Artemisinic acid glycoconjugate compounds, process for preparation and use thereof

Abstract

The present invention relates to artemisinic acid glycoconjugate compounds. More particularly, the present invention relates to a glycoconjugate compound of formula (I) and a process for the preparation of artemisinic acid glycoconjugate compound of formula (I) by using 1,3-dipolar cycloaddition chemistry starting from artemisinic acid. ##STR00001##

Claims

1. A compound of formula (I); ##STR00015## wherein X is selected from O or N; and Y is selected from D-Glucose, D-Galactose, L-Rhamnose, Maltose, D-Glucal, D-Galactal, Lactose, arabinose, D-galactosamine, D-glucosamine, D-mannosamine, D-mannose, D-Xylose, and D-fructose.

2. The compound of formula (I) as claimed in claim 1, wherein said compound of formula (I) is selected from the group consisting of a) (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (4); b) (2R,3S,4S,5R,6R)-2-((benzoyloxy)methyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (5); c) (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (6); d) (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (7); e) (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (8); f) (2R,3R,4S,5R,6R)-2-((benzoyloxy)methyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (9); and g) (2R,3R,4R,5S,6S)-2-(4-(((2-((1R,4R,4aS,8aR)-4,7-Dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (10).

3. A pharmaceutical composition comprising the compound of claim 1 or pharmaceutically acceptable salt thereof and pharmaceutically acceptable carrier.

4. A process for the synthesis of compound of formula (I) comprising the steps of: a) reacting artemisinic acid with propargyl alcohol or propargyl amine to afford acrylate (2) or acrylamide (3) respectively; and b) subjecting the acrylate (2) or acrylamide (3) of step (a) to 1, 3-dipolar cycloaddition with sugar-azides to afford compound of formula (I).

5. The process as claimed in claim 4, wherein said step (a) comprises stirring the reaction mixture of artemisinic acid, propargyl alcohol and 4-Dimethylaminopyridine (DMAP) in a solvent at a temperature of 0 C. for a period in the range of 1 to 2 hrs followed by further stirring the reaction mixture at a temperature in the range of 25 to 30 C. for a period in the range of 10 to 12 hrs to afford corresponding acrylate compound (2).

6. The process as claimed in claim 4, wherein said step (a) comprises adding propargyl amine to the reaction mixture of artemisinic acid, (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate) {HATU}, N,N-Diisopropylethylamine (DIPEA) in a solvent at a temperature in the range of 25 to 30 C. followed by stirring the reaction mixture at temperature in the range of 25 to 30 C. for the period in the range of 5 to 7 hrs to afford corresponding acrylamide compound (3).

7. The process as claimed in claim 4, wherein said step (b) comprises adding N,N-Diisopropylethylamine (DIPEA) and Copper(I) iodide (CuI) to the reaction mixture of sugar-azide and acrylate compound 2 or acrylamide compound 3 in a solvent followed by stirring the reaction mixture at a temperature in the range of 25 to 30 C. for a period in the range of 8 to 10 hrs to afford compound of formula (I).

8. The process as claimed in claim 7, wherein said sugar-azides are protected or free sugar azides, selected from azides of D-Glucose, D-Galactose, L-Rhamnose, Maltose, D-Glucal, D-Galactal, Lactose, arabinose, D-galactosamine, D-glucosamine, D-mannosamine, D-mannose, D-Xylose, and D-fucose.

9. The process as claimed in claim 8, wherein said sugar-azide are selected from the group consisting of D-Glucosyl azide, D-Galactosyl azide, L-Rhamnosyl azide, Maltosyl azide, D-Glucal azide, Lactosyl azide and D-mannose azide.

10. The process as claimed in claim 4, wherein the solvents used for step a and b are selected from dichloromethane, dimethylformamide, tetrahydrofuran, chloroform, and carbon tetrachloride.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

(2) In view of the above, the present invention provides an artemisinic acid glycoconjugate compound of formula (I) and a process for the synthesis thereof by using 1,3-dipolar cycloaddition chemistry starting from artemisinic acid.

(3) In an embodiment, the present invention provides an artemisinic acid glycoconjugate compound of formula (I);

(4) ##STR00003##
Wherein,
X is selected from O or N;
Y is selected from various protected or free sugars like D-Glucose, D-Galactose, L-Rhamnose, Maltose, D-Glucal, D-Galactal, Lactose, arabinose, D-galactosamine, D-glucosamine, D-mannosamine, D-mannose, D-Xylose, D-fucose or the like.

(5) In preferred embodiment, the artemisinic acid glycoconjugate compound of formula (I) is selected from a) (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (4); b) (2R,3S,4S,5R,6R)-2-((benzoyloxy)methyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (5); c) (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (6); d) (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (7); e) (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (8); or f) (2R,3R,4S,5R,6R)-2-((benzoyloxy)methyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (9) and g) (2R,3R,4R,5S,6S)-2-(4-(((2-((1R,4R,4aS,8aR)-4,7-Dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (10).

(6) ##STR00004## ##STR00005##

(7) The present invention provides a method for the preparation of 12-O-artemisinic acid glycoconjugates and 12-N-artemisinic acid glycoconjugates by using 1,3-dipolar cycloaddition chemistry starting from artemisinic acid. First the artemisinic acid is propargylated and subjected to 1, 3-dipolar cycloaddition reaction with various sugar azides to furnish the desired title compounds.

(8) In one embodiment of the present invention, the present invention provides a process for the synthesis of artemisinic acid glycoconjugate compound of formula (I) comprising the steps of: a) Reacting artemisinic acid with propargyl amine or propargyl alcohol to afford acrylate (2) or acrylamide (3) and b) Subjecting the acrylate (2) or acrylamide (3) of step (a) to 1, 3-dipolar cycloaddition with sugar-azides to afford compound of formula (I).

(9) In preferred embodiment, the step (a) comprises stirring the reaction mixture of artemisinic acid, propargyl alcohol and 4-Dimethylaminopyridine (DMAP) in a solvent at a temperature 0 C. for a period in the range of 1 to 2 hrs followed by further stirring the reaction mixture of at temperature in the range of 25 to 30 C. for a period in the range of 10 to 12 hrs to afford corresponding acrylate compound (2).

(10) In another preferred embodiment, the step (a) comprises adding propargyl amine to the reaction mixture of artemisinic acid, (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) {HATU}, N,N-Diisopropylethylamine (DIPEA) in a solvent at a temperature in the range of 25 to 30 C. followed by stirring the reaction mixture at a temperature in the range of 25 to 30 C. for a period in the range of 5 to 7 hrs to afford corresponding acrylamide compound (3).

(11) In one embodiment of the present invention, the step (b) comprises adding N,N-Diisopropylethylamine (DIPEA) and Copper(I) iodide (CuI) to the reaction mixture of sugar-azide and acrylate compound 2 or acrylamide compound 3 in a solvent followed by stirring the reaction mixture at a temperature in the range of 25 to 30 C. for a period in the range of 8 to 10 hrs to afford compound of formula (I).

(12) In preferred embodiment, the sugar-azide is selected from various protected or free sugar azides like D-Glucose, D-Galactose, L-Rhamnose, Maltose, D-Glucal, D-Galactal, Lactose, arabinose, D-galactosamine, D-glucosamine, D-mannosamine, D-mannose, D-Xylose and D-fucose.

(13) In more preferred embodiment, the sugar-azide is selected from D-Glucosyl azide, D-Galactosyl azide, L-Rhamnosyl azide, Maltosyl azide, D-Glucal azide, Lactosyl azide and D-mannose.

(14) In one embodiment of the present invention, the reaction steps (a) and (b) are carried out under argon atmosphere.

(15) The solvents of step (a) or (b) are selected from dichloromethane, dimethylformamide, tetrahydrofuran, chloroform or carbon tetrachloride.

(16) The process for the synthesis of compound of formula (I) is as depicted in scheme in below:

(17) ##STR00006##

(18) The scheme 1 shows the process for the synthesis of compound of formula (I), wherein the sugar is selected from various protected or free sugars like D-Glucose, D-Galactose, L-Rhamnose, Maltose, D-Glucal, D-Galactal, Lactose, arabinose, D-galactosamine, D-glucosamine, D-mannosamine, D-mannose, D-Xylose, D-fucose or the like.

(19) In yet another embodiment, the present invention provides a pharmaceutical composition comprising artemisinic acid glycoconjugate compound of formula (I) or pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

(20) The carrier is typically used when the composition is prepared, and includes, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybezoate, talcum, magnesium stearate, mineral oil, or the like.

(21) The composition can additionally comprise stability improving material, viscosity improving or adjusting material, solubility improving material, sweetener, dye, palatability improving material, osmotic pressure variable salt, buffer solution, antioxidant, and so on.

(22) The pharmaceutical compositions of the present invention can be oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

(23) Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

(24) Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.

(25) In the present invention, the pharmaceutically acceptable salt can include a pharmaceutically acceptable acid addition salt. The pharmaceutically acceptable acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid, or phosphorous acid, and nontoxic organic acids such as aliphatic mono- and di-carboxylates, phenyl-substituted alkanoate, hydroxyl alkanoate, and alkandioate, aromatic acids, and aliphatic and aromatic sulfuric acids.

(26) In another embodiment, the present invention provides a pharmaceutical composition comprising compound of formula (I) or pharmaceutically acceptable salt thereof for preventing and treating cancer or fungal infection comprising administering an effective dose of the pharmaceutical composition to a mammal.

(27) Further, the present invention provides a method of treating cancer or fungal infection, which is characterized by administering an effective dose of pharmaceutical composition to a mammal.

(28) Typically, the inventive pharmaceutical composition is administered in the form of a unit dose containing its effective ingredient at an amount between about 1 mg and about 500 mg. The total dose per day of the inventive pharmaceutical composition is within a range from about 1 mg to about 500 mg, and preferably from about 1 mg to about 300 mg. However, in comprehensive consideration of the situation of a patient, and in consideration of the activity of an administered medication, a specific dose beyond such a range can be administered. An optimal dose administered under a specific situation must be decided experimentally.

(29) The inventive compounds can be administered once or several times at a dose. Preferably, a dose per day is administered once or twice per day. The inventive compounds can be administered alone or in conjunction with a pharmaceutically acceptable carrier and excipient. The inventive pharmaceutical composition can be formulated into excipient known in the art as well as a pharmaceutically acceptable carrier and diluents. This formulation can take the form of a unit dose by a method known in the pharmaceutical field for convenience.

(30) The inventive pharmaceutical composition can be used in conjunction with one or more other therapeutically useful materials, for instance other anti-cancer or anti-malarial drugs.

(31) In one embodiment of the present invention provides use of artemisinic acid glycoconjugate compound of formula (I) as anti-cancer agent, anti-fungal agent, anti-malarial agent, antipyretic agent, antibacterial agent, or imaging agent. Further, the present invention provides artemisinic acid glycoconjugate compound of formula (I) which shows allelopathy effect or anti-adipogenesis effect.

(32) In still another embodiment, the present invention provides study of anticancer activity of compound (I) against MCF-7 (breast cancer cell line) with a IC.sub.50 value. In preferred embodiment, compound (2R,3R,4R,5S,6S)-2-(4-(((2-((1R,4R,4aS,8aR)-4,7-Dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (10) shows anticancer activity against MCF-7 (breast cancer cell line) with a IC.sub.50 value of 42+/4 M.

(33) ##STR00007##
Many compounds of formula (I) are prepared and studied anticancer activities of these compounds against MCF7 (breast cancer cell line). Results of anticancer activities are summarized in below Table 1.

(34) TABLE-US-00001 TABLE 1 In vitro anticancer activities of glycoconjugates against MCF7 (breast cancer cell line). Compound code Compound IC50 mol KTK-PA-1 >100 KTK-PA-2 7 >100 KTK-PA-3 8 >100 KTK-PA-4 9 >100 KTK-PA-5 >100 KTK-PA-6 >100 KTK-PA-7 >100 KTK-PA-8 >100 KTK-PA-9 >100 KTK-PA-10 >100 KTK-PA-11 >100 KTK-PA-12 >100 KTK-AA-1 >100 KTK-AA-2 4 >100 KTK-AA-3 >100 KTK-AA-4 6 >100 KTK-AA-5 >100 KTK-AA-6 5 >100 KTK-AA-7 10 42 2 KTK-AA-8 >100 KTK-AA-9 >100 KTK-AA-10 >100 KTK-AA-11 >100 KTK-AA-12 >100 KTK-AA- Artimisinic acid 1 >100
From table 1, it is observed that compound 10 shows unexpected enhancement in anticancer activity over artimisinic acid, IC50 value is more than 50% less when compared with artimisinic acid, thus providing very potent anticancer activity against breast cancer cell lines. Further, a library of compounds of formula (I) are undergoing screening and testing against more cell lines for various pharmacological activities.

(35) In one embodiment, the present invention provides a method for treating cancer or fungal infection, wherein said method comprises administering to the subject a therapeutically effective amount of artemisinic acid glycoconjugate compound of formula (I) or pharmaceutically acceptable salt thereof.

EXAMPLES

(36) Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.

Example 1: Synthesis of prop-2-yn-1-yl 2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylate (2)

(37) A solution of Artemisinic acid (AA) (325 mg, 1 equiv), propargyl alcohol (80 L, 1 equiv.) and DMAP (17 mg, 0.1 equiv) in a dry DCM (5 mL) was cooled to 0 C. and then treated with EDC.HCl (1.2 equiv). The reaction mixture was stirred at the same temperature (0 C.) for 2 h and then at 25 C. for a 10 h. After completion of reaction (TLC), the reaction mixture concentrated in vacuo, the residue was taken up in EtOAc and water. The organic layers were collected and washed with saturated NH.sub.4Cl solution, and dried over Na.sub.2SO.sub.4. The solvent was concentrated in vacuo and subjected to flash chromatography to give compound 2 (354 mg) in 94% yield.

(38) .sup.1H NMR (CHLOROFORM-d, 500 MHz): =6.35 (s, 1H), 5.49 (s, 1H), 4.97 (br. s, 1H), 4.78-4.68 (m, 2H), 2.73-2.69 (m, 1H), 2.50-2.57 (m, 1H), 2.47-2.46 (m, 1H), 1.94-1.83 (m, 2H), 1.78-1.67 (m, 2H), 1.58 (s, 3H), 1.55-1.49 (s, 1H), 1.45-1.30 (m, 4H), 1.10-1.02 (m, 1H), 0.89 (d, J=5.7 Hz, 3H). .sup.13C NMR (CHLOROFORM-d, 126 MHz): =166.3, 142.6, 134.9, 125.2, 120.2, 77.8, 74.8, 52.1, 42.3, 41.3, 37.9, 35.2, 27.5, 26.4, 25.9, 25.5, 23.7, 19.7. HRMS (ESI) m/z found 295.1661 [M+Na].sup.+ (calcd for C.sub.18H.sub.24O.sub.2Na, 295.1669).

Example 2: Synthesis of 2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)-N-(prop-2-yn-1-yl)acrylamide (3)

(39) To a solution of artemisinic acid (1), (100 mg, 1 equiv), HATU (191 mg, 1.2 equiv) and DIPEA (87 L, 1.2 equiv) in DMF (10 mL) was added propargyl amine (32 L, 1.2 equiv) at 25 C. under a argon atmosphere. The mixture was stirred for 6 h. After completion of reaction (TLC), the solvent was removed in vacuo and the resulting oil residue was diluted with DCM (15 mL) and extracted with water. The combined DCM layers were dried over Na.sub.2SO.sub.4 filtered and concentrated. The crude product was purified by flash chromatography to give compound 3 (109 mg) in 95% yield.

(40) .sup.1H NMR (500 MHz, CDCl.sub.3) =6.29 (brs, 1H), 5.63 (s, 1H), 5.15 (s, 1H), 4.96 (s, 1H), 4.16-4.11 (m, 1H), 4.03-3.98 (m, 1H), 2.74 (d, J=12.6 Hz, 1H), 2.42 (s, 1H), 2.23 (t, J=2.7 Hz, 1H), 2.15 (s, 1H), 1.90-1.85 (m, 2H), 1.75-1.66 (m, 2H), 1.57 (s, 3H), 1.56-1.49 (m, 1H), 1.44-1.37 (m, 3H), 1.29-1.23 (m, 1H), 0.86 (d, J=5.3 Hz, 3H). .sup.13C NMR (125 MHz, CDCl.sub.3) =169.5, 148.7, 135.1, 120.2, 116.6, 79.6, 71.6, 42.6, 41.2, 37.7, 35.0, 29.4, 27.5, 26.4, 25.4, 25.3, 23.7, 19.7. HRMS (ESI): m/z calcd for [M+H].sup.+, C.sub.18H.sub.26ON 272.2009 found 272.2004.

Example 3: General Procedure for the Synthesis of Artemisinic Acid Glycoconjugates

(41) To a stirred solution of various sugar-azides (1 equiv) and compound 2 (1 equiv) or compound 3 (1 equiv) in a dry DCM (5 mL), DIPEA (1 equiv) and CuI (0.5 equiv) were added under argon atmosphere. The solution was stirred at 25 C. for 8 to 10 h. After completion of the reaction (TLC), the reaction mixture diluted with DCM (10 mL) and washed with water, the DCM layer dried over anhydrous Na.sub.2SO.sub.4, concentrated in vacuo. The crude residue was subjected to flash chromatography to give various artemisinic glycoconjugates in excellent yields (85-98%).

I. (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (4)

(42) ##STR00008##

(43) To a stirred solution of various galactosyl azide (1 equiv) and compound 2 (1 equiv) in a dry DCM (5 mL), DIPEA (1 equiv) and CuI (0.5 equiv) were added under argon atmosphere. The solution was stirred at 25 C. for 10 h. After completion of the reaction (TLC), the reaction mixture diluted with DCM (10 mL) and washed with water, the DCM layer dried over anhydrous Na.sub.2SO.sub.4, concentrated in vacuo. The crude residue was subjected to flash chromatography to give artemisinic glycoconjugates in excellent yields (94%).

(44) .sup.1H NMR (400 MHz, CDCl.sub.3): =7.90 (s, 1H), 6.30 (s, 1H), 5.84 (d, J=9.2 Hz, 1H), 5.54-5.49 (m, 2H), 5.44 (s, 1H), 5.33-5.30 (m, 1H), 5.26-5.17 (m, 2H), 4.94 (br. s., 1H), 4.24-4.16 (m, 2H), 4.12-4.08 (m, 2H), 2.67 (d, J=11.0 Hz, 1H), 2.51 (br. s., 1H), 2.19 (s, 3H), 2.00 (s, 3H), 1.97 (s, 3H), 1.89-1.87 (m, 1H), 1.82 (s, 3H), 1.72-1.64 (m, 2H), 1.54 (br. s., 3H), 1.38-1.28 (m, 3H), 1.26-1.19 (m, 3H), 0.85 (d, J=4.9 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): =170.3, 170.0, 169.8, 168.9, 166.9, 143.6, 142.7, 134.8, 125.2, 122.5, 120.2, 86.2, 74.0, 70.7, 67.8, 66.9, 61.2, 60.3, 57.5, 42.3, 41.3, 37.9, 35.2, 27.5, 26.3, 25.9, 25.5, 23.6, 20.6, 20.4, 20.1, 19.7; HRMS (ESI): m/z calcd for [M+H].sup.+, C.sub.32H.sub.44O.sub.11N.sub.3 646.3699 found 646.2970.

II. (2R,3S,4S,5R,6R)-2-((benzoyloxy)methyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (5)

(45) ##STR00009##

(46) .sup.1H NMR (400 MHz, CDCl.sub.3): =8.14-8.12 (m, 3H), 8.02-8.00 (m, 2H), 7.81-7.77 (m, 4H), 7.70-7.66 (m, 1H), 7.58-7.54 (m, 3H), 7.47-7.40 (m, 4H), 7.32-7.24 (m, 4H), 6.32-6.18 (m, 4H), 5.91-5.88 (m, 1H), 5.47 (br s, 1H), 5.39 (d, J=12.8 Hz, 1H), 5.24 (d, J=12.8 Hz, 1H), 5.01 (br. s., 1H), 4.72-4.64 (m, 2H), 4.53-4.49 (m, 1H), 2.74-2.71 (m, 1H), 2.58 (br. s., 1H), 1.90-1.69 (m, 5H), 1.59 (br. s., 3H), 1.55-1.50 (m, 1H), 1.43-1.36 (m, 4H), 0.89 (d, J=4.5 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): =167.0, 166.0, 165.4, 165.3, 164.8, 142.8, 134.9, 133.9, 133.7, 133.5, 133.4, 129.9, 129.8, 129.7, 129.1, 128.9, 128.8, 128.5, 128.4, 128.1, 125.1, 120.3, 86.5, 74.7, 71.7, 68.8, 68.0, 62.0, 57.6, 42.3, 41.4, 38.0, 35.2, 27.6, 26.4, 25.9, 25.6, 23.7, 19.7; HRMS (ESI) m/z: calcd for [M+Na].sup.+, C.sub.52H.sub.51O.sub.11N.sub.3Na 916.3416 found 916.3408.

III. (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (6)

(47) ##STR00010##

(48) .sup.1H NMR (500 MHz, CDCl.sub.3): =7.84 (s, 1H), 6.27 (s, 1H), 5.89-5.87 (m, 1H), 5.42-5.39 (m, 3H), 5.28-5.18 (m, 3H), 4.92 (br. s., 1H), 4.28-4.24 (m, 1H), 4.12-4.09 (m, 1H), 4.02-3.99 (m, 1H), 2.65 (d, J=11.8 Hz, 1H), 2.49 (br. s., 1H), 2.03 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H), 1.83-1.80 (m, 4H), 1.71-1.63 (m, 3H), 1.52 (br. s., 3H), 1.37-1.27 (m, 4H), 1.18-1.23 (m, 2H), 0.83 (d, J=5.7 Hz, 3H); .sup.13C NMR (125 MHz, CDCl.sub.3): =170.5, 169.9, 169.3, 168.7, 166.9, 143.7, 142.7, 134.8, 125.1, 122.2, 120.2, 85.6, 75.1, 72.6, 70.3, 67.7, 61.5, 57.5, 42.3, 41.3, 37.9, 35.2, 29.6, 27.5, 26.3, 25.8, 25.5, 23.6, 20.6, 20.5, 20.0, 19.7; HRMS (ESI) m/z: calcd for [M+Na].sup.+, C.sub.32H.sub.43O.sub.11N.sub.3Na 668.2790 found 668.2880.

IV. (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (7)

(49) ##STR00011##

(50) .sup.1H NMR (500 MHz, CDCl.sub.3) =7.81 (s, 1H), 6.81 (m, 1H), 5.86 (d, J=8.8 Hz, 1H), 5.59 (s, 1H), 5.43-5.38 (m, 2H), 5.21 (t, J=9.5 Hz, 1H), 5.13-5.10 (m, 1H), 4.98 (br. s., 1H), 4.59 (dd, J=15.3, 6.1 Hz, 1H), 4.48 (dd, J=15.3, 5.7 Hz, 1H), 4.26 (dd, J=12.6, 5.0 Hz, 1H), 4.14-4.06 (m, 1H), 4.03-4.00 (m, 1H), 2.77-2.72 (m, 1H), 2.44 (br. s., 1H), 2.05 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H), 1.91-1.86 (m, 2H), 1.81 (s, 3H), 1.76-1.72 (m, 1H), 1.68-1.66 (m, 1H), 1.56 (s, 3H), 1.40-1.37 (m, 3H), 1.25-1.22 (m, 2H), 0.85 (d, J=5.7 Hz, 3H); .sup.13C NMR (125 MHz, CDCl.sub.3) =170.5, 169.9, 169.3, 168.7, 148.8, 145.6, 135.1, 121.0, 120.3, 116.4, 85.7, 75.0, 72.6, 70.5, 67.7, 61.5, 42.5, 41.2, 37.8, 35.0, 34.9, 27.5, 26.4, 25.5, 25.2, 23.7, 20.6, 20.5, 20.4, 20.1, 19.7; HRMS (ESI) m/z: calcd for [M+H].sup.+, C.sub.32H.sub.45O.sub.10N.sub.4 645.3130 found 645.3109.

V. (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (8)

(51) ##STR00012##

(52) .sup.1H NMR (500 MHz, CDCl.sub.3) =7.86 (s, 1H), 6.82-6.81 (m, 1H), 5.83 (d, J=9.2 Hz, 1H), 5.60 (s, 1H), 5.52-5.48 (m, 2H), 5.26 (dd, J=10.3, 3.0 Hz, 1H), 5.12 (s, 1H), 4.97 (br. s., 1H), 4.66 (dd, J=15.3, 6.3 Hz, 1H), 4.39 (dd, J=15.3, 5.3 Hz, 1H), 4.25 (t, 6.5 Hz, 1H), 4.20-4.16 (m, 1H), 4.13-4.06 (m, 1H), 2.77-2.72 (m, 1H), 2.43 (br. s., 1H), 2.20 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H), 1.89-1.85 (m, 2H), 1.83 (s, 3H), 1.73-1.65 (m, 2H), 1.55 (s, 3H), 1.42-1.37 (m, 3H), 1.28-1.22 (m, 2H), 0.85 (d, J=5.3 Hz, 3H); .sup.13C NMR (125 MHz, CDCl.sub.3) =170.3, 170.1, 169.8, 168.9, 148.7, 145.2, 135.0, 121.2, 120.3, 116.5, 86.2, 74.0, 70.7, 68.0, 66.9, 61.2, 42.5, 41.2, 37.8, 35.0, 34.8, 27.5, 26.4, 25.4, 25.3, 23.7, 20.6, 20.6, 20.5, 20.1, 19.7; HRMS (ESI) m/z: calcd for [M+H].sup.+, C.sub.32H.sub.45O.sub.10N.sub.4 645.3130 found 645.3113.

VI. (2R,3R,4S,5R,6R)-2-((benzoyloxy)methyl)-6-(4-((2-((1R,4R,4aS,8aR)-4,7-dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acrylamido)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (9)

(53) ##STR00013##

(54) .sup.1H NMR (400 MHz, CDCl.sub.3) =8.01-7.98 (m, 3H), 7.93-7.91 (m, 2H), 7.82-7.80 (m, 2H), 7.76-7.74 (m, 2H), 7.53-7.48 (m, 2H), 7.43-7.33 (m, 6H), 7.29-7.25 (m, 4H), 6.68 (s, 1H), 6.29 (d, J=9.2 Hz, 1H), 6.15 (t, J=9.5 Hz, 1H), 5.99-5.94 (m, 1H), 5.89-5.84 (m, 1H), 5.54 (s, 1H), 5.08 (s, 1H), 5.01 (s, 1H), 4.64-4.63 (m, 2H), 4.52-4.49 (m, 3H), 2.79-2.78 (m, 2H), 2.49 (s, 1H), 2.03 (s, 1H), 1.91-1.85 (m, 2H), 1.70-1.67 (m, 1H), 1.59 (s, 3H), 1.40 (m, 2H), 1.26-1.23 (m, 2H), 0.88 (d, J=4.9 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) =169.9, 166.1, 165.6, 165.1, 164.6, 148.8, 135.1, 133.7, 133.6, 133.5, 133.3, 129.9, 129.8, 129.7, 129.3, 128.5, 128.4, 128.3, 128.0, 121.1, 120.3, 116.4, 86.1, 75.5, 73.0, 71.2, 68.9, 62.8, 42.6, 41.2, 38.6, 37.8, 35.0, 27.6, 26.4, 25.4, 25.3, 23.7, 19.7; HRMS (ESI) m/z: calcd for [M+H].sup.+, C.sub.52H.sub.53O.sub.10N.sub.4 893.3756 found 893.3749.

VII. (2R,3R,4R,5S,6S)-2-(4-(((2-((1R,4R,4aS,8aR)-4,7-Dimethyl-1,2,3,4,4a,5,6,8a-octahydronaphthalen-1-yl)acryloyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (10)

(55) ##STR00014##

(56) Colorless solid; Yield: 90%, m.p.: 89-92 C.; Rf=0.40 (40% EtOAc-petroleum ether); flash chromatography eluting with 25% EtOAc-petroleum ether; []D26=+26.62 (c 1.7, CHCl.sub.3); 1H NMR (500 MHz, CDCl.sub.3): =7.84 (s, 1H), 6.30-6.27 (m, 1H), 6.14 (s, 1H), 5.95-5.80 (m, 1H), 5.66-5.65 (m, 1H), 5.45-5.43 (m, 1H), 5.34-5.29 (m, 1H), 5.23-5.20 (m, 1H), 5.18-5.15 (m, 1H), 4.92 (s, 1H), 3.86-3.81 (m, 1H), 2.69-2.64 (m, 1H), 2.49 (brs, 1H), 2.14 (s, 1H), 2.06 (s, 3H), 2.03-2.01 (m, 4H), 1.95 (s, 3H), 1.91-1.82 (m, 2H), 1.74-1.65 (m, 2H), 1.55 (s, 3H), 1.39-1.38 (m, 2H), 1.32 (d, J=6.5 Hz, 3H), 1.22-1.20 (m, 2H), 0.86 (d, J=5.3 Hz, 3H); .sup.13C NMR (125 MHz, CDCl.sub.3): =169.8, 169.8, 169.1, 167.0, 143.6, 142.9, 142.7, 142.7, 134.9, 125.2, 125.1, 124.0, 123.1, 120.1, 120.1, 84.6, 83.8, 73.9, 70.7, 70.0, 69.6, 69.2, 68.8, 68.4, 60.3, 57.6, 57.5, 42.3, 41.4, 41.3, 37.9, 35.2, 27.5, 26.3, 25.8, 25.5, 23.6, 20.7, 20.5, 20.5, 20.3, 19.7, 17.5, 17.1, 14.1; HRMS (ESI) m/z: calcd for C.sub.30H.sub.42O.sub.9N.sub.3 [M+H].sup.+: 588.2916, found 588.2903

Example 4: Study of Anticancer Activity Against MCF-7 (Breast Cancer Cell Line)

(57) Methodology

(58) Compounds were dissolved in DMSO (Sigma) to prepare 50 mM concentrations stock solutions. All the further dilutions were also made in DMSO. During the treatment, the final concentration of DMSO was maintained <0.02%.

(59) Antibodies

(60) Anti-Caspase 9 and Anti-Caspase 3 antibodies were purchased from Cell Signaling and Anti-Tubulin antibody was procured from Sigma, Goat anti-rabbit HRP conjugated secondary antibody was purchased from Bio-Rad, and Goat anti-mouse HRP conjugated secondary antibody was purchased from Cell Signaling.

(61) Cell Culture

(62) Breast cancer cell line MCF7, maintained at NCCS, a national resource for providing cell lines, was grown in DMEM (GIBCO), MBA-MB-231 in RPMI (GIBCO) with 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin and MCF10A in DMEM/F12 (Gibco) containing, horse Serum (10% final), EGF (20 ng/ml), Hydrocortisone (0.5 mg/ml), Cholera Toxin (100 ng/ml), Insulin (10 g/ml) and Penicillin/Streptomycin mix (1 ml/100 ml) at 37 C. in a humid, 5% CO.sub.2 regulated incubator.

(63) Growth Inhibition by Cytotoxicity Assay

(64) The cytotoxic effect of the compounds was determined using MTT (3-(4, 5 dimethylthiazol-2-yl)-2-5 diphenyltetrazolium bromide) assay. Cells were seeded (410.sup.3 per well) in 96 well plates. After 24 hours of seeding, cells were exposed with varying concentrations (0-100 M) of respective compounds for 48 hours in triplicates. Then, MTT solution (20 L of 5 mg/mL stock for each well of 96 well plate) was added and further incubated for 3.5 hours in humid 5% CO.sub.2 incubator. Media containing MTT solution was then replaced by MTT solvent (iso-propanol, HCl and Triton X-100), incubated for 15 min at room temperature with gentle shaking for complete dissolution of Formazan. Absorbance was measured at 570 nm using a Thermo Scientific Multiskan GO Elisa plate reader and IC 50 was calculated by routine methods. All experiments were carried out at least in triplicate, and the percentage of viable cells was calculated as the mean with respect to the controls.

(65) Table 1 shows the results for anticancer activity.

(66) TABLE-US-00002 Compound Code Compound IC50 mol KTK-PA-1 >100 KTK-PA-2 7 >100 KTK-PA-3 8 >100 KTK-PA-4 9 >100 KTK-PA-5 >100 KTK-PA-6 >100 KTK-PA-7 >100 KTK-PA-8 >100 KTK-PA-9 >100 KTK-PA-10 >100 KTK-PA-11 >100 KTK-PA-12 >100 KTK-AA-1 >100 KTK-AA-2 4 >100 KTK-AA-3 >100 KTK-AA-4 6 >100 KTK-AA-5 >100 KTK-AA-6 5 >100 KTK-AA-7 Compound 10 42 2 KTK-AA-8 >100 KTK-AA-9 >100 KTK-AA-10 >100 KTK-AA-11 >100 KTK-AA-12 >100 KTK-AA- Artimisinic acid 1 >100

ADVANTAGES OF THE INVENTION

(67) The present invention provide novel artemisinic acid glycoconjugate of formula (I) a which is used as anti-cancer agent, anti-malarial agent or imaging agent. The number of novel 12-O-artemisinic acid glycoconjugates and 12-N-artemisinic acid glycoconjugates are synthesized starting from artemisinic acid by a simple, environment friendly and economic process.