0 Hz) than the expected value ([J.sub.4,5] = ~10.0 Hz) was due the presence of a five-membered isopropylidene ring fused to the six-membered rhamnopyranoside ring. This clearly indicates the slight distortion of the pyranose ring from regular [sup.1][C.sub.4] conformation as shown in the Figure 1. Similar distortion of the pyranose ring from regular [sup.1][C.sub.4] conformation was also observed for compound 5. It was evident from the Table 1 that coupling constants of compounds 8-14 were in good agreement with regular [sup.1][C.sub.4] conformation with C-5 substituent (-C[H.sub.3]) equatorially oriented [(5S) configuration].

Antimicrobial activities

The results of the in vitro inhibition zone against the selected Gram-positive bacteria due to the effect of the rhamnopyranosides (2, 5-14) are mentioned in Table 2. It was observed from Table 2 that the tested chemicals are less effective against these Gram-positive organisms. Only 2,3-di-O-octanoate 11 exhibited considerable inhibition against these pathogens. Inhibition zone against the selected Gram-negative bacteria due to the effect of the chemicals (2, 5-14) are presented in Table 3. Like Gram-positive organisms, these rhamnopyranosides didn't show considerable inhibition against the tested Gram-negative pathogens. Although 4-O-palmitoyl-2,3-di-O-octanoate 11 was found to be more effective against these Gram-negative pathogens.

Structure activity relationship (SAR)

It was evident from Table 2-4 that incorporation of palmitoyl group increased the antimicrobial potentiality of rhamnopyranoside 2. Again, these acylated rhamnopyranoside derivatives (2, 5-14) were more active against fungal pathogens than that of bacterial organisms. An important observation was that, compounds 5 and 6 showed poorer toxicity than that of compounds 7-14 against these pathogens. This is probably due to the slight distortion in [sup.1][C.sub.4] conformation of 5 and 6. While compounds 7-14 having regular [sup.1][C.sub.4] conformation showed much better antimicrobial potentiality. Again, compounds 2, 5, 6 and 7 contain more hydroxyl groups than that of compounds 8-14. Compounds 8-14 having fewer or no hydroxyl groups showed much better antimicrobial potentiality. Here the hydrophobicity of the molecules increased gradually from compound 5, 7 to 8-14. The hydrophobicity of materials is an important parameter with respect to such bioactivity as toxicity or alteration of membrane integrity, because it is directly related to membrane permeation [16]. Hunt also proposed that the potency of aliphatic alcohols is directly related to their lipid solubility through the hydrophobic interaction between alkyl chains from alcohols and lipid regions in the membrane [17]. We believe that a similar hydrophobic interaction might occur between the acyl chains of glucofuranoses accumulated in the lipid like nature of the bacteria membranes. As a consequence of their hydrophobic interaction, bacteria lose their membrane permeability, ultimately causing death of the bacteria [16-18].

It was observed from Table 2-4 that 4-O-palmitoyl-2,3-di-0-octanoate 11 exhibited excellent activity against both bacterial and fungal pathogens which was, in some cases, higher than that of the standard antibiotics. This led us to conclude that incorporation of 4-0-palmitoyl group in rhamnopyranoside frame work along with 2,3-di-0octanoyl group increased the antimicrobial potentiality of the rhamnopyranoside 2.

4. CONCLUSION

Thus, we have successfully synthesized methyl 4-0-palmitoyl-[alpha]-L-rhamnopyranoside (7) in reasonably good yield from methyl [alpha]-L rhamnopyranoside (2) via blocking-deblocking technique. A number of 2,3-di-0-acyl substituted derivatives (8-14) of 7 were also prepared for biological evaluation. All the rhamnopyranosides (2, 5- 14) were employed as test chemicals for in vitro antibacterial and antifungal functionality test. The structure activity relationship (SAR) study revealed that incorporation of 4-0-palmitoyl group in rhamnopyranoside frame work along with 2,3-di-O-octanoyl group increased the antimicrobial potentiality of the rhamnopyranoside 2.

5. ACKNOWLEDGMENTS

The author highly acknowledges the financial support from Bangladesh University Grants Commission (UGC, 2010-2011). The author is also indebted to Dr. M.S. Rahman and Mr. D.C. Debnath, University of Chittagong for screening antimicrobial activities.

6. REFERENCES AND NOTES

[1] Schaffer, R. In: The Carbohydrates (vol. 1A, 2nd edn). Pigman W.; Horton, D., Eds, New York: Academic Press, 1972, pp 69-111.

[2] Lang, S.; Wullbrandt, D. Appl. Microbiol. Biotechnol. 1999, 51, 22. [CrossRef]

[3] Bauer, J.; Brandenburg, K.; Zahringer, U.; Rademann, J. Chem. Eur. J. 2006, 12, 7116. [CrossRef]

[4] Kucukislamoglu, M.; Yayli, N.; Senturk, H. B.; Genc, H. Turk J. Chem. 2000, 24, 191.

[5] Gohar, A.; Gedara, S. R.; Baraka, H. N. J. Med. Plants Res. 2009, 3, 424.

[6] Qin,L.; Ming, L.; Mabry, T. J.; Dixon, R. A. Phytochemistry 1994, 36, 229. [CrossRef]

[7] Tsai, Y. F.; Yang, J. T.; Chen, J. D.; Lin, C. H. Acta Cryst. E 2007, 63, 3772. [CrossRef]

[8] Andary, C.; Wylde, R.; Laffite, C.; Privat, G.; Winternitz, F. Phytochemistry 1982, 21, 1123. [CrossRef]

[9] Marin, L. M. L.; Laneelle, M. A.; Prome, D.; Laneelle, G.; Prome, J. C.; Daffe, M. Biochemistry 1992, 31, 11106. [CrossRef]

[10] Lazar, L.; Csavas, M.; Borbas, A.; Gyemant, G.; Liptak, A. Arkivoc 2004, vii, 196. [CrossRef]

[11] Kabir, A. K. M. S.; Matin, M. M.; Hossain, A. J. Bangladesh Chem. Soc. 2003, 16, 85.

[12] Haines, A. H. Carbohydr. Res. 1969, 10, 466. [CrossRef]

[13] Bauer, A. W.; Kirby, W. M. M.; Sherris, J. C.; Turck, M. Am. J. Clin. Pathol. 1966, 45, 493.

[14] Grover R. K.; Moore, J. D. Phytopathol. 1962, 52, 876.

[15] Liptak, A.; Fugedi, P.; Nanasi, P. Carbohydr. Res. 1978, 65, 209. [CrossRef]

[16] Kim, Y. M.; Farrah, S.; Baney, R. H. Int. J. Antimicrob. Agents 2007, 29, 217. [CrossRef]

[17] Hunt, W. A. Adv. Exp. Med. Biol. 1975, 56, 95. [CrossRef]

[18] Judge, V.; Narasimhan, B.; Ahuja, M.; Sriram, D.; Yogeeswari, P.; Clercq, E. D.; Pannecouque, C.; Balzarini, J.Med. Chem. 2013, 9, 53. [CrossRef].

Mohammed M. Matin *

Organic Research Laboratory, Department of Chemistry, University of Chittagong, Chittagong-4331, Bangladesh.

* Corresponding author. E-mail: mahbubchem@cu. ac .bd

Article history: Received: 31 December 2013; revised: 09 March 2014; accepted: 11 March 2014. Available online: 02 April 2014.

Table 1. Coupling constants of compounds 5-14.

Compound              coupling constants (Hz)
no.
           [J.sub.2,3]   [J.sub.3,4]   [J.sub.4,5]

5              5.8           6.9           --
6              --           10.0           7.0
7              3.3           9.6           9.6
8              3.3          10.1           9.9
9              2.6           9.8           9.8
10             3.0          10.1           9.9
11             3.1          10.0          10.0
12             3.3          10.0          10.0
13             3.2          10.0           9.9
14             2.9           9.6           9.6

Table 2. Inhibition against Gram-positive organisms by the
rhamnopyranosides.

                       Diameter of zone of inhibition
                        in mm (50 [micro]g.dw/disc)

Compound no.        Bacillus             Bacillus
                     cereus             megaterium

2                      NF                   NF
5                      7                    6
6                      7                    4
7                      NF                   5
8                      7                    7
9                      5                    6
10                     NF                   4
11                    * 23                  17
12                     5                    NF
13                     NF                   6
14                     8                    NF
** Ampicillin         * 22                 * 19

                       Diameter of zone of inhibition
                        in mm (50 [micro]g.dw/disc)

Compound no.         Bacillus            Staphylococcus
                     subtilis                aureus

2                       NF                     NF
5                       NF                     6
6                       8                      NF
7                       5                      8
8                       6                      NF
9                       8                      NF
10                      5                      8
11                     * 25                   * 20
12                      8                      4
13                      8                      6
14                      7                      4
** Ampicillin          * 25                   * 21

NB. NF indicates not found, dw means dry weight,

"**" indicates standard antibiotic, "*" shows good inhibition.

Table 3. Inhibition against Gram-negative organisms by the
rhamnopyranosides.

                     Diameter of zone of inhibition in
                         mm (50 [micro]g.dw/disc)

Compound        Escherichia    INABAET    Pseomodomonas
no.                 coli       (vibrio)      species

2                    NF           NF           NF
5                    8            9             6
6                    NF           NF            6
7                    NF           6            NF
8                    8            7             4
9                    NF           NF            6
10                   NF           9             9
11                  * 21         * 26         * 21
12                   8            NF           NF
13                   10           NF            8
14                   7            9             5
** Ampicillin       * 25         * 24          17

                     Diameter of zone of inhibition in
                        mm (50 [micro]g.dw/disc)

Compound        Salmonella   Salmonella    Shigella
no.             paratyphi      typhi      dysenteriae

2                   NF           NF           NF
5                   6            6             7
6                   7            6             8
7                   7            5             5
8                   9            NF            8
9                   7            6             7
10                  NF           NF           NF
11                 * 20         * 19          14
12                  7            9             5
13                  9            6             6
14                  NF           8            NF
** Ampicillin      * 35          13          * 35

NB. NF indicates not found, dw means dry weight,

"**" indicates standard antibiotic, "*" shows good inhibition.
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Date:Jan 1, 2014
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