Chimeric adjuvant

Abstract

The invention relates to chimeric adjuvants of formula I formed by conjugation of Mincle and NOD agonists. These conjugates combine the desirable properties of each agonist to provide an effective adjuvant compound.

Description

[0001] CHIMERIC ADJUVANT

[0002] 1. FIELD OF THE INVENTION

[0003] The present invention relates to chimeric adjuvants formed by conjugation of Mincle and NOD agonists. These conjugates combine the desirable properties of each agonist to provide an effective adjuvant compound.

[0004] 2. BACKGROUND TO THE INVENTION

[0005] The innate immune system provides the first line of host defence against invading pathogens. Innate immune cells contain a limited number of evolutionarily conserved pattern recognition receptors (PRRs) that interact with pathogen-associated molecular patterns (PAMPs) leading to the production of cytokines and chemokines that shape the type and duration of the immune response, including immunological memory.

[0006] Consequently, effective vaccines not only need an antigen, against which immunological memory will be raised, but also adjuvants to engage innate immune cell receptors. The exact structure of each antigen and PAMP determines the immune response. Moreover, since pathogens contain multiple PAMPs, which the host recognizes and responds to in an orchestrated manner, targeting a combination of PAMPs, rather than a single entity, may enhance vaccine efficacy (Cao, 2016) (Janine K Tom, 2019) (Bob J Iganacio, 2018).

[0007] This enhanced vaccine efficacy is often attributed to adjuvant synergism, which not only enhances the immune response but can also reduce adjuvant loading and toxicity (Janine K Tom, 2019). Adverse reactions induced by immunological adjuvants occur through an immunological-based mechanism so that immunostimulatory effects necessary to increase the effectiveness of the vaccine can also lead to undesirable effects. Unfortunately, for immunological adjuvants, the concentration needed to obtain desired pharmacological effects may approach or even exceed the concentration at with toxicity becomes in issue.

[0008] Synergistic combinations of PAMPs employed as adjuvants in clinical trials have focused on the Toll-like receptor (TLR)-4 adjuvant monophosphoryl lipid A (MPL) in combination with the TLR-9 ligand CpG, the TLR-3 ligand poly(I:C), or the Macrophage inducible C- type lectin (Mincle) ligand monomycolyl glycerol (MMG).

[0009] Other studies exploring the synergistic effects of PAMPs have also predominantly focused on combinations of TLR ligands, or TLR ligands with nucleotide-binding oligomerization domain (NOD)-2 PAMPs, stimulator of interferon genes (STING) agonists or the Mincle agonist trehalose dibehenate (TDB). The results have been variable. Studies into the co-administration of selected Mincle and NOD agonists have indicated a synergistic effect with respect to some immunological responses (Amir I. Tukhvatulin, 2020) (Jean-Yves Dube, 2020).

[0010] Chimeric adjuvants, in which two or more PAMPs are incorporated into one molecule, have the potential to increase the likelihood of synergistic immunological effects. Vaccine efficacy may be enhanced by co-delivery of the conjugated PAMPs to the same cell and 'cross-talk' between them (Cao, 2016) (Janine K Tom, 2019) (Vincent Pavot, 2014) (Amir I. Tukhvatulin, 2020). However, due to technical and design challenges, very few chimeric adjuvants have been prepared (Vincent Pavot, 2014) (Alice Guthjahr, 2020) (Gijs G. Zorn, 2019).

[0011] NOD ligands have long been known for their adjuvanticity, but these ligands can be toxic (F. Ellous, 1974) (C. A. Dinarello, 1978) (Chedid., 1983), thereby necessitating careful application or structural modifications (P. Lefrancier, 1987).

[0012] The only Mincle-NOD chimeric adjuvant prepared to date consists of trehalose dimycolate (TDM) conjugated to a muramyl dipeptide (MDP) ligand using a succinic acid linker (Hidenaru Ishida, 1989). Unfortunately, the adjuvanticity of this compound was not demonstrated. Disappointingly, the conjugation of TDB to immunogenic Mycobacterium tuberculosis peptides ESAT61-20 and TB10.43-11 resulted in a vaccine construct that failed to be effective (Cameron C. Hanna, 2022) (Hanna, 2020). This was surprising given that a ESAT61-20 / TB10.43-ll-Pam2Cys conjugate provided protection against M. tuberculosis in murine vaccination study (A. S. Ashhurst, 2019), and that TDB, which is a synthetic derivative of the major cell wall component of M. tuberculosis, exhibits potent Mincle-mediated adjuvant activity (Yamasaki, 2008).

[0013] Accordingly, it is not possible to predict how combinations of, or chimeric adjuvants of NOD and Mincle agonists will act with respect to immune system function, however, the use of NOD and Mincle agonists as adjuvants shows potential if a way of utilising their activity in a safe way that avoids toxic effects can be found.

[0014] Accordingly, the importance of effective vaccine adjuvants compels further investigation of combinations and conjugates of PAMPs. It is therefore an object of the invention to provide a conjugate of Mincle and NOD agonists that goes at least some way to alleviating the problems set out above, and / or that will at least provide the public with a useful choice.

[0015] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0016] 3. SUMMARY OF THE INVENTION

[0017] The invention provides a chimeric adjuvant comprising Mincle and NOD2 ligands connected via a pH dependent hydrolysable linker group.

[0018] In one aspect the invention provides a compound of Formula I wherein

[0019] X is selected from O or NH;

[0020] Ai and A2 are each independently selected from:

[0021] -phenyl-, -(Ci2-C42)alkoxyphenyl-, -(Ci2-C42)alkylphenyl-, -(Ci2-C42)alkylene-, -(C12- C42)alkenylene-, -(C12- C42)alkynylene-, -(Ci2-C42)alkoxy(Ci2-C42)alkyl-, each of which may be optionally substituted with one or more substituents selected from:

[0022] -hydroxy, oxo, -(Ce-Cio)aryl, -(Ci-Cis)alkyl, -(Ci-Cis)alkaryl, -(Ci-Cis)alkoxy, -(Ci- Cisjalkenyloxy, -(Ci-Cis)alkynyloxy, carboxy, cyano, -(Ci-Cis)alkoxycarbonyl, (Ci- Cisjalkylthio, -(Ci-Cis)alkenylthio, -(Ci-Cis)alkynylthio, -(Ci-Cis)alkylsulfonyl, -(Ci- Cisjalkenylsuflonyl, -(Ci-Cis)alkynylsulphonyl, halo, halo(Ci- Cisjalkoxy, halo(Ci- Cisjalkenyloxy, halo(Ci-Cis)alkynyloxy, -CONRR^nd -NRR1; wherein heterocyclyl is optionally substituted with one or two groups independently selected from -(Ci-Cis)alkyl, -hydroxy, -(Ci-Cis)alkoxy, -(Ci-Cis)alkylthio, -(Ci- Cisjalkylsulfonyl, halo and -CONRR1, wherein each R is independently selected from H, -(Ci-Cis)alkyl, hydroxy(Ci- Cisjalkyl and - (Ci-Ci8)alkoxy(Ci-Cis)alkyl and each R1is independently selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl, - (Ci-Ci8)alkoxy(Ci-Cis)alkyl and heterocyclyl; or Ai is selected from the above options and A2 is absent;

[0023] Li and L2 are each absent or independently selected from: wherein each R3is independently selected from H or-(Ci-Ci2)alkyl (preferably Ci-ealkyl, more preferably methyl, ethyl); provided that at least one of Li or L2 must be present;

[0024] Y is absent or selected from -(Ci-Ci2)aryl-, -(Ci-Ci2)alkylene-, -(Ci-Ci2)alkoxylene-, and - (Ci-Ci2)alkoxy(Ci-Ci2)alkyl-, each of which may be optionally substituted with one or more substituents selected from hydroxy (-OH), thiol (-SH), seleno, oxo, -aryl, -(Ci-Cis)alkoxy, -(Ci-Cis)alkenyloxy, -(Ci-Cis)alkynyloxy, carboxy, cyano, -(Ci-Cis)alkoxycarbonyl, -(Ci-Cis)alkylthio, - (Ci-Cis)alkenylthio, -(Ci-Cis)alkynylthio, -(Ci-Cis)alkylsulfonyl, -(Ci- Cis)alkenylsuflonyl, -(Ci-Cis)alkynylsulphonyl, halo, halo(Ci-Cis)alkoxy, halo(Ci- Cis)alkenyloxy, halo(Ci-Cis)alkynyloxy, -CONR4R5and -NR4R5; wherein R4is selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl or-(Ci- Ci8)alkoxy(Ci-Ci8)alkyl and R5is selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl, -(Ci-Cis)alkoxy(Ci-

[0025] Cis)alkyl and heterocyclyl, wherein heterocyclyl is optionally substituted with one or two groups independently selected from -(Ci-Cis)alkyl, hydroxy, -(Ci-Cis)alkoxy, -(Ci-Cis)alkylthio, -(Ci- Cis)alkylsulfonyl, halo and -CONR6R7, wherein R6and R7are independently selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl or -(Ci-Ci8)alkoxy(Ci-Cis)alkyl; with the proviso that Y is absent when one of Li or L2 is absent;

[0026] W is a MDP ligand selected from the group consisting of: a) wherein R6is selected from H, -C(O)CHs, and -C(O)CH2OH, preferably -C(O)CHs; each R7is independently selected from H and -(Ci-Cs)alkyl, preferably H;

[0027] R8is selected from H and (Ci-Ce)alkyl, preferably H or C4-alkyl; and indicates the point of attachment to A2, or to L2 where A2 is absent; and

[0028]

[0029] Ill wherein R9is selected from -H, -C(O)CH3, and -C(O)CH2OH, and R10is selected from -H, and -C(O)CnH(2n+i), wherein n = 1-22, preferably n = 15 or 17; and indicates the point of attachment to A2 or to L2 where A2 is absent.

[0030] In one embodiment X is O.

[0031] In one embodiment Ai and A2 are independently selected from:

[0032] -phenyl-, -(Ci2-C42)alkoxyphenyl-, and -(Ci2-C42)alkylphenyl-, each of which may be optionally substituted with one or more substituents selected from the group consisting of -hydroxy, -(Ce-Cio)aryl, -(Ci-Cis)alkyl and -(Ci-Cis)alkoxy.

[0033] In one embodiment A2 is absent, and Ai is selected from: -phenyl-, -(C12- C42)alkoxyphenyl-, and -(Ci2-C42)alkylphenyl-, each of which may be optionally substituted with one or more substituents selected from the group consisting of - hydroxy, -(Ce-Cio)aryl, -(Ci-Cis)alkyl and -(Ci-Cis)alkoxy.

[0034] In one embodiment A2 is absent, and Ai is -(Ci2-C42)alkoxyphenyl- which is optionally substituted with one or more substituents selected from the group consisting of - hydroxy, -(Ce-Cio)aryl, -(Ci-Cis)alkyl and -(Ci-Cis)alkoxy, preferably -(Ci-Cis)alkoxy. In one embodiment A2 is absent, and Ai is -(Ci7)alkoxy(octadecanyloxy)phenyl-. In one embodiment A2 is absent, and Ai is -(Ci7)alkoxyphenyl-.

[0035] In one embodiment Li is

[0036] In one embodiment Y is -(Ci-Ci2)alkylene-.

[0037] In one embodiment Y is -(CH2)3-.

[0038] R3O R3

[0039] In one embodiment L2 is selected from wherein R3is selected from H or -(Ci-Ci2)alkyl.

[0040] R30 .N J

[0041] In one embodiment L2 is , wherein R3is selected from H, methyl or ethyl. 3 0 .N J

[0042] In one embodiment L2 is , wherein R3is methyl.

[0043] In another aspect the invention provides a pharmaceutical composition comprising a compound of Formula I and one or more pharmaceutically acceptable excipients.

[0044] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein that have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0045] 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The invention will now be described by way of example only and with reference to the drawings in which:

[0047] Figure 1 is a set of graphs showing that chimeric adjuvants bind and signal through Mincle and NOD2. A) NFAT-GFP 2B4-reporter cells expressing mMincle or hMincle were stimulated using plates coated with TDB, (1), (20), (4) or (5) at a concentration of 0.1 or 1 nmol / well for 18 h. The cells were then harvested and examined by flow cytometry for green fluorescent protein (GFP) expression. Data represents the mean of two experiments performed in duplicate (mean ± SEM), B) Molecular modelling demonstrating proposed binding mode of (4) to hMincle, C) HEK-293 cells expressing either murine or human NOD2 were stimulated with (3) (10 or 100 j-iM) or TDB, (1), (20), (4) or (5) (5 or 50 ptM) or combinations of (3) + (1) or (3) + (20) (10 or 100 ptM (3) + 5 or 50 ptM (l) / (20)) for 20 h. Supernatants were removed and IL-8 production analysed using ELISA. Data represents the mean ± SEM of three independent experiments conducted in triplicate. Statistical significance was determined using oneway ANOVA, with Dunnett's multiple comparisons test compared to iPrOH control, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0048] Figure 2 A) shows the rates of hydrolysis of (4) at acidic pH to release (21). The rates of hydrolysis at the specified pH conditions were determined by measuring the change in concentration of (4) using UV-HPLC-MS over time at 37 °C.

[0049] B) NFAT-GFP 2B4-reporter cells expressing mMincle were stimulated using plates coated with TDB, (1), (4) and (21) at a concentration of 0.1 or 1 nmol / well for 18 h. The cells were then harvested and examined by flow cytometry for GFP expression. Data represents the mean of three experiments performed in duplicate (mean ± SEM).

[0050] Figure 3 shows how chimeric adjuvants of the invention induce cytokine and chemokine production by BMDMs in vitro. C57BL / 6 BMDMs were incubated with solubilized ligands TDB, (1), (20), (4), (5) (1 nmol / well), (3) (2 nmol / well), combinations of (3) + (1) or (20), or LPS (100 ng / mL). At 48 h IL-lp, IL-6, IL-23 (Fig. 3A), TNF-a and MIP-2 (Fig. 3B) levels in the supernatant were measured by ELISA. Data represents the mean±SEM of three independent experiments. *P< 0.05, **P< 0.01, ***P< 0.001, ***P< 0.0001 using Dunnett's multiple comparisons test one-way ANOVA compared to untreated control or Sidak’s multiple comparisons test to compare the mean between groups.

[0051] Figure 4 shows that vaccination using chimeric adjuvants enhances antibody production and memory recall response while reducing toxicity. A) Blood was taken from mice and the anti-OVA whole IgG titre for weeks 3 to 6 were calculated using OVA-specific antibody titres determined by ELISA. ECso were defined by plotting the absorbance at 450 nm against log of serum concentration. Antibody titration curves were plotted using GraphPad Prism9 (GraphPad Software). B) At week 8, mice were culled, splenocytes were removed, and the memory recall response to OVA was measured. Statistical significant was calculated using the student's t-test. Statistical significance compared to no adjuvant#P< 0.05,##P<0.01,###P<0.005, between groups *P< 0.05, **P< 0.01. Figure 5 is a graph showing mean (± SE) antibody responses to M. haemolytica whole cell antigens in sheep vaccinated with antigens alone (control group) or antigens formulated with adjuvants. Arrows denote timing of the two vaccinations. Significant differences (P <0.05) in antibody responses between vaccine groups are indicated by different letters.

[0052] Figure 6 is a graph showing mean (± SE) serum antibody responses to M. ovipneumoniae whole cell antigens in sheep vaccinated with antigens alone (control group) or antigens formulated with adjuvants. Arrows denote timing of the two vaccinations. Significant differences (P <0.05) in antibody responses between vaccine groups are indicated by different letters.

[0053] Figure 7 is a graph showing mean (+SE) IFN-y responses to M. ovipneumoniae (MO) and M. haemolytica (MH) whole cell antigens at 6 weeks (2 weeks after the second vaccination). *higher than control group (P <0.05) (one-way ANOVA on loglO transformed data).

[0054] Figure 8 is a graph showing mean (+SE) IL-17 responses to M. haemolytica (MH) and M. ovipneumoniae (MO) whole cell antigens at 6 weeks (2 weeks after the second vaccination). *higher than control group (P <0.05) (one-way ANOVA).

[0055] Figure 9 shows induction of cytokine production IL-ip (A), IL-6 (B), IL-8 (C) and IL-23 (D) by chimeric adjuvants (4) and (5) in a human PBMC assay. Cells were stimulated with LPS (100 ng), TDB, (4), (5) or (1) (4 or 40 mM) or (3) (8 or 80 mM).

[0056] 5. DETAILED DESCRIPTION OF THE INVENTION

[0057] 5.1 Definitions and abbreviations

[0058] As used herein the term "comprising" means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

[0059] The term "about" as used herein means a reasonable amount of deviation of the modified term such that the end result is not significantly changed. For example, when applied to a value, the term should be construed as including a deviation of+ / - 5% of the value.

[0060] The term "alkyl" means any saturated hydrocarbon radical having up to 30 carbon atoms and includes any C1-C26, C1-C22, Ci-Cis, C1-C10, or C1-C7 alkyl group, and is intended to include cyclic (including fused bicyclic) alkyl groups (sometimes referred to herein as "cycloalkyl"), straight-chain and branched-chain alkyl groups, and straight or branched chain alkyl groups substituted with cyclic alkyl groups. Examples of alkyl groups include: methyl group, ethyl group, n-propyl group, / so-propyl group, cyclopropyl group, n-butyl group, / so-butyl group, sec-butyl group, t-butyl group, n-pentyl group, 1,1- dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1- ethylpropyl group, 2-ethylpropyl group, n-hexyl group, cyclohexyl group, cyclooctyl group, and l-methyl-2-ethylpropyl group.

[0061] The term "alkenyl" means any hydrocarbon radical having at least one double bond, and having up to 30 carbon atoms, and includes any C2-C26, C2-C22, C2-C18, C2-C10, or C2-C7, alkenyl group, and is intended to include both straight- and branched-chain alkenyl groups. Examples of alkenyl groups include: ethenyl group, n-propenyl group, isopropenyl group, n-butenyl group, iso-butenyl group, sec-butenyl group, t-butenyl group, n-pentenyl group, 1,1-dimethylpropenyl group, 1,2-dimethylpropenyl group, 2,2- dimethylpropenyl group, 1-ethylpropenyl group, 2-ethylpropenyl group, n-hexenyl group and l-methyl-2-ethylpropenyl group.

[0062] The term "alkynyl" means any hydrocarbon radical having at least one triple bond, and having up to 30 carbon atoms, and includes any C2-C26, C2-C22, C2-C18, C2-C10, or C2-C7, alkenyl group, and is intended to include both straight- and branched-chain alkenyl groups. Examples of alkenyl groups include: ethynyl group, n-propynyl group, iso- propynyl group, n-butynyl group, iso-butynyl group, sec-butynyl group, t-butynyl group, n-pentynyl group and the like.

[0063] The term "halo" includes F, Cl, Br and I.

[0064] The term "cyano" means -CN.

[0065] The term "hydroxy" means -OH.

[0066] The term "thio" means -SH.

[0067] The term "carbonyl" means -C(=O)-.

[0068] The term "sulfonyl" means -S(O2)-.

[0069] The term "heterocyclyl" means means a saturated or unsaturated non-aromatic ring system containing 3 or more ring atoms, of which one or more is a heteroatom. In some embodiments, the heteroatom is nitrogen, oxygen, or sulfur. In some embodiments, the heterocycle group contains one, two, three, or four heteroatoms. In some embodiments, heterocycle groups include mono-, bi- and tricyclic rings having from 3 to 20, 3 to 16, from 3 to 14, from 3 to 12, from 3 to 10, from 3 to 8, or from 3 to 6 ring atoms. Heterocycle groups include partially unsaturated and saturated ring systems, for example, imidazolinyl and imidazolidinyl. Heterocycle groups include fused and bridged ring systems containing a heteroatom, for example, quinuclidyl. Heterocycle groups include, but are not limited to, aziridinyl, azetidinyl, azepanyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, isoxazolidinyl, morpholinyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetra hydrofuranyl, tetrahydrothienyl, thiadiazolidinyl, and trithianyl. The prefix "x-y membered", wherein x and y are each an integer, when used in combination with the term "heterocycle" refers to the number of ring atoms in the heterocycle group. In some embodiments "heterocycle" groups may be substituted with one or more optional substituents as described herein.

[0070] The term "alkoxy" means an O-alkyl group, where alkyl as defined above. The terms "alkenyloxy" and "alkynyloxy" have analogous meanings.

[0071] The term "antigen" refers to any substance capable of inducing a specific immune response and of reacting with the products of that response. Antigens may be molecules such as toxins and proteins, or parts of bacteria and / or tissue cells.

[0072] The term "immunologic adjuvant" means a substance that, when incorporated into, or administered in conjunction with, a vaccine composition, acts to accelerate, prolong or enhance the antigen-specific immune response to the vaccine.

[0073] The term "pharmaceutically acceptable excipient" means a carrier, diluent or vehicle with which the therapeutic is administered, that is not unduly toxic. Pharmaceutically acceptable excipients have been approved by relevant government regulatory agencies. Excipients include but are not limited to sterile liquids such as water and oils, including animal, vegetable, synthetic or petroleum oils, saline solutions, aqueous dextrose and glycerol solutions, starch glucose, lactose, sucrose, gelatin, sodium stearate, glycerol monostearate, sodium chloride, propylene glycol, ethanol, wetting agents, emulsifying agents, binders, dispersants, thickeners, lubricants, pH adjusters, solubilizers, softening agents, surfactants and the like. The compositions of the invention can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders and sustained- release formulations. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 18thEd., Gennaro, ed. (Mack Publishing Co. 1990). The pharmaceutically acceptable excipient is present in a composition of the invention in an amount that does not impair the activity of the compound of the invention.

[0074] As used herein, the term "substituted" is intended to mean that one or more hydrogen atoms in the group indicated is replaced with one or more independently selected suitable substituents, provided that the normal valency of each atom to which the substituent / s are attached is not exceeded, and that the substitution results in a stable compound. Suitable substituents include the optional substituents indicated herein. For the purposes of the invention, any reference to the disclosed compounds includes all possible formulations, configurations, and conformations, for example, in free form (e.g. as a free acid or base), in the form of salts or hydrates, in the form of isomers (e.g. cis / trans isomers), stereoisomers such as enantiomers, diastereomers and epimers, in the form of mixtures of enantiomers or diastereomers, in the form of racemates or racemic mixtures, or in the form of individual enantiomers or diastereomers. Specific forms of the compounds are described in detail herein.

[0075] Asymmetric or chiral centers may exist in the compounds of the invention. Asymmetric or chiral centers may be designated as (R) or (S), depending on the configuration of substituents in three dimensional space at the chiral atom. All stereochemical isomeric forms of the compounds, including diastereomeric, enantiomeric, and epimeric forms, as well as d-isomers and l-isomers, and mixtures thereof, including enantiomerically enriched and diastereomerically enriched mixtures of stereochemical isomers, are included herein.

[0076] Individual enantiomers can be prepared synthetically from commercially available enantiopure starting materials or by preparing enantiomeric mixtures and resolving the mixture into individual enantiomers. Resolution methods include conversion of the enantiomeric mixture into a mixture of diastereomers and separation of the diastereomers by, for example, recrystallization or chromatography, and any other appropriate methods known in the art. Starting materials of defined stereochemistry may be commercially available or made and, if necessary, resolved by techniques well known in the art.

[0077] The compounds of the invention may also exist as conformational or geometric stereoisomers, including cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers. All such stereoisomers and any mixtures thereof are within the scope of the invention.

[0078] Also within the scope of the invention are any tautomeric isomers or mixtures thereof of the compounds of the invention. As would be appreciated by those skilled in the art, a wide variety of functional groups and other structures may exhibit tautomerism. Examples include, but are not limited to, keto / enol, imine / enamine, and thioketone / enethiol tautomerism.

[0079] The compounds of the invention may also exist as isotopologues and isotopomers, wherein one or more atoms in the compounds are replaced with different isotopes. Suitable isotopes include, for example, 1H, 2H (D), 3H (T), 12C, 13C, 14C, 160, and 180. Procedures for incorporating such isotopes into the compounds will be apparent to those skilled in the art. Isotopologues and isotopomers of the compounds are also within the scope of the invention. Also within the scope of the invention are pharmaceutically acceptable salts of the compounds of the invention. Such salts include, acid addition salts, base addition salts, and quaternary salts of basic nitrogen-containing groups.

[0080] Acid addition salts can be prepared by reacting compounds, in free base form, with inorganic or organic acids. Examples of inorganic acids include, but are not limited to, hydrochloric, hydrobromic, nitric, sulfuric, and phosphoric acid. Examples of organic acids include, but are not limited to, acetic, trifluoroacetic, propionic, succinic, glycolic, lactic, malic, tartaric, citric, ascorbic, maleic, fumaric, pyruvic, aspartic, glutamic, stearic, salicylic, methanesulfonic, benzenesulfonic, isethionic, sulfanilic, adipic, butyric, and pivalic. Base addition salts can be prepared by reacting compounds, in free acid form, with inorganic or organic bases. Examples of inorganic base addition salts include alkali metal salts, alkaline earth metal salts, and other physiologically acceptable metal salts, for example, aluminium, calcium, lithium, magnesium, potassium, sodium, or zinc salts. Examples of organic base addition salts include amine salts, for example, salts of trimethylamine, diethylamine, ethanolamine, diethanolamine, and ethylenediamine.

[0081] Quaternary salts of basic nitrogen-containing groups in the compounds may be prepared by, for example, reacting the compounds with alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides, dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfates, and the like.

[0082] N-Oxides of the compounds of the invention are also within the scope of the present invention.

[0083] The compounds of the invention may form or exist as solvates with various solvents. If the solvent is water, the solvate may be referred to as a hydrate, for example, a monohydrate, a di- hydrate, or a tri-hydrate. All solvated forms and unsolvated forms of the compounds are within the scope of the invention.

[0084] The terms "administering" or "administration" refer to placement of the composition or compound of the invention into a subject by a method appropriate to result in an immune response. The dosage form is selected and used as appropriate depending on the therapeutic purpose and the subject. The dose of the composition of the invention may be selected depending on the therapeutic purpose and the characteristics of the subject including their species, age, sex, general health and disease progression. In general, for human subjects, the compound of the invention may be administered in a dose of 0.01 to 100 mg, preferably 0.1 to 50 mg per day, per kg of body weight, either once or divided over several administrations.

[0085] A "therapeutically effective amount" (or "effective amount") is an amount sufficient to effect beneficial or desired results, including clinical results, but not limited thereto. A therapeutically effective amount can be administered in one or more administrations by various routes of administration. The therapeutically effective amount of the compound to be administered to a subject depends on, for example, the purpose for which the compound is administered, mode of administration, nature and dosage of any coadministered compounds, and characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight and tolerance to drugs. A person skilled in the art will be able to determine appropriate dosages having regard to these any other relevant factors.

[0086] A "subject" refers to a human or a non-human animal, preferably a vertebrate that is a mammal. Non-human mammals include, but are not limited to; livestock, such as, cattle, sheep, swine, deer, and goats; sport and companion animals, such as, dogs, cats, and horses; and research animals, such as, mice, rats, rabbits, and guinea pigs. Preferably, the subject is a human.

[0087] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0088] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the disclosure and the claims, "and / or" means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.

[0089] 5.2 Preparation of the chimeric adjuvants of the invention

[0090] It has been previously determined that long-chain lipidated derivatives of the natural product brartemicin such as (1) are potent Mincle agonists. Accordingly, Mincle agonist (lipidated brartemicin derivative) (1) was combined with NOD2 agonist (MDP) (3) to provide chimeric adjuvants of the invention (4) and (5).

[0091] In (4) an MDP ligand (3) is attached to each lipid chain of the brartemicin derivative via an acid labile linker. In (5) the brartemicin derivative attached to each of the MDP ligands (3) additionally includes a second lipid chain.

[0092] The synthesis of (4) and (5) is set out in Example 1. Other compounds of the invention can be prepared using a modification of the synthesis provided. The linkage between the MDP ligand and brartemicin units can be accomplished via a variety of linker strategies. Thiol-modified MDP ligand and brartemicin unit can be combined to form a disulfide linked conjugate. An esterification reaction between a carboxy-modified MDP ligand and an alcohol on brartemicin, or an alcohol on the MDP ligand and a carboxy-modified brartemicin, can be used to produce an ester linkage. An aldehyde or ketone modified MDP ligand can be condensed with an amine modified brartemicin unit, or vice versa, to form an imine linkage. An oxyamine modified brartemicin can be condensed with an MDP ligand to form an oxyamine linkage. The condensation of a carboxy-modified MDP ligand and an amine modified brartemicin, or vice versa, can be used to produce an amide linkage. A sulfonyl halide modified MDP ligand can be reacted with a hydroxy or amino group on brartemicin, or vice versa, to form a sulfonate or sulfonamide linkage, respectively. The condensation of a carboxy-modified MDP ligand and a thiol modified brartemicin, or vice versa, can be used to produce a thioester linkage. A click reaction can be used to conjugate an azide modified MDP ligand to an alkyne modified brartemicin, or vice versa, to form a triazole linkage. A phosphate modified brartemicin can be condensed with an alcohol or amine modified MDP ligand, or vice versa, to form a phosphate or phosphoramidate linkage, respectively. Phosphate modified MDP ligand and brartemicin unit can be condensed to form a pyrophosphate linkage. A cyanate modified MDP ligand can be condensed with an amine or alcohol functionalized brartemicin, or vice versa, to form a urethane or carbamate linkage, respectively. A chloroformate or activated carbonate ester of an MDP ligand can be condensed with a hydroxy or amino group on brartemicin, or vice versa, to form a carbonate or carbamate linkage, respectively. Ether linkages can be made through the reaction of a haloalkyl or sulfonyl modified MDP ligand with a hydroxy group on the brartemicin unit, or vice versa. Amine linkages can be made through the reaction of a haloalkyl or sulfonyl modified MDP ligand with an amine modified brartemicin unit, or vice versa. Carbonyl linked conjugates can be made through aldol condensation of a ketone functionalized MDP ligand and an aldehyde modified brartemicin.

[0093] 5.3 Properties of the chimeric adjuvants of the invention

[0094] Key in the design of the chimeric adjuvants of the invention was the incorporation of a pH-dependent hydrolyzable linker. While Mincle can tolerate a wide variety of lipophilic ligands, a limitation of incorporating an MDP ligand in a vaccine adjuvant is its unwanted toxicity (F. Ellous, 1974) (C. A. Dinarello, 1978) (Chedid., 1983). Toxicity is thought to relate to excessive cytokine production (Ogure, Inohara, & Benito, 2001), although other modes of toxicity, including targeting of mitochondria (El-Jamal, Bahr, & Echtay, 2009) have been suggested.

[0095] Facilitating the intracellular release of the MDP ligand allows NOD2 to be targeted using lower concentrations of MDP ligand, thus limiting the toxicity. Moreover, any uncleaved chimeric adjuvant is likely to be less toxic, as illustrated in studies where lipophilic MDP derivatives have been designed to reduce the toxicity of the parent compound (Marian M. J. H. P. Willems G. G., 2016).

[0096] To determine the mode of action of the chimeric adjuvants of the invention, their hydrolytic stability and ability to signal through Mincle and NOD2 were measured. Table 1 summarizes the agonist activity of the compounds used in the experiments set out in the Examples section.

[0097] Table 1: Summary of Mincle and NOD2 agonist compounds

[0098]

[0099] Using C18Brar-MDP (4), the inventors demonstrated that the adjuvant was remarkably stable at physiological pH (pH = 7, t> / 2> 1 year). This indicates that it is predominantly the chimeric adjuvants that signal through Mincle. Example 2 explores the signaling pathways of the chimeric adjuvants of the invention. Chimeric adjuvants (4) and (5) exhibited strong signaling through both murine and human Mincle, with molecular modelling suggesting that (4) can bind to Mincle in an analogus manner to other trehalose glycolipids. If any chimeric adjuvant were hydrolysed before engagement with surface expressed Mincle, the hydrolysed product can also signal through Mincle.

[0100] The chimeric adducts can be internalized by cells through either Mincle-dependent or independent pathways. Upon acidification of the lysosome, the release of MDP is dramatically enhanced, as illustrated by tv2= 2.5 days (pH = 4.4) for (4) in Example 3.

[0101] The observed hydrolysis rates of (4), which are in agreement with previous studies, (Decout, et al., 2017) also indicate that significant amounts of (4) and (5) would be present at 20 h, the timepoint when IL-8 production from the NOD2 cells was measured in Example 2.

[0102] The lower levels of IL-8 produced by mN0D2 cells in response to (4) and (5) compared to (3) alone and, in particular (3) + (1) or (3) + (20), also supports the notion that ligand hydrolysis is occurring intracellularly, and that if (4) and (5) are ligands for mN0D2, they have less affinity for mN0D2 than (3).

[0103] In contrast, the similar levels of IL-8 observed for chimeric adjuvants (4) and (5) compared to (3) alone when using hNOD2 reporter cells may indicate that these chimeric ligands more readily activate hNOD2. Others have previously demonstrated that less toxic MDP derivatives that incorporate lipids or peptides can signal through NOD2 (Marian M. J. H. P. Willems G. G., 2016) (Marian M. J. H. P. Willems G. G., 2014).

[0104] Example 4 demonstrated that (4) and (5) have the potential to be effective adjuvants. Both chimeric adjuvants activate BMDMs, as illustrated by the significant release of cytokines, such as IL-10 and IL-23 compared to untreated cells (see Figure 3).

[0105] The ability of (4) and (5) to act as adjuvants was also demonstrated using in vivo immunisation assays in Example 5, where OVA was used as a model antigen. In these studies, a very low concentration of (3) was used (0.08 mmol per dose) to mitigate any unwarranted toxicity of this compound. This necessitated the use of a low dose of the chimeric adjuvants so that their adjuvanticity could be compared to an equivalent dose of the co-administered PAMPs. Accordingly, neither (1) nor (3) led to a statistically significant increase in IgG compared to antigen alone, despite (1) previously augmenting antigen-specific IgG responses in vivo. (Foster, 2018). However, targeting multiple PAMPs significantly improved IgG titres compared to antigen alone. This was even more remarkable given that only low doses (0.04 mmol) of chimeric adjuvants (4) and (5) were used showing the surprising potency of the chimeric compounds of the present invention. This allows the compounds to be administered at much lower doses to achieve the same effect, thereby avoiding the toxicity issues associated with higher doses of the MDP ligand (3) alone or when co administered with brartemicin derivative (1).

[0106] Differences in relative IgG titres were observed when comparing (4) and (5) against their respective co-administered ligands. Chimeric adjuvant (4) led to higher IgG titres than the co-administered (1) + (3). A significant difference was seen between the two groups at weeks 4, 5 and 6. These antibody responses correlated to a strong T-cell mediated memory response, as indicated by the significant increase in IFN-y by splenocytes in an ex vivo recall response assay. In contrast, while the co-administration of (20) + (3) led to significantly higher OVA-specific IgG titres at weeks 4 and 5 compared to the administration of (5), this did not correlate to a T-cell-mediated memory response.

[0107] Moreover, the co-administration of (20) + (3) led to undesirable reactogenicity suggesting inflammation rather than improved vaccine immunity when the two PAMPs were added as individual compounds. In comparison, administration of (5) led to a significant IFN-y memory recall response, while local reactogenicity and inflammation was almost abolished. Again, this demonstrates that the use of the chimeric adjuvant compounds of this present invention can mitigate any unwanted toxicity associated with the use of the singular PAMPs.

[0108] In Example 6, the chimeric adjuvants of the invention were tested against their component PAMPs with respect to their ability to stimulate antibody and T-cell responses to Mannheimia haemolytica and Mycoplasma ovipneumoniae whole cell antigens.

[0109] The combination of (20) + (3) tended to stimulate stronger antibody responses to M. haemolytica whole antigens than the other adjuvants including (1) + (3). Compound (20) alone or combined with (3) appeared to be more optimal at stimulating antibody responses to M. ovipneumoniae whole cell antigens than (1) combined with (3).

[0110] To determine T-cell responses, the levels of two cytokines, IFN-y and L-17A were measured in antigen-stimulated blood cultures. Interferon-y (IFN-y), a Thl signature cytokine is a major proinflammatory cytokine, mainly produced by activated T- lymphocytes. Interleukin 17A (IL-17 or IL-17A) is a pro-inflammatory cytokine produced by T helper 17 cells. IL-17 interacts with the type I cell surface receptor IL-17R, activates several signalling cascades that, in turn, led to the induction of chemokines. Chemokines recruit immune cells, such as monocytes and neutrophils, to the site of inflammation following an invasion of the body by pathogens.

[0111] All the four brartemicin adjuvant systems in the study ((1), (20), (4) and (5)) stimulated significant IFN-y responses to M. ovipneumoniae but there was considerable animal-to-animal variation in these results with some animals not responding. Neither the various brartemicin adjuvants nor Quil-A stimulated appreciable IFN-y responses to M. haemolytica. All the adjuvants promoted some IL-17A response.

[0112] The conjugation of MDP to a brartemicin derivative to provide chimeric adjuvants of the invention (4) and (5) improved ligand adjuvanticity by enhancing antibody titres, reducing injection site inflammation, and enhancing a T-cell mediated memory response while lowering dose requirements. Insomuch, targeting Mincle and NOD2 can augment and enhance the adjuvanticity of the individual PAMPs and reduce inflammation that can lead to unwanted inflammatory or autoimmune responses. The dual targeting of these PAMPs also correlates to lower doses of adjuvant being required for protection, as illustrated by the remarkable adjuvanticity of (4) and (5) when administered in a very low single dose of 40 nmol.

[0113] In summary, the chimeric adjuvants (4) and (5) show increased antibody production compared to brartemicin derivative (1) or MDP (3) alone, or brartemicin derivative (1) co-administered (+) with MDP (3), as seen in the antibody response graphs in weeks 3 to 6 (see Figure 4A). Compound (20) co-administered (+) with MDP (3) led to high antibody responses, however, this did not translate into an immune memory response, as seen by the lack of IFN-y production for 20 + 3 (see Figure 4B). When these compounds were conjugated (5), the memory recall response was significantly higher. The combination of (20) + (3) also led to the greatest number of vaccination site reactions (see Figure 4C, and Table 4), which is indicative of an inflammatory response rather than improved immunity.

[0114] This is also shown in Example 6 where sheep were vaccinated using the combination of PAMPs. The dose of inventive conjugate (5) in sheep was 0.7 mg / dose (Figures 5, 6, 7 and 8). In the same study (20) and (1) were used at 3.75 mg / dose and MDP added at 0.05 mg / dose. Although the dose of (5) was significantly lower than that of (20) or (1), the antibody responses and cytokine responses were comparable. The vaccination site reactions were especially large for (20) (tables 3 and 4), while the vaccination site reactions for (5) were greatly reduced. Taken together conjugate (5) of the present invention reduced the side-effects and enables the dose of the adjuvant to be reduced, while maintaining a good immune response. This is both surprising and was not expected when looking at the use of the two components individually.

[0115] Accordingly, the chimeric adjuvants of the invention have application as immunomodulators that can be used to prevent or treat infections caused by pathogens, as well as cancer and other diseases. The compounds and compositions of the invention, when used in conjunction with antigens, can also function as immunologic adjuvants.

[0116] In one aspect the invention provides a pharmaceutical composition comprising a compound of formula I and one or more pharmaceutical acceptable excipients.

[0117] In one embodiment, the pharmaceutical composition additionally comprises an antigen.

[0118] In one embodiment, the pharmaceutical composition additionally comprises a pathogen associated molecular pattern (PAMP).

[0119] In one aspect the invention provides a method of enhancing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula I. In one aspect the invention provides a method of enhancing an immune response to an antigen in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula, in conjunction with the antigen.

[0120] In one aspect, the invention provides a method of inducing or enhancing Thl-mediated immunity in a subject, the method comprising providing administering to the subject a therapeutically effective amount of a compound of Formula I.

[0121] In one aspect, the invention provides a method of inducing or enhancing Thl-mediated immunity to an antigen in a subject, the method providing administering to the subject a therapeutically effective amount of a compound of Formula I, simultaneously, sequentially or separately with the antigen.

[0122] In one aspect, the invention provides a method of inducing or enhancing Thl7-mediated immunity in a subject, the method comprising providing administering to the subject a therapeutically effective amount of a compound of Formula I.

[0123] In one aspect, the invention provides a method of inducing or enhancing Thl7- medicated immunity to an antigen in a subject, the method providing administering to the subject a therapeutically effective amount of a compound of Formula I, simultaneously, sequentially or separately with the antigen.

[0124] In the above aspects:

[0125] In one embodiment the subject is a mammal selected from human and non-human mammals. In one embodiment, the subject is immune-compromised human. In one embodiment the immune-compromised subject is selected from the group consisting of newborns, infants, children under 12 years of age, the elderly, HIV sufferers and people taking immunosuppressants.

[0126] In one embodiment, the subject is a non-human mammal, preferably a companion animal or livestock animal.

[0127] In one embodiment, the antigen is selected from the group consisting of a live attenuated microorganism or antigenic parts thereof, an inactivated or dead microorganism or antigenic parts thereof, an inactivated toxin produced by or derived from, viral or bacterial proteins or antigenic fragments thereof, viral or bacterial DNA or antigenic parts thereof, toxoids, and combinations thereof.

[0128] In one embodiment, the antigen is selected from the group consisting of an antigen against human immunodeficiency virus (HIV), tuberculosis, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV), influenza, pneumonia, meningitis, rotavirus, tetanus, Leishmaniasis, anthrax, human papillomavirus (HPV), measles, rubella, chicken pox, mumps, shingles, polio, pertussis, yellow fever, rabies, tetanus, dengue, typhoid and Japanese encephalitis.

[0129] A person skilled in the art will be able to choose the appropriate mode of administration of the medicament with reference to the literature and as described herein.

[0130] In one embodiment, administration is local or systemic administration. In one embodiment, administration is intranasal, epidermal, and transdermal, oral or parenteral.

[0131] In one embodiment oral administration comprises application of a liquid, gel, creme, ointment, lotion or slurry. In one embodiment oral administration comprises delivery of an oral dosage form. In one embodiment the oral dosage form is a solid oral dosage form. In one embodiment the solid oral dosage form comprises a powder, a granule, a tablet, a pill, a capsule or a lozenge or combination thereof.

[0132] In one embodiment the oral dosage form is a liquid dosage form. In one embodiment the liquid dosage form is aqueous suspension, an aqueous solution, a non-aqueous suspension or a non-aqueous solution.

[0133] In one embodiment the oral dosage from comprises an additional ingredient selected from the group consisting of thickeners, flavoring agents, diluents, emulsifiers, dispersing aids and binders.

[0134] In one embodiment, parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.

[0135] In one embodiment, administration is transient administration. In one embodiment transient administration comprises administration of a compound of the invention or a pharmaceutical composition as described herein for a sufficient period of time to provide a treatment or achieve a therapeutic result.

[0136] A particular and effective dosage regime according to a method of the invention will be dependent on the desired effect and on the responsiveness of the treated subject to the course of treatment. An effective treatment may last from several hours to several days to several months or longer, or until an acceptable therapeutic outcome is affected or assured or until an acceptable reduction of the infection is observed.

[0137] An optimal dosing schedule (s) may be calculated from drug accumulation as measured in the body of a treated subject. It is believed to be within the skill of persons in the art to be able to easily determine optimum and / or suitable dosages, dosage formulations and dosage regimes. Of course, the optimum dosages may vary depending on the relative potency of the compound of the invention or pharmaceutical composition comprising the compound. In general, dosage is from 0.0001 g to 99 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, but not limited thereto.

[0138] Specifically contemplated as embodiments of the above aspects of the invention related to uses of the compounds of the invention in the manufacture of medicaments, and to compositions for use are all of the embodiments encompassed herein by the method of treatment aspects set out above.

[0139] The compounds of the invention described as having been prepared in the Examples are specifically contemplated for use in the methods of treatment, manufacture of medicaments and compositions for use in said methods.

[0140] Additionally, the following embodiments of the invention are also contemplated for the aspects of the invention above that are the uses of the compounds of the invention in the manufacture of medicaments, and the compositions for use.

[0141] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents; or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0142] The invention will now be illustrated in a non-limiting way by reference to the following examples.

[0143] 6. EXAMPLES

[0144] Example 1: Synthesis of chimeric adjuvants of the invention

[0145] The synthesis of (4) commenced via the KzCOs-mediated coupling of 4-hydroxylbenzoate (6) to tosylated alkyne (7) to give ester 8 in excellent yield (Scheme 1A). Tosylate 7 was prepared in 3-steps and 50% overall yield from 1-bromohexadecene (9). Hydrolysis of 8 under the agency of NaOH then gave carboxylate 10 in excellent yield. Following the preparation of TMS-trehalose 12 from a,a'-trehalose (11) (Khan, 2011), this was conjugated to acid 10 to give the fully protected glycolipid, as evidenced by HRMS (HRMS (ESI) calc. [C82Hi46)Oi5Si6+NH4]+= 1556.96156; obs. : 1556. 96154), the presence of a single anomeric signal (Stocker B. L., 2014), and an HMBC between trehalose 6-Ha(4.53 ppm) and Hb (4.24 ppm) and13C of the ester carbonyl (166.4 ppm). Removal of the TMS groups then occurred readily under the agency of Dowex-H+to give the fully deprotected sugar 13 in quantative yield. To conjugate MDP to the alkyne- functionalised brartemicin scaffold, azide-functionalised oxyamine liner 14, itself prepared in 3-steps and 91% overall yield (S. Munneke J. R., 2015), was conjugated to MDP to give azide-functionalised MDP 15. A copper-mediated click reaction between 15 and the brartemicin scaffold 13 then provided C18Brar-MDP (4) in good yield following purification by silica gel, C18 reverse phase, and P2 size exclusion chromatography to ensure complete removal of any residual copper salts.

[0146] Scheme 1A. Synthesis of chimeric NOD-2-Mincle agonist (4)

[0147] 18 19 20

[0148] Scheme 1C. Synthesis of control dilipid-Brartemicin (20)

[0149] In an analogous manner, (5) was prepared by first coupling benzoate 16 (Foster, 2018) to tosylate 7 (Scheme IB). The ensuing alkylated methyl ester was then hydrolysed, the resulting carboxylate conjugated to TMS-trehalose 12, and the TMS groups then removed under the agency of Dowex-H+to give the dilipidated brartemicin core 17. A copper-mediated click reaction between 17 and MDP-azide 15 then afforded C18Brar- dilipid (5). For the purposes of subsequent immunological comparison, a non-conjugated dilipid was also prepared (Scheme 1C). To this end, benzylated trehalose 18 (Khan, 2011) was conjugated to dilipid 19, whereby the latter was prepared via the K2CO3- mediated alkylation of methyl 2-hydroxy-4-(octadecyloxy)benzoate (Foster, 2018) with 1-bromooctadecane. Following esterification, the resulting protected trehalose glycolipid was hydrogenated using Pd(OH)2 / C to give the target di-lipidated brartemicin derivative (20).

[0150] Synthesis of MDP-amide-C18Brardilipid

[0151] Scheme ID. Synthesis of chimeric adjuvant (23).

[0152]

[0153] N-[Muramyl dipeptide]-4-azidobutanamide (22).

[0154] To a solution of hydroxylammonium chloride (2.5 equiv.) in methanol (2 mL / mmol of sugar) at room temperature was added sodium methoxide (2.5 equiv.) and the solution stirred for 20 mins, at which point MDP (1 equiv.) was added followed by the addition of distilled water (4 mL / mmol) and the solution stirred at 40 °C for 18 h. The reaction was cooled to r.t. and concentrated under reduced pressure. The residue was dissolved in DMSO (2 mL / mmol), the acylsilane (1.5 equiv.), and concentrated sulfuric acid (1 equiv.) were added to the reaction mixture and the solution stirred at r.t. overnight. The reaction was neutralised by the addition of sat. aq. NaHCCh and either lyophilized and purified by C18 reversed phase chromatography or loaded directly onto a size exclusion column (BioGel, P-2, 1200 x 18 mm) and eluted with 0.1 M NH4HCO3 aq. solution. Lyophilisation of the product fractions gave 22 in 79% yield. HRMS(ESI) calcd. for [CzsHssNsOn+H] = 603.2733; obsd. : 603.2711.

[0155] MDP-amide-C18Brardilipid (23) To a solution of (22) (19 mg, 0.032 mmol) and (17) (20 mg, 0.0126 mmol) in DMSO (100 |jL) and CH2CI2 (100 |jL) under argon was added a solution of CUSO4.5H2O (6.9 mg, 0.028 mmol) and sodium ascorbate (12.5 mg, 0.063 mmol) in H2O (100 pL) under argon and the resulting mixture was stirred at 40 °C for 6 h. The solvent was then removed by lyophilisation and purified by flash column chromatography (CH2Cl2 / MeOH / pyridine, 100 / 0 / 0 50 / 49 / 1, v / v / v and concentrated under reduce pressure to afford compound

[0156] 23 as a colourless oil (10.0 mg, 0.0035 mmol, 28%). R- = 0.30 (CH2Cl2:MeOH, 60:40, v / v) stained using 20% H2SO4 in ethanol. 1H NMR (500 MHz, d5-pyridine) 6 8.19 (d, J = 8.7, 2H, H-5' or 6'), 7.68 (s, 2H, H-19"), 6.72 (s, 2H, H-3'), 6.48 (d, J = 8.7, 2H, H-5' or 6'), 5.89 (d, Ji,2= 3.0 Hz, 2H, H-l), 5.15 (m, 2H, H-5), 5.07 - 4.98 (m, 4-H, H-6a and H- 6b), 4.88 - 4.76 (m, 8H, H-l"", MA-CH-7", isoglut-CH, and Ala-CH), 4.75 - 4.52 (m, 4H, H-3, H-2"") 4.47 - 4.07 (m, H-14, H-2, H-4, H-l'", H-6a"", H-6b"" and H-3""), 4.03 - 3.89 (m, 12H, H-4"", H-5"", H-8' and H-l"), 2.73 (m, 4H, H-17"), 2.42 - 2.25 (m, 8H, H-2'" and H-3'"), 2.15 (s, 6H, NAc), 1.84 (m, 4H, H-9' or H-2"), 1.74- 1.51 (m, H-20, H- 16", H-9' or H-2", AlaCH3, MA-CH3), 1.50-1.13 (m, H-112, H-10' to 24', H-3" to H-15"), 0.81 (t, J24',25- = 6.3 Hz, 6H, H-25'); HRMS(ESI) calcd. for [Ci46H248Ni6O39]2+= 1425.3969; obsd. : 1425.3997.

[0157] Example 2: Chimeric adjuvants signal through Mincle and NOD2

[0158] To determine whether the chimeric adjuvants of the invention signaled through Mincle, the inventors stimulated nuclear factor of activated T cells (NFAT) green fluorescent protein (GFP) reporter cells expressing murine Mincle (mMincle) or human Mincle (hMincle) coupled to FcRy (R-J. Eveline, 2019) using plates coated with control lipidated brartemicin derivative (1), control dilipid (20), and the corresponding chimeric adjuvants 4 and 5, respectively. TDB was used as a positive control, with cells expressing FcRy as negative controls. All ligands resulted in the production of GFP by the reporter cells at both concentrations of ligand (0.1 nmol / well, 1 nmol / well) (Figure 1A). Similar results were observed when using hMincle reporter cells (Figure 1A). Molecular docking of (4) into hMincle confirmed that binding of the conjugate to Mincle can occur in an analogous manner to C18Brartemicin (Figure IB). (Foster, 2018) As anticipated, the trehalose moiety anchors the ligand in the CRD of Mincle through binding of the 3- and 4-hydroxys to the Ca2+ion and the lipid chain interacts with a hydrophobic region, including Phel98 and Leul76 side chains. The lipid chain is long enough so that the polar MDP does not interfere with binding of the lipid chain in the hydrophobic groove and instead MDP interacts with surrounding water molecules and polar amino acids.

[0159] The ability of the chimeric adjuvants 4 and 5 to signal through NOD2 was explored using HEK cell lines expressing murine or human NOD2 (Figure 1C). The level of IL-8 being produced by the cell line upon stimulation with the ligands was then determined by ELISA. As anticipated, the Mincle ligands alone (TDB, 1, and 20) did not signal through NOD2, while the coadministration of (3) + 1 and MDP (3) + 20 led to significant IL-8 production at both concentrations of (3). By comparison, chimeric adjuvants (4) (Figure 1C) and (5) led to lower levels of IL-8 by the mN0D2 cells, particularly when lower concentrations of the ligands were used. When using hN0D2 cells, the response to the chimeric adjuvants was similar to that elicited by the co-administration of the respective NOD2 and Mincle PAMPs (Figure 1C).

[0160] Example 3: Chimeric ligand for Mincle and NOD2 is cleaved at lysosomal pH to release MDP

[0161] The inventors have previously demonstrated that the hydrolysis of the oxyamine linker occurs at lysosomal pH (S. Munneke J. R., 2015) (S. Munneke J. C., 2017) (S. Munneke E. M., 2017). To determine whether there is any effect of the increased steric bulk of the chimeric adjuvants on the rate of hydrolysis, (4) was placed in water (pH = 7.0), 0.1 M NaOAc / AcOH buffer (pH = 4.4, lysosomal pH), and 0.1 M TFA (pH = 1). The hydrolysis reaction is shown in scheme 2.

[0162] Scheme 2: Hydrolysis of chimeric adjuvants

[0163] Hydrolysis rates were then determined by HPLC, with half lives of t2= 1 year, t2= 2.5 days, and t> / 2= 1 hour at pH = 7.0, 4.4 and 1.0, respectively (Figure 2A). This is consistent with inventors' previous observations and suggests that the hydrolysis of the chimeric adjuvants would be significantly accelerated within the lysosomal compartments of cells. The hydrolyzed product, (21), was also synthesized and determined to be a ligand for mMincle, as demonstrated using the NFAT-GFP reporter cells (Figure 2B).

[0164] Example 4: Chimeric adjuvants activate murine BMDMs in vitro. The functional immune response to the chimeric adjuvants of the invention was assessed by determining cytokine production by murine bone marrow derived macrophages (BMDMs) in vitro. The activity of the brartemicin conjugates was assessed using a 'solubilised' BMDM assay (Khan, 2011) (Stocker B. L., 2019).

[0165] To this end, (1), (20), (3), (4) and (19), along with the co-administered (1) + (3) and (20) + (3), were added to BMDMs and the level of IL-lp, IL-6, IL-23, TNF-a and MIP-2 was measured by ELISA at 48 h (Figures 3A and 3B). Conjugates (4) and (5) were able to signal and activate BMDMs, as illustrated by significant levels of IL-lp and IL-23 for both (4) and (5) compared to untreated cells. The stimulation of BMDMs with (5) also led to significant increases in IL-6 and MIP-2. The IL-23 and IL-6 response to (5) was greater than that observed in response to (4).

[0166] Example 5: Chimeric adjuvants demonstrate enhanced in vivo adjuvanticity compared to co-administered adjuvants

[0167] The in vivo adjuvanticity of the chimeric adjuvants of the invention was assessed using a murine in vivo OVA-immunization assay. C57BL / 6 mice (n = 6) were vaccinated with OVA alone, OVA with either (1), (3), chimeric adjuvants (4) or (5), or with combinations of (3) and either (1) or (20) at day 0 The mice were challenged with OVA again 1 week later. Throughout the course of the experiments, mice were monitored according to normal animal husbandry protocols with weight gain and inflammation at the site of the vaccination (tail base) being measured. Anti-OVA IgG titres were then determined at week 3, 4, 5 and 6. No weight loss was observed across the six-week period post immunisation for all the vaccination combinations, however co-administration of (5) + (3) resulted in all six mice having inflamed skin at the tail base 1 week post immunization, with 2 / 6 mice exhibiting a residual scab at 2 weeks post infection At 4 weeks post immunization, 1 / 6 mice exhibited inflamed skin. For (5), one mouse exhibited local inflammation at 4 weeks post immunization.

[0168] Regarding IgG titres, the co-administration of (3) and (20) led to the highest antibody titres at each of the four time points (Figure 4A). This response was statistically significant compared to all other brartemicin adjuvants, including (5). Notwithstanding, the reactogenicity associated with (3) + (20) would likely limit the application of this adjuvant combination. Chimeric adjuvant (5) led to a significant increase in IgG tires at weeks 3, 4 and 5 compared to no adjuvant. In contrast, (4) led to significantly greater antibody titres compared to the co-administration of (1) + (3) at weeks 4, 5 and 6. There was no statistical difference between the observed IgG titres for (4) and (5) across the course of the study. It should also be noted that in these studies the concentration of (1) was chosen so that equal molar amounts of (4) and co-administered (1) + (3) could be used while attempting to mitigate any undesired toxic effects of MDP. At week eight an ex vivo recall response assay using splenocytes was performed to determine the extent of memory response elicited by each adjuvant. In this assay, the cells were subsequently stimulated with OVA and the production of IFN-y, IL-17 and IL- 21 measured by ELISA after 72 h. While no IL-17 or IL-21 was observed upon restimulation of the splenocytes with 300 mg / mL of OVA, good levels of IFN-y were observed (Figure 4B). The greatest response was observed in mice vaccinated with chimeric adjuvants (4) and (5), as well as those given (1) + (3). Notably, (5) led to significantly higher levels of IFN-y compared to the co-administration (5) + (3). For (5) + (3), there was no significant increase in IFN-y compared to antigen alone.

[0169] Example 6: Sheep vaccination

[0170] 1. Sheep and vaccination groups

[0171] Seventy-two male sheep (Romney cross wethers ~12 months age) were selected for a trial to compare the ability of chimeric adjuvants in promoting antigen-specific immune responses to organisms that cause ovine pneumonia. These animals were selected from a larger group of 90 sheep to eliminate animals with high pre-existing antibody responses to M. haemolytica and M. ovipnuemoniae antigens. Animal ethics approval (AE14991) for the trial was obtained from the Grassland's Animal Ethics committee. Research ACVM approval (No. A011644) was obtained from the Ministry for Primary Industries.

[0172] The vaccine groups are shown in Table 2.

[0173] Table 2: Vaccine groups and adjuvants Vaccine antigens: M. haemolytica X387 was obtained from MSD Animal Health. This had been inactivated with formalin and was used at 25% v / v in each vaccine dose. M. ovipnuemoniae were field isolates #16, 90, 103 obtained by AgResearch (Bridgeman, 2020). Cultured mycoplasma cells were inactivated using H2O2 and used at a total of 1.8 mg cellular protein per vaccine dose.

[0174] Adjuvants: The adjuvants were stored at 4 °C until used. Each vial contained adjuvants made up to 37.5 mL of mineral oil / Tween-80 / PBS, 9: 1 :40 (6.75 mL Mineral oil, 0.75 mL Tween-80, 30 mL PBS). The adjuvants were vortexed well before combining with the antigens to formulate the vaccines. Adjuvants (1) and (20) were used at 3.75 mg / dose. MDP (3) was used at 0.05 mg / dose. The chimeric adjuvant (5) was used at 0.7 mg per dose. Each 2.5 mL dose of vaccine was prepared by mixing 1.25 mL adjuvant solution with 1.25 mL antigen / PBS). Quil-A (purified lyophilised saponin) (Brenntag, distributed by InvivoGen, CA, USA) was made up fresh in distilled H2O as a 5 mg / ml stock, filter- sterilised through a 0.22 mm filter and used at 1.5 mg per dose.

[0175] Formulated vaccines were stored at 4 °C until used (within 18 h).

[0176] 2. Vaccination and collection of blood samples

[0177] Sheep were vaccinated subcutaneously in the neck twice at a 4-week interval The vaccination sites were monitored daily for 3 days after each vaccination and weekly after through the duration of the trial, including up to 4 weeks after the second vaccination (week 8 of trial).

[0178] Blood samples (1 tube for serum) were obtained from the 72 sheep at each time point. In addition, one tube of heparinised blood was collected at time 0 and 6 weeks for measurement of T-cell responses in whole blood cultures.

[0179] 3. Measurement of antibody, cytokine responses and gene expression Serum antibody: Antigen-specific serum antibodies (IgG) at weeks 0, 4, 6, 9 and 12 were measured by ELISA as described previously (Lynch, 2021).

[0180] T-cell assays

[0181] IFN-y

[0182] T-cell responses were determined by measurement of IFN-y and IL-17 release in antigen-stimulated whole blood cultures (blood samples diluted 1: 1 with RPMI media) at 0 and 6 weeks. Whole blood cultures were incubated for 40 hours with M. haemolytica and M. ovipneumoniae whole cell antigens (final concentration of 10 mg / ml protein). The non-specific immune stimulator pokeweed mitogen PWM (final concentration of 2.5 mg / ml) was used as a positive control and PBS (10 mM, pH 7.3) was added to wells as a negative control. Following incubation, the plates were centrifuged and the concentration of IFN-y in the plasmas was measured by ELISA (Bovigam kit, Prionics). An ovine IFN-y standard (Kingfisher Biotech, St. Paul, USA) was included and titrated and the concentration of IFN-y was calculated from the standard curve.

[0183] IL-17A

[0184] ELISA protocols for ovine-specific IL-17A were developed and optimised in-house using capture, detection antibodies and recombinant ovine IL-17A as standards (Kingfisher Biotech, MN, USA) according to the manufacturer's instructions. Briefly, ELISA plates (Nunc™; Thermo Fisher Scientific) were coated overnight at room temperature with 50 pL / well of capture antibody (2 pg / mL protein) in PBS, 10 mM, pH 7.4. The plates were washed with PBS + Tween 20 (0.5%) (PBST) and blocked for 1 h at room temperature with PBS containing 4% (w / v) BSA (100 mL / well) at 37 °C on shaking. Following blocking, the plates were washed again with PBST. Ovine IL-17A standards and undiluted samples (50 pL / well) were added and the plates were incubated for 1 h at 37 °C. Following incubation, the plates were washed with PBST and incubated for 1 h at 37 °C with biotin conjugated detection antibody diluted at 1 :4,000 in blocking buffer (50 mL / well). Following washing with PBST, the plates were incubated for 30 min at 37°C with streptavidin diluted at 1 :500 in blocking buffer (50 mL / well). Following washing, 50 pL / well of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (BD Biosciences was added, and the plates incubated 20 min at room temperature in the dark. The reactions were stopped with addition of 50 pL / well of 0.5 M H2SO4 and absorbance read at 450 nm using a microplate reader (VERSAmax, Molecular Devices). The concentration of IL-17A in each sample was calculated from the standard curve.

[0185] Gene expression

[0186] The cells from the cultured blood samples obtained at week 6 were homogenised in TRIzol reagent (Invitrogen) and stored at -20 °C for determining gene expression in immune cells in peripheral blood mononuclear cells using Nanostring.

[0187] Statistical analyses

[0188] A repeated measurements model was used to analysis antibody responses which has fixed effects of Group, Week and their interaction, and random effect of Animal. For modelling the results, a multiple comparison of predicted means was performed with p- value adjusted by "BH" method (Benjamini, 1995). The analyses were conducted using packages "predictmeans" and "Ime4" in R 4.0.5 (Team, 2021).

[0189] IFN-y and IL-17A responses were analysed using One-way ANOVA (Minitab 17). P values less than 0.05 were considered statistically significant. 4. Effects on Human Cells

[0190] The use of human leukocytes (PBMCs) from healthy donors with written informed consent was approved by New Zealand Northern A Health and Disability Ethics Committee (approval number 15 / NTA / 178). Human monocytes were purified from whole blood by negative selection using RosetteSep Human Monocyte Enrichment Cocktail (StemCell) according to the manufacturer's instructions. Following isolation, the cells were adjusted to a final concentration of 1 x 106cells / mL in complete RPMI (10% FCS, 1% PenStrep) and stimulated with compounds at the specified concentrations. After 24 hours, the supernatants were collected and analysed for cytokine production. The data represents the mean ± SEM of 4 repeats. Conjugates 4 and 5 induced significantly greater IL-ip, IL-6 and IL-8 compared to either 1 or 3 alone, or to TDB. IL-23 production (an indicator of Thl7 skewing) was induced in response to 4 and 5, and this was greater than that produced in response to TDB, or 1, and comparable to that induced by 3 alone.

[0191] 5. Results

[0192] Vaccination site reactions

[0193] Animals were monitored daily for three days and then weekly for three weeks after both the 1stand 2ndvaccinations for any development of lumps or lesions at the vaccination sites (Table 3).

[0194] In general, the adjuvants that promoted the strongest antibody responses produced the more pronounced vaccination site reactions. However, the reactions were considered mild or moderate in nature. Following the first vaccination, a proportion of animals vaccinated with antigens formulated with (5), the combination of (1) and (3), or (20) combined with (3) developed lumps at the vaccination site. After the second vaccination, all adjuvants including Quil-A produced a lump in at least one animal at the second vaccination site. Some of these lumps developed into abscesses which healed during the time course of the trial. No lumps were observed at the vaccination sites of animals vaccinated with antigens alone. Table 3: Vaccination site reactions in animals after the 1stand 2ndvaccinations

[0195] *Lumps 5 mm in size or greatera2,b4,C1 lump developed into an abscess. These abscesses healed during the time course of the trial.

[0196] In a second test, animals were monitored for reactions for 4 weeks post vaccination (see Table 4). In both experiments, the use of compound 20, both alone and in combination with (3), led to the greatest number of vaccination site reactions, indicating both the toxicity of (20) and (20) + (3) and that this was inducing an inflammatory response in the animals rather than improved immunity. However, the chimeric adjuvants (4) and (5) showed a significant reduction in vaccination site reaction, showing that the chimeric compounds had an unexpected reduction in the toxicity level of the Mincle ligands, and that when combined with the other data, is leading to an increased immune response.

[0197] Table 4: Proportion of animals with Vaccination site reactions a3 mice moderate, 3 mice severe, all mice healed by week three. bl mouse severe, healed by week six.

[0198] Antibody responses

[0199] Serum antibody responses in the vaccinated sheep were measured by ELISA at weeks 0, 4, 6, 9 and 12. Antigen-specific responses to M. haemolytica and M. ovipneumoniae whole cell antigens are shown in Figures 5 and 6 respectively.

[0200] The sheep administered vaccines formulated with adjuvants produced higher serum antigen-specific responses to M. haemolytica and M. ovipneumoniae post-vaccination than responses in the control group (antigens without adjuvant). Interestingly, the antibody responses to M. haemolytica were delayed in the animals given (1) + (3) (Figure 5). Animals vaccinated with antigens alone produced a maximum response to M. haemolytica after the first vaccination at week 4 but these responses were not boosted after the second vaccination and antibody levels in serum declined during the remainder of the trial.

[0201] In general, the (20) adjuvant either alone or in combination with (3) stimulated higher levels of antigen-specific antibody than (1) combined with (3). The combination of (20) and (3) stimulated the highest antibody responses to M. haemolytica and M. ovipnuemoniae whole cell antigens compared to all the other adjuvants at 6-12 weeks. Compounds (1) and (20) were used at a dose of 3.75 mg / dose. In contrast, chimeric adjuvant (5) was used at a lower dose of 0.7 mg / dose. In addition, in general the different C18Brar dilipid adjuvants ((20), (20) + (3) and (5)) produced higher levels of antibody to M. haemolytica antigens than Quil-A at weeks 6-12. These differences were statistically significant for (20) + (3) and (5) at weeks 9 and 12 (P <0.05). Both (20) + (3) and (5) also stimulated higher levels of antibody to M. ovipneumoniae antigens than Quil-A at weeks 9 and 12, although these differences were not statistically significant.

[0202] 4.3 IFN-y and IL-17A responses

[0203] IFN-y responses to M. haemolytica and M. ovipneumoniae whole cell antigens at week 6 are shown in Figure 7. All adjuvants promoted IFN-y responses to the M. ovipnuemoniae antigens but not to M. haemolytica antigens. Mean IFN-y responses were higher in the groups vaccinated with M. ovipneumoniae whole cell antigens formulated with the various adjuvants compared to responses in the control (antigens alone) group (P <0.05). There was considerable animal-to-animal variation in IFN-y responses and a proportion of animals within each group did not respond.

[0204] IL-17A responses to M. haemolytica and M. ovipneumoniae whole cells antigens are shown in Figure 8. All the adjuvants promoted higher IL-17A responses compared to the antigen alone (control) groups. Quil-A and (20) promoted the strongest IL-17A responses to M. haemolytica and M. ovipnuemoniae whole cells antigens. Mean IL-17A responses were higher in animals administered vaccines formulated with (20) or Quil-A compared to responses in the control group (P <0.05). There were minimal IFN-y and IL- 17 responses to both M. haemolytica and M. ovipnuemoniae antigens in all groups prior to vaccination (week 0, data not shown).

[0205] 4.3 IL-ip, IL-6, IL-8 and IL-23 responses in human cells

[0206] Human leukocytes exhibited increased stimulation in response to compounds (4) and (5) compared to (1) and (3), as shown in Figure 9. Specifically, markers of immune activation; IL-ip (a pro-inflammatory cytokine produced by the inflammasome) in Figure 9A, IL-6 (critical to immune cell mediation, including neutrophil recruitment and B cell growth) in Figure 9B, IL-8 (attracts and activates neutrophils in infection) in Figure 9C and IL-23 (macrophage activation and the induction of IL-17) in Figure 9D. Chimeric adjuvants (4) and (5) induced significantly greater IL-ip, IL-6 and IL-8 compared to either (1) or (3) alone. IL-23 production (an indicator of Thl7 skewing) was induced in response to chimeric adjuvants (4) and (5), and this was greater than that produced in response to TDB (positive control), or 1, and comparable to that induced by (3) alone. The results show that the chimeric compounds of the present invention (4) and (5) invoke a greater production of immune related interleukin production in human leukocytes when compared to brartemicin derivative (1), TDB (control) and the NOD2 agonist (MDP) (3) alone. REFERENCES

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Claims

WHAT WE CLAIM:

1. A compound of Formula IX is selected from O or NH;Ai and Az are each independently selected from:-phenyl-, -(Ci2-C42)alkoxyphenyl-, -(Ci2-C42)alkylphenyl-, -(Ci2-C42)alkylene-, -(C12- C42)alkenylene-, -(C12- C42)alkynylene-, -(Ci2-C42)alkoxy(Ci2-C42)alkyl-, each of which may be optionally substituted with one or more substituents selected from:-hydroxy, oxo, -(Ce-Cio)aryl, -(Ci-Cis)alkyl, -(Ci-Cis)alkaryl, -(Ci-Cis)alkoxy, -(Ci- Cis)alkenyloxy, -(Ci-Cis)alkynyloxy, carboxy, cyano, -(Ci-Cis)alkoxycarbonyl, (Ci- Cis)alkylthio, -(Ci-Cis)alkenylthio, -(Ci-Cis)alkynylthio, -(Ci-Cis)alkylsulfonyl, -(Ci- Cis)alkenylsuflonyl, -(Ci-Cis)alkynylsulphonyl, halo, halo(Ci- Cis)alkoxy, halo(Ci- Cis)alkenyloxy, halo(Ci-Cis)alkynyloxy, -CONRR^nd -NRR1; wherein heterocyclyl is optionally substituted with one or two groups independently selected from -(Ci-Cis)alkyl, -hydroxy, -(Ci-Cis)alkoxy, -(Ci-Cis)alkylthio, -(Ci- Cis)alkylsulfonyl, halo and -CONRR1, wherein each R is independently selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl and - (Ci-Ci8)alkoxy(Ci-Cis)alkyl and each R1is independently selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl, -(Ci- Ci8)alkoxy(Ci-Cis)alkyl and heterocyclyl; or Ai is selected from the above options and A2 is absent;Li and L2 are each absent or independently selected from:wherein each R3is independently selected from H or-(Ci-Ci2)alkyl (preferably Ci-ealkyl, more preferably methyl, ethyl); provided that at least one of Li or L2 must be present; Y is absent or selected from -(Ci-Ci2)aryl-, -(Ci-Ci2)alkylene-, -(Ci-Ci2)alkoxylene-, and - (Ci-Ci2)alkoxy(Ci-Ci2)alkyl-, each of which may be optionally substituted with one or more substituents selected from hydroxy (-OH), thiol (-SH), seleno, oxo, -aryl, -(Ci-Cis)alkoxy, -(Ci-Cis)alkenyloxy, - (Ci-Cis)alkynyloxy, carboxy, cyano, -(Ci-Cis)alkoxycarbonyl, -(Ci-Cis)alkylthio, -(Ci- Cis)alkenylthio, -(Ci-Cis)alkynylthio, -(Ci-Cis)alkylsulfonyl, -(Ci-Cis)alkenylsuflonyl, - (Ci-Cis)alkynylsulphonyl, halo, halo(Ci-Cis)alkoxy, halo(Ci-Cis)alkenyloxy, halo(Ci- Ci8)alkynyloxy, -CONR4R5and -NR4R5; wherein R4is selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl or-(Ci- Ci8)alkoxy(Ci-Cis)alkyl andR5is selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl, -(Ci-Cis)alkoxy(Ci- Cis)alkyl and heterocyclyl, wherein heterocyclyl is optionally substituted with one or two groups independently selected from -(Ci-Cis)alkyl, hydroxy, -(Ci-Cis)alkoxy, -(Ci-Cis)alkylthio, -(Ci- Cis)alkylsulfonyl, halo and -CONR6R7, wherein R6and R7are independently selected from H, -(Ci-Cis)alkyl, hydroxy(Ci-Cis)alkyl or -(Ci-Ci8)alkoxy(Ci-Cis)alkyl; with the proviso that Y is absent when one of Li or L2 is absent;W is a MDP ligand selected from the group consisting of: a)II wherein R6is selected from H, -C(O)CH3, and -C(O)CH2OH, preferably -C(O)CH3; each R7is independently selected from H and -(Ci-C3)alkyl, preferably H;R8is selected from H and (Ci-Ce)alkyl, preferably H or C4-alkyl; and indicates the point of attachment to A2, or to L2 where A2 is absent; and b)wherein R9is selected from -H, -C(O)CH3, and -C(O)CH2OH, and R10is selected from -H, and -C(O)CnH(2n+i), wherein n = 1-22, preferably n = 15 or 17; and indicates the point of attachment to A2, or to L2 where A2 is absent2. A compound of claim 1 wherein X is O.

3. A compound of claim 1 or claim 2 wherein Ai and A2 are each independently selected from: -phenyl-, -(Ci2-C42)alkoxyphenyl-, and -(Ci2-C42)alkylphenyl-, each of which may be optionally substituted with one or more substituents selected from the group consisting of -hydroxy, -(Ce-Cio)aryl, -(Ci-Cis)alkyl and -(Ci- Cis)alkoxy.

4. A compound of claim 1 or claim 2 wherein A2 is absent, and Ai is selected from: - phenyl-, -(Ci2-C42)alkoxyphenyl-, and -(Ci2-C42)alkylphenyl-, each of which may be optionally substituted with one or more substituents selected from the group consisting of -hydroxy, -(Ce-Cio)aryl, -(Ci-Cis)alkyl and -(Ci-Cis)alkoxy.

5. A compound of claim 1 or claim 2 wherein A2 is absent, and Ai is -(C12- C42)alkoxyphenyl- which is optionally substituted with one or more substituentsselected from the group consisting of -hydroxy, -(C6-Cio)aryl, -(Ci-Cis)alkyl and - (Ci-Cis)alkoxy, preferably -(Ci-Cis)alkoxy.

6. A compound of claim 5 wherein A2 is absent and Ai is -(Ci?)alkoxy(octadecanyloxy)phenyl-.

7. A compound of claim 5 wherein A2 is absent and Ai is -(Ci?)alkoxyphenyl-.

8. A compound of any one of claims 1-7 wherein Li is9. A compound of any one of claims 1-8 wherein Y is -(Ci-Ci2)alkylene-.

10. A compound of claim 9 wherein Y is -(CH2)3-. 3 011. A compound of any one of claims 1-10 wherein L2 is selected fromandRI3wherein R3is selected from H or -(Ci-Ci2)alkyl.R3012. A compound of claim 11 wherein L2 is, wherein R3is selected from H, methyl or ethyl.R30 .N J13. A compound of claim 12 wherein L2 is , wherein R3is methyl.

14. A pharmaceutical composition comprising a compound of any one of claims 1-13 and one or more pharmaceutically acceptable excipients.

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