MURAMYL DIPEPTIDES AND PROCESS FOR PREPARATION THEREOF

Abstract

The present invention relates to Muramyl dipeptide compounds having adjuvant activity. The present invention also discloses the process for the preparation of Muramyl dipeptide compound and their intermediates. The immuno-modulating properties of the Muramyl dipeptide compound and their use as NOD2 agonistic adjuvants in vaccine formulations is also disclosed.

Claims

1-10. (canceled)

11. A muramyl dipeptide compound according to general formula (I): ##STR00037## where: R.sub.1 is selected from substituted or unsubstituted (C.sub.3-C.sub.6)cycloalkyl, 3 to 6 membered heterocycloalkyl with one or more hetero atoms selected from nitrogen and oxygen, or ##STR00038## and R.sub.2 is a C.sub.1 to C.sub.18 aliphatic chain.

12. The muramyl dipeptide compound according to claim 11, wherein R.sub.1 is selected from the group consisting of ##STR00039##

13. The muramyl dipeptide compound according to claim 11, selected from group consisting of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(cyclopropanecarboxamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid; (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(cyclobutanecarboxamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid; (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(cyclopentanecarboxamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid; (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(cyclohexanecarboxamido)-2,5-dihydroxy 6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid; (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(8-(tetradecanoyloxy)octanamido)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid; (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(8-(dodecanoyloxy)octanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid; (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(8-(octanoyloxy)octanamido)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid; and (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(tetrahydrofuran-2-carboxamido)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid.

14. An advanced intermediate of the muramyl dipeptide compound according to claim 11, comprising O-benzyl protecting groups on D-glutamic acid, an O-benzyl on an anomeric hydroxyl group, and a benzylidine protecting group on a 5,6 diol of a sugar moiety, the advanced intermediate having formula (II): ##STR00040## where: R.sub.1 is selected from substituted or unsubstituted (C.sub.3-C.sub.6)cycloalkyl, 3 to 6 membered heterocycloalkyl with one or more hetero atoms selected from nitrogen and oxygen, or ##STR00041## where R.sub.2 is a C.sub.1 to C.sub.18 aliphatic chain.

15. A process for preparing the muramyl dipeptide compound according to claim 11, the process comprising: (a) performing a diazotransfer of a compound (7): ##STR00042## with trifluoromethanesulfonic azide to obtain (3R,4R,5S,6R)-3-azido-6-(hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triol as a compound (8): ##STR00043## (b) performing an anomeric benzylation of the compound (8) in the presence of a Lewis acid to obtain (2R,3S,4R,5R,6S)-5-azido-6-(benzyloxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol as a compound (9): ##STR00044## (c) performing a benzylidene protection of the compound (9) in the presence of an organic acid with benzaldehyde dimethylacetal to obtain (2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-ol as a compound (10): ##STR00045## (d) performing an O-alkylation of the compound (10) in the presence of a metal hydride with S-(2)-chloropropionic acid to obtain (R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid as a compound (11): ##STR00046## (e) peptide coupling the compound (11) with L-alanyl-D-isoglutamine benzyl ester trifluoroacetate in the presence of a first coupling agent and a first base to obtain (R)-benzyl 5-amino-4-((S)-2-((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate as a compound (12): ##STR00047## (f) reducing the compound (12) with triphenylphosphine to obtain benzyl (R)-5-amino-4-((S)-2-((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-amino-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate as a compound (13): ##STR00048## (g) acid and amine coupling the compound (13) with a cycloalkyl carboxylic acid or mixed ester carboxylic acid C(O)R.sub.1 where R.sub.1 is selected from substituted or unsubstituted (C.sub.3-C.sub.6) cycloalkyl, 3 to 6 membered heterocvcloalkyi with one or more hetero atoms selected from nitrogen and oxygen, or ##STR00049## where R.sub.2 is a C.sub.1 to C.sub.18 aliphatic ester, in the presence of a second coupling agent and a second base to obtain a compound (14): ##STR00050## and (h) performing a hydrogenolysis of the compound (14) to obtain the muramyl dipeptide compound.

16. The process according to claim 15, wherein the Lewis acid of (b) is selected from the group consisting of BF.sub.3, AlCl.sub.3, ZnCl.sub.2, p-toluenesulfonic acid, chlorosulfonic acid, camphorsulfonic acid, HCl, acetyl chloride, Amberlite and zeolite, and clay.

17. The process according to claim 15, wherein the acid of (c) is selected from the group consisting of p-toluenesulfonic acid, camphorsulfonic acid, and ZnCl.sub.2.

18. The process according to claim 15, wherein the metal hydride of (d) is selected from the group consisting of sodium hydride, potassium hydride, and calcium hydride.

19. The process according to claim 15, wherein: the first coupling agent of (e) and the second coupling agent of (g) are selected from the group consisting of EDCI/HOBt, DCC/DMAP, DIC/DMAP, and T3P; and the first base in (e) and the second base in (g) are diisopropylethylamine.

20. A prophylactic vaccine composition comprising: a muramyl dipeptide compound according to claim 11; and an antigen selected from inactivated or live attenuated infectious pathogens or subunits thereof either recombinant or derived from a natural pathogen, a conjugate vaccine antigen, or a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0063] FIG. 1 represents synthesis of dipeptide.

[0064] FIG. 2 represents synthesis of Muramyl dipeptide of general formula-I.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention provides Muramyl dipeptide of general Formula-I, with reduced pyrogenicity, while maintaining a considerable effective function as vaccine adjuvants.

##STR00021## [0066] wherein, R.sub.1 is selected from substituted or unsubstituted (C.sub.3-C.sub.6) cycloalkyl, 3 to 6 membered heterocycloalkyl with one or more hetero atoms selected from Nitrogen and Oxygen or

##STR00022## [0067] R.sub.2 is selected from group consisting of C.sub.1 to Cis aliphatic chain.

[0068] In another aspect, the present invention provides a process for preparation of Muramyl dipeptide of general Formula-I. The present process has advantages over the prior art in terms of reduced number of steps to achieve the synthesis of key intermediate obviating redundant acylation, selective of anomeric hydroxyl group, deacylation followed by activation and its subsequent deacylation, thereby generating less waste and improving the overall efficiency and yield of the process compared to previous processes, offering better atom economy. The invention relates to process for the preparation Muramyl dipeptide of general Formula-I which are depicted in FIG. 1 and FIG. 2.

[0069] L-alanyl-D-isoglutamine benzyl ester (dipeptide) obtained in FIG. 1 is used for the synthesis of Muramyl dipeptide of general Formula-I as shown in FIG. 2.

[0070] The Muramyl dipeptide of general formula-I, wherein, R.sub.1 is selected from substituted or unsubstituted alkyl, (C.sub.3-C.sub.6) cycloalkyl, 3 to 6 membered heterocycloalkyl with one or more hetero atoms selected from Nitrogen and Oxygen or

##STR00023##

wherein R.sub.2 is selected from group consisting of C.sub.1 to Cis Aliphatic chain, is synthesized in FIG. 2.

[0071] In another aspect, the invention provides a process for the synthesis of muramyl dipeptide of Formula-I involving the reaction steps as depicted in above FIG-1 for the synthesis of the L-alanyl-D-isoglutamine benzyl ester and FIG. 2 for the synthesis of novel N-glycolyl peptidoglycan derivatives of Structural Formula-I.

[0072] In yet another embodiment, the invention provides a novel N-glycolyl peptidoglycan intermediate of Structural formula-II

##STR00024## [0073] wherein R.sub.1 is selected from substituted or unsubstituted (C.sub.3-C.sub.6) cycloalkyl, 3 to 6 membered heterocycloalkyl with one or more hetero atoms selected from nitrogen and oxygen or,

##STR00025## [0074] wherein R.sub.2 is selected from group consisting of C.sub.1 to C.sub.18 aliphatic chain.

[0075] FIG. 1: Provides the process for the synthesis of dipeptide according to the following steps.

[0076] Step-a: [Compound 2] To a stirred mixture of D-Glutamic acid [Compound 1] and anhydrous sodium sulphate (Na.sub.2SO.sub.4) suspended in benzyl alcohol, added boron trifluoride. diethyl ether (BF.sub.3. E.sub.2O) and the suspension is stirred at room temperature (RT) followed by diluting with absolute THF and filtering with the aid of charcoal. Treatment with triethylamine, followed by concentration and precipitation and washing provides (R)-2-amino-5-(benzyloxy)-5-oxopentanoic acid [Compound 2].

[0077] Step-b: [Compound 3] is prepared by providing a solution of the [compound 2], adding with Di-teritiary butyl dicarbonate (Boc).sub.2O in dioxane and water at a low temperature (0-5 C.) and basified with sodium bicarbonate (NaHCO.sub.3), stirring overnight. Then the solvent is removed under reduced pressure and the residue is diluted with water, and washed with Ethyl Acetate (EtOAc), adjusted to pH 2-3 with aqueous HCl solution and extracted with EtOAc followed by further processing to get(R)-5-(benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid[Compound 3].

[0078] Step-c: [Compound 4] is synthesized by providing [Compound 3] in dry Tetrahydrofuran (THF), adding ethyl chloroformate and triethylamine at a low temperature such as 0 C. The reaction mixture is stirred at low temperature, and then cooled to low temperature (such as 15 C.) followed by addition of a methanolic solution of ammonia and further cooling to one hour at minus temperature and is added with ethyl acetate. After washing the organic phase with water followed by washing with brine solution, drying, removing the solvent in vacuum and purification)-benzyl-5-amino-4-((tert-butoxy carbonyl) amino)-5-oxopentanoate [Compound 4] is obtained.

[0079] Step-d: [Compound 5]i-Butoxy carbonyl-D-isoglutamine benzyl ester [Compound 4] is dissolved in cold trifluoroacetic acid and the resultant solution is stirred at RT and then Trifluoroacetic acid is removed and the residue is triturated with Diethyl Ether (Et.sub.2O) to obtain oily D-isoglutamine benzyl ester trifluoroacetate. Separately, to t-butoxycarbonyl-L-alanine in dry THF, 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDCI) and Hydroxy benzotriazole (HOBt) are added and stirred at RT. To this solution, D-isoglutamine benzyl ester trifluoroacetate (dissolved in THF) is added followed by N, N-Diisopropylethylamine (DIPEA). After stirring and concentrating the residue is extracted with EtOAc, washed and dried to obtain a residue. After further washing and recrystallization it provides benzyl-5-amino-4-((5)-2-((tert-butoxycarbonyl)amino) propanamido)-5-oxopentanoate [Compound 5].

[0080] Step-e: [Compound 6]benzyl-5-amino-4-((5)-2-((tert-butoxycarbonyl)amino) propanamido)-5-oxopentanoate [Compound 5] is dissolved in cold trifluoroacetic acid and dichloromethane and the resultant solution is stirred at RT and then Trifluoroacetic acid is removed with toluene by making azeotropic mixture and the residue was triturated with Diethyl Ether (Et.sub.2O) to obtain oily benzyl(R)-5-amino-4-((S)-2-aminopropanamido)-5-oxopentanoatetrifluoroacetate[Compound 6]

[0081] FIG. 2 provides the process for the synthesis of muramyl dipeptide of general formula-I, according to the following steps.

[0082] Step-(f): Diazo transfer of glucosamine hydrochloride[Compound 8].

[0083] Step (f) involves Diazo transfer of Formula 7 with a suitable azide transferring agent in the presence of an organic base with a suitable solvent, to obtain a compound of Formula-8 as schematically presented below;

##STR00026##

[0084] To the mechanically stirred solution of sodium azide in dry acetonitrile add triflic anhydride at 0 C. in 5Ltr RBF and kept it stirring for 30 minutes insitu triflic azide generated. To the mechanically stirred solution of glucosamine hydrochloride [Compound 7] copper sulphate. pentahydrate and amine base in the second 5Ltr RBF at 0 C. while stirring. After 15 min to the 2.sup.nd RBF at 0 C. triflic azide was added then kept it rt for 10 hrs. after completion of reaction. The reaction mixture was extracted in EtOAc (3100 ml) then combined organic fraction concentrated under reduced pressure to afford the required dark green liquid compound and further color removed through charcoal decolorization on filteration then dissolved in methanol and poured into diethyl ether to afford white solid compound it is used for next step without further purification. In one embodiment the organic amine base used is a triethylamine. The reaction mixture is diluted with Ethyl acetate washed successively with water, dried with sodium sulphate (Na.sub.2So.sub.4), and then filtered and the filtrate is evaporated to dryness. The filtrate is diluted with methanol and poured into diethyl ether to solidify the compound 8.

[0085] Step-(g): Anomeric benzylation of 2-Azido-D-Glucosamine [Compound 9]

[0086] Step (g) involves anomeric benzylation of a compound 8 to obtain a compound of Formula 9 as schematically presented below:

##STR00027##

[0087] Anomeric benzylation takes place by treating the compound 8 with alcohol while stirring. The reaction may be carried out in the presence of Lewis acid at high temperature Preferably in one embodiment stirring is done at 60 C. and evaporation are done at 50 C. In another embodiment reaction carried out in benzyl alcohol as solvent and Lewis acid is boron trifluoride dietherate. The reaction mixture is poured into Et.sub.2O and then stirred at 0 C. residue formed and filtered. Preferably the alcoholic solvent used is isopropanol. Further crystallization affords white crystalline solid. The solid obtained may be washed with an alcoholic solvent such as isopropanol and followed by diethyl ether.

[0088] Step-(h): Installing a 4,6-O-benzylidene protecting group [Compound 10].

[0089] Step (h) involves benzylidene protection of the compound of Formula 9 to obtain a compound of Formula 10 as schematically presented below;

##STR00028##

[0090] This benzylidene protection is performed by treating a compound with structural Formula-9 with dimethyl benzaldehyde acetal. The reaction can be performed in the presence of Organic acid. In one embodiment the Organic acid used is a p-toulene sulfonic acid.

[0091] The mixture stirred at room temperature for 4 to 8 hours, preferably for 6 hours and then stirred at rt, preferably dissolve a small amount of 5% EtOAc/n-Hexane followed by stirring at room temperature for 30 minutes at rt.

[0092] After proper stirring, the reaction residue filtered on Buchner funnel under continuous suction followed by washing with 5% EtOAc/n-Hexane of solvents, and water. The precipitate washed with water once under continuous suction until getting free white powder. The white powder is dissolved with EtOAc and slowly added n-Hexane until precipitate out filtered on a filter paper. After drying Benzyl 2-azido-4, 6-O-benzylidene-2-deoxy-D-glucopyranoside [Compound 10] is obtained. The drying can be carried out in vacuum at room temperature over phosphorous pentoxide (P.sub.2O.sub.5)

[0093] Step-(i): O-Alkylation [Compound 11].

[0094] Step-(i) involves O-alkylation of the compound of Formula 10 by treating with (S)-(2)-chloropropionic acid in presence of an appropriate solvent to obtain a compound 11

##STR00029##

[0095] The solvent in O-Alkylation step (i) is dry DMF. Any other similar inert high boiling solvents can also be used. The reaction can be performed in the presence of metal hydride such as sodium hydride, potassium hydride, calcium hydride etc. In one preferred embodiment, the metal hydride used in step-(i) is sodium hydride. Initially metal hydride is added at 0 C. in dry DMF under stirring for 10 minutes compound IV added slowly then after 30 min (S)-(2)-chloropropionic acid in dry DMF more diluted was added slowely through dropping funnel. After completion of reaction, the reaction mixture poured into crushed ice and adjusted ph 2-3 until forms precipitate then filtered it out. The solid compound recrystalized in 10% MeOH/DCM to give O-alkylated monosaccharide Compound 11 is obtained as white solid.

[0096] Step-(j): Peptide Coupling [Compound 12].

[0097] Step-(j) involves the coupling of peptides by treating the compound of Formula 11 with L-alanyl-D-isoglutamine benzyl ester trifluoroacetate to obtain a compound Formula 12 as schematically presented below;

##STR00030##

[0098] The coupling takes place under standard carbodiimide coupling conditions such as in the presence of 1-Ethyl-3-(-3-dimethyl aminopropyl) carbodiimide (EDCI) and Hydroxybenzotriazol (HOBt).Step-(j) can be performed in the presence of a base. In one embodiment the base is N.N-Diisopropylethylamine (DIPEA). The reaction mixture is stirred at RT for 12 to 18 hours, preferably 15 hours. After concentrating, the residue can be extracted with an organic solvent such as chloroform and followed by washing the organic layer with sat bicarbonate solution, dried and evaporated. The residue obtained can be re-crystalized in alcohol/DCM solvent such as DCM, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment 5% DCM/Methanol mixture is used for re-crystallization.

[0099] Step-(k): Azide reduction to amine [Compound 13].

[0100] Transformation of amine from azide by using compound of Formula 12 with triphenylphoshine in a suitable solvent system to obtain a compound of Formula 13 as schematically presented below;

##STR00031##

[0101] The Transformation of amine from azide carried out in presence of trialkylphoshine in THF/H.sub.2O in the portion of (3:1) then Transformation of amine from azide The reaction mixture is stirred at RT for 12 to 18 hours, preferably 16 hours. After concentrating, the residue can be dissolved in DCM and added Diethylether to afford compound VII; Step-(k) can be performed in the presence of Phosphine ligand.

[0102] In one embodiment the phoshine ligand can be triphenylphosphine (PPh.sub.3)

[0103] In one embodiment the reducing agents can be THF/H.sub.2O in the portion of (3:1)

[0104] Step-(l): general procedure for acid amine coupling

[0105] Step-(l) involves the coupling of alkyl or alicyclic carboxylic acid by treating the compound of Formula 13 with suitable coupling agent to obtain a compound of Formula 14 as schematically presented below;

##STR00032## [0106] wherein R.sub.1 is selected from substituted or unsubstituted (C.sub.3-C.sub.6) cycloalkyl, 3 to 6 membered heterocycloalkyl with one or more hetero atoms selected from Nitrogen and Oxygen or

##STR00033## [0107] wherein R.sub.2 is selected from group consisting of C.sub.1 to C.sub.18 aliphatic chain.

[0108] The coupling takes place under standard carbodiimide coupling conditions such as in the presence of 1-Propanephosphonic acid anhydride (T3P). Step-(1) can be performed in the presence of a base. In one embodiment the base is N.N-Diisopropylethylamine (DIPEA). The reaction mixture is stirred at RT for 12 to 18 hours, preferably 15 hours. After concentrating, the residue can be extracted with an organic solvent such as chloroform and followed by washing the organic layer with sat bicarbonate solution, dried and evaporated. The residue obtained can be recrystalized in solvent such as chloroform, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment chloroform-methanol mixture is used for recrystalization.

[0109] Step-(m): Removal of protection to obtain structural formula-I

[0110] Step-(m) involves hydrogenolysis De-protection is carried out by treating the compound of Formula 14 with acetic acid, water and EtOAc in suitable metal catalyst for removal of protecting groups to obtain the desired compound as represented by ##structural Formula-I ##.

##STR00034## [0111] wherein R.sub.1 is selected from substituted or unsubstituted (C.sub.3-C.sub.6) cycloalkyl, 3 to 6 membered heterocycloalkyl with one or more hetero atoms selected from Nitrogen and Oxygen or

##STR00035## [0112] wherein R.sub.2 is selected from group consisting of C.sub.1 to C.sub.18 aliphatic chain.

[0113] In one embodiment the acid used in step-(m) for deprotection is glacial acetic acid. The de-protection can be performed in the presence of a catalyst. In one embodiment the catalyst used in step-(m) is palladium black. To a solution of compound Formula-14 dissolved in acetic acid added with catalyst. And the compound subjected to hydrogenolysis for 12 h to 48 h.

[0114] The catalyst is filtered off, and, after addition of water, the filtrate is evaporated under diminished pressure by azeotroping with toluene. The residue can be dissolved in water and lyophilized. In one embodiment the residue is dissolved in a small volume of water and then applied to a column of Sephadex LH-20. The purified fractions can be lyophilized.

[0115] In a preferred embodiment of the present invention the following compounds of formula I are prepared wherein R.sub.1 is selected from 15b to 15i.

##STR00036##

EXAMPLES

[0116] The Muramyl Dipeptide compound its intermediates, the process for their synthesis and evaluation of these Muramyl Dipeptide compounds as an adjuvant are further explained and demonstrated by way of below non-limiting examples.

[0117] The reaction steps shown in above FIG-1 are further described in below experimental procedure step-a to step-f.

Example 1

[0118] The synthesis of the compound of formula I involves the following steps:

FIG. 1: Experimental Procedure for the Synthesis of L-Alanyl-D-Isoglutamine Benzyl Ester:

[0119] The novel Muramyl Dipeptide derivatives as described and disclosed in this invention contains an L-alanyl-D-isoglutamine dipeptide entity. The L-alanyl-D-isoglutamine benzyl ester required for the final preparation of novel Muramyl Dipeptide derivatives according to the present invention was synthesized by the method as shown in above reaction FIG. 1 and as described below

Step-a): Preparation of (R)-2-amino-5-(benzyloxy)-5-oxopentanoic acid [Compound 2]

[0120] A mixture of D-Glutamic acid [Compound 1](4.0 g, 27.2 mmols) and anhydrous. Na.sub.2SO.sub.4 (4.0 g) was suspended in benzyl alcohol (50 mL, 484 mmol) and BF.sub.3. Et.sub.2O (54%, 7.4 ml, 54.4 mmols) was added by means of a syringe. The suspension was stirred at RT for 14 hrs. The mixture was diluted with absolute THF (150 mL) and filtered with the aid of charcoal. The clear filtrate was treated with Et.sub.3N (8.2 mL, 59.2 mmols) and concentrated under vacuum until a slurry residue was formed.

[0121] The viscous residue was triturated with EtOAc (200 mL) and the precipitated solid was isolated by suction and washed with additional solvent to afford [compound 2] as a white solid (6.04 g, yield 93%, with respect to the starting material).

Step-b): Preparation of (R)-5-(benzyloxy)-2-(tert-butoxy carbonyl amino)-5-oxopentanoic acid [Compound 3]

[0122] To a solution of the [compound 2](6.0 g, 25.3 mmol) in dioxane and water (1:1, 40 mL) at 0 C. was added Boc.sub.2O (6.62 g, 30.36 mmol) and the mixture was stirred overnight (15 hours). The solvent was removed under reduced pressure and the residue was diluted with water (30 mL), basified with Na.sub.2CO.sub.3 and washed with EtOAc (3100 mL). The aqueous layer was adjusted to pH 2-3 with a 5M aqueous HCl solution and extracted with EtOAc (4100 mL). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure to afford (R)-5-(benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid [compound 3](8.19 g, 95%) as a viscous colourless oil. with the following NMR characteristics. 1H NMR (500 MHz, CDCl3) 7.42-7.25 (m, 5H), 5.25 (br, 1H), 5.12 (s, 2H), 4.32 (t, J=24.5 Hz, 1H), 2.57-2.42 (m, 2H), 2.29-2.19 (m, 1H), 2.02 (dd, 1H), 1.44 (s, 9H) ppm; ESI-MASS m/z: m/z Calculated 237, found 260 [M+Na]+.

Step-c): Preparation of (R)-benzyl 5-amino-4-((tert-butoxy carbonyl) amino)-5-oxopentanoate [Compound 4]

[0123] To a solution of above acid [Compound 3](7.0 g, 20.8 mmol) in THF (15 mL) were added ethyl chloroformate (2.7 mL, 28.36 mmol) and triethylamine (4.21 mL, 30.25 mmol) at 0 C. The reaction mixture was stirred at 0 C. for 0.5 hrs, then cooled to 15 C. followed by addition of a methanolic solution of ammonia (25 mL, 4.0 M) was added. Then the reaction mixture was stirred at 15 C. for another 1.5 hrs and diluted with ethyl acetate (200 mL). The organic phase was washed with water (100 mL3) and then followed by washing with brine solution (100 mL) and dried over anhydrous sodium sulphate. The solvent was removed in vacuum and the residue was purified by flash chromatography to furnish the desired [compound 4](6.63 g, 90%) as a white solid. characterized by NMR. 1H NMR (400 MHz, CDCl3) 7.39-7.29 (m, 5H), 6.35 (br, 1H), 5.72 (br, 1H), 5.11 (s, 2H), 3.7 (s, 2H), 2.62-2.40 (m, 2H), 2.31-1.94 (m, 2H), 1.43 (s, 9H) ppm; ESI-MASS m/z: Calculated 236, found 237 [M+H]+.

Step-d): (R)-benzyl 5-amino-4-((S)-2-((tert-butoxy carbonyl)amino) propanamido)-5-oxopentanoate [Compound 5]

[0124] t-Butoxycarbonyl-D-isoglutamine benzyl ester [Compound 4](6.5 g, 19.3 mmols) was dissolved in cold trifluoroacetic acid (4 mL in 16 mL DCM) and the resulting solution was stirred at room temperature for 15 mins. Trifluoroacetic acid was then removed and the residue was triturated with Et.sub.2O. This D-isoglutamine benzyl ester trifluoroacetate was dried over on KOH pellets followed by using trap.

[0125] To t-butoxycarbonyl-L-alanine (4.012 g, 21.23 mmols) in dry THF, EDCI (4.46 g, 23.36 mmols) and HOBt (3.57 g, 23.36 mmols). were added at 0 C. After stirring at RT for 30 mins, to this solution D-isoglutamine benzyl ester trifluoroacetate (dissolved in THF) was added followed by DIPEA (7.06 ml, 40.53 mmols). The reaction was stirred at RT for 15 hrs, the solution was then concentrated and the residue extracted with EtOAc (500 ml). The EtOAc layer was washed successively with 5% NaHCO.sub.3, 10% citric acid, and water (200 ml3), then dried over Na.sub.2SO.sub.4 and evaporated. The residue was triturated with petroleum ether giving crystals which were recrystallized from EtOAc-petroleum ether to yield [compound 5](6.37 g, yield 81%) as a white solid with the following NMR Characterization.

[0126] 1H NMR (400 MHz, CDCl3): 7.39-7.30 (m, 5H), 6.81 (br, 1H), 5.78 (br, 1H), 5.20 (d, J=3.2 Hz, 1H), 5.12 (ABq, J=12.3 Hz, 2H), 4.49 (dd, J=10.1, 7.6 Hz, 1H), 4.07 (dq, J=7.2, 3.2 Hz, 1H), 2.56 (dt, J=16.9, 7.2 Hz, 1H), 2.22 (m, 1H), 2.48 (dt, J=17.0, 6.7 Hz, 1H), 2.26-2.19, (m, 1H), 2.05-1.98 (m, 1H), 1.41 (s, 9H), 1.32 (d, J=7.1 Hz, 3H) ppm; ESI-MASS m/z: Calculated 407, found [M+Na]+430

[0127] Step-e): benzyl (R)-5-amino-4-((S)-2-aminopropanamido)-5-oxopentanoate trifluoroacetate [Compound 6](t-Butoxycarbonyl-L-alanyl-D-isoglutamine benzyl ester (25 g, 84.2 mmols) was dissolved in cold trifluoroacetic acid (20 mL) and DCM (80 mL) the resulting solution was stirred at room temperature for 30 mins. Trifluoroacetic acid was then removed by making azeotropic mixture with touelene and the residue was triturated with Et.sub.2O to obtain L-alanyl-D-isoglutamine benzyl ester trifluoroacetate [compound 6]. This L-alanyl-D-isoglutamine benzyl ester trifluoroacetate was dried over KOH pellets followed by using a trap. To afford oily compound 6 following NMR Characterization.

FIG. 2:

[0128] The synthesized peptide fragment of compound 6 as shown in example-1 used to couple with Sugar fragment of compound 11 which has shown in FIG. 2

Step-f): Preparation of (3R,4R,5S,6R)-3-azido-6-(hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triol (8)

[0129] A typical experimental procedure for the preparation of triflyl azide was as following: To the mechanically stirred suspension of sodium azide (43.6 g, 67.0 mmol) in 600 mL of acetonitrile was cooled in ice bath. Then triflic anhydride (157 g, 55.6 mmol) was added to the mixture through addition funnel during 45 min while stirring. After the reaction was maintained for 2 h in ice bath, the TfN.sub.3-containing solution (filtration of the salts can be done if necessary) was added directly to the mechanically stirring solution of amine solution for the subsequent diazotransfer reaction.

[0130] General procedure for the reaction of triflyl azide with amines. For organic soluble substrates, 100 g of [Compound 7] was dissolved in 250 mL of acetonitrile. In case of saline substrates, water was employed instead. Then 1% equiv of CuSO.sub.4 and 2 equiv of NEt.sub.3 per substrate amine were added into the solution while stirring. The mixture was cooled in an ice bath for a while, acetonitrile solution of triflyl azide (1.2 equiv per amino group, based on the amount of triflic anhydride used in the preparation of TfN.sub.3) was then added into the mixture dropwise. The reaction mixture was allowed to warm to room temperature. Generally speaking, a homogeneous solution could be obtained after the addition of triflyl azide, and the reaction normally went to completion within 12 h. The solvent was removed under reduced pressure. The residue was Recrystallized by dissolving in DCM then poured into n-hexane to afford (3R,4R,5S,6R)-3-azido-6-(hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triol [Compound 8](108 g, 94%) as white solid characterized by 1H-NMR and MASS-ESI 1H NMR (500 MHz, CD3OD) 1H NMR (500 MHz, CDCl3) 5.06-5.01 (d, J=3.7 Hz, 1H, anomeric) 4.77-4.72 (d, J=12.0 Hz, 1H), 4.62 (d, J=12.0 Hz, 1H), 4.32-4.20 (m, 2H), 3.91 (m, 1H), 3.74-3.71 (t, J=10.4 Hz, 1H), 3.54-3.50 (t, J=9.3 Hz, 1H) ppm; ESI-MS: m/z Calcd. for C.sub.6H.sub.11N.sub.3O.sub.5 205 [M+Na]+: 228:

Step-g): Preparation of (2R,3S,4R,5R,6S)-5-azido-6-(benzyloxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol [Compound 9]

Anomeric benzylation of 2-azido D-Glucosamine (8)

[0131] To the mechanically stirred suspension of 2-azido D-glucosamine [compound 8](85 g, 0.39 mmol) and Acetyl chloride (27.4 mL, 0.39 mmol) was slowly added to a in anhydrous benzyl alcohol (200 mL) under nitrogen atmosphere. The suspension was stirred at room temperature for 2 h, heated to 50 C. for 8h, and then cooled to room temperature. The resulting yellow solution was slowly poured onto Et.sub.2O (3L) in ice-water bath, and the mixture was vigorously stirred for 2 h at 0 C. The precipitate was recovered by filtration and dried under vacuum to afford (2R, 3S,4R,5R,6S)-5-azido-6-(benzyloxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol [Compound 9](118 g, 96%) as a white solid characterized by 1H-NMR and MASS-ESI 1H NMR (500 MHz, CD3OD) 1H NMR (500 MHz, CDCl3) 7.51-7.46 (m, 2H), 7.43-7.31 (m, 3H), 5.00-4.95 (d, J=3.7 Hz, 1H, anomeric), 4.77-4.73 (d, J=12.0 Hz, 1H), 4.62 (d, J=12.0 Hz, 1H), 4.32-4.20 (m, 2H), 3.91 (m, 1H), 3.74 (t, J=10.4 Hz, 1H), 3.54 (t, J=9.3 Hz, 1H), 3.31-3.29 (dd, J=10.0, 3.7 Hz, 1H) ppm; ESI-MASS m/z: m/z Calculated. C.sub.13H.sub.17N.sub.3O.sub.5 295 [M+Na]+318 [M+K]+; found: 334.12

Step-h): Preparation of (2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-ol [Compound 10]

Benzylidene protection of compound (9)

[0132] (2R,3S,4R,5R,6S)-5-azido-6-(benzyloxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol [Compound 9](80 g, 0.271 mol) was added to a mechanically stirred mixture of dimethyl benzaldehyde acetal (16.5 ml, 1.35 mol in dry ACN). and PTSA (10 g, 0.27 mol) were added then This mixture was stirred at room temperature for 2 hours, then stirred at rt for 2 hours to dissolve a small amount of remaining solid, then stirred at room temperature for 6 hours. The well-stirred reaction solution was now diluted with petroleum ether (300 ml), absolute ethanol (EtOH) for an amount of 100 ml) and 45 ml of water (H.sub.2O). The reaction mixture was stirred for 1 day at room temperature, then stored at 0 C. for 2 days. The curdy white precipitate so formed was collected on a coarse glass frit funnel, drained thoroughly, then washed by re-suspension in absolute EtOH (approx. 100 ml). The white finely divided solid was drained thoroughly, re-suspended in diethyl ether (approx. 500 ml), re-drained thoroughly, and then dried in vacuum at room temperature over phosphorus pentoxide (P.sub.2O.sub.5) to get (2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-ol [Compound 10](99 g, yield 95%) as a white solid characterized by 1H-NMR and MASS-ESI 1H NMR (500 MHz, CDCl3) 1H NMR (500 MHz, CDCl3) 7.51-7.46 (m, 2H), 7.43-7.31 (m, 8H), 5.54 (s, 1H), 5.00 (d, J=3.7 Hz, 1H, anomeric), 4.77 (d, J=12.0 Hz, 1H), 4.62 (d, J=12.0 Hz, 1H), 4.32-4.20 (m, 2H), 3.91 (m, 1H), 3.74 (t, J=10.4 Hz, 1H), 3.54 (t, J=9.3 Hz, 1H), 3.31 (dd, J=10.0, 3.7 Hz, 1H), 2.69 (s, 1H) ppm; ESI-MS: m/z Calcd. for C.sub.20H.sub.21N.sub.3O.sub.5[M 30+H]+: 384.

Step-i): preparation of (R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid [Compound 11]

O-Alkylation: of compound (10)

[0133] To the mechanically stirred solution of Sodium hydride 60% dispersion in mineral oil, (50 g, 0.130 mol) was washed with hexanes (50 mL) three time to remove mineral oil and was suspended in 1Ltr anhydrous DMF under argon. [Compound 10](35 g, 1.56 mol) was added into the above solution slowly, followed by (S)-2-chloropropanoic acid (2.2 mL, 20.5 mmol) added dropwise over 30 min through addition funnel in 150 mL anhydrous DMF The resulting mixture was stirred vigorously at room temperature until there was no gas evolution. Then it was stirred at rt for 14 h stirring). The reaction mixture was cooled down to room temperature, after completion of reaction quenched with 200 mL of DI water and acidified with 1N HCl to pH=3. It forms precipitate then filtered and washed with 30% ethyl acetate/n-Hexane (21Ltr), and dried under continuous vacuum until dry to afforded product solid (R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid [Compound 11](54 g, 91%) as an off white solid characterized by 1H-NMR and MASS-ESI; .sup.1H NMR (500 MHz, CDCl3): 7.50-7.41 (m, 2H), 7.41-7.31 (m, 8H), 5.56 (s, 1H), 5.06 (d, J=3.7 Hz, 1H, anomeric), 4.75 (d, J=11.9 Hz, 1H), 4.62 (d, J=11.9 Hz, 1H), 4.50 (q, J=13.9, 6.9 Hz, 1H), 4.23 (dd, J=10.3, 4.8 Hz, 1H), 4.05 (t, J=9.5 Hz, 1H), 3.90 (m, 1H), 3.74 (t, J=10.3 Hz, 1H), 3.64 (t, J=9.3 Hz, 1H), 3.42-3.38 (m, 1H), 1.47 (d, J=6.9 Hz, 3H) ppm; ESI-MASS: m/z Calcd. for C.sub.23H.sub.25N.sub.3O.sub.7 [M+H]+: 456.

Step-j): (R)-benzyl-5-amino-4-((S)-2-((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate [compound 12]

Peptide Coupling of Azido Muramic Acid (11)

[0134] Compound 6 (25 g) dissolved in THF (100 mL) with DIPEA (22 mL 123 mmol) was treated with Compound 11 (30 g, 61.9 mmol) in dry THF (500 mL) and EDC-HCl (1.27 g, 92.9 mmol). A catalytic amount of HOBt was added to the solution. The solution was stirred 8 h at room temperature. After the complete consumption of the starting material, the solvent was removed and the residue was extracted with EtOAc (500 mL) and washed with 1N HCl (250 mL2) and saturated NaHCO.sub.3 (250 mL2) then brine (500 mL2). The extract was dried over Na.sub.2SO.sub.4, concentrated, filtered, and recrystalized in 5% DCM/Methanol into give (R)-benzyl-5-amino-4-((S)-2-((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate [Compound 12](38 g, 93%) as a white solid. .sup.1H NMR (400 MHz, CDCl3): 7.65 (t, J=8.8 Hz, 1H), 7.46-7.40 (m, 2H), 7.40-7.30 (m, 12H), 7.15 (d, J=8.0 Hz, 1H), 6.79 (s, 1H), 5.55 (s, 1H), 5.45 (s, 1H), 5.04 (d, J=2.4 Hz, 2H, anomeric), 4.76 (d, J=10.0 Hz, 1H), 4.62 (d, J=11.7 Hz, 1H), 4.46 (td, J=8.3, 4.6 Hz, 1H), 4.31-4.19 (m, 3H), 3.96-3.85 (m, 2H), 3.75 (t, J=10.3 Hz, 1H), 3.61 (t, J=9.3 Hz, 1H), 3.41 (dd, J=10.1, 3.7 Hz, 1H), 2.58 (m, 1H), 2.45 (m, 1H), 2.27-2.16 (m, 1H), 2.02 (m, 1H), 1.39 (d, J=7.0 Hz, 3H), 1.35 (d, J=6.7 Hz, 3H) ppm; ESI-MASS: m/z Calcd. for C.sub.38H.sub.44N.sub.6O.sub.10 [M+H].sup.+: 745.

[0135] Step-k):(R)-benzyl-5-amino-4-((S)-2-((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-amino-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopetanoate (13): To the mechanically stirred solution of [compound 12](20 g, 9.41 mmol) in tetrahydrofuran (200 mL), was added triphenylphosphine (8.4 g, 32.35 mmol) and water (70 mL). The reaction mixture was stirred at 60 C. for 16 hours. After the completetion of the starting material, solvent was removed and the residue was extracted with CH.sub.2Cl.sub.2 (2100 mL) and brine (50 mL2). The extract was dried over MgSO.sub.4, concentrated, and disolved in DCM then diethyl ether were added till precipitate out the compound followed by decanting excess triphenylphoshine decanted which disolved in diethyl ether and smae repeated once to give white solid, benzyl (R)-4-((S)-2-((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-alkaamido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-amino-5-oxopentanoate to afford [Compound 13](17.8 g, 92%) as a white solid.characterized by .sup.1HNMR, MASS-ESI .sup.1H NMR (500 MHz, CDCl.sub.3): 7.39-7.33 (m, 2H), 7.34-7.23 (m, 15H), 7.04 (d, J=8.1 Hz, 1H), 6.74 (s, 1H), 5.50 (s, 2H), 5.23 (s, 1H), 5.02 (d, J=7.2 Hz, 2H), 4.78 (d, J=4.0 Hz, 1H), 4.68 (d, J=11.7 Hz, 2H), 4.44 (d, J=11.7 Hz, 1H), 4.39 (m, 1H), 4.23 (m, 1H), 4.17 (m, 1H), 3.76 (m, 1H), 3.67 (t, J=10.2 Hz, 1H), 3.59-3.50 (m, 3H), 2.49 (m, 1H), 2.39 (m, 1H), 2.14 (m, 1H), 1.95 (m, 1H), 1.34 (d, J=7.0 Hz, 3H), 1.29 (d, J=7.0 Hz, 3H) ppm; ESI-MASS: m/z Calcd. for C.sub.38H.sub.46N.sub.4O.sub.10 718 [M+H].sup.+: 719.

The Synthesized Compound 13 as Shown in Example-1 (FIG. 2) Used to Coupled with Cycloalkyl Carboxylic Acid or Mixed Ester Carboxylic Acid to Obtain Compounds (14a to 14h) which as Described in Step-l Given Below

Step-1): General Procedure for Acid Amine Coupling

[0136] A mechanically stirred solution of cyclo alkyl carboxylic acid or mixed ester carboxylic acid (13 g) in anhydrous tetrahydrofuran (100 mL) T3P (7.5 mL, 25 mmol and DIPEA (9 mL, 50.4 mmol) were added and stirred 30 min at 0 C. and compound 13 (12 g) in (50 mL) anhydrous THF were added through addition funnel to the stirring reaction mixture then keep it stirring at RT for 14 h. After the consumption of the starting material, solvent was removed and the residue was extracted with EtOAc (150 mL) and washed with 1N HCl (20 mL2) then brine (wash 50 mL2). The extract was dried over MgSO.sub.4, concentrated, and recrystalized in (5% MeOH in DCM) to give benzyl(R)-4-((S)-2-((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-alkaamido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-amino-5-oxopentanoate to afford (14a to 14h) in the ranges of the yields (90-95%) as white solid compounds.

[0137] i).benzyl(R)-4-((S)-2-((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7-acetamido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-amino-5-oxopentanoate (14a 11.8 g, 92%) as a white solid characterized by .sup.1HNMR, MASS-ESI .sup.1H NMR (300 MHz, CDCl3): 7.45-7.37 (m, 2H), 7.34-7.22 (m, 13H), 5.52 (s, 1H), 5.04 (s, 2H), 4.94-4.87 (d, J=3.7 Hz, 1H), 4.49-4.32 (dd, J=57.8, 11.9 Hz, 1H), 4.30 (dd, J=8.9, 4.7 Hz, 1H), 4.22-4.03 (m, 3H), 3.83-3.53 (m, 3H), 3.29 (s, 1H), 2.34-2.31 (s, 3H), 2.29-2.21 (m, 2H), 2.12 (dt, J=12.4, 7.6 Hz, 1H), 1.84 (dt, J=14.2, 8.4 Hz, 1H), 1.41 (td, J=7.8, 4.0 Hz, 1H), 1.39-1.27 (dd, 6H) ppm; ESI-MASS: m/z Calcd. for C.sub.40H.sub.48N.sub.4O.sub.11 760.33 found [M+Na].sup.+: 783.

[0138] ii). 2.7.2: benzyl (4R)-5-amino-4-((2S)-2-((2R)-2-(((2R,4aR,7R,8R,8aS)-(benzyloxy)-7-(cyclopropanecarboxamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate (14b, 12.4 g, 94%) as a white solid characterized by 1H NMR, MASS-ESI, 1H NMR (300 MHz, CDCl3) 7.48-7.37 (m, 2H), 7.36-7.20 (m, 13H), 5.56-5.50 (s, 1H), 5.10-5.02 (s, 2H), 4.98-4.93 (d, J=3.7 Hz, 1H), 4.72-4.62 (d, J=11.9 Hz, 1H), 4.52-4.44 (d, J=11.9 Hz, 1H), 4.36-4.27 (dd, J=8.9, 4.7 Hz, 1H), 4.24-4.06 (m, 3H), 3.86-3.52 (m, 5H), 2.45-2.28 (m, 2H), 2.24-2.06 (m, 1H), 1.96-1.75 (m, 1H), 1.50-1.37 (m, 1H), 1.35-1.20 (dd, J=10.9, 9.7 Hz, 6H), 0.87-0.78 (m, 2H), 0.75-0.62 (m, 2H) ppm; ESI-MASS: m/z Calcd. for C.sub.38H.sub.46N.sub.4O.sub.11 786. found [M+H].sup.+ 787, [M+Na].sup.+809

[0139] iii). benzyl (4R)-5-amino-4-((2S)-2-((2R)-2-(((2R,4aR,7R,8R,8aS)-6-(benzyloxy)-7-(cyclobutanecarboxamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate (14c, 12.5 g, 93%) as a white solid characterized by 1HNMR, MASS-ESI, 1H NMR (300 MHz, CDCl3) 7.50-7.38 (m, 2H), 7.35-7.21 (m, 13H), 5.55-5.49 (s, 1H), 5.08-5.03 (s, 2H), 4.99-4.94 (d, J=3.7 Hz, 1H), 4.72-4.61 (d, J=11.9 Hz, 1H), 4.51-4.43 (d, J=11.9 Hz, 1H), 4.37-4.28 (dd, J=8.9, 4.7 Hz, 1H), 4.25-4.05 (m, 3H), 3.87-3.51 (m, 5H), 3.20-3.15 (m, 1H), 2.46-2.27 (m, 2H), 2.25-2.07 (m, 1H), 1.95-1.74 (m, 1H), 1.42-1.25 (m, 10H), 0.92-0.77 (m, 2H) ppm; ESI-MASS: m/z Calcd. for. C.sub.43H.sub.52N.sub.4O.sub.11, 800.36 found [M+H].sup.+ 801., [M+Na].sup.+824

[0140] iv). benzyl (4R)-5-amino-4-((2S)-2-((2R)-2-(((2R,4aR,7R,8R,8aS)-6-(benzyloxy)-7-(cyclopentanecarboxamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate (14d, 13 g, 95%) as a white solid characterized by 1HNMR, MASS-ESI, 1H NMR (400 MHz, CDCl3) 7.52-7.36 (m, 4H), 7.34-7.19 (m, 11H), 5.54-5.49 (s, 1H), 5.09-5.02 (s, 2H), 4.98-4.95 (d, J=3.7 Hz, 1H), 4.73-4.60 (d, J=11.9 Hz, 1H), 4.54-4.42 (d, J=11.9 Hz, 1H), 4.36-4.27 (dd, J=8.9, 4.7 Hz, 1H), 4.27-4.04 (m, 3H), 3.88-3.50 (m, 5H), 2.47-2.26 (m, 3H), 2.27-2.06 (m, 1H), 1.94-1.75 (m, 1H), 1.45-1.27 (m, 10H), 1.15-0.79 (m, 4H) ppm; ESI-MASS: m/z Calcd. for. C.sub.44H.sub.54N.sub.4O.sub.11, 814.38, found [M+H].sup.+ 815,[M+K].sup.+854.

[0141] v). benzyl (4R)-5-amino-4-((2S)-2-((2R)-2-(((2R,4aR,7R,8R,8aS)-6-(benzyloxy)-7-(cyclohexanecarboxamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate (14e, 13.1 g, 94%) as a white solid characterized by 1H NMR (500 MHz, CDCl3) 7.51-7.37 (m, 4H), 7.34-7.19 (m, 11H), 5.53-5.48 (s, 1H), 5.08-5.01 (s, 2H), 4.97-4.95 (d, J=3.7 Hz, 1H), 4.72-4.61 (d, J=11.9 Hz, 1H), 4.53-4.42 (d, J=11.9 Hz, 1H), 4.35-4.28 (dd, J=8.9, 4.7 Hz, 1H), 4.25-4.03 (m, 4H), 3.89-3.49 (m, 4H), 2.46-2.25 (m, 3H), 2.27-2.06 (m, 1H), 1.94-1.79 (m, 1H), 1.74-1.51 (m, 4H), 1.47-1.29 (m, 10H), 1.18-0.90 (m, 2H) ppm; ESI-MASS: m/z Calcd. for. C.sub.45H.sub.56N.sub.4O.sub.11, 828.39, found [M+H].sup.+ 829.20, [M+Na].sup.+852

[0142] vi). 8-(((2R,4aR,7R,8R,8aS)-8-(((R)-1-(((S)-1-(((R)-1-amino-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)oxy)-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl)amino)-8-oxooctyl tetradecanoate (14f, 16.5 g, 92%) as a white solid characterized by 1HNMR, MASS-ESI, 1H NMR (500 MHz, CDCl3): 7.86 (s, 1H), 7.45-7.40 (m, 3H), 7.37-7.27 (m, 12H), 7.13 (d, J=6.1 Hz, 1H), 6.90 (s, 1H), 6.29 (d, J=8.8 Hz, 1H), 5.54 (s, 1H), 5.08 (s, 2H), 4.93 (d, J=3.8 Hz, 1H), 4.71 (d, J=11.7 Hz, 1H), 4.51-4.43 (m, 2H), 4.30 (m, 1H), 4.23 (dd, J=10.2, 4.7 Hz, 1H), 4.15 (t, J=6.7 Hz, 1H), 4.04 (t, J=6.7 Hz, 2H), 3.86 (m, 1H), 3.76 (t, J=10.2 Hz, 1H), 3.67 (m, 1H), 2.56 (m, 1H), 2.45 (m, 1H), 2.27 (t, J=7.5 Hz, 2H), 2.21 (m, 1H), 2.16-2.08 (m, 2H), 2.02 (m, 1H), 1.62-1.57 (m, 3H), 1.53 (m, 1H), 1.38 (d, J=7.0 Hz, 3H), 1.34 (d, J=6.7 Hz, 3H), 1.30-1.23 (m, 28H), 0.88 (t, J=6.9 Hz, 3H) ppm; ESI-MASS: m/z Calcd. for. C.sub.60H.sub.86N.sub.4O.sub.13 1070.61 found [M+H].sup.+ 1071.

[0143] vii).8-(((2R,4aR,7R,8R,8aS)-8-(((R)-1-(((S)-1-(((R)-1-amino-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)oxy)-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl)amino)-8-oxooctyl dodecanoate (14 g, 15.8 g, 90%) as a white solid characterized by 1HNMR, MASS-ESI, 1H NMR (500 MHz, CDCl3): 7.86 (s, 1H), 7.45-7.38 (m, 3H), 7.38-7.27 (m, 14H), 7.12 (d, J=6.1 Hz, 1H), 6.91 (s, 1H), 6.29 (d, J=8.8 Hz, 1H), 5.54 (s, 1H), 5.08 (s, 2H), 4.93 (d, J=3.8 Hz, 1H), 4.71 (d, J=11.7 Hz, 1H), 4.49 (d, J=11.7 Hz, 1H), 4.30 (m, 1H), 4.24 (dd, J=10.2, 4.7 Hz, 1H), 4.16 (t, J=6.7 Hz, 1H), 4.04 (t, J=6.7 Hz, 2H), 3.87 (m, 1H), 3.77 (t, J=10.2 Hz, 1H), 3.72-3.62 (m, 2H), 2.57 (m, 1H), 2.46 (m, 1H), 2.28 (t, J=7.5 Hz, 2H), 2.21 (m, 1H), 2.17-2.07 (m, 2H), 2.02 (m, 1H), 1.63-1.56 (m, 3H), 1.54-1.51 (m, 1H), 1.39-1.37 (d, J=7.0 Hz, 3H), 1.35-1.33 (d, J=6.7 Hz, 3H), 1.30-1.24 (m, 24H), 1.11-0.88 (t, J=6.9 Hz, 3H) ppm; ESI-MASS: m/z Calcd. for.C.sub.58H.sub.82N.sub.4O.sub.13 1043 [M+H].sup.+:1044

[0144] viii).8-(((2R,4aR,7R,8R,8aS)-8-(((R)-1-(((S)-1-(((R)-1-amino-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)oxy)-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl)amino)-8-oxooctyl octanoate (14h, 15.3 g, 93%) as a white solid characterized bym HNMR, MASS-ESI, 1H NMR (500 MHz, CDCl3): 7.47-7.42 (m, 3H), 7.39-7.29 (m, 14H), 7.16 (d, J=7.9 Hz, 1H), 7.10 (d, J=6.0 Hz, 1H), 6.89 (s, 1H), 6.13 (d, J=9.2 Hz, 1H), 5.56 (s, 1H), 5.35 (s, 1H), 5.10-5.09 (s, 2H), 4.91 (d, J=3.8 Hz, 1H), 4.73 (d, J=11.7 Hz, 1H), 4.49 (d, J=11.6 Hz, 1H), 4.31 (m, 1H), 4.24 (dd, J=10.0, 4.5 Hz, 1H), 4.11 (m, 1H), 4.01 (m, 1H), 3.87 (m, 1H), 3.78 (d, J=10.1 Hz, 1H), 3.69-3.64 (m, 2H), 2.57 (m, 1H), 2.46 (m, 1H), 2.27-1.97 (m, 6H), 1.66 (s, 3H), 1.58-1.51 (m, 4H), 1.39 (d, J=7.1 Hz, 3H), 1.35 (d, J=6.7 Hz, 2H), 1.27-1.24 (m, 15H), 1.10-0.88 (t, J=6.6 Hz, 3H) ppm; ESI-MASS: m/z Calcd. for.C.sub.54H.sub.74N.sub.4O.sub.13 986 [M+H].sup.+: 987.

The Synthesized Compound 14a to 14h as Shown in Example-1 (FIG. 2) Used to Hydrogenolysis to Obtain Compounds (15a to 15i) as Shown Below in Step-m

Step-m): General Procedure for Hydrogenolysis

[0145] To a stirred solution of [compound 14](10 g, 1.65 mmol), THF (50 mL), acetic acid (20 mL) and water (100 mL) was added 10% Pd/C (3.5 g) added as catalyst. The resulting mixture was hydrogenated at room temperature by using a hydrogen-reacter for 16 h and filtered through a celite cake. After evaporation followed by azotropization then the residue was disolved in HPLC MeOH and diethyl ether were added untill precipitate stirred for 30 min to free of then ether decanted the process repeated once to afford compounds 15a to 15i in the ranges of yields (90-95%) as a white solid compounds

[0146] Example-2: The synthesis of the title compound (4R)-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-amino-5-oxopentanoic acid (15a, 5.9 g, 92%) given as example 2 involves all the steps for the synthesis of peptide as given in FIG. 1 of example 1 and the specific sugar fragment prepared as explained in FIG. 2 of example 1 and their successive acid amine coupling and deprotection as given under example 1 and the final compound was obtained as a white solid characterized by .sup.1HNMR, MASS-ESI, .sup.1H NMR (400 MHz, D2O): 5.11 (d, J=3.2 Hz, 1H), 4.51-4.49 (d, J=8.0 Hz, 1H), 4.42-4.31 (dd, J=13.4, 6.7 Hz, 1H), 4.20-4.12 (m, 2H), 3.79-3.54 (m, 4H), 3.45-3.32 (m, 1H), 2.32-2.27 (s, 3H), 2.24-2.21 (m, 2H), 2.15-1.90 (m, 1H), 1.89 (d, J=25.3 Hz, 1H), 1.59-1.48 (d, 3H), 1.33-1.27 (d, 3H) ppm; ESI-MASS: m/z Calcd. for C.sub.19H.sub.32N.sub.4O.sub.11 492.3 [M+Na].sup.+: 515.3.

[0147] Example-3: (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R, 5S, 6R)-3-(cyclopropanecarboxamido)-2, 5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (15b, 6.2 g, 94%) as a white solid characterized by .sup.1H NMR, MASS-ESI, .sup.1H NMR (400 MHz, CD3OD) 5.13-5.08 (d, J=3.2 Hz, 1H), 4.52-4.48 (d, J=8.0 Hz, 1H), 4.45-4.37 (dd, J=13.4, 6.7 Hz, 1H), 4.24-4.12 (m, 2H), 3.78-3.57 (m, 4H), 3.42-3.32 (m, 1H), 2.31-2.17 (m, 1H), 2.14-1.99 (m, 1H), 1.90-1.77 (m, 2H), 1.60-1.47 (m, 1H), 1.40-1.25 (m, 6H), 0.84-0.71 (m, 2H), 0.69-0.61 (m, 2H) ppm; ESI-MS: m/z Calcd for C.sub.21H.sub.34N.sub.4O.sub.11 518.32, found [M+H].sup.+ 519, [M+Na].sup.+541. [M+K].sup.+557.

[0148] Example-4: (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(cyclobutanecarboxamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (15c, 6.1 g, 92%) as a white solid characterized by 1H NMR, MASS-ESI, .sup.1H NMR (400 MHz, CD3OD) 5.12-5.07 (d, J=3.2 Hz, 1H), 4.51-4.47 (d, J=8.0 Hz, 1H), 4.44-4.36 (dd, J=13.4, 6.7 Hz, 1H), 4.25-4.11 (m, 2H), 3.77-3.56 (m, 4H), 3.41-3.33 (m, 1H), 3.15-3.13 (m, 1H), 2.31-2.16 (m, 1H), 2.15-1.98 (m, 1H), 1.91-1.76 (m, 2H), 1.62-1.48 (m, 1H), 1.41-1.23 (m, 10H), 0.90-0.75 (m, 2H) ppm; ESI-MS: m/z Calcd for C.sub.22H.sub.36N.sub.4O.sub.11 532.24, found [M+H].sup.+ 534.

[0149] Example-5: (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(cyclopentanecarboxamido)-2, 5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (15d, 6.4 g, 95%) as a white solid characterized by .sup.1H NMR, MASS-ESI, .sup.1H NMR (400 MHz, CD3OD) 5.11-5.06 (d, J=3.2 Hz, 1H), 4.51-4.46 (d, J=8.0 Hz, 1H), 4.43-4.35 (dd, J=13.4, 6.7 Hz, 1H), 4.24-4.11 (m, 2H), 3.76-3.55 (m, 4H), 3.40-3.32 (m, 1H), 2.55-2.35 (m, 2H), 2.30-2.25 (m, 2H), 2.14-2.10 (m, 3H), 2.05-1.47 (m, 6H), 1.44-1.25 (m, 6H), ppm; ESI-MS: m/z Calcd for C.sub.23H.sub.38N.sub.4O.sub.11 546.25, found [M+K].sup.+584.

[0150] Example-6: (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(cyclohexanecarboxamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (15e, 6.3 g, 93%) given as example 2 involves all the steps for the synthesis of peptide as given in FIG. 1 of example 1 and the specific sugar fragment prepared as explained in FIG. 2 of example 1 and their successive acid amine coupling and deprotection as given under example 1 and the final compound was obtained as a white solid characterized by .sup.1H NMR, MASS-ESI, .sup.1H NMR (400 MHz, CD3OD) 5.10-5.05 (d, J=3.2 Hz, 1H), 4.51-4.45 (d, J=8.0 Hz, 1H), 4.42-4.34 (dd, J=13.4, 6.7 Hz, 1H), 4.25-4.10 (m, 2H), 3.75-3.54 (m, 4H), 3.41-3.31 (m, 1H), 2.52-2.45 (m, 2H), 2.34-2.25 (m, 2H), 2.16-1.98 (m, 2H), 1.95-1.74 (m, 3H), 1.71-1.49 (m, 6H), 1.50-1.25 (m, 6H), 1.17-1.10 (m, 2H) ppm; ESI-MS: m/z Calcd for C.sub.24H.sub.40N.sub.4O.sub.11 560.27, found, [M+K].sup.+599.

[0151] Example-7:(4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(8-(tetradecanoyloxy)octanamido)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (15f, 7 g, 93%) given as example 2 involves all the steps for the synthesis of peptide as given in FIG. 1 of example 1 and the specific sugar fragment prepared as explained in FIG. 2 of example 1 and their successive acid amine coupling and deprotection as given under example 1 and the final compound was obtained as a white solid characterized by .sup.1HNMR, MASS-ESI, .sup.1H NMR (500 MHz, D.sub.2O): 7.53 (br, 1H), 5.15-5.08 (m, 1H), 4.55-4.44 (m, 2H), 4.37-4.16 (m, 3H), 4.01-3.45 (m, 5H), 2.57-2.31 (m, 6H), 2.22-2.08 (m, 2H), 1.99-1.78 (m, 4H), 1.75-1.53 (m, 2H), 1.46-1.26 (m, 34H), 1.16 (t, J=7.1 Hz, 3H) ppm; ESI-MS: m/z Calcd. for C.sub.39H.sub.70N.sub.4O.sub.13 802.5 found [M+H].sup.+ 803.6, [M+Na].sup.+825.7

[0152] Example-8: (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-(8-(dodecanoyloxy)octanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (15 g, 6.8 g, 91%) given as example 2 involves all the steps for the synthesis of peptide as given in FIG. 1 of example 1 and the specific sugar fragment prepared as explained in FIG. 2 of example 1 and their successive acid amine coupling and deprotection as given under example 1 and the final compound was obtained as a white solid characterized by: .sup.1H NMR (500 MHz, CD.sub.3OD): 5.21-5.08 (m, 1H), 4.56-4.42 (m, 3H), 4.38-3.99 (m, 7H), 2.58-2.10 (m, 8H), 1.74-1.25 (m, 34H), 1.10-0.88 (t, J=7.2 Hz, 3H) ppm; ESI-MASS: m/z Calcd. for C.sub.37H.sub.66N.sub.4O.sub.13 774 [M+H].sup.+: 775.

[0153] Example-9: (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(8-(octanoyloxy)octanamido)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (15h, 6.5 g, 90%) given as example 2 involves all the steps for the synthesis of peptide as given in FIG. 1 of example 1 and the specific sugar fragment prepared as explained in FIG. 2 of example 1 and their successive acid amine coupling and deprotection as given under example 1 and the final compound was obtained as a white solid characterized by: .sup.1H NMR (500 MHz, CD.sub.3OD): 5.28-5.10 (m, 1H), 4.56-4.26 (m, 4H), 4.39-3.85 (m, 6H), 2.58-2.10 (m, 8H), 1.75-1.08 (m, 26H), 1.12-0.87 (t, J=7.1 Hz, 3H) ppm; ESI-MASS: m/z Calcd. for C.sub.33H.sub.58N.sub.4O.sub.13 718 [M+H].sup.+: 719.

[0154] Example-10:(4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(tetrahydrofuran-2-carboxamido)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid(15i, 5.2 g, 91%) given as example 2 involves all the steps for the synthesis of peptide as given in FIG. 1 of example 1 and the specific sugar fragment prepared as explained in FIG. 2 of example 1 and their successive acid amine coupling and deprotection as given under example 1 and the final compound was obtained as a white solid characterized by: .sup.1H NMR (300 MHz, CD.sub.3OD) 5.14-5.06 (d, J=3.2 Hz, 1H), 4.78-4.62 (m, 1H), 4.58-4.45 (d, J=11.9 Hz, 1H), 4.42-4.23 (m, 3H), 4.15-4.06 (m, 1H), 3.86-3.78 (m, 5H), 3.58-3.41 (m, 2H), 2.44-2.27 (m, 2H), 2.23-2.05 (m, 3H), 1.97-1.74 (m, 3H), 1.44-1.23 (dd, J=10.9, 9.7 Hz, 6H); ESI-MASS: m/z Calcd. for C.sub.19H.sub.32N.sub.4O.sub.11 546.3 [M+Na].sup.+: 469.3

Example-11 Biological Activity: Evaluation of Adjuvant activity

Sources of Biologicals

[0155] Balb-C mice and cell lines for in vivo and in vitro immunopharmacological assay were purchased from the commercial sources. IAEC permission has been obtained for the procurement and use of animals

TABLE-US-00001 Name of the biological resource Source of access Name Contact Details Balb-C Mice Centre for Balb-C Mice Centre for Cellular & Molecular Cellular & IAEC IICT/66/2016 Biology Habsiguda, Metro Molecular Station, , India. Contact: Animal Biology, House, CSIR-CCMB, Tarnaka, Hyderabad, Hyderabad 500007. CPCSEA Reg. India No. 20/GO/RBi/S/1999/ CPCSEA Recombinant Purchased from Polyvalent dengue Prospec,Protein technologies, antigens the Prospec, antigen (DENV), Israel represented by Sailendra K Protein HBsAg surface Ray, LabproIndia, Plot no26, technologies, antigen, and Japanese Room No. 302, 3.sup.rd Israel Encephalitis virus Floor,Nacharam, Hyderabad- (JEV) antigen 500076, Tel 040-27275604

[0156] Adjuvanticity of 15b was evaluated using antigens (OVA, HBsAg, DENV and JEV) in BALB/c mice modelFrom the immunized mice retro-orbital sampling or retro-orbital blood was collected on 14th and 28.sup.th day before sacrifice. Serum was separated by centrifugation at 12,500 rpm for 5 min. Transferred to a clean centrifuge tube and store at 80 until used.

[0157] The plates were coated with antigen in carbonate buffer. Plates were incubated at 4 C. overnight. They were then washed 3 times with PBS/Tween, and non-specific binding sites were blocked by adding 200 l of blocking solution. Plates were incubated at room temperature for 1 h. Then 3 times wash is done, diluted standards and samples to desired concentrations in blocking solution were added to the plates. Incubate at 37 C. for 1 h or at 4 C. overnight. Plates were washed 3 times with PBS/Tween. Avidin-Horseradish Peroxidase (Av-HRP) was diluted and added. Incubated at room temperature for 30 min. Plates were washed 3 times with PBS/Tween. OPD Substrate was added and plates were incubated at room temperature (4-30 min) for colour development. The colour reaction was stopped by adding 50 l of stop solution. Optical density (OD) was read at 492 nm.

[0158] Serum anti-antigens (OVA, HBsAg, DENV and JEV) IgG titer. antigen specific IgG was assayed by indirect ELISA and titers obtained after booster immunization reveals that the production of anti-antigen antibodies was strongly enhanced in mice treated with conjugates in comparison with antigen alone.

In Vivo Cytokine Estimation

[0159] ELISA was carried out by Bio Legend capture and detection antibodies. Briefly, 96-well plates were coated with capture antibody dissolved in coating buffer per well incubated overnight at 4 C. Wells were blocked with BSA for 1 h at RT. After blocking, 50 L/well of serum was added and incubated for 3 h. After washing, Biotinylated secondary antibody was added along with enzyme. Plates were incubated for 1 h at RT. Then plates were washed and TMB substrate solution was added. The reaction was stopped after 30 min with a stopping solution. Absorbance was measured at 450 nm with a plate reader.

Immunophenotyping

[0160] Staining for extracellular markers Staining was as per the manufacturer's protocol and run on a BD FACS Verse flow cytometer. Compensation was established using BD Biosciences compensation beads. Post-acquisition flow cytometry analysis was performed using FACS Suite software.

In Vivo Splenocyte Proliferation

[0161] Splenocytes were seeded into 96-well flat-bottom microtiter plates having 110.sup.5 cells/well in 100 mL complete RPMI-1640 medium. Plates were incubated at 37 C. with 5% CO2. After 48 h, 20 mL MTT solution (5 mg/mL) was added to each well and left to incubate for next 4 h. Untransformed MTT (180 mL) was removed from each well by pipetting. A total of 180 mL of DMSO was added and the absorbance was evaluated in an ELISA reader at 630 nm on the multimode reader (Infinite 200 Pro, Switzerland) after 15 min

TABLE-US-00002 TABLE 1 In vitro Hemolysis assay % Hemolysis Dis. Water 100 0.94 PBS 0 MDP 0.450128 0.014 15b 2.30179 0.147

TABLE-US-00003 TABLE 2 In vitro splenocyte proliferation assay EXVIVO CC MDP 15b MTT ConA LPS ConA LPS ConA LPS 100 g 1.00- 0.9124- 1.1569- 1.431967- 1.10- 0.9568- 2.14 1.54 1.78 1.97 1.98 1.54 10 g 1.05- 1.00- 1.1- 1.0- 1.47 1.74 1.75 1.74 1 g 1.02- 0.95- 1.08- 1.02- 1.76 1.84 1.74 1.46

TABLE-US-00004 TABLE 3 IgG titer with OVA antigen IgG OVA 100 g 6400-12800 MDP 20 g 42666.67-75241.21 15b 1 g 1600-124000 10 g 204800-416000 100 g 115000-320000

TABLE-US-00005 TABLE 4 Cytokine assay on restimulation of splenocytes with OVA antigen IFN (pg/ml) IL-1(pg/ml) IL-4(pg/ml) 15b 80.6- 36.1- 56.35- 1 g 88.24 41.95 61.47 15b 75.06667- 38.23333- 25.16667- 10 g 82.14 45.27 31.74 15b 68.6- 23.06667- 25.1- 100 g 75.45 29.47 29.47

TABLE-US-00006 TABLE 5 IgG titer with HBsAg antigen HBsAg-15b HBsAg alone 15b 1 g 51200-102400 10 g 25600-34570 102400-240000

TABLE-US-00007 TABLE 6 Post HBsAg immunization In vivo splenocyte proliferation assay Cells alone HBsAg LPS Con A HBsAg alone 100 100 100 100 20 130.6- 131.87- 111.6- 163.56- g 145.74 140.24 124.58 175.38 15b 123.5- 125.07- 165.8- 176.5- 1 g 142.87 135.87 175.34 181.54 15b 115.5- 124.30- 151.66- 188.42- 10 g 125.76 134.84 162.47 194.28 15b 142.1- 141.19- 164.9- 173.3- 100 g 149.25 148.27 175.24 149.24

TABLE-US-00008 TABLE 7 Post HBsAg immunization In vivo cytokine assay IFN IL-1 IL-4 IL-6 IL-10 (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) HBSAG 75.28- 12.43- 22.43- 93.23- 25.5- 84.28 19.47 34.78 110.87 31.84 MDP 97.28- 33.6- 22.63- 207.3- 49- 110.54 37.48 27.58 278-28 55.84 15b 80.6 16.6- 23.73- 137.83- 22.7- 1 g 20.75 29.48 145.89 29.48 15b 75.06667 19.23- 30.46- 197.2- 22.33- 10 g 28.47 39.27 201.48 29.48 15b 68.6 29.86- 25.8- 380.85- 30.63- 100 g 37.48 34.87 395.75 38.54

TABLE-US-00009 TABLE 8 Post HBsAg immunization Immunophenotyping CD4 CD8 HBsAg 21.64-28.47 10.26-14.38 MDP 17.11-19.48 5.9-9.57 15b -1 G 24.48-29.48 12.71-19.48 15b -10 G 25.47-29.48 12.67-18.54

TABLE-US-00010 TABLE 9 IgG titer with Dengue MDP IgG DENGUE (20 g) 15b 1 g 40000- 160000- 80000- 80000 240000 160000 10 g 640000- 950000

TABLE-US-00011 TABLE 10 Post Denv immunization in vivo cytokine assay IL-1 IL-2 IL-6 IL-10 TNF (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) Dengue 2.65- 15.1- 346.44- 44.95- 27.56- alone 5.95 19.47 374.47 49.28 34.28 MDP 7.25- 35.65- 203.36- 75.8- 55.5- 20 g 9.48 39.27 227.15 89.27 60.27 15b 18.05- 5- 297.08- 55.2- 15.5- 1 g 20.48 9 304.84 61.28 21.48 15b 8.75- 9.25- 257.50- 49.7- 32.8- 10 g 10.57 10.84 274.58 54.84 47.28

TABLE-US-00012 TABLE 11 Post Denv immunization In vivo splenocyte proliferation assay Cells alone Cells Cells + LPS Cells + Con-A UT 100 100 100 100 Dengue antigen 111.69- 120.15- 115.63- 96.25- 115.24 125.48 119.24 104.57 MDP 104.48- 110.45- 115.6- 123.53- 109.54 115.48 119.84 128.48 15b 99.26- 111.72- 122.4- 115.61- 1 g 104.65 116.24 127.35 120.48 15b 115- 121.72- 126.9- 136.72- 10 g 119 127.24 134.85 140.27

TABLE-US-00013 TABLE 12 Post Denv immunization Immunophenotyping CD4 CD8 CD19 CD80 CD 28 DEV 34.37-39.5 22.03-27.48 33.92-39.48 5.2-9.8 3.3-8.4 MDP 41.25-46.5 16.39-21.84 39.65-42.57 4.42-7.5 5.33-9.4 15b -1 G 36.26-41.8 22.26-29.48 30.57-35.48 6.3-8.4 3.65-7.48 15b -10 G 38.74-43.84 22.92-27.84 36.19-41.28 6.11-11.4 3.68-8.41

TABLE-US-00014 TABLE 13 IgG titer with JEV antigen JEV alone MDP-20 g 15b 1 g 64000-128000 10 g 16000-32000 128000-320000 8000-16000 100 g 40000-80000

TABLE-US-00015 TABLE 14 Post JEV immunization In vivo cytokine assay IL-1 IL-2 IFN IL-6 IL-10 (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) JEV alone 62.7-8.41 15.0-20.41 1.9-2.4 256.8-274.01 63.7-64.24 MDP 20 g 62.8-67.54 13.88-19.47 5.01-7.37 149.2-154.27 44.96-47.58 15b 1 g 63.369.48 13.77-19.81 4.21-6.37 157.05-160.47 48-51.02 15b 10 g 61.9-67.25 12.17-15.34 11.03-13.05 233.3-245.27 52.3-54.28 15b 100 g 70.1-73.48 23.97-27.45 3.06-6.48 279.85-281.47 57.5-59.48

TABLE-US-00016 TABLE 15 Post JEV immunization In vivo splenocyte proliferation assay Normal Restimulated LPS Con A UT 100 100 100 100 JEV alone 109.84-111.24 117.9-120.48 106.3-109.54 134.4-139.27 MDP 20 g 98.56-101.02 93.3-97.28 85.1-89.24 141.95-144.28 15b 1 g 118.6-120.48 116.5-119.27 118.2-120.01 117.5-119.48 15b 10 g 111.38-115.78 114.2-117.24 104.6-109.57 124.55-126.85

TABLE-US-00017 TABLE 16 Post JEV immunization Immunophenotyping CD4 CD8 JEV 40.78-45.27 16.31-19.24 MDP 43.45-48.28 19.05-21.48 15b -1 G 39.75-42.58 14.56-19.54 15b -10 G 41.86-43.28 14.78-17.25

[0162] Table-17 and Table 18: Immunopharmacological evaluation of compound 15c and 15 d carried out using OVA antigen as per protocols described in Example-10.

TABLE-US-00018 TABLE 17 IgG OVA 100 g 6400-12800 MDP 20 g 42666.67-75241.21 15c 1 g 25000-102400 10 g 10000-85000 100 g 5500-75000

TABLE-US-00019 TABLE 18 IgG OVA 100 g 6400-12800 MDP 20 g 42666.67-75241.21 15d 1 g 3200-12400 10 g 3200-41600 100 g 6400-84000

TABLE-US-00020 Table-19 and Table-20: Immunopharmacological evaluation of compound 15e and 15f was carried out using OVA antigen using protocols as described in Example-10. IgG OVA 100 g 6400-12800 MDP 20 g 42666.67-75241.21 15e 1 g 1600-12400 10 g 1600-41600 100 g 12800-96000 IgG OVA 100 g 6400-12800 MDP 20 g 42666.67-75241.21 15f 1 g 8000-25400 10 g 5000-41600 100 g 200000-960000

TABLE-US-00021 TABLE 21 Immunopharmacological evaluation of compound 15f was carried out with JEV antigen using protocols as described in Example-10. IgG Titer JEV 40000-49000 MDP 160000-320000 15f-5 G 80000-160000 15f -20 G 160000-640000 15f -50 G 640000-9560000

TABLE-US-00022 TABLE 22 Immunophenotyping post JEV immunization IL-1 IL-2 IL-4 IL-10 IL-12 (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) JEV 545-625 45-60 90-110 349-374 95-110 MDP 1467-1725 89-97 145-157 2000-2500 140-154 15f-5 G 745-845 41-52 134-155 243-294 124-147 15f-20 G 1059-1124 52-64 138-146 398-421 175-194 15f-50 G 1487-1634 69-76 148-164 424-487 136-147

TABLE-US-00023 TABLE 23 In vitro splenocyte proliferation assay % MDPO Myr Proliferation 1000 G/ml 65.24 500 G/ml 95.87 250 G/ml 120.54 125 G/ml 135.24

TABLE-US-00024 TABLE 24 Post immunization In vivo cytokine assay IL-1 IL-2 IL-4 IL-10 IL-12 TNF (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) JEV 545-625 45-60 90-110 349-374 95-110 545-672 MDP 1467-1725 89-97 145-157 2000-2500 140-154 645-694 15f-5 745-845 41-52 134-155 243-294 124-147 624-654 G 15f-20 1059-1124 52-64 138-146 398-421 175-194 898-942 G 15f-50 1487-1634 69-76 148-164 424-487 136-147 1024-1124 G

TABLE-US-00025 TABLE 25 In vivo splenocyte proliferation assay JEV LPS Con A JEV 120-135 140-155 110-124 MDP 145-155 189-194 194-210 15f-5 G 139-147 124-134 125-141 15f-20 G 149-162 128-137 134-142 15f-50 G 155174 184-192 245-264

[0163] Table-26 and Table-27: Immunopharmacological evaluation of compound 15 g and 15h was carried out with OVA antigen using biological resources and protocols as described in Example-10.

TABLE-US-00026 TABLE 26 IgG OVA 100 g 6400-12800 MDP 20 g 42666.67-75241.21 15g 1 g 25000-124000 10 g 10000-60000 100 g 5000-35000

TABLE-US-00027 TABLE 27 IgG OVA 100 g 6400-12800 MDP 20 g 42666.67-75241.21 15 h 1 g 8750-64000 10 g 10000-50000 100 g 3750-25000

TABLE-US-00028 TABLE-28 Immunopharmacological evaluation of compound 15i was carried out with OVA antigen using biological resources and protocols as described in Example-10. IgG OVA 100 g 6400-12800 MDP 20 g 42666.67-75241.21 15i 1 g 4800-50000 10 g 102400-560000 100 g 119466-484000

[0164] From all the above in vitro and in vivo immunological evaluation data of 15b against Ovalbumin, DEV, JEV and HBsAg antigens, and 15f against JEV antigen, it could be concluded that the adjuvant molecule was non-toxic and non-hemolytic even at higher concentrations and has elicited a significant antibody titer, along with cytokine production against most of the antigens tested. A potent humoral and cell mediated immune response was observed which is very much required characteristic for an effective adjuvant.

[0165] In an embodiment the present invention also provides a prophylactic vaccine composition comprising: a muramyl dipeptide compound of general formula-I and an antigen is selected from inactivated or live attenuated infectious pathogens their subunit, either natural pathogen derived or recombinant, a conjugate vaccine antigen or a combination thereof.

[0166] The following vaccine compositions with muramyl dipeptide compound of general formula-I are prepared wherein the ratio of antigen: muramyl dipeptide compound is 1:0.1-1.

TABLE-US-00029 Vaccine compositions DEV and muramyl dipeptide compound JEV and muramyl dipeptide compound HBsAg and muramyl dipeptide compound