Abstract
Novel -linked 5a–e and -linked glycopyranosides 7a–e were synthesized in high yield from commercially available L-tartaric acid containing two asymmetric centers and axis of symmetry. The compound L-tartaric acid was completely protected and then partially hydrolyzed to get the monoester, which upon treatment with different amino and hydroxyl derivatives of glycopyranoses gave the desired amides and esters. The synthesized derivatives were purified by chromatography and characterized by spectroanalytical techniques. The structure of compound 7c in the series was supported by X-ray analysis. Leishmanicidal activities of compounds 5a–e and 7a–e were investigated which showed moderate to good activities.
1. Introduction
Leishmania has long been known to human beings especially in African and Indian populations since the mid eighteenth century [1–3]. In 1903, Leishman and Donovan discovered the protozoan in spleen tissues of patients in India, which is now known as Leishmania donovani [4, 5]. Nicolle and Comte reported infection by Leishmania in dogs in 1908 [6]. Leishmaniasis is a vector-borne disease carried by sand fly and caused by protozoa of the genus Leishmania. Recent reports confirm that people in about 88 countries are affected by Leishmania and 350 million are at risk, and more or less 2 million new cases are reported each year [7]. In spite of the socioeconomical significance of this tropical infectious disease, the discovery of new potential drugs against it is underway [8, 9].
Glycolipid and glycoprotein oligosaccharides present on cell surfaces are known to play an important role in various biological processes, that is, cellular recognition, adhesion, tumor metastasis, and cell growth regulation [10]. One major class of glycoprotein oligosaccharides consists of -oligosaccharides linked to asparagine by an amide bond [11, 12]. Amides are usually prepared by thermolysis of carboxylic acids with amines, by coupling of carboxylic acids and amines in the presence of a coupling reagent [13] or by prior conversion of the carboxylic acid into a more electrophilic derivative [14]. The concept to add a chiral aglycone moiety such as tartaric acid to prepare glycosides is an active area of research. L-tartaric acid is cheap and readily available starting material; it has two chiral centers with two carboxylic acid groups which can be easily derivatized. Derivatives of tartaric acid containing ester and amide linkages have promisingly useful biological applications, for example, anti-inflammatory, antifungal, thrombin inhibitors, antimicrobial, and -secretase inhibitory activities [15–20].
Hence, glyco derivatives of tartaric acid have potential as curative agents for various infectious diseases. Herein, we report an efficient and scalable synthesis of -linked (5a–e) and -linked glycopyranosides (7a–e) through anomeric - and -acylation, respectively, using commercially available L-tartaric acid as a starting material.
2. Experimental
All chemicals were of highest purity available and used as supplied. D-glucose, D-mannose, D-galactose, D-glucosamine, and D-galactosamine hydrochloride were purchased from Fluka and Sigma Aldrich. Dry solvents like methanol, dichloromethane, chloroform, and n-hexane were obtained by distillation using standard procedures or by passage through a column of anhydrous alumina using equipment from anhydrous engineering (University of Bristol, UK) based on Grubbs’ design. Reactions under anhydrous conditions were carried out under nitrogen gas using three-way stopcock and rubber septa.
Liquid reagents, solutions, or solvents were added via syringe or cannula through rubber septa. Solid reagents were added via Schlenk type adapters. All reactions were monitored by TLC on Kieselgel 60 F254 (Merck); ethyl acetate/n-hexane and methanol/chloroform were used as eluent. Chromatograms were detected under UV light ( 254 and 365 nm) and by charring with 10% sulfuric acid in ethanol, ninhydrin, and vanillin, respectively. Column chromatography was performed using silica gel [Merck, 230−400 mesh (40−63 μm)]. Extracts were concentrated under reduced pressure using both rotary evaporator (bath temperature up to 40°C) at a pressure of either 15 mmHg (diaphragm pump) or 0.1 mmHg (oil pump), as appropriate and a high vacuum line at room temperature. Melting points were determined in degree Celsius (°C) using Gallenkamp digital melting point apparatus and are uncorrected. IR spectra were recorded on a Bruker IFS 66 (FT-IR), Nicolet 205 FT-IR; Nicolet 360 smart orbit (ATR); Thermo Scientific Nicolet 6700 FT-IR and Shimadzu Fourier Transform Infrared Spectrophotometer Model 270. Solid samples were taken in KBr pellets and oils were used in NaCl cells for recording their spectra. 1HNMR spectra were recorded on an NMR Bruker apparatus at 300 MHz and Varian 400 MHz INOVA instrument. 13C NMR spectra were recorded on NMR Bruker apparatus at 75 MHz and Varian 100 MHz INOVA instrument. Chemical shifts are quoted in parts per million from SiMe4 or residual solvent proton signals and coupling constants given in Hertz. Multiplicities are abbreviated as b (broad), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or combinations thereof. Positive ion Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectra were recorded using an HP-MALDI instrument using gentisic acid as matrix material. Optical rotations were measured on an ATAGO, AP-100 Automatic polarimeter. Single crystal X-ray diffraction data were collected on a Bruker Smart APEX II, CCD detector diffractometer [21]. Data reductions were performed using the SAINT program. The structures were solved by direct methods [22] and refined by full-matrix least squares on F2 by using the SHELXTL-PC package [23]. The figures were plotted with the aid of ORTEP program [24].
2.1. General Procedure for the Synthesis of N-Linked Glycopyranosides (5a–e)
In a round bottom flask (100 mL), monoester 1 (0.61 g, 3 mmol), EDC (0.74 g, 3 mmol × 1.3 eq.), and catalytic amount of DMAP were placed under nitrogen. CH2Cl2 (30 mL) was added as a solvent. After 30 min glycopyranosyl amine 4a–e (3 mmol) was added and the mixture was stirred for 18 h. The byproduct urea was removed by extraction of the reaction mixture with ethyl acetate or chloroform and water (30 mL × 2). The crude product was purified by column chromatography using ethyl acetate : hexane (3 : 7) as eluent.
2.2. (2R,3R,5R,6R)-2-(Acetoxymethyl)-6-((4R,5R)-5-(methoxycarbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxamido)tetrahydro-2H-pyran-3,4,5-triyl Triacetate 5a
Yield: 72%. Colorless oil = 12.5. (IR υ cm−1): 3347 (NH), 2955 (CH), 1742 (COOCH3), 1701 (CONH), 1299 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 7.18 (d, J = 9.3 Hz, NH), 5.23, 5.18, 5.05, 4.96 (pseudo t, J ~ 9.2 Hz in each, CH), 4.65 (d, J = 5.4 Hz, CH), 4.59 (d, J = 5.4 Hz, CH), 4.30 (dd, J = 12.5, 3.6 Hz, CH), 4.21 (ddd, J = 10.4, 3.6, 2.1 Hz, CH), 3.95 (dd, J = 12.5, 2.1 Hz, CH), 3.75 (s, OCH3), 1.94, 1.92, 1.91, 1.89 (s, CH3), 1.56 (s, CH3), 1.50 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 171.2 (CONH), 170.7 (COOCH3), 170.0, 169.9, 169.5, 169.2 (CO), 113.3 (qt C), 81.8 (CH), 76.3 (CH), 75.2 (CH), 73.3, 72.5, 70.0, 67.8 (CH), 61.2 (CH), 52.4 (OCH3), 25.9, (CH3), 25.2 (CH3), 20.2, 20.1 (3), (CH3). HRMS-ESI for C22H31NO14 Na: [M + Na]+ calcd: 556.1745, found: 556.1621. Anal. Calc. For C22H31NO14: C, 49.53; H, 5.86; N, 2.63; O, 41.99 Found C, 49.43; H, 5.56; N, 2.78; O, 42.10.
2.3. (2R,3R,5R,6R)-2-(Acetoxymethyl)-6-((4R,5R)-5-(methoxycarbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxamido)tetrahydro-2H-pyran-3,4,5-triyl Triacetate 5b
Yield: 69%. Colorless oil = 13.3. (IR υ cm−1): 3337 (NH), 2925 (CH), 1732 (COOCH3), 1703 (CONH), 1292 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 7.47 (d, J = 9.3 Hz, NH), 5.34, 5.31, 5.24 (pseudo t, J = 10.0, Hz in each, CH), 4.95 (t, J = 3.9 Hz, CH), 4.93 (d, J = 5.5 Hz, CH), 4.80 (d, J = 5.5 Hz, CH), 4.28 (dd, J = 12.4, 3.5 Hz, CH), 4.10 (ddd, J = 10.3, 3.6, 2.1 Hz, CH), 3.95 (dd, J = 12.4, 3.5 Hz, CH), 3.77 (s, OCH3), 2.34, 2.23, 2.13, 1.99 (s, CH3), 1.52 (s, CH3), 1.46 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 172.2 (CONH), 170.9 (COOCH3), 170.1, 169.6, 169.3, 169.1 (CO), 114.3 (qt C), 81.4 (CH), 76.5 (CH), 75.1 (CH), 73.1, 72.3, 70.1, 67.9 (CH), 61.8 (CH), 52.9 (OCH3), 25.5 (CH3), 25.1 (CH3), 20.1, 20.0 (3) (CH3). HRMS-ESI for C22H31NO14 Na: [M + Na]+ calcd: 556.1745, found: 556.1627. Anal. Calc. For C22H31NO14: C, 49.53; H, 5.86; N, 2.63; O, 41.99 Found C, 49.43; H, 5.56; N, 2.78; O, 42.10.
2.4. (2R,3R,5R,6R)-2-(Acetoxymethyl)-6-((4R,5R)-5-(methoxycarbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxamido)tetrahydro-2H-pyran-3,4,5-triyl Triacetate 5c
Yield: 65%. Colorless oil = 27.1. (IR υ cm−1): 3339 (NH), 2928 (CH), 1736 (COOCH3), 1700 (CONH), 1290 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 7.37 (d, J = 9.3 Hz, NH), 5.42, 5.40, 5.36 (pseudo t, J ~ 6.5 Hz in each, CH), 4.98 (t, J = 11.9 Hz, CH), 4.97 (d, J = 5.6 Hz, CH), 4.90 (d, J = 5.6 Hz, CH), 4.48 (dd, J = 12.7, 3.4 Hz, CH), 4.12 (ddd, J = 10.4, 3.6, 2.1 Hz, CH), 3.92 (dd, J = 12.4, 3.5 Hz, CH), 3.84 (s, OCH3), 2.11, 2.10, 2.09, 1.99 (s, CH3), 1.54 (s, CH3), 1.48 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 171.8 (CONH), 170.2 (COOCH3), 170.3, 169.5, 169.4, 169.2 (CO), 114.2 (qt C), 81.5 (CH), 76.1 (CH), 75.2 (CH), 73.3, 72.2, 70.1, 67.5 (CH), 61.9 (CH), 52.5 (OCH3), 26.5 (CH3), 25.7 (CH3), 20.4, 19.5 (3) (CH3). HRMS-ESI for C22H31NO14 Na: [M + Na]+ calcd: 556.1745, found: 556.1643. Anal. Calc. For C22H31NO14: C, 49.53; H, 5.86; N, 2.63; O, 41.99 Found C, 49.43; H, 5.56; N, 2.78; O, 42.10.
2.5. (2R,3S,5R,6R)-5-Acetamido-2-(acetoxymethyl)-6-((4R,5R)-5-(methoxycarbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxamido)tetrahydro-2H-pyran-3,4-diyl Diacetate 5d
Yield: 67%. Gel like. = 43.3. (IR υ cm−1): 3343 (NH), 2948 (CH), 1746 (COOCH3), 1710 (CONH), 1293 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 7.69 (d, J = 9.5 Hz, NH), 7.57 (d, J = 9.3 Hz, NH), 5.43 (t, J = 10.0 Hz, 1H), 5.41-5.32 (m, CH), 5.37, 4.99 (pseudo t, J ~ 10.0 Hz in each, CH), 4.96 (d, J = 5.5 Hz, CH), 4.94 (d, J = 5.5 Hz, CH), 4.44 (dd, J = 12.7, 3.4 Hz, CH), 4.22 (ddd, J = 10.4, 3.6, 2.1 Hz, CH), 3.91 (dd, J = 12.4, 3.5 Hz, CH), 3.86 (s, OCH3), 2.21, 2.20, 2.19, 2.13 (s, CH3), 1.57 (s, CH3), 1.49 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 172.8 (CONH), 171.2 (COOCH3), 170.2, 169.8, 169.5, 169.1 (CO), 114.1 (qt C), 81.8 (CH), 76.1 (CH), 75.3 (CH), 73.2, 72.1, 70.0, 67.8 (CH), 61.4 (CH), 52.2 (OCH3), 26.2 (CH3), 25.8 (CH3), 20.3, 19.3 (3) (CH3). HRMS-ESI for C22H32N2O13 Na: [M + Na]+ calcd: 555.1904, found: 555.1619. Anal. Calc. For C22H32N2O13: C, 49.62; H, 6.06; N, 5.26; O, 39.06 Found C, 49.32; H, 6.16; N, 5.21; O, 39.16.
2.6. (2R,3S,5R,6R)-5-Acetamido-2-(acetoxymethyl)-6-((4R,5R)-5-(methoxycarbonyl)-2,2-dimethyl-1,3-dioxolane-4-carboxamido)tetrahydro-2H-pyran-3,4-diyl Diacetate 5e
Yield: 68%. Gel like. = 47.1. (IR υ cm−1): 3341 (NH), 2942 (CH), 1745 (COOCH3), 1711 (CONH), 1290 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 7.67 (d, J = 9.6 Hz, NH), 7.55 (d, J = 9.3 Hz, NH), 5.41 (t, J = 10.8 Hz, CH), 5.40 (m, CH), 5.38, 4.97 (pseudo t, J ~ 6.9 Hz in each, CH), 4.93 (d, J = 5.6 Hz, CH), 4.91 (d, J = 5.5 Hz, CH), 4.41 (dd, J = 12.7, 3.4 Hz, CH), 4.21 (ddd, J = 10.4, 3.6, 2.1 Hz, CH), 3.90 (dd, J = 12.4, 3.5 Hz, CH), 3.83 (s, OCH3), 2.23, 2.22, 2.20, 2.16 (s, CH3), 1.56 (s, CH3), 1.46 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 172.2 (CONH), 171.3 (COOCH3), 170.5, 169.5, 169.2, 169.1 (CO), 113.4 (qt C), 81.6 (CH), 76.4 (CH), 75.2 (CH), 73.4, 72.3, 70.1, 67.3, (CH), 62.4 (CH), 53.2 (OCH3), 26.2 (CH3), 25.7 (CH3), 20.7, 19.5 (3) (CH3). HRMS-ESI for C22H31NO14 Na: [M + Na]+ calcd: 555.1904, found: 555.1719. Anal. Calc. For C22H32N2O13: C, 49.62; H, 6.06; N, 5.26; O, 39.06 Found C, 49.32; H, 6.16; N, 5.21; O, 39.16.
2.7. General Procedure for the Synthesis of -Linked Glycopyranosides (7a–e)
Hydrazinium acetate (1.10 g, 12 mmol) was added to a stirred solution of anomeric mixture of acetylated monosaccharides 1a–e (3.12 g, 8 mmol) in DMF (40 mL). The reaction mixture was heated at 55°C for 30 min under a nitrogen atmosphere. After completion, the reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with water and brine (40 × 2), dried over anhydrous MgSO4. The solvent was removed under reduced pressure and the crude product was used without further purification. To the resulting hemiacetal (1.04 g, 3 mmol), monoester 1 (0.61 g, 3 mmol), EDC (0.74 g, 3 mmol × 1.3 eq.), and catalytic amount of DMAP were added under nitrogen with CH2Cl2 (30 mL). The reaction mixture was stirred for 18 h; the byproduct urea was removed by extraction of the reaction mixture with ethyl acetate or chloroform and water (30 mL × 3). The crude product was purified by column chromatography using ethyl acetate: n-hexane (3 : 7) as eluent.
2.8. (4R,5R)-4-Methyl-5-((2S,3R,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)-2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylate 7a
Yield: 72%. Colorless oil = 26.5. (IR υ cm−1): 2991 (CH), 1742 (CO), 1366 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 6.45 (d, J = 4.0 Hz, 1H, H-1α), 5.56 (d, J = 8.0 Hz, 1H, H-1β), 5.48, 5.45, 4.93 (pseudo t, J ~ 9.8 Hz in each, CH), 4.91 (d, J = 6.3 Hz, CH), 4.88 (d, J = 6.3 Hz, CH), 4.32 (dd, J = 12.5, 3.6 Hz, CH), 4.12 (ddd, J = 10.4, 3.6, 2.1 Hz, CH), 3.93 (dd, J = 12.5, 2.1 Hz, CH), 3.86 (s, OCH3), 2.19, 2.17, 2.12, 2.04 (s, CH3), 1.55 (s, CH3), 1.46 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 172.6 (COOCH3), 171.2, 169.5, 169.3, 169.0 (CO), 113.4 (qt C), 92.3 (CH), 92.1 (CH), 76.3 (CH), 75.4 (CH), 74.2, 73.3, 72.1, 70.3 (CH), 56.6 (CH), 53.4 (OCH3), 26.6 (CH3), 25.9 (CH3), 20.3 (2), 19.8, 19.3 (CH3). HRMS-ESI for C22H30O15 Na: [M + Na]+ calcd: 557.1585, found: 557.1452. Anal. Calc. For C22H30O15: C, 49.44; H, 5.66; O, 44.90 Found C, 49.39; H, 5.70; O, 44.81.
2.9. (4R,5R)-4-Methyl-5-((2S,3R,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)-2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylate 7b
Yield: 74%. Colorless gel = 21.6. (IR υ cm−1): 2993 (CH), 1744 (CO), 1361 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 6.47 (d, J = 3.9 Hz, 1H, H-1α), 5.53 (d, J = 8.3 Hz, 1H, H-1β), 5.47, 4.93 (pseudo t, J ~ 6.9 Hz in each, CH), 5.50 (t, J = 8.9 Hz, CH), 4.94 (d, J = 6.3 Hz, CH), 4.88 (d, J = 6.3 Hz, CH), 4.27 (dd, J = 12.5, 3.6 Hz, CH), 4.14 (ddd, J = 10.4, 3.6, 2.1 Hz, CH), 3.96 (dd, J = 12.5, 2.1 Hz, CH), 3.86 (s, OCH3), 2.19, 2.17, 2.12, 2.04 (s, CH3), 1.55 (s, CH3), 1.45 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 172.3 (COOCH3), 171.7, 169.4, 169.3, 169.1 (CO), 113.3 (qt C), 92.7 (CH), 92.4 (CH), 76.2 (CH), 75.6 (CH), 74.4, 73.5, 72.2, 70.2, (CH), 56.5 (CH), 53.2 (OCH3), 25.6 (CH3), 24.9 (CH3), 20.6 (2), 19.7, 19.5 (CH3). HRMS-ESI for C22H30O15 Na: [M + Na]+ calcd: 557.1585, found: 557.1466. Anal. Calc. For C22H30O15: C, 49.44; H, 5.66; O, 44.90 Found C, 49.39; H, 5.70; O, 44.81.
2.10. (4R,5R)-4-Methyl-5-((2R,3R,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)-2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylate 7c
Yield: 70%. White foam m.p = 151–153°C. = 17.3. (IR υ cm−1): 2995 (CH), 1745 (CO), 1336 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 6.42 (d, J = 3.7 Hz, 1H, H-1α), 5.51 (d, J = 4.0 Hz, 1H, H-1β), 5.46, 5.43 (pseudo t, J ~ 5.6 Hz in each, CH), 4.96 (t, J = 9.8 Hz, CH), 4.93 (d, J = 5.3 Hz, CH), 4.89 (d, J = 5.3 Hz, CH), 4.26 (dd, J = 12.5, 3.6 Hz, CH), 4.13 (ddd, J = 10.4, 3.6, 2.1 Hz, CH), 3.93 (dd, J = 12.5, 2.1 Hz, CH), 3.76 (s, OCH3), 2.15, 2.14, 2.12, 2.11 (s, CH3), 1.56 (s, CH3), 1.46 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 171.3 (COOCH3, 170.7, 169.6, 169.3, 169.1 (CO), 113.4 (qt C), 92.4 (CH), 92.1 (CH), 76.1 (CH), 75.6 (CH), 74.2, 73.2, 72.1, 70.5 (CH), 56.7 (CH), 53.3 (OCH3), 26.6 (CH3), 25.8 (CH3), 20.2 (2), 19.8, 19.4 (CH3). HRMS-ESI for C22H30O15 Na: [M + Na]+ calcd: 557.1585, found: 557.1475. Anal. Calc. For C22H30O15: C, 49.44; H, 5.66; O, 44.90 Found C, 49.39; H, 5.70; O, 44.88.
2.11. (4R,5R)-4-((2S,3R,5S,6R)-3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)-5-methyl-2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylate 7d
Yield: 67%. Colorless oil = 45.0. (IR υ cm−1): 3342 (NH), 2981 (CH), 1739 (CO), 1360 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 7.87 (d, J = 9.5 Hz, NH), 6.49 (d, J = 3.7 Hz, 1H, H-1α), 5.57 (d, J = 8.3 Hz, 1H, H-1β), 5.46 (m, CH) 5.43, 4.96 (pseudo t, J ~ 7.4 Hz in each, CH), 4.94 (d, J = 6.3 Hz, CH), 4.83 (d, J = 6.3 Hz, CH), 4.37 (dd, J = 12.5, 3.6 Hz, CH), 4.23 (ddd, J = 10.4, 3.6, 2.1 Hz, CH), 3.93 (dd, J = 12.5, 2.1 Hz, CH), 3.87 (s, OCH3), 2.13, 2.11, 2.10, 2.04 (s, CH3), 1.57 (s, CH3), 1.49 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 171.6 (COOCH3), 170.3, 169.3, 169.2, 169.0 (CO), 113.6 (qt C), 92.6 (CH), 92.2 (CH), 76.2 (CH), 75.1 (CH), 74.5, 73.3, 72.6, 70.1 (CH), 57.9 (CH), 53.5 (OCH3), 26.4 (CH3), 25.6 (CH3), 20.1 (2), 19.5, 19.2, (CH3). HRMS-ESI for C22H31NO14 Na: [M + Na]+ calcd: 556.1745, found: 556.1614. Anal. Calc. For C22H31NO14: C, 49.53; H, 5.86; N, 2.63; O, 41.99 Found C, 49.55; H, 5.76; N, 2.60; O, 41.90.
2.12. (4R,5R)-4-((2S,3R,5S,6R)-3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)-5-methyl-2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylate 7e
Yield: 63%. Colorless oil = 33.3. (IR υ cm−1): 3348 (NH), 2990 (CH), 1741 (CO), 1356 (C-O-C). 1HNMR (400 MHz, CDCl3): δ (ppm): 7.86 (d, = 9.7 Hz, NH), 6.52 (d, = 3.9 Hz, 1H, H-1α), 5.67 (d, = 8.7 Hz, 1H, H-1β), 5.46 (m, CH), 5.43, 4.96 (pseudo t, ~ 6.0 Hz in each, CH), 4.93 (d, J = 5.4 Hz, CH), 4.82 (d, = 5.3 Hz, CH), 4.38 (dd, = 12.5, 3.6 Hz, CH), 4.33 (ddd, = 10.4, 3.6, 2.1 Hz, CH), 3.93 (dd, = 12.5, 2.1 Hz, CH), 3.85 (s, OCH3), 2.14, 2.12, 2.10, 2.06 (s, CH3), 1.56 (s, CH3), 1.46 (s, CH3). 13C NMR (100 MHz, CDCl3): δ (ppm): 171.4 (COOCH3), 170.3, 169.5, 169.4, 169.2 (CO), 114.4 (qt C), 93.4 (CH), 92.7 (CH), 76.3 (CH), 75.2 (CH), 74.5, 73.4, 72.6, 70.1 (CH), 58.7 (CH), 53.5 (OCH3), 26.6 (CH3), 25.6 (CH3), 20.1 (2), 19.4, 19.1 (CH3). HRMS-ESI for C22H31NO14 Na: [M + Na]+ calcd: 556.1745, found: 556.1616. Anal. Calc. For C22H31NO14: C, 49.53; H, 5.86; N, 2.63; O, 41.99 Found C, 49.55; H, 5.81; N, 2.60; O, 41.91.
3. Results and Discussion
As outlined in synthetic Schemes 1 and 2, our proposed strategy was to transform five different glycopyranoses to glycopyranosyl amines 4a–e which would serve as nucleophiles. In sugars, the anomeric position is comparatively more reactive due to the presence of the endocyclic oxygen atom. Owing to its higher reactivity, microwave assisted Kochetkov amination was tried. The Kochetkov amination is used to introduce amino functionality at the anomeric position in unprotected sugars [25]. However, when glucose was reacted under microwave with Kochetkov reagents, that is, ammonium carbonate in DMSO, a mixture of amino and untreated glucose was obtained after prolonged lipophilization. In the case of glucosamine and galactosamine hydrochlorides, which already contain amino functionality, to ensure chemoselectivity in coupling with tartaric acid, a relative long route was adopted involving protecting the entire hydroxyl and amino functionalities on these sugars. Thus per acetylation [26], followed by anomeric bromination [27], azide formation [28] and reduction of azides afforded the target acetylated glycopyranosyl amines 4a–e.
Following the same coupling procedure with increased time (Table 2), -linked glycopyranosides 7a–e were also prepared in 63–74% yields, starting from acetylated monosaccharides 1a–e, Scheme 3.
It was observed that during per acetylation, a mixture of both α and β anomers is formed in a 3 : 1 ratio. In the bromination step, the thermodynamically more stable α anomer was obtained exclusively. In the resultant bromo analogue when left for longer time (two days), slight decomposition was observed. Therefore the product was used in the next step immediately. The reaction of bromide 3a with sodium azide in DMF proceeded in low yield; however under sonication, the azide analogue was produced in excellent yield. The substitution involves inversion of configuration at the anomeric site and thus the α-glycopyranosyl bromide gave the corresponding -glycopyranosyl azide. The azide intermediates were reduced immediately under catalytic hydrogenation in the next step. The hydrogenation reaction furnished the desired amines in quantitative yield, which were stable when completely dried under vacuum. Once a series of glycopyranosyl amines 4a–e were generated, the next step was to synthesize the monoester 1 starting from commercially available L-tartaric acid. Both the hydroxyl and carboxyl groups were protected followed by partial hydrolysis with one equivalent of lithium hydroxide; however the yield was variable. By the use of a stronger base sodium hydroxide, the yield was improved. It is important to mention that the formation of monoester 1 from the diester does not depend on which ester group of the two is cleaved as both groups are homotopic and therefore monohydrolysis of any of these groups will lead to identical molecules. The same strategy was then used for all of the compounds in the series.
With glycopyranosyl amines and monoester in hand, it was decided to use coupling reagents to form peptides 5a–e. Different coupling conditions were tried to generate the target amides, Table 1. DCC in the presence of a DMAP catalyst furnished the target amide in low yield. EDC when used in 1.3 equivalents for 12 h afforded the amides in good yield and the reaction was reproducible. Once coupling conditions were optimized, the same coupling conditions were used for the rest of the series to furnish the respective amides, Table 1.
The coupling reaction was high yielding and the compounds were purified by column chromatography. IR spectral data of the compounds 5a–e exhibited the appearance of characteristic (NH) stretching bands in the range of 3347–3331 cm−1 and disappearance of NH2 stretching. Two C=O stretching bands ranging from 1742 to 1736 and 1701 to 1692 cm−1 were assigned to ester and amide, respectively. 1HNMR spectral data shows characteristic doublets for the NH protons in the range of 7.69–7.18 ppm with a coupling constant of 9.6–9.3 Hz. The anomeric protons appeared as pseudotriplets in the range of 5.42–5.36 ppm with coupling constant of 12.2–6.5 Hz which confirms the formation of the β-anomer. In 13C NMR spectra, peaks ranging from 172.8–171.2 ppm were assigned to C=O (amide) and 170.9–170.2 ppm to C=O (ester), respectively. Elemental analysis of the glycopyranosyl amides 5a–e confirmed the structures of the compounds. The exact mass of the compounds was confirmed by HRMS-ESI.
The anomeric acetyl group was hydrolyzed selectively using hydrazinium acetate, in DMF [29–31]; the resulting hemiacetal was washed with water and brine and used without further purification. To the crude hemiacetal, monoester was added using EDC as a coupling agent. When compound 6a was coupled with monoester 1 using 1.3 eq. EDC, DMAP with increased reaction time afforded compound 7a in 72% yield. Once the reaction conditions were optimized, the same conditions were used for the rest of the series to furnish the respective esters, Table 2.
The synthesized compounds were purified by column chromatography and characterized by spectroscopic techniques. IR spectral data of the compounds 7a–e showed characteristic (C=O) stretching bands ranging from 1745 to 1739 cm−1 in addition to C-O-C stretching in 1366–1356. The NH stretching bands 7d-e appeared in 3348–3342 cm−1. 1HNMR spectral data showed doublets 7d-e, for the NH protons in the range of 7.87–7.86 ppm with coupling constant of 9.7–9.5 Hz. Bothα- andβ-anomers were found to be 1 : 3 except compound 7c where α andβ ratio was 1 : 1 as confirmed by 1HNMR spectroscopy. The anomeric protons appeared as doublets in the range of 5.67–5.43 ppm with coupling constant of 8.3–8.0 Hz for theβ-anomers and in 6.52–6.42 ppm with coupling constant of 4.0–3.7 Hz for theα-anomers, respectively. The coupling constant of anomeric hydrogen for mannopyranosyl 7c was 4.0 Hz in both the anomers. Finally the synthesis of glycopyranosides was supported by one of the crystal structure forα-anomer, O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-(4R,5R)-2,2-dimethyl-1,3-dioxolane-4,5-dicarboxylate 7c by X-ray crystallography (Figure 1).
Crystal system = monoclinic. Torsion angles for compound 7c are C5-O1-C1-O13 = −62.9(2)°, C5-O1-C1-C2 = 54.9(2)°, C15-O13-C1-O1 = −76.4(2) (2)°, C15-O13-C1-C2 = 160.28(19)°, C13-O8-C2-C3 = −103.8(2)°, C13-O8-C2-C1 = 136.00(19)°, O1-C1-C2-O8 = 73.3(2)°, O13-C1-C2-O8 = −165.60(16)°, O1-C1-C2-C3 = −46.9(2)°, O13-C1-C2-C3 = 74.2(2)°, C11-O6-C3-C4 = −149.8(2)°, C11-O6-C3-C2 = 88.7(3)°, O8-C2-C3-O6 = 49.8(2)°, C1-C2-C3-O6 = 167.17(17)°, O8-C2-C3-C4 = −69.0(2)°, C1-C2-C3-C4 = 48.4(3)°, C9-O4-C4-C3 = −119.6(3)°, C9-O4-C4-C5 = 123.8(3)°, O6-C3-C4-O4 = 67.2(2)°, C2-C3-C4-O4 = −172.34(18)°, O6-C3-C4-C5 = −176.24(17)°, C2-C3-C4-C5 = −55.8(2)°, C1-O1-C5-C6 = 176.43(19)°, C1-O1-C5-C4 = −61.4(2)°, O4-C4-C5-O1 = 176.08(18)°, C3-C4-C5-O1 = 60.3(2)°, O4-C4-C5-C6 = −65.2(3)°, C3-C4-C5-C6 = 179.0(2)°, C7-O2-C6-C5 = −153.1(2)°, O1-C5-C6-O2 = −68.2(3)°, C4-C5-C6-O2 = 172.1(2)°, C6-O2-C7-O3 = 1.9(5)°, C6-O2-C7-C8 = −175.2(3)°, C4-O4-C9-O5 = −7.1(6)°, C4-O4-C9-C10 = 174.8(3)°, C3-O6-C11-O7 = 3.7(4)°, C3-O6-C11-C12 = −175.5(2)°, C2-O8-C13-O9 = 6.4(3)°, C2-O8-C13-C14 = −171.9(2)°, C1-O13-C15-O12 = −5.2(4)°, C1-O13-C15-C16 = 173.8(2)°, C19-O15-C16-C15 = 131.8(4)°, C19-O15-C16-C17 = 11.0(5)°, O12-C15-C16-O15 = −7.6(4)°, O13-C15-C16-O15 = 173.5(2)°, O12-C15-C16-C17 = 108.9(4)°, O13-C15-C16-C17 = −70.1(3)°, C19-O14-C17-C18 = 130.6(5)°, C19-O14-C17-C16 = 11.5(6)°, O15-C16-C17-O14 = −13.4(5)°, C15-C16-C17-O14 = −133.2(4)°, O15-C16-C17-C18 = −131.8(3)°, C15-C16-C17-C18 = 108.4(3)°, C22-O11-C18-O10 = −2.3(9)°, C22-O11-C18-C17 = 178.0(4)°, O14-C17-C18-O10 = −31.2(7)°, C16-C17-C18-O10 = 82.9(6)°, O14-C17-C18-O11 = 148.5(4)°, C16-C17-C18-O11 = −97.4(4)°, C17-O14-C19-O15 = −5.3(7)°, C17-O14-C19-C21 = 109.3(8)°, C17-O14-C19-C20 = −123.6(6)°, C16-O15-C19-O14 = −4.3(6)°, C16-O15-C19-C21 = −121.1(8)°, and C16-O15-C19-C20 = 113.7(6)°.
4. Material and Methods
4.1. Antileishmanial Activity
Antileishmanial activities of compounds 5a–e and 7a–e were assayed by Zhai et al.’s method (1999) [32] using a preestablished culture of Leishmania. Triple-N media slants were overlaid with 199 media for leishmanial growth. NNN medium was prepared by mixing 4 g of agar in 100 mL of distilled water. The mixture was then dissolved and sterilized by autoclaving at 121°C and then allowed to cool to 55°C. 15–20 mL of defibrinated sheep blood was aseptically added to the mixture with gentle rolling with glass beads; 1 ampoule of gentamicin was mixed in blood and mixed with the agar mixture. Slopes of culture medium were prepared by dispensing 2-3 mL of the blood agar mixture into sterile tubes that were then set in a slant position until the agar completely solidified. To prepare 199 medium different constituents were mixed in 1000 mL of distilled water. The pH was adjusted to 7.4 and medium was filtered, sterilized, and kept at 37°C for 24 h to check sterility. Preestablished culture of Leishmania tropica KWH23 was inoculated in 199 medium in Triple-N medium slants and incubated at 24°C for 6-7 days and the results are summarized in Table 3.
5. Results
All the synthesized N-linked 5a–e and O-linked glycopyranosides 7a–e were tested for their antileishmanial activity using Leishmania tropica KWH23 promastigotes for in vitro screening. The results are shown in Table 3. Compounds 5a, 5b, and 5d showed good activity while the compounds 5c and 5e exhibited low activity.
Among the synthesized O-linked glycopyranosides 7a–7e, compounds 7c and 7e showed good activity; 7b and 7d showed moderate activity. Compound 7a showed nonsignificant activity.
6. Conclusion
Most of the synthesized compounds 5a–e and 7a–e showed moderate to good activities against Leishmania tropica KWH23 promastigotes. Compounds 5a, 5b, and 5d were the most active. Among the synthesized compounds, 7a–7e, 7c, and 7e were more active than 7a, 7b, and 7d. Drug resistance has been reported in various species of Leishmania against various antileishmanial drugs like antimonials, amphotericin B, pentamidine, miltefosine, and so forth. In this scenario these synthetic compounds may be further explored for research in medicinal chemistry and drug designing. These compounds may prove to be good candidates against leishmaniasis. A number of other new compounds can also be synthesized while following the same N- and O-acylation synthetic routes. Such compounds can act as valuable glycodrugs as many of the glycosides and their derivatives have been reported to be biologically active compounds.
Conflicts of Interest
The authors confirm that this article’s content has no conflicts of interest.
Acknowledgments
This work was financially supported by Higher Education Commission (HEC), Pakistan, under International Research Support Initiative Program (IRSIP) and National Research Program (NRPU) for Universities.
Supplementary Materials
Supplementary data include NMR (1H and 13C), IR, and MS spectra of the synthesized compounds (5a–5d and 7a–7e). 1HNMR of compound 5a. 13CNMR of compound 5a. ESI-MS of compound 5a. FT-IR of compound 5a. 1HNMR of compound 5b. 13CNMR of compound 5b. ESI-MS of compound 5b. 1HNMR of compound 5c. 13CNMR of compound 5c. 1HNMR of compound 5d. 13CNMR of compound 5d. ESI-MS of compound 5d. 1HNMR of compound 7a. 13CNMR of compound 7a. ESI-MS of compound 7a. 1HNMR of compound 7b. 13CNMR of compound 7b. ESI-MS of compound 7b. ESI-MS of compound 7b backup. 1HNMR of compound 7c, new. 1HNMR of compound 7c. 13CNMR of compound 7c. 13CNMR of compound 7b, new. ESI-MS of compound 7c. Mass analysis of compound 7c. 1HNMR of compound 7d. 13CNMR of compound 7d. ESI-MS of compound 7d. Mass analysis of compound 7d. 1HNMR of compound 7e. 13CNMR of compound 7e. (Supplementary Materials)