Indian Patents. 253810:NOVEL GL YCOSYL-D-FRUCTOSE DERIVATIVES AS ANTIHYPERLIPIMEMIC AGENT

Title of Invention	NOVEL GL YCOSYL-D-FRUCTOSE DERIVATIVES AS ANTIHYPERLIPIMEMIC AGENT
Abstract	The present invention provides a novel glycosyl-D-fructoses of general formula I, and isomers thereof. The compound of general formula I is used as antihyperlipidemic agent in terms of either lowering triglyceride (TG) concentration and/or by increasing HDL-cholestero! and total cholesterol ratio (H/C).

Full Text	FIELD OF THE INVENTION The present invention relates to a novel glycosyl-D-fructoses of formula I and isomers thereof, wherein, R1, R2,R3,R4,R5,R6 is hydrogen, hydroxyl or ester amide, alkyl and alkyl ether protected or unprotected monosaccharide, disaccharide, anhydro sugar, olefinic sugar, deoxy sugar, modified sugar and mixture thereof, R7 is hydrogen, methyl or CH2OR10; R8 is hydrogen or methyl; R9 and R10 is hydrogen or glycosyl moiety More particularly it relates to the glycosyl-D-fructoses, polymorphic forms, salts con.aining such compounds and compositions having such compounds as active ingredients. It also relates to the process of preparation of such compounds. BACKGROUND OF THE INVENTION Glycosyl-D-fructoses, also termed as "fructo oligosaccharides" comprises a large class of oligosaccharides, which have been isolated from the various honeydews. Most of these compounds are water-soluble and crystalline in nature having low optical rotations and have a slightly sweet to very sweet taste. D-Raffinose and D-melezitose are the most commonly found fructo oligosaccharides isolated from the natural sources. Most of the fructo oligosaccharides isolated have been put to various pharmaceutical uses such as bifidus factor [Okhusa, Toshifumi; Ozaki, Yoshinori; Satochifumi; Miken, Katasuhika; Ikeda, Hiroshi. Digestion, 1995, 56, 415-20] and anti-cariogenic agents [Mikuni, Katsuhiko; Fujita, Takateru; Osawa, Takeshi; Aoyama, Yoko; Hara, Kozo; Hashimoto, Hitoshi. JP 03 083 992]. Among all isolated fructo oligosaccharides, raffinose, lactosucrose and melezitose have been used in health food materials [Hayakawa, Kunihiko; Omura, Kazutaka; Mizutani, Jun; Shirayanagi, Satoru; Kato, Yasuhito; Ikeda, Nagisa. JP 03 151 854], [Uchida, Yukio; Iritani, Satoshi; Mayake, Toshio. JP 07 298 833], as low cariogenic sugar substitute [Biton Jacques; Michael-Jean, Marc; Le Beller, Dominique; Pelenc, Vincent; Paul, Francois; Monsan, Pierre, F; Gelf, Gerard. Ann. NY. Acad. Sci. 1995, 750 (Enzyme Engineering XII), 312-14], as well as food substituents for various animals [Satoya, Hiroaki. JP 05 003 758] and [Kihara, Minoru; Mino, Riyuusuke. JP 07 203 868]. They have been used as osteoporosis preventing agents [Ohta, Atsutane; Takizawa, Toshio. EP 0753302]. They have also been used as prophylaxis for hepatopathy and as laxatives [Suga, Tatsuhiko; Kobayashi, Yoichi; Sonoike, Yoshiko; Terajima, Tsuneo; Mutai, Masahiko. JP 62 220 169]. Sulfated Raffinose derivatives have also been reported as antiarteriosclerotic agent [Hosang, Markus; Iberg, Niggi; Tschopp Thomas, Beat; Wessel-Hans, Peter. EP 504645]. Raffinose and Melibiose have also been used as carcinostatic Pharmaceuticals [Watanabe, Atsushi; Nukada, Akira. JP 57 077 620]. Lactosucrose has been used as an active ingredient in atopic dermatitis [Watanabe, Atsushi; Nukada, Akira. JP 11 124 390]. Coronary heart disease is a multifactorial disease in which the incidence and severity are affected by the lipid profile, the presence of diabetes and the sex of the subject. Coronary heart disease (CHD) remains the leading cause of death in the industrialized countries. The primary cause of CHD is arteriosclerotic, a disease characterized by the deposition of lipids, including cholesterol, in the arterial vessel wall, resulting in a narrowing of the vessel passages and ultimately hardening of the vascular system. There are two types of cholesterol mainly present in the human body, among which the harmful cholesterol is low-density lipoprotein (LDL) cholesterol. Several clinical studies have established that lowering of LDL-cholesterol in mammal is an effective way to treat and prevent heart attacks, sudden death and angina. Lowering LDL-cholesterol is now one of the primary objectives of medicinal chemists for treating patients who have hypercholesterolemia, or who have a tendency to develop cardiovascular diseases such as coronary heart disease. It is therefore recommended that the compounds which increase high-density lipoprotein-cholesterol (HDL) and therefore HDL-cholesterol and total cholesterol ratio (H/C) are effective curative agents against hypercholesterolemia. Recently it has been reported that, lactic acid bacteria present in the body produce an abundant variety of exopolysaccharides (EPS's), which provide an important contribution to human health by acting as prebiotic substrates, nutraceuticals, cholesterol lowering agents or immunomodulants [Van, Hijum; Sacha Adrianus Fokke Taco Groningen; Van, Geel-Schutten; Gerritdina, Hendrika; Dijkhuizen; Lubbert; Rahaoui; Hakim. US 6,730,502]. These compounds are also synthesized using extracellular sucrase enzymes, fructosyltransferases and glucosyltransferases. These fructosyltransferases have already been used for the chemoenzymatic preparation of various fructo-oligosaccharides. For example maltosylfructose has been prepared by using levansucrase from Bacillus subtilis [Jimenez-Estrada, M; Cassani, J; Lopez-Munguia, A. Biocatal. Biotransform. 1999,16 (6), 475-485], A number of reports appeared in the literature for the use of fructose containing oligosaccharides as prebiotic substrates [ACS Symposium Series, 2003, 849 (oligosaccharide in food and agriculture), 76-89], nutraceuticals. However their use in the treatment of metabolic disorders like hypercholesterolemia has not been explored. As a part of the ongoing research program to develop antihyperlipidemic agents, the present inventors set out to explore the pharmaceutical potentiality of several glycosyl-D-fructoses as antihyperlipidemic agents. A series of chemically synthesized and commercially available novel fructose containing oligosaccharides have been evaluated for their antihyperlipidemic activity. SUMMARY OF THE INVENTION Accordingly the present invention relates to a novel glycosyl-D-fructoses having the general formula I and isomers thereof, formula I wherein R1, R2,R3,R4,R5,R6 is hydrogen, hydroxyl or ester amide ,alkyl and alkyl ether protected or unprotected monosaccharide, disaccharide, anhydro sugar, olefinic sugar, deoxy sugar, modified sugar and mixture thereof, R7 is hydrogen, methyl or CH2OR10; R8 is hydrogen or methyl; R9 and R10 is hydrogen orglycosyl moiety In an embodiment of the invention the representative compounds of general formula I are: la= (3-D-galactopyranosyl (1 ->4)-(3-D-glucopyranosyl-(1 ->3)-D-fructose lb= α-D-glucopyranosyl-(1-4)-(3-D-glucopyranosyl-(1-3)-D-fructose lc= β-D-glucopyranosyl-(1-4)-(3-D-glucopyranosyl-(1-3)-D-fructose ld= β-D-galactopyranosyl-(1 ->3)-D-fructose le=α -D-mannopyranosyl-(1 ->3)-D-fructose lf=β-D-arabinopyranosyl-(1 ->3)-D-fructose lg= β-D-xylopyranosyl-(1 ->3)-D-fructose lh= β-D-ribopyranosyl-(1 ->3)-D-fructose li= 6-deoxy-β-L-mannopyranosyl-(1 ->3)-D-fructose lj= α-D-galactopyranosyl-(1 ->6)-(3-D-glucopyranosyl-(1 ->2)-D-fructose (D-raffinose pentahydrate) lk=α-D-glucopyranosyl-(1->3)-p-D-fructofuranosyl-a-D-glucopyranoside (D-melezitose monohydrate). In another embodiment of the invention the modified sugar is selected from the group consisting of glucose, galactose, mannose, arabinose, xylose, ribose, rhamnose, fructose, tagatose, digitose, N-acetyl neuraminic acid, maltose, cellobiose, lactose, melibiose and gentiobiose. In yet another embodiment the glycosyl moieties is selected from the group consisting of D-glucose, D-galactose, D-mannose, D-arabinose, D-xylose, D-ribcse, L-rhamnose, L-fucose, tagatose, digitose, A/-acetyl neuraminic acid, D-maltose, D-cellobiose, D-lactose, D-glucosamine, D-galactosamine, D-fucosamine, D-mannosamine. In yet another embodiment the compound of general formula I is effective against lowering triglycerides (TG) upto 52%. In yet embodiment the compound is effective for increasing in HDL-cholesterol and total cholesterol ratio (H/C) by 60%. In yet another embodiment the compound of formula I and isomers thereof are used for the treatment of hyperlipidemia. In yet another embodiment the compound of formula I and isomers thereof are used as cholesterol lowering agent. In yet another embodiment the compound of formula I and isomers thereof are used as high density lipoprotein-cholesterol (HDL) increasing agent. In yet another embodiment the compound of formula I and isomers thereof are used as triglyceride lowering agent. Accordingly the present invention provides a process for the preparation of compound of general formula I and isomers thereof wherein R1, R2,R3,R4,R5,R6 is hydrogen, hydroxyl or ester amide ,alkyl and alkyl ether protected or unprotected monosaccharide, disaccharide, anhydro sugar, olefinic sugar, deoxy sugar, modified sugar and mixture thereof, R7 is hydrogen, methyl or CH2OR10; R8 is hydrogen or methyl; R9 and R10 is hydrogen or glycosyl moiety A series of fructose containing novel oligosaccharides have been prepared by conventional glycosylation techniques involving coupling of several per-O-acetylated thioglycoside donors (III, wherein R1-R8 have the same meaning as compound of formula I) with suitably protected fructose acceptor. In order to so, a series of per-O-acetylated thioethyl glycosides (III, wherein R1-R8 have the same meaning as compound of formula I) of several mono- and di-saccharides have been prepared from commonly available unprotected sugars following conventional acetylation followed by thioglycosylation. 1,2:4,5-Di-O-isopropylidene-D-fructose derivative was used as glycosyl acceptor. The glycosylation reaction was performed in the presence of N-iodosuccinimide (NIS) and trifluoromethane sulfonic acid as thioglycoside activator. The protected fructose oligosaccharide derivatives (V, wherein R1-R8 have the same meaning as compound of formula I) thus obtained were deprotected following convention deprotection techniques to furnish VI, wherein R1-R8 have the same meaning as compound of formula I. In an embodiment of the invention is a process for the preparation of glycosyl-D-fructoses of general formula I comprising of: a) reacting the per-O-acetylated sugar of formula II wherin R1-R10 have the same meaning as compound of formula I and sulphur containing alkanol compound in the ratio of 1:1 in the presence of a dry organic solvent under inert atmosphere and cooling the above said reaction mixture to a temperature of about 0 °C and further adding borontrifluoride diethyletherate under stirring, for a period of about 5 hrs to obtain the desired product of formula III, wherin R1-R10 have the same meaning as compound of formula I b) reacting suspension of D-fructose in presence of dry acetone with concentrated sulphuric acid under stirring for a period of about 2hrs at a temperature of about 25°C and cooling the reaction mixture cooling the reaction mixture and quenching in 50%(w/v) of sodium hydroxide to a temperature of about 0°C , followed by purifying by known method and . drying to obtain the crude mass , further washing with alkaline sodium bicarbonate solution and purifying by known chromatographic methods to obtain the desired product of formula IV, c) mixing the above said reaction product of formula III and the above said reaction product of formula IV in the mmolar ratio of 1:1 in presence of dry organic solvent, followed by, i) adding molecular sieves of 4A of weight 500mg in argon atmosphere under stirring at a temperature of about 25°C for a time period of about 30 minutes, further adding N-iodosuccinimide and cooling the reaction mixture to a temperature of about -4°C, ii) cooling the above said reaction mixture and adding trifluoromethanesulphonic acid to obtain the resultant solution, and stirring the aforesaid resultant solution for a period of about -40°C for a period of about 1 hour and purifying the reaction mixture by known chromatographic methods to obtain the desired product of formula V, wherein R1-R10 have the same meaning as compound of formula I iii) reacting the above said reaction product obtained in step (ii) with trifluroacetic acid and water in the ratio of 2:1 in presence of an organic solvent, followed by stirring at a temperature of about 25°C for a period of about 3 hours, purifying the above said reaction mixture obtained by known chromatographic methods to obtain the desired product of formula I. In yet another embodiment of the invention the representative compounds of the general formula I and isomers thereof are: la=β -D-galactopyranosyl(1 ->4)-3-D-gluco-pyranosyl-(1 ->3)-(3-D-fructose lb= β-D-glucopyranosyl-(1 ->4)-p-d-glucopyranosyl(1 ->3)-d-fructose lc= β-D-glucopyranosyl-(1 ->4)-(3-d-glucopyranosyl-(1 ->3)-d-fructose ld= β-D-galactopyranosyl-(1 ->3)-d-fructose le= α-D-mannopyranosyl-(1 ->3)-d-fructose lf=β-D-arabinopyranosyl-(1->3)-d-fructose lg= β-D-xylopyranosyl-(1->3)-d-fructose lh= β-D-ribopyranosyl-(1->3)-d-fructose li= 6-deoxy-β-l-mannopyranosyl-(1->3)-d-fructose Accordingly the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of general formula I as claimed in claim 1 or compound 3j and 3k optionally along with pharmaceutically acceptable diluent and or carrier. In an embodiment of the present invention the composition is useful in the treatment of hyperlipidemia, cholesterol lowering agent, used as high density lipoprotein-cholesterol (HDL) increasing agent, triglyceride lowering agent. In still another embodiment the dose of the compound of general formula I is 25 mg/kg to 100mg/kg. Another feature of the present invention is a method of treating a subject having hyperlipidemia, comprising administering to the subject a pharmaceutically effective amount of a composition containing substituted compound of formula I, or compound 3j a and 3k. In yet another embodiment of the invention wherein the compound composition is administered orally. In yet another embodiment of the invention wherein the pharmaceutically acceptable amount of compound of formula I is in the range of 25 mg per kg to 100 mg per kilogram of the body weight of subject per day. DETAILED DESCRIPTION OF THE INVENTION The present invention provides antihyperlipidemic properties of novel glycosyl-D-fructoses of general formula I, and their isomers. salts containing such compounds, compositions containing these compounds and methods for using such compounds and compositions for the treatment of antihyperlipidemia. As such, in a particular, the present invention provides novel compounds having the general formula I and isomers thereof. In an embodiment of the invention is a process for the preparation of glycosyl-D-fructoses of general formula I comprising of: a) reacting the per-O-acetylated sugar of formula II wherin R1-R8 have the same meaning as compound of formula I and sulphur containing alkanol compound in the ratio of 1:1 in the presence of a dry organic solvent under inert atmosphere and cooling the above said reaction mixture to a temperature of about 0 °C and further adding borontrifluoride diethyletherate under stirring, for a period of about 5 hrs to obtain the desired product of formula III, wherin R1-R8 have the same meaning as compound of formula I b) reacting suspension of D-fructose in presence of dry acetone with concentrated sulphuric acid under stirring for a period of about 2hrs at a temperature of about 25°C and cooling the reaction mixture cooling the reaction mixture and quenching in 50%(w/v) of sodium hydroxide to a temperature of about 0°C , followed by purifying by known method and drying to obtain the crude mass , further washing with alkaline sodium bicarbonate solution and purifying by known chromatographic methods to obtain the desired product of formula IV, c) mixing the above said reaction product of formula III and the above said reaction product of formula IV in the molar ratio of 1:1 in presence of dry organic solvent, followed by, i) adding molecular sieves of 4A of weight 500mg-2 g in argon atmosphere under stirring at a temperature of about 25°C for a time period of about 30 minutes, further adding N-iodosuccinimide and cooling the reaction mixture to a temperature of about -4°C, ii) cooling the above said reaction mixture and adding trifluoromethanesulphonic acid to obtain the resultant solution, and stirring the aforesaid resultant solution at -40°C for a period of about 1 hour and purifying the reaction mixture by known chromatographic methods to obtain the desired product of formula V, wherein R1-R8 have the same meaning as compound of formula I reacting the above said reaction product obtained in step (ii) with trifluroacetic acid and water in the ratio of 2:1 in presence of an organic solvent, followed by stirring at a temperature of about 25°C for a period of about 3 hours, purifying the above said reaction mixture obtained by known chromatographic methods to obtain the desired product of formula I. In yet another embodiment of the invention the per-O-acetyl sugars compound of formula V is selected from the group consisting of 2,3,4,6-Tetra-O-acetyl-β-D- galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-pD-glucopyranosyl-(1->3)-1,2:4,5-di- 0-isopropylidene-D-fructopyranose (Va); 2,3,4,6-Tetra-O-acetyl-β-D- glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-β~-D-glucopyranosyl-(1->3)-1,2:4,5-di-O- isopropylidene-D-fructopyranose (Vb); 2,3,4,6-Tetra-O-acetyl-β-D- glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl-(1->3)-1,2:4,5-di-O- isopropylidene-D-fructopyranose (Vc); 2,3,4,6-Tetra-O-acetyl-p-D- galactopyranosyl-(1 -»3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-ose (Vd); 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene- D-fructopyran-ose (Ve); 2,3,4-Tri-O-acetyl-p-D-arabinopyranosyl-(1->3)-1,2:4,5- di-O-isopropylidene-D-fructopyranose (Vf); 2,3,4-Tri-O-acetyl-β-D-xylopyranosyl- (1-»3)-1,2:4,5-di-0-isopropylidene-D-fructopyranose (Vg); 2,3,4-Tri-O-acetyl-β- D-ribopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vh); 2,3,4-Tri-O-acetyl-6-deoxy-α-L-mannopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vi). In yet another embodiment the sulphur containing alkanol is selected from ethanethiol, thiophenol, p-thiocresol, m-thiocresol, o-thiocresol, 2-mercaptobenzothiazole, 2-mercaptobenzimidazole, 2-mercaptopyridine, 2-mercaptopiperidine. In yet another embodiment the organic solvent used is selected from dichloromethane, chloroform, dichloroethane, tetrahydrofuran, diethyl ether, acetonitrile, 1,4-dioxane, dimethyl formamide, toluene and benzene. In yet another embodiment of the invention the product obtained of formula III is selected from the group consisting of ethyl 2,3,4,6-tetra-O-acetyl-p-D-galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (Ilia), ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (1Mb), ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-(1->4)-2,3,5-tri-0-acetyl-1-thio-p~-D-glucopyranoside (Illc), ethyl 2,3,4,6-tetra-O-acetyl-1-thio-p-D-galactopyranoside (Hid), ethyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-mannopyranoside (Ille), ethyl 2,3,4-tri-O-acetyl-1-thio-(3-D-arabinopyranoside (Illf), ethyl 2,3,4-tri-O-acetyl-1-thio-β-D-xylopyranoside (Illg) ethyl 2,3,4-tri-O-acetyl-1-thio-β-D-ribopyranoside (Illh), ethyl 2,3,4-tri-O-acetyl-1-thio-6-deoxy-a-L-mannopyranoside(llli). In yet another embodiment the product obtained of formula V is selected from the group consisting of 2,3,4,6-Tetra-O-acetyl-p-D-galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-βD-glucopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Va); 2,3,4,6-Tetra-O-acetyl-p-D-glucopyranosyl-(1 ->4)-2,3,6-tri- O-acetyl-β-D-glucopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D- fructopyranose (Vb); 2,3,4,6-Tetra-0-acetyl-β-D-glucopyranosyl-(1 ->4)-2,3,6-tri- O-acetyl-β-D-glucopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D- fructopyranose (Vc); 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1 -»3)-1,2:4,5- di-O-isopropylidene-D-fructopyran-ose (Vd); 2,3,4,6-Tetra-O-acetyl-a-D- mannopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-ose (Ve); 2,3,4-Tri-O-acetyl-β-D-arabinopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vf); 2,3,4-Tri-O-acetyl-p-D-xylopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vg); 2,3,4-Tri-O-acetyl-p-D-ribopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vh); 2,3,4-Tri-O-acetyl-6-decxy-a-L-mannopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vi)-. The invention is further illustrated by the following examples, which should not however be construed to limit the scope of the present invention. EXAMPLE 1 GENERAL PREPARATION OF THIOETHYL GLYCOSIDES [III (a-i)] To a solution of per-O-acetylated sugars [II (a-i)] (1.0 mmol) in dry CH2CI2 (5.0 rnL), ethanethiol (1.50 mmol) was added under inert atmosphere. The reaction mixture was cooled to 0°C and borontrifluoride diethyletherate (2.0 mmol) was added to it and the reaction mixture was stirred at 0°C for 5 hrs. The progress of the reaction was monitored by thin layer chromatography over silica gel coated plates. After completion of the reaction, the reaction mixture was diluted with CH2Cl2, washed with aq. sodium bicarbonate solution and water in succession. The organic layer was dried over anhydrous sodium sulphate and concentrated to dryness under reduced pressure. The crude reaction mixture was purified over Si02 using hexane-EtOAc as eluant to furnish desired ethylthioglycosides [III (a-i)] (Table 1). (Table Removed) EXAMPLE 2 PREPARATION OF 1,2:4,5-DI-O-ISOPROPYLIDENE-D-FRUCTOPYRANOSE [IV] To a suspension of D-fructose (30 g) in dry acetone (600 ml) was added cone. H2SO4 (2.9 ml) and the reaction mixture was allowed to stir for 2 hrs. at room temperature. After completion of the reaction as monitored by thin layer chromatography over silica gel coated plates the reaction mixture was cooled to 0°C and quenched with aq. 50% NaOH solution. The reaction mixture was evaporated to dryness to a crude mass. The crude mass was diluted with CH2Cl2 (1000 ml) and the organic layer was washed with aq. sodium bicarbonate solution and water in succession. The organic layer was dried over anhydrous sodium sulphate and concentrated to dryness under reduced pressure. The crude reaction mixture was purified over Si02 using hexane-EtOAc as eluant to furnish desired 1,2:4,5-di-O-isopropylidene-D-fructose [IV]. Yield: 76%; m.p. 119°C; [α]D25-145 (c 1.0, CHCI3). EXAMPLE 3 GENERAL GLYCOSYLATION PROTOCOL To a mixture of ethyl per-O-acetyl-1-thio-D-D-glycopyranoside [Ml(a-i)] (1.1 mmol) and 1,2:4,5-di-O-isopropylidene-D-fructopyranose [IV] (1.0 mmol) in dry CH2CI2 (5 ml) was added molecular sieves 4A (500 mg) and flushed with argon. After stirring at room temperature for 30 min, AModosuccinimide (1.21 mmol) was added and the reaction mixture was cooled to -40°C. To the cooled reaction mixture trifluoromethanesulphonic acid (0.1 mmol) was injected and the resulting solution was stirred at -40°C for 1 hour. The progress of the reaction was monitored by thin layer chromatography over silica gel coated plates. After completion of the reaction, the reaction mixture was diluted with CH2Cl2 and filtered through a celite bed. The filtrate thus obtained was washed with aq. sodium thiosulphate, aq. sodium bicarbonate solution and water in succession. The organic layer was dried over anhydrous sodium sulphate and the resulting reaction mixture was concentrated to dryness under reduced pressure. The crude reaction mixture was purified over SiO2 using hexane-EtOAc as eluant to furnish desired per-O-acetyl-D-glycopyranosyl-(1->3)-1,2:4,5-di-O- isopropylidene-D-fructopyranose [V(a-i)]. GENERAL DEPROTECTION PROTOCOL FOR THE PREPARTION OF UNPROTECTED FRUCTOSE OLIGOSACCHARIDES [l(a-i)] To a solution of per-O-acetyl-β~-D-glycopyranosyl-(1->3)-1,2:4,5-di-0-isopropylidene-D-fructopyranose [V(a-i)] (1.0 mmol) in CH2CI2 (5.0 ml) was added trifluroacetic acid (10.0 ml), and water (5.0 ml). The reaction mixture was stirred at room temperature for 3 hrs. The reaction was monitored by thin layer chromatography over silica gel coated plates. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3x20mL) to furnish desired per-O-acetyl-D-glycopyranosyl-(1->3)-D-fructose. To a solution of crude per-O-acetyl-βD-glycopyranosyl-(1->3)-D-fructose thus obtained in dry methanol (10.0 ml), sodium methoxide (50 mg) was added and the reaction mixture was stirred at room temperature for 4 hrs. The reaction was monitored by thin layer chromatography over silica gel coated plates. After completion of the reaction, Amberlite IR 120 (H+) resin was added to the reaction mixture with constant stirring till the solution becomes neutral. The resulting reaction mixture was cotton filtered and the filtrate was concentrated to dryness under reduced pressure. The crude reaction mixture was purified over SiO2 using chloroform-MeOH (2:1) as eluant to furnish desired p~-D-glycopyranosyl-(1-»3)-D-fructose [l(a-i)]. EXAMPLE 3a PREPARATION OF p-D-GALACTOPYRANOSYL-(1->4)-p-D- GLUCOPYRANOSYL-(1-»3) D-FRUCTOSE (la): 2,3,4,6-Tetra-O-acetyl-p-D-galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-βD-glucopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Va) was prepared by following the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-p-D-galactopyranosyl-(1-»4)-2,3,6-tri-O-acetyl-1-thio-p-D-glucopyranoside (Ilia) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 72%. [p]D25 - 25 (c 1.0, CHCI3). Compound la was prepared by following the general deprotection protocol as mentioned in Example 3-2. Yield: 50% in two steps. [(3]D25 + 2.1 (c 1.0, CHCI3). EXAMPLE 3b PREPARATION OF β-D-GLUCOPYRANOSYL-(1->4)-β~-D-GLUCOPYRANOSYL (1->3)-D-FRUCTOSE (Ib): 2,3,4,6-Tetra-0-acetyl-β-D-glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-3"-D-glucopyranosyl-(1-»3)-1,2:4,5-di-0-isopropylidene-D-fructopyranose (Vb) was prepared by using the generalized glycosylation protocol as given in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-p-D-glucopyranosyl-(1-»4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (Illb) as donor and 1,2:4,5-di-O-isopropylidene-β-D-fructopyranose (IV) as acceptor. Yield: 75%. [β]D25 + 7.2 (c 1.0, CHCI3). Compound Ib was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 58% in two steps. [β]D25 + 8.6 (c 1.0, CHCI3). EXAMPLE 3c PREPARATION OF β-D-GLUCOPYRANOSYL-(1->4)-p-D- GLUCOPYRANOSYL-(1->3)-D-FRUCTOSE (Ic) 2,3,4,6-Tetra-O-acetyl-p-D-glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-βD-glucopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vc) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-p-D-glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-1-thio-p-D-glucopyranoside (Illc) as donor and 1,2:4,5-di-O-isopropylidene-D-frctopyranose (IV) as acceptor. Yield: 73%. [β]D25: - 19.5 (c 1.0, CHCI3). Compound Ic was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 52% in two steps. [β]D25 - 1.8 (c 1.0, CHCI3). EXAMPLE 3d PREPARATION OF β-D-GALACTOPYRANOSYL-(1-»3)-D-FRUCTOSE (Id): 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-ose (Vd) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-1-thio-β -D-galactopyranoside (Mid) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 73%. [β]D25 - 17.4 (c 1.0, CHCI3). Compound Id was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 51% in two steps.[β ]D25 + 3.3 (c 1.0, CHCI3). EXAMPLE 3e PREPARATION OF α-D-MANNOPYRANOSYL-(1-»3)-D-FRUCTOSE (le) 2,3,4,6-Tetra-O-acetyl-β-D-mannopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-ose (Ve) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-mannopyranoside (llle) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 74%. [β]D25 - 5.4 (c 1.0, CHCI3). Compound le was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 56% in two steps. [βD25 - 2.6 (c 1.0, CHCI3). EXAMPLE 3f PREPARATION OF β-D-ARABINOPYRANOSYL-(1-»3)-D-FRUCTOSE (If) 2,3,4-Tri-O-acetyl-β-D-arabinopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vf) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4-tri-O-acetyl-1-thio-β-D-arabinopyranoside (Illf) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 76%. [β]D25: - 33.3 (c 1.0, CHCI3). Compound If was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 55% in two steps. [β]D25 - 11.5° (c: 1.0, CHCI3). EXAMPLE 3g PREPARATION OF β-D-XYLOPYRANOSYL-(1-»3)-D-FRUCTOSE (Ig) 2,3,4-Tri-O-acetyl-β-D-xylopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vg) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4-tri-O-acetyl-1-thio-p-D-xylopyranoside (Illg) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 77%. [β]D25 - 41.1 (c 1.0, CHCI3). Compound Ig was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 58% in two steps, [pfo25- 5.4 (c 1.0, CHCI3). EXAMPLE 3h PREPARATION OF β-D-RIBOPYRANOSYL-(1->3)-D-FRUCTOSE (Ih) 2,3,4-Tri-O-acetyl-β-D-ribopyranosyl-(1->3)-1,2:4,5-di-0-isopropylidene-D-fruccopyranose (Vh) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4-tri-O-acetyl-1-thio-p-D-ribopyranoside (Illh) as donor and 1,2:4,5-di-0-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 78%. [β]D25 - 52.5 (c 1.0, CHCI3). Compound Ih was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 53% in two steps. [β]D25 - 6.6 (c 1.0, CHCI3). EXAMPLE 3i PREPARATION OF 6-DEOXY-β-L-MANNOPYRANOSYL-(1->3)-D-FRUCTOSE (li) 2,3,4-Tri-O-acetyl-6-deoxy-β-L-mannopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructo-pyranose (Vi) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4-tri-O-acetyl-1-thio-6-deoxy-D-L-mannopyranoside (Illi) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 76%. [β]fo25 - 47.4 (c 1.0,CHCI3). Compound li was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 57% in two steps. [α]D25 - 2.7 (c 1.0, CHCI3). EXAMPLE 4 D-Raffinose (Ij; α-D-galactopyranosyl-(1->6)-β-D-glucopyranosyl-(1-»2)-D-fructose) is a commercially available compound (CAS Number 17629-30-0); procured from Lancaster (Catalogue number 8465) and evaluated for antihyperlipidemic activity as received. EXAMPLE 5 D-melezitose (Ik; α-D-glucopyranosyl-(1-»3)-β-D-fructofuranosyl-a-D- gluoopyranoside) is a commercially available compound (CAS Number 10030-67-8); procured from Lancaster (Catalogue number 6606) and evaluated for antihyperlipidemic activity as received. The activity testing illustrated in example 6, which however should not be construed to the limit the scope of invention. EXAMPLE 6 ANTIHYPERLIPIDEMIC ACTIVITY OF COMPOUNDS (la-Ik) Male golden Syrian hamsters weighing 120-130g were divided into hyperlipidemic and hyperlipidemic plus drug treated groups. Each group consisted of the eight animals. Hyperlipidemia was produced by feeding with high fat diet (HFD). Hyperlipidemic hamsters had a free access to the HFD and water ad lib during the entire period of the experiment. The test samples were given orally at dose of 100mg/kg using water as a drug vehicle from day 4 to day 10 (7 days) in the HFD hamsters. Normal hamsters fed with HFD and given drug vehicle (water) only served as control animals. Body weight and diet intake of each animal group was recorded daily to check the effect of the drug on food intake and body weight of the animals. At the end of experiment i.e. on the 10th day, the blood of the non fasted animals were withdrawn in two sets of tubes in which one set contains 120)j\| NaF (4.5 mg/ ml) and tubes were cooled to 0°C for 15 min. The cold plasma was separated and biological analysis of the plasma without NaF was performed on the same day for triglycerides (TG), total cholesterol (TC) and High Density Lipoprotein-Cholesterol (HDL) using commercially available enzymatic diagnostic kits. Similarly the plasma containing NaF was assayed for glycerol (GLY) and free fatty acids (FFA) using Synchron CX-5 Clinical System Beckmann Coulter Instrument. The data was analyzed for its significance on Prism Software. Among the total eleven compounds tested, Compounds la, Ic, Id, Ij and Ik were found to possess antihyperlipidemic activity in terms of lowering triglyceride (TG) concentration and increase in HDL-cholesterol and total cholesterol ratio (H/C). Compound Ik was found to possess maximum lipid lowering activity at the dose of 100 mg. per kg. body weight as compared to control animals. Animals treated orally with this compound showed 51% lowering in TG concentration, significant increase of the plasma HDL by 59% thereby increase of HDL-cholesterol and total cholesterol ratio (H/C) by 38%. Biological activities of 11 Compounds are exemplified as follows, but the scope of the invention is not limited to these compounds (Table 2). (Table Removed)Values are mean ± SD of 8 animals. P We claim: 1. Novel glycosyl-D-fructose derivatives having the general formula I, (Formula Removed) wherein R1, R2,R3,R4,R5,R6 is hydrogen, hydroxyl or ester amide ,alkyl and alkyl ether protected or unprotected monosaccharide, disaccharide, anhydro sugar, olefinic sugar, deoxy sugar, modified sugar and mixture thereof, R7 is hydrogen, methyl or CH2OR10; R8 is hydrogen or methyl; R9 and R10 is hydrogen or glycosyl moiety and alkyl or acyl protecting groups. 2. Novel glycosyl-D-fructose derivatives as claimed in claim 1, wherein the representative compounds of general formula I are: la= (3-D-galactopyranosyl (1→4)-ß-◘D-gluco-pyranosyl-(1→3)-ß-D-fructose lb= ß-D-glucopyranosyl-(1→4)-ß-d-glucopyranosyl(1→3)-d-fructose lc= ß-D-glucopyranosyl-(1→4)-ßN◘-d-glucopyranosyl-(1→3)-d-fructose ld= ß-D-galactopyranosyl-(1→3)-d-fructose le= ß-D-mannopyranosyl-(1→3)-d-fructose lf=ß-D-arabinopyranosyl-(1→3)-d-fructose lg= ß-D-xylopyranosyl-(1→3)-d-fructose lh= ß-D-ribopyranosyl-(1→3)-d-fructose li= 6-deoxy-ß-l-mannopyranosyl-(1→3)-d-fructose 3. A compound as claimed in claim 1, wherein the modified sugar is selected from the group consisting of glucose, galactose, mannose, arabinose, xylose, ribose, rhamnose, fructose, tagatose, digitose, N-acetyl neuraminic acid, maltose, cellobiose, lactose, melibiose and gentiobiose. 4. A compound as claimed in claim 1, wherein the glycosyl moieties is selected from the group consisting of D-glucose, D-galactose, D-mannose, D-arabinose, D-xylose, D-ribose, L-rhamnose, L-fucose, tagatose, digitose, N-acetyl neuraminic acid, D-maltose, D-cellobiose, D-lactose, D-glucosamine, D-galactosamine, D-fucosamine, D-mannosamine. 5. A process for the preparation of novel glycosyl-D-fructose derivatives, comprising the steps of: a) reacting the per-O-acetylated sugar of formula II and sulphur containing alkanol compound in the ratio of 1:1 in the presence of an dry organic solvent under inert atmosphere and cooling the above said reaction mixture to a temperature of about 0°C and further adding borontrifluoride diethyletherate under stirring, for a period of about 5 hrs to obtain the desired product of formula III, b) reacting suspension of D-fructose in presence of dry acetone with concentrated sulphuric acid under stirring for a period of about 2hrs at a temperature of about 25°C and cooling the reaction mixture and quenching in 50%w/v of sodium hydroxide at a temperature of about 0°C , followed by purifying by known method and drying to obtain the crude mass , further washing with alkaline sodium bicarbonate solution and purifying by known chromatographic methods to obtain the desired product of formula IV, c) mixing the above said reaction product of formula III and the above said reaction product of formula IV in the mmolar ratio of 1:1 in presence of dry organic solvent, followed by, i) adding molecular sieves of 4A of weight 500mg in argon atmosphere under stirring at a temperature of about 25°C for a time period of about 30 minutes, further adding N-iodosuccinimide and cooling the reaction mixture to a temperature of about -4°C, ii) cooling the above said reaction mixture and adding trifluoromethanesulphonic acid to obtain the resultant solution, and stirring the aforesaid resultant solution for a period of about -40°C for a period of about 1 hour and purifying the reaction mixture by known chromatographic methods to obtain the desired product of formula V, iii) reacting the above said reaction product obtained in step (ii) with trifluroacetic acid and water in the ratio of 2:1 in presence of an organic solvent, followed by stirring at a temperature of about 25°C for a period of about 3 hours, purifying the above said reaction mixture obtained by known chromatographic methods to obtain the desired product of formula I. 6. A process as claimed in claim 5, wherein the per-O-acetyl sugars is selected from the group consisting of 2,3,4-tri-O-acetyl-ß-D-ribopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose, 2,3,4,6-Tetra-O-acetyl-ß-D-mannopyranosyl,1,2:4,5-di-0-isopropylidene-D-fructopyran-ose,2,3,4-Tri-O-acetyl-ß-D-arabinopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose, 2,3,4-Tri-O-acetyl-ß-D-arabinopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose,2,3,4-Tri-0-acetyl-ß-D-xylopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose,2,3,4-Tri-O-acetyl-ß-D-ribopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose. 7. A process as claimed in claim 5, wherein the sulphur containing alkanol is selected from ethanethiol, thiophenol, p-thiocresol,m-thiocresol,o-thiocresol,2-mercaptobenzothiazole, 2-mercaptobenzimidazole, 2-mercaptopyridine,2-mercaptopiperidine. 8. A process as claimed in claim 5, wherein the organic solvent used is selected from dichloromethane, chloroform, dichloroethane, tertrahydrofuran, diethyl ether, acetonitrile,1,4- dioxane, dimethyl formamide, toluene and benzene. 9. A process as claimed in claim 5, wherein the product obtained of formula III is selected from the group consisting of ethyl 2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-1-thio-ß-D-glucopyranoside (IlIa), ethyl 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl-(1→4)-2,3,6-tri-O-acetyl-1-thio-ß-D-glucopyranoside(lllb), ethyl 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl-(1→4)-2,3,6-tri-O-acetyl-1-thio-ß-DD-glucopyranoside (lllc), ethyl 2,3,4,6-tetra-O-acetyl-1-thio-ß-D-galactopyranoside (IIId), ethyl 2,3,4,6-tetra-O-acetyl-1-thio-ß-D-mannopyranoside (IlIe), ethyl 2,3,4-tri-O-acetyl-1-thio-(3-D-arabinopyranoside (lllf), ethyl 2,3,4-tri-O-acetyl-1-thio-ß-D-xylopyranoside (lllg) ethyl 2,3,4-tri-O-acetyl-1-thio-ß-D-ribopyranoside (lllh), ethyl 2,3,4-tri-O-acetyl-1-thio-6-deoxy-α -L-mannopyranoside(llli) 10. A process as claimed in claim 5, wherein the product obtained of formula V is selected from the group consisting of2,3,4,6-Tetra-O-acetyl-ß-D- galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-ß-◘D-glucopyranosyl-(1→3)- 1,2:4,5-di-O-isopropylidene-D-fructopyranose (Va), 2,3,4,6-Tetra-O-acetyl-(3-D-glucopyranosyl-(1→4)-2,3,6-tri-O-acetyl-ß◘ D-glucopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vb), 2,3,4,6-Tetra-O-acetyl-ß-D-glucopyranosyl-(1→4)-2,3,6-tri-O-acetyl-ß-D-glucopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vc), 2,3,4,6-Tetra-O-acetyl-ß-D-galactopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyran- ose (Vd), 2,3,4,6-Tetra-O-acetyl-ß-D-mannopyranosyl-(1→3)-1,2:4,5-di- 0-isopropylidene-D-fructopyran-ose(Ve),2,3,4-Tri-0-acetyl-ß-D- arabinopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vf), 2,3,4-Tri-0-acetyl-(3-D-xylopyranosyl-(1 →3)-1,2:4,5-di-O- isopropylidene-D-fructopyranose (Vg), 2,3,4-Tri-0-acetyl-ß-D- ribopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vh), 2,3,4-Tri-0-acetyl-6-deoxy-ß-L-mannopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructo-pyranose(Vi).

Full Text

FIELD OF THE INVENTION
The present invention relates to a novel glycosyl-D-fructoses of formula I and isomers thereof,
wherein, R1, R2,R3,R4,R5,R6 is hydrogen, hydroxyl or ester amide, alkyl and alkyl ether protected or unprotected monosaccharide, disaccharide, anhydro sugar, olefinic sugar, deoxy sugar, modified sugar and mixture thereof,
R7 is hydrogen, methyl or CH2OR10;
R8 is hydrogen or methyl;
R9 and R10 is hydrogen or glycosyl moiety
More particularly it relates to the glycosyl-D-fructoses, polymorphic forms, salts con.aining such compounds and compositions having such compounds as active ingredients. It also relates to the process of preparation of such compounds.
BACKGROUND OF THE INVENTION
Glycosyl-D-fructoses, also termed as "fructo oligosaccharides" comprises a large class of oligosaccharides, which have been isolated from the various honeydews. Most of these compounds are water-soluble and crystalline in nature having low optical rotations and have a slightly sweet to very sweet taste.
D-Raffinose and D-melezitose are the most commonly found fructo oligosaccharides isolated from the natural sources. Most of the fructo oligosaccharides isolated have been put to various pharmaceutical uses such as bifidus factor [Okhusa, Toshifumi; Ozaki, Yoshinori; Satochifumi; Miken,
Katasuhika; Ikeda, Hiroshi. Digestion, 1995, 56, 415-20] and anti-cariogenic agents [Mikuni, Katsuhiko; Fujita, Takateru; Osawa, Takeshi; Aoyama, Yoko; Hara, Kozo; Hashimoto, Hitoshi. JP 03 083 992]. Among all isolated fructo oligosaccharides, raffinose, lactosucrose and melezitose have been used in health food materials [Hayakawa, Kunihiko; Omura, Kazutaka; Mizutani, Jun; Shirayanagi, Satoru; Kato, Yasuhito; Ikeda, Nagisa. JP 03 151 854], [Uchida, Yukio; Iritani, Satoshi; Mayake, Toshio. JP 07 298 833], as low cariogenic sugar substitute [Biton Jacques; Michael-Jean, Marc; Le Beller, Dominique; Pelenc, Vincent; Paul, Francois; Monsan, Pierre, F; Gelf, Gerard. Ann. NY. Acad. Sci. 1995, 750 (Enzyme Engineering XII), 312-14], as well as food substituents for various animals [Satoya, Hiroaki. JP 05 003 758] and [Kihara, Minoru; Mino, Riyuusuke. JP 07 203 868]. They have been used as osteoporosis preventing agents [Ohta, Atsutane; Takizawa, Toshio. EP 0753302]. They have also been used as prophylaxis for hepatopathy and as laxatives [Suga, Tatsuhiko; Kobayashi, Yoichi; Sonoike, Yoshiko; Terajima, Tsuneo; Mutai, Masahiko. JP 62 220 169]. Sulfated Raffinose derivatives have also been reported as antiarteriosclerotic agent [Hosang, Markus; Iberg, Niggi; Tschopp Thomas, Beat; Wessel-Hans, Peter. EP 504645]. Raffinose and Melibiose have also been used as carcinostatic Pharmaceuticals [Watanabe, Atsushi; Nukada, Akira. JP 57 077 620]. Lactosucrose has been used as an active ingredient in atopic dermatitis [Watanabe, Atsushi; Nukada, Akira. JP 11 124 390].
Coronary heart disease is a multifactorial disease in which the incidence and severity are affected by the lipid profile, the presence of diabetes and the sex of the subject. Coronary heart disease (CHD) remains the leading cause of death in the industrialized countries. The primary cause of CHD is arteriosclerotic, a disease characterized by the deposition of lipids, including cholesterol, in the arterial vessel wall, resulting in a narrowing of the vessel passages and ultimately hardening of the vascular system. There are two types of cholesterol mainly present in the human body, among which the harmful cholesterol is low-density lipoprotein (LDL) cholesterol. Several clinical studies have established that lowering of LDL-cholesterol in mammal is an effective way to treat and prevent
heart attacks, sudden death and angina. Lowering LDL-cholesterol is now one of the primary objectives of medicinal chemists for treating patients who have hypercholesterolemia, or who have a tendency to develop cardiovascular diseases such as coronary heart disease. It is therefore recommended that the compounds which increase high-density lipoprotein-cholesterol (HDL) and therefore HDL-cholesterol and total cholesterol ratio (H/C) are effective curative agents against hypercholesterolemia.
Recently it has been reported that, lactic acid bacteria present in the body produce an abundant variety of exopolysaccharides (EPS's), which provide an important contribution to human health by acting as prebiotic substrates, nutraceuticals, cholesterol lowering agents or immunomodulants [Van, Hijum; Sacha Adrianus Fokke Taco Groningen; Van, Geel-Schutten; Gerritdina, Hendrika; Dijkhuizen; Lubbert; Rahaoui; Hakim. US 6,730,502]. These compounds are also synthesized using extracellular sucrase enzymes, fructosyltransferases and glucosyltransferases. These fructosyltransferases have already been used for the chemoenzymatic preparation of various fructo-oligosaccharides. For example maltosylfructose has been prepared by using levansucrase from Bacillus subtilis [Jimenez-Estrada, M; Cassani, J; Lopez-Munguia, A. Biocatal. Biotransform. 1999,16 (6), 475-485],
A number of reports appeared in the literature for the use of fructose containing oligosaccharides as prebiotic substrates [ACS Symposium Series, 2003, 849 (oligosaccharide in food and agriculture), 76-89], nutraceuticals. However their use in the treatment of metabolic disorders like hypercholesterolemia has not been explored. As a part of the ongoing research program to develop antihyperlipidemic agents, the present inventors set out to explore the pharmaceutical potentiality of several glycosyl-D-fructoses as antihyperlipidemic agents. A series of chemically synthesized and commercially available novel fructose containing oligosaccharides have been evaluated for their antihyperlipidemic activity.
SUMMARY OF THE INVENTION
Accordingly the present invention relates to a novel glycosyl-D-fructoses having the general formula I and isomers thereof,
formula I
wherein R1, R2,R3,R4,R5,R6 is hydrogen, hydroxyl or ester amide ,alkyl and alkyl ether protected or unprotected monosaccharide, disaccharide, anhydro sugar, olefinic sugar, deoxy sugar, modified sugar and mixture thereof,
R7 is hydrogen, methyl or CH2OR10;
R8 is hydrogen or methyl;
R9 and R10 is hydrogen orglycosyl moiety
In an embodiment of the invention the representative compounds of general formula I are:
la= (3-D-galactopyranosyl (1 ->4)-(3-D-glucopyranosyl-(1 ->3)-D-fructose
lb= α-D-glucopyranosyl-(1-4)-(3-D-glucopyranosyl-(1-3)-D-fructose
lc= β-D-glucopyranosyl-(1-4)-(3-D-glucopyranosyl-(1-3)-D-fructose
ld= β-D-galactopyranosyl-(1 ->3)-D-fructose
le=α -D-mannopyranosyl-(1 ->3)-D-fructose
lf=β-D-arabinopyranosyl-(1 ->3)-D-fructose
lg= β-D-xylopyranosyl-(1 ->3)-D-fructose
lh= β-D-ribopyranosyl-(1 ->3)-D-fructose
li= 6-deoxy-β-L-mannopyranosyl-(1 ->3)-D-fructose
lj= α-D-galactopyranosyl-(1 ->6)-(3-D-glucopyranosyl-(1 ->2)-D-fructose (D-raffinose pentahydrate)
lk=α-D-glucopyranosyl-(1->3)-p-D-fructofuranosyl-a-D-glucopyranoside (D-melezitose monohydrate).
In another embodiment of the invention the modified sugar is selected from the group consisting of glucose, galactose, mannose, arabinose, xylose, ribose, rhamnose, fructose, tagatose, digitose, N-acetyl neuraminic acid, maltose, cellobiose, lactose, melibiose and gentiobiose.
In yet another embodiment the glycosyl moieties is selected from the group consisting of D-glucose, D-galactose, D-mannose, D-arabinose, D-xylose, D-ribcse, L-rhamnose, L-fucose, tagatose, digitose, A/-acetyl neuraminic acid, D-maltose, D-cellobiose, D-lactose, D-glucosamine, D-galactosamine, D-fucosamine, D-mannosamine.
In yet another embodiment the compound of general formula I is effective against lowering triglycerides (TG) upto 52%.
In yet embodiment the compound is effective for increasing in HDL-cholesterol and total cholesterol ratio (H/C) by 60%.
In yet another embodiment the compound of formula I and isomers thereof are used for the treatment of hyperlipidemia.
In yet another embodiment the compound of formula I and isomers thereof are used as cholesterol lowering agent.
In yet another embodiment the compound of formula I and isomers thereof are used as high density lipoprotein-cholesterol (HDL) increasing agent.
In yet another embodiment the compound of formula I and isomers thereof are used as triglyceride lowering agent.
Accordingly the present invention provides a process for the preparation of compound of general formula I and isomers thereof

wherein R1, R2,R3,R4,R5,R6 is hydrogen, hydroxyl or ester amide ,alkyl and alkyl ether protected or unprotected monosaccharide, disaccharide, anhydro sugar, olefinic sugar, deoxy sugar, modified sugar and mixture thereof,
R7 is hydrogen, methyl or CH2OR10;
R8 is hydrogen or methyl;
R9 and R10 is hydrogen or glycosyl moiety
A series of fructose containing novel oligosaccharides have been prepared by conventional glycosylation techniques involving coupling of several per-O-acetylated thioglycoside donors (III, wherein R1-R8 have the same meaning as compound of formula I) with suitably protected fructose acceptor. In order to so, a series of per-O-acetylated thioethyl glycosides (III, wherein R1-R8 have the same meaning as compound of formula I) of several mono- and di-saccharides have been prepared from commonly available unprotected sugars following conventional acetylation followed by thioglycosylation. 1,2:4,5-Di-O-isopropylidene-D-fructose derivative was used as glycosyl acceptor. The glycosylation reaction was performed in the presence of N-iodosuccinimide (NIS) and trifluoromethane sulfonic acid as thioglycoside activator. The protected fructose oligosaccharide derivatives (V, wherein R1-R8 have the same meaning as compound of formula I) thus obtained were deprotected following convention deprotection techniques to furnish VI, wherein R1-R8 have the same meaning as compound of formula I.
In an embodiment of the invention is a process for the preparation of glycosyl-D-fructoses of general formula I comprising of:
a) reacting the per-O-acetylated sugar of formula II wherin R1-R10 have the
same meaning as compound of formula I and sulphur containing alkanol
compound in the ratio of 1:1 in the presence of a dry organic solvent
under inert atmosphere and cooling the above said reaction mixture to a
temperature of about 0 °C and further adding borontrifluoride
diethyletherate under stirring, for a period of about 5 hrs to obtain the
desired product of formula III, wherin R1-R10 have the same meaning as
compound of formula I
b) reacting suspension of D-fructose in presence of dry acetone with
concentrated sulphuric acid under stirring for a period of about 2hrs at a
temperature of about 25°C and cooling the reaction mixture cooling the
reaction mixture and quenching in 50%(w/v) of sodium hydroxide to a
temperature of about 0°C , followed by purifying by known method and
. drying to obtain the crude mass , further washing with alkaline sodium bicarbonate solution and purifying by known chromatographic methods to obtain the desired product of formula IV,
c) mixing the above said reaction product of formula III and the above said
reaction product of formula IV in the mmolar ratio of 1:1 in presence of dry
organic solvent, followed by,
i) adding molecular sieves of 4A of weight 500mg in argon atmosphere under stirring at a temperature of about 25°C for a time period of about 30 minutes, further adding N-iodosuccinimide and cooling the reaction mixture to a temperature of about -4°C,
ii) cooling the above said reaction mixture and adding trifluoromethanesulphonic acid to obtain the resultant solution, and stirring the aforesaid resultant solution for a period of about -40°C for a period of about 1 hour and purifying the reaction mixture by known chromatographic methods to obtain the desired product of formula V, wherein R1-R10 have the same meaning as compound of formula I
iii) reacting the above said reaction product obtained in step (ii) with trifluroacetic acid and water in the ratio of 2:1 in presence of an organic solvent, followed by stirring at a temperature of about 25°C for a period of about 3 hours, purifying the above said reaction mixture obtained by known chromatographic methods to obtain the desired product of formula I.
In yet another embodiment of the invention the representative compounds of the general formula I and isomers thereof are:
la=β -D-galactopyranosyl(1 ->4)-3-D-gluco-pyranosyl-(1 ->3)-(3-D-fructose
lb= β-D-glucopyranosyl-(1 ->4)-p-d-glucopyranosyl(1 ->3)-d-fructose
lc= β-D-glucopyranosyl-(1 ->4)-(3-d-glucopyranosyl-(1 ->3)-d-fructose
ld= β-D-galactopyranosyl-(1 ->3)-d-fructose
le= α-D-mannopyranosyl-(1 ->3)-d-fructose
lf=β-D-arabinopyranosyl-(1->3)-d-fructose
lg= β-D-xylopyranosyl-(1->3)-d-fructose
lh= β-D-ribopyranosyl-(1->3)-d-fructose
li= 6-deoxy-β-l-mannopyranosyl-(1->3)-d-fructose
Accordingly the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of general formula I as claimed in claim 1 or compound 3j and 3k optionally along with pharmaceutically acceptable diluent and or carrier.
In an embodiment of the present invention the composition is useful in the treatment of hyperlipidemia, cholesterol lowering agent, used as high density lipoprotein-cholesterol (HDL) increasing agent, triglyceride lowering agent.
In still another embodiment the dose of the compound of general formula I is 25 mg/kg to 100mg/kg.
Another feature of the present invention is a method of treating a subject having hyperlipidemia, comprising administering to the subject a pharmaceutically effective amount of a composition containing substituted compound of formula I, or compound 3j a and 3k.
In yet another embodiment of the invention wherein the compound composition is administered orally.
In yet another embodiment of the invention wherein the pharmaceutically acceptable amount of compound of formula I is in the range of 25 mg per kg to 100 mg per kilogram of the body weight of subject per day.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides antihyperlipidemic properties of novel glycosyl-D-fructoses of general formula I, and their isomers. salts containing such compounds, compositions containing these compounds and methods for using such compounds and compositions for the treatment of antihyperlipidemia.
As such, in a particular, the present invention provides novel compounds having the general formula I and isomers thereof.
In an embodiment of the invention is a process for the preparation of glycosyl-D-fructoses of general formula I comprising of:
a) reacting the per-O-acetylated sugar of formula II wherin R1-R8 have the
same meaning as compound of formula I and sulphur containing alkanol
compound in the ratio of 1:1 in the presence of a dry organic solvent under
inert atmosphere and cooling the above said reaction mixture to a
temperature of about 0 °C and further adding borontrifluoride
diethyletherate under stirring, for a period of about 5 hrs to obtain the
desired product of formula III, wherin R1-R8 have the same meaning as
compound of formula I
b) reacting suspension of D-fructose in presence of dry acetone with
concentrated sulphuric acid under stirring for a period of about 2hrs at a
temperature of about 25°C and cooling the reaction mixture cooling the
reaction mixture and quenching in 50%(w/v) of sodium hydroxide to a
temperature of about 0°C , followed by purifying by known method and
drying to obtain the crude mass , further washing with alkaline sodium
bicarbonate solution and purifying by known chromatographic methods to obtain the desired product of formula IV,
c) mixing the above said reaction product of formula III and the above said reaction product of formula IV in the molar ratio of 1:1 in presence of dry organic solvent, followed by,
i) adding molecular sieves of 4A of weight 500mg-2 g in argon atmosphere under stirring at a temperature of about 25°C for a time period of about 30 minutes, further adding N-iodosuccinimide and cooling the reaction mixture to a temperature of about -4°C,
ii) cooling the above said reaction mixture and adding trifluoromethanesulphonic acid to obtain the resultant solution, and stirring the aforesaid resultant solution at -40°C for a period of about 1 hour and purifying the reaction mixture by known chromatographic methods to obtain the desired product of formula V, wherein R1-R8 have the same meaning as compound of formula I
reacting the above said reaction product obtained in step (ii) with trifluroacetic acid and water in the ratio of 2:1 in presence of an organic solvent, followed by stirring at a temperature of about 25°C for a period of about 3 hours, purifying the above said reaction mixture obtained by known chromatographic methods to obtain the desired product of formula I.
In yet another embodiment of the invention the per-O-acetyl sugars compound of
formula V is selected from the group consisting of 2,3,4,6-Tetra-O-acetyl-β-D-
galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-pD-glucopyranosyl-(1->3)-1,2:4,5-di-
0-isopropylidene-D-fructopyranose (Va); 2,3,4,6-Tetra-O-acetyl-β-D-
glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-β~-D-glucopyranosyl-(1->3)-1,2:4,5-di-O-
isopropylidene-D-fructopyranose (Vb); 2,3,4,6-Tetra-O-acetyl-β-D-
glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl-(1->3)-1,2:4,5-di-O-
isopropylidene-D-fructopyranose (Vc); 2,3,4,6-Tetra-O-acetyl-p-D-
galactopyranosyl-(1 -»3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-ose (Vd);
2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-
D-fructopyran-ose (Ve); 2,3,4-Tri-O-acetyl-p-D-arabinopyranosyl-(1->3)-1,2:4,5-
di-O-isopropylidene-D-fructopyranose (Vf); 2,3,4-Tri-O-acetyl-β-D-xylopyranosyl-
(1-»3)-1,2:4,5-di-0-isopropylidene-D-fructopyranose (Vg); 2,3,4-Tri-O-acetyl-β-
D-ribopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vh);
2,3,4-Tri-O-acetyl-6-deoxy-α-L-mannopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vi).
In yet another embodiment the sulphur containing alkanol is selected from ethanethiol, thiophenol, p-thiocresol, m-thiocresol, o-thiocresol, 2-mercaptobenzothiazole, 2-mercaptobenzimidazole, 2-mercaptopyridine, 2-mercaptopiperidine.
In yet another embodiment the organic solvent used is selected from dichloromethane, chloroform, dichloroethane, tetrahydrofuran, diethyl ether, acetonitrile, 1,4-dioxane, dimethyl formamide, toluene and benzene.
In yet another embodiment of the invention the product obtained of formula III is selected from the group consisting of ethyl 2,3,4,6-tetra-O-acetyl-p-D-galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (Ilia), ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (1Mb), ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-(1->4)-2,3,5-tri-0-acetyl-1-thio-p~-D-glucopyranoside (Illc), ethyl 2,3,4,6-tetra-O-acetyl-1-thio-p-D-galactopyranoside (Hid), ethyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-mannopyranoside (Ille), ethyl 2,3,4-tri-O-acetyl-1-thio-(3-D-arabinopyranoside (Illf), ethyl 2,3,4-tri-O-acetyl-1-thio-β-D-xylopyranoside (Illg) ethyl 2,3,4-tri-O-acetyl-1-thio-β-D-ribopyranoside (Illh), ethyl 2,3,4-tri-O-acetyl-1-thio-6-deoxy-a-L-mannopyranoside(llli).
In yet another embodiment the product obtained of formula V is selected from the group consisting of 2,3,4,6-Tetra-O-acetyl-p-D-galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-βD-glucopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Va); 2,3,4,6-Tetra-O-acetyl-p-D-glucopyranosyl-(1 ->4)-2,3,6-tri-
O-acetyl-β-D-glucopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-
fructopyranose (Vb); 2,3,4,6-Tetra-0-acetyl-β-D-glucopyranosyl-(1 ->4)-2,3,6-tri-
O-acetyl-β-D-glucopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-
fructopyranose (Vc); 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1 -»3)-1,2:4,5-
di-O-isopropylidene-D-fructopyran-ose (Vd); 2,3,4,6-Tetra-O-acetyl-a-D-
mannopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-ose (Ve); 2,3,4-Tri-O-acetyl-β-D-arabinopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vf); 2,3,4-Tri-O-acetyl-p-D-xylopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vg); 2,3,4-Tri-O-acetyl-p-D-ribopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vh); 2,3,4-Tri-O-acetyl-6-decxy-a-L-mannopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vi)-.
The invention is further illustrated by the following examples, which should not however be construed to limit the scope of the present invention.
EXAMPLE 1
GENERAL PREPARATION OF THIOETHYL GLYCOSIDES [III (a-i)]
To a solution of per-O-acetylated sugars [II (a-i)] (1.0 mmol) in dry CH2CI2 (5.0 rnL), ethanethiol (1.50 mmol) was added under inert atmosphere. The reaction mixture was cooled to 0°C and borontrifluoride diethyletherate (2.0 mmol) was added to it and the reaction mixture was stirred at 0°C for 5 hrs. The progress of the reaction was monitored by thin layer chromatography over silica gel coated plates. After completion of the reaction, the reaction mixture was diluted with CH2Cl2, washed with aq. sodium bicarbonate solution and water in succession. The organic layer was dried over anhydrous sodium sulphate and concentrated to dryness under reduced pressure. The crude reaction mixture was purified over Si02 using hexane-EtOAc as eluant to furnish desired ethylthioglycosides [III (a-i)] (Table 1).
(Table Removed)
EXAMPLE 2
PREPARATION OF 1,2:4,5-DI-O-ISOPROPYLIDENE-D-FRUCTOPYRANOSE [IV]
To a suspension of D-fructose (30 g) in dry acetone (600 ml) was added cone. H2SO4 (2.9 ml) and the reaction mixture was allowed to stir for 2 hrs. at room temperature. After completion of the reaction as monitored by thin layer chromatography over silica gel coated plates the reaction mixture was cooled to
0°C and quenched with aq. 50% NaOH solution. The reaction mixture was evaporated to dryness to a crude mass. The crude mass was diluted with CH2Cl2 (1000 ml) and the organic layer was washed with aq. sodium bicarbonate solution and water in succession. The organic layer was dried over anhydrous sodium sulphate and concentrated to dryness under reduced pressure. The crude reaction mixture was purified over Si02 using hexane-EtOAc as eluant to furnish desired 1,2:4,5-di-O-isopropylidene-D-fructose [IV]. Yield: 76%; m.p. 119°C; [α]D25-145 (c 1.0, CHCI3).
EXAMPLE 3
GENERAL GLYCOSYLATION PROTOCOL
To a mixture of ethyl per-O-acetyl-1-thio-D-D-glycopyranoside [Ml(a-i)] (1.1 mmol)
and 1,2:4,5-di-O-isopropylidene-D-fructopyranose [IV] (1.0 mmol) in dry CH2CI2
(5 ml) was added molecular sieves 4A (500 mg) and flushed with argon. After
stirring at room temperature for 30 min, AModosuccinimide (1.21 mmol) was
added and the reaction mixture was cooled to -40°C. To the cooled reaction
mixture trifluoromethanesulphonic acid (0.1 mmol) was injected and the resulting
solution was stirred at -40°C for 1 hour. The progress of the reaction was
monitored by thin layer chromatography over silica gel coated plates. After
completion of the reaction, the reaction mixture was diluted with CH2Cl2 and
filtered through a celite bed. The filtrate thus obtained was washed with aq.
sodium thiosulphate, aq. sodium bicarbonate solution and water in succession.
The organic layer was dried over anhydrous sodium sulphate and the resulting
reaction mixture was concentrated to dryness under reduced pressure. The
crude reaction mixture was purified over SiO2 using hexane-EtOAc as eluant to
furnish desired per-O-acetyl-D-glycopyranosyl-(1->3)-1,2:4,5-di-O-
isopropylidene-D-fructopyranose [V(a-i)].
GENERAL DEPROTECTION PROTOCOL FOR THE PREPARTION OF UNPROTECTED FRUCTOSE OLIGOSACCHARIDES [l(a-i)]
To a solution of per-O-acetyl-β~-D-glycopyranosyl-(1->3)-1,2:4,5-di-0-isopropylidene-D-fructopyranose [V(a-i)] (1.0 mmol) in CH2CI2 (5.0 ml) was added trifluroacetic acid (10.0 ml), and water (5.0 ml). The reaction mixture was stirred at room temperature for 3 hrs. The reaction was monitored by thin layer chromatography over silica gel coated plates. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3x20mL) to furnish desired per-O-acetyl-D-glycopyranosyl-(1->3)-D-fructose. To a solution of crude per-O-acetyl-βD-glycopyranosyl-(1->3)-D-fructose thus obtained in dry methanol (10.0 ml), sodium methoxide (50 mg) was added and the reaction mixture was stirred at room temperature for 4 hrs. The reaction was monitored by thin layer chromatography over silica gel coated plates. After completion of the reaction, Amberlite IR 120 (H+) resin was added to the reaction mixture with constant stirring till the solution becomes neutral. The resulting reaction mixture was cotton filtered and the filtrate was concentrated to dryness under reduced pressure. The crude reaction mixture was purified over SiO2 using chloroform-MeOH (2:1) as eluant to furnish desired p~-D-glycopyranosyl-(1-»3)-D-fructose [l(a-i)].
EXAMPLE 3a
PREPARATION OF p-D-GALACTOPYRANOSYL-(1->4)-p-D-
GLUCOPYRANOSYL-(1-»3) D-FRUCTOSE (la):
2,3,4,6-Tetra-O-acetyl-p-D-galactopyranosyl-(1->4)-2,3,6-tri-O-acetyl-βD-glucopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Va) was prepared by following the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-p-D-galactopyranosyl-(1-»4)-2,3,6-tri-O-acetyl-1-thio-p-D-glucopyranoside (Ilia) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 72%. [p]D25 - 25 (c 1.0, CHCI3).

Compound la was prepared by following the general deprotection protocol as mentioned in Example 3-2. Yield: 50% in two steps. [(3]D25 + 2.1 (c 1.0, CHCI3).
EXAMPLE 3b
PREPARATION OF β-D-GLUCOPYRANOSYL-(1->4)-β~-D-GLUCOPYRANOSYL (1->3)-D-FRUCTOSE (Ib):
2,3,4,6-Tetra-0-acetyl-β-D-glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-3"-D-glucopyranosyl-(1-»3)-1,2:4,5-di-0-isopropylidene-D-fructopyranose (Vb) was prepared by using the generalized glycosylation protocol as given in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-p-D-glucopyranosyl-(1-»4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (Illb) as donor and 1,2:4,5-di-O-isopropylidene-β-D-fructopyranose (IV) as acceptor. Yield: 75%. [β]D25 + 7.2 (c 1.0, CHCI3).
Compound Ib was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 58% in two steps. [β]D25 + 8.6 (c 1.0, CHCI3).
EXAMPLE 3c
PREPARATION OF β-D-GLUCOPYRANOSYL-(1->4)-p-D-
GLUCOPYRANOSYL-(1->3)-D-FRUCTOSE (Ic) 2,3,4,6-Tetra-O-acetyl-p-D-glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-βD-glucopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vc) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-p-D-glucopyranosyl-(1->4)-2,3,6-tri-O-acetyl-1-thio-p-D-glucopyranoside (Illc) as donor and 1,2:4,5-di-O-isopropylidene-D-frctopyranose (IV) as acceptor. Yield: 73%. [β]D25: - 19.5 (c 1.0, CHCI3).
Compound Ic was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 52% in two steps. [β]D25 - 1.8 (c 1.0, CHCI3).
EXAMPLE 3d
PREPARATION OF β-D-GALACTOPYRANOSYL-(1-»3)-D-FRUCTOSE (Id):
2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-ose (Vd) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-1-thio-β -D-galactopyranoside (Mid) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 73%. [β]D25 - 17.4 (c 1.0, CHCI3).
Compound Id was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 51% in two steps.[β ]D25 + 3.3 (c 1.0, CHCI3).
EXAMPLE 3e
PREPARATION OF α-D-MANNOPYRANOSYL-(1-»3)-D-FRUCTOSE (le)
2,3,4,6-Tetra-O-acetyl-β-D-mannopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-ose (Ve) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-mannopyranoside (llle) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 74%. [β]D25 - 5.4 (c 1.0, CHCI3).
Compound le was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 56% in two steps. [βD25 - 2.6 (c 1.0, CHCI3).
EXAMPLE 3f
PREPARATION OF β-D-ARABINOPYRANOSYL-(1-»3)-D-FRUCTOSE (If)
2,3,4-Tri-O-acetyl-β-D-arabinopyranosyl-(1-»3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vf) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4-tri-O-acetyl-1-thio-β-D-arabinopyranoside (Illf) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 76%. [β]D25: - 33.3 (c 1.0, CHCI3).
Compound If was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 55% in two steps. [β]D25 - 11.5° (c: 1.0, CHCI3).
EXAMPLE 3g
PREPARATION OF β-D-XYLOPYRANOSYL-(1-»3)-D-FRUCTOSE (Ig)
2,3,4-Tri-O-acetyl-β-D-xylopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vg) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4-tri-O-acetyl-1-thio-p-D-xylopyranoside (Illg) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 77%. [β]D25 - 41.1 (c 1.0, CHCI3).
Compound Ig was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 58% in two steps, [pfo25- 5.4 (c 1.0, CHCI3).
EXAMPLE 3h
PREPARATION OF β-D-RIBOPYRANOSYL-(1->3)-D-FRUCTOSE (Ih)
2,3,4-Tri-O-acetyl-β-D-ribopyranosyl-(1->3)-1,2:4,5-di-0-isopropylidene-D-fruccopyranose (Vh) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4-tri-O-acetyl-1-thio-p-D-ribopyranoside (Illh) as donor and 1,2:4,5-di-0-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 78%. [β]D25 - 52.5 (c 1.0, CHCI3).
Compound Ih was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 53% in two steps. [β]D25 - 6.6 (c 1.0, CHCI3).
EXAMPLE 3i
PREPARATION OF 6-DEOXY-β-L-MANNOPYRANOSYL-(1->3)-D-FRUCTOSE (li)
2,3,4-Tri-O-acetyl-6-deoxy-β-L-mannopyranosyl-(1->3)-1,2:4,5-di-O-isopropylidene-D-fructo-pyranose (Vi) was prepared by using the generalized glycosylation protocol as mentioned in Example 3.1, using ethyl 2,3,4-tri-O-acetyl-1-thio-6-deoxy-D-L-mannopyranoside (Illi) as donor and 1,2:4,5-di-O-isopropylidene-D-fructopyranose (IV) as acceptor. Yield: 76%. [β]fo25 - 47.4 (c 1.0,CHCI3).
Compound li was prepared by following the generalized deprotection protocol as mentioned in Example 3.2. Yield: 57% in two steps. [α]D25 - 2.7 (c 1.0, CHCI3).
EXAMPLE 4
D-Raffinose (Ij; α-D-galactopyranosyl-(1->6)-β-D-glucopyranosyl-(1-»2)-D-fructose) is a commercially available compound (CAS Number 17629-30-0); procured from Lancaster (Catalogue number 8465) and evaluated for antihyperlipidemic activity as received.
EXAMPLE 5
D-melezitose (Ik; α-D-glucopyranosyl-(1-»3)-β-D-fructofuranosyl-a-D-
gluoopyranoside) is a commercially available compound (CAS Number 10030-67-8); procured from Lancaster (Catalogue number 6606) and evaluated for antihyperlipidemic activity as received.
The activity testing illustrated in example 6, which however should not be construed to the limit the scope of invention.
EXAMPLE 6
ANTIHYPERLIPIDEMIC ACTIVITY OF COMPOUNDS (la-Ik)
Male golden Syrian hamsters weighing 120-130g were divided into hyperlipidemic and hyperlipidemic plus drug treated groups. Each group consisted of the eight animals. Hyperlipidemia was produced by feeding with high fat diet (HFD). Hyperlipidemic hamsters had a free access to the HFD and water ad lib during the entire period of the experiment. The test samples were given orally at dose of 100mg/kg using water as a drug vehicle from day 4 to day 10 (7 days) in the HFD hamsters.
Normal hamsters fed with HFD and given drug vehicle (water) only served as control animals. Body weight and diet intake of each animal group was recorded daily to check the effect of the drug on food intake and body weight of the animals. At the end of experiment i.e. on the 10th day, the blood of the non fasted animals were withdrawn in two sets of tubes in which one set contains
120)j| NaF (4.5 mg/ ml) and tubes were cooled to 0°C for 15 min. The cold plasma was separated and biological analysis of the plasma without NaF was performed on the same day for triglycerides (TG), total cholesterol (TC) and High Density Lipoprotein-Cholesterol (HDL) using commercially available enzymatic diagnostic kits. Similarly the plasma containing NaF was assayed for glycerol (GLY) and free fatty acids (FFA) using Synchron CX-5 Clinical System Beckmann Coulter Instrument. The data was analyzed for its significance on Prism Software.
Among the total eleven compounds tested, Compounds la, Ic, Id, Ij and Ik were found to possess antihyperlipidemic activity in terms of lowering triglyceride (TG) concentration and increase in HDL-cholesterol and total cholesterol ratio (H/C).
Compound Ik was found to possess maximum lipid lowering activity at the dose of 100 mg. per kg. body weight as compared to control animals. Animals treated orally with this compound showed 51% lowering in TG concentration, significant increase of the plasma HDL by 59% thereby increase of HDL-cholesterol and total cholesterol ratio (H/C) by 38%.
Biological activities of 11 Compounds are exemplified as follows, but the scope of the invention is not limited to these compounds (Table 2).

(Table Removed)Values are mean ± SD of 8 animals. P

We claim:
1. Novel glycosyl-D-fructose derivatives having the general formula I,
(Formula Removed)
wherein R1, R2,R3,R4,R5,R6 is hydrogen, hydroxyl or ester amide ,alkyl and alkyl ether protected or unprotected monosaccharide, disaccharide, anhydro sugar, olefinic sugar, deoxy sugar, modified sugar and mixture thereof,
R7 is hydrogen, methyl or CH2OR10;
R8 is hydrogen or methyl;
R9 and R10 is hydrogen or glycosyl moiety and alkyl or acyl protecting groups.
2. Novel glycosyl-D-fructose derivatives as claimed in claim 1, wherein the
representative compounds of general formula I are:
la= (3-D-galactopyranosyl (1→4)-ß-◘D-gluco-pyranosyl-(1→3)-ß-D-fructose
lb= ß-D-glucopyranosyl-(1→4)-ß-d-glucopyranosyl(1→3)-d-fructose
lc= ß-D-glucopyranosyl-(1→4)-ßN◘-d-glucopyranosyl-(1→3)-d-fructose
ld= ß-D-galactopyranosyl-(1→3)-d-fructose
le= ß-D-mannopyranosyl-(1→3)-d-fructose
lf=ß-D-arabinopyranosyl-(1→3)-d-fructose
lg= ß-D-xylopyranosyl-(1→3)-d-fructose
lh= ß-D-ribopyranosyl-(1→3)-d-fructose
li= 6-deoxy-ß-l-mannopyranosyl-(1→3)-d-fructose
3. A compound as claimed in claim 1, wherein the modified sugar is selected from the group consisting of glucose, galactose, mannose, arabinose, xylose, ribose, rhamnose, fructose, tagatose, digitose, N-acetyl neuraminic acid, maltose, cellobiose, lactose, melibiose and gentiobiose.
4. A compound as claimed in claim 1, wherein the glycosyl moieties is selected from the group consisting of D-glucose, D-galactose, D-mannose, D-arabinose, D-xylose, D-ribose, L-rhamnose, L-fucose, tagatose, digitose, N-acetyl neuraminic acid, D-maltose, D-cellobiose, D-lactose, D-glucosamine, D-galactosamine, D-fucosamine, D-mannosamine.
5. A process for the preparation of novel glycosyl-D-fructose derivatives, comprising the steps of:
a) reacting the per-O-acetylated sugar of formula II and sulphur containing alkanol compound in the ratio of 1:1 in the presence of an dry organic solvent under inert atmosphere and cooling the above said reaction mixture to a temperature of about 0°C and further adding borontrifluoride diethyletherate under stirring, for a period of about 5 hrs to obtain the desired product of formula III,
b) reacting suspension of D-fructose in presence of dry acetone with concentrated sulphuric acid under stirring for a period of about 2hrs at a temperature of about 25°C and cooling the reaction mixture and quenching in 50%w/v of sodium hydroxide at a temperature of about 0°C , followed by purifying by known method and drying to obtain the crude mass , further washing with alkaline sodium
bicarbonate solution and purifying by known chromatographic methods to obtain the desired product of formula IV,
c) mixing the above said reaction product of formula III and the above said reaction product of formula IV in the mmolar ratio of 1:1 in presence of dry organic solvent, followed by,
i) adding molecular sieves of 4A of weight 500mg in argon atmosphere under stirring at a temperature of about 25°C for a time period of about 30 minutes, further adding N-iodosuccinimide and cooling the reaction mixture to a temperature of about -4°C,
ii) cooling the above said reaction mixture and adding trifluoromethanesulphonic acid to obtain the resultant solution, and stirring the aforesaid resultant solution for a period of about -40°C for a period of about 1 hour and purifying the reaction mixture by known chromatographic methods to obtain the desired product of formula V,
iii) reacting the above said reaction product obtained in step (ii) with trifluroacetic acid and water in the ratio of 2:1 in presence of an organic solvent, followed by stirring at a temperature of about 25°C for a period of about 3 hours, purifying the above said reaction mixture obtained by known chromatographic methods to obtain the desired product of formula I.
6. A process as claimed in claim 5, wherein the per-O-acetyl sugars is selected from the group consisting of 2,3,4-tri-O-acetyl-ß-D-ribopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose, 2,3,4,6-Tetra-O-acetyl-ß-D-mannopyranosyl,1,2:4,5-di-0-isopropylidene-D-fructopyran-ose,2,3,4-Tri-O-acetyl-ß-D-arabinopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose, 2,3,4-Tri-O-acetyl-ß-D-arabinopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose,2,3,4-Tri-0-acetyl-ß-D-xylopyranosyl,
1,2:4,5-di-O-isopropylidene-D-fructopyranose,2,3,4-Tri-O-acetyl-ß-D-ribopyranosyl, 1,2:4,5-di-O-isopropylidene-D-fructopyranose.
7. A process as claimed in claim 5, wherein the sulphur containing alkanol is
selected from ethanethiol, thiophenol, p-thiocresol,m-thiocresol,o-thiocresol,2-mercaptobenzothiazole, 2-mercaptobenzimidazole, 2-mercaptopyridine,2-mercaptopiperidine.
8. A process as claimed in claim 5, wherein the organic solvent used is selected from dichloromethane, chloroform, dichloroethane, tertrahydrofuran, diethyl ether, acetonitrile,1,4- dioxane, dimethyl formamide, toluene and benzene.
9. A process as claimed in claim 5, wherein the product obtained of formula III
is selected from the group consisting of ethyl 2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-1-thio-ß-D-glucopyranoside (IlIa), ethyl 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl-(1→4)-2,3,6-tri-O-acetyl-1-thio-ß-D-glucopyranoside(lllb), ethyl 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl-(1→4)-2,3,6-tri-O-acetyl-1-thio-ß-DD-glucopyranoside (lllc), ethyl 2,3,4,6-tetra-O-acetyl-1-thio-ß-D-galactopyranoside (IIId), ethyl 2,3,4,6-tetra-O-acetyl-1-thio-ß-D-mannopyranoside (IlIe), ethyl 2,3,4-tri-O-acetyl-1-thio-(3-D-arabinopyranoside (lllf), ethyl 2,3,4-tri-O-acetyl-1-thio-ß-D-xylopyranoside (lllg) ethyl 2,3,4-tri-O-acetyl-1-thio-ß-D-ribopyranoside (lllh), ethyl 2,3,4-tri-O-acetyl-1-thio-6-deoxy-α -L-mannopyranoside(llli)
10. A process as claimed in claim 5, wherein the product obtained of formula
V is selected from the group consisting of2,3,4,6-Tetra-O-acetyl-ß-D-
galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-ß-◘D-glucopyranosyl-(1→3)-
1,2:4,5-di-O-isopropylidene-D-fructopyranose (Va), 2,3,4,6-Tetra-O-acetyl-(3-D-glucopyranosyl-(1→4)-2,3,6-tri-O-acetyl-ß◘ D-glucopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vb), 2,3,4,6-Tetra-O-acetyl-ß-D-glucopyranosyl-(1→4)-2,3,6-tri-O-acetyl-ß-D-glucopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vc), 2,3,4,6-Tetra-O-acetyl-ß-D-galactopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyran-
ose (Vd), 2,3,4,6-Tetra-O-acetyl-ß-D-mannopyranosyl-(1→3)-1,2:4,5-di-
0-isopropylidene-D-fructopyran-ose(Ve),2,3,4-Tri-0-acetyl-ß-D-
arabinopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose
(Vf), 2,3,4-Tri-0-acetyl-(3-D-xylopyranosyl-(1 →3)-1,2:4,5-di-O-
isopropylidene-D-fructopyranose (Vg), 2,3,4-Tri-0-acetyl-ß-D-
ribopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructopyranose (Vh), 2,3,4-Tri-0-acetyl-6-deoxy-ß-L-mannopyranosyl-(1→3)-1,2:4,5-di-O-isopropylidene-D-fructo-pyranose(Vi).

Documents:

609-del-2006-abstract.pdf

609-DEL-2006-Claims-(28-06-2012).pdf

609-del-2006-claims.pdf

609-DEL-2006-Correspondence Others-(28-06-2012).pdf

609-del-2006-correspondence-others 1.pdf

609-del-2006-correspondence-others.pdf

609-del-2006-description (complete).pdf

609-del-2006-description (provisional).pdf

609-del-2006-drawings.pdf

609-del-2006-form-1.pdf

609-del-2006-form-18.pdf

609-del-2006-form-2.pdf

609-del-2006-form-3.pdf

609-del-2006-form-5.pdf

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Patent Number

253810

Indian Patent Application Number

609/DEL/2006

PG Journal Number

35/2012

Publication Date

31-Aug-2012

Grant Date

27-Aug-2012

Date of Filing

08-Mar-2006

Name of Patentee

COUNCIL OF SCIENTIFIC & INDISTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	PALLAVI TIWARI	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226001, INDIA.
2	ANJU PURI	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226001, INDIA.
3	ANUP KUMAR MISRA	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226001, INDIA.
4	RAMESH CHANDER	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226001, INDIA.
5	GEETIKA BHATIA	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226001, INDIA.

PCT International Classification Number

C129P 12/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA