DESC:FIELD OF THE INVENTION
The invention relates to a novel muramyl dipeptide derivative compound of structural Formula-VIII and a process for synthesis thereof.

BACKGROUND OF THE INVENTION
Muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine) is a synthetic immunoreactive peptide consisting of N-acetyl muramic acid attached to a short amino acid chain of L-alanyl-D-isoglutamine (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 Freund’s Complete Adjuvant (FCA), one of the most potent and widely used adjuvants in animal experimental models. Muramyl peptides are well known for their immunomodulatory properties. Muramyl peptides are derived from bacteria and are products thereof. Muramyl dipeptides are the smallest, biologically active components of bacterial cell walls. The biological activities of MDP and its derivatives find application as effective adjuvants and may be used for boosting the potency of drugs and vaccines. Muramyl peptides derivatives have been proved to show significant immunomodulatory properties, via one of the PRRs (Pattern Recognition Receptors), nucleotide-binding oligomerization domain 2 (NOD2) receptor (Girardin S. et al.,. 2003. J Biol Chem. 278(11): 8869-72;F. Coulombe et al., 2012 PloS ONE, 7 (5): Article ID e36734). Muramyldipeptides activate macrophages and other cells of the immune system to kill cancer cells (I. 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, till date a series of MDP derivatives have been designed, synthesized and tested with the aim of increasing specific functions, while suppressing pyrogenicity. There are many reported inventions or publications related to synthesis of Muramyl dipeptide and its derivatives and also on their use as adjuvants (Namba et al., 1997. Vaccine, 15(4):405-13; WO1996001645; US 4395399; US 7173107 B2).

N-glycolyl Glucosaminylmuramyl dipeptide (GMDP), is a derivative of Muramyl dipeptide (MDP). GMDP was originally developed in the 1970s at the Shemyakin Institute for Bio-organic Chemistry, Moscow. GMDP has been shown to stimulate both innate and adaptive immune responses. GMDP also activates macrophages and release cytokines and colony stimulating factors (CSFs), which in-turn stimulate the differentiation of hemopoietic cells to clear infections (Australasian Biotechnology, Volume 6 Number 4, July/August 1996,pp. 223-229). GMDP in comparison to MDP, shows higher immuno-adjuvant activity and less pyrogenic effect. Hence, GMDP derivatives are more potential immunotherapeutics over MDP derivatives (Andronova T.M., et al., 1991. Review. fImmunology 4, 1). Hence, this molecule is been widely used in immuno-therapeutic 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).

GMDP-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. immuno stimulation and prevention of infections complicating post-traumatic, post-operative, post-chemotherapeutic and post- radiotherapeutic patienthood. Other areas are treatment of infectious diseases, as tuberculosis, human cervical papilomavirus, ophthalmic herpetic infections, psoriasis and treatment of ulcerous and inflammation processes (WO2007045192).

Below shown structure of general Formula-X represents chemical structures of compounds MDP and GMDP.

Formula-X

There are many inventions related to Muramyl peptide compound and its derivatives. They are used as an adjuvant and therapeutic agent also.

There are many inventions related to Muramyl peptide compound and its derivatives. Mostly, they have been used as an adjuvant and sometimes as therapeutic agents as well.

WO1996001645 A1 relates to the use of Muramyl peptide compounds, particularly N-acetyl-D-glucosaminyl-(ß1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine for the treatment or prophylaxis of inflammatory dermatological conditions such as psoriasis and in the treatment or prophylaxis of immune-related diseases of the skin and mucous membranes.

US 7173107 B2 discloses glycopeptides and preparation thereof which is stereospecific synthesis of a glycopeptide using a triply orthogonal protection scheme is described, in particular, the synthesis of N-acetylglucosaminyl-ß-[1,4]-N-acetylmuramylmonopeptide and derivatives thereof. The glycopeptide is useful for the preparation of MDP derivatives and related compounds having a glucosaminyl-ß-[1,4]-N-acetylmuramic acid disaccharide core.

WO2007045192 relates to glucosaminylmuramic acid (2-amino-2-deoxy-ß-D-gluco- pyranosyl-(1?4)-N-acetylmuramic acid) derivatives, method of their synthesis, and their use for the synthesis of glucosaminylmuramylglycopeptides, i.e. disaccharide analogues of muramylglycopeptides. US 4395399 disclose different glycopeptides and their preparations.

However, despite improvements seen in several newly developed MDP derivatives, yet there is a continuous need for compounds with further reduced side-effects and increased adjuvant activity for the purpose of either therapeutic or prophylactic use in vaccine formulations. Based on the literature, researchers should critically focus on the balance between efficacy and side effects, while designing or synthesizing new compounds. There is a need to develop more effective and improved MDP derivatives, which would reduce the side effects associated with known MDP Derivatives. This invention describes novel MDP derivatives with reduced pyrogenicity while maintaining a considerable effective functionality as vaccine adjuvants. In this patent application, inventors have developed and thereby disclosed new MDP derivatives, not known earlier.

OBJECTS OF THE INVENTION
The primary object of the invention is to provide novel muramyl dipeptide derivatives useful as vaccine adjuvants.

Another objective of the invention is to provide a process for the synthesis of the novel muramyl dipeptide derivative compound.

A further object of the invention is to provide novel muramyl dipeptide derivative compound as an adjuvant for pharmaceutical preparations and vaccine formulations.

SUMMARY OF THE INVENTION
Accordingly in one aspect, the invention provides a novel muramyl dipeptide derivative compound as shown below in Formula-VIII:

In another aspect the invention provides a process for the synthesis of the novel muramyl dipeptide derivative compound of formula-VIII comprising the steps as shown in below Scheme A and Scheme B:

SCHEME FOR THE SYNTHESIS OF MURAMYL PEPTIDE DERIVATIVE

SCHEME-A

SCHEME-B

In a further aspect, the invention provides use of novel muramyl dipeptide derivative (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid as an adjuvant in an adjuvant composition to be used in the pharmaceutical preparations and/or vaccine formulations.

The novel muramyl dipeptide derivative compound represented by the structural Formula-VIII is now named as MDP-AA, which is earlier named as MDP-SE. Wherever in the specification MDP-AA and MDP-SE are mentioned, both the representative names of Formula-VIII corresponds to the same MDP derivative compound i.e. MDP-SE = MDP-AA.

Structural Formula-VIII (MDP-SE = MDP-AA)

In one aspect the invention provides a compound of Structural Formula-VIII

Wherein, ‘n’ can be any natural number(s), and:
wherein R or R1 is selected from the group consisting of a hydrogen, heteroatom substitutued alkyl (with both linear and branched chains), cycloalkyl, arylalkyl, heteroaryl represented by CH3, CH2CH3, CONH2, COOH, CH2OH, CH(CH3)OH, CH(CH(CH3), CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, p-hydroxy benzyl, CH(OH)CH3, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2COOH, CH2CH2COOH, and;
wherein ‘ ’ is selected from the group consisting cyclic amino acids such as proline, homoproline, pipecolinic acid, 4-hydroxy L-proline, Azetidine carboxylic acid, piperazic acid.

In another aspect, the invention provides a process for preparation of structural formula-VIII comprising the steps of:
a. anomeric benzylation of a compound with structural formula-I by treating with benzyl alcohol as represented below to obtain a compound with a structural formula-II;


b. undergoing benzyledene protection of compound with structural formula-II by treating compound with structural formula II with dry benzaldehyde to obtain compound with structural formula-III;

c. undergoing deacylation of compound with structural formula-III by treating compound with structural formula-III with absolute ethanol in presence of potassium hydroxide to obtain a compound with structural formula-IV;


d. undergoing amino acid coupling of compound of compound with structural formula IV with a suitable amino acid coupling conjugate in presence of a basic solvent, the said solvent selected from dry pyridine, di-isopropyl ethyl amine, tri-ethylamine, and N, N-dimethyl amino pyridine (DMAP) in dichloromethane to obtain a compound with structural formula-V
wherein, ‘n’ can be any natural number(s);
wherein R or R1 is selected from the group consisting of a hydrogen, heteroatom substitutued alkyl (with both linear and branched chains), cycloalkyl, arylalkyl, heteroaryl represented by CH3, CH2CH3, CONH2, COOH, CH2OH, CH(CH3)OH, CH(CH(CH3), CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, p-hydroxy benzyl, CH(OH)CH3, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2COOH, CH2CH2COOH, and;
wherein ‘ ’ is selected from the group consisting cyclic amino acids such as proline, homoproline, pipecolinic acid, 4-hydroxy L-proline, Azetidine carboxylic acid, piperazic acid;

e. undergoing O-alkylation of compound with structural formula-V by treating compound with structural formula-V with L-2-chloropropanoic acid in presence of an inert high boiling solvent such as dry di-oxane to obtain a compound with structural formula-VI;


f. undergoing peptide coupling of compound with structural formula VI by treating compound with structural formula VI with L-alanyl-D-isoglutamine benzyl ester under standard carbodiimide coupling conditions to obtain a compound with structural formula-VII,;


g. subjecting compound with structural formula-VII by treatment with glacial acetic acid for removal of protecting groups to obtain the desired compound as represented by structural formula-VIII (MDP-SE).

In another aspect the invention provides novel intermediate compounds.

In one embodiment the invention provides a compound with structural formula V as represented below:
wherein,

In one embodiment the invention provides a compound with structural formula VI as represented below:
wherein,

In one embodiment the invention provides a compound with structural formula VII as represented below:
wherein,
In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid. The compound is characterized by IR (n in cm-1): 3321, 2927, 1625, 1573, 1538, 1311, 1271, 1088.

The compound further characterized by 1H-NMR (300 MHz, DMSO-d6) : d 4.54-4.44 (m, 2H), 4.32-4.2 (m,2H ), 4.15-4.08 (m,2H ), 2.90-2.83 (m,2H ), 2.13-2.23 (m, 2H), 2.04-1.98 (m,2H ), 1.96-1.86 (m, 1H), 1.83-1.66 (m, 2H), 1.30 (d, J = 6.561, 3H), 1.23 (d, J = 6.866,3H).

The melting point of the compound is between 157-159oC[a]25D +22.6o (c 0.2, water).

In one embodiment the invention provides a process for preparation of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid comprising the steps of:

(a) anomeric benzylation of N-acetyl D-Glucosamine to obtain Benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside;
(b) benzylidene protection of Benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside to obtain benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranoside;
(c) deacylation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranoside to obtain benzyl-2-amino-4,6-O-benzylidene-2-deoxy-a-D-mannopyranoside;
(d) amino acid coupling of benzyl-2-amino-4,6-O-benzylidene-2-deoxy-a-D-mannopyranoside with an amino acid coupling conjugate, the said amino acid coupling conjugate is a combination of N-(tert-Butoxycarbonyl)-O-benzyl-L-serine and N, N-dicyclohexylcarbodiimide in presence of a solvent 4-(N, N-dimethylamino)pyridine to obtain tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate;
(e) o-alkylation of tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate with L-2-chloropropionic acid in presence of a solvent dry dioxane to obtain (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid;
(f) peptide coupling of (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid with L-alanyl-D-isoglutamine benzyl ester trifluoroacetate to obtain (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate;
(g) deprotecting (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate in presence of glacial acetic acid to obtain (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid.

In one embodiment the invention provides a compound tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate characterized IH NMR: 7.48-6.97 (m, 10 H), 5.45 (s, 1H), 4.82 (d, 1H), 4.75 (s, 2 H), 4.64-4.45 (m, 1H), 3.89 (t, 1H), 3.85-3.72 (m, 2H), 3.56 (m, 4H), 1.83-1.65 (m, 10 H; ESI-MS- 526(M++1).

In one embodiment the invention provides a compound (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid characterized by IH NMR: 7.47-6.95 (m, 10 H), 5.45 (s, 1H), 5.2-5.0 (d, 1H), 4.75 (d, 1 H), 4.64-4.52 (m, 2H), 4.49-4.23(m, 2H), 4.25-3.41(m, 2H), 3.89 (t, 1H), 3.85-3.72 (m, 2H), 3.56 (m, 4H), 1.64-0.95 (s, 10 H; ESI-MS- 598(M++1).

In one embodiment the invention provides a compound (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate characterized by IH NMR: 8.42- 7.95(m, 6H), 7.65(d,2H), 7.45-6.85(m,7H), 6.05(s,1H), 5.5(s,1H), 4.85(d, 1H), 4.75-4.45(m,6H), 4.42-3.41(m,5H), 2.25-1.95(m,5H), 1.45-0.95(m,18H): ESI-MS- 882(M++1).

In the above processes in step (d), the suitable amino acid coupling agent is selected from N-benzyl alanine, N-benzyl valine, N-benzyl Asparagine, N-benzyl (N-Boc) Lysine, and N-benzyl (O-tertiary Butyl) Tyrosine.

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-aminopropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-ala) characterized by 1H-NMR (300 MHz): 5.32 (d, J = 6.3 Hz , 1H), 4.41 (m, 2H), 4.28 (t, J = 6.2 Hz, 2H), 3.96-3.04 (m, 8H), 2.36 (m, 2H), 2.09 (t, J = 2.9 Hz, 2H), 1.47 (d, J = 5.7 Hz, 3H,), 1.32 (d, J = 6.7 Hz, 3H), 1.26 (d, J = 5.9 Hz, 3H) ). Mass for C20H35N5O11: m/z 522 [M +1]+.

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-methylbutanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid(MDP-Val) characterized by 1H-NMR (300 MHz): 5.48(d, J = 6.4 Hz, 1H), 4.56 (m, 2H), 4.23 (t, J = 6.2 Hz 2H), 4.12-3.15 (m, 8H), 2.24 (m, 2H), 2.13 (t, J = 2.9 Hz, 2H), 1.82 (m, 1H), 1.46 (d, J = 5.5 Hz, 3H), 1.33 (d, 3H, J = 6.3 Hz), 0.96 (d, J = 4.8 Hz, 6H). Mass m/z; 550(M+1)+ .

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2,4-diamino-4-oxobutanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-asparagine) charecterized by 1H-NMR (300 MHz): 5.38 (d, J = 6.4 Hz , 1H), 4.43 (m, 2H), 4.29 (t, J = 6.2 Hz, 2H), 3.96-3.04 (m, 8H), 2.83-2.62 (m, 2H), 2.35 (m, 2H), 2.15 (t, J = 2.9 Hz, 2H), 1.46 (d, J = 5.3 Hz, 3H), 1.39 (d, J = 6.6 Hz, 3H), 1.26 (d, J = 5.9 Hz, 3H) Mass m/z 564.(M)+.

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2,6-diaminohexanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)pro panamido)propanamido)-5-oxopentanoic acid (MDP-Lys) characterized by 1H-NMR (300 MHz): 5.34 (d, J = 6.3 Hz , 1H), 4.43 (m, 2H), 4.27 (t, J = 6.1 Hz, 2H), 3.98-3.21 (m, 8H), 2.33 (m, 2H), 2.24 (t, J = 2.6 Hz, 2H), 1.79 (m, 2H), 1.43 (d, J = 5.7 Hz, 3H,), 1.33 (d, J = 6.7 Hz, 3H), 1.54 (m, 2H), 1.32 (m, 2H), 1.26 (d, J = 5.9 Hz, 3H) Mass: m/z 601(M+Na)+ .

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2-amino-3-(4-hydroxyphenyl)propanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-Tyrosine) characterized by 1H-NMR (300 MHz): 7.21 (m, 2H), 6.63 (m, 2H), 5.46 (d, J =6.4 Hz , 1H), 4.31 (m, 2H), 4.21 (t, J = 6.2 Hz 2H), 3.96-3.04 (m, 10H), 2.31 (m, 2H), 2.17 (t, J = 2.9 Hz, 2H), 1.57 (d, J = 5.6 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H) Mass m/z 636 [M + Na]+.

In the above processes in step (f) the L-alanyl-D-isoglutamine benzyl ester is (R)-benzyl-5-amino-4-((S)-2-((ter-butoxy carbonyl) amino) propanamido)-5-oxopentanooate.

The above compound with structural formula-VIII is non-toxic, non-pyrogenic and safe to be used as an adjuvant in vaccine formulations along with a suitable vaccine antigen.

In another aspect the invention provides a vaccine formulation comprising the compound with structural formula VIII as an adjuvant.

The vaccine antigen may be 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.

In one embodiment the vaccine formulation the vaccine formulation is selected from a bacterial vaccine, a viral vaccine or any potential infectious pathogens against mammals.

BRIEF DESCRIPTION OF THE FIGURES
Figure 1: represents dose response curve of MDP-SE. X-axis represents concentration in log scale and Y-axis represents % response. Y-axis values were normalized by taking smallest value or low absorbance as 0% and largest value or high absorbance as 100%. This graph indicates EC50 as 64.23µg/ml, the concentration at which adjuvant showing half maximal response. Each data set point is represented as a Mean±SD, which was obtained from three independent experiments done in duplicates.

Figure 2: represents cell toxicity of MDP-SE. X-axis represents concentration in log scale and Y-axis represents % toxicity. This graph indicates IC50 as 213µg/ml. The concentration required to cause the cell death was found to be 99.22µg/ml. Each data set point is represented as a Mean±SD, which was obtained from three independent experiments done in duplicates

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel muramyl dipeptide derivative compound and a process for synthesis thereof. The novel muramyl dipeptide derivative compound of the invention is (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid as shown below in Formula-VIII:

The novel muramyl dipeptide derivative compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid of Formula-VIII is synthesized sequentially by below shown reaction steps mentioned separately in Scheme-A and Scheme-B:

SCHEME FOR THE SYNTHESIS OF NOVEL MURAMYL PEPTIDE DERIVATIVES

SCHEME-A
The L-alanyl-D-isoglutamine benzyl ester of novel muramyl dipeptide compound is synthesised by the method as shown in below reaction Scheme-B:

SCHEME-B

The novel muramyl dipeptide derivative compound represented by the structural Formula-VIII is now named as MDP-AA, which is earlier named as MDP-SE. Wherever in the specification MDP-AA and MDP-SE are mentioned, both the representative names of Formula-VIII corresponds to the same MDP derivative compound i.e. MDP-SE = MDP-AA.

Structural Formula-VIII (MDP-SE = MDP-AA)

In a further aspect, the invention provides use of novel muramyl dipeptide derivative (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid as an adjuvant in an adjuvant composition to be used in the pharmaceutical preparations and/or vaccine formulations.

In one aspect the invention provides a compound of Structural Formula-VIII

Wherein, ‘n’ can be any natural number(s), and:
wherein R or R1 is selected from the group consisting of a hydrogen, heteroatom substitutued alkyl (with both linear and branched chains), cycloalkyl, arylalkyl, heteroaryl represented by CH3, CH2CH3, CONH2, COOH, CH2OH, CH(CH3)OH, CH(CH(CH3), CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, p-hydroxy benzyl, CH(OH)CH3, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2COOH, CH2CH2COOH, and;
wherein ‘ ’ is selected from the group consisting cyclic amino acids such as proline, homoproline, pipecolinic acid, 4-hydroxy L-proline, Azetidine carboxylic acid, piperazic acid.

In another aspect, the invention provides a process for preparation of structural formula-VIII comprising the steps of:
a. anomeric benzylation of a compound with structural formula-I by treating with benzyl alcohol as represented below to obtain a compound with a structural formula-II;


b. undergoing benzyledene protection of compound with structural formula-II by treating compound with structural formula II with dry benzaldehyde to obtain compound with structural formula-III;

c. undergoing deacylation of compound with structural formula-III by treating compound with structural formula-III with absolute ethanol in presence of potassium hydroxide to obtain a compound with structural formula-IV;


d. undergoing amino acid coupling of compound of compound with structural formula IV with a suitable amino acid coupling conjugate in presence of a basic solvent, the said solvent selected from dry pyridine, di-isopropyl ethyl amine, tri-ethylamine, and N, N-dimethyl amino pyridine (DMAP) in dichloromethane to obtain a compound with structural formula-V
wherein, ‘n’ can be any natural number(s);
wherein R or R1 is selected from the group consisting of a hydrogen, heteroatom substitutued alkyl (with both linear and branched chains), cycloalkyl, arylalkyl, heteroaryl represented by CH3, CH2CH3, CONH2, COOH, CH2OH, CH(CH3)OH, CH(CH(CH3), CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, p-hydroxy benzyl, CH(OH)CH3, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2COOH, CH2CH2COOH, and;
wherein ‘ ’ is selected from the group consisting cyclic amino acids such as proline, homoproline, pipecolinic acid, 4-hydroxy L-proline, Azetidine carboxylic acid, piperazic acid;

e. undergoing O-alkylation of compound with structural formula-V by treating compound with structural formula-V with L-2-chloropropanoic acid in presence of an inert high boiling solvent such as dry di-oxane to obtain a compound with structural formula-VI;


f. undergoing peptide coupling of compound with structural formula VI by treating compound with structural formula VI with L-alanyl-D-isoglutamine benzyl ester under standard carbodiimide coupling conditions to obtain a compound with structural formula-VII,;


g. subjecting compound with structural formula-VII by treatment with glacial acetic acid for removal of protecting groups to obtain the desired compound as represented by structural formula-VIII (MDP-SE).

In another aspect the invention provides novel intermediate compounds.

In one embodiment the invention provides a compound with structural formula V as represented below:
wherein,

In one embodiment the invention provides a compound with structural formula VI as represented below:
wherein,
In one embodiment the invention provides a compound with structural formula VII as represented below:
wherein,
In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid. The compound is characterized by IR (n in cm-1): 3321, 2927, 1625, 1573, 1538, 1311, 1271, 1088.

The compound further characterized by 1H-NMR (300 MHz, DMSO-d6) : d 4.54-4.44 (m, 2H), 4.32-4.2 (m,2H ), 4.15-4.08 (m,2H ), 2.90-2.83 (m,2H ), 2.13-2.23 (m, 2H), 2.04-1.98 (m,2H ), 1.96-1.86 (m, 1H), 1.83-1.66 (m, 2H), 1.30 (d, J = 6.561, 3H), 1.23 (d, J = 6.866,3H).

The melting point of the compound is between 157-159oC[a]25D +22.6o (c 0.2, water).

In one embodiment the invention provides a process for preparation of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid comprising the steps of:
(a) anomeric benzylation of N-acetyl D-Glucosamine to obtain Benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside;

(b) benzylidene protection of Benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside to obtain benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranoside;

(c) deacylation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranoside to obtain benzyl-2-amino-4,6-O-benzylidene-2-deoxy-a-D-mannopyranoside;

(d) amino acid coupling of benzyl-2-amino-4,6-O-benzylidene-2-deoxy-a-D-mannopyranoside with an amino acid coupling conjugate, the said amino acid coupling conjugate is a combination of N-(tert-Butoxycarbonyl)-O-benzyl-L-serine and N, N-dicyclohexylcarbodiimide in presence of a solvent 4-(N, N-dimethylamino)pyridine to obtain tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate;

(e) o-alkylation of tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate with L-2-chloropropionic acid in presence of a solvent dry dioxane to obtain (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid;

(f) peptide coupling of (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid with L-alanyl-D-isoglutamine benzyl ester trifluoroacetate to obtain (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate;

(g) deprotecting (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate in presence of glacial acetic acid to obtain (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid.

In one embodiment the invention provides a compound tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate characterized IH NMR: 7.48-6.97 (m, 10 H), 5.45 (s, 1H), 4.82 (d, 1H), 4.75 (s, 2 H), 4.64-4.45 (m, 1H), 3.89 (t, 1H), 3.85-3.72 (m, 2H), 3.56 (m, 4H), 1.83-1.65 (m, 10 H; ESI-MS- 526(M++1).

In one embodiment the invention provides a compound (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid characterized by IH NMR: 7.47-6.95 (m, 10 H), 5.45 (s, 1H), 5.2-5.0 (d, 1H), 4.75 (d, 1 H), 4.64-4.52 (m, 2H), 4.49-4.23(m, 2H), 4.25-3.41(m, 2H), 3.89 (t, 1H), 3.85-3.72 (m, 2H), 3.56 (m, 4H), 1.64-0.95 (s, 10 H; ESI-MS- 598(M++1).

In one embodiment the invention provides a compound (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate characterized by IH NMR: 8.42- 7.95(m, 6H), 7.65(d,2H), 7.45-6.85(m,7H), 6.05(s,1H), 5.5(s,1H), 4.85(d, 1H), 4.75-4.45(m,6H), 4.42-3.41(m,5H), 2.25-1.95(m,5H), 1.45-0.95(m,18H): ESI-MS- 882(M++1).

In the above processes in step (d), the suitable amino acid coupling agent is selected from N-benzyl alanine, N-benzyl valine, N-benzyl Asparagine, N-benzyl (N-Boc) Lysine, and N-benzyl (O-tertiary Butyl) Tyrosine.

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-aminopropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-ala) characterized by 1H-NMR (300 MHz): 5.32 (d, J = 6.3 Hz , 1H), 4.41 (m, 2H), 4.28 (t, J = 6.2 Hz, 2H), 3.96-3.04 (m, 8H), 2.36 (m, 2H), 2.09 (t, J = 2.9 Hz, 2H), 1.47 (d, J = 5.7 Hz, 3H,), 1.32 (d, J = 6.7 Hz, 3H), 1.26 (d, J = 5.9 Hz, 3H) ). Mass for C20H35N5O11: m/z 522 [M +1]+.

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-methylbutanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid(MDP-Val) characterized by 1H-NMR (300 MHz): 5.48(d, J = 6.4 Hz, 1H), 4.56 (m, 2H), 4.23 (t, J = 6.2 Hz 2H), 4.12-3.15 (m, 8H), 2.24 (m, 2H), 2.13 (t, J = 2.9 Hz, 2H), 1.82 (m, 1H), 1.46 (d, J = 5.5 Hz, 3H), 1.33 (d, 3H, J = 6.3 Hz), 0.96 (d, J = 4.8 Hz, 6H). Mass m/z; 550(M+1)+ .

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2,4-diamino-4-oxobutanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-asparagine) charecterized by 1H-NMR (300 MHz): 5.38 (d, J = 6.4 Hz , 1H), 4.43 (m, 2H), 4.29 (t, J = 6.2 Hz, 2H), 3.96-3.04 (m, 8H), 2.83-2.62 (m, 2H), 2.35 (m, 2H), 2.15 (t, J = 2.9 Hz, 2H), 1.46 (d, J = 5.3 Hz, 3H), 1.39 (d, J = 6.6 Hz, 3H), 1.26 (d, J = 5.9 Hz, 3H) Mass m/z 564.(M)+.

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2,6-diaminohexanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)pro panamido)propanamido)-5-oxopentanoic acid (MDP-Lys) characterized by 1H-NMR (300 MHz): 5.34 (d, J = 6.3 Hz , 1H), 4.43 (m, 2H), 4.27 (t, J = 6.1 Hz, 2H), 3.98-3.21 (m, 8H), 2.33 (m, 2H), 2.24 (t, J = 2.6 Hz, 2H), 1.79 (m, 2H), 1.43 (d, J = 5.7 Hz, 3H,), 1.33 (d, J = 6.7 Hz, 3H), 1.54 (m, 2H), 1.32 (m, 2H), 1.26 (d, J = 5.9 Hz, 3H) Mass: m/z 601(M+Na)+ .

In one embodiment the invention provides a compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2-amino-3-(4-hydroxyphenyl)propanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-Tyrosine) characterized by 1H-NMR (300 MHz): 7.21 (m, 2H), 6.63 (m, 2H), 5.46 (d, J =6.4 Hz , 1H), 4.31 (m, 2H), 4.21 (t, J = 6.2 Hz 2H), 3.96-3.04 (m, 10H), 2.31 (m, 2H), 2.17 (t, J = 2.9 Hz, 2H), 1.57 (d, J = 5.6 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H) Mass m/z 636 [M + Na]+.

In the above processes in step (f) the L-alanyl-D-isoglutamine benzyl ester is (R)-benzyl-5-amino-4-((S)-2-((ter-butoxy carbonyl) amino) propanamido)-5-oxopentanooate.

The above compound with structural formula-VIII is non-toxic, non-pyrogenic and safe to be used as an adjuvant in vaccine formulations along with a suitable vaccine antigen.

In another aspect the invention provides a vaccine formulation comprising the compound with structural formula VIII as an adjuvant.

The vaccine antigen may be 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.

In one embodiment the vaccine formulation the vaccine formulation is selected from a bacterial vaccine, a viral vaccine or any potential infectious pathogens against mammals.

The reaction steps shown in above Scheme-A and Scheme-B are further described in below experimental examples:

EXAMPLES
Example 1.1: Scheme-B: Experimental procedure for the synthesis of L-alanyl-D-isoglutamine benzyl ester:
The novel MDP derivative as described and disclosed in this invention contains a L-alanyl-D-isoglutamine benzyl ester. The L-alanyl-D-isoglutamine benzyl ester of novel muramyl dipeptide compound of this invention is synthesised by the method as shown in above reaction Scheme-B:

Example-1.1.1: (Step-a): Preparation of (R)-2-amino-5-(benzyloxy)-5-oxopentanoic acid [Compound 2]
A mixture of D-Glutamic acid [Compound 1] (4.0 g, 27.2mmols) and anhy.Na2SO4 (4.0 g) was suspended in benzyl alcohol (50 ml, 484 mmol) and BF3. Et2O (54%, 7.4 ml, 54.4 mmols) was added by means of a syringe. The suspension was stirred at RT for 15 hrs. The mixture was diluted with absolute THF (150 ml) and filtered with the aid of charcoal. The clear filtrate was treated with Et3N (8.2 ml, 59.2mmols) and concentrated under vacuum until a slurry was formed. The viscous residue was triturated with EtOAc (200 ml) and the precipitated solid was isolated by suction and washed with additional solvent to afford compound 2 as a white solid. (6.04 g, yield 94%).

Example-1.1.2 (Step-b): Preparation of (R)-5-(benzyloxy)-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid [Compound 3]
To a solution of the compound 2 (6.0 g, 25.3mmol) in dixoane and water (1 :1 , 40 mL) Boc2O (6.62 g, 30.36 mmol) was added at 0 °C and the mixture was stirred overnight. The solvent was removed under reduced pressure and the residue was diluted with water (30 mL), basified with Na2C03 and washed with EtOAc (3 × 100 mL). The pH of the aqueous layer was adjusted to 2-3 with a 5M aqueous HCI solution and the crude compound 3 was extracted with EtOAc (4 × 100 mL) and washed with brine, dried over Na2SO4 simultaneously while removing solvent under reduced pressure to afford a pure (8.19 g, 96%), viscous colour less oil (R)-5-(benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid [Compound 3]. 1H NMR(CDCl3): 7.38-7.31(m, 5H), 5.13 (s, 1H), 2.61-2.41 (m, 2H), 2.23-1.99(m, 2H), 1.43 (s, 9H). ESI MS:,338(M++1)

Example-1.1.3 (Step-c): Preparation of (R)-benzyl 5-amino-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate [Compound 4]
To a solution of above acid Compound 3 (7.0 g, 20.8 mmol)in THF (15 mL) ethyl chloroformate (2.7 mL, 28.36mmol) and triethylamine (4.21 mL, 30.25mmol) were added at 0 oC . The reaction mixture was stirred at 0 oC for 0.5 hrs, then cooled to -15oC followed by the addition of methanolic solution of ammonia (25 mL, 4.0 M). Then the reaction mixture was stirred at -15oC for another 1.5 hrs and diluted with ethyl acetate (200 mL). The organic phase was washed with water (100 mL × 3) and brine (100 mL), and dried over anhydrous sodium sulphate. The solvent was removed in vacuum and the residue was purified by flash chromatography to furnish the desired compound 4 (6.63g, 95%) as a white solid.
1H NMR (300 MHz, CDCl3): d 7.35-7.30(m, 5H), 5.12 (S, 2H), 3.70 (s, 2H), 2.58-2.42 (m, 2H), 2.31-2.19(m,2H) 1.43(s, 9H); ESI MS: 337(M+).

Example-1.1.4 (Step-d):(R)-benzyl 5-amino-4-((S)-2-((tert-butoxycarbonyl)amino)propanamido)-5-oxopentanoate[Compound 5]
t-Butoxycarbonyl-D-isoglutamine benzyl ester Compound 4 (6.5 g, 19.3 mmols) was dissolved in cold trifluoroacetic acid (20 mL) and the resultant solution was stirred at room temperature for 15 minutes. Trifluoroacetic acid was then removed and the residue was triturated with Et2O. This oily D-isoglutamine benzyl ester trifluoroacetate was dried over NaOH pellets.

To t-butoxycarbonyl-L-alanine (4.012 g, 21.23mmols) in dry THF, EDCI (4.46 g, 23.36mmols) and HOBt (3.57 g, 23.36mmols) were added and stirred at RT for 30 minutes. Later, D-isoglutamine benzyl ester trifluoroacetate (dissolved in THF) was added followed by DIPEA (7.06ml, 40.53mmols). The reaction was stirred at RT for 15 hrs, the solution was then concentrated and the residue extracted with EtOAc (500ml). The EtOAc layer was washed successively with 5% NaHCO3, 10% citric acid, and water (200ml × 3), then dried over Na2SO4 and evaporated. The residue was triturated with petroleum ether to obtain crystals which were again recrystallized from EtOAc-petroleum ether to get a white solid compound 5 (6.37 g, yield 81%).
1H-NMR (300 MHz, CDCl3) : d 7.39-7.29 (m, 5H), 5.82 (s, 1H), 5.13-5.10(m,1H), 4.07 (m, 1H), 2.60-2.42 (m, 2H), 2.27-1.97 (m, 2H), 1.32 (d, 3H) ; ESI MS : m/z 308 [M + H]+.

Example 1.2: Scheme-A:
Example-1.2.1: Preparation of Benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside [Compound II]
Anomeric benzylation of N-acetyl D-Glucosamine [Compound I]
A mixture of 2-acetamido-2-deoxy-D-glucopyranose (I) (15.0 g, 0.068 mol), amberlite IR 120 [H]+) ion exchange resin (15.0 g) in benzyl alcohol (125 mL) was stirred at 80 °C for 3.5 hours. The reaction mixture was filtered & the filtrate was evaporated under reduced pressure at 90 C°. The residue was taken up in hot isopropanol (60 mL) and filtered. The filtrate was left to form white solid crystals. These white crystals were washed twice with cold isopropanol (20 mL) and twice with ether (200mL) to give benzyl 2-acetamido-2-deoxy-D-glucopyranoside Compound II (5.62 g, 27%).

Example-1.2.2: Preparation of Benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranoside [Compound III]
Benzylidene protection-
To a well-stirred mixture of zinc chloride (5.0 g, 36.82 mmol) in dry benzaldehyde (16.5 ml) was added benzyl 2-acetamido-2-deoxy-D-glucopyranoside (compound II) (4.98 g, 16.01 mol). This mixture was stirred at room temperature for 20 hours, then stirred at 40°C for 4 hours to dissolve a small amount of remaining solid, then stirred at room temperature for 18 hours. The well-stirred reaction solution was now diluted with petroleum ether (30 ml), absolute EtOH (10 ml) and H2O (15 ml). The reaction mixture was stirred for 2 days at room temperature, then stored at 0°C for 11 days. The curdy white precipitate was collected on a coarse glass frit funnel, drained thoroughly, then washed by re-suspension in absolute EtOH (approx. 50 ml). The white finely divided solid was drained thoroughly, re-suspended in diethyl ether (approx. 50 ml), re-drained thoroughly, and then dried in vacuum at room temperature over P2O5 to yield compound III (4.17 g, 65.4%).
1HNMR(CDCl3): 7.37-7.7.82(m, 10H), 5.5(s, 1H), 4.2-4.9(m, 4H), 3.3-3.8(m, 4H), 2.5(s, 3H), ESI MS: 400(M+ +1)

Example-1.2.3: Preparation of Benzyl-2-amino-4,6-O-benzylidene-2-deoxy-a-D-mannopyranoside[Compound IV]:
Deacylation-
A mixture of compound III (2.8 g, 6.7 mmol), KOH (12 g), and 95% EtOH(40 mL) was refluxed under N2 for 10 hr and then poured into hot water (150 mL). The resulting lumps of crude product were broken up. The suspension was stirred at -5oC overnight. The product was filtered and was re-crystallized from EtOH–water with charcoal decolourization followed by re-crystallization from THF-i-Pr2O. to yield whit solid compound IV(2.3 g, 95%)
1H NMR(CDCl3): 7.37-7.7.82(m, 10H), 5.5(s, 1H), 4.9-3.3(m, 8H), ESI MS: 358(M+ +1).

Procedure for the synthesis of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid(Compound having the designated Formula VIII):

This product was synthesized as described below.

Example-1.2.4: Preparation of tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate [Compound V] (Amino Acid Coupling):
To a solution of compound IV (1.07g, 3.0 mmol) N-(tert-Butoxycarbonyl)-O-benzyl-L-serine (1.033 g, 3.5 mmol), 4-(N,N-dimethylamino)pyridine (0.010 g, 0.08 mmol), and N,N-dicyclohexylcarbodiimide (0.67 g, 3.3 mmol) were sequentially added in distilled dichloromethane (100 mL) and the mixture was stirred at room temperature for 4 hrs. The solid was removed by filtration, and the filtrate was diluted with dichloromethane and washed successively with 1N aqueous acetic acid, saturated aqueous sodium bicarbonate, and water, dried (MgSO4), filtered, and the filtrate evaporated to dryness. The solid was purified by flash chromatography on silica gel, using dichloromethane–methanol as eluent, to give compound V (1.53 g, 81%).
IH NMR: 7.48-6.97 (m, 10 H), 5.45 (s, 1H), 4.82 (d, 1H), 4.75 (s, 2 H), 4.64-4.45 (m, 1H), 3.89 (t, 1H), 3.85-3.72 (m, 2H), 3.56 (m, 4H), 1.83-1.65 (m, 10 H; ESI-MS- 526(M++1)

Example-1.2.5: Preparation of (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid [Compound VI]
(O-Alkylation):
To a stirred solution of compound V (1.44 g, 2.28mmol) in dry dioxane (30 ml) at 95°C was added sodium hydride (2.5 g) (60% oil suspension). After 1 hour, the temperature was raised to 65°C and then a solution of L-2-chloropropionic acid (1.6 g) in a small volume of dioxane was added. After 1 hour, an additional 1 g of sodium hydride was added, and heating with stirring at 65°C was continued overnight. Water (150 ml) was carefully added to the cooled reaction mixture. A dark-colored lower layer which developed was discarded, and the upper layer was filtered, partially concentrated, and diluted with water (100 ml). The aqueous mixture was extracted with diethyl ether, and the aqueous layer acidified to pH ~3 at 0°C and extracted with chloroform (100 ml ×3). The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to give the desired product VI as a white solid (1.36 g, 85%).
IH NMR: 7.47-6.95 (m, 10 H), 5.45 (s, 1H), 5.2-5.0 (d, 1H), 4.75 (d, 1 H), 4.64-4.52 (m, 2H), 4.49-4.23(m, 2H), 4.25-3.41(m, 2H), 3.89 (t, 1H), 3.85-3.72 (m, 2H), 3.56 (m, 4H), 1.64-0.95 (s, 10 H; ESI-MS- 598(M++1).

Example-1.2.6: Preparation of (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate [Compound VII] (Peptide coupling):

t-Butoxycarbonyl-L-alanyl-D-isoglutamine benzyl ester i.e., compound 5 (0.98 g, 2.42mmols) was dissolved in cold trifluoroacetic acid (10 mL) and the resulting solution was stirred at room temperature for 15 mins. Trifluoroacetic acid was then removed and the residue was triturated with Et2O. This L-alanyl-D-isoglutamine benzyl ester trifluoroacetate was dried over NaOH pellets.

EDCI (0.63 g, 3.3 mmols) and HOBt (0.504 g, 3.3 mmols) was added to compound VI (1.55 g, 2.2mmols) in dry THF. After stirring at RT for 30 mins, L-alanyl-D-isoglutamine benzyl ester trifluoroacetate (dissolved in THF) was added followed by DIPEA (1.05 mL, 6.05 mmols). The reaction was stirred at RT for 15 hr, the solution was then concentrated and the residue extracted with CHCl3 (50mL). The CHCl3 layer was washed successively with 5% NaHCO3, 10% citric acid, and water (20 mL × 3), then dried over Na2SO4 and evaporated. The residue obtained was purified on silica gel column by elution with the chloroform-methanol mixture to give compound VII (1.64 g, 75%).
1H NMR: 8.42- 7.95(m, 6H), 7.65(d,2H), 7.45-6.85(m,7H), 6.05(s,1H), 5.5(s,1H), 4.85(d, 1H), 4.75-4.45(m,6H), 4.42-3.41(m,5H), 2.25-1.95(m,5H), 1.45-0.95(m,18H): ESI-MS- 882(M++1).

Example-1.2.7: Preparation of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid [Compound with Formula VIII] (Removal of protecting groups):
Trifluoroacetic acid (1mL) was added dropwise to a solution of compound VII (1.47 g, 0.9 mmol) in methylene chloride (4mL), under nitrogen at 0 oC, and the reaction mixture stirred for 1.5 h then concentrated to dryness in vacuum. The resultant residue was repeatedly dissolved in toluene (2-3 mL) and the solvent removed to give trifluoroacetate as an orange oil.

To a solution of this compound in glacial acetic acid (50 mL) was added palladium black (150 mg) and the compound was hydrogenolyzed for 3 to 5 days in the usual way, the progress of the hydrogenolysis being monitored by t.l.c. using any of the aforementioned solvent systems. The catalyst was filtered off, and, after addition of water (30 mL), the filtrate was evaporated under diminished pressure. The residue was dissolved in a small volume of 0.1M acetic acid and then applied to a column (2.5 ×85 cm) of Sephadex LH-20 which was developed with the same solvent. The fractions corresponding to the main peak were pooled and lyophilized. The lyophilized material was re-chromatographed under the same conditions, to yield compound of Formula-VIII (MDP-SE) as fine, white crystals (0.39 g, 50%),
1H-NMR (300 MHz, DMSO-d6) : d 4.54-4.44 (m, 2H), 4.32-4.2 (m,2H ), 4.15-4.08 (m,2H ), 2.90-2.83 (m,2H ), 2.13-2.23 (m, 2H), 2.04-1.98 (m,2H ), 1.96-1.86 (m, 1H), 1.83-1.66 (m, 2H), 1.30 (d, J = 6.561, 3H), 1.23 (d, J = 6.866,3H);
Mass for C20H35O12N5Na: m/z Calculated 560, found 560 [M + Na]+.

Example 2: Preparation of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-aminopropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-ala) [Compound with Formula VIII]
Title compound defined in example 2 was prepared by reacting compound IV in scheme-A (refer Example 1.2.4) with N-benzyl alanine and the ensuing intermediate V was subjected to O-alkylation with 3-chloropropanoic acid to get the intermediate VI which was further coupled with protected dipeptide entity to afford compound VII. Final global deprotection of compound VII afford final target compound VIII (MDP-Ala)
White solid; 1H-NMR (300 MHz): 5.32 (d, J = 6.3 Hz , 1H), 4.41 (m, 2H), 4.28 (t, J = 6.2 Hz, 2H), 3.96-3.04 (m, 8H), 2.36 (m, 2H), 2.09 (t, J = 2.9 Hz, 2H), 1.47 (d, J = 5.7 Hz, 3H,), 1.32 (d, J = 6.7 Hz, 3H), 1.26 (d, J = 5.9 Hz, 3H)
). Mass for C20H35N5O11: m/z 522 [M +1]+.
Example-3: Preparation (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-methylbutanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid(MDP-Val) [Compound with Structural Formula VIII]
Title compound defined in example 3 was prepared by reacting compound IV in scheme-A with N-benzyl valine and the ensuing intermediate V was subjected to O-alkylation with 3-chloropropanoic acid to get the intermediate VI which was further coupled with protected dipeptide entity to afford compound VII. Final global deprotection of compound VII afford final target compound VIII (MDP-Val)
White solid; 1H-NMR (300 MHz): 5.48(d, J = 6.4 Hz, 1H), 4.56 (m, 2H), 4.23 (t, J = 6.2 Hz 2H), 4.12-3.15 (m, 8H), 2.24 (m, 2H), 2.13 (t, J = 2.9 Hz, 2H), 1.82 (m, 1H), 1.46 (d, J = 5.5 Hz, 3H), 1.33 (d, 3H, J = 6.3 Hz), 0.96 (d, J = 4.8 Hz, 6H). Mass m/z; 550(M+1)+ .

Example-4: Preparation of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2,4-diamino-4-oxobutanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-asparagine) [Compound with Structural Formula VIII]
Title compound defined in example 4 was prepared by reacting compound IV in scheme-A with N-benzyl Asparagine and the ensuing intermediate V was subjected to O-alkylation with 3-chloropropanoic acid to get the intermediate VI which was further coupled with protected dipeptide entity to afford compound VII. Final global deprotection of compound VII afford final target compound VIII (MDP-Asp).
White solid; 1H-NMR (300 MHz): 5.38 (d, J = 6.4 Hz , 1H), 4.43 (m, 2H), 4.29 (t, J = 6.2 Hz, 2H), 3.96-3.04 (m, 8H), 2.83-2.62 (m, 2H), 2.35 (m, 2H), 2.15 (t, J = 2.9 Hz, 2H), 1.46 (d, J = 5.3 Hz, 3H), 1.39 (d, J = 6.6 Hz, 3H), 1.26 (d, J = 5.9 Hz, 3H)
ESI MS 564.(M)+.

Example-5: Preparation of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2,6-diaminohexanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)pro panamido)propanamido)-5-oxopentanoic acid (MDP-Lys) [Compound with Structural Formula VIII]
Title compound defined in example 5 was prepared by reacting compound IV in scheme-A with N-benzyl (N-Boc) Lysine and the ensuing intermediate V was subjected to O-alkylation with 3-chloropropanoic acid to get the intermediate VI which was further coupled with protected dipeptide entity to afford compound VII. Final global deprotection of compound VII afford final target compound VIII (MDP-Lys).
White solid; 1H-NMR (300 MHz): 5.34 (d, J = 6.3 Hz , 1H), 4.43 (m, 2H), 4.27 (t, J = 6.1 Hz, 2H), 3.98-3.21 (m, 8H), 2.33 (m, 2H), 2.24 (t, J = 2.6 Hz, 2H), 1.79 (m, 2H), 1.43 (d, J = 5.7 Hz, 3H,), 1.33 (d, J = 6.7 Hz, 3H), 1.54 (m, 2H), 1.32 (m, 2H), 1.26 (d, J = 5.9 Hz, 3H)
ESI MS 601(M+Na)+

Example-6: Preparation of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2-amino-3-(4-hydroxyphenyl)propanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-Tyrosine) [Compound with Structural Formula VIII]
Title compound defined in example 6 was prepared by reacting compound IV in scheme-A with N-benzyl (O-tertiary Butyl) Tyrosine and the ensuing intermediate V was subjected to O-alkylation with 3-chloropropanoic acid to get the intermediate VI which was further coupled with protected dipeptide entity to afford compound VII. Final global deprotection of compound VII afford final target compound VIII (MDP-Tyr).
White solid; 1H-NMR (300 MHz): 7.21 (m, 2H), 6.63 (m, 2H), 5.46 (d, J =6.4 Hz , 1H), 4.31 (m, 2H), 4.21 (t, J = 6.2 Hz 2H), 3.96-3.04 (m, 10H), 2.31 (m, 2H), 2.17 (t, J = 2.9 Hz, 2H), 1.57 (d, J = 5.6 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H)
ESI MS 636(M+ +Na)

Likewise, different amino acid conjugates depicted in formula VIII were generated by introducing various protected amino acids on the intermediate V of the scaffold followed by O-alylation of the ensuing intermediate VI, peptide coupling of intermediate VII followed by final complete deprotection of pretecting groups to achieve the molecular diversity presented as structural formula VIII.

Example-7: In-vitro Evaluation of MDP-SE
Muramyl dipeptide molecules have the ability to bind to NOD2 receptors that are present on the surface of the immune cells, so as to stimulate immune response. NOD2 (Nucleotide oligomerization domain) receptor is an intracellular pattern recognition receptor (PRRs) recognizes muramyl dipeptide derivatives and stimulates cascade of signalling pathways to induce immune response. To demonstrate whether the MDP-SE synthesized in the present invention stimulates NOD2 receptors, HEK – Blue Human NOD2 reporter cell lines purchased from Invivogen, California, USA. These cells were prepared by co-transfection of human NOD2 gene and codon-optimized SEAP (secreted embryonic alkaline phosphatase) reporter gene into HEK 293 cells. Upon stimulation of cells with muramyl dipeptide derivative, NFkB gets activated, which inturn secrets SEAP and it was measured in cell supernatant. Experimental details of this in-vitro assay are given as example 7.1.

Example 7.1: NOD2 Specific Reporter Assay: HEK – Blue Human NOD2 reporter cells (5x104 / well) were plated and cultured overnight in a humidified CO2 incubator. Next day, cells were treated with various concentrations of MDP derivative (MDP-SE, 0.002µM to 2000µM) and cultured for 24-72 hrs. Supernatant was collected and treated with Quanti-blue detection reagent and incubated for 15-30 min. Absorbance was read at 630nm. Dose response curve was generated by plotting concentration of adjuvant on X-axis and % response on the Y-axis as shown in Figure-1. To generate dose response curve, highest absorbance shown at particular adjuvant concentration was taken as 100% and the least absorbance was taken as 0% response and effective concentration at 50% (EC50) response was determined from the dose response curve.

In the present invention, MDP-SE shows maximum 100% response with a high absorbance at 1000µg/ml concentration (reached plateau at this concentration in sigmoidal curve), whereas, it shows 0% response with a low absorbance at 0.01µg/ml concentration. These results indicated EC50 as 64.23µg/ml (concentration at which MDP-SE showed half maximal response)

These results indicate MDP-SE ability to recognize or stimulate NOD2 receptors. Presence of secreted cytokine levels in the cell supernatant determines the adjuvant ability to initiate signaling cascade to secrete cytokines, which in turn determines the efficiency of an adjuvant to stimulate immune response and act as an adjuvant.

Further, the use of immunomodulatory molecules as adjuvants poses safety concerns. To evaluate the toxicity of MDP-SE, two in-vitro assays namely cytotoxicity & pyrogenicity assays were performed, those were described hereafter as examples as in examples 7.2 & 7.3.

Example 7.2: Cytotoxicity Assay: J774.2A cells (2x104 / well) were plated and incubated overnight. Next day, cells were treated with various concentrations of MDP-SE (0.005mg/ml to 0.5mg/ml) and cultured for 24hrs. Supernatant was discarded and cells were treated with 100µl of MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (5mg/ml) and incubated for 3-4 hrs until formozan crystals were formed. Later, supernatant was discarded and crystals were dissolved with DMSO for 5-10 min. Absorbance was read at 540nm.
Figure 2 represents cell toxicity effect of MDP-SE. X-axis represents concentration in log scale and Y-axis represents % toxicity. This graph indicated IC50 as 213µg/ml, the concentration at which MDP-SE causes 50% cell death or toxicity effect.

MTT assays were performed to identify the in-vitro cytotoxicity of the various adjuvants. MDP derivative found to be safe at a stock concentration of 2-5 mg/ml. In the above example 2.1, after collection of supernatant for reporter assay, cells were washed with phenol free DMEM and also treated with MTT to determine the cytotoxicity effect in human cell lines.

Generally, pyrogenicity of any molecule can be measured by the over production of pro-inflammatory cytokines IL-1ß, IL-6, and TNFa, following an activation of monocytes in-vitro, which in turn, could be an indicative of increased reactogenicity in-vivo. Hence, in the present invention, MDP-SE was tested for In-vitro pyrogenicity. Experimental details have been mentioned as below as an example 7.3. Inorder to determine whether the MDP-SE synthesized in the present invention, is pyrogenic or not, in-vitro pyrogen test was performed using Peripheral blood mononuclear cells (PBMCs).

Example 7.3: In-vitro pyrogen test: Frozen Peripheral blood mononuclear cells (PBMCs) were revived and plated (0.5 x 106 cells/ml, 100µl) in 96 well round bottom cell culture plate. Cells were supplemented with RPMI 1640 medium (Gibco Laboratories, Grand Island, N.Y.) containing fetal bovine serum (10%), glutamine (2 mM), and pencillin-streptomycin. Cells further stimulated with LPS (10ng/ml) or endotoxin (1EU/ml, 0.5EU/ml, 0.25EU/ml, 0.125EU/ml) or MDP-SE (10 or 100µg/ml). Cells stimulated with LPS or endotoxin were incubated for 4hrs where as cells stimulated with MDP derivatives of the present invention kept for incubation in humidified CO2 chamber at 37ºC for 24hrs. After stimulation, cell culture supernatant was collected and cytokines (such as IL-1ß, IL-6 or TNF-a) were measured by ELISA. Concentrations each cytokine produced by PBMCs in the presence of reference standard endotoxin at 0.5 EU/ml was measured in triplicates. Threshold levels were calculated for each cytokine, using the cytokine levels produced by the reference standard endotoxin at 0.5EU/ml (Marina et al., Vaccine 30 (2012) 4859– 4865).

Example 7.3.1: Determination of IL-1beta by ELISA: To determine Interleukin 1ß, Enzyme Linked Immunosorbent Assay (ELISA) was performed according to the instruction manual (ebioscience, cat#88-7010). Briefly, capture antibody (cat#14-7018-67, eBioscience) was first diluted 1:250 using coating buffer and added 100 µL to each well in 96-well microplate. Plates were incubated overnight at 2-8 °C. Coated plates were then washed with wash buffer (PBST). After washing, these plates were blocked using 1x assay diluent for 1hr at room temperature followed by washing with PBST. A series of 2 fold dilutions were made from top standard ranging from 4 to 500 pg/mL, to make the standard curve. Similarly, 4fold dilutions (1:4, 1:16 & 1:64) of cell supernatant obtained from PBMCs stimulation with MDP-SE were prepared. Aliquot (100 µL) of diluted standards or cell supernatants were added to well in duplicates and the plate was incubated at room temperature for 2hrs. After washing the plate, 100 µL/well of detection antibody (cat#33-7016-67) diluted in 1X Assay diluent was added and incubated at room temperature for 1hr. Later, 100 µL/well of Avidin-HRP* diluted in 1X Assay diluent was added and incubated at room temperature for 30 minutes. Finally, after washes, 100 µL of substrate solution was added to each well and incubated at RT for 15 minutes. Reaction was stopped by the addition of 50 µL of 2N H2SO4 to each well and the plate was read at 450 nm.

Example 7.3.2: Determination of IL-6 by ELISA: To determine Interleukin 6 (IL-6), Enzyme Linked Immunosorbent Assay (ELISA) was performed according to the instruction manual (ebioscience, cat#88-7066). Briefly, capture antibody (cat# 14-7069-67, eBioscience) was first diluted 1:250 using coating buffer and 100 µL was added to each well in 96-well microplate. Plates were either incubated overnight at 2-8 °C. Coated plates were then washed four times (350 µL/well) with 1x wash buffer previously prepared by dilution of a 20x concentrate with deionized water. After washing, plates were inverted and blotted on absorbent paper to remove any residual buffer. These plates were blocked using assay diluent (250 µL/well) for 60 minutes at room temperature followed by four times wash (350 µL/well) with 1x wash buffer. A series of 2fold dilutions of were made from top standard ranging from 0 to 200 pg/mL, to make the standard curve. Similarly, 4fold dilutions (1:4, 1:16 & 1:64) of cell supernatant obtained from PBMCs stimulation with MDP-SE were prepared. Aliquot (100 µL) of diluted standards or cell supernatants were added to well in duplicates and the plate was incubated at room temperature for 2hrs. After washing the plate, 100 µL/well of detection antibody (cat# 33-7068-67, eBioscience) diluted in 1X Assay diluent was added and incubated at room temperature for 1hr. Later, 100 µL/well of Avidin-HRP* diluted in 1X Assay Diluent was added and incubated at room temperature for 30 minutes. Finally, after washes, 100 µL of Substrate Solution was added to each well and incubated at RT for 15 minutes. Reaction was stopped by the addition of 50 µL of stop solution to each well and the plate was read at 450 nm.

Example 7.3.3: Determination of TNF-alpha by ELISA: To determine Tissue Necrosis Factor -alpha (TNF-a), Enzyme Linked Immunosorbent Assay (ELISA) was performed according to the instruction manual (ebioscience, cat#88-7346). Briefly, capture antibody (cat# 14-7348-67, eBioscience) was first diluted 1:250 using coating buffer and 100 µL was added to each well in 96-well microplate. Plates were either incubated overnight at 2-8 °C. Coated plates were then washed four times (350 µL/well) with 1x wash buffer previously prepared by dilution of a 20x concentrate with deionized water. After washing, plates were inverted and blotted on absorbent paper to remove any residual buffer. These plates were blocked using assay diluent (250 µL/well) for 60 minutes at room temperature followed by four times wash (350 µL/well) with 1x wash buffer. A series of 2fold dilutions of were made from top standard ranging from 4 to 500 pg/mL, to make the standard curve. Similarly, 4fold dilutions (1:4, 1:16 & 1:64) of cell supernatant obtained from PBMCs stimulation with MDP-SE were prepared. Aliquot (100 µL) of diluted standards or cell supernatants were added to well in duplicates and the plate was incubated at room temperature for 2hrs. After washing the plate, 100 µL/well of detection antibody (cat# 33-7349-67, eBioscience) diluted in 1X Assay diluent was added and incubated at room temperature for 1hr. Later, 100 µL/well of Avidin-HRP* diluted in 1X Assay Diluent was added and incubated at room temperature for 30 minutes. Finally, after washes, 100 µL of Substrate Solution was added to each well and incubated at RT for 15 minutes. Reaction was stopped by the addition of 50 µL of Stop Solution to each well and the plate was read at 450 nm.

Threshold levels for each cytokine were found to be as follows 370pg/ml for IL-1ß, 3110pg/ml for IL-6 & 753pg/ml for TNF-a. MDP-SE, when tested at 10 & 100µg/ml concentration, cytokine (IL-6 , IL-1ß & TNF-a) levels were found to be within the threshold value. Based on these results, it was observed that the MDP-SE is not pyrogenic at 100µg/ml concentration, tested in human PBMCs.

The compound of the present invention with the Structural Formla VIII (i.e. MDP-AA), in particular has been found to have a pronounced immunomodulating activity, when it is used in combination with various vaccine antigen(s). The vaccine antigens for combination with the vaccine adjuvant of the present invention may be formulated with suitable vaccine antigens including but not limited to other antigens namely subunit malarial antigens comprising and all stages of life cycle, for example any form of circumsporozoite protein (CSP), rPvRII, rMsPs, rPfs25 transmission blocking antigens, rPvs25, 27, rPfF2, GLURP, liver stage antigens, inactivated rabies antigen, chikungunya virus antigen etc. The vaccine antigens of the present invention for formulation with the novel adjuvant of the present invention MDP-AA may be selected from recombinant Human papillomavirus antigen (including any and all different serotypes of HPV infection), Japanese encephalitis virus antigen, inactivated rabies antigen, hepatitis A antigen, hepatitis B antigen, hepatitis E antigen (including subunit virus like particles), ebola virus antigen any arbovirus including but not limited to zika virus, dengue vaccine antigen, diphtheria-tetanus-pertussis ( either whole cell pertusis or acellular pertusis like DTaP, Tdap), Haemophilus influenzae type b (Hib), including any and all known form of vaccine antigens causing meningococcal infections namely Nisseria meningitidis A, Nisseria meningitidis C Nisseria meningitidis Y, Nisseria meningitidis W135, Nisseria meningitidis X and Nisseria meningitidis B. The vaccine antigens of this present invention for formulation with this novel adjuvant MDP-MN may also include Streptococcus pneumoniae antigens for example streptococcal pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. Vaccine antigens such as Salmonella typhi Vi polysaccharide (either plain or conjugated), Salmonella enteritidis, non-typhoidal salmonella, Salmonella paratyphi, Herpes simplex virus antigen, human immunodeficiency virus antigen, cytomegalovirus antigen, H1N1 virus antigen, or tuberculosis vaccines are also included under the ambit of the present invention. Any potential live-attenuated vaccine antigen for formulation with the novel vaccine adjuvant MDP-MN of the present invention include MMR (Measles, Mumps, Rubella), chicken pox, oral polio (Sabin), influenza (the seasonal flu H1N1 virus), rotavirus and yellow fever, west nile fever vaccine. Vaccine antigens such as chikungunya virus, chandipura virus, or other forms of inactivated vaccine antigens such as inactivated polio-Salk, live attenuated sabin polio virus type I, II and III, Hepatitis A which are either heat-inactivated or chemically inactivated particles of the pathogen are also within the scope of the present invention for formulation with the novel adjuvant of the present invention. The antigens as mentioned hereinabove of the present invention may be present singly or in combinations thereof.
,CLAIMS:
1. A compound of Structural Formula-VIII

Wherein, ‘n’ can be any natural number(s), and:
wherein R or R1 is selected from the group consisting of a hydrogen, heteroatom substitutued alkyl (with both linear and branched chains), cycloalkyl, arylalkyl, heteroaryl represented by CH3, CH2CH3, CONH2, COOH, CH2OH, CH(CH3)OH, CH(CH(CH3), CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, p-hydroxy benzyl, CH(OH)CH3, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2COOH, CH2CH2COOH, and;
wherein ‘ ’ is selected from the group consisting cyclic amino acids such as proline, homoproline, pipecolinic acid, 4-hydroxy L-proline, Azetidine carboxylic acid, piperazic acid.

2. A process for preparation of structural formula-VIII as claimed in claim 1 comprising the steps of :
a. anomeric benzylation of a compound with structural formula-I by treating with benzyl alcohol as represented below to obtain a compound with a structural formula-II;


b. undergoing benzyledene protection of compound with structural formula-II by treating compound with structural formula II with dry benzaldehyde to obtain compound with structural formula-III;

c. undergoing deacylation of compound with structural formula-III by treating compound with structural formula-III with absolute ethanol in presence of potassium hydroxide to obtain a compound with structural formula-IV;


d. undergoing amino acid coupling of compound of compound with structural formula IV with a suitable amino acid coupling conjugate in presence of a basic solvent, the said solvent selected from dry pyridine, di-isopropyl ethyl amine, tri-ethylamine, and N, N-dimethyl amino pyridine (DMAP) in dichloromethane to obtain a compound with structural formula-V
wherein, ‘n’ can be any natural number(s);
wherein R or R1 is selected from the group consisting of a hydrogen, heteroatom substitutued alkyl (with both linear and branched chains), cycloalkyl, arylalkyl, heteroaryl represented by CH3, CH2CH3, CONH2, COOH, CH2OH, CH(CH3)OH, CH(CH(CH3), CH2CH(CH3)2, CH(CH3)CH2CH3, CH2C6H5, p-hydroxy benzyl, CH(OH)CH3, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2COOH, CH2CH2COOH, and;
wherein ‘ ’ is selected from the group consisting cyclic amino acids such as proline, homoproline, pipecolinic acid, 4-hydroxy L-proline, Azetidine carboxylic acid, piperazic acid;

e. undergoing O-alkylation of compound with structural formula-V by treating compound with structural formula-V with L-2-chloropropanoic acid in presence of an inert high boiling solvent such as dry di-oxane to obtain a compound with structural formula-VI;


f. undergoing peptide coupling of compound with structural formula VI by treating compound with structural formula VI with L-alanyl-D-isoglutamine benzyl ester under standard carbodiimide coupling conditions to obtain a compound with structural formula-VII,;


g. subjecting compound with structural formula-VII by treatment with glacial acetic acid for removal of protecting groups to obtain the desired compound as represented by structural formula-VIII (MDP-SE).

3. A compound with structural formula V as represented below:
wherein,

4. A compound with structural formula VI as represented below:
wherein,
5. A compound with structural formula VII as represented below:
wherein,
6. A compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid.

7. The compound as claimed in claim 6, characterized by IR (n in cm-1): 3321, 2927, 1625, 1573, 1538, 1311, 1271, 1088.

8. The compound as claimed in claim 6, further characterized by 1H-NMR (300 MHz, DMSO-d6) : d 4.54-4.44 (m, 2H), 4.32-4.2 (m,2H ), 4.15-4.08 (m,2H ), 2.90-2.83 (m,2H ), 2.13-2.23 (m, 2H), 2.04-1.98 (m,2H ), 1.96-1.86 (m, 1H), 1.83-1.66 (m, 2H), 1.30 (d, J = 6.561, 3H), 1.23 (d, J = 6.866,3H).

9. The compound as claimed in claim 6, whose melting point is between 157-159oC[a]25D +22.6o (c 0.2, water).

10. A process for preparation of (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid comprising the steps of:
(a) anomeric benzylation of N-acetyl D-Glucosamine to obtain Benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside;

(b) benzylidene protection of Benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside to obtain benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranoside;

(c) deacylation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranoside to obtain benzyl-2-amino-4,6-O-benzylidene-2-deoxy-a-D-mannopyranoside;

(d) amino acid coupling of benzyl-2-amino-4,6-O-benzylidene-2-deoxy-a-D-mannopyranoside with an amino acid coupling conjugate, the said amino acid coupling conjugate is a combination of N-(tert-Butoxycarbonyl)-O-benzyl-L-serine and N, N-dicyclohexylcarbodiimide in presence of a solvent 4-(N, N-dimethylamino)pyridine to obtain tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate;

(e) o-alkylation of tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate with L-2-chloropropionic acid in presence of a solvent dry dioxane to obtain (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid;

(f) peptide coupling of (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid with L-alanyl-D-isoglutamine benzyl ester trifluoroacetate to obtain (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate;

(g) deprotecting (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate in presence of glacial acetic acid to obtain (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-hydroxypropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid.

11. A compound tert-butyl((2S)-3-(benzyloxy)-1-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-8-hydroxy-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-7-yl) amino)-1-oxopropan-2-yl) carbamate characterized IH NMR: 7.48-6.97 (m, 10 H), 5.45 (s, 1H), 4.82 (d, 1H), 4.75 (s, 2 H), 4.64-4.45 (m, 1H), 3.89 (t, 1H), 3.85-3.72 (m, 2H), 3.56 (m, 4H), 1.83-1.65 (m, 10 H; ESI-MS- 526(M++1).

12. A compound (2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanoic acid characterized by IH NMR: 7.47-6.95 (m, 10 H), 5.45 (s, 1H), 5.2-5.0 (d, 1H), 4.75 (d, 1 H), 4.64-4.52 (m, 2H), 4.49-4.23(m, 2H), 4.25-3.41(m, 2H), 3.89 (t, 1H), 3.85-3.72 (m, 2H), 3.56 (m, 4H), 1.64-0.95 (s, 10 H; ESI-MS- 598(M++1).

13. A compound (4R)-benzyl-5-amino-4-((2S)-2-((2R)-2-(((4aR,6S,7R,8R,8aS)-6-(benzyloxy)-7-((S)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propanamido)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5-oxopentanoate characterized by IH NMR: 8.42- 7.95(m, 6H), 7.65(d,2H), 7.45-6.85(m,7H), 6.05(s,1H), 5.5(s,1H), 4.85(d, 1H), 4.75-4.45(m,6H), 4.42-3.41(m,5H), 2.25-1.95(m,5H), 1.45-0.95(m,18H): ESI-MS- 882(M++1).

14. The process as claimed in claim 2(d) and 10(d), wherein the suitable amino acid coupling agent is selected from N-benzyl alanine, N-benzyl valine, N-benzyl Asparagine, N-benzyl (N-Boc) Lysine, and N-benzyl (O-tertiary Butyl) Tyrosine.

15. A compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-aminopropanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-ala) characterized by 1H-NMR (300 MHz): 5.32 (d, J = 6.3 Hz , 1H), 4.41 (m, 2H), 4.28 (t, J = 6.2 Hz, 2H), 3.96-3.04 (m, 8H), 2.36 (m, 2H), 2.09 (t, J = 2.9 Hz, 2H), 1.47 (d, J = 5.7 Hz, 3H,), 1.32 (d, J = 6.7 Hz, 3H), 1.26 (d, J = 5.9 Hz, 3H) ). Mass for C20H35N5O11: m/z 522 [M +1]+.

16. A compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((S)-2-amino-3-methylbutanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid(MDP-Val) characterized by 1H-NMR (300 MHz): 5.48(d, J = 6.4 Hz, 1H), 4.56 (m, 2H), 4.23 (t, J = 6.2 Hz 2H), 4.12-3.15 (m, 8H), 2.24 (m, 2H), 2.13 (t, J = 2.9 Hz, 2H), 1.82 (m, 1H), 1.46 (d, J = 5.5 Hz, 3H), 1.33 (d, 3H, J = 6.3 Hz), 0.96 (d, J = 4.8 Hz, 6H). Mass m/z; 550(M+1)+ .

17. A compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2,4-diamino-4-oxobutanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-asparagine) charecterized by 1H-NMR (300 MHz): 5.38 (d, J = 6.4 Hz , 1H), 4.43 (m, 2H), 4.29 (t, J = 6.2 Hz, 2H), 3.96-3.04 (m, 8H), 2.83-2.62 (m, 2H), 2.35 (m, 2H), 2.15 (t, J = 2.9 Hz, 2H), 1.46 (d, J = 5.3 Hz, 3H), 1.39 (d, J = 6.6 Hz, 3H), 1.26 (d, J = 5.9 Hz, 3H) Mass m/z 564.(M)+.

18. A compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2,6-diaminohexanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)pro panamido)propanamido)-5-oxopentanoic acid (MDP-Lys) characterized by 1H-NMR (300 MHz): 5.34 (d, J = 6.3 Hz , 1H), 4.43 (m, 2H), 4.27 (t, J = 6.1 Hz, 2H), 3.98-3.21 (m, 8H), 2.33 (m, 2H), 2.24 (t, J = 2.6 Hz, 2H), 1.79 (m, 2H), 1.43 (d, J = 5.7 Hz, 3H,), 1.33 (d, J = 6.7 Hz, 3H), 1.54 (m, 2H), 1.32 (m, 2H), 1.26 (d, J = 5.9 Hz, 3H) Mass: m/z 601(M+Na)+ .

19. A compound (4R)-5-amino-4-((2S)-2-((2R)-2-(((3R,4R,5S,6R)-3-((R)-2-amino-3-(4-hydroxyphenyl)propanamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (MDP-Tyrosine) characterized by 1H-NMR (300 MHz): 7.21 (m, 2H), 6.63 (m, 2H), 5.46 (d, J =6.4 Hz , 1H), 4.31 (m, 2H), 4.21 (t, J = 6.2 Hz 2H), 3.96-3.04 (m, 10H), 2.31 (m, 2H), 2.17 (t, J = 2.9 Hz, 2H), 1.57 (d, J = 5.6 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H) Mass m/z 636 [M + Na]+.

20. The process as claimed in claim 2(f) and 10(f), wherein the L-alanyl-D-isoglutamine benzyl ester is (R)-benzyl-5-amino-4-((S)-2-((ter-butoxy carbonyl) amino) propanamido)-5-oxopentanooate.

21. The compound with structural formula VIII as claimed in clam 1, wherein the compound with structural formula-VIII is non-toxic, non-pyrogenic and safe to be used as an adjuvant in vaccine formulations along with a suitable vaccine antigen.
22. A vaccine formulation comprising the compound with structural formula VIII as an adjuvant.

23. The vaccine antigen as claimed in claim 15, wherein the vaccine antigen may be 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.

24. The vaccine formulation as claimed in claims 15 and 16, wherein the vaccine formulation is selected from a bacterial vaccine, a viral vaccine or any potential infectious pathogens against mammals.