Cyclic glycolipopeptide vaccine adjuvants and the process for preparation thereof

Abstract

The present invention relates a cyclic glycolipopeptide compounds useful as adjuvants in immunogenic compositions for vaccination. The cyclic glycolipopeptides bear 1,2,3- triazole or C4-homologue moieties on glucose unit of a NOD2 agonistic peptide moiety and have high potential of immuno-modulating properties for use as adjuvants in vaccine formulations. The present invention discloses a process for the preparation of these cyclic glycolipopeptide analogues and their intermediates.

Description

CYCLIC GLYCOLIPOPEPTIDE VACCINE ADJUVANTS AND THE PROCESS FOR PREPARATION THEREOF FIELD OF THE INVENTION

[0001] The present invention relates generally to adjuvants and immunogenic compositions useful for vaccination. The present invention particularly relates to cyclic glycolipopeptide analogues having high potential of immuno-modulating properties for use as adjuvants in vaccine formulations.

[0002] The present invention more particularly relates to cyclic glycolipopeptides bearing 1,2,3- triazole and C4-homologue moieties on glucose unit of a NOD2 agonistic peptide moiety. The present invention also relates to a process for the preparation of these cyclic glycolipopeptide analogues and their intermediates. BACKGROUND OF THE INVENTION

[0003] Several naturally occuring glycoproteins and glycopeptides exhibit powerful immune modulatory properties and often used as adjuvants for vaccines, synthetic vaccines and in cancer immunotherapy. Of particular interest are the glycoproteins / peptides bearing glucosamine moiety which play important role in regulating the immune system. While some of these glycoproteins / peptides are tumor cell tumor surface markers, many N-acyl glycopreteins are the part of peptidoglycan moiety of several gram positive and gram-negative bacteria and hence are highly antigenic in nature. Muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D- isoglutamine)- a synthetic immunoreactive peptide consisting of N-acetyl muramic acid attached to a short amino acid chain of L-Ala-D-isoGln. It was first identified in bacterial cell wall peptidoglycan as an active component in Freund's Complete Adjuvant (FCA). In 1974, MDP was discovered to be the minimal structure required for the efficacy of FCA, one of the most potent and widely used adjuvants in animal experimental models. Muramyl dipeptide derivatives have been proved to show significant immunomodulatory properties, via one of the PRRs (Pattern Recognition Receptors), nucleotide-binding oligomerization domain 2 (NOD2) receptor (GirardinS. et al.,.2003. J Biol Chem.278(11): 8869-72; F. Coulombe et al, 2012 PloS ONE, 7 (5): Article ID e36734). Muramyl dipeptides activate macrophages and other cells of the immune system to kill cancer cells (7. Jakopin, 2013. Current Medicinal Chemistry, 20 (16): 2068- 2079; Ogawa et. al., 2011.CurrBioact Compd. 7(3): 180-197), however, it is also reported to be pyrogenic in nature. In order to reduce its pyrogenic effect, and improve the activity, a series of MDP derivatives have been designed, synthesized and tested till date. There are many reported inventions or publications related to the synthesis of Muramyl dipeptide and its derivatives (Namba et al., 1997. Vaccine, 15(4):405-13; WO1996001645; US 4395399; US 7173107B2). N-glycolyl muramyl dipeptide, GMDP (N-glycolyl glucosaminyl-N- acetylmuramyl-L-alanyl-D- isoglutamine), Murabutide are few examples of Muramyl dipeptide derivative, which, show to have higher immunoadjuvant activity and less pyrogenic effect, compared to MDP. Several other N-acyl derivatives of MDP are reported to be better immuno- therapeutics compared to parent compound (Andronova T.M., et al., 1991. Review. Immunology 4,1). Hence, this molecule has been widely used in immunotherapeutic approaches, especially to treat chronic infections, autoimmune diseases and cancer (L. I. Rostovtseva et al., 1981. Russian Journal of Bioorganic Chemistry, 7 (12):1843- 1858).

[0004] MDP-based compound Likopid™ is the first immunotherapeutic of the muramyl glycopeptide type introduced to the clinical practice. Likopid™ was developed and registered by a Russian company Peptek as an immunotherapeutic with broad applicability, e.g. immune stimulation and prevention of infections complicating post-traumatic, post-operative, post-chemotherapeutic and post- radiotherapeutic patient hood. Other areas are the treatment of infectious diseases, like tuberculosis, human cervical papillomavirus, ophthalmic herpetic infections, psoriasis and treatment of ulcerous and inflammation processes (WO2007045192). Below shown Formula-Z represents the general chemical structure of muramyl dipeptides(MDPs).Formula-Z: St d GMDP

[0005] There are many inventions related to Muramyl peptide compounds and its derivatives. Although their application as therapeutic agents is known, clinical use of MDP derivatives as adjuvants in vaccine formulations has not been realized so far eventhough several candidates are in various phases of clinical trials.

[0006] WO1996001645Al relates to the use of Muramyl peptide compounds, particularly N-acetyl-D-glucosaminyl-(pi-4)-N-acetylmuramyl-L-alanyl-D- isoglutamine (MDP), for the treatment of inflammatory dermatological conditions such as psoriasis and in the treatment of immune-related diseases of the skin and mucous membranes.

[0007] US7173107B2 discloses glycopeptides and preparation thereof, which covers the stereospecific synthesis of a glycopeptide following a triplyorthogonal protection scheme in particular, the synthesis of N-acetylglucosaminyl-P-[l,4]-N-acetylmuramylmonopeptide and derivatives thereof. The glycopeptides are shown to be useful for the preparation of MDP and related compounds having a glucosaminyl- P- [l,4]-N-acetylmuramic acid disaccharide core.

[0008] WO2007045192 relates to glucosaminylmuramic acid (2-amino-2-deoxy-P-D-gluco- pyranosyl-(l 4)-N-acetylmuramic acid) derivatives, method of their synthesis, and their use for the synthesis of glucosaminylmuramyl glycopeptides, i.e. disaccharide analogues of muramyl glycopeptides. US 4395399 discloses different glycopeptides and their preparations.

[0009] However, despite improvements seen in several newly developed MDPderivatives, yet there is a continuous need for novel compounds with further reduced side-effects and increased adjuvant activity for the purpose of either therapeutic or prophylactic use in vaccine formulations. As adjuvant action is antigen specific, in order to evoke qualitatively specific immune response, a variety of alternate glycopeptides structures that are more efficacious and least toxic with high degree of complimetarity with specific antigens need to be generated. Hence it is critical to focus on the balance between efficacy and side effects while designing or synthesizing new compounds. Development of more efficacious and improved glycopeptides adjuvants, with reduced side effects associated with known MDP derivatives, is highly needed. In view of the importance of Glucosamine derived glycolipopeptides as immune modulators, disclosed herein is a series of novel cyclic glycolipopeptides bearing 1,2,3- triazoly and C4-homologue moieties on glucose unit to a NOD2 agonistic peptide moiety. OBJECTS OF THE INVENTION

[0010] The primary object of the invention is to provide cyclic glycolipopeptide analogues having adjuvant activity.

[0011] Another object of the invention is to provide a process for the preparation of these cyclic glycolipopeptide compounds.

[0012] Yet another object of the invention is to provide cyclic glycolipopeptide intermediate compounds for the synthesis cyclic 1,2,3-triazole and C4-homologue glycolipopeptide compounds.

[0013] A further object of the invention is to provide safe use of cyclic glycolipopeptide compounds as adjuvants for pharmaceutical preparations and vaccine formulations.

[0014] Still, a further object is to provide a vaccine formulation comprising cyclic glycolipopeptide compounds. SUMMARY OF THE INVENTION

[0015] Accordingly, the present invention provides a cyclic glycolipopeptidecompound of formula-I, wherein,R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedand substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; G is selected from 2.

[0016] In a preferred embodiment, the cyclic glycolipopeptide compound is selected from the group consisting of,wherein, R is selected from the group consisting of H , R1is selected from thegroup consisting of substituted or unsubstituted C4-C14 linear or branched alkyl, C3- C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and x and y are independently selected form 1, 2 and 3.

[0017] In a more preferred embodiment, the cyclic glycolipopeptide compound is selected from the group consisting of; a) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-26-(hydroxymethyl)-4,7- dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9-diaza-1(1,4)- triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(19); b) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-26-((hexanoyloxy)methyl)-22,25- dihydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9- diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21a); c) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-4,7-dimethyl-5,8,11- trioxo-26-((tetradecanoyloxy)methyl)-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9- diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21b); d) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-4,7-dimethyl-26-(((3- methylbutanoyl)oxy)methyl)-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa- 6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21c); e) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-26-(((cyclopropanecarbonyl)oxy)methyl)- 22,25-dihydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12- dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21d);f) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-18-(hydroxymethyl)- 2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (28); g) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18-((butyryloxy)methyl)-16,19- dihydroxy-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30a); h) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-18- ((octanoyloxy)methyl)-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30b); i) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxo-18-((tetradecanoyloxy)methyl)hexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30c); j) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-18-(((3- methylbutanoyl)oxy)methyl)-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30d); k) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18- (((cyclopropanecarbonyl)oxy)methyl)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30e); l) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18-((benzoyloxy)methyl)-16,19- dihydroxy-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30f); m) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-18-(((4- methoxybenzoyl)oxy)methyl)-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30g); and n) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxo-18-(((4-(trifluoromethyl)benzoyl)oxy)methyl)hexadecahydro-11H,16H- pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30h).

[0018] The present invention provides a process for the preparation of a cyclic glycolipopeptide compound of wherein,R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedor branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl;

[0019] and x is selected form 1, 2 and 3, comprising the steps of: a. coupling of dipeptide by treating the compound of formula VI with propargyl L-alanyl-D-isoglutamine benzyl ester trifluoroacetate (6) in an acid amine coupling conditions to obtain a compound of formula VII, wherein x is as defined above;b nd of formula VII by treating with CuSO4.5H2O catalyst in presence of sodium ascorbate, THF / H2O (4:1) solvent to obtain the compound of formula VIII, wherein x is as defined above;presence of a organic acid TFA and water solvent to obtain the compound of formula IX, wherein x is as defined above;d. hydrogenolysis of the compound of formula IX in presence of a Pd / C catalyst and a THF / H2O (4:1) solvent to obtain the compound of formula- I, wherein R is H and x is as defined above;(R1COCl) and a pyridine solvent to obtain the compound of formula XI wherein R x is as defined above; andhydrogen gas and a THF / H2O solvent to obtain the compound of formula- I’.

[0020] In an embodiment of the process of the present invention the coupling is carried out in presence of EDC.HCl, HOBt,THF, DIPEA at 0oC-RT.

[0021] In an embodiment of the present invention the intermediate compound for the preparation of cyclic glycolipopeptide compound of formula-I’, selected from the group consisting of:nd p p p p p yclic glycolipopeptide compound of formula-I’’wherein, R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedor branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and y is selected form 1, 2 and 3, comprising the steps of:

[0023] A process for the preparation of a cyclic glycolipopeptide compound of formula-II’’, the process comprising the steps of: a. coupling of dipeptide by treating the compound of formula VI with allyl L-alanyl-D-isoglutamine benzyl ester trifluoroacetate (9) in an acid amine coupling conditions to obtain a compound of formula XII, wherein y is as defined above; b.PPh3and THF / H2O (4:1) solvent to obtain a compound of formula-XIII), wherein y is as defined above;c. presence T3P reagent, DIPEA and THF as solvent to obtain a compound of formula-XIV), wherein y is as defined above;catalyst and DCM solvent to obtain a compound of formula-XV), wherein y is as defined above;e. benzylidene deprotection of the compound of formula-XV in presence of TFA / H2O (4:1) solvent to obtain a compound of formula-XVI), wherein y is as defined above;f. double bond reduction of the compound of formula-XVI with a NaBH4 reducing agent in presence of nickel dichloride hexahydrate (NiCl2.6H2O) catalyst andTHF / MeOH solvent to obtain a compound of formula-XVII), wherein y is as defined above;using H2gas and THF / H2O (4:1) solvent to obtain a compound of formula-I, wherein R is H;in presence pyridine as solvent to obtain the compound of formula XIX, wherein y is as defined above; andi. hydrogenation of the compound of formula-XIX with Palladium on carboncatalyst in presence H2 gas and THF / H2O (4:1) solvent to obtain the compound of Formula-II’’.

[0024] In an embodiment of the present invention the intermediate compound for the preparation of cyclic glycolipopeptide compound of Formula-II’’ is selected from the group consisting of:wherein

[0025] In an embodiment of the present invention the cyclic glycolipopeptide compounds is an adjuvant useful in pharmaceutical preparations and vaccine formulations with an antigen.

[0026] The present invention provides a vaccine formulation comprising the cyclic glycolipopeptide compound of Formula-I as defined above, as an adjuvant and a suitable antigen derived from bacterial, viral or any potential infectious pathogensagainst mammals, selected from a group consisting of a live attenuated vaccine antigen, inactivated vaccine antigen, subunit vaccine antigen, a conjugate vaccine antigen, and recombinant vaccine antigen or any combinations thereof.

[0027] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Abbreviations

[0028] Abbreviations used in the present invention process have the following meaning or have been elaborated as follows: L-Ala-D-isoGln : L-alanyl-D-isoglutamine EtOAc: Ethyl acetate Na2SO4:Sodium Sulphate BF3.E2O: Borantrifluoro etherate Boc2O: Di-tert-butyl dicarbonate THF: Tetrahydrofuran DCM: Dichloromethane ACN: Acetonitrile EtOAc: Ethyl acetate Et3N: Triethylamine DIPEA: N,N-Diisopropylethylamine EDCI: l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide THF-i-Pr2O: Tetrahydroforan-Diisopropylether DMF: Dimethylformamide P2O5 : Phousphorus pentoxide KOH: Potassium hydroxideT3P: propanephosphonic acid anhydride HOBt: Hydroxy benzotriazole NaH: Sodium hydride TFA: Trifluoroaceticacid PTSA: P-tolune sulphonic acid DMSO: Dimethylsulphoxide MDP: Muramyl dipeptide ELISA: Enzyme Linked Immunosorbent Assay NOD2: Nucleotide Oligomerization Domain PRR: Pattern Recognition Receptors PAMP: Pathogen associated membrane Patterns PBS: Phosphate-buffered saline BSA: Bovine serum albumin TMB: 3,3′,5,5′-Tetramethylbenzidine FACs: Fluorescence-activated cell sorting DETAILED DESCRIPTION OF THE INVENTION

[0029] For better understanding the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0030] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0031] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[0032] Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0033] The term “consisting of’ means the embodiment necessarily includes the listed components only and no other unlisted components are present. Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub- ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. The term “between” should be understood as being inclusive of the limits.

[0034] The term “alkyl” refers to a saturated hydrocarbon chain having the specified number of carbon atoms. In an aspect of the present disclosure, alkyl refers to an alkyl group having 4–14 carbon atoms. Alkyl groups may be straight or branched chained groups which may be optionally substituted. Representative branched alkyl groups have one, two, or three branches. Preferred alkyl groups include, without limitation, methyl, ethyl, n-propyl, and isopropyl. One or more hydrogens of the alkyl groups may be optionally replaced with deuterium such as CD3, CHD2, CDH2, CH2CD3, CD2CH3 and the like.

[0035] The term “cycloalkyl” used herein refers to a saturated non-aromatic carbocyclic ring with 3 to 10 carbon atoms, may be optionally substituted by one or more substituents. Examples of cycloalkyl includes but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0036] The term “aryl” refers to the aromatic ring radical comprising at least 6 carbon atoms with extended conjugation of pi electrons. In an aspect of the present disclosure,the aryl ring comprises 6 to 15 carbon atoms. Furthermore, the aryl ring may be substituted with ne or substituents selecetde from C1-10 alkyl, C1-10 alkoxy, and trifloro alkyl

[0037] The present invention provides a cyclic glycolipopeptide compound of formula- I,

[0038] wherein,R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedand substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; G is selected from 2.

[0039] In a preferred embodiment, the cyclic glycolipopeptide compound is selected from the group consisting of,nd wherein, R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstituted C4-or branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and x and y are independently selected form 1, 2 and 3.

[0040] The cyclic glycolipopeptide compounds of structural formula-I’ and II’’have reduced pyrogenicity, while maintaining a considerable effective function as vaccine adjuvants. The present invention provides the process for preparing the cyclic glycolipopeptide compounds. Additionally, the starting compound, propargyl L- alanyl-D-isoglutamine benzyl ester used in the synthesis of the cyclic 1,2,3- triazole glycolipopeptide compounds and allyl L-alanyl-D-isoglutamine benzyl ester used in the synthesis of the C4-homologue glycolipopeptide compounds are synthesized.

[0041] SCHEME-A and B provide the synthesis of the reactants / starting compounds propargylated L-alanyl-D-isoglutamine benzyl ester and allylated L-alanyl-D- isoglutamine benzyl ester respectively.p y y p g - acid [Compound 1] and anhydrous sodium sulphate (Na2SO4) suspended in benzyl alcoholand Boron trifluoride. Diethyl Ether (BF3. E2O), To this mixture, is added and the suspension is stirred at room temperature (RT) followed by diluting with absolute THF and filtering with the aid of charcoal. Further treatment with triethylamine, concentrating, precipitating followed by washing it provides (R)-2-amino- 5- (benzyloxy)-5-oxopentanoic acid [Compound 2].

[0043] [Compound 3] is prepared by providing a solution of the [compound 2], adding with Di-teritiary butyl dicarbonate (Boc)2O in dioxane and water at a low temperature such as 0°C and basified with Sodium bicarbonate (NaHCO3) 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 Compound 3.

[0044] [Compound 4] is synthesized by providing [Compound 3] in dry dichloromethane (DCM), K2CO3adding propargyl bromide at 0°C. The reaction mixture is stirred at rt for 4h. After disappear all starting material as indicated by thin- layer chromatography reaction mixture filtered, then filtrate extracted in DCM and organic phase with water followed by washing with brine solution, dried on sodium sulphate, removing the solvent in vacuum and purified through Column chromatography to furnish Compound 4 as crystalline solid.

[0045] [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 toluene and distilled to obtain oily D-isoglutamine benzyl ester trifluoroacetate. Separately, to t- butoxycarbonyl-L-alanine in dry THF, l-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 aresidue. After further washing and recrystallization it provides Compound 5 as white solid.

[0046] [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 (Et2O) to obtain syrupy type compound 6

[0047] [Compound 7] is synthesized by providing [Compound 3] in dry Toluene and N, N-Diisopropylethylamine (DIPEA) has taken followed by addition of allyl bromide at 70 °C. The reaction mixture is stirred at rt for 8h. After complete consumption of all starting material as indicated by thin-layer chromatography reaction mixture filtered, then filtrate extracted in DCM and water followed by washing with brine solution, dried on sodium sulphate, removing the solvent in vacuum organic, and purified through column chromatography to afford compound 7 as oily compound.

[0048] [Compound 8] allylated i-Butoxy carbonyl-D-isoglutamine benzyl ester [Compound 7] 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 (Et2O) to obtain oily D-isoglutamine benzyl ester trifluoroacetate. Separately, to t-butoxycarbonyl-L-alanine in dry THF, l-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 compound 8 as colorless syrup.

[0049] [Compound 9] allyl benzyl-5-amino-4-((5)-2-((tert-butoxycarbonyl) amino) propanamido)-5-oxopentanoate [Compound 8] 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 wastriturated with toluene to obtain syrupy [Compound 6]

[0050] The synthesis of the cyclic glycolipopeptide compounds is shown in Scheme C and Scheme D. The synthesis of cyclic glycolipopeptide compounds (19, 21a to 21d) of formula-I’ is carried out as shown in scheme-C and (28, 30a to 30h) of formula- II’’ is carried out as shown in scheme-D.

[0051] In the scheme D, R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedor branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl;

[0052] and y is selected form 1, 2 and 3.

[0053] In the scheme D, R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedorbranched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl;

[0054] and y is selected form 1, 2 and 3.

[0055] The cyclic 1,2,3-triazole glycolipopeptide and cyclic C4-homologue glycolipopeptide compounds synthesized are listed below. a) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-26-(hydroxymethyl)-4,7- dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9-diaza-1(1,4)- triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(19); b) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-26-((hexanoyloxy)methyl)-22,25- dihydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9- diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21a); c) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-4,7-dimethyl-5,8,11- trioxo-26-((tetradecanoyloxy)methyl)-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9- diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21b); d) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-4,7-dimethyl-26-(((3- methylbutanoyl)oxy)methyl)-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa- 6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21c); e) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-26-(((cyclopropanecarbonyl)oxy)methyl)- 22,25-dihydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12- dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21d); f) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-18-(hydroxymethyl)- 2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (28);g) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18-((butyryloxy)methyl)-16,19- dihydroxy-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30a); h) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-18- ((octanoyloxy)methyl)-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30b); i) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxo-18-((tetradecanoyloxy)methyl)hexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30c); j) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-18-(((3- methylbutanoyl)oxy)methyl)-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30d); k) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18- (((cyclopropanecarbonyl)oxy)methyl)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30e); l) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18-((benzoyloxy)methyl)-16,19- dihydroxy-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30f); m) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-18-(((4- methoxybenzoyl)oxy)methyl)-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro- 11H,16H-pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30g); and n) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxo-18-(((4-(trifluoromethyl)benzoyl)oxy)methyl)hexadecahydro-11H,16H- pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30h).

[0056] In further aspects of the invention, the cyclic glycolipopeptide compounds of the present invention are evaluated for its activity. The safety of the cyclic glycolipopeptide compounds of the present invention is established through cytotoxicity and pyrogenicity assays. In another aspect, the invention provides use of cyclic glycolipopeptide compounds as an adjuvant to be used in the pharmaceutical preparations and vaccine formulations. In further embodiments of the invention, the in- vivo immunogenicity of the glycolipopeptide compounds of the present invention is shown to effectuate a number of folds increase in antibody titers when used as an adjuvant with various vaccine antigens, thereby capable of generating both humoral and cell-mediated immune response.

[0057] The cyclic glycolipopeptide compounds of Structural formula-I’ is prepared by a process comprising the following steps.

[0058] a. Anomeric benzylation of a compound of Formula-I to obtain a compound of Formula-II; Reagents and conditions: BnOH, CH3COCl, 50oC, 12h. b.compound Formula-III; Reagents and conditions: PTSA, PhCH(OMe)2, ACN 0oC-RT, 7h.c. De-aylating the compound of Formula-III to obtain a compound of Formula- IV; Reagents and conditions: KOH, EtOH, 90oC, 10h. d.azide transferring agent in presence of an appropriate solvent, to obtain a compound of Formula- V; Reagents and conditions: CuSO4.5H2O, Et3N, TfN3, ACN 0oC-RT,10h. e.ethyl (S)-2- Chloropropionic acid in presence of a suitable base and solvent to obtain a compound of Formula-VI; Reagents and conditions: S-(-)-2-Chloropropanoic acid, NaH, Dry DMF, 0oC-RT, 14h. f.L-alanyl-D-isoglutamine benzyl ester trifluoroacetate in a suitable acid amine coupling conditions to obtain a compound of Formula-VII, Reagents andconditions: Compound 6, EDC.HCl, HOBt,THF, DIPEA 0oC-RT, 10h. g.VII by treating with suitable catalyst in suitable solvent to obtain the desired compound as represented by Formula-VIII; Reagents and conditions: CuSO4.5H2O, Sodium ascorbate,THF / H2O (4:1), RT, 8h.suitable acid and solvent to obtain the desired compound as represented by Formula-IX; Reagents and conditions: TFA / H2O (4:1), RT, 2h.a and solvent to obtain the desired compound as represented by Formula-X; Reagents and conditions: H2,10% Pd / C, THF / H2O (4:1), RT, 20h.suitable solvent to obtain the desired compound as represented by Formula-XI; Reagents and conditions: Pyridine, R1COCl, -10oC, 2h.and solvent to obtain the desired compound as represented by formula-I’; Reagents and conditions: H2, Pd / C, THF / H2O (4:1), RT, 24h.

[0059] Wherein R is selected from the group consisting of H , R1is selected from the group consisting of substituted orC4-C14 linear or branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the groupconsisting of alkyl, alkoxy and trifloro alkyl; and x and y are independently selected form 1, 2 and 3.

[0060] The invention provides the process for preparing the cyclic glycolipopeptide compounds of Structural formula-II’’ comprising the steps following steps. l. Coupling a dipeptide by treating the compound of Formula-VI with allyl L- alanyl-D-isoglutamine benzyl ester trifluoroacetate in a suitable acid amine coupling conditions to obtain a compound of Formula-XII; Reagents and conditions: compound 9, EDC.HCl, HOBt,THF, DIPEA 0oC-RT, 16h. m.and suitable solvent to obtain a compound of Formula-XIII; Reagents and conditions: THF / H2O (4:1), PPh3, 14h, 60oC.n. Coupling of the compound of Formula-XIII with acrylic acid in a suitable solvent to obtain a compound of Formula-XIV; Reagents and conditions: T3P, THF, DIPEA, Acrylic acid, RT, 16h.nd suitable solvent to obtain a compound of Formula-XV; Reagents and conditions: DCM, IIndGeneration Grubbs, 50oC, 20h.organic acid and solvent to obtain a desired compound of Formula-XVI; Reagents and conditions: TFA / H2O (4:1), RT, 2h.reducing agent in presence of metal dichloride catalyst and suitable solvent to obtain a desired compound of Formula-XVII; Reagents and conditions: NaBH4, NiCl2.6H2O, THF / MeOH, 0oC, 4h.id and solvent to obtain a desired compound of Formula-XVIII; Reagents and conditions: H2, 10% Pd / C, THF / H2O (4:1), RT, 22h.suitable solvent to obtain a desired compound of Formula-XIX; Reagents and conditions: Pyridine, R1COCl, -10oC, 2h.solvent to obtain a desired compound of formula-I’’; Reagents and conditions: H2, Pd / C, THF / H2O (4:1), RT, 22h.

[0062] R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedor branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and x and y are independently selected form 1, 2 and 3.

[0063] The detailed process for the synthesis of cyclic glycolipopeptide compounds of formula-I’ and I’’ as depicted in above Scheme-C and D are provided below. Process for preparation of cyclic 1,2,3-triazole glycopeptide compounds of structural formula-I’ Step-a: Anomeric benzylation of N-acetyl D-Glucosamine (I):

[0064] Step (a) involves anomeric benzylation of a compound of Formula-I to obtain a compound of Formula-II as schematically presented below:

[0065] I withbenzyl alcohol with stirring. The reaction may be carried out in the presence of AcCl at 50°C. Preferably in one embodiment stirring is done at 80°C and evaporation are done at 50°C.

[0066] The reaction mixture is poured into Et2O 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 ether. Step-b: Benzylidene protection

[0067] Step (b) involves benzylidene protection of the compound of Formula-II to obtain a compound of Formula-III as schematically presented below.

[0068] with structural Formula-II with dimethyl benzaldehyde acetal. The reaction can be performed in the presence of organic Sulfonic acid. In one embodiment the organic sulfonic acid used is a p-toluene sulfonic acid.

[0069] 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 10% EtOAc / n-Hexane followed by stirring at room temperature for 30 minutes at rt.

[0070] After proper stirring, the reaction residue filtered on Buchner funnel under continuous suction followed by washing with 10% EtOAc / n-Hexane of solvents, and water

[0071] The precipitate washed with water once under continuous suction until getting free powder.

[0072] In one embodiment the white powder is dissolved with EtOAc and slowly added n-Hexane until precipitate out filtered on a filter paper.

[0073] After drying Benzyl 2-acetamido-4, 6-O-benzylidene-2-deoxy-D- glucopyranoside [Compound III] is obtained. The drying can be carried out in vacuum at room temperature over phosphorous pentoxide(P2O5). Step-c: Deacylation

[0074] Step (c) involves deacylation the compound of Formula- III to obtain a compound of Formula- IV, as schematically presented below;such as absolute ethanol in the presence of a base followed by refluxing. The base may be potassium hydroxide (KOH), sodium hydroxide (NaOH), lithium hydroxide (LiOH), calcium hydroxide (CaOH) etc.

[0076] In one embodiment the base used is potassium hydroxide (KOH). Then refluxing is performed under Nitrogen (N2) for 12-16hrs, preferably 14hrs and then poured into hot water. The suspension is stirred at low temperature (such as -5°C) overnight for 14 to 18 hours, preferably 16 hours. The obtained product is filtered and recrystallized. The recrystallization can be performed from a solvent such as water, alcohol such as ethanol, tetrahydrofuran (THF), diisopropyl ether or mixture thereof. In one embodiment product is recrystallized from EtOH-water, followed by recrystallization from tetrahydrofuran and di-isopropyl ether (THF andi-Pr2O). Step-d: Azide-installation

[0077] Step (d) involves Azide installation of Formula-IV with a suitable azide transferring agent in the presence of an organic base with a suitable solvent, to obtain a compound of Formula-V as schematically presented below;

[0078] nitrile add triflic anhydride at 0 o C. in one RBF and keep it stirring for 30 minutes insitu triflic azide generated. To the magnetically stirred solution of amine compound 4, copper sulphate. pentahydrate and amine base in the second RBF at 0°C was added while stirring. After 15min to the 2ndRBF at 0°C. triflic azide was added then kept rt for 10hrs. The reaction mixture was extracted in EtOAc (3x100ml) then combined organic fraction concentrated under reduced pressure to afford the required white sticky product after flash chromatography. In one embodiment the organic amine base used is a triethylamine

[0079] In yet another embodiment, the reaction mixture is diluted with Ethyl acetate washed successively with water, dried with sodium sulphate (Na2SO4), and then filtered and the filtrate is evaporated to dryness to give the compound of Formula-V. Chromatographic purification can be performed such as flash chromatography on silica gel. Step-e: O-Alkylation

[0080] Step-(e) involves O-alkylation of the compound of Formula-V by treating with ethyl (S)-α-trifluorosulfonyloxy propionate in presence of an appropriate solvent to obtain a compound of Formula- VI;

[0081] The solvent in O-Alkylation step (e) is dry THF. 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-(e) is sodium hydride.

[0082] In another hand, the metal hydride is added at 0°C in dry DMF under stirring for 10 minutes then then (S)-2-chloropropanoic acid in dry DMF was added dropwise in the duration of 30 min followed by cannulation After the addition, the solvent was removed and the residue was extracted with EtOAc and brine The extract was dried over anhydrous Na2SO4, concentrated, filtered, and purified by CC (EtOAc in hexane, silica gel) to give O-alkylated monosaccharide Compound VI is obtained as white solid. Step-f: Peptide Coupling to obtain structural formula-VII

[0083] Step-(f) involves the coupling of peptides by treating the compound of Formula VII with L-alanyl-D-isoglutamine benzyl ester trifluoroacetate to obtain a compound of Formula-VIII as schematically presented below;A is coupled with a compound of Formula-VI. Compound 6 (5-benzyl 1-(prop-2-yn-1-yl) (tert-butoxycarbonyl)-L-alanyl-D-glutamate) is dissolved in a solvent with 20% TFA and the resulting solution is stirred at room temperature for a few minutes. A solvent is then removed and to the residue toluene were added to get azeotropic mixture has removed under reduced pressure to obtain 5-benzyl 1-(prop-2-yn-1-yl) ((S)-2-((2,2,2- trifluoroacetyl)- λ4-azanyl) propanoyl)-D-glutamate.

[0085] The solvent in step (f) may be selected from trifluoroacetic acid (TFA) anddichloromethane (DCM) or mixture thereof. In one embodiment the solvent in step-(g) involves both TFA and DCM.

[0086] 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).

[0087] Step-(f) can be performed in the presence of a base.

[0088] In one embodiment the base is N.N-Diisopropylethylamine (DIPEA).

[0089] In one embodiment the solvent used is Tetrahydrofuran (THF).

[0090] 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 NaHCO3 solution, dried and evaporated.

[0091] The residue obtained can be purified on a silica gel column by elution with the solvent such as chloroform, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment chloroform-methanol mixture is used for elution. Step-g: procedure for 1, 3-cycloaddition to obtain structural formula-VIII

[0092] Step-(g) involves intra 1, 3-cycloaddition of the compound of Formula-VII by treating with an internal alkyne to obtain a compound of Formula- VIII;or Dioxane / H2O in (4:1) ratio. Thetetrahydrofuran (THF) and water (H2O). The intra 1, 3-takes place under standard copper catalysed conditions such as in the presence of copper (II) sulphate pentahydrate (CuSO4.5H2O) and sodium ascorbate.

[0094] Step-(g) can be performed in the presence of a copper (II) catalysed.

[0095] In one embodiment the reagent used is sodium ascorbate.

[0096] In one embodiment the catalyst used is copper (II) sulphate pentahydrate

[0097] The reaction mixture is stirred at RT for 2 to 6 hours, preferably 5 hours. After concentrating, the residue can be extracted with an organic solvent such as DCM and followed by washing the organic layer with brain solution, dried and evaporated.

[0098] The residue obtained can be purified on silica gel column by elution with the solvent such as dichoromethane, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichoromethane-methanol mixture is used for elution. Step-h: Removal of benzilydine to obtain structural formula-IX

[0099] De-protection of benzilydene is carried out by treating the compound of formula-VIII with organic acid in presence of suitable solvent to obtain the desired compound as represented by compound of formula-IXin (4:1) ratio

[0101] The solvent in step (h) may be selected from trifluoroacetic acid (TFA) and water (H2O)

[0102] The benzylidene cleavage takes place in presence of organic acid and water conditions such as in the presence of trifluoroacetic acid (TFA) and water (H2O).

[0103] Step-(i) can be performed in the presence of trifluoroacetic acid.

[0104] In one embodiment the solventt used is water.

[0105] The reaction mixture is stirred at RT for 2 to 3 hours, preferably 2 hours. After concentrating, the residue can be extracted with an organic solvent such as DCM andfollowed by washing the organic layer with sat NaHCO3 solution, dried and evaporated.

[0106] The residue obtained can be purified on a silica gel column by elution with a solvent such as dichoromethane, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichoromethane-methanol mixture is used for elution. Step-i: deprotection of benzyl groups to obtain structural formula-X

[0107] De-protection is carried out by treating the compound of formula-IX with organic acid in presence of palladium catalysed to remove of all protecting groups to obtain the desired compound as represented by compound of formula-X. X is the compound of formula-I wherein, R is H and y is 1, 2 or 3.glacial acetic acid.

[0109] The de-protection can be performed in the presence of a metal catalyst. In one embodiment the catalyst used in step-(i) is palladium black.

[0110] To a solution of compound formula-VIII, dissolved in organic acid added with palladium catalyst. And the compound subjected to hydrogenolysis for 12 h to 48 h..

[0111] The catalyst is filtered off, and, after addition of water, the filtrate is evaporated under diminished pressure. The residue obtained can be purified on a silica gel column by elution with a solvent such as dichoromethane, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichoromethane-methanol mixture is used for elution. Step-j: Re-acylation of compound-IX to obtain structural formula-XI

[0112] Re-acylation is carried out by treating the compound of formula-IX withorganic acid chlorides (R1COCl) in presence of suitable base as solvent to obtain the desired compound as represented by compound of formula-XIor Triethylamine or DIPEA

[0114] In one embodiment the base selected as Pyridine

[0115] The re-acylation takes place in presence of organic base conditions such as in the presence of Pyridine as base and solvent.

[0116] Step-(j) can be performed in the presence of acid chlorides.

[0117] In one embodiment the solvent used is Pyridine.

[0118] The reaction mixture is stirred at -10 °C for 2 to 3 hours, preferably 2 hours. After concentrating, the residue can be extracted with an organic solvent such as DCM and followed by washing the organic layer with 1N HCl solution, dried and evaporated.

[0119] The residue obtained can be purified on a silica gel column by elution with a solvent such as dichoromethane, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichoromethane-methanol mixture is used for elution. Step-k: Hydrogenation of compound-XI to obtain Formula-I’

[0120] De-protection is carried out by treating the compound of formula-XI with organic acid in presence of palladium catalysed to remove of all protecting groups to obtain the desired compound as represented by compound of formula-I’.wherein,R is selected from the group consisting of H and , R1is selected from the group consisting of substituted or unsubstituted or branched alkyl, C3-C6cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and x is selected form 1, 2 and 3.

[0121] In one embodiment the solvent used in step-(K) for deprotection is Tetrahydrofuran (THF) and water (H2O). The de-protection can be performed in the presence of a metal catalyst. In one embodiment the catalyst used in step-(h) is palladium black. To a solution of compound formula-XI, dissolved in THF / H2O added with palladium black catalyst. And the compound subjected to hydrogenolysis for 12h to 48h.

[0122] The catalyst is filtered off, and, after addition of water, the filtrate is evaporated under diminished pressure. The residue obtained can be purified on a silica gel column by elution with a solvent such as dichoromethane, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichoromethane-methanol mixture is used for elution.

[0123] In another aspect, the invention provides a process for the preparation of cyclic glycolipopeptide with C4-homologue compounds of structural formula-I’’ step wise as depicted below

[0124] .The process comprises the following steps (l) to (t): Step-l: Peptide Coupling to obtain structural formula-XII

[0125] Step-(l) involves the coupling of peptide fragment 9 by treating the compound of formula-VI with allyl L-alanyl-D-isoglutamine benzyl ester trifluoroacetate to obtain a compound of formula-XII as schematically presented below;heme-B is coupled with a compound of formula-VI, (1-allyl 5-benzyl (tert-butoxycarbonyl)-L- alanyl-D-glutamate) is dissolved in a solvent with 20% TFA and the resulting solution is stirred at room temperature for a few minutes. A solvent is then removed and to the residue toluene were added to get azeotropic mixture has removed under reduced pressure to obtain 1-allyl 5-benzyl ((S)-2-((2,2,2-trifluoroacetyl)-λ4- azanyl)propanoyl)-D-glutamate.

[0127] The solvent in step (l) may be selected from trifluoroacetic acid (TFA) and dichloromethane (DCM) or mixture thereof. In one embodiment the solvent in step-(l) involves both TFA and DCM. 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).

[0128] Step-(l) can be performed in the presence of a base.

[0129] In one embodiment the base is N.N-Diisopropylethylamine (DIPEA).

[0130] In one embodiment the solvent used is Tetrahydrofuran (THF).

[0131] 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 NaHCO3solution, dried and evaporated.

[0132] The residue obtained can be purified on a silica gel column by elution with the solvent such as chloroform, alcohols such as methanol, ethanol etc. or mixture thereof.

[0133] In one embodiment chloroform-methanol mixture is used for elution. Step-m: Transformation of azide to amine to obtain structural formula-XIII

[0134] Conversion of azide to amine is carried out by treating the compound of formula-XII with triphenylphoshine in presence of suitable solvent to obtain the desired compound as represented by compound of formula-XIII.or Methanol / water

[0136] The solvent in step (m) may be selected Tetrahydrofuran / water

[0137] The transformation of azide to amine takes place in presence of organic triarylphoshine conditions such as in the presence of Pyridine as base and solvent.

[0138] In one embodiment the solvent used is Tetrahydrofuran / water.

[0139] The reaction mixture is stirred at 50 °C for 10 to 20 hours, preferably 16 hours. After concentrating, the residue can be extracted with an organic solvent such as DCM and followed by washing the organic layer with brain solution, dried and evaporated.

[0140] The residue obtained can be purified on a silica gel column by elution with a solvent such as dichloromethane, alcohols such as methanol, ethanol etc. or mixture thereof.

[0141] In one embodiment dichloromethane-methanol mixture is used for elution. Step-n: coupling of acrylic acid to obtain structural formula-XIV

[0142] Coupling of acrylic acid is carried out by treating the compound of formula- XIII with acrylic acid in presence of suitable coupling reagent and solvent to obtain the desired compound as represented by compound of formula-XIV.

[0143] To a solution of a compound XIII in Tetrahydrofuran or 1,4-dioxane or dichloromethane or dimethyl formamide. The solvent in step (n) may be selected Tetrahydrofuran

[0144] The coupling of acrylic acid takes place in presence of organic couplind reagent conditions such as in the presence of T3P and base.

[0145] In one embodiment the base used is N, N-diisopropylethylamine.

[0146] The reaction mixture is stirred at RT for 10 to 16 hours, preferably 14 hours. After concentrating, the residue can be extracted with an organic solvent such as DCM and followed by washing the organic layer with sat NaHCO3solution, dried and evaporated.

[0147] The residue obtained can be purified on a silica gel column chromatography by elution with the solvent such as dichloromethane, alcohols such as methanol, ethanol etc. or mixture thereof.

[0148] In one embodiment dichloromethane-methanol mixture is used for elution. Step-o: Ring closing metathesis to obtain structural formula-XV

[0149] Ring closing Cross-metathesis is carried out by treating the compound of formula-XIV with Grubbs catalyst in presence of suitable solvent to obtain the desired compound as represented by compound of formula-XV.dioxane or dichloromethane or Toluene. The solvent in step (o) may be selected from dichloromethane

[0151] The ring closing cross metathesis takes place in the presence of Grubb’s catalyst conditions.

[0152] In one embodiment the catalyst used is IIndGeneration grubb’s catalyst.

[0153] The reaction mixture is stirred at 50 for 10 to 16 hours, preferably 14 hours. After concentrating, the residue can be extracted with an organic solvent such as DCM and dried, evaporated.

[0154] The residue obtained can be purified on a silica gel column chromatography by elution with the solvent such as dichloromethane, alcohols such as methanol, ethanol etc. or mixture thereof.

[0155] In one embodiment dichloromethane-methanol mixture is used for elution. Step-p: Removal of benzilydene to obtain structural formula-XVI

[0156] De-protection of benzilydene is carried out by treating the compound of formula-XV with organic acid in presence of suitable solvent to obtain the desired compound as represented by compound of formula-XVIin (4:1) ratio

[0158] The solvent in step (p) can be selected trifluoroacetic acid (TFA) and water (H2O)

[0159] The benzylidene cleavage takes place in presence of organic acid and water conditions such as in the presence of trifluoroacetic acid (TFA) and water (H2O).

[0160]

[0161] Step-(p) can be performed in the presence of trifluoroacetic acid.

[0162] In one embodiment the solvent used is water.

[0163] The reaction mixture is stirred at RT for 2 to 4 hours, preferably 3 hours. After concentrating, the residue can be extracted with an organic solvent such as 10% CHCl3 / MeOH and followed by washing the organic layer with sat NaHCO3solution,dried and evaporated.

[0164] The residue obtained can be purified on a silica gel column by elution with the solvent such as chloroform, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichloromethane-methanol mixture is used for elution. Step-q: Reduction of doble bond to obtain structural formula-XVII

[0165] Reduction of doble bond is carried out by treating the compound of formula- XVI with reducing agent in presence of suitable solvent to obtain the desired compound as represented by compound of formula-XVIIor methanol.

[0167] The solvent in step (q) may be selected from methanol.

[0168] The selective reduction of double bond takes place in presence of metal dichloride hexahydrate. conditions such as in the presence of nickel dichloride hexahydrate and methanol.

[0169] Step-(q) can be performed in the presence of sodium tetraborohydrate.

[0170] In one embodiment the solvent used is methanol.

[0171] The reaction mixture is stirred at RT for 2 to 4 hours, preferably 3 hours. After concentrating, the residue can be extracted with an organic solvent such as 15% CHCl3 / MeOH and followed by washing the organic layer with brain solution, dried and evaporated.

[0172] The residue obtained can be purified on a silica gel column by elution with the solvent such as chloroform, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichloromethane-methanol mixture is used for elution Step-r: Hydrogenation of compound-XVII to obtain structural formula-XVIII

[0173] De-protection is carried out by treating the compound of formula-XVIII with organic acid in presence of palladium catalysed to remove of all protecting groups to obtain the desired compound as represented by compound of formula-XVIII. XVII is the compound of formula-I’ wherein, R is H and y is selected form 1, 2 and 3.glacial acetic acid.

[0175] The de-protection can be performed in the presence of a metal catalyst.

[0176] In one embodiment the catalyst used in step-(r) is palladium black.

[0177] To a solution of compound formula-XVII, dissolved in organic acid added with palladium catalyst. And the compound subjected to hydrogenolysis for 12 h to 28 h.

[0178] The catalyst is filtered off, and, after addition of water, the filtrate is evaporated under diminished pressure. The residue obtained can be purified on a silica gel column by elution with the solvent such as dichloromethane, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichloromethane-methanol mixture is used for elution. Step-s: Re-acylation of compound-XVII to obtain structural formula-XIX

[0179] Re-acylation is carried out by treating the compound of formula-XVII with organic acid chlorides (R1COCl) in presence of suitable base as solvent to obtain the desired compound as represented by compound of formula-XIXIPEA

[0181] The solvent in step (s) may be selected as Pyridine

[0182] The re-acylation takes place in the presence of organic base conditions such as in the presence of Pyridine as base as well as solvent.

[0183] Step-(s) can be performed in the presence of acid chlorides.

[0184] In one embodiment the solvent used is Pyridine.

[0185] The reaction mixture is stirred at -10 °C for 2 to 3 hours, preferably 2 hours. After concentrating, the residue can be extracted with an organic solvent such as DCM and followed by washing the organic layer with 1N HCl solution, dried and evaporated.

[0186] The residue obtained can be purified on a silica gel column by elution with a solvent such as dichloromethane, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichloromethane-methanol mixture is used for elution. Step-t: deprotection of all groups to obtain formula-I’’

[0187] De-protection is carried out by treating the compound of formula-XIX with organic acid in presence of palladium catalysed to remove of all protecting groups to obtain the desired compound as represented by compound of formula-I’’

[0188] wherein, R , R1is selected from the group consisting of substitutedor unsubstituted C4-C14 linear or branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and y is selected form 1, 2 and 3,

[0189] In one embodiment the solvent used in step-(t) for deprotection is Tetrahydrofuran (THF) and water (H2O).

[0190] The de-protection can be performed in the presence of a metal catalyst. In one embodiment the catalyst used in step-(t) is palladium black.

[0191] To a solution of compound formula-XIX, dissolved in THF / H2O added with palladium black catalyst. And the compound subjected to hydrogenolysis for 12 h to 48 h.

[0192] The catalyst is filtered off, and, after addition of water, the filtrate is evaporated under diminished pressure. The residue obtained can be purified on a silica gel column by elution with the solvent such as dichloromethane, alcohols such as methanol, ethanol etc. or mixture thereof. In one embodiment dichloromethane-methanol mixture is used for elution.

[0193] The cyclic glycolipopeptide compounds of the present invention are evaluated for its activity. The safety of the cyclic glycolipopeptide compounds of the present invention is established through cytotoxicity and pyrogenicity assays.

[0194] In a further aspect for the invention the vaccine formulations are prepared which comprises antigen selected from a live attenuated vaccine antigen, inactivated vaccine antigen, subunit vaccine antigen, a conjugate vaccine antigen, and recombinant vaccine antigen or any combinations thereof.

[0195] Optionally, the formulation may comprise one or more of pharmaceutically acceptable excipients, diluents and other additives as may be required to formulate the vaccine formulation. Non-limiting, the above vaccine formulation may be a bacterial vaccine, a viral Vaccine or any potential infectious pathogens against mammals.EXAMPLES:

[0196] The cyclic glycolipopeptides compounds, their synthesis and its intermediate compounds used for synthesis and evaluation of these cyclic glycolipopeptide compounds as an adjuvant are further explained and demonstrated by way of below non-limiting examples. The reaction steps shown in above Scheme-A are further described in below experimental examples Example 1: SCHEME-A: Experimental procedure for the synthesis of propargyl L-alanyl-D-isoglutamine benzyl ester:

[0197] The cyclic 1, 2, 3-triazole glycolipopeptide compounds as described and disclosed in this invention contain a propargyl L-alanyl-D-isoglutamine benzyl ester entity. Which required for the final preparation of cyclic 1,2,3-triazole glycolipopeptide compounds according to the present invention was synthesized by the method as shown in above reaction Scheme-A and examples as described below Example- 1.1: Preparation of (R)-2-amino-5-(benzyloxy)-5- oxopentanoic acid [2]

[0198] A mixture of D-Glutamic acid Compound 1 (4.0 g, 0.340mols) and anhy. Na2SO4 (30 g) was suspended in benzyl alcohol (500mL) and BF3. Et2O (54%, 21ml, 0.170mols) was added by means of a syringe. The suspension was stirred at RT for 14 hrs. The mixture was diluted with absolute THF (150mL) and filtered with the aid of charcoal. The clear filtrate was treated with Et3N (93mL, 0.680mols) and concentrated under vacuum until a slurry residue was formed. The viscous residue was triturated with EtOAc (500 mL) and the precipitated solid was isolated by suction and washed with additional solvent to afford compound 2 as a white solid (75 g, 93%) yield with respect to the trace amount starting material). Example-1.2: Preparation of (R)-5-(benzyloxy)-2-(tert-butoxy carbonyl amino)-5- oxopentanoic acid [3]

[0199] To a solution of the compound 2 (30 g, 0.126mols) in dioxane and water (1:1, 500mL) and NaHCO3 (24g, 0.506mols) were added at 0 °C then Boc2O (43mL,0.189mols) was added and the mixture was stirred overnight for 15 hours. The solvent was removed under reduced pressure and the residue was diluted with water (200 mL), then washed with EtOAc (3 x 100 mL). The aqueous layer was adjusted to pH 2-3 with a 1N HCl solution and extracted with EtOAc (3 x 100 mL). The combined organic extracts were washed with brine, dried over Na2SO4and the solvent was removed under reduced pressure to afford (R)-5-(benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid compound 3 (28g, 96%) as a viscous colourless oil. with the following NMR characteristics.1H NMR (500 MHz, CDCl3) δ 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 Calculated for C17H23NO6:m / z 337.15, found 260 [M + H]+338.50. Example-1.3: Preparation of 1-allyl 5-benzyl (tert-butoxycarbonyl)-L-alanyl-D- glutamate [4]:

[0200] To the stirring solution of compound 3 (20 g, 0.0593mols) in dry DCM (200 mL) and K2CO3(12g, 0.0890mols) were added, then propargyl broomide (9 mL, 0.118mols) were added at 0°C. The reaction mixture was stirred at 0oC for 6hrs, after completion of all starting material as indicated by thin-layer chromatography. The reaction mixture filtered and extracted in DCM (3×100 mL) organic phase was washed with water then followed by washing with brine solution (60mL) 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 (19g, 86%) as a white solid. characterized by NMR.1H NMR (400 MHz, CDCl3) δ 7.39 – 7.30 (m, 5H), 5.12 (s, 2H), 4.78 – 4.65 (m, 2H), 4..42 – 4.33 (m, 1H), 2.55 – 2.39 (m, 3H), 2.29 – 2.17 (m, 1H), 1.99 (td, J = 14.7, 8.3 Hz, 1H), 1.43 (s, 9H)ppm; LRMS (ESI) Calculated for C20H25NO6: m / z 375.16, Found [M+Na]+398.25. Example-1.4: preparation of 5-benzyl 1-(prop-2-yn-1-yl) (tert-butoxycarbonyl)- L-alanyl-D-glutamate[5]:

[0201] t-Butoxycarbonyl-D-isoglutamine benzyl ester compound 4 (10 g, 0.0266mols) was dissolved in (20%) trifluoroacetic acid and DCM (50mL) and the resulting solutionwas stirred at room temperature for 15mins. Trifluoroacetic acid was then removed, and the residue was triturated with toluene. This D-isoglutamine benzyl ester trifluoroacetate was dried over on KOH pellets followed by using trap. To the stirring solution of t-butoxycarbonyl-L-alanine (5 g, 0.0266mols) in dry THF (200 mL), EDC.HCl (10g, 0.0533mols) and HOBt (6 g, 0.0400mols) were added at 0°C After stirring at RT for 30mins, to this solution D-isoglutamine benzyl ester trifluoroacetate dissolved in THF was added followed by DIPEA (9.6 mL, 0.0533mols), then the reaction was stirred at RT for 15hrs, the solution was then concentrated and the residue extracted with EtOAc (200 mL). The EtOAc layer was washed successively with 5% NaHCO3, 10% citric acid, and water, then dried over Na2SO4 and evaporated. The residue was triturated with petroleum ether giving crystals which were recrystallized from EtOAc-petroleum ether to yield compound 5 (10g, yield 84%) as a white solid with the following characterized by NMR;1H NMR (500 MHz, CDCl3) δ 7.39 – 7.30 (m, 5H), 6.92 (br, 1H), 5.12 (s, 2H), 4.92 (br, 1H), 4.75 – 4.67 (m, 2H), 4.64 (td, J = 8.0, 5.1 Hz, 1H), 4.18 (br, 1H), 2.53 – 2.39 (m, 3H), 2.30 – 2.22 (m, 1H), 2.09 – 1.99 (m, 1H), 1.44 (s, 9H), 1.34 (d, J = 7.1 Hz, 3H)ppm; LRMS (ESI) Calculated for C23H30N2O7 : m / z 446.20, Found [M+Na]+469. Example-1.5: 5-benzyl 1-(prop-2-yn-1-yl) (tert-butoxycarbonyl)-L-alanyl-D- glutamate trifluoroacetate [6]

[0202] Compound 5 propargyl t-Butoxycarbonyl-L-alanyl-D-isoglutamine benzyl ester (2g, 1.87mmols) which was dissolved in (20%) trifluoroacetic acid (4mL) and dichloromethane (16mL) the resulting solution was stirred at room temperature for 30 mins. Trifluoroacetic acid was then removed, and the residue was triturated with toluene to obtain propargyl L-alanyl-D-isoglutamine benzyl ester trifluoroacetate compound 6. was dried over KOH pellets followed by using a trap. To afford oily compound 6 (1.3 g, 86%) which is used for next step without further purification. Example-2: SCHEME-B: Experimental procedure for the synthesis of allyl L-alanyl-D-isoglutamine benzyl ester:

[0203] The cyclic glycolipopeptide with C4-homologue compounds as described and disclosed in this invention contains an allyl L-alanyl-D-isoglutamine benzyl ester entity. Which required for the final preparation of cyclic glycolipopeptide with C4-homologue compounds according to the present invention was synthesized by the method as shown in above reaction Scheme-B and examples as described below Example-2.1: Preparation of 1-allyl 5-benzyl (tert-butoxycarbonyl)-D-glutamate [7]

[0204] To a solution of above acid compound 3 (10 g, 0.0296mols) in dry toluene (150mL) and DIPEA (10.6mL, 0.0593mols) were added, then allyl broomide (3.8 mL, 0.0445 mols) were added at 0°C. The reaction mixture was stirred allowed to stirr at 70oC for 10hrs, after completion of all starting material as indicated by thin-layer chromatography. The reaction mixture was extracted in DCM (3×100 mL) organic phase was washed with water then followed by washing with 1N HCl solution (50 mL) and dried over anhydrous sodium sulphate. The solvent was removed in vacuum and the residue was purified by column chromatography in 4% MeOH / DCM used as eluent to furnish the desired compound 7 (9.5g, 86%) as syrup compound. Characterized by NMR.1H NMR (400 MHz, CDCl3) δ 7.35 – 7.25 (m, 5H), 5.87 (ddd, J = 16.3, 11.0, 5.8 Hz, 1H), 5.31 (dd, J = 17.2, 1.3 Hz, 1H), 5.23 (d, J = 10.4 Hz, 1H), 5.09 (s, 2H), 4.63 (s, 2H), 4.59 (d, J = 5.8 Hz, 1H), 2.51 – 2.34 (m, 2H), 2.24 – 2.12 (m, 1H), 1.93 (td, J = 14.7, 8.2 Hz, 1H), 1.42 (s, 9H)ppm; LRMS (ESI) Calculated for C20H27NO6 : m / z 377.18, Found [M+Na]+400.30. Example-2.2 : preparation of 1-allyl 5-benzyl (tert-butoxycarbonyl)-L-alanyl-D- glutamate

[0205] [8] Compound 7 (8 g, 0.0212mols) was dissolved in (20%) trifluoroacetic acid and DCM (20mL) and the resulting solution was stirred at room temperature for 15mins. Trifluoroacetic acid was then removed and the residue was triturated with toluene. This allyl D-isoglutamine benzyl ester trifluoroacetate was dried over on KOH pellets followed by using trap. To the stirring solution of t-butoxycarbonyl-L-alanine(3.4g, 0.0180mols) dissolved in dry THF (60mL), EDC.HCl (6.9 g, 0.0361mols) and HOBt (4.1g, 0.0270mols) were added at 0°C After stirring at RT for 30mins, to this solution allyl D-isoglutamine benzyl ester trifluoroacetate (5g, 0.0180mols) dissolved in THF was added followed by DIPEA (8.1 mL, 0.0451mols), then the reaction was stirred at RT for 15hrs, the solution was then concentrated and the residue extracted with EtOAc (200 mL). The EtOAc layer was washed successively with sat. NaHCO3, 10% citric acid, and water (100 mL), then dried over Na2SO4 and evaporated. The crude compound was purified by column chromatography in 3% MeOH / DCM to furnish desired compound 8 which were yield to (7g, 87%) as a oily with the following characterized by NMR;1H NMR (400 MHz, CDCl3) δ 7.38 – 7.31 (m, 5H), 5.94 – 5.83 (m, 1H), 5.35 – 5.22 (m, 2H), 5.11 (s, 2H), 5.04 (d, J = 6.2 Hz, 1H), 4.66 – 4.59 (m, 3H), 2.54 – 2.36 (m, 2H), 2.32 – 2.20 (m, 1H), 2.08 – 1.97 (m, 1H), 1.43 (s, 9H), 1.35 (d, J = 7.1 Hz, 1H)ppm; LRMS (ESI) Calculated for C23H32N2O7 : m / z 448.22, Found [M+Na]+471.35. Example-2.3:1-allyl 5-benzyl (tert-butoxycarbonyl)-L-alanyl-D-glutamate trifluoroacetate [9]:

[0206] Compound 8 t-Butoxycarbonyl-L-alanyl-D-isoglutamine benzyl ester (5g, 0.0111mols) which was dissolved in (20%) trifluoroacetic acid (4mL) and dichloromethane (16mL) the resulting solution was stirred at room temperature for 45 mins. Trifluoroacetic acid was then removed, and the residue was triturated with toluene to obtain allyl L-alanyl-D-isoglutamine benzyl ester trifluoroacetate compound 9. This allyl L-alanyl-D-isoglutamine benzyl ester trifluoroacetate was dried over KOH pellets followed by using a trap. To afford syrupy compound 9 (3 g, 78%) which is used for next step without further purification. Example-3: SCHEME-C Example-3.1: Preparation of N-((2S,3R,4R,5S,6R)-2-(benzyloxy)-4,5-dihydroxy- 6-(hydroxymethyl) tetrahydro-2H-pyran-3-yl) acetamide

[0011] Anomeric benzylation of N-acetyl D-Glucosamine (11):

[0207] Acetyl chloride (27.4mL, 0.39mmol) was slowly added to a suspension of N-acetyl D-glucosamine compound 10 (85g, 0.39mmol) in anhydrous benzyl alcohol (500 mL) under nitrogen atmosphere. The suspension was stirred at room temperature for 2 h, heated to 50 °C for 10 h, and then cooled to room temperature. The resulting yellow solution was slowly poured onto Et2O (2L) in ice-water bath, and the mixture was vigorously stirred for 2h at 0 °C. The precipitate was recovered by filtration and dried under vacuum to – afford N-((2S,3R,4R,5S,6R)-2-(benzyloxy)-4,5-dihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-3-yl) acetamide Compound 8 (113 g, 94%) as a white solid characterized by NMR.1H NMR (400 MHz, CD3OD) δ 7.45 – 7.28 (m, 5H), 4.79 (d, J = 12.0 Hz, 1H), 4.54= 12.0 Hz, 1H), 3.95 (d, J = 3.6 Hz, 1H), 3.92 (d, J = 3.6 Hz, 1H), 3.87 (dd, J = 11.5, 1.8 Hz, 1H), 3.77 (d, J = 3.8 Hz, 1H), 3.76 – 3.71 (m, 1H), 3.71 – 3.68 (m, 1H), 3.45 – 3.37 (m, 1H), 1.99 (s, 3H)ppm; ESI-MASS Calculated :m / z 334.1 found: 357.12 [M+Na]+Example-3.2: Preparation of N-((2R,4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8- hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl)acetamide Benzylidene protection (12):

[0208] Benzyl 2-acetamido-2-deoxy-D-glucopyranoside Compound 11 (50 g, 0.149mols) was added to a well-stirred mixture of PTSA (2.8g, 0.0149mol) in dry ACN and dimethyl benzaldehyde acetal (45 mL, 0.299mols). This reaction mixture was vigorously stirred at room temperature for 5 hrs, then stirred at rt for 2 hours to dissolve a small amount of remaining solid. The well-stirred reaction solution was now diluted with petroleum ether (1 L) till it precipitate out, then filtered on Buchner funnel and washed with 20% EtOH / n-Hexane (500 mL) under continuous vacuum to furnish white finely divided solid compound 12 to yield (54 g, 91%) as fluffy soid with the following NMR characteristics.1H NMR (300 MHz, CD3OD) δ 7.52 – 7.22 (m, 10H), 5.55 (s, 1H), 4.90 (d, J = 3.7 Hz, 1H), 4.70 (d, J = 11.8 Hz, 1H), 4.47 (d, J = 11.8 Hz, 1H), 4.20 (dd, J = 9.8, 4.4 Hz, 1H), 4.06 (dd, J = 10.4, 3.7 Hz, 1H), 3.81 (ddd, J = 31.1, 17.8, 9.9 Hz, 3H), 3.55 (t, J = 9.2 Hz, 1H), 1.94 (s, 3H)ppm; LRMS (ESI) Calculated : m / z 399, found [M+H]+400.15, [M+Na]+422.23. Example-3.3:(2R,4aR,6S,7R,8R,8aS)-7-amino-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d] [1,3]dioxin-8-ol Deacylation:(13)

[0209] A mixture of Compound 12 (40 g, 0.100mols) dissolved in KOH in 95% EtOH was stirred at 0°C for 10 min then shifted to heating at 100°C for 16hrs in an inert atmosphere. After complete conversion to amine the hot solution i.e., the reaction mixture was added to the hot water in conical flask carefully and it was kept in the fridge for overnight then the resulting lumps of crude product were broken up. The precipitate formed was filtered and washed with hexane thoroughly then it was recrystallized from EtOH-water with charcoal decolorization followed by recrystallization from tetrahydrofuran and di-isopropyl ether (THF andi-Pr2O) to get the required compound 13 to yield (29 g, 82%) as light-yellow solid which is characterized by NMR.1H NMR (400 MHz, CDCl3): δ 7.49 (m, 2H), 7.41 – 7.31 (m, 8H), 5.55 (s, 1H), 4.92 (d, J = 7.2 Hz, 1H, anomeric), 4.75 (d, J = 11.7 Hz, 1H), 4.53 (d, J = 11.7 Hz, 1H), 4.24 (dd, J = 10.1, 4.8 Hz, 1H), 3.89 (m, 1H), 3.76 (m, 2H), 3.49 (t, J = 9.3 Hz, 1H), 2.81 (dd, J = 9.7, 3.7 Hz, 1H), 1.96 (br, 2H) ppm; ESI-MS Calcd. for C20H23NO5 : m / z 357 found [M + H]+: 358. Example-3.4: Preparation of (2R,4aR,6S,7R,8R,8aS)-7-azido-6-(benzyloxy)-2- phenylhexahydropyrano[3,2-d][1,3]dioxin-8-ol (14) Azide installation:

[0210] A suspension of sodium azide (2.6 g, 40.33 mmol) in 8 mL of acetonitrile was cooled in ice bath. Then triflic anhydride (9.4 g, 33.61 mmol) was added by a syringe during 5 min while stirring under argon. After the reaction was maintained for 2 h in ice bath, the in situ generated triflyl azide TfN3 (5.9 g, 33.72 mmol) solution acetonitrile solution of triflyl azide (5.9 g, 33.72 mmol) was then cannulated drop wise into amine compound 13 (10g, 28.01mmol) in 100 mL acetonitrile containing CuSO4.5H2O (1.4g, 5.60mmol) and triethylamine (5.7 g, 56.02 mmol) for the subsequent diazotransfer reaction. The reaction mixture was allowed to warm to room temperature for 10 h. After completion of reaction, the solvent was removed under reduced pressure. Theresidue upon dilution with ethyl acetate (100 mL), the reaction mixture was washed with 5% HCl (50 mL), a saturated solution of NaHCO3(70 mL) and brine (50 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography (3% MeOH / DCM, on silica gel) to afford compound 14 as an off white fluffy solid (9.1 g, 84%).1H NMR (500 MHz, CDCl3) δ 7.51 – 7.46 (m, 2H), 7.43 – 7.31 (m, 8H), 5.54 (s, (d, J = 3.7Hz, 1H), 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 Calcd. for C20H21N3O5: m / z 383.50 found [M + H]+384.18. Example-3.5: 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 (15) O-Alkylation:

[0211] Sodium hydride 60% dispersion in mineral oil, (3.2g, 83.9 mmol) was washed with hexanes (3 mL) three time to remove mineral oil and was suspended in 80 mL DMF under argon. Compound 6 (8 g, 20.8 mmol) was added into the above solution slowly, followed by (S)-2-chloropropanoic acid (2.2 mL, 20.5mmol,) addition dropwise. In 40 mL dry DMF The resulting mixture was stirred vigorously at room temperature until there was no gas evolution. Then it was allowed stirr at rt for 14 h (max speed of stirring), after completion of reaction quenched with 60 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 (3 × 100 mL), and dried under continuous vacuum until dry to afforded compound 15 to yield (8.3 g, 87 %) as an off white solid.1H NMR (500 MHz, CDCl3): δ 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. Calculated for C23H25N3O7: m / z 455 found [M + H]+456 Example-3.6: 5-benzyl 1-(prop-2-yn-1-yl) ((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7- azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8- yl)oxy)propanoyl)-L-alanyl-D-glutamate (16)

[0212] A solution of the peptide fragment 6 from Scheme-A as disclosed in Example 1.5 (4.6g, 13.1mmol) in THF (50mL) with DIPEA (4 mL 21.9mmol) was treated with compound 15 (5 g, 10.9mmol) in dry THF (50 mL) and EDC· HCl (4.2 g, 21.9mmol), HOBt (2.5 g, 16.4mmol) was added to the solution. The solution was stirred 10h at room temperature. After the complete consumption of the starting material as monitored by thin-layer chromatography, the solvent was removed, and the residue was extracted with CH2Cl2(100 mL) and brine (50 mL). The extract was dried over Na2SO4, concentrated, filtered, and purified by column chromatography (3% MeOH in chloroform, silica gel) to furnish compound 16 (7 g, 81%) as a white fluffy solid.1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 7.3 Hz, 1H), 7.46 – 7.42 (m, 2H), 7.41 – 7.29 (m, 13H), 7.05 (d, J = 7.9 Hz, 1H), 5.56 (s, 1H), 5.13 – 5.09 (m, 3H), 4.77 (d, J = 11.7 Hz, 1H), 4.68 (dd, J = 5.3, 2.5 Hz, 2H), 4.64 – 4.57 (m, 2H), 4.48 (t, J = 7.2 Hz, 1H), 4.32 (q, J = 6.8 Hz, 1H), 4.25 (dd, J = 10.2, 4.8 Hz, 1H), 3.94 – 3.86 (td, J = 9.5, 4.3 Hz, 2H), 3.75 (t, J = 10.3 Hz, 1H), 3.61 (t, J = 9.3 Hz, 1H), 3.42 (dd, J = 10.1, 3.7 Hz, 1H), 2.53 – 2.36 (m, 3H), 2.30 – 2.19 (m, 1H), 2.08 – 1.97 (m, 1H), 1.41 (dd, J = 8.3, 7.0 Hz, 6H) ppm; LRMS (ESI) Calculated for C41H45N5O11 : m / z 783.31, Found [M+Na]+806.60, [M+K]+822.55. Example-3.7: procedure for the synthesis of compound

[0017]

[0213] To a mixture of compound 16 (5 g, 6.3mmol), CuSO4.5H2O (790 mg 3.1mmol), Sodium ascorbate (1.26 g, 6.3mmol), dissolved in 2.5 Ltr of THF / H2O (4:1) was stirred for 8h at rt until the TLC shows consumption of starting material. Reaction mixture was then directly vacuum dried and redissolved in CH2Cl2. Organic layer was washed with water and aqueous layer extracted with DCM (3x100 mL). Combined all organic layer was dried on Na2SO4, evaporated and then purified by chromatography on silicagel with 6% MeOH / DCM used as eluents to afford intra cyclised triazol compound 17 to yield (4.6 g, 92%) as white solid. Which is by characterisation by NMR.1H NMR (400 MHz, DMSO-d6) δ 8.64 (d, J = 6.4 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H), (s, 1H),7.51 (dd, J = 7.8, 1.6 Hz, 2H), 7.45 – 7.22 (m, 11H), 7.12 (d, J = 6.9 Hz, 2H), 5.78 (s, 1H), 5.57 (d, J = 13.7 Hz, 1H), 5.12 (s, 2H), 5.03 (d, J = 3.5 Hz, 1H), 4.94 – 4.87 (m, 2H), 4.66 (d, J = 12.3 Hz, 1H), 4.45 (dd, J = 10.7, 8.8 Hz, 1H), 4.38 – 4.31 (m, 2H), 4.30 – 4.18 (m, 3H), 3.96 (t, J = 8.9 Hz, 1H), 3.91 – 3.82 (m, 2H), 2.57 (t, J = 7.5 Hz, 2H), 2.14 – 1.92 (m, 2H), 1.15 (d, J = 7.2 Hz, 3H), 1.08 (d, J = 6.5 Hz, 3H)ppm; LRMS (ESI) Calculated for C41H45N5O11: m / z 783.31, Found [M+H]+784.60, [M+NA]+806.60, [M+K]+822.55. Example-3.8: procedure for the synthesis of compound (18):

[0214] To the magnetically stirring solution of compound 17 (3 g, 3.7 mmol) in a mixture of TFA (16 mL), water (4.0 mL), then stirred at room temperature for 2 h. after consumption of all stating material as monitored by thin-layer chromatography. The reaction mixture nutralised with sat NaHCO3and extracted with 10% MeOH / DCM and the solvent was removed under reduced pressure, then the resulting crude reaction mixture was purified by column chromatography (7% MeOH in DCM on silica gel) to furnish compound 18 (2.3g, 86%) as white solid in good yield. Which is characterized by NMR, MASS-ESI.1H NMR (400 MHz, DMSO-d6) δ 8.63 (d, J = 6.5 Hz, 1H), 8.11 (d, J = 7.2 Hz, 1H), 7.74 (s, 1H), 7.41 – 7.19 (m, 8H), 7.11 (d, J = 7.0 Hz, 2H), 5.61 (dd, J = 32.4, 10.4 Hz, 2H), 5.12 (s, 2H), 4.90 (d, J = 13.5 Hz, 1H), 4.85 (d, J = 3.4 Hz, 1H), 4.65 – 4.56 (m, 3H), 4.31 (d, J = 12.6 Hz, 1H), 4.26 – 4.20 (m, 1H), 4.19 – 4.12 (m, 1H), 3.77 – 3.69 (m, 1H), 3.63 – 3.50 (m, 3H), 2.57 (t, J = 7.5 Hz, 2H), 2.14 – 1.93 (m, 2H), 1.17 (d, J = 7.2 Hz, 3H), 1.11 (d, J = 6.5 Hz, 3H)ppm; LRMS (ESI) Calculated for C34H41N5O11: m / z 695.28, Found [M+H]+696.70, [M+Na]+718.70. Example-3.9: procedure for the removal of protecting group for the synthesis of compounds

[0019]

[0215] To a solution of compound 18 (200 mg, 0.28mmol) in THF (8 mL) and H2O (2 mL) was taken into 50mL Rbf, then 10% Pd / C were added as catalytic amount. The resulting reaction mixture was hydrogenated at RT by using a hydrogen-filled balloon for 20h after complete consumption of all starting material as indicated by thin-layer chromatography. The reaction mixture was filtered through a celite cake. Collected filtrate evaporation by azotroping with toluene, the residue was redissolved in pure methanol and diethyl ether were added untill it forms white precipitate then filter the white solid dried under continuous vacuum till dryness to afford compound 19 in yield (125mg, 83%) as white solid.1H NMR (400 MHz, D2O) δ 8.04 (s, 1H), 5.57 (d, J = 11.8 Hz, 1H), 5.20 (d, J = 8.4 Hz, 1H), 5.09 (d, J = 9.4 Hz, 1H), 4.52 – 4.35 (m, 2H), 4.27 – 4.12 (m, 2H), 4.10 – 4.03 (dd, J = 10.3, 7.8 Hz, 1H), 4.01 – 3.89 (m, 1H), 3.88 – 3.68 (m, 3H), 2.55 (t, J = 6.3 Hz, 2H), 2.34 – 2.04 (m, 2H), 1.39 – 1.25 (m, 6H)ppm; LRMS (ESI) Calculated for C20H29N5O11 : m / z 515.18, Found [M+H]+516.15. Example-4: General procedure for the synthesis of compounds (20a to 20d)

[0216] To the magnetically stirring solution of compound 18 (200 mol%) in a anhydrous pyridine (5 mL) at – 10 °C stirred for 10min, then acid chlorides (2 mol%) were added drop wise over 5min then reaction allowed to stirr at the same temperature for 2 h. after consumption of all stating material as indicated by thin-layer chromatography. The reaction mixture nutralised with 1N HCl and extracted with 10% MeOH / DCM and the solvent was removed under reduced presure. The resulting crude reaction mixture was purified by Columnn chromatography (5% MeOH in DCM on silica gel) to give compounds 21a to 21d as white solid (85 - 90%) in a quantitative yields. Example-4.1: ((11S,22S,23R,24R,25S,26R,4R,7S,10R, Z)-22-(benzyloxy)-10-(3- (benzyloxy)-3-oxopropyl)-25-hydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26- tetrahydro-11H,22H-3,12-dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)- pyranacyclotridecaphane-26-yl)methyl hexanoate (20a)

[0217] Compound 18 (140mg, 0.2mmol) and (a) hexanoyl chloride (268 μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-4 to provide compound 20a (137mg, 86%) in yield;1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.37 – 7.22 8H), 7.11 – 7.06 (m,2H), 6.67 (d, J = 7.2 Hz, 1H), 5.27 (d, J = 12.8 Hz, 1H), 5.13 (s, 2H), 5.10 (d, J = 12.8 Hz, 1H), 4.99 (d, J = 3.6 Hz, 1H), 4.81 (dd, J = 10.7, 3.6 Hz, 1H), 4.63 (d, J = 11.8 Hz, 1H), 4.53 (dd, J = 12.3, 4.6 Hz, 1H), 4.44 – 4.18 (m, 7H), 4.02 – 3.95 (m, 1H), 3.58 (dd, J = 14.9, 9.1 Hz, 1H), 2.57 – 2.47 (m, 2H), 2.40 (t, J = 7.5 Hz, 2H), 2.27 – 2.15 (m, 2H), 1.71 – 1.61 (m, 2H), 1.35 – 1.18 (m, 10H), 0.89 (t, J = 5.6 Hz, 3H)ppm; LRMS (ESI) Calculated for C40H51N5O12 : m / z 793.35, Found [M+H]+794.85, [M+Na]+816.80. Example-4.2: ((11S,22S,23R,24R,25S,26R,4R,7S,10R,Z)-22-(benzyloxy)-10-(3- (benzyloxy)-3-oxopropyl)-25-hydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26- tetrahydro-11H,22H-3,12-dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)- pyranacyclotridecaphane-26-yl)methyl tetradecanoate (20b)

[0218] Compound 18 (140mg, 0.2mmol) and (b) myristoyl chloride (500 μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-4 to provide compound 20b (159mg, 87%) in yield;1H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.32 – 7.15 (m, 8H), 7.02 (dd, J = 6.5, 3.0 Hz, 2H), 6.37 (d, J = 6.7 Hz, 1H), 5.24 (d, J = 12.8 Hz, 1H), 5.10 – 5.02 (m, 3H), 4.92 (d, J = 3.6 Hz, 1H), 4.72 (dd, J = 10.8, 3.6 Hz, 1H), 4.57 (d, J = 11.9 Hz, 1H), 4.52 (dd, J = 12.5, 4.2 Hz, 1H), 4.39 – 4.10 (m, 6H), 4.04 (d, J = 6.0 Hz, 1H), 3.90 (d, J = 8.6 Hz, 1H), 3.49 (dd, J = 15.7, 9.1 Hz, 1H), 2.45 (t, J = 7.6 Hz, 2H), 2.34 (t, J = 7.6 Hz, 2H), 2.20 – 2.00 (m, 2H), 1.64 – 1.52 (m, 2H), 1.30 – 1.12 (m, 26H), 0.81 (t, J = 6.9 Hz, 3H)ppm; LRMS (ESI) Calculated for C48H67N5O12: m / z 905.47, Found [M+2]+907.00. Example-4.3: ((11S,22S,23R,24R,25S,26R,4R,7S,10R,Z)-22-(benzyloxy)-10-(3- (benzyloxy)-3-oxopropyl)-25-hydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)- pyranacyclotridecaphane-26-yl)methyl 3-methyl butanoate (20c)

[0219] Compound 18 (140mg, 0.2mmol) and (c) 3-methylbutyryl chloride (240μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-4 to provide compound 20c (138mg, 88%) in yield;1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.29 – 7.12 , 6.99 (dd, J = 6.5,2.9 Hz, 1H), 6.80 (d, J = 7.0 Hz, 1H), 5.17 (d, J = 12.8 Hz, 1H), 5.05 (s, 2H), 5.00 (d, J = 12.5 Hz, 1H), 4.90 (d, J = 3.6 Hz, 1H), 4.74 (dd, J = 10.7, 3.5 Hz, 1H), 4.55 (d, J = 11.8 Hz, 1H), 4.42 (dd, J = 12.2, 5.0 Hz, 2H), 4.35 – 4.14 (m, 6H), 3.93 (dd, J = 10.0, 2.6 Hz, 1H), 3.52 (dd, J = 17.1, 8.2 Hz, 1H), 2.52 – 2.38 (m, 2H), 2.20 (d, J = 7.0 Hz, 2H), 2.16 – 1.99 (m, 3H), 1.21 (d, J = 6.5 Hz, 3H), 1.11 (d, J = 6.9 Hz, 3H), 0.89 (d, J = 6.6 Hz, 6H)ppm; LRMS (ESI) Calculated for C39H49N5O12: m / z 779.33, Found [M+H]+780.65, [M+H]+802.60. Example-4.4: ((11S,22S,23R,24R,25S,26R,4R,7S,10R,Z)-22-(benzyloxy)-10-(3- (benzyloxy)-3-oxopropyl)-25-hydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26- tetrahydro-11H,22H-3,12-dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)- pyranacyclotridecaphane-26-yl)methyl cyclopropanecarboxylate (20d)

[0220] Compound 18 (140mg, 0.2mmol) and (d) cyclopropanecarbonyl chloride (200 μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-4 to provide compound 20d (132mg, 86%) in yield;1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.28 – 7.14 (m, 8H), 7.03 – 6.99 (m, 2H), 6.69 (d, J = 7.2 Hz, 1H), 5.21 (d, J = 12.8 Hz, 1H), 5.05 (s, 2H), 5.01 (d, J = 12.5 Hz, 1H), 4.91 (d, J = 3.6 Hz, 1H), 4.75 (dd, J = 10.8, 3.6 Hz, 1H), 4.56 (d, J = 11.9 Hz, 1H), 4.43 (dd, J = 12.3, 4.6 Hz, 1H), 4.38 – 4.12 (m, 6H), 3.93 – 3.87 (m, 1H), 3.50 (dd, J = 17.1, 8.3 Hz, 1H), 2.51 – 2.38 (m, 2H), 2.17 – 1.97 (m, 2H), 1.67 – 1.59 (m, 1H), 1.21 (d, J = 6.5 Hz, 3H), 1.12 (d, J = 7.0 Hz, 3H), 0.98 – 0.81 (m, 4H)ppm; LRMS (ESI) Calculated for C38H49N5O12 : m / z 763.30, Found [M+H]+764.60, [M+H]+786.55. Example-5: General procedure for the synthesis of compounds [21a to 21d]

[0221] To a solution of compound 18 (100mol %), THF (15mL), H2O (3mL) and 10% Pd / C (20 mol%) was added. The resulting reaction mixture was hydrogenated at rt by using a hydrogen-filled balloon for 24h after complete consumption of all starting material as indicated by thin-layer chromatography and the reaction mixtured was filtered through a celite cake. Collected filtrate evaporation by azotroping with toluene, the residue was dissolved in pure methanol and diethyl ether were added untill it forms white precipitate then filter the white solid under continuous vacuum dryness to afford compound 21a to 21d (85 – 90%) in good yields Example-5.1: 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-26-((hexanoyloxy)methyl)- 22,25-dihydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12- dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid (21a)

[0222] Compound 20a (80mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 5 to provide compound 21a (54mg, 88%) in yield;1H NMR (400 MHz, CD3OD) δ 7.88 (s, 1H), 5.59 (d, J = 13.2 Hz, 1H), 5.42 (d, J = 12.8 Hz, 1H), 5.13 (dd, J = 15.2, 8.1 Hz, 1H), 5.00 (dd, J = 14.4, 7.1 Hz, 1H), 4.65 (dd, J = 10.4, 3.8 Hz, 1H), 4.50 – 4.41 (m, 1H), 4.36 (dd, J = 8.9, 6.0 Hz, 1H), 4.33 – 4.23 (m, 2H), 4.22 – 4.11 (m, 2H), 4.05 – 3.99 (m, 1H), 3.76 – 3.69 (m, 1H), 3.67 – 3.58 (m, 1H), 2.46 (dd, J = 16.1, 7.4 Hz, 2H), 2.37 (t, J = 7.4 Hz, 2H), 2.20 (td, J = 13.4, 6.6 Hz, 1H), 2.04 (td, J = 15.4, 7.4 Hz, 1H), 1.69 – 1.60 (m, 2H), 1.38 – 1.23 (m, 10H), 0.93 (t, J = 6.8 Hz, 3H)ppm; LRMS (ESI) Calculated for C26H39N5O12 : m / z 613.25, Found [M+H]+614.25, [M+Na]+633.55. Example-5.2: 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-4,7- dimethyl-5,8,11-trioxo-26-((tetradecanoyloxy)methyl)-23,24,25,26-tetrahydro- 11H,22H-3,12-dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10- yl)propanoic acid (21b)

[0223] Compound 20b (90mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 5 to provide compound 21b (63mg, 87%) in yield;1H NMR (400 MHz, CD3OD) δ 7.87 (s, 1H), 5.60 (d, J = 13.2 Hz, 1H), 5.43 (d, J = 12.8 Hz, 1H), 5.12 (dd, J = 10.4, 8.2 Hz, 1H), 4.99 (dd, J = 15.8, 7.3 Hz, 1H), 4.68 – 4.62 (m, 1H), 4.49 – 4.41 (m, 1H), 4.39 – 4.24 (m, 3H), 4.22 – 4.11 (m, 2H), 4.02 (dd, J = 10.4, 8.5 Hz, 1H), 3.76 – 3.70 (m, 1H), 3.67 – 3.57 (m, 1H), 2.49 – 2.40 (m, 2H), 2.37 (t, J = 7.3 Hz, 2H), 2.24 – 2.13 (m, 1H), 2.10 – 1.98 (m, 1H), 1.68 – 1.58 (m, 2H), 1.37 – 1.22 (m, 26H), 0.90 (t, J = 6.8 Hz, 3H)ppm; LRMS (ESI) Calculated for C34H55N5O12: m / z 725.38, Found 725. Example-5.3: 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-4,7- dimethyl-26-(((3-methylbutanoyl)oxy)methyl)-5,8,11-trioxo-23,24,25,26- tetrahydro-11H,22H-3,12-dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)- pyranacyclotridecaphane-10-yl)propanoic acid (21c)

[0224] Compound 20c (80mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 5 to provide compound 21c (51mg, 86%) in yield;1H NMR (400 MHz, CD3OD) δ 7.90 (s, 1H), 5.60 (d, J = 13.6 Hz, 1H), 5.44 (d, J = 12.8 Hz, 1H), 5.14 – 5.09 (m, 1H), 5.01 (dd, J = 14.2, 10.8 Hz, 1H), 4.69 – 4.62 (m, 2H), 4.48 (td, J = 12.3, 2.0 Hz, 1H), 4.36 (dd, J = 9.1, 6.0 Hz, 1H), 4.33 – 4.23 (m, 2H), 4.22 – 4.13 (m, 2H), 4.02 (dd, J = 10.5, 8.5 Hz, 1H), 3.76 – 3.58 (m, 1H), 2.48 – 2.39 (m, 2H), 2.26 (d, J = 4.3 Hz, 2H), 2.22 – 1.99 (m, 3H), 1.35 – 1.24 (m, 6H), 0.98 (d, J = 6.6 Hz, 6H)ppm; LRMS (ESI) Calculated for C25H37N5O12: m / z 599.24, Found [M+H]+600.45, [M+Na]+622.45. Example-5.3:3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-26- (((cyclopropanecarbonyl)oxy)methyl) -22,25-dihydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12- dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid (21d)

[0225] Compound 20d (80mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 5 to provide compound 21d (50mg, 86%) in yield;1H NMR (400 MHz, CD3OD) δ 7.90 (s, 1H), 5.62 – 5.58 (d, J = 13.5 Hz, 1H), 5.43 (d, J = 12.7 Hz, 1H), 5.18 – 5.09 (m, 1H), 5.04 – 4.96 (m, 1H), 4.71 – 4.63 (m, 1H), 4.49 – 4.34 (m, 2H), 4.32 – 4.23 (m, 2H), 4.22 – 4.11 (m, 2H), 4.02 (dd, J = 10.4, 8.5 Hz, 1H), 3.77 – 3.59 (m, 2H), 2.52 – 2.42 (m, 2H), 2.20 (td, J = 13.6, 6.6 Hz, 1H), 2.04 (dt, J = 7.7, 5.6 Hz, 1H), 1.72 – 1.65 (m, 1H), 1.35 – 1.23 (m, 6H), 0.99 – 0.89 (m, 4H)ppm; LRMS (ESI) Calculated for C24H33N5O12: m / z 583.21, Found [M+H]+584.30, [M+Na]+610.10. Example-6: SCHEME-D Example-6.1: 1-allyl 5-benzyl ((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7- azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8- yl)oxy)propanoyl)-L-alanyl-D-glutamate (22)

[0226] A solution of compound 15 (5g, 10.9mmol) in dry THF (50 mL) was treated with EDC·HCl (4.2 g, 21.9mmol), HOBt (2.5g, 16.4mmol) and then peptide fragment 9 (4.6 g, 13.1mmol), from Scheme-B as disclosed in Example 2.3, which is dissolved in THF (20 mL ), DIPEA (1.7mL, 9.89mmol)) were cannulated into to the stirring solution of activated acid. Then the reaction mixture allowed to stir at RT for 16h. After the complete consumption of the starting material as monitored by thin-layer chromatography, solvent was removed under vacuo and the residue was extracted with CH2Cl2 (100 mL) and brine (2x50 mL). The extract was passed over anhydrous MgSO4, then concentrated, and crude compound purified by column chromatography (5% MeOH / CH2Cl2, on silica gel) to furnish desired compound 22 (7.5 g, 87%) as a white solid. Which is characterized by NMR.1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 7.3 Hz, 1H), 7.47 – 7.41 (m, 2H), 7.40 – 7.30 (m, 13H), 6.97 (d, J = 7.9 Hz, 1H), 5.92 – 5.81 (m, 1H), 5.56 (s, 1H), 5.33 – 5.20 (m, 2H), 5.13 – 5.08 (m, 3H), 4.77 (d, J = 11.8 Hz, 1H), 4.64 – 4.57 (m, 4H), 4.48 (p, J = 7.0 Hz, 1H), 4.32 (q, J = 6.8 Hz, 1H),4.24 (dd, J = 10.2, 4.8 Hz, 1H), 3.94 – 3.86 (m, 2H), 3.75 (t, J = 10.3 Hz, 1H), 3.60 (t, J = 9.3 Hz, 1H), 3.41 (dd, J = 10.1, 3.7 Hz, 1H), 2.52 – 2.34 (m, 2H), 2.30 – 2.19 (m, 1H), 2.07 – 1.96 (m, 1H), 1.41 (dd, J = 6.9, 5.1 Hz, 6H)ppm; LRMS (ESI) Calculated for C41H47N5O11 : m / z 785.32, Found [M+H]+786.60, [M+Na]+808.65. Example-6.2: 1-allyl 5-benzyl ((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7- amino-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8- yl)oxy)propanoyl)-L-alanyl-D-glutamate (23)

[0227] To the mechanically stirring solution of compound 22 (7g, 8.91mmol) in tetrahydrofuran (50 mL), and water (10 mL) at room temperature triphenylphosphine (3.5 g, 13.3 mmol) were added. The reaction mixture was stirred at 60 °C for 14 hours. After the completion of the starting material as monitored by thin-layer chromatography, solvent was removed, and the residue was extracted with CH2Cl2(2x100 mL) and brine (2x50 mL). The extract was dried over Na2SO4, concentrated, and redissolved in diethyl ether then n-pentane were added till precipitate out the compound followed by decanting excess triphenylphoshine decanted which dissolved in diethyl ether and same repeated once to afford white solid compound 23 (5.5 g, 82%) as a white solid1H NMR (400 MHz, CDCl3) δ 7.68 – 7.64 (m, 1H), 7.49 – 7.42 (m, 2H), 7.39 – 7.30 (m, 12H), 7.07 (d, J = 8.1 Hz, 1H), 5.93 – 5.81 (m, 1H), 5.57 (s, 1H), 5.34 – 5.21 (ddd, J = 13.8, 11.6, 1.3 Hz, 2H), 5.10 (s, 2H), 4.84 (d, J = 3.8 Hz, 1H), 4.74 (d, J = 4.1 Hz, 1H), 4.64 – 4.57 (m, 3H), 4.56 – 4.49 (m, 2H), 4.35 (q, J = 6.8 Hz, 1H), 4.24 (dd, J = 9.9, 4.6 Hz, 1H), 3.83 (td, J = 9.3, 4.6 Hz, 1H), 3.74 (t, J = 10.2 Hz, 1H), 3.62 – 3.58 (m, 2H), 2.91 (dd, J = 9.4, 4.1 Hz, 1H), 2.52 – 2.35 (m, 2H), 2.29 – 2.18 (m, 1H), 2.05 – 1.94 (m, 1H), 1.45 (d, J = 6.9 Hz, 3H), 1.35 (d, J = 7.1 Hz, 3H)ppm; LRMS (ESI) Calculated for C41H49N3O11: m / z 759.33, Found [M+H]+760.60, [M+Na]+782.65.

[0228] Example-6.3: 1-allyl 5-benzyl ((R)-2-(((2R,4aR,6S,7R,8R,8aS)-7- acrylamido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8- yl)oxy)propanoyl)-L-alanyl-D-glutamate(24) Compound 23 (5 g, 6.58 mmol)dissolved in anhydrous tetrahydrofuran (100 mL), then DIPEA (2.4 mL, 13.1mmol) and propane phosphonic acid anhydride (3.9 mL, 13.1mmol) were added at 0 °C, after 10min then acrylic acid (1.3 mL, 19.7mmol) were added drop wise. The reaction mixture was allowed to stir at RT for 16 hrs. After the completion of the starting material as monitored by thin-layer chromatography, solvent was removed and the residue was extracted with CH2Cl2 (2x100 mL) and washed with sat NaHCO3 (50 mL). The extract was dried over Na2SO4, concentrated, and the crude residue purified through column chromatography in 4% MeOH / DCM used as eluent to afford compound 24 (4.5 g, 84%) as a white solid;1H NMR (400 MHz, CDCl3) δ 7.70 – 7.63 (m, 1H), 7.49 – 7.44 (m, 2H), 7.39 –12H), 7.00 – 6.88 (m, 2H), 6.40 – 6.25 (m, 1H), 6.13 – 6.01 (m, 1H), 5.90 – 5.76 (m, 1H), 5.68 (dd, J = 10.2, 1.4 Hz, 1H), 5.57 (s, 1H), 5.31 – 5.18 (m, 1H), 5.08 (s, 2H), 5.00 (d, J = 3.9 Hz, 1H), 4.70 (d, J = 11.7 Hz, 1H), 4.59 – 4.55 (m, 2H), 4.49 (d, J = 11.7 Hz, 1H), 4.43 – 4.34 (m, 1H), 4.32 – 4.20 (m, 2H), 4.11 (q, J = 6.7 Hz, 1H ), 3.93 – 3.84 (m, 1H), 3.80 – 3.66 (m, 2H), 2.48 – 2.33 (m, 2H), 2.26 – 2.16 (m, 1H), 2.04 – 1.94 (m, 1H), 1.45 (d, J = 6.9 Hz, 3H), 1.35 (d, J = 7.1 Hz, 3H)ppm; LRMS (ESI) Calculated for C44H51N3O12: m / z 813.34, Found [M+H]+814.35, [M+Na]+836.33. Example-6.4: benzyl 3-((2R,4aR,6S,6aR,14R,17S,20R,21aR,21bS,Z)-6- (benzyloxy)-17,20-dimethyl-8,13,16,19-tetraoxo-2-phenyl- 4,4a,6a,7,8,11,13,14,15,16,17,18,19,20,21a,21b-hexadecahydro-6H- [1,3]dioxino[4',5':5,6]pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-14- yl)propanoate (25)

[0229] To the magnetically stirring solution of compound 24 (3 g, 3.6 mmol) in 3 Ltr anhydrous dichloromethane at 50 °C, IIndgeneration grubbs catalyst (30mol%, 254mg) were added and then allowed to stir at the same temperature for 20 hrs. After consumption of all stating material as monitored by thin-layer chromatography. The reaction mixture was extracted with 10% MeOH / DCM and the solvent was removed under reduced presure. The resulting crude reaction mixture was purified by columnchromatography (6% MeOH in CHCl3 on silica gel) to furnish the desired compound 25 to yield (2.2g, 78%) as white solid. Which is characterised by NMR.1H NMR (400 MHz, CDCl3) δ 7.47 – 7.18 (m, 15H), 6.64 (d, J = 15.4 Hz, 1H), 6.20 (d, J = 14.5 Hz, 1H), 5.57 (s, 1H), 5.10 (s, 2H), 4.99 (s, 1H), 4.89 (d, J = 14.7 Hz, 1H), 4.67 (d, J = 12.2 Hz, 2H), 4.47 (dd, J = 19.2, 10.0 Hz, 2H), 4.28 – 4.12 (m, 3H), 3.95 (t, J = 9.6 Hz, 1H), 3.89 – 3.64 (m, 4H), 2.49 (t, J = 7.1 Hz, 2H), 2.20 – 1.95 (m, 2H), 1.30 (dd, J = 12.5, 6.7 Hz, 6H)ppm; LRMS (ESI) Calculated for C42H47N3O12 : m / z 785.31, Found [M+H]+786.60, [M+Na]+808.60. Example-6.5: benzyl 3-((2R,5S,8R,15aR,16S,18R,19S,19aR,Z)-16-(benzyloxy)-19- hydroxy-18-(hydroxymethyl)-2,5-dimethyl-3,6,9,14-tetraoxo- 2,3,4,5,6,7,8,9,14,15,15a,18,19,19a-tetradecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoate (26)

[0230] To the magnetically stirring solution of compound 25 (2 g, 2.5 mmol) in a mixture of TFA (16 mL), water (4.0 mL), then stirred at room temperature for 2 h. after consumption of all stating material as monitored by thin-layer chromatography. The reaction mixture neutralised with sat NaHCO3 and extracted with 15% MeOH / DCM and the solvent was removed under reduced pressure, then the resulting crude reaction mixture was purified by column chromatography (9% MeOH in DCM on silica gel) to furnish compound 26 (1.5g, 88%) as white solid in good yield. Which is characterized by NMR.1H NMR (400 MHz, CD3OD) δ 7.39 – 7.21 (m, 10H), 6.61 (dt, J = 15.6, 2.9 Hz, 1H), 6.05 (dt, J = 15.6, 2.2 Hz, 1H), 5.14 (s, 2H), 4.97 (d, J = 3.5 Hz, 1H), 4.85 – 4.82 (m, 2H), 4.73 (d, J = 12.0 Hz, 1H), 4.57 (dd, J = 9.3, 6.2 Hz, 1H), 4.49 (d, J = 11.9 Hz, 1H), 4.28 – 4.20 (m, 2H), 3.86 – 3.79 (m, 2H), 3.76 – 3.64 (m, 3H), 3.47 – 3.40 (m, 1H), 2.54 (td, J = 7.4, 1.9 Hz, 2H), 2.25 – 2.15 (m, 1H), 2.10 – 1.99 (m, 1H), 1.35 (dd, J = 7.0, 4.2 Hz, 6H)ppm; LRMS (ESI) Calculated for C35H43N3O12: m / z 697.28, Found [M+H]+698.55, [M+Na]+720.50. Example-6.6: benzyl 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-19-hydroxy-18-(hydroxymethyl)-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoate (27)

[0231] To the magnetically stirring solution of compound 26 (1.5g, 2.1mmol) in anhydrous MeOH / THF (50 mL), NiCl2.6H2O (1.3 g, 5.3 mmol) were added at 0 °C, then stirred for 15min until solvent turns to dark greenish color, then NaBH4 (40mg, 1.0mmol) were added portion wise the reaction mixture turns to black color has been observed, then reaction mixture allowed to stirred at 0 °C for 4h. After complete reduction of starting material as monitored by thin-layer chromatography, solvent removed under reduced pressure then crude reaction mixture dissolved in 10% MeOH / DCM and extracted in DCM (2x50 mL) and water. All collected organic fractions dried over anhydrous Na2SO4, then concentrated under reduced pressure. The resulting crude reaction mixture was purified by column chromatography (7% MeOH / DCM on silica gel) to afford compound 27 Tto yield (1.3 g, 86%) as white solid in good yield. Characterized by NMR; 1H NMR (400 MHz, CDCl3+CD3OD) δ 7.95 (d, J = 5.8 Hz, 1H), 7.35 – 7.20 (m, 10H), 5.18 (d, J = 3.0 Hz, 1H), 5.08 (s, 2H), 4.56 (d, J = 11.9 Hz, 1H), 4.45 (d, J = 11.9 Hz, 1H), 4.41 – 4.31 (m, 2H), 4.20 – 4.14 (m, 1H), 4.13 – 4.00 (m, 2H), 3.71 – 3.63 (m, 3H), 3.59 – 3.41 (m, 3H), 2.44 (t, J = 7.2 Hz, 2H), 2.33 – 2.24 (m, 1H), 2.23 – 2.10 (m, 2H), 2.00 (dt, J = 15.1, 7.9 Hz, 1H), 1.91 – 1.81 (m, 2H), 1.30 (dd, J = 8.9, 7.2 Hz, 6H)ppm; LRMS (ESI) Calculated for C35H45N3O12 : m / z 699.30, Found [M+H]+700.55, [M+Na]+722.55. Example-6.7: 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19- dihydroxy-18-(hydroxymethyl)-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (28)

[0232] To a solution of compound 27 (200 mg, 0.28mmol) in THF (8 mL) and H2O (2 mL) was taken into 50mL Rbf, then 10% Pd / C were added as catalytic amount. The resulting reaction mixture was hydrogenated at RT by using a hydrogen-filledballoon for 22h after complete consumption of all starting material as indicated by thin-layer chromatography. The reaction mixture was filtered through a celite cake. Collected filtrate evaporation by azotroping with toluene, the residue was redissolved in pure methanol and diethyl ether were added untill it forms white precipitate then filter the white solid dried under continuous vacuum till dryness to afford compound 28 in yield (115mg, 77%) as white solid.Which is characterized by NMR.1H NMR (400 MHz, CD3OD) δ 5.39 (d, J = 2.6 Hz, 1H), 4.52 – 4.44 (m, 1H), 4.39 – 4.26 (m, 2H), 4.23 – 4.11 (m, 2H), 3.81 – 3.64 (m, 3H), 3.62 – 3.56 (m, 2H), 3.49 – 3.41 (m, 1H), 2.50 – 2.33 (m, 3H), 2.28 – 2.08 (m, 2H), 2.05 – 1.84 (m, 3H), 1.38 (dd, J = 6.9, 5.8 Hz, 6H)ppm; LRMS (ESI) Calculated for C21H33N3O12 : m / z 519.20, Found [M+Na]+542.45. Example-7: General procedure for the synthesis of compounds (29a to 29h)

[0233] To the magnetically stirring solution of compound 27 (200 mol%) in a anhydrous pyridine (5 mL) at – 10 °C stirred for 10min then acid chlorides (2 mol%) were added drop wise over 5min then reaction allowed to stirr at the same temperature for 2h. after consumption of all stating material as indicated by thin-layer chromatography. The reaction mixture nutralised with 1N HCl and extracted with 10% MeOH / DCM and the solvent was removed under reduced presure. The resulting crude reaction mixture was purified by Columnn chromatography (5% MeOH in DCM on silica gel) to give compounds 29a to 29h as white solid (85 - 90%) in a quantitative yields. Example-7.1: ((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-8-(3- (benzyloxy)-3-oxopropyl)-19-hydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-18-yl)methyl butyrate (29a)

[0234] Compound 27 (140mg, 0.2mmol) and (i) butyryl chloride (212 μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-7 to provide compound 29a (133mg, 86%) in yield;1H NMR (400 MHz, CDCl3+CD3OD) δ 7.37 – 7.22 (m, 10H), 5.22 (d, J = 3.4 Hz, 1H), 5.07 (s, 2H), 4.56(d, J = 11.8 Hz, 1H), 4.46 (d, J = 11.8 Hz, 1H), 4.42 – 4.29 (m, 2H), 4.26 – 4.13 (m, 3H), 4.12 – 3.98 (m, 2H), 3.69 (dd, J = 10.2, 3.5 Hz, 1H), 3.65 – 3.58 (m, 2H), 3.48 – 3.37 (m, 3H), 2.44 (t, J = 7.2 Hz, 2H), 2.33 – 2.25 (m, 3H), 2.22 – 2.08 (m, 2H), 2.00 (dt, J = 14.4, 7.3 Hz, 1H), 1.90 – 1.79 (m, 2H), 1.59 (dt, J = 14.8, 7.4 Hz, 2H), 1.31 (dd, J = 6.6, 5.4 Hz, 6H), 0.90 (t, J = 7.4 Hz, 3H)ppm; LRMS (ESI) Calculated for C39H51N3O13 : m / z 769.34, Found [M+H]+770.65, [M+Na]+792.60. Example-7.2: ((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-8-(3- (benzyloxy)-3-oxopropyl)-19-hydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-18-yl)methyl octanoate (29b)

[0235] Compound 27 (140mg, 0.2mmol) and (ii) octanoyl chloride (324 μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-7 to provide compound 29b (142mg, 86%) in yield;1H NMR (400 MHz, CDCl3+CD3OD) δ 7.96 (d, J = 5.7 Hz, 1H), 7.34 – 7.19 (m, 10H), 5.24 (d, J = 3.4 Hz, 1H), 5.08 (s, 2H), 4.56 (d, J = 11.8 Hz, 1H), 4.46 (d, J = 11.8 Hz, 1H), 4.42 – 4.31 (m, 2H), 4.25 – 4.13 (m, 3H), 4.12 – 3.99 (m, 2H), 3.69 (dd, J = 9.6, 5.0 Hz, 1H), 3.64 – 3.61 (m, 1H), 3.43 (dt, J = 18.5, 8.9 Hz, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.34 – 2.26 (m,, 3H), 2.22 – 2.09 (m, 2H), 2.00 (dt, J = 14.8, 8.0 Hz, 1H), 1.89 – 1.81 (m, 2H), 1.62 – 1.52 (m, 2H), 1.34 – 1.16 (m, 16H), 0.81 (t, J = 7.4 Hz, 3H)ppm; LRMS (ESI) Calculated for C43H59N3O13 : m / z 825.40, Found [M+H]+826.70, [M+Na]+826.75. Example-7.3: ((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-8-(3- (benzyloxy)-3-oxopropyl)-19-hydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-18-yl)methyl tetradecanoate (29c)

[0236] Compound 27 (140mg, 0.2mmol) and (iii) myristoyl chloride (490μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-7 to provide compound 29c (158mg, 87%) in yield;1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 5.7 Hz, 1H), 7.38 – 7.23 (m, 10H), 6.73 (d, J = 6.6 Hz, 1H), 5.34(d, J = 3.4 Hz, 1H), 5.13 (s, 2H), 4.60 – 4.54 (m, 3H), 4.53 – 4.42 (m 2H), 4.29 (p, J = 6.8 Hz, 1H), 4.20 – 4.07 (m, 2H), 3.99 (dd, J = 12.3, 1.9 Hz, 1H), 3.85 – 3.77 (m, 1H), 3.69 (d, J = 5.5 Hz, 1H), 3.59 – 3.50 (m, 2H), 3.38 (td, J = 9.4, 5.7 Hz, 1H), 2.57 – 2.44 (m, 2H), 2.38 (t, J = 7.1 Hz, 2H), 2.34 – 2.25 (m, 2H), 2.22 – 2.05 (m, 2H), 2.02 – 1.80 (m, 2H), 1.67 – 1.57 (m, 2H), 1.35 (dd, J = 6.8, 3.8 Hz, 6H), 1.31 – 1.21 (m, 20H), 0.88 (t, J = 6.9 Hz, 3H)ppm; LRMS (ESI) Calculated for C49H71N3O13 : m / z 909.49, Found [M+H]+910.90, [M+Na]+938.85. Example-7.4: ((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-8-(3- (benzyloxy)-3-oxopropyl)-19-hydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-18-yl)methyl 3-methylbutanoate (29d)

[0237] Compound 27 (140mg, 0.2mmol) and (iv) 3-methylbutanoyl chloride (240μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-7 to provide compound 29d (136mg, 87%) in yield;1H NMR (400 MHz, CDCl3+CD3OD) δ 7.33 – 7.25 (m, 9H), 7.24 – 7.19 (dd, J = 8.4, 3.9 Hz, 1H), 5.22 (d, J = 3.5 Hz, 1H), 5.08 (s, 1H), 4.57 (d, J = 11.8 Hz, 1H), 4.46 (d, J = 11.8 Hz, 1H), 4.42 – 4.30 (m, 2H), 4.19 (dd, J = 12.0, 5.4 Hz, 3H), 4.13 – 4.00 (m, 2H), 3.69 (dd, J = 10.4, 3.5 Hz, 1H), 3.62 (dd, J = 8.6, 4.8 Hz, 1H), 3.44 (dt, J = 18.5, 8.8 Hz, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.35 – 2.26 (m, 1H), 2.20 (d, J = 7.1 Hz, 2H), 2.17 – 2.10 (m, 1H), 2.09 – 1.95 (m, 2H), 1.90 – 1.80 (m, 2H), 1.30 (dd, J = 6.6, 5.3 Hz, 6H), 0.91 (d, J = 2.5 Hz, 6H)ppm; LRMS (ESI) Calculated for C40H53N3O13: m / z 783.35, Found [M+H]+784.65, [M+Na]+806.65. Example-7.5: ((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-8-(3- (benzyloxy)-3-oxopropyl)-19-hydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-18-yl)methyl cyclopropanecarboxylate (29e)

[0238] Compound 27 (140mg, 0.2mmol) and (v) cyclopropanecarbonyl chloride (200 μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-7 to provide compound 29e (133mg, 87%) in yield;1H NMR (400 MHz, CDCl3 + CD3OD) δ 7.34 – 7.26 (m, 9H), 7.25 – 7.20 (m, 1H), 5.22 (d, J = 3.4 Hz, 1H), 5.09 (s, 2H), 4.59 (d, J = 11.8 Hz, 1H), 4.47 (d, J = 11.9 Hz, 1H), 4.43 – 4.32 (m, 2H), 4.23 – 4.17 (m, 3H), 4.13 – 4.07 (m, 2H), 3.71 (dd, J = 10.4, 3.4 Hz, 1H), 3.68 – 3.63 (m, 1H), 3.47 (dt, J = 18.6, 8.8 Hz, 2H), 2.46 (t, J = 7.4 Hz, 2H), 2.38 – 2.28 (m, 1H), 2.24 – 2.10 (m, 2H), 2.07 – 1.95 (m, 1H), 1.92 – 1.81 (m, 2H), 1.68 – 1.61 (m, 1H), 1.31 (dd, J = 6.6, 5.4 Hz, 6H), 0.99 – 0.94 (m, 2H), 0.90 – 0.83 (m, 2H)ppm; LRMS (ESI) Calculated for C39H49N3O13 : m / z 767.32, Found [M+H]+768.65, [M+Na]+790.60. Example-7.6: ((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-8-(3- (benzyloxy)-3-oxopropyl)-19-hydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-18-yl)methyl benzoate (29f)

[0239] Compound 27 (140mg, 0.2mmol) and (vi) benzoyl chloride (280μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-7 to provide compound 29f (139mg, 86%) in yield;1H NMR (400 MHz, CDCl3+ CD3OD) δ 8.19 (d, J = 5.6 Hz, 1H), 8.01 (d, J = 8.2 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1H), 7.42 (dd, J = 8.0, 5.4 Hz, 2H), 7.36 – 7.18 (m, 10H), 5.24 (d, J = 3.3 Hz, 1H), 5.10 (s, 2H), 4.61 (d, J = 11.8 Hz, 1H), 4.49 (d, J = 11.9 Hz, 1H), 4.47 – 4.39 (m, 2H), 4.36 – 4.32 (m, 2H), 4.21 (q, J = 7.1 Hz, 1H), 4.15 – 4.03 (m, 2H), 3. 84 – 3.69 (m, 2H), 3.56 (dd, J = 7.5, 2.5 Hz, 2H), 2.48 (t, J = 7.4 Hz, 2H), 2.39 – 2.28 (m, 2H), 2.23 – 2.11 (m, 2H), 2.02 (dt, J = 14.8, 7.9 Hz, 2H), 1.93 – 1.79 (m, 2H), 1.33 (dd, = 6.8, 3.8 Hz, 6H)ppm; LRMS (ESI) Calculated for C42H49N3O13: m / z 803.32, Found [M+Na]+826.60. Example-7.7: ((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-8-(3- (benzyloxy)-3-oxopropyl)-19-hydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-18-yl)methyl 4-methoxybenzoate (29g)

[0240] Compound 27 (140mg, 0.2mmol) and (vii) Anisoyl chloride (340μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-7 to provide compound 29g (145mg, 87%) in yield;1H NMR (400 MHz, CDCl3 + CD3OD) δ 7.94 (d, J = 5.6 Hz, 2H), 7.34 – 7.18 (m, 10H), 6.87 (d, J = 8.2, 2H), 5.25 (d, J = 3.4 Hz, 1H), 5.07 (s, 2H), 4.57 (d, J = 11.8 Hz, 1H), 4.51 – 4.41 (m, 2H), 4.39 – 4.30 (m, 3H), 4.21 – 4.13 (m, 1H), 4.11 – 3.98 (m, 2H), 3.81 (s, 3H), 3.76 – 3.69 (m, 2H), 3.56 – 3.48 (m, 3H), 2.48 – 2.39 (m, 2H), 2.34 – 2.25 (m, 1H), 2.22 – 2.08 (m, 2H), 2.06 – 1.93 (m, 1H), 1.90 – 1.80 (m, 2H), 1.28 (dd, J = 6.8, 3.8 Hz, 6H)ppm; LRMS (ESI) Calculated for C43H51N3O14 : m / z 833.33, Found [M+H]+834.60, [M+Na]+856.65. Example-7.8: ((2R,5S,8R,15aR,16S,18R,19S,19aR)-16-(benzyloxy)-8-(3- (benzyloxy)-3-oxopropyl)-19-hydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-18-yl)methyl 4- (trifluoromethyl)benzoate (29h)

[0241] Compound 27 (140mg, 0.2mmol) and (viii) 4-(trifluoromethyl)benzoyl chloride (416μL, 0.002mmol) in a pyridine (5 mL) were treated according to general protocol as given in example-7 to provide compound 29h (155mg, 89%) in yield;1H NMR (400 MHz, CDCl3+CD3OD) δ 8.20 (d, J = 5.4 Hz, 1H), 8.14 (d, J = 8.1 Hz, 2H), 7.69 (d, J = 8.3 Hz, 2H), 7.35 – 7.20 (m, 10H), 5.25 (d, J = 3.3 Hz, 1H), 5.10 (s, 2H), 4.60 (d, J = 11.9 Hz, 1H), 4.55 – 4.49 (m,, 2H), 4.48 (d, J = 4.3 Hz, 1H), 4.45 – 4.39 (m, 2H), 4.21 (q, J = 7.0 Hz, 1H), 4.15 – 4.02 (m, 2H), 3.84 – 3.69 (m, 2H), 3.55 (dd, J = 7.6, 2.3 Hz, 1H), 2.47 (t, J = 7.4 Hz, 2H), 2.40 – 2.29 (m, 1H), 2.23 – 2.11 (m, 2H), 2.07 – 1.96 (m, 1H), 1.94 – 1.80 (m, 2H), 1.33 (dd, J = 6.8, 3.8 Hz, 6H)ppm; LRMS (ESI) Calculated for C43H48F3N3O13 : m / z 871.31, Found [M+H]+871.65, [M+Na]+894.65. Example-8: General procedure for the synthesis of compounds [30a to 30h]

[0242] To a solution of compound 29a to 29h (100mol %), THF (15mL), H2O (5mL) and 10% Pd / C (20 mol%) was added. The resulting reaction mixture was hydrogenated at rt by using a hydrogen-filled balloon for 22h after complete consumption of all starting material as indicated by thin-layer chromatography and the reaction mixtured was filtered through a celite cake. Collected filtrate evaporation by azotroping with toluene, the residue was dissolved in pure methanol and diethyl ether were added untill it forms white precipitate then filter the white solid under continuous vacuum dryness to afford compounds 30a to 30h (85 – 90 %) in good yields Example-8.1: 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18-((butyryloxy)methyl)- 16,19-dihydroxy-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H- pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30a)

[0243] Compound 29a (76mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 8 to provide compound 30a (51mg, 86%) in yield;1H NMR (400 MHz, CDCl3+ CD3OD) δ 5.42 (d, J = 2.6 Hz, 1H), 4.51 – 4.44 (m, 1H), 4.37 – 4.25 (m, 3H), 4.24 – 4.14 (m, 3H), 3.93 (dd, J = 9.9, 3.0 Hz, 1H), 3.62 – 3.54 (m, 1H), 3.53 – 3.39 (m, 2H), 2.48 – 2.40 (m, 1H), 2.40 – 2.28 (m, 4H), 2.26 – 2.16 (m, 1H), 2.15 – 2.08 (m, 1H), 2.05 – 1.88 (m, 3H), 1.69 – 1.57 (m, 2H), 1.37 (dd, J = 7.0, 1.7 Hz, 6H), 0.94 (t, J = 7.4 Hz, 3H)ppm; LRMS (ESI) Calculated for C25H39N3O13 : m / z 589.24, Found [M+Na]+612.50. Example-8.2: 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5- dimethyl-18-((octanoyloxy)methyl)-3,6,9,14-tetraoxohexadecahydro-11H,16H- pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30b)

[0244] Compound 29b (82mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 8 to provide compound 30b (56mg, 87%) in yield;1H NMR (400 MHz, CDCl3 +CD3OD) δ 5.42 (d, J = 2.3 Hz, 1H), 4.48 (q, J = 6.6 Hz, 1H), 4.39 – 4.25 (m, 3H), 4.24 – 4.13 (m, 3H), 3.94 (dd, J = 10.0, 2.9 Hz, 1H), 3.62 – 3.53 (m, 2H), 3.52 – 3.40 (m, 1H), 2.49 – 2.43 (m, 1H), 2.41 – 2.30 (m, 4H), 2.26 – 2.08 (m, 2H), 2.06 – 1.87 (m, 2H), 1.66 – 1.56 (m, 2H), 1.37 (dd, J = 13.0, 6.4 Hz, 6H), 1.33 – 1.25 (m, 8H), 0.88 (t, J = 7.3 Hz, 3H)ppm; LRMS (ESI) Calculated for C29H47N3O13: m / z 645.31, Found [M+Na]+668.50. Example-8.3: 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5- dimethyl-3,6,9,14-tetraoxo-18-((tetradecanoyloxy)methyl)hexadecahydro- 11H,16H-pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30c )

[0245] Compound 29c (90mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 8 to provide compound 30c (81mg, 89%) in yield;1H NMR (400 MHz, CDCl3+CD3OD) δ 5.42 (d, J = 3.2 Hz, 1H), 4.48 (q, J = 6.6 Hz, 1H), 4.40 – 4.25 (m, 3H), 4.24 – 4.11 (m, 3H), 3.93 (dd, J = 9.9, 2.8 Hz, 1H), 3.63 – 3.53 (m, 2H), 3.51 – 3.40 (m, 1H), 2.50 – 2.43 (m, 1H), 2.39 (t, J = 6.9 Hz, 2H), 2.33 ( t, J = 7.1 Hz, 2H), 2.26 – 2.08 (m, 2H), 2.06 – 1.87 (m, 3H), 1.65 – 1.56 (m, 2H), 1.37 ( dd, J = 7.0, 1.7 Hz, 6H), 1.33 – 1.23 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H)ppm; LRMS (ESI) Calculated for C35H50N3O13: m / z 729.40, Found [M+Na]+752.70. Example-8.4: 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5- dimethyl-18-(((3-methylbutanoyl)oxy)methyl)-3,6,9,14-tetraoxohexadecahydro- 11H,16H-pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30d)

[0246] Compound 29d (78mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 8 to provide compound 30d (52mg, 86%) in yield;1H NMR (400 MHz, CDCl3+ CD3OD) δ 5.37 (d, J = 2.3 Hz, 1H), 4.52 – 4.43 (m, 1H), 4.41 – 4.26 (m, 3H), 4.22 –4.14 (m, 3H), 3.94 (ddd, J = 10.0, 5.1, 2.0 Hz, 1H), 3.61 – 3.55 (m, 2H), 3.46 – 3.40 (m, 1H), 2.36 (t, J = 6.9 Hz, 2H), 2.26 – 2.16 (m, 4H), 2.14 – 2.04 (m, 2H), 2.04 – 1.91 (m, 2H), 1.38 (dd, J = 13.0, 6.8 Hz, 6H), 0.98 (d, J = 6.6 Hz, 6H)ppm; LRMS (ESI) Calculated for C26H41N3O13 : m / z 603.26, Found [M+Na]+626.50. Example-8.5:3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18- (((cyclopropanecarbonyl)oxy)methyl)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30e)

[0247] Compound 29e (76mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 8 to provide compound 30e (50mg, 85%) in yield;1H NMR (400 MHz, CD3OD) δ 5.37 (d, J = 3.6 Hz, 1H), 4.48 (q, J = 6.7 Hz, 1H), 4.39 – 4.26 (m, 3H), 4.24 – 4.16 (m, 3H), 3.97 – 3.91 (m, 1H), 3.58 (dd, J = 11.5, 5.2 Hz, 2H), 3.49 – 3.41 (m, 1H), 2.48 – 2.38 (m, 3H), 2.26 – 2.10 (m, 2H), 2.05 – 1.95(m, 2H), 1.94 – 1.85 (m, 1H), 1.70 – 1.62 (m, 1H), 1.38 (dd, J = 6.9, 5.8 Hz, 1H), 0.97 – 0.87 (m, 4H)ppm; LRMS (ESI) Calculated for C25H37N3O13: m / z 587.23, Found [M+Na]+610.45. Example-8.6: 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18- ((benzoyloxy)methyl)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30f)

[0248] Compound 29f (80mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 8 to provide compound 30f (54mg, 87%) in yield;1H NMR (400 MHz, CDCl3 + CD3OD) δ 8.02 (d, J = 1.4 Hz, 2H), 7.58 (t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 5.44 (d, J = 2.7 Hz, 1H), 4.61 – 4.44 (m, 3H), 4.36 – 4.24 (m, 2H), 4.20 – 4.14 (m, 2H), 4.08 (dd, J = 9.9, 2.8 Hz, 1H), 3.68 – 3.55 (m, 3H), 2.49 – 2.40 (m, 1H), 2.37 (t, J = 6.3 Hz, 2H), 2.28 – 2.08 (m, 2H), 2.05 – 1.86 (m, 3H), 1.37 (dd, J = 13.0, 6.4 Hz,6H)ppm; LRMS (ESI) Calculated for C28H37N3O13 : m / z 623.23, Found [M+Na]+646.45. Example-8.7: 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-18- (((4-methoxybenzoyl) oxy)methyl)-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30g)

[0249] Compound 29g (83mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 8 to provide compound 30g (57mg, 87%) in yield;1H NMR (500 MHz, CD3OD) δ 8.01 (d, J = 8.1 Hz, 2H), 6.82 (d, J = 8.2, 2H),J = 2.6 Hz, 1H), 4.62 (dd, J = 11.7, 2.0 Hz, 1H), 4.51 – 4.45 (m, 2H), 4.38 – 4.27 (m, 2H), 4.22 – 4.16 (m, 2H), 4.13 – 4.07 (dd, J = 9.9, 2.8 Hz, 1H), 3.79 (s, 3H), 3.66 – 3.62 (m, 2H), 3.59 – 3.53 (m, 1H), 2.50 – 2.43 (m, 1H), 2.42 – 2.39 (t, J = 6.3 Hz, 2H), 2.26 – 2.11 (m, 3H), 2.04 – 1.95 (m, 2H), 1.94 – 1.85 (m, 1H), 1.37 (dd, J = 7.0, 1.7 Hz, 6H)ppm; LRMS (ESI) Calculated for C29H39N3O14: m / z 653.24, Found [M+Na]+676.50. Example-8.8: 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5- dimethyl-3,6,9,14-tetraoxo-18-(((4-(trifluoromethyl) benzoyl)oxy)methyl)hexadecahydro-11H, 16H-pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30h)

[0250] Compound 29h (87mg, 0.1mmol), THF (15mL) and H2O (3mL) were added 10% Pd / C (20mol%) were treated according to general procedure as given in example- 8 to provide compound 30h (61mg, 88%) in yield;1H NMR (500 MHz, CDCl3 + CD3OD) δ 8.23 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H), 5.41 (d, J = 2.6 Hz, 1H), 4.61 (dd, J = 11.7, 2.0 Hz, 1H), 4.54 – 4.47 (m, 2H), 4.38 – 4.27 (m, 2H), 4.22 – 4.16 (m, 2H), 4.13 – 4.07 (dd, J = 9.9, 2.8 Hz, 1H), 3.66 – 3.62 (m, 2H), 3.61 – 3.55 (m, 1H), 2.49 – 2.44 (m, 1H), 2.42 – 2.39 (t, J = 6.3 Hz, 2H), 2.27 – 2.10 (m, 3H), 2.05 –1.96 (m, 2H), 1.95 – 1.88 (m, 1H), 1.38 (dd, J = 12.8, 6.3 Hz, 6H)ppm; LRMS (ESI) Calculated for C29H36F3N3O13: m / z 691.22, Found [M+Na]+714.45. Biological evaluation: Name of Source of Village / Town / District State Name Contact the access Panchayat Taluk Details et, ,

[0251] Balb-C mice and cell lines for In vitro immunopharmacological assay were purchased from the commercial sources. IAEC permission has been obtained for the procurement and use of animals IICT / 76 / 2021. Example-9 Example-9.1: Hemolysis

[0252] The ability of these analogues in disturbing the cell membrane integrity and causing lysis of the cell was accessed using red blood cells. All the analogues at a concentration of 1000µg / ml were incubated with RBC collected from mice and the % hemolysis was estimated according to standard protocol, using MDP as standard. The data obtained was mean of three individual experiments as represented in table -1 Table 1: Effect of MDP analogues on hemolysis Group % Hemolysis30b 10.217± 0.0171 30c 1367 ± 00011 [02 to thestandard adjuvant MDP. Example-9.2: Cytotoxicity assay:

[0254] RAW macrophages were cultured using DMEM media to confluency. 2×104cells / well were seeded and incubated with 20μg of test compounds and standard MDP for 48 hrs at 37oC with 5% CO2. After incubation 20μl MTT reagent was added to check the viability of cells. The addition of MTT reagent was followed by 2 hrs incubation at 37oC with 5 % CO2. Untransformed MTT was replaced by DMSO and absorbance was read at 570nm. Table 2: Effect of MDP analogues on cytotoxicity Group %Viability10µg 122.3333 1µg 112454510µg 122.7273 1µg 1134848

[0255] The analogues have not shown any cytotoxicity compared to the standard Example-9.3: In vitro cytokine estimation

[0256] RAW macrophages were cultured using DMEM media to confluency. 2×104cells / well were seeded and incubated with 20μg of test compounds and standard MDP for 48 hrs at 37oC with 5% CO2. After incubation cell supernatant was collected and 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 µL / 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 nmwith a plate reader. Table 4: Effect of MDP analogues on cytokine response Cytokines (pg / mL) Analogue IL-6 TNF-α 6 4

[0257] All themmatory cytokine response up on stimulating RAW macrophages.

[0258] From all the above in vitro data can confirm the non-cytotoxic nature and immune stimulating potential of the novel analogues. The adjuvanticity of these novel analogues will be evaluated using quadrivalent split influenza virus antigen in BALB / c mouse model through intramuscular route. The immunological end points like antibody response, cellular response and cytokines will be quantified from the immunized groups in comparison to standard MDP.EXAMPLE 10: Adjuvanticity of cyclic glycopeptide analogues with split influenza antigen Immunizations

[0259] All the compounds were studied in vivo in BALB / c mice. Female BALB / c mice (6–8 weeks) were purchased from RCC, Hyderabad. All animal experiments were approved by the local ethical committee and animals were kept in accordance with CPCSEA guidelines with an IAEC No IICT / 59 / 2022. They were immunized with different groups according to various concentrations of analogues. Administration of Vaccine

[0260] The adjuvant molecules under invention along with Influenza antigen are generally administered by injection, particularly intramuscular route. The primary vaccination is followed by booster vaccination was given after two weeks with the same. The vaccine was formulated with antigen alone or with emulsion, various adjuvants like MDP and their analogues. The adjuvanticity induced by each of the vaccines is determined by antibody titer. Preparation and immunization of the vaccine

[0261] Male BALB / c mice were immunized subcutaneously with different groups according to various concentrations of (conjugates) preferably 2 to 80 µg / dose, more preferably 6 to 50 µg / dose, most preferably 8 to 40 µg / dose, along with PBS and antigen control. A booster dose was given on 14thday and sacrificed 2 weeks after booster. EXAMPLE 10.1: Antibody titer against influenza antigen

[0262] From the immunized mice retro-orbital sampling or retro-orbital blood was collected on 14th and 28thday 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.

[0263] The plates were coated with antigen in carbonate buffer. Plates were incubated at 4oC overnight. They were then washed 3 times with PBS / Tween, and non-specificbinding sites were blocked by adding 200μl 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. TMB Substrate was added and plates were incubated at room temperature (4-30 min) for color development. The color reaction was stopped by adding 50μl of stop solution. Optical density (OD) was read at 450 nm.

[0264] Table 5 shows the serum anti-Influenza IgG titer. Influenza specific IgG was assayed by indirect ELISA and titers obtained after booster immunization reveals that the production of anti- flu antibodies was strongly enhanced in mice treated with conjugates in comparison with antigen alone. Table 5: Humoral response of cyclic glycopeptide analogues Group Dose IgG (titer)21b 10-30µG 80000-160000 21c 10-30µG 2133333- 480000 *Titers are anti HBsAg, and Ch.... EXAMPLE 10.2: In vivo Splenocyte proliferation

[0265] Splenocytes were seeded into 96-well flat-bottom microtiter plates having 1x105cells / 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 (180μL) was removed from each well by pipetting. A total of 180 μL of DMSO was added and the absorbance was evaluated in an ELISA reader at 630nm on the multimode reader (Infinite 200 Pro, Switzerland) after 15 min. Table 6: Effect of cyclic glycopeptide analogues on splenocyte proliferation AG MDP 30e (10-30μg) 21c (10-30μg) 21d (10-30μg) ntionelicit a synergistic immune response with the various viral, protozoan, recombinant and subunit antigens such as Japanese Encephalitis Virus, HIV (Human Immunodeficiency Virus), HCV (Hepatitis C Virus), Different malarial protozoans (P. falciparum, P. malaria, P. ovale and P.vivax), Dengue, chikungunya, Ebola, SAR-CoV-2(COVID-19), Hantavirus, Influenza, Papillomavirus, Zika, Measles, Mumps and also effective with many malignant sarcomas and carcinomas derived antigens. These analogues principally boost the antigen-specific antibody and Th1 / Th2 cytokine response required for the development of respective vaccines with or without appropriate vaccine delivery systems.

Claims

We Claim:

1. A cyclic glycolipopeptide compound of formula-I,wherein, R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedand substituted or unsubstituted aryl, wherein one or more substituents selected from the group consisting of alkyl, alkoxy and trifloro alkyl; G is selected from 2.

2. The cyclicwherein the compound is selected from the group consisting of,R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedor branched alkyl, C3-C6cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and x and y are independently selected form 1, 2 and 3.

3. The compound as claimed in claim 1, wherein the compound is selected from the group consisting of; a) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-26-(hydroxymethyl)-4,7- dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9-diaza-1(1,4)- triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(19); b) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-26-((hexanoyloxy)methyl)-22,25- dihydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9- diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21a); c) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-4,7-dimethyl-5,8,11- trioxo-26-((tetradecanoyloxy)methyl)-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa-6,9- diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21b); d) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-22,25-dihydroxy-4,7-dimethyl-26-(((3- methylbutanoyl)oxy)methyl)-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12-dioxa- 6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21c); e) 3-((11S,23R,24R,25S,26R,4R,7S,10R,Z)-26-(((cyclopropanecarbonyl)oxy)methyl)- 22,25-dihydroxy-4,7-dimethyl-5,8,11-trioxo-23,24,25,26-tetrahydro-11H,22H-3,12- dioxa-6,9-diaza-1(1,4)-triazola-2(3,4)-pyranacyclotridecaphane-10-yl)propanoic acid(21d); f) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-18-(hydroxymethyl)- 2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (28);g) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18-((butyryloxy)methyl)-16,19- dihydroxy-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30a); h) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-18- ((octanoyloxy)methyl)-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30b); i) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxo-18-((tetradecanoyloxy)methyl)hexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30c); j) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-18-(((3- methylbutanoyl)oxy)methyl)-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30d); k) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18- (((cyclopropanecarbonyl)oxy)methyl)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30e); l) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-18-((benzoyloxy)methyl)-16,19- dihydroxy-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro-11H,16H-pyrano[4,3- k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30f); m) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-18-(((4- methoxybenzoyl)oxy)methyl)-2,5-dimethyl-3,6,9,14-tetraoxohexadecahydro- 11H,16H-pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30g); and n) 3-((2R,5S,8R,15aR,16S,18R,19S,19aR)-16,19-dihydroxy-2,5-dimethyl-3,6,9,14- tetraoxo-18-(((4-(trifluoromethyl)benzoyl)oxy)methyl)hexadecahydro-11H,16H- pyrano[4,3-k][1,10]dioxa[4,7,13]triazacycloheptadecin-8-yl)propanoic acid (30h).

4. A process for the preparation of a cyclic glycolipopeptide compound of formula-I’ as claimed in claim 2,wherein, R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedor branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and x is selected form 1, 2 and 3, the process comprising the steps of: a. coupling of dipeptide by treating the compound of formula VI with propargyl L-alanyl-D-isoglutamine benzyl ester trifluoroacetate (6) in an acid amine coupling condition to obtain a compound of formula VII, wherein x is as defined above;b. - of formula VII by treating with CuSO4.5H2O catalyst in presence of sodium ascorbate, THF / H2O (4:1) solvent to obtain the compound of formula VIII, wherein x is asdefined above;acid TFA and water solvent to obtain the compound of formula IX, wherein x is as defined above;and a THF / H2O (4:1) solvent to obtain the compound IX (formula-I, wherein R is H) and x is as defined above;and a pyridine solvent to obtain the compound of formula XI wherein R x is as defined above; andn gas and a THF / H2O solvent to obtain the compound of formula-I’R , R1is selected from the group consisting of substituted or unsubstituted C4-or branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and x is selected from 1, 2 or 3, 5. The process as claimed in claim 4, wherein the coupling is carried out in presence of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), hydroxybenzotriazole (HOBt), tetrahydrofuran (THF), N,N-diisopropylethylamine (DIPEA) at a temperature in a range of 0oC to room temperature (RT).

6. An intermediate compound for the preparation of cyclic glycolipopeptide compound of formula-I’, selected from the group consisting of:, , wherei 7. A process for the preparation of a cyclic glycolipopeptide compound of formula-I’’wherein, R is selected from the group consisting of H , R1is selected from the group consisting of substituted or unsubstitutedor branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl; and y is selected form 1, 2 and 3, comprising the steps of: a. coupling of dipeptide by treating the compound of formula VI with allyl L- alanyl-D-isoglutamine benzyl ester trifluoroacetate (9) in an acid amine coupling conditions to obtain a compound of formula XII, wherein y is as defined above;b. conv osphine PPh3and THF / H2O (4:1) solvent to obtain a compound of formula-XIII), wherein y is as defined above;of T3P reagent, DIPEA and THF as solvent to obtain a compound of formula-XIV), wherein y is as defined above;d. cross metathesis of the compound of formula-XIV with suitable grubbs catalyst and DCM solvent to obtain a compound of formula-XV), wherein y is as defined above;e ce of TFA / H2O (4:1), solvent to obtain a compound of formula-XVI), wherein y is as defined above;agent in presence of nickel dichloride hexahydrate (NiCl2.6H2O) catalyst and THF / MeOH solvent to obtain a compound of formula-XVII), wherein y is as defined above;using H2 gas and THF / H2O (4:1) solvent to obtain a compound of formula-XVII (formula-I’’, wherein R is H);COCl) in presence pyridine as solvent to obtain the compound of formula XIX, wherein y is as defined above; andcatalyst in presence H2 gas and THF / H2O (4:1) solvent to the compound of Formula-I’’,wherein, R is , R1is selected from the group consisting of substituted or unsubstitutedlinear or branched alkyl, C3-C6 cycloalkyl, and substituted or unsubstituted aryl, wherein the substituent is selected from the group consisting of alkyl, alkoxy and trifloro alkyl;and y is selected form 1, 2 and 3, 8. An intermediate compound for the preparation of cyclic glycolipopeptide compound of Formula-I’’ as claimed in claim 2, wherein the compound of Formula-I” is selected from the group consisting of:

9. The cyclic glycolipopeptide compound of Formula-I as claimed in claim 1, wherein the compound is an adjuvant useful in pharmaceutical preparations and vaccine formulations with an antigen.

10. A vaccine formulation comprising the cyclic glycolipopeptide compound of Formula-I as claimed in claim 1 as an adjuvant and a suitable antigen derived from bacterial, viral or any potential infectious pathogens against mammals, wherein the antigen is selected from a group consisting of a live attenuated vaccine antigen, inactivated vaccine antigen, subunit vaccine antigen, a conjugate vaccine antigen, and recombinant vaccine antigen or any combinations thereof.

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