Unsymmetrical trehalose compounds and uses thereof

Abstract

Disclosed herein are unsymmetrical trehalose compounds, methods of preparation, and methods of use thereof, for example as vaccine adjuvants.

Description

UNSYMMETRICAL TREHALOSE COMPOUNDS AND USES THEREOF RELATED APPLICATION(S)

[0001] This application claims priority to U.S. Provisional Application No.63 / 684,094, filed August 16, 2024, which is hereby incorporated by reference in its entirety. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under contract numbers HHSN272201400050C and HHSN75N93023C00047 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The present disclosure relates to unsymmetrical trehalose compounds, methods of preparation, and methods of use thereof, for example as vaccine adjuvants. BACKGROUND

[0004] Globally, there are several disease-causing pathogens for which limited to no prophylaxis is available. Among these diseases, many fall into a class of bacterial and fungal pathogens for which Th17 mediated immunity has been implicated in disease protection, including Mycobacterium tuberculosis (Mtb), Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Aspergillus fumigatus, and others. While antigens are available for some of these pathogens, the development of Th17-inducing adjuvants has lagged. Currently adjuvant systems available and approved for humans induce primarily either a Th2 (aluminum salts, emulsions) or Th1 (MPL, CPG) type immune response.

[0005] Currently, the only adjuvant available for human use that promotes a Th17-mediated immune response is CAF01. This adjuvant is a cationic liposome that includes a synthetic Mincle receptor ligand (trehalose dibehenate) formulated with dimethyldioctadecylammonium. However, to date this adjuvant has not progressed beyond Phase I clinical trials.

[0006] Th17 has been implicated in several autoimmune diseases and thus novel immunemediators impacting these pathways (agonist and antagonists) could have beneficial impacts in autoimmunity and related disorders.

[0007] Additional adjuvants with improved efficacy, pharmacology, and safety attributes are needed to advance new vaccines and therapeutics in the areas of infectious disease, as well as autoimmunity and cancer. SUMMARY

[0008] In one aspect, disclosed are compounds of formula (I), or pharmaceutically acceptable salts thereof, (I), wherein: R1is wherein: X1is O or NRX, where RXis hydrogen or C1-4alkyl; R1a, R1cand R1eare each independently –OH or hydrogen and R1band R1dare each independently tert-butyl or trifluoromethyl; or, alternatively, R1aand R1eare each hydrogen, R1band R1dare each –OC1-12alkyl, and R1cis hydrogen or –OC1-12alkyl; R2is a five-membered heteroaryl having 1, 2, or 3 heteroatoms independently selected from the group consisting of N, O, and S, where the five-membered heteroaryl is substituted with –CH2-R3or ; or, alternatively, R2is –R3;R3 is , wherein:R4is methyl or hydrogen; Xaand Xbare each independently O, S, NH, NC1-4alkyl, or CH2; Xcis O, , S, NH, or NC1-4alkyl; n is 2-28; m is 2-28; p is 0 or 1; and Yaand Ybare each independently methyl or –N(RY)2, where RYis hydrogen or C1-4alkyl; or, alternatively, Yais , and Ybis methyl.

[0009] Also disclosed herein are adjuvant compositions comprising the compounds of formula (I) or pharmaceutically acceptable salts thereof, vaccine compositions comprising the compounds, methods for inducing an enhanced immune response in a subject using the compounds and compositions, and methods for inducing or enhancing immunogenicity of an antigen using the compounds and compositions.

[0010] Another aspect of the disclosure provides compounds of formula (I), or pharmaceutically acceptable salts or compositions thereof, for use in methods of inducing an enhanced immune response or inducing or enhancing immunogenicity of an antigen.

[0011] Another aspect of the disclosure provides use of compounds of formula (I), or pharmaceutically acceptable salts or compositions thereof, in the manufacture of a medicament for inducing an enhanced immune response or inducing or enhancing immunogenicity of an antigen.

[0012] In another aspect, are disclosed methods of preparing compounds of formula (I) and intermediates useful in the preparation of compounds of formula (I).Other aspects and embodiments of the disclosure will become apparent in light of the following description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1A graphically shows peripheral blood mononuclear cell (PBMC) cytokine responses from UM-1182-1186 where each compound was dissolved and serially diluted in ethanol (EtOH) and then dried to the bottom of a tissue culture plate. Dotted lines indicate additional control compounds for response reference.

[0014] FIG. 1B graphically shows PBMC cytokine responses from UM-1182-1186 where each compound was dissolved in dimethyl sulfoxide (DMSO) and serially diluted in media. Dotted lines indicate additional control compounds for response reference.

[0015] FIG.2A graphically shows human PBMC (hPBMC) cytokine responses from example unsymmetrical trehalose compounds UM1183-1186 compared to symmetrical trehalose compound UM-1024 and positive controls (trehalose 6,6′-dimycolate (TDM) and trehalose dibehenate (TDB)).

[0016] FIG.2B graphically shows hPBMC cytokine responses from example unsymmetrical trehalose compounds UM1187-1190 compared to symmetrical trehalose compound UM-1024 and positive controls (TDM and TDB).

[0017] FIG.2C graphically shows hPBMC cytokine responses from example unsymmetrical trehalose compounds UM1183-1186 compared to symmetrical trehalose compounds UM-1052 and positive controls (TDM and TDB).

[0018] FIG.2D graphically shows hPBMC cytokine responses from example unsymmetrical trehalose compounds UM1187-1190 compared to symmetrical trehalose compounds UM-1052 and positive controls (TDM and TDB).

[0019] FIG. 3A graphically shows day 28 post-secondary injection M72-specific IgG antibody concentrations. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s Least Significant Difference (LSD) for multiple comparisons.

[0020] FIG. 3B graphically shows day 28 post-secondary injection M72-specific IgG1 antibody concentrations. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0021] FIG. 3C graphically shows day 28 post-secondary injection M72-specific IgG2a antibody concentrations. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0022] FIG.4A graphically shows post-tertiary injection (day 77) draining lymph nodes (dLN) M72-specific IFNγ production. Asterisks indicate statistical significance as determined by one- way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0023] FIG.4B graphically shows post-tertiary injection (day 77) dLN M72-specific IL-17A production. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0024] FIG. 4C graphically shows post-tertiary injection (day 77) dLN M72-specific IL-5 production. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0025] FIG. 5A graphically shows IL-6 response in humans comparing dose response and different activity of three different C-Type Lectin Receptor (CLR) compound families (UM-1024 as representative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). All compounds were formulated on 50 nm diethylenetriamine-functionalized silica nanoparticles (DSNP-50s).

[0026] FIG.5B graphically shows IL-6 response in wild-type mice comparing dose response and different activity of three different CLR compound families (UM-1024 as representative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). All compounds were formulated on DSNP-50s.

[0027] FIG. 5C graphically shows IL-6 response in knockout (KO) mice comparing dose response and different activity of three different CLR compound families (UM-1024 as representative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). All compounds were formulated on DSNP-50s.

[0028] FIG.6A graphically shows day 42 (14 post-secondary) injection, serum M72-specific IgG antibody concentrations in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 as representative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one- way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0029] FIG.6B graphically shows day 42 (14 post-secondary) injection, serum M72-specific IgG1 antibody concentrations in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 as representative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one- way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0030] FIG.6C graphically shows day 42 (14 post-secondary) injection, serum M72-specific IgG2a antibody concentrations in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 as representative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one- way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0031] FIG.7A graphically shows post-secondary injection (day 42) draining lymph node T- cell response following re-stimulation with M72 cytokine production of IFNγ in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 as representative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0032] FIG.7B graphically shows post-secondary injection (day 42) draining lymph node T- cell response following re-stimulation with M72 cytokine production of IL-17A in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 as representative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0033] FIG.7C graphically shows post-secondary injection (day 42) draining lymph node T- cell response following re-stimulation with M72 cytokine production of IL-5 in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 asrepresentative compound for “branched BRAT” family, UM-1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0034] FIG. 8A graphically shows day 42 post-secondary M72-specific splenic cytokine production of IFNγ in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 as representative compound for “branched BRAT” family, UM- 1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0035] FIG. 8B graphically shows day 42 post-secondary M72-specific splenic cytokine production of IL-17A in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 as representative compound for “branched BRAT” family, UM- 1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0036] FIG. 8C graphically shows day 42 post-secondary M72-specific splenic cytokine production of IL-5 in response to different doses of CLR adjuvants from multiple different compound families (UM-1024 as representative compound for “branched BRAT” family, UM- 1089 as representative compound for “CLICK” family, and UM-1189 as representative compound for “BRICK” family). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0037] FIG.9 graphically shows comparison of hydrodynamic size of DSNP-50 formulations with various asymmetric Mincle agonists (UM-1189, UM-5240, UM-5249, UM-5257) versus symmetric BRAT Mincle agonist UM-1098, showing that changes in structure seem to lend themselves to smaller particle sizes and improved formulation properties.

[0038] FIG. 10 graphically shows DSNP-50 formulations with the asymmetric Mincle agonists (UM-1189, UM-5238, UM-5249) to demonstrate excellent formulation reproducibility.

[0039] FIG.11 graphically shows comparison of Mincle agonist / DSNP-50 formulation sizes vs their sizes in the vaccine formulations.

[0040] FIG. 12 graphically shows IL-6 response of hPBMCs three lead Mincle agonistsformulated in liposomes. Liposomes were titrated in a 96-well plate and hPBMCs (n=2 donors) were added to compounds in RPMI with 5% autologous plasma and incubated for 24 hr. Supernatants were tested for IL-6 activity by ELISA (RnD Systems). The graph is cut off at 30uM due to >50% cell death at higher concentrations.

[0041] FIG.13 graphically shows characteristics of Squalene / DOTAP-based O / W emulsions loaded with 2 mM UM-1098 or UM-1189. Particle size (reported as Z-average, in nm), polydispersity index (PdI) and zeta potential (in mV) were measured using a Dynamic light scattering instrument. For statistical analysis, t-Test: Two-Sample Assuming Unequal Variances was used, * p < 0.05, ** p < 0.001.

[0042] FIG. 14 graphically shows hPBMC IL-6 cytokine responses from UM-1183 through UM-1190. (Top) UM-1052 BRAT-based BRICK compounds UM-1183 through UM-1186. UM- 1024 BRAT-based BRICK compounds UM-1187 through UM-1190. Both sets were compared to their respective symmetric BRAT, TDM and TDB.

[0043] FIG.15 graphically shows IL-6 cytokine induction from hPBMCs for UM-1189 and parent compounds. Adjuvant-absorbed SNP formulations were titrated. PBMCs (n=2) were added to compounds in RPMI with 5% autologous plasma and incubated 24 hr. Supernatants were tested for IL-6 activity by ELISA (RnD Systems). New lots are designated by closed symbols and old lots with dotted lines and open symbols.

[0044] FIG. 16 graphically shows IL-6 response of hPBMCs to plate coated and silica nanoparticle formulated: Compounds were dissolved in EtOH, titrated down a 96-well plate and allowed to dry overnight (top row). Compounds formulated on silica nanoparticles were titrated down the plate immediately prior to addition of cells (bottom row). hPBMCs (n=3) were added in RPMI with 5% autologous plasma and incubated for 24 hr. Supernatants were tested for IL-6 activity by ELISA (RnD Systems).

[0045] FIG. 17 graphically shows IL-6 response of hPBMCs to plate coated and silica nanoparticle formulated UM-5249: Compounds were dissolved in EtOH, titrated down a 96-well plate and allowed to dry overnight (top row). Compounds formulated on silica nanoparticles were titrated down the plate immediately prior to addition of cells (bottom row). hPBMCs (n=3) were added in RPMI with 5% autologous plasma and incubated for 24 hr. Supernatants were tested for IL-6 activity by ELISA (RnD Systems).

[0046] FIG. 18 graphically shows IL-6 response of hPBMCs to plate coated formulatedasymmetric compounds: Compounds were dissolved in EtOH, titrated down a 96-well plate and allowed to dry overnight. hPBMCs were added in RPMI with 5% autologous plasma and incubated for 24 hr. Supernatants were tested for IL-6 activity by ELISA (RnD Systems).

[0047] FIG.19A graphically shows Day 42 (14 post-secondary) injection, serum M72-specific IgG antibody concentrations. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0048] FIG.19B graphically shows Day 42 (14 post-secondary) injection, serum M72-specific IgG1 antibody concentrations. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0049] FIG. 19C graphically shows Day 42 (14 post-secondary) injection, serum M72- specific IgG2a antibody concentrations. Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0050] FIG. 20A graphically shows day 42 post-secondary M72-specific splenic cytokine production of IFNγ in response to different doses of CLR adjuvants from multiple different compound families (BRAT, CLICK and BRICK). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0051] FIG. 20B graphically shows day 42 post-secondary M72-specific splenic cytokine production of IL-17A in response to different doses of CLR adjuvants from multiple different compound families (BRAT, CLICK and BRICK). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0052] FIG. 20C graphically shows day 42 post-secondary M72-specific splenic cytokine production of IL-5 in response to different doses of CLR adjuvants from multiple different compound families (BRAT, CLICK and BRICK). Asterisks indicate statistical significance as determined by one-way ANOVA followed by uncorrected Fisher’s LSD for multiple comparisons.

[0053] FIG. 21A graphically shows hindlimb weight bearing of the mice post-primary vaccination. The dotted line indicates where the average mouse range for differences in hindlimb weight bearing was on day 0 across mice in all groups. Normality and lognormality tests for each day were run separately and then an ordinary on-way ANOVA (p>0.05).

[0054] FIG. 21B graphically shows hindlimb weight bearing of the mice post-secondary vaccination. The dotted line indicates where the average mouse range for differences in hindlimb weight bearing was on day 0 across mice in all groups. Normality and lognormality tests for eachday were run separately and then an ordinary on-way ANOVA (p>0.05).

[0055] FIG. 22 graphically shows post-tertiary M72-specific splenic cytokine production in response to asymmetric UM-1189. Open circles are from Minkle KO C57 / Bl6 mice and closed squares are from wt. C57 / Bl6 mice. Normality and lognormality tests were run on the groups and a Kruskal-Wallis test was run for significance (p>0.05).

[0056] FIG. 23 graphically shows post-tertiary M72-specific splenic cytokine production in response to different asymmetric CLR adjuvants. Normality and lognormality tests were run on the groups and a Kruskal-Wallis test was run for significance (p>0.05).

[0057] FIG. 24 graphically shows post-tertiary M72-specific splenic cytokine production in response to different asymmetric CLR adjuvants. Normality and lognormality tests were run on the groups and a Kruskal-Wallis test was run for significance (p>0.05).

[0058] FIG.25 graphically shows hindlimb weight bearing of the mice post-primary and post- secondary vaccination. The dotted line indicates where the average mouse range for differences in hindlimb weight bearing was on day 0 across mice in all groups. Normality and lognormality tests for each day were run separately and then an ordinary on-way ANOVA (p>0.05). DETAILED DESCRIPTION

[0059] Described herein is a class of compounds that may act as Th17-inducing adjuvants, which may be useful for targeting diseases of significant medical burden for which no effective vaccine is available. The compounds may also be useful in the treatment of cancer.

[0060] The C-Type Lectin Receptor (CLR) family contains many diverse members that have different substrates, adaptor proteins, downstream signaling pathways, and cell type expression profiles. While some members of this family are purely phagocytic receptors, most are known to activate intracellular signaling networks that induce functional changes within the cell, such as modulation of transcription, endocytosis / phagocytosis, and / or cell adhesion and migration. One of the best characterized of these signaling cascade-inducing receptors is Mincle (CLEC4e).

[0061] Mincle must be coupled to an ITAM-containing adaptor molecule, FcRγ, to initiate downstream signaling, and ligand binding is Ca2+-dependent. However, the ligands for the Mincle receptor are distinct from those for other CLR receptors such as Dectin-1 and Dectin-2, and include the mycobacterial glycolipid trehalose-6,6’-dimycolate (TDM), its synthetic analog trehalose dibehenate (TDB), and many α-mannose-containing lipids found in various fungi and Candidastrains. These ligands can also be found in many pathogenic organisms including M. tuberculosis, S. mansoni, and T. rubrum. Additionally, the use of TDB and subsequent Mincle-induced signaling was shown to be necessary for the Th1 / Th17 adjuvanting properties in some pre-clinical vaccine models.

[0062] Using an approach described herein for the identification of novel Th17-inducing CLR agonists as vaccine adjuvants, which can query single CLRs on the surface of myeloid cells using specifically designed molecules and follow their effects through signaling pathways and subsequent cytokine / biomarker induction downstream, new biologically active compounds have been identified that may serve as a basis for a new class of Th-17-inducing adjuvants. Furthermore, mechanism of action (MOA) studies with lead CLR agonists / antagonists in combination with Toll- Like Receptor (TLR) agonists can used to evaluate cross-talk between CLRs and TLRs through the evaluation of (1) intracellular signaling pathways, (2) biomarker upregulation, and (3) in vivo cytokine / biomarker induction.

[0063] Compounds described herein may represent a new class of Th17-inducing adjuvants for vaccines targeting bacterial and fungal pathogens causing a significant burden of disease in humans, certain cancers, and autoimmune diseases. 1. Definitions

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0065] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0066] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0067] The term “immune response” includes any response associated with immunity including, but not limited to, increases or decreases in cytokine expression, production or secretion (e.g., IL-1, IL-6, IL-17, TNFα expression, production or secretion), cytotoxicity, immune cell migration, antibody production and / or immune cellular responses.

[0068] The term “monotherapy,” as used herein, means that only a single drug or therapeutic agent is administered.

[0069] The phrase “modulating an immune response” or “modulation of an immune response” or “modulate an immune response” includes upregulation, potentiating, stimulating, enhancing or increasing an immune response, as defined herein.

[0070] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0071] The term “alkoxy,” as used herein, refers to a group –O–alkyl. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert- butoxy.

[0072] The term “alkyl,” as used herein, means a straight or branched, saturated hydrocarbon chain. The term “lower alkyl” or “C1-6alkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term “C1-4alkyl” means a straight or branched chain hydrocarbon containing from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, and n-decyl.

[0073] The term “alkenyl,” as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon double bond.

[0074] The term “alkylene,” as used herein, refers to a divalent group derived from a straight or branched chain saturated hydrocarbon. Representative examples of alkylene include, but are not limited to, -CH2-, -CD2-, -CH2CH2-, -C(CH3)(H)-, -C(CH3)(D)-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-.

[0075] The term “amide,” as used herein, means -C(O)NR- or -NRC(O)-, wherein R may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl.

[0076] The term “amino,” as used herein, means –NRxRy, wherein Rxand Rymay be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl. In the case of an aminoalkyl group or any other moiety where amino appends together two other moieties, amino may be –NRx–, wherein Rxmay be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl.

[0077] The term “aryl,” as used herein, refers to a phenyl or a phenyl appended to the parent molecular moiety and fused to a cycloalkane group (e.g., the aryl may be indan-4-yl), fused to a 6-membered arene group (i.e., the aryl is naphthyl), or fused to a non-aromatic heterocycle (e.g., the aryl may be benzo[d][1,3]dioxol-5-yl). The term “phenyl” is used when referring to a substituent and the term 6-membered arene is used when referring to a fused ring. The 6-membered arene is monocyclic (e.g., benzene or benzo). The aryl may be monocyclic (phenyl) or bicyclic (e.g., a 9- to 12-membered fused bicyclic system).

[0078] The term “halogen” or “halo,” as used herein, means Cl, Br, I, or F.

[0079] The term “heteroaryl,” as used herein, refers to an aromatic monocyclic heteroatom- containing ring (monocyclic heteroaryl) or a bicyclic ring system containing at least one monocyclic heteroaromatic ring (bicyclic heteroaryl). The term “heteroaryl” is used herein to refer to a heteroarene when present as a substituent. The monocyclic heteroaryl are five or six membered rings containing at least one heteroatom independently selected from the group consisting of N, O and S (e.g.1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). The five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds. The bicyclic heteroaryl is an 8- to 12-membered ring system and includes a fused bicyclic heteroaromatic ring system (i.e., 10π electron system) such as a monocyclic heteroaryl ring fused to a 6-membered arene (e.g., quinolin-4-yl, indol-1-yl), a monocyclic heteroaryl ring fused to a monocyclic heteroarene (e.g., naphthyridinyl), and a phenyl fused to a monocyclic heteroarene (e.g., quinolin-5-yl, indol-4-yl). A bicyclic heteroaryl / heteroarene group includes a 9-membered fused bicyclic heteroaromatic ring system having four double bonds and at least one heteroatom contributing a lone electron pair to a fully aromatic 10π electron system, such as ring systems with a nitrogen atom at the ring junction (e.g., imidazopyridine) or a benzoxadiazolyl. A bicyclic heteroaryl also includes a fused bicyclic ring system composed of one heteroaromatic ring and one non-aromatic ring such as a monocyclic heteroaryl ring fused to a monocyclic carbocyclic ring (e.g., 6,7-dihydro-5H- cyclopenta[b]pyridinyl), or a monocyclic heteroaryl ring fused to a monocyclic heterocycle (e.g., 2,3-dihydrofuro[3,2-b]pyridinyl). The bicyclic heteroaryl is attached to the parent molecular moiety at an aromatic ring atom. Other representative examples of heteroaryl include, but are not limited to, indolyl (e.g., indol-1-yl, indol-2-yl, indol-4-yl), pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl (e.g., pyrazol-4-yl), pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl (e.g., triazol-4-yl), 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl (e.g., thiazol-4-yl), isothiazolyl, thienyl, benzimidazolyl (e.g., benzimidazol-5-yl), benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, furanyl, oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl (e.g., indazol-4-yl, indazol-5-yl), quinazolinyl, 1,2,4-triazinyl, 1,3,5- triazinyl, isoquinolinyl, quinolinyl, imidazo[1,2-a]pyridinyl (e.g., imidazo[1,2-a]pyridin-6-yl), naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl, and thiazolo[5,4-d]pyrimidin-2-yl.

[0080] The term “heterocycle” or “heterocyclic,” as used herein, means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The term “heterocyclyl” is used herein to refer to a heterocycle when present as a substituent. The monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. The five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocyclyls include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3- dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, 2-oxo-3-piperidinyl, 2-oxoazepan-3-yl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, oxepanyl, oxocanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, tetrahydrothiopyranyl, thiadiazolinyl, thiadiazolidinyl, 1,2- thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1- dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a 6-membered arene, or a monocyclic heterocycle fused to a monocyclic cycloalkane (e.g., 7- to 12-membered fused bicyclic heterocyclyl ring system such as hexahydro-2H-cyclopenta[b]furanyl, octahydro-3aH-cyclohepta[b]furanyl, or 3- oxabicyclo[3.1.0]hexanyl), or a monocyclic heterocycle fused to a monocyclic cycloalkene, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a monocyclic heterocycle fused to a monocyclic heteroarene, or a spiro heterocycle group (e.g., a 7- to 12-membered spiro heterocyclyl ring system such as 2-oxaspiro[3.3]heptanyl, 3-oxaspiro[5.5]undecanyl, 6- oxaspiro[2.5]octanyl, or 5-oxaspiro[2.4]heptanyl), or a bridged heterocycle ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., a 6- to 10-membered bridged bicyclic heterocyclyl ring system such as 7- oxabicyclo[2.2.1]heptanyl or 2-oxabicyclo[2.1.1]hexanyl), or an alkenylene bridge of two, three,or four carbon atoms. The bicyclic heterocyclyl is attached to the parent molecular moiety at a non-aromatic ring atom (e.g., indolin-1-yl). Representative examples of bicyclic heterocyclyls include, but are not limited to, chroman-4-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothien- 2-yl, 1,2,3,4-tetrahydroisoquinolin-2-yl, 2-azaspiro[3.3]heptan-2-yl, 2-oxa-6-azaspiro[3.3]heptan- 6-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), azabicyclo[3.1.0]hexanyl (including 3-azabicyclo[3.1.0]hexan-3-yl), 2,3-dihydro-1H-indol-1-yl, isoindolin-2-yl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, tetrahydroisoquinolinyl, 7- oxabicyclo[2.2.1]heptanyl, hexahydro-2H-cyclopenta[b]furanyl, 2-oxaspiro[3.3]heptanyl, 3- oxaspiro[5.5]undecanyl, 6-oxaspiro[2.5]octan-1-yl, and 3-oxabicyclo[3.1.0]hexan-6-yl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a 6-membered arene, or a bicyclic heterocycle fused to a monocyclic cycloalkane, or a bicyclic heterocycle fused to a monocyclic cycloalkene, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro- 2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza- adamantane (1-azatricyclo[3.3.1.13,7]decane), and oxa-adamantane (2- oxatricyclo[3.3.1.13,7]decane). The monocyclic, bicyclic, and tricyclic heterocyclyls are connected to the parent molecular moiety at a non-aromatic ring atom.

[0081] The term “hydroxyl” or “hydroxy,” as used herein, means an -OH group.

[0082] The term “hydroxyalkyl,” as used herein, means at least one -OH group, is appended to the parent molecular moiety through an alkylene group, as defined herein.

[0083] Terms such as “alkyl,” “cycloalkyl,” “alkylene,” etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance ( e.g., “C1-4alkyl,” “C3-6cycloalkyl,” “C1-4alkylene”). These designations are used as generally understood by those skilled in the art. For example, the representation “C” followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, “C3alkyl” is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in “C1-4,” the members of the group that follows may have any number of carbon atoms falling within the recited range. A “C1-4alkyl,” for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0084] The term “substituted” refers to a group that may be further substituted with one or more non-hydrogen substituent groups. Substituent groups include, but are not limited to, halogen, =O (oxo), =S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, - COOH, ketone, amide, carbamate, and acyl.

[0085] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0086] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0087] ABBREVIATIONS Me is methyl; Et is ethyl; MeOH is methanol; EtOH is ethanol; OAc is acetate; Ac is acetyl; EtOAc is ethyl acetate; KOTMS is potassium trimethylsilanolate; Py is pyridine; p-TSA is p-Toluene sulfonic acid; THF is tetrahydrofuran; TFA is trifluoroacetic acid; Tf is –SO₂CF₃; Tf2O is triflic anhydride;TMSOTf is trimethylsilyl trifluoromethanesulfonate; TBAI is tetrabutylammonium iodide; DMSO is dimethyl sulfoxide; DMF is N,N-dimethylformamide; DCM is dichloromethane; DCC is N,N′-dicyclohexylcarbodiimide; DMAP is 4-dimethylaminopyridine; BSA is trimethylsilyl (E)-N-(trimethylsilyl)acetimidate; FBS is fetal bovine serum; DMEM is Dulbecco's Modified Eagle Medium; MsCl is methanesulfonyl chloride; NMR is nuclear magnetic resonance spectroscopy; ESI is electrospray ionization; TMS is trimethyl silane; DIAD is diisopropyl azodicarboxylate; TBAF is tetrabutylammonium fluoride; HPLC-CAD is high-performance liquid chromatography-charged aerosol detector; HRMS is high-resolution mass spectrometry; EDCI-MeI is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; rt, RT, or r.t. is room temperature; eq, eq., or equiv is equivalent(s); sat. is saturated; soln. is solution; UV is ultraviolet; TLC is thin layer chromatography; TDB is trehalose dibehenate; TDM is trehalose 6,6′-dimycolate; TPP is triphenyl phosphine; wt. is weight; CLR is C-Type Lectin Receptor; DSNP or D-SNP is diethylenetriamine-functionalized silica nanoparticle;PBMC is peripheral blood mononuclear cell; hPBMC is human peripheral blood mononuclear cell; CCR2 is CC chemokine receptor 2; CCL2 is CC chemokine ligand 2; and CCR5 is CC chemokine receptor 5. 2. Compounds

[0088] In one aspect, disclosed is a compound of formula (I), wherein R1and R2are as defined herein.

[0089] Optionally substituted cyclic groups (i.e., unsubstituted or substituted cyclic groups), such as optionally substituted aryl, heteroaryl, etc., are composed of a ring system and the ring system's optional substitution. Accordingly, a “ring system” refers to the base molecular structure formed by the constituent ring atoms, including any hydrogens required to satisfy the valency of the ring atoms. A ring system may be defined independently of its substituents. Thus, where only the ring system of an optionally substituted cyclic group is redefined with a more specific definition, any optional substitution of the original optionally substituted cyclic group remains for the new more specifically defined ring system. For example, an optionally substituted 5- to 12- membered heteroaryl may be further defined by specifying the ring system of the optionally substituted 5- to 12-membered heteroaryl is a 5- to 6-membered heteroaryl (i.e., 5- to 6-membered heteroaryl ring system), in which case the optional substitution of the original optionally substituted 5- to 12-membered heteroaryl continues to apply to the 5- to 6-membered heteroaryl ring system, unless otherwise expressly indicated.

[0090] Where heterocyclic and heteroaromatic ring systems are defined to "contain" or as "containing" specified heteroatoms (e.g., 1-3 heteroatoms independently selected from the group consisting of O, N, and S), any ring atoms of the heterocyclic and heteroaromatic ring systems that are not one of the specified heteroatoms are carbon atoms.

[0091] In the following, numbered embodiments of the compounds are disclosed. The first embodiment is denoted E1, and subsequent embodiments are denoted E1.1, E1.2, E1.3, E1.4, E2, E3, E4, E5, etc.

[0001] E1. A compound of formula (I), or a pharmaceutically acceptable salt thereof,,X1is O or NRX, where RXis hydrogen or C1-4alkyl; R1a, R1cand R1eare each independently –OH or hydrogen and R1band R1dare each independently tert-butyl or trifluoromethyl; or, alternatively, R1aand R1eare each hydrogen, R1band R1dare each –OC1-12alkyl, and R1cis hydrogen or –OC1-12alkyl; R2is a five-membered heteroaryl having 1, 2, or 3 heteroatoms independently selected from the group consisting of N, O, and S, where the five-membered heteroaryl is substituted withR4is methyl or hydrogen; Xaand Xbare each independently O, S, NH, NC1-4alkyl, or CH2;n is 2-28; m is 2-28; p is 0 or 1; andYaand Ybare each independently methyl or –N(RY)2, where RYis hydrogen or C1-4alkyl; or, alternatively, Yais , and Ybis methyl.

[0092] E1.1. The compound of E1, or a pharmaceutically acceptable salt thereof, wherein p is 0.

[0093] E1.2. The compound of E1, or a pharmaceutically acceptable salt thereof, wherein p is 1.

[0094] E1.3 The compound of any one of E1-E1.2, or a pharmaceutically acceptable salt thereof, wherein R4is methyl.

[0095] E1.4. The compound of any one of E1-E1.2, or a pharmaceutically acceptable salt thereof, wherein R4is hydrogen.

[0096] E2. The compound of any one of E1-E1.4, or a pharmaceutically acceptable salt thereof, wherein X1is O.

[0097] E3. The compound of any one of E1-E1.4, or a pharmaceutically acceptable salt thereof, wherein X1is NRXand RXis hydrogen.

[0098] E4. The compound of any one of E1-E3, or a pharmaceutically acceptable salt thereof, wherein R1aand R1care each independently –OH or hydrogen, R1band R1dare each tert- butyl, and R1eis hydrogen.

[0099] E5. The compound of E4, or a pharmaceutically acceptable salt thereof, wherein R1ais –OH or hydrogen and R1cis hydrogen.

[0100] E5.1. The compound of E5, or a pharmaceutically acceptable salt thereof, wherein R1ais –OH.

[0101] E5.2. The compound of E5, or a pharmaceutically acceptable salt thereof, wherein R1ais hydrogen.

[0102] E6. The compound of any one of E1-E3, or a pharmaceutically acceptable salt thereof, wherein R1a, R1c, and R1eare each hydrogen and R1band R1dare each –OC1-12alkyl.

[0103] E6.1. The compound of E6, or a pharmaceutically acceptable salt thereof, wherein R1band R1dare each –OC2-8alkyl.

[0104] E7. The compound of any one of E1-E3, or a pharmaceutically acceptable salt thereof, wherein R1aand R1eare each hydrogen and R1b, R1c, and R1dare each –OC1-12alkyl.

[0105] E7.1. The compound of E7, or a pharmaceutically acceptable salt thereof, wherein R1b, R1c, and R1dare each –OC2-8alkyl.

[0106] E8. The compound of any one of E1-E7.1, or a pharmaceutically acceptable salt thereof, wherein R1is , or .

[0107] E8.1. The compound of E8, or a pharmaceutically acceptable salt thereof, wherein R1is , , or .

[0108] E9. The compound of any one of E1-E8.1, or a pharmaceutically acceptable salt thereof, wherein R2is the five-membered heteroaryl.

[0109] E9.1. The compound of E9, or a pharmaceutically acceptable salt thereof, wherein the five-membered heteroaryl is triazolyl, imidazolyl, oxazolyl, or thiazolyl.

[0110] E10. The compound of E9 or E9.1, or a pharmaceutically acceptable salt thereof, wherein the five-membered heteroaryl is triazolyl.

[0111] E10.1. The compound of E10, or a pharmaceutically acceptable salt thereof, wherein R2is , , , or OC1-28alkyl N N N (OC1-28alkyl)0-1.

[0112] E10.2. The compound of E10 or E10.1, or a pharmaceutically acceptable salt 3 thereof, wherein R2is or .

[0113] E11. The compound of any one of E10-10.2, or a pharmaceutically acceptable salt thereof, wherein R2is .

[0114] E12. The compound of any one of E1-E8, or a pharmaceutically acceptable salt thereof, wherein R2is –R3.

[0115] E13. The compound of any one of E1-E12, or a pharmaceutically acceptable salt thereof, wherein Xcis O or .

[0116] E13.1. The compound of E13, or a pharmaceutically acceptable salt thereof, wherein Xcis O.

[0117] E13.2. The compound of E13, or a pharmaceutically acceptable salt thereof, wherein Xcis .

[0118] E14. The compound of any one of E1-E13.2, or a pharmaceutically acceptable salt thereof, wherein Xaand Xbare each O or CH2.

[0119] E14.1. The compound of E14, or a pharmaceutically acceptable salt thereof, wherein Xaand Xbare each O.

[0120] E14.2. The compound of E14, or a pharmaceutically acceptable salt thereof, wherein Xaand Xbare each CH2.

[0121] E15. The compound of any one of E12-E14.2, or a pharmaceutically acceptable salt thereof, wherein R3is

[0122] E15.1. The compound of E15, or a pharmaceutically acceptable salt thereof, wherein R3is

[0123] E15.2. The compound of E15 or E15.1., or a pharmaceutically acceptable salt thereof, wherein R3is:, , , , , or .

[0124] E15.3. The compound of E15, or a pharmaceutically acceptable salt thereof, wherein R3is .

[0125] E15.4. The compound of E15.3, or a pharmaceutically acceptable salt thereof, O O wherein R3is .

[0126] E16. The compound of any one of E1-E15.2, or a pharmaceutically acceptable salt thereof, wherein n is 2-14.

[0127] E16.1. The compound of E16, or a pharmaceutically acceptable salt thereof, wherein n is 4-10.

[0128] E17. The compound of any one of E1-E16.1, or a pharmaceutically acceptable salt thereof, wherein m is 2-14.

[0129] E17.1. The compound of E17, or a pharmaceutically acceptable salt thereof, wherein m is 4-10.

[0130] E18. The compound of any one of E1-E17.1, or a pharmaceutically acceptable salt thereof, wherein Yaand Ybare each methyl.

[0131] E19. The compound of any one of E1-E17.1, or a pharmaceutically acceptable salt thereof, wherein Yais –N(RY)2and Ybis methyl.

[0132] E20. The compound of any one of E1-E17.1, or a pharmaceutically acceptable salt thereof, wherein Yais , and Ybis methyl.

[0133] E21. The compound of E1, or a pharmaceutically acceptable salt thereof, wherein the compound is: , , ,, or .

[0134] E21.1. The compound of E21, or a pharmaceutically acceptable salt thereof, wherein the compound is: , ,, , ,, , , ,, , , , , 35, , , , ,, , or .

[0135] E21.2. The compound of E21, or a pharmaceutically acceptable salt thereof, wherein the compound is: ,, , , or .

[0136] E22. An adjuvant composition comprising an effective amount of a compound of any one of E1-E21.2, or a pharmaceutically acceptable salt thereof.

[0137] E23. A method for inducing an enhanced immune response in a subject, comprising administering to the subject an effective amount of a compound of any one of E1-E21.2, or a pharmaceutically acceptable salt thereof, or the adjuvant composition of E22.

[0138] E23.1. The method of E23, comprising administering to the subject the adjuvant composition of E22.

[0139] E24. A vaccine composition comprising: (a) an antigen; and (b) an adjuvant composition comprising an effective amount of a compound of any one of E1-E21.2, or a pharmaceutically acceptable salt thereof.

[0140] E25. A method for inducing or enhancing the immunogenicity of an antigen in a subject, comprising administering to the subject a vaccine composition comprising an antigen and an adjuvant composition comprising an effective amount of a compound of any one of E1-E21.2, or a pharmaceutically acceptable salt thereof.

[0141] E26. An immunomodulatory composition comprising an effective amount of a compound of any one of E1-E21.2, or a pharmaceutically acceptable salt thereof.

[0142] E27. The immunomodulatory composition of E26, further comprising at least one additional adjuvant or immunostimulant.

[0143] E28. A method of modulating an immune response in a subject, comprising administering to the subject, an effective amount of a compound of any one of E1-E21.2, or a pharmaceutically acceptable salt thereof, or the immunomodulatory composition of E26 or E27.

[0144] E28.1. A method of modulating an immune response in a subject, comprising administering to the subject the immunomodulatory composition of E26 or E27.

[0145] E29. The method of E28 or E28.1, wherein the immunomodulatory composition is administered as a monotherapy.

[0146] E30. The method of any one of E28-E29, wherein the immune response in the subject is increased.

[0147] E31. The method of any one of E28-E30, wherein the subject is suffering from cancer, an autoimmune disorder, or an infectious disease.

[0148] E32. A method of preparing a compound of any one of E1-E21.2, or a pharmaceutically acceptable salt thereof, the method comprising reacting a compound of formula (ii):(ii) with (CH3)3Si–N3in the presence of a phosphine and an azodicarboxylate to produce a compound of formula (iii): (iii), wherein: PG, at each occurrence, is a hydroxyl protecting group, wherein the hydroxyl protecting group is a trimethyl silyl, a benzyl, or a benzoyl group. (CH3)3Si–N3may also be referred to as “trimethylsilyl azide” or “TMS azide.”

[0149] E32.1. The method of E32, wherein the phosphine is selected from the group consisting of triphenyl phosphine, tris(p-methoxyphenyl)phosphine, tricyclohexylphosphine, trimethyl phosphine, triethyl phosphine, tri-n-propylphosphine, tri-n-butylphosphine, triisopropylphosphine, and tris(trimethylsilyl)phosphine.

[0150] E32.2. The method of E32 or E32.1, wherein the azodicarboxylate is selected from the group consisting of diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD), and di-tert-butyl azodicarboxylate (DBAD).

[0151] E32.3. The method of any one of E32-E32.2, wherein the molar ratio of the phosphine to the compound of formula (iii) is at least 1:1.

[0152] E32.4. The method of any one of E32-E32.3, wherein the molar ratio of the azodicarboxylate to the compound of formula (iii) is at least 1:1.

[0153] E32.5. The method of any one of E32-E32.4, wherein the molar ratio of (CH3)3Si– N3to the compound of formula (iii) is at least 1:1.

[0154] E32.6. The method of any one of E32-E32.5, wherein the molar ratio of the phosphine to the compound of formula (iii) is no greater than 10:1.

[0155] E32.7. The method of any one of E32-E32.6, wherein the molar ratio of the azodicarboxylate to the compound of formula (iii) is no greater than 10:1.

[0156] E32.8. The method of any one of E32-E32.7, wherein the molar ratio of (CH3)3Si– N3to the compound of formula (iii) is no greater than 10:1.

[0157] E33. The method of any one of E32-E32.8, wherein reacting the compound of formula (ii) with (CH3)3Si–N3in the presence of the phosphine and an azodicarboxylate comprises: adding the azodicarboxylate to a solution comprising the compound of formula (ii), the phosphine, and an aprotic organic solvent, to produce a first reaction mixture; and adding the (CH3)3Si–N3to the first reaction mixture to produce a second reaction mixture.

[0158] E33.1. The method of E33, wherein the aprotic organic solvent is selected from the group consisting of toluene, tetrahydrofuran, dichloromethane, acetonitrile, dimethyl formamide, and 1,2-dimethoxyethane.

[0159] E33.2. The method of E33 or E33.1, wherein the aprotic organic solvent is toluene.

[0160] E34. The method of any one of E32-E33.2, wherein the phosphine is triphenyl phosphine or trimethyl phosphine.

[0161] E34.1. The method of E34, wherein the phosphine is triphenyl phosphine.

[0162] E34.2. The method of E34, wherein the phosphine is trimethyl phosphine.

[0163] E35. The method of any one of E32-E34.2, wherein the azodicarboxylate is diisopropyl azodicarboxylate.

[0164] E36. The method of any one of E32-E35, further comprising: reacting the compound of formula (iii) with a propargyl reagent of formula (iv): (iv), in the presence of a copper (II) source and a reducing agent; or, alternatively, in the presence of a copper (I) source, to produce a compound of formula (v): (v); and removing the –Si(CH3)3 group to produce a compound of formula (vi):(vi).

[0165] E36.1. The method of E36, wherein the compound of formula (iii) is reacted with the propargyl reagent of formula (iv) in the presence of a copper (II) source and a reducing agent.

[0166] E36.2. The method of E36, wherein the compound of formula (iii) is reacted with the propargyl reagent of formula (iv) in the presence of a copper (I) source.

[0167] E36.3. The method of E36 or E36.2, wherein the copper (I) source is selected from the group consisting of copper (I) iodide (CuI), copper (I) bromide (CuBr), and copper (I) chloride (CuCl).

[0168] E37. The method of E36 or E36.1, wherein the copper (II) source is copper sulfate (CuSO₄).

[0169] E38. The method of any one of E36, E36.1, or E37, wherein the reducing agent is sodium ascorbate.

[0170] E39. The method of any one of E36-E38, further comprising: reacting the compound of formula (vi) with a triflating agent to provide a compound of formula (vii): (vii).

[0171] E40. The method of E39, wherein the triflating agent is triflic anhydride or trimethylsilyl trifluoromethanesulfonate.

[0172] E41. The method of E39 or E40, further comprising reacting the compound of formula (vii) with a salt of formula (viii): (viii),to produce a compound of formula (ix): (ix), wherein G1is .

[0173] E41.1. The method of E41, wherein the compound of formula (vii) is reacted with a salt of formula (viii) in the presence of a crown ether.

[0174] E41.2. The method of E41.1, wherein the crown ether is 18-crown-6.

[0175] E42. A compound of formula (iii): (iii), wherein: PG, at each occurrence, is a hydroxyl protecting group, wherein the hydroxyl protecting group is a trimethyl silyl, a benzyl, or a benzoyl group.

[0176] E42.1. The compound of E41, wherein PG, at each occurrence, is a trimethyl silyl.

[0177] E43. A method of preparing the compound of E42 or E42.1, the method comprising: reacting a compound of formula (ii)(ii) with (CH3)3Si–N3in the presence of a phosphine and an azodicarboxylate.

[0178] E43.1. The method of E43, wherein the phosphine is selected from the group consisting of triphenyl phosphine, tris(p-methoxyphenyl)phosphine, tricyclohexylphosphine, trimethyl phosphine, triethyl phosphine, tri-n-propylphosphine, tri-n-butylphosphine, triisopropylphosphine, and tris(trimethylsilyl)phosphine.

[0179] E43.2. The method of E43 or E43.1, wherein the azodicarboxylate is selected from the group consisting of diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD), and di-tert-butyl azodicarboxylate (DBAD).

[0180] E43.3. The method of any one of E43-E43.2, wherein the molar ratio of the phosphine to the compound of formula (iii) is at least 1:1.

[0181] E43.4. The method of any one of E43-E43.3, wherein the molar ratio of the azodicarboxylate to the compound of formula (iii) is at least 1:1.

[0182] E43.5. The method of any one of E43-E43.4, wherein the molar ratio of (CH3)3Si– N3to the compound of formula (iii) is at least 1:1.

[0183] E43.6. The method of any one of E43-E43.5, wherein the molar ratio of the phosphine to the compound of formula (iii) is no greater than 10:1.

[0184] E43.7. The method of any one of E43-E43.6, wherein the molar ratio of the azodicarboxylate to the compound of formula (iii) is no greater than 10:1.

[0185] E43.8. The method of any one of E43-E43.7, wherein the molar ratio of (CH3)3Si– N3to the compound of formula (iii) is no greater than 10:1.

[0186] E44. The method of any one of E43-E43.8, wherein reacting the compound of formula (ii) with (CH3)3Si–N3in the presence of the phosphine and an azodicarboxylate comprises: adding the azodicarboxylate to a solution comprising the compound of formula (ii), the phosphine, and an aprotic organic solvent, to produce a first reaction mixture; and adding the (CH3)3Si–N3 to the first reaction mixture to produce a second reaction mixture.

[0187] E44.1. The method of E44, wherein the aprotic organic solvent is selected from the group consisting of toluene, tetrahydrofuran, dichloromethane, acetonitrile, dimethyl formamide, and 1,2-dimethoxyethane.

[0188] E44.2. The method of E44 or E44.1, wherein the aprotic organic solvent is toluene.

[0189] E45. The method of E43-E44.2, wherein the phosphine is triphenyl phosphine or trimethyl phosphine.

[0190] E45.1. The method of E45, wherein the phosphine is triphenyl phosphine.

[0191] E45.2. The method of E45, wherein the phosphine is trimethyl phosphine.

[0192] E46. The method of any one of E43-E45.2, wherein the azodicarboxylate is diisopropyl azodicarboxylate.

[0193] The compound may exist as a stereoisomer wherein asymmetric or chiral centers are present. The stereoisomer is “R” or “S” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The disclosure contemplates various stereoisomers and mixtures thereof and these are specifically included within the scope of this invention. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. In the compounds disclosed herein, a chiral atom depicted or described without a specific stereochemical configuration (e.g., a straight bond, not wedged or dashed bond, HC(OH)(CH3)(CH2CH3)) encompasses any stereochemical configuration at the chiral atom.

[0194] Individual stereoisomers of the compounds may be prepared synthetically from commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods.

[0195] It should be understood that the compound may possess tautomeric forms, as well as geometric isomers, and that these also constitute embodiments of the disclosure.

[0196] In the compounds of formula (I), and any subformulas, any "hydrogen" or "H," whether explicitly recited or implicit in the structure, encompasses hydrogen isotopes1H (protium) and2H (deuterium). Accordingly, any group comprising one or more hydrogen atoms encompasses corresponding deuterium-labeled versions of the group. For example, “–CH2–” encompasses “–

[0197] The present disclosure also includes isotopically-labeled compounds (e.g., deuterium labeled), where an atom in the isotopically-labeled compound is specified as a particular isotope of the atom. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F, and36Cl, respectively. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron-emitting isotopes that can be incorporated in compounds of formula (I) are11C,13N,15O, and18F.

[0198] Isotopically-enriched forms of compounds of formula (I), or any subformulas, may generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically- enriched reagent in place of a non-isotopically-enriched reagent. The extent of isotopic enrichment can be characterized as a percent incorporation of a particular isotope at an isotopically-labeled atom (e.g., % deuterium incorporation at a deuterium label). a. Pharmaceutically Acceptable Salts

[0199] The disclosed compounds may exist as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the compounds which are water or oil-soluble or dispersible, suitable for treatment of disorders without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the compounds or separately by reacting an amino group of the compounds with a suitable acid. For example, a compound may be dissolved in a suitable solvent, such as but not limited to methanoland water and treated with at least one equivalent of an acid, like hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. The amino groups of the compounds may also be quaternized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0200] Basic addition salts may be prepared during the final isolation and purification of the disclosed compounds by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N- methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N- dibenzylphenethylamine, 1-ephenamine and N,N’-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. b. General Synthesis

[0201] Compounds of formula (I) may be prepared by synthetic processes or by metabolic processes. Preparation of the compounds by metabolic processes includes those occurring in the human or animal body (in vivo) or processes occurring in vitro.

[0202] In various implementations, compounds of formula (I) may be prepared as shown in Scheme 1, below.Scheme 1A.

[0203] As shown in Scheme 1A, trehalose may be subjected to suitable protection conditions to provide protected trehalose i wherein PG is a hydroxyl protecting group, the hydroxyl group being a trimethyl silyl (TMS), a benzyl, or a benzoyl group—examples of which can be found in PGM Wuts and TW Greene, in Greene’s book titled Protective Groups in Organic Synthesis (4thed.), John Wiley & Sons, NY (2006). Example benzyl groups include, without limitation, benzyl (Bn), p-methoxybenzyl (PMB), 4-methoxyphenylmethyl (MPM), 3,4-dimethoxybenzyl (DMB), o-nitrobenzyl (ONB), and p-nitrobenzyl (PNB). Example benzoyl groups include, withoutlimitation, benzoyl (Bz), p-methoxybenzoyl (PMBz), p-nitrobenzoyl (PNBz), m-chlorobenzoyl, and 2,4-dinitrobenzoyl (DNBz).

[0204] Protected trehalose i may then be subjected to suitable primary alcohol deprotection conditions (e.g., K2CO3, MeOH) to provide compounds of formula ii. Compounds of formula ii may be reacted with N3-Si(CH3)3under suitable Mitsunobu conditions (e.g., in the presence of a phosphine, such as TPP or PMe3, and an azodicarboxylate, such as DIAD, in an aprotic solvent, such as toluene) to provide azides of formula iii. Other example Mitsunobu conditions include those described in Rao, M.L. et al., Org. Biomol. Chem.10 (45), 9090-9098, 2012.

[0205] Azides of formula iii may then be reacted with a propargyl reagent of formula iv under suitable click reaction conditions (e.g., in the presence of a copper (II) source, such as CuSO₄, and a reducing agent, such as sodium ascorbate, in a solvent, such as a mixture of dioxane and water) to provide a compound of formula v. Compounds of formula v may then be subjected to suitable primary alcohol deprotection conditions (e.g., in the presence of K2CO3in MeOH) to provide compounds of formula vi. Compounds of formula vi may then be reacted with a suitable triflating agent (e.g., Tf2O or TfOTMS) to provide triflate vii. Triflate vii may then be reacted with a salt of formula viii under suitable SN2 reaction conditions (e.g., in the presence of a crown ether such as 18-crown-6) to provide a compound of formula ix. Compounds of formula ix may then be deprotected under suitable deprotection conditions (e.g., in the presence of an acidic resin (e.g., Dowex-H+resin) and MeOH) to provide compounds of formula x.

[0206] In some implementations, compounds of formula (I) may be synthesized as shown in Scheme 1B, below.Scheme 1B.

[0207] As shown in Scheme 1B, TMS-protected trehalose compounds of formula A’ may be subjected to suitable Mitsunobu reaction conditions (e.g., TPP, DIAD, and TMSN3) to provide an azide intermediate of formula B’. Azide intermediates of formula B’ may be reacted with an alkyne intermediate of formula iv to provide −CH2-R3substituted triazole intermediates. After reacting the remaining unprotected alcohol group with triflic anhydride to provide a triflate and reacting the triflate with a potassium salt of formula viii, the TMS groups may be deprotected under suitable conditions to provide unsymmetrical trehalose compounds of formula E’.

[0208] Alternatively, in some implementations, compounds of formula (I) may be prepared as shown in Schemes 2-4, below.Scheme 2.

[0209] As shown in Scheme 2, alternatively, compounds of formula ii may be reacted with a compound of formula xi or xiii, respectively, under suitable coupling conditions (e.g., EDC-MeI or DCC and solvent, such as DCM) to provide compounds formula xii or xiii. Then, compounds of formula xi or xiii may be reacted with a compound of formula xiii or xi, respectively, under suitable coupling conditions (e.g., EDC-MeI or DCC and solvent, such as DCM) to provide compounds of formula xv. Scheme 3.

[0210] As shown in Scheme 3, alternatively, compounds of formula xii may be reacted with a suitable triflating agent (e.g., Tf2O or TfOTMS) to provide triflate xii-Tf. Triflate xii-Tf may then be reacted with a salt of formula viii under suitable SN2 reaction conditions (e.g., in the presence of a crown ether such as 18-crown-6) to provide a compound of formula xv.Scheme 4.

[0211] As shown in Scheme 4, compounds of formula xv may then be deprotected under suitable deprotection conditions (e.g., in the presence of an acidic resin (e.g., Dowex-H+resin) and MeOH) to provide compounds of formula xvi.

[0212] Example propargyl reagents of formula iv, e.g., where Xaand Xbare each O, may be prepared as shown in Scheme 5, below. Scheme 5.

[0213] As shown in Scheme 6, diol compounds of formula xvii may be subjected to suitable ketal protection conditions (e.g., p-TSA and acetone) to provide ketal intermediate xviii. Ketal intermediate xviii may then be reacted with propargyl bromide under suitable SN2 reaction conditions (e.g., Williamson ether conditions, such as NaH, TBAI, and solvent (e.g., THF)) to provide propargyl intermediate xix. Then, propargyl intermediate xix may be subjected to suitable ketal deprotection conditions (e.g., in the presence of an acidic resin (e.g., Dowex-H+resin) and MeOH) to provide propargyl diol intermediate xx. Propargyl diol intermediate xx may be reacted with xxi under suitable SN2 reaction conditions (e.g., Williamson ether conditions, such as NaH,TBAI, and solvent (e.g., THF)), then reacted with xxii under suitable SN2 reaction conditions (e.g., Williamson ether conditions, such as NaH, TBAI, and solvent (e.g., THF)), to provide propargyl reagents of formula xxiii.

[0214] Example salts of formula viii may be synthesized as shown in Scheme 5, below. Scheme 6.

[0215] As shown in Scheme 6, compounds of formula xiii may be reacted under suitable potassium salt formation conditions (e.g., in the presence of KOTMS and a solvent, such as THF) to provide salts of formula viii.

[0216] Other compounds of formula (I) and corresponding reagents may be prepared in a similar manner.

[0217] The compounds and intermediates may be isolated and purified by methods well- known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds can include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperature with an optional pretreatment with activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described for instance in “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), by Furniss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM202JE, England.

[0218] A disclosed compound may have at least one basic nitrogen whereby the compound can be treated with an acid to form a desired salt. For example, a compound may be reacted with an acid at or above room temperature to provide the desired salt, which is deposited, and collected by filtration after cooling. Examples of acids suitable for the reaction include, but are not limited to tartaric acid, lactic acid, succinic acid, as well as mandelic, atrolactic, methanesulfonic, ethanesulfonic, toluenesulfonic, naphthalenesulfonic, benzenesulfonic, carbonic, fumaric, maleic, gluconic, acetic, propionic, salicylic, hydrochloric, hydrobromic, phosphoric, sulfuric, citric, hydroxybutyric, camphorsulfonic, malic, phenylacetic, aspartic, or glutamic acid, and the like.

[0219] Reaction conditions and reaction times for each individual step can vary depending onthe particular reactants employed and substituents present in the reactants used. Specific procedures are provided in the Examples section. Reactions can be worked up in the conventional manner, e.g. by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. Starting materials, if not commercially available, can be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar compounds, or techniques that are analogous to the above described schemes or the procedures described in the synthetic examples section.

[0220] Routine experimentations, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the invention. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in PGM Wuts and TW Greene, in Greene’s book titled Protective Groups in Organic Synthesis (4thed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of the compounds of the invention can be accomplished by methods analogous to those described in the synthetic schemes described hereinabove and in specific examples.

[0221] When an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization or enzymatic resolution).

[0222] Similarly, when a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the above procedures using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or intermediates using a standard procedure such as chromatographic separation.

[0223] It can be appreciated that the synthetic schemes and specific examples as described areillustrative and are not to be read as limiting the scope of the invention as it is defined in the appended claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are included within the scope of the claims. c. Biological Activity

[0224] The compounds disclosed herein, including compounds of formula (I), may have biological activity that makes them useful as immunologic adjuvants or immunomodulators. For example, the compounds may stimulate the immune system’s response to a co-administered antigen. In some embodiments, the compounds may modulate the immune response when administered as a monotherapy. In some embodiments, the compounds may have activity as Th- 17 stimulating adjuvants.

[0225] In some embodiments, the compounds may have activity as partial agonists whereby inhibiting the activity of endogenous or exogenous ligands reducing inflammation or treating an underlying disease state, for example an autoimmune disease.

[0226] In some embodiments, the compounds may simulate the production of cytokines in a sample or when administered to a subject. The compounds may stimulate the production of Th17- type cytokines. Exemplary cytokines include IL-6, IL-1β, IL-23, and TNFα. Such activity may be tested according to established methods. For example, the levels of such cytokines may be measured in samples of peripheral blood mononuclear cells (PBMCs) after exposure to the compounds.

[0227] The compounds disclosed herein, including compounds of formula (I) may also have biological activity that makes them useful as cytotoxic compounds, for example for treatment of cancer. In some embodiments, the compounds may inhibit or reduce the growth or proliferation of cancer cells. Such activity can be determined according to established methods. 3. Compositions

[0228] The disclosed compounds may be incorporated into pharmaceutical compositions, adjuvant compositions, and vaccine compositions that may be suitable for administration to a subject (such as a patient, which may be a human or non-human).a. Pharmaceutical Compositions

[0229] The disclosed compounds may be incorporated into pharmaceutical compositions. The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the agent. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual.

[0230] A therapeutically effective amount is also one in which any toxic or detrimental effects of a compound of the invention (e.g., a compound of formula (I)) are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0231] For example, a therapeutically effective amount of a compound of formula (I), may be about 0.001 mg / kg to about 1000 mg / kg, 0.01 mg / kg to about 1000 mg / kg, 0.1 mg / kg to about 1000 mg / kg, 1 mg / kg to about 1000 mg / kg, about 5 mg / kg to about 950 mg / kg, about 10 mg / kg to about 900 mg / kg, about 15 mg / kg to about 850 mg / kg, about 20 mg / kg to about 800 mg / kg, about 25 mg / kg to about 750 mg / kg, about 30 mg / kg to about 700 mg / kg, about 35 mg / kg to about 650 mg / kg, about 40 mg / kg to about 600 mg / kg, about 45 mg / kg to about 550 mg / kg, about 50 mg / kg to about 500 mg / kg, about 55 mg / kg to about 450 mg / kg, about 60 mg / kg to about 400 mg / kg, about 65 mg / kg to about 350 mg / kg, about 70 mg / kg to about 300 mg / kg, about 75 mg / kg to about 250 mg / kg, about 80 mg / kg to about 200 mg / kg, about 85 mg / kg to about 150 mg / kg, and about 90 mg / kg to about 100 mg / kg.

[0232] The pharmaceutical compositions and formulations may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non- toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0233] Thus, the compounds and their physiologically acceptable salts may be formulated for administration by, for example, solid dosing, eye drop, in a topical oil-based formulation, injection, inhalation (either through the mouth or the nose), implants, or oral, buccal, sublingual, parenteral, or rectal administration. Techniques and formulations may generally be found in “Remington's Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.

[0234] The route by which the disclosed compounds are administered and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).

[0235] Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions.

[0236] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.

[0237] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma. The amount oflubricant(s) in a systemic or topical composition is typically about 5 to about 10%.

[0238] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50%.

[0239] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10%.

[0240] Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%.

[0241] Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.

[0242] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1%.

[0243] Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%.

[0244] Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.

[0245] Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.

[0246] Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.

[0247] Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.

[0248] Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and theTWEENS from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington's Pharmaceutical Sciences, 15th Ed.1975, pp.335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.

[0249] Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% of an active compound (e.g., a compound of formula (I)) and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.

[0250] Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives. The oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.

[0251] Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.

[0252] Capsules (including implants, time release and sustained release formulations) typically include an active compound (e.g., a compound of formula (I)), and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise a disclosed compound, and preferably glidants such as silicon dioxide to improve flow characteristics. Implants can be of the biodegradable or the non-biodegradable type.

[0253] The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of thisinvention.

[0254] Solid compositions may be coated by conventional methods, typically with pH or time- dependent coatings, such that a disclosed compound is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.

[0255] Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non- effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include a disclosed compound and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.

[0256] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[0257] The disclosed compounds can be topically administered. Topical compositions that can be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions include: a disclosed compound (e.g., a compound of formula (I)), and a carrier. The carrier of the topical composition preferably aids penetration of the compounds into the skin. The carrier may further include one or more optional components.

[0258] The amount of the carrier employed in conjunction with a disclosed compound is sufficient to provide a practical quantity of composition for administration per unit dose of thecompound. Techniques and compositions for making dosage forms useful in the methods of this invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0259] A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.

[0260] The carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.

[0261] Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95%.

[0262] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) in a topical composition is typically about 0% to about 95%.

[0263] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinationsthereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95%.

[0264] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectant(s) in a topical composition is typically 0% to 95%.

[0265] The amount of thickener(s) in a topical composition is typically about 0% to about 95%.

[0266] Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically-modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a topical composition is typically 0% to 95%.

[0267] The amount of fragrance in a topical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1%.

[0268] Suitable pH adjusting additives include HCl or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition. b. Adjuvant Compositions and Vaccine Compositions

[0269] The compounds may also be incorporated into adjuvant compositions and vaccine compositions. The vaccine compositions may further include an antigen. Suitable antigens include microbial pathogens, bacteria, viruses, proteins, glycoproteins lipoproteins, peptides, glycopeptides, lipopeptides, toxoids, carbohydrates, and tumor-specific antigens. Mixtures of two or more antigens may be employed.

[0270] The adjuvant and vaccine compositions may include an “effective amount” of the disclosed compound. In the context of an adjuvant or vaccine composition, an “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result (e.g., to potentiate an immune response to one or more antigens). The immune response can be measured, for example, by measuring antibody titers against an antigen, assessing the ability of a vaccine containing the compound to immunize a host in response to a disease or antigen challenge, etc. For example, administering an “effective amount” of a compound or compositionto a subject increases one or more antibody titers by 10% or more over a nonimmune control, by 20% or more over a nonimmune control, by 30% or more over a nonimmune control, by 40% or more over a nonimmune control, by 50% or more over a nonimmune control, by 50% or more over a nonimmune control, by 70% or more over a nonimmune control, by 80% or more over a nonimmune control, by 90% or more over a nonimmune control, or by 100% or more over a nonimmune control.

[0271] Vaccine preparation is a well-developed art and general guidance in the preparation and formulation of vaccines is readily available from any of a variety of sources. One such example is New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A.1978.

[0272] The vaccine compositions of the present disclosure may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be present, either incorporated into a fusion polypeptide or as a separate compound, within the vaccine composition. Polypeptides may, but need not, be conjugated to other macromolecules as described, for example, within U.S. Pat. Nos. 4,372,945 and 4,474,757. Vaccine compositions may generally be used for prophylactic and therapeutic purposes.

[0273] In one embodiment, the antigen in a vaccine composition is a peptide, polypeptide, or immunogenic portion thereof. An “immunogenic portion,” as used herein is a portion of a protein that is recognized (i.e., specifically bound) by a B cell and / or T cell surface antigen receptor. Such immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of an antigenic protein or a variant thereof.

[0274] Immunogenic portions of antigen polypeptides may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and / or T cell lines or clones. As used herein, antisera and antibodies are “antigen-specific” if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well known techniques. An immunogenic portion of a protein is a portion that reacts with such antisera and / or T cells at a level that is not substantially less than the reactivity of the fulllength polypeptide (e.g., in an ELISA and / or T cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example,125I-labeled Protein A.

[0275] Peptide and polypeptide antigens may be prepared using any of a variety of well-known techniques. Recombinant polypeptides encoded by DNA sequences may be readily prepared from isolated DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.

[0276] Portions and other variants of a protein antigen having less than about 100 amino acids, and generally less than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See, Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer's instructions.

[0277] Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3’ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5’ end of a DNA sequence encodingthe second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

[0278] A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No.4,935,233 and U.S. Pat. No.4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

[0279] In another embodiment, a compound or adjuvant composition described herein may be used in the preparation of DNA-based vaccine compositions. Illustrative vaccines of this type contain DNA encoding one or more polypeptide antigens, such that the antigen is generated in situ. The DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In one preferred embodiment, the DNA is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which typically involves the use of a non-pathogenic(defective), replication competent virus. Illustrative systems are disclosed, for example, in Fisher- Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos.4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art.

[0280] Alternatively, the DNA may be “naked,” as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a polypeptide component if desired.

[0281] Moreover, it will be apparent that a vaccine may contain pharmaceutically acceptable salts of the desired polynucleotide, polypeptide and / or carbohydrate antigens. For example, such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).

[0282] The adjuvant system may exhibit strong adjuvant effects when administered over a wide range of dosages and a wide range of ratios.

[0283] The amount of antigen in each vaccine dose is generally selected as an amount which induces an immunoprotective response without significant adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise about 1-1000 μg of protein, most typically about 2-100 μg, preferably about 5-50 μg. Of course, the dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered.

[0284] The immunogenic activity of a given amount of a vaccine composition can be readily determined, for example by monitoring the increase in titer of antibody against the antigen used in the vaccine composition (Dalsgaard, K. Acta Veterinia Scandinavica 69:1-40 (1978)). Anothercommon method involves injecting CD-1 mice intradermally with various amounts of a vaccine composition, later harvesting sera from the mice and testing for anti-immunogen antibody, e.g., by ELISA. These and other similar approaches will be apparent to the skilled artisan.

[0285] The antigen can be derived and / or isolated from essentially any desired source depending on the infectious disease, autoimmune disease, condition, cancer, pathogen, or a disease that is to be treated with a given vaccine composition. By way of illustration, the antigens can be derived from viral sources, such as influenza virus, feline leukemia virus, feline immunodeficiency virus, Human HIV-1, HIV-2, Herpes Simplex virus type 2, Human cytomegalovirus, Hepatitis A, B, C or E, Respiratory Syncytial virus, human papilloma virus rabies, measles, or hoof and mouth disease viruses. Illustrative antigens can also be derived from bacterial sources, such as anthrax, diphtheria, Lyme disease, malaria, tuberculosis, Leishmaniasis, T. cruzi, Ehrlichia, Candida, etc., or from protozoans such as Babeosis bovis or Plasmodium. The antigen(s) will typically be comprised of natural or synthetic amino acids, e.g., in the form of peptides, polypeptides, or proteins, can be comprised of polysaccharides, or can be mixtures thereof. Illustrative antigens can be isolated from natural sources, synthesized by means of solid phase synthesis, or can be obtained by way of recombinant DNA techniques.

[0286] In another embodiment, tumor antigens may be used in the vaccine compositions for the prophylaxis and / or therapy of cancer. Tumor antigens are surface molecules that are differentially expressed in tumor cells relative to non-tumor tissues. Tumor antigens make tumor cells immunologically distinct from normal cells and provide diagnostic and therapeutic targets for human cancers. Tumor antigens have been characterized either as membrane proteins or as altered carbohydrate molecules of glycoproteins or glycolipids on the cell surface. Cancer cells often have distinctive tumor antigens on their surfaces, such as truncated epidermal growth factor, folate binding protein, epithelial mucins, melanoferrin, carcinoembryonic antigen, prostate- specific membrane antigen, HER2-neu, which are candidates for use in therapeutic cancer vaccines. Because tumor antigens are normal or related to normal components of the body, the immune system often fails to mount an effective immune response against those antigens to destroy the tumor cells. To achieve such a response, the adjuvant systems described herein can be utilized. As a result, exogenous proteins can enter the pathway for processing endogenous antigens, leading to the production of cytolytic or cytotoxic T cells (CTL). This adjuvant effect facilitates the production of antigen specific CTLs which seek and destroy those tumor cells carrying on theirsurface the tumor antigen(s) used for immunization. Illustrative cancer types for which this approach can be used include prostate, colon, breast, ovarian, pancreatic, brain, head and neck, melanoma, leukemia, lymphoma, etc.

[0287] In one embodiment, the antigen present in the vaccine composition is not a foreign antigen, but a self-antigen, i.e., the vaccine composition is directed toward an autoimmune disease. Examples of autoimmune diseases include type 1 diabetes, conventional organ specific autoimmunity, neurological disease, rheumatic diseases / connective tissue disease, autoimmune cytopenias, and related autoimmune diseases. Such conventional organ specific autoimmunity may include thyroiditis (Graves + Hashimoto's), gastritis, adrenalitis (Addison's), ovaritis, primary biliary cirrhosis, myasthenia gravis, gonadal failure, hypoparathyroidism, alopecia, malabsorption syndrome, pernicious anemia, hepatitis, anti-receptor antibody diseases and vitiligo. Such neurological diseases may include schizophrenia, Alzheimer's disease, depression, hypopituitarism, diabetes insipidus, sicca syndrome and multiple sclerosis. Such rheumatic diseases / connective tissue diseases may include rheumatoid arthritis, systemic lupus erythematous (SLE) or Lupus, scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis, ulcerative colitis, Crohn's disease, vasculitis, psoriatic arthritis, exfoliative psoriatic dermatitis, pemphigus vulgaris, Sjögren's syndrome. Other autoimmune related diseases may include autoimmune uvoretinitis, glomerulonephritis, post myocardial infarction cardiotomy syndrome, pulmonary hemosiderosis, amyloidosis, sarcoidosis, aphthous stomatitis, and other immune related diseases, as presented herein and known in the related arts.

[0288] In one embodiment, the antigen may be covalently bonded to an adjuvant such as the compound of formula I to produce a discrete molecule which may exhibit an enhanced adjuvanting effect on the antigen, which may be greater than the adjuvanting effect attainable in the absence of such covalent bonding, as in a mixture of components (i.e., the antigen and a compound of formula (I)). The covalent bonding can be achieved by reaction through functional groups; for example, in the case of the compound of formula I through a carboxylic acid group, a hydroxyl group or an aldehyde functionality. A further enhanced adjuvanting effect may be attained for such covalently bonded antigen by incorporating a mineral salt adjuvant with such compounds. The mineral salt adjuvant preferably comprises aluminum hydroxide or aluminum phosphate, although other known mineral salt adjuvants, such as calcium phosphate, zinc hydroxide or calcium hydroxide, may be used.

[0289] The adjuvant may include other polynucleotides and / or polypeptides. It will be apparent that a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts may be prepared from pharmaceutically acceptable non- toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).

[0290] The vaccine compositions may be formulated for any appropriate manner of administration, and thus administered, including for example, topical, oral, nasal, intravenous, intravaginal, epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, subcutaneous, intramuscular administration, or via inhalation. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.

[0291] In one illustrative embodiment, the vaccine formulations are administered to the mucosae, in particular to the oral cavity, and preferably to a sublingual site, for eliciting an immune response. Oral cavity administration may be preferred in many instances over traditional parenteral delivery due to the ease and convenience offered by noninvasive administration techniques. Moreover, this approach further provides a means for eliciting mucosal immunity, which can often be difficult to achieve with traditional parenteral delivery, and which can provide protection from airborne pathogens and / or allergens. An additional advantage of oral cavity administration is that patient compliance may be improved with sublingual vaccine delivery, especially for pediatric applications, or for applications traditionally requiring numerous injections over a prolonged period of time, such as with allergy desensitization therapies.

[0292] The vaccine compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and / or preservatives. Alternatively, vaccine compositions may be formulated as a lyophilisate. Compounds may also be encapsulated within liposomes using wellknown technology.

[0293] The vaccine compositions may also comprise other adjuvants or immunoeffectors. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham); mineral salts (for example, aluminum, silica, kaolin, and carbon); aluminum salts such as aluminum hydroxide gel (alum), AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), and Al(OH)3; salts of calcium (e.g, Ca3(PO4)2), iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polynucleotides (for example, poly IC and poly AU acids); polyphosphazenes; cyanoacrylates; polymerase-(DL-lactide-co-glycoside); biodegradable microspheres; liposomes; lipid A and its derivatives; monophosphoryl lipid A; wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella); bovine serum albumin; diphtheria toxoid; tetanus toxoid; edestin; keyhole-limpet hemocyanin; Pseudomonal Toxin A; choleragenoid; cholera toxin; pertussis toxin; viral proteins; and Quil A. Aminoalkyl glucosamine phosphate compounds can also be used (see, e.g., WO 98 / 50399, U.S. Pat. No.6,113,918 (which issued from U.S. Ser. No. 08 / 853,826), and U.S. Ser. No.09 / 074,720). In addition, adjuvants such as cytokines (e.g., GM- CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants. Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule. Other known immunostimulatory macromolecules which can be used include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4′,4-diaminodiphenylmethane-3,3′-dicarboxylic acid and 4- nitro-2-aminobenzoic acid (See, Sela, M., Science 166: 1365-1374 (1969)) or glycolipids, lipids or carbohydrates.

[0294] Within the vaccine compositions provided herein, the adjuvant composition is preferably designed to induce an immune response predominantly of the Th17 type. High levels of Th17-type cytokines (e.g., IL-6, IL-1β, IL-23, and TNFα) may favor the induction of cell mediated immune responses to an administered antigen. Following administration of a vaccine asprovided herein, a patient may support an immune response that includes Th17-type responses. In some embodiments, in which a response is predominantly Th17-type, the level of Th17-type cytokines will increase to a greater extent than the level other cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see, Mosmann and Coffman, Ann. Rev. Immunol.1989, 7: 145-173.

[0295] The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology (see, e.g., Coombes et al., Vaccine 14:1429-1438, 1996) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly(lactide- co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see, e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94 / 20078, WO / 94 / 23701 and WO 96 / 06638). The amount of active compound contained within a sustained release formulation will vary depending upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[0296] Any of a variety of known delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and / or maintenance of the T cell response, to have anti-target effects per se and / or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0297] Certain embodiments may use dendritic cells or progenitors thereof as antigen- presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see, Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated. As an alternative to dendritic cells, secreted vesicles antigen- loaded dendritic cells (called exosomes) may be used within a vaccine (see, Zitvogel et al., Nature Med.4:594-600, 1998).

[0298] Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and / or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and / or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.

[0299] Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

[0300] APCs may generally be transfected with a polynucleotide encoding an antigenpolypeptide (or portion or other variant thereof) such that the antigen polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells, and the adjuvants described herein, may then be used for therapeutic purposes. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97 / 24447, or the gene gun approach described by Mahvi et al., Immunology and Cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the antigen polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

[0301] In one embodiment, the vaccine composition may comprise a liposome vesicle comprising the compound of formula I. Liposomes are generally produced from phospholipids or other lipid substances. Procedures for the preparation of liposomes are well known to those of skill in the art. Any lipid capable of forming vesicles that comprises the compound of formula I can be employed. For clinical application, it is desirable that the lipid be non-toxic, physiologically acceptable, and metabolizable. Common bilayer forming lipids having clinical potential are phospholipids, fatty acids, sphingolipids, glycosphingolipids, and steroids. Glycerol containing phospholipids are the most commonly used component of liposome formulations having clinical utility. One commonly used example is phosphatidylcholine or lecithin. The steroid cholesterol and its derivatives are often included as components of liposomal membranes. The tendency of liposomes to aggregate and fuse can be controlled by the inclusion of small amounts of acidic or basic lipids in the formulation. The properties of liposomes containing phospholipids are determined by the chemistry of the phospholipid. Important considerations are the hydrocarbon chain length, degree of unsaturation of the hydrocarbon chain, degree of branching of the hydrocarbon chain, and temperature of the system.

[0302] Multilamellar liposomes can be created by depositing a mixture of lipids as a thin filmby evaporation under reduced pressure followed by dispersion with an excess volume of aqueous buffer containing the antigen with or without organic solvents. Another method is to mix the aqueous phase containing the antigen with small unilamellar liposomes followed by lyophilization. The multilamellar liposomes are formed when the lyophilized product is rehydrated, usually with a small amount of distilled water. The small unilamellar liposomes to be used in this process are produced by dispersing the lipids in an aqueous medium followed by a mechanical means of dispersion such as sonication, use of a high pressure device, or a solvent injection method. Large and intermediate sized unilamellar liposomes can also be produced by conventional techniques including detergent dialysis, extrusion through small pore size membranes under high pressure, freeze thawing followed by slow swelling, dehydration followed by rehydration and dilution, or dialysis of lipids in the presence of chaotropic ions. The size of the liposomes can be made more uniform by fractionation procedures such as centrifugation or size exclusion chromatography, homogenization, or capillary pore membrane extrusion. 4. Methods of Use

[0303] The disclosed compounds and compositions may be used in various methods, including methods for modulating an immune response in a subject, methods of inducing or enhancing immunogenicity of an antigen in a subject, and related methods. The disclosed compounds and compositions may also be used in methods of treating cancer and related methods. The disclosed compounds and compositions may also be used in methods of treatment and prevention of an autoimmune disorder or an infectious disease. a. Modulating Immune Response

[0304] The disclosed compounds and compositions may be used in methods of modulating the immune response in a subject, comprising administering to the subject an effective amount of a compound described herein, an adjuvant composition described herein, or an immunomodulatory composition described herein.

[0305] In some embodiments, disclosed compounds and compositions may be used to treat an inflammatory or autoimmune condition by modifying or modulating the immune response

[0306] In some embodiments, disclosed compounds and compositions may be used in a method of inducing an enhanced immune response in a subject. In some embodiments, theenhanced immune response is an immune response of a Th17-type or a Th17 / Th1-tpe. In some embodiments, administration of the compound or composition may induce a Th17-type immune response and not a Th1-type or Th2-type immune response.

[0307] The enhanced immune response may be induced by co-administering the compound or composition with an antigen. Suitable antigens include microbial pathogens, bacteria, viruses, proteins, glycoproteins lipoproteins, peptides, glycopeptides, lipopeptides, toxoids, carbohydrates, and tumor-specific antigens. Mixtures of two or more antigens may be employed.

[0308] In some embodiments, the disclosed compounds and compositions may be administered as a monotherapy. In other embodiments, the disclosed compositions may futher contain at least one additional adjuvant or immunostimulant. b. Inducing or Enhancing Immunogenicity of an Antigen

[0309] The disclosed compounds and compositions may be used in methods of inducing or enhancing immunogenicity of an antigen in a subject, comprising administering to the subject a vaccine composition comprising the antigen and an adjuvant composition comprising an effective amount of a compound or composition described herein.

[0310] Suitable antigens include microbial pathogens, bacteria, viruses, proteins, glycoproteins lipoproteins, peptides, glycopeptides, lipopeptides, toxoids, carbohydrates, and tumor-specific antigens. Mixtures of two or more antigens may be employed. c. Treatment of Cancer and Related Methods

[0311] The disclosed compounds and compositions may be used in methods of treating cancer, or in methods of reducing or inhibiting the proliferation of cancer cells, the methods comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a composition described herein.

[0312] The methods can be used with any cancer cell or in a subject having any type of cancer, for example those described by the National Cancer Institute. Exemplary cancers may include the following: digestive / gastrointestinal cancers such as anal cancer; bile duct cancer; extrahepatic bile duct cancer; appendix cancer; carcinoid tumor, gastrointestinal cancer; colon cancer; colorectal cancer including childhood colorectal cancer; esophageal cancer including childhood esophageal cancer; gallbladder cancer; gastric (stomach) cancer including childhood gastric(stomach) cancer; hepatocellular (liver) cancer including adult (primary) hepatocellular (liver) cancer and childhood (primary) hepatocellular (liver) cancer; pancreatic cancer including childhood pancreatic cancer; sarcoma, rhabdomyosarcoma; islet cell pancreatic cancer; rectal cancer; and small intestine cancer; endocrine cancers such as islet cell carcinoma (endocrine pancreas); adrenocortical carcinoma including childhood adrenocortical carcinoma; gastrointestinal carcinoid tumor; parathyroid cancer; pheochromocytoma; pituitary tumor; thyroid cancer including childhood thyroid cancer; childhood multiple endocrine neoplasia syndrome; and childhood carcinoid tumor; eye cancers such as intraocular melanoma; and retinoblastoma; musculoskeletal cancers such as Ewing's family of tumors; osteosarcoma / malignant fibrous histiocytoma of the bone; childhood rhabdomyosarcoma; soft tissue sarcoma including adult and childhood soft tissue sarcoma; clear cell sarcoma of tendon sheaths; and uterine sarcoma; breast cancer such as breast cancer including childhood and male breast cancer and breast cancer in pregnancy; neurologic cancers such as childhood brain stem glioma; brain tumor; childhood cerebellar astrocytoma; childhood cerebral astrocytoma / malignant glioma; childhood ependymoma; childhood medulloblastoma; childhood pineal and supratentorial primitive neuroectodermal tumors; childhood visual pathway and hypothalamic glioma; other childhood brain cancers; adrenocortical carcinoma; central nervous system lymphoma, primary; childhood cerebellar astrocytoma; neuroblastoma; craniopharyngioma; spinal cord tumors; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; and childhood supratentorial primitive neuroectodermal tumors and pituitary tumor; genitourinary cancers such as bladder cancer including childhood bladder cancer; renal cell (kidney) cancer; ovarian cancer including childhood ovarian cancer; ovarian epithelial cancer; ovarian low malignant potential tumor; penile cancer; prostate cancer; renal cell cancer including childhood renal cell cancer; renal pelvis and ureter, transitional cell cancer; testicular cancer; urethral cancer; vaginal cancer; vulvar cancer; cervical cancer; Wilms tumor and other childhood kidney tumors; endometrial cancer; and gestational trophoblastic tumor; Germ cell cancers such as childhood extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; head and neck cancers such as lip and oral cavity cancer; oral cancer including childhood oral cancer; hypopharyngeal cancer; laryngeal cancer including childhood laryngeal cancer; metastatic squamous neck cancer with occult primary; mouth cancer; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer including childhood nasopharyngeal cancer; oropharyngeal cancer; parathyroid cancer; pharyngeal cancer;salivary gland cancer including childhood salivary gland cancer; throat cancer; and thyroid cancer; hematologic / blood cell cancers such as a leukemia (e.g., acute lymphoblastic leukemia including adult and childhood acute lymphoblastic leukemia; acute myeloid leukemia including adult and childhood acute myeloid leukemia; chronic lymphocytic leukemia; chronic myelogenous leukemia; and hairy cell leukemia); a lymphoma (e.g., AIDS-related lymphoma; cutaneous T cell lymphoma; Hodgkin's lymphoma including adult and childhood Hodgkin's lymphoma and Hodgkin's lymphoma during pregnancy; non-Hodgkin's lymphoma including adult and childhood non-Hodgkin's lymphoma and non-Hodgkin's lymphoma during pregnancy; mycosis fungoides; Sezary syndrome; Waldenstrom's macroglobulinemia; and primary central nervous system lymphoma); and other hematologic cancers (e.g., chronic myeloproliferative disorders; multiple myeloma / plasma cell neoplasm; myelodysplastic syndromes; and myelodysplastic / myeloproliferative disorders); lung cancer such as non-small cell lung cancer; and small cell lung cancer; respiratory cancers such as adult malignant mesothelioma; childhood malignant mesothelioma; malignant thymoma; childhood thymoma; thymic carcinoma; bronchial adenomas / carcinoids including childhood bronchial adenomas / carcinoids; pleuropulmonary blastoma; non-small cell lung cancer; and small cell lung cancer; skin cancers such as Kaposi's sarcoma; Merkel cell carcinoma; melanoma; and childhood skin cancer; AIDS-related malignancies; other childhood cancers, unusual cancers of childhood and cancers of unknown primary site; and metastases of the aforementioned cancers. 5. Kits

[0313] In one aspect, the disclosure provides kits comprising at least one disclosed compound or a pharmaceutically acceptable salt thereof, or a composition comprising the compound or a pharmaceutically acceptable salt thereof, and one or more of: (a) at least one antigen; and (b) instructions for administering the compound or composition.

[0314] In some embodiments, the at least one disclosed compound and the at least one antigen are co-formulated. In some embodiments, the at least one disclosed compound and the at least one antigen are co-packaged. The kits can also comprise compounds and / or products co-packaged, co- formulated, and / or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosedcompound and / or product and another component for delivery to a patient.

[0315] The disclosed kits can be employed in connection with disclosed methods of use.

[0316] The kits may further include information, instructions, or both that use of the kit will provide increased immunity against certain pathogens in mammals (particularly humans). The information and instructions may be in the form of words, pictures, or both, and the like. In addition or in the alternative, the kit may include the compound, a composition, or both; and information, instructions, or both, regarding methods of administration of compound, or of the composition, preferably with the benefit of treating or preventing medical conditions in mammals (e.g., humans).

[0317] The compounds and processes of the disclosure may be better understood by reference to the following examples, which are intended as an illustration of and not a limitation upon the scope of the disclosure. Examples

[0318] General Experimental. All reagents and solvents were used as received. Reactions were monitored by TLC-analysis on Merck Silica gel 60 F254 plates and visualized by UV at 254 nm and dipping in vanillin (vanillin / water / ethanol / sulfuric acid, 0.2 g:5 mL:5 mL:1 mL) or phosphomolybdic acid in ethanol (PMA) and developed with heat. All compounds were confirmed to be >95% pure by NMR and HPLC-CAD analysis.1H and13C NMR spectra were recorded on an Agilent or Bruker 400 MHz instrument and were referenced to TMS or a solvent peak. High- resolution HPLC-MS analysis was obtained on an Agilent 6520 Q-TOF mass spectrometer utilizing an electrospray ionization source in positive or negative mode. Chromatography was performed on Grace®or Biotage®automated medium pressure chromatography instruments with preloaded Buchi silica gel cartridges. Human and mouse Mincle expressing HEK cells were obtained from Invivogen (San Diego, CA). Cells were cultured according to the manufacturer’s instructions in DMEM with 10% FBS, 50 U / mL penicillin, 50 mg / mL streptomycin, 100 mg / mL Normocin, 2 mM L-glutamine, 30 µg / mL blasticidin, 1 µg / mL puromycin, and 1x HEK-Blue™ CLR Selection. 2,2’,3,3’,4,4’,6-Heptakis-O-trimethylsilyl-α,α-D-trehalose, 2-methyl-2-((prop-2- yn-1-yloxy)methyl)propane-1,3-diol and lipidated compounds [1-(3-(hexyloxy)-2-methyl-2- ((prop-2-yn-1-yloxy)methyl)propoxy)hexane; 1-(3-(heptyloxy)-2-methyl-2-((prop-2-yn-1- yloxy)methyl)propoxy)heptane; 1-(2-methyl-3-(octyloxy)-2-((prop-2-yn-1-yloxy)methyl)propoxy)octane; 1-(2-methyl-3-(nonyloxy)-2-((prop-2-yn-1- yloxy)methyl)propoxy)nonane] were prepared using the literature method without any modification. The analytical data of all final compounds are described below. 1. Example Syntheses – Part 1 General Procedures

[0319] Various example general procedures for preparing unsymmetrical trehalose compounds are described below.

[0320] In the following schemes “Ar” denotes:. General procedure for selective 6-silyl deprotection

[0321] Globally silylated trehalose compound F’ (1 mmol) was dissolved in anhydrous methanol under a nitrogen atmosphere at −15 °C. A solution of potassium carbonate (K₂CO₃, 1.2 mmol) in methanol (10 V) was added slowly to the reaction mixture while maintaining the temperature. The mixture was stirred for 3-4 hours at 0 °C. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 30% ethyl acetate in heptane, visualized with a vanillin stain. The reaction was neutralized with acetic acid (10 mL). The product was extracted with n-heptane (500 mL × 2), and the combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄). After filtration, the solvent was removed under reduced pressure using a rotary evaporator. The crude residue was further purified by flash chromatography (EtOAc:Heptane, 1:4) to provide G’.General Procedure for diester formation using peptide coupling reagents

[0322] To a stirred mixture of 2,2’,3,3’,4,4’,6-heptakis-O-trimethylsilyl-α,α-D-trehalose G’ (1 mmol), aryl carboxylic acid H’ (2.2 mmol) and DMAP (1.5 mmol) in anhydrous DCM (10 mL) was added DCC (2 eq.; 2 mmol) or EDCI-MeI (3 mmol) at 0 °C for 30 min and then at room temperature overnight. The reaction mixture was diluted with water, extracted it with DCM and washed the organic layer with aq.1N NaOH solution. The combined organic layer was dried over MgSO4and reduced in vacuo. The crude mixture was subjected to chromatography using the Biotage® system with a 12 g silica column and a zero to 20% ethyl acetate in heptane gradient to provide I’.

[0323] To a stirred solution of I’ (1 mmol) in anhydrous CH2Cl2(20 mL) was added MsCl (2.5 eq.) and Et3N (2.5 eq.) at 0 °C. After stirring for 2 hrs, water (10 mL) was added. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4. After filtration and concentration, the mesylate G’-Ms was obtained quantitatively. The mesylate trehalose was combined with sodium azide (3.51 mmol) in DMF (7 V) was heated for 6 hrs at 65 °C. The hot reaction mixture was poured with vigorous stirring into ice-cooled water (10 mL) and neutralized with acetic acid. The residue was extracted with EtOAc and the product was purified using column chromatography on silica gel to provide azido trehalose J’.General procedure for triazole formation

[0324] A reaction mixture containing azido trehalose J’ (1 mmol), requisite alkyne compound K’ (1.2 mmol), CuSO4.5H2O (10 mol%) and sodium ascorbate (20 mol%) in dioxane (15 mL) / H2O (3 mL) was prepared stirred at room temperature for 24 hours than extracted with ethyl acetate. After that, the organic layer was washed with 1 M HCl (3 x 50 mL), then with 1 M NH4OH (3 x 50 mL), and then with water. The organic layer was dried over MgSO4and reduced in vacuo. The crude was purified by flash chromatography with DCM and methanol (8:2) to provide L’.

[0325] The silyl intermediate L’ (1 mmol) was dissolved in equal amount of methylene chloride and methanol (5 V) and treated with Dowex® 50WX8 resin (2 mg / mmol) with magnetic stirring. Upon consumption of the starting material as determined by TLC (20% methanol in methylene chloride and charring with vanillin stain) the reaction was filtered, concentrated and chromatographed on a silica column eluting with a 40% to 80% methylene chloride to methanol gradient to provided desired product M’.Preparation and Characterization of Example Intermediates: ((2R,3R,4S,5R,6R)-6-(((2R,3R,4S,5R,6R)-6-(Hydroxymethyl)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-bis(pentyloxy)benzoate:

[0326] To a stirred mixture of 2,2’,3,3’,4,4’,6-Heptakis-O-trimethylsilyl-α,α-D-trehalose (1 eq.; 1 mmol), aryl carboxylic acid (1.2 eq.; 2.2 mmol) and DMAP (1.5 eq.; 1.5 mmol) in anhydrous DCM (10 mL) was added DCC (2 eq.; 2 mmol) or EDCI-MeI (3 eq.; 3 mmol) at 0 °C for 30 min and then at room temperature overnight. The reaction mixture was diluted with water, extracted it with DCM and washed the organic layer with aq.1N NaOH solution. The combined organic layer was dried over MgSO4and reduced in vacuo. The crude mixture was subjected to chromatography using the Biotage®system with a 12 g silica column and a zero to 20% ethyl acetate in heptane gradient. The product was obtained as white powder (53%).1H NMR (400 MHz, CHLOROFORM- d) d 7.18 (d, J = 2.32 Hz, 2H), 6.66 (s, 1H), 4.92 - 4.97 (m, 2H), 4.57 - 4.63 (m, 1H), 4.24 - 4.31 (m, 1H), 4.07 - 4.14 (m, 1H), 3.82 - 4.02 (m, 8H), 3.60 - 3.72 (m, 3H), 3.49 (d, J = 9.17 Hz, 3H), 1.76 - 1.85 (m, 4H), 1.36 - 1.48 (m, 8H), 0.91 - 0.98 (m, 6H), 0.12 - 0.20 (m, 54H);13C NMR (101 MHz, CHLOROFORM-d) d 166.38; 160.09; 131.77; 107.83; 106.53; 94.91; 94.68; 94.42; 73.68; 73.54; 73.37; 71.89; 71.84; 71.53; 70.93; 70.78; 68.31; 63.60; 63.65; 61.91; 28.84; 28.14; 22.42; 14.00; 1.09; 0.90; 0.25; 0.23; 0.16; HRMS (ESI+, m / z) calcd for (C47H94O14Si6+ NH4)+requires 1068.5597, found: 1069.5583. ((2R,3R,4S,5R,6R)-6-(((2R,3R,4S,5R,6R)-6-(Hydroxymethyl)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-di-tert-butyl-2- hydroxybenzoate:

[0327] To a stirred solution of protected trehalose in anhydrous DCM (10 mL / g) and pyridine (6 eq. for each OH) were added triflic anhydride (2.5 eq. for each OH) at −5 °C dropwise. The reaction mixture was allowed to warm gradually at room temperature stirred for 30 min. After this the mixture was diluted with DCM and washed with cold 1 M HCl, aq. NaHCO3and then with water. The organic layer was evaporated under reduced pressure and used it for next step without any purification. The mixture of potassium salt of an aryl acid (1.2 eq.) [synthesis (1.3 mmol of acid and 0.75 mL of KOTMS were dissolved in THF and stirred for 10 min and then solvent was removed under reduced pressure and used without purification)] triflate (0.5 eq. ) and 18-crown-6 (0.5 eq. ) were heated in toluene at 70 °C for 12 hrs. After this reaction mixture was diluted with DCM and washed the organic layer with water and then with 1 N aq. NaOH solution. The organic layer was dried over MgSO4and reduced in vacuo. The crude mixture was subjected to chromatography using the Biotage®system with a 12 g silica column and a zero to 20% ethyl acetate in heptane gradient. The product was obtained as white powder (48%).1H NMR (400 MHz, CHLOROFORM-d) d 11.04 (s, 1H), 7.59 (d, J2.32 Hz, 1H), 7.37 (d, J = 2.32 Hz, 1H), 4.77 (dd, J = 3.00, 15.22 Hz, 2H), 4.49 - 4.61 (m, 1H), 4.03 - 4.10 (m, 1H), 3.90 - 3.98 (m, 1H), 3.63 - 3.84 (m, 3H), 3.51 (d, J = 9.54 Hz, 3H), 3.26 (d, J = 9.17 Hz, 3H), 1.26 (s, 9H), 1.15 (s, 9H), -0.11 - 0.09 (m, 54H);13C NMR (101 MHz, CHLOROFORM-d) d 170.83, 159.10, 140.30, 137.33, 130.55, 123.51, 111.34, 94.75, 73.41, 72.82, 71.88, 71.34, 71.06, 63.15, 61.62, 35.14, 34.23, 31.87, 31.31, 29.37, 29.01, 22.69, 14.11, 1.06, 0.85, 0.021, 0.01; HRMS (ESI+, m / z) calcd for (C45H90O13Si6+ H)+requires 1007.507, found: 1007.512. ((2R,3R,4S,5R,6R)-6-(((2R,3R,4S,5R,6R)-6-(((Methylsulfonyl)oxy)methyl)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-di-tert-butyl-2- hydroxybenzoate

[0328] To a stirred solution of 3,5-di-tert-butyl-2-hydroxybenzoate protected trehalose (1 mmol) in anhydrous CH2Cl2(20 mL) was added MsCl (2.5 eq.) and Et3N (2.5 eq.) at 0 °C. After stirring for 2 hrs, water (10 mL) was added. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4. After filtration and concentration, the mesylate was obtained quantitatively as a white solid. The compound was used for next step without further purification.1H NMR (400 MHz, CHLOROFORM-d) d 11.21 (s, 1H), 7.76 (d, J = 2.45 Hz, 1H), 7.55 (d, J = 2.45 Hz, 1H), 4.93 (dd, J = 3.00, 15.83 Hz, 2H), 4.70 - 4.77 (m, 1H), 4.30 - 4.33 (m, 1H), 4.21 - 4.25 (m, 1H), 3.89 - 4.10 (m, 4H), 3.65 - 3.70 (m, 2H), 3.45 (d, J = 8.93 Hz, 3H), 3.01 - 3.03 (m, 3H), 1.43 (s, 9H), 1.32 (s, 9H), 0.07 - 0.28 (m, 54H);13C NMR (101 MHz, CHLOROFORM-d) d 170.79, 159.10, 140.33, 137.37, 130.60, 123.46, 111.29, 94.90, 94.64, 73.40, 72.72, 72.57, 71.77, 71.42, 70.80, 69.13, 37.45, 36.65, 35.15, 34.23, 31.32, 29.36, 1.04, 0.07; HRMS (ESI+, m / z) calcd for (C46H92O15SSi6+ H)+requires 1085.4845, found: 1085.4841. ((2R,3R,4S,5R,6R)-6-(((2R,3R,4S,5R,6R)-6-(((Methylsulfonyl)oxy)methyl)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-bis(pentyloxy)benzoate

[0329] To a stirred solution of 3,5-bis(pentyloxy)benzoate protected trehalose (1 mmol) inanhydrous CH2Cl2(20 mL) was added MsCl (2.5 eq.) and Et3N (2.5 eq.) at 0 °C. After stirring for 2 hrs, water (10 mL) was added. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4. After filtration and concentration, the mesylate was obtained quantitatively as a white solid. The compound was used for next step without further purification.1H NMR (400 MHz, CHLOROFORM-d) d 6.98 - 7.13 (m, 2H), 6.49 - 6.62 (m, 1H), 5.05 - 5.14 (m, 1H), 4.78 - 4.91 (m, 1H), 4.52 - 4.56 (m, 1H), 4.21 - 4.26 (m, 1H), 3.85 - 3.95 (m, 7H), 3.53 - 3.62 (m, 2H), 3.35 - 3.44 (m, 3H), 3.02 - 3.08 (m, 2H), 2.93 - 2.96 (m, 3H), 1.73 (m, 4H), 1.32 - 1.39 (m, 8H), 0.86 (d, J = 3.30 Hz, 6H), -0.12 - 0.17 (m, 54H). HRMS (ESI+, m / z) calcd for (C48H96O16SSi6+ H)+requires 1129.5107, found: 1129.5110. ((2R,3S,4S,5R,6R)-6-(((2R,3R,4S,5S,6R)-6-(Azidomethyl)-3,4,5-trihydroxytetrahydro-2H- pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3,5- bis(pentyloxy)benzoate

[0330] The stirred mixture of mesylate trehalose (0.85 g, 0.76 mmol) and sodium azide (0.25 g, 3.51 mmol) in DMF (7 mL) was heated for 6 hrs at 65 °C. The hot reaction mixture was poured with vigorous stirring into ice-cooled water (10 mL). [Note: the crude product contains mixture of deprotected TMS trehalose products with azide group]. The crude mixture product was precipitated out which was filtered, dried, and used without further purification for silyl deprotection. The product obtained is white powder (0.35; 72%). HRMS (ESI+, m / z) calcd for (C29H45O13N3+ NH4)+requires 661.3291, found: 661.3279. ((2R,3S,4S,5R,6R)-6-(((2R,3R,4S,5S,6R)-6-(Azidomethyl)-3,4,5-trihydroxytetrahydro-2H- pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3,5-di-tert-butyl-2- hydroxybenzoate

[0331] The stirred mixture of mesylate trehalose (0.85 g, 0.76 mmol) and sodium azide (0.25 g, 3.51 mmol) in DMF (7 mL) was heated for 6 hrs at 65 °C. The hot reaction mixture was poured with vigorous stirring into ice-cooled water (10 mL). [Note: the crude product contains mixture of deprotected TMS trehalose products with azide group]. The crude mixture product was precipitated out which was filtered, dried, and used without further purification for silyl deprotection. The product obtained is white powder (0.32; 74%). HRMS (ESI+, m / z) calcd for (C27H41O12N3+ NH4)+requires 617.3028, found: 617.3021. Preparation and Characterization of Example Unsymmetrical Trehalose Compounds

[0332] General Procedure for Click Chemistry: The reaction mixture containing azido trehalose (1 mmol), lipidated propargyl compound (1.2 mmol), CuSO4.5H2O (52 mg, 10 mol%), sodium ascorbate (0.79 mg, 20 mol%) in dioxane (15 mL) / H2O (3 mL) was stirred at room temperature for 24 hours than extracted with ethyl acetate. After that, the organic layer was washed with 1 M HCl (3 x 50 mL), then with 1 M NH4OH (3 x 50 mL), and then with water. The organic layer was dried over MgSO4and reduced in vacuo. The crude was purified by flash chromatography with DCM and methanol (8:2).

[0333] The following compounds were prepared and characterized according to the general procedure above. ((2R,3S,4S,5R,6R)-6-(((2R,3R,4S,5S,6R)-6-((4-((3-(hexyloxy)-2-((hexyloxy)methyl)-2- methylpropoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2- yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3,5-di-tert-butyl-2-hydroxybenzoate (UM-1187):

[0334] The targeted compound was prepared according to the general procedure for click chemistry (white powder; 54 mg; 87%).1H NMR (400 MHz, METHANOL-d4) d 8.05 - 8.46 (m, 1H), 7.81 (d, J = 2.08 Hz, 1H), 7.59 (d, J = 2.08 Hz, 1H), 5.16 (d, J = 3.18 Hz, 1H), 4.77 (br. s., 2H), 4.67 (d, J = 11.37 Hz, 4H), 4.41 (d, J = 6.24 Hz, 1H), 4.20 (d, J = 9.17 Hz, 2H), 3.78 - 3.91 (m, 2H), 2.96 - 3.61 (m, 19H), 1.50 (d, J = 6.72 Hz, 4H), 1.45 (s, 9H), 1.27 - 1.36 (m, 22H), 0.87 - 0.95 (m, 9H);13C NMR (101 MHz, METHANOL-d4) d 172.50; 160.23; 141.97; 138.37; 131.66; 125.09; 112.73; 95.19; 74.69; 74.45; 73.28; 73.20; 72.33; 71.81; 71.50; 65.71; 42.10; 36.22; 35.33; 32.99; 32.08; 30.82; 30.07; 27.19; 23.88; 18.23; HRMS (ESI+, m / z) calcd for (C47H79O15N3+ NH4)+requires 943.5849, found: 943.6001. ((2R,3S,4S,5R,6R)-6-(((2R,3R,4S,5S,6R)-6-((4-((3-(heptyloxy)-2-((heptyloxy)methyl)-2- methylpropoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2- yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3,5-di-tert-butyl-2-hydroxybenzoate (UM-1188):

[0335] The targeted compound was prepared according to the general procedure for click chemistry (white powder; 55 mg; 85%).1H NMR (400 MHz, METHANOL-d4) d 7.93 - 8.02 (m, 1H), 7.82 (d, J = 2.20 Hz, 1H), 7.60 (d, J = 2.08 Hz, 1H), 5.17 (d, J = 3.55 Hz, 1H), 4.75 - 4.86 (m, 2H), 4.67 (s, 1H), 4.57 (br. s., 3H), 4.36 - 4.44 (m, 1H), 4.22 (br. s., 2H), 3.80 - 3.88 (m, 2H),3.40 - 3.50 (m, 3H), 3.34 (br. s., 12H), 3.21 (br. s., 3H), 3.16 (s, 1H), 1.50 (d, J = 3.91 Hz, 4H), 1.45 (s, 9H), 1.27 - 1.37 (m, 26H), 0.84 - 0.94 (m, 9H);13C NMR (101 MHz, METHANOL-d4) d 175.52; 160.24; 141.99; 138.39; 131.68; 125.11; 112.74; 95.20; 74.70; 74.45; 73.25; 72.66; 72.35; 71.94; 71.52; 65.71; 42.08; 36.22; 35.35; 33.21; 32.08; 30.86; 30.07; 27.48; 23.86; 18.22; 14.62; HRMS (ESI+, m / z) calcd for (C49H83O15N3+ NH4)+requires 971.6162, found: 971.6442. ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((4-((2-methyl-3- (octyloxy)-2-((octyloxy)methyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H- pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-di-tert-butyl-2-hydroxybenzoate (UM- 1189):

[0336] The targeted compound was prepared according to the general procedure for click chemistry (white powder; 49 mg; 83%).1H NMR (400 MHz, METHANOL-d4) d 7.94 (s, 1H), 7.80 (d, J = 2.32 Hz, 1H), 7.59 (d, J = 2.32 Hz, 1H), 5.16 (d, J = 3.55 Hz, 1H), 4.78 (d, J = 3.55 Hz, 2H), 4.64 - 4.70 (m, 1H), 4.55 (s, 3H), 4.35 - 4.42 (m, 1H), 4.15 - 4.26 (m, 2H), 3.83 (d, J = 5.14 Hz, 2H), 3.36 - 3.48 (m, 4H), 3.31 - 3.35 (m, 12H), 3.19 - 3.22 (m, 3H), 3.15 (s, 1H), 1.42 - 1.52 (m, 13H), 1.24 - 1.37 (m, 29H), 0.85 - 0.94 (m, 9H);13C NMR (400 MHz, METHANOL-d4) d 170.96; 158.70; 140.44; 136.83; 130.12; 123.56; 111.19; 93.63; 73.14; 72.89; 71.69; 71.09; 70.79; 70.41; 69.69; 64.17; 64.00; 40.52; 34.68; 33.80; 31.64; 30.52; 29.09; 28.52; 25.97; 22.35; 16.68; 13.08; HRMS (ESI+, m / z) calcd for (C51H87O15N3 + NH4)+requires 999.6475, found: 999.6483. ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((4-((2-methyl-3- (nonyloxy)-2-((nonyloxy)methyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H- pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-di-tert-butyl-2-hydroxybenzoate (UM- 1190):

[0337] The targeted compound was prepared according to the general procedure for click chemistry (white powder; 51 mg; 82%).1H NMR (400 MHz, METHANOL-d4) d 7.97 (s, 1H), 7.82 (d, J = 2.45 Hz, 1H), 7.60 (d, J = 2.32 Hz, 1H), 5.17 (d, J = 3.67 Hz, 1H), 4.79 (d, J = 3.55 Hz, 2H), 4.67 (s, 1H), 4.57 (s, 3H), 4.37 - 4.44 (m, 1H), 4.17 - 4.28 (m, 2H), 3.80 - 3.88 (m, 2H), 3.40 - 3.50 (m, 3H), 3.38 (s, 3H), 3.32 - 3.36 (m, 11H), 3.22 (br. s., 3H), 3.16 (s, 1H), 1.48 - 1.53 (m, 4H), 1.45 (s, 9H), 1.29 - 1.36 (m, 32H), 0.86 - 0.96 (m, 9H) ;13C NMR (101 MHz, METHANOL-d4) d 172.29; 160.03; 141.76; 138.16; 131.44; 124.90; 112.53; 94.98; 74.48; 74.21; 70.03; 72.43; 72.13; 71.73; 71.30; 65.50; 52.35; 41.86; 36.01; 35.13; 33.02; 31.87; 30.70; 30.63; 30.53; 30.38; 29.87; 27.31; 23.69; 18.03; 14.42 ; HRMS (ESI+, m / z) calcd for (C53H91O15N3+ NH4)+requires 1027.6788, found: 1027.6781. ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((4-((2-methyl-3- (nonyloxy)-2-((nonyloxy)methyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H- pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-bis(pentyloxy)benzoate (UM-1183):

[0338] The targeted compound was prepared according to the general procedure for click chemistry (white powder; 77 mg; 79%).1H NMR (400 MHz, DMSO-d6) d 7.94 (s, 1H), 7.01 (d, J = 2.08 Hz, 2H), 6.73 (br. s., 1H), 4.87 (d, J = 3.30 Hz, 7H), 4.53 - 4.69 (m, 2H), 4.33 - 4.50 (m,3H), 3.86 - 4.26 (m, 8H), 3.49 - 3.70 (m, 2H), 2.95 - 3.30 (m, 12H), 2.63 - 2.78 (m, 2H), 1.64 - 1.76 (m, 4H), 1.14 - 1.48 (m, 24H), 0.72 - 0.97 (m, 15H);13C NMR (101 MHz, METHANOL-d4) d 160.03; 144.14; 131.84; 107.37; 93.67; 72.86; 71.60; 70.78; 67.96; 31.45; 29.20; 28.96; 28.91; 28.49; 27.08; 25.85; 25.37; 22.30; 22.09; 17.51; 14.13; HRMS (ESI+, m / z) calcd for (C49H83O16N3+ NH4)+requires 987.6112, found: 987.6098. ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((4-((2-methyl-3- (nonyloxy)-2-((nonyloxy)methyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H- pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-bis(pentyloxy)benzoate (UM-1184):

[0339] The targeted compound was prepared according to the general procedure for click chemistry (white powder; 73 mg; 75%).1H NMR (400 MHz, DMSO-d6) d 7.94 (s, 1H), 7.03 (d, J = 2.20 Hz, 2H), 6.74 (s, 1H), 5.33 - 5.40 (m, 1H), 5.21 - 5.27 (m, 1H), 5.04 (t, J = 5.38 Hz, 2H), 4.85 - 4.98 (m, 3H), 4.56 - 4.68 (m, 2H), 4.45 (s, 3H), 3.97 (dd, J = 4.28, 6.11 Hz, 8H), 3.52 - 3.67 (m, 2H), 3.06 - 3.29 (m, 11H), 2.95 - 3.03 (m, 1H), 2.70 - 2.78 (m, 2H), 1.66 - 1.75 (m, 4H), 1.17 - 1.57 (m, 28H), 0.76 - 0.94 (m, 15H);13C NMR (101 MHz, METHANOL-d4) d 160.33; 131.70; 107.36; 105.84; 96.85; 73.21; 73.06; 73.01; 72.72; 71.71; 70.84; 70.58; 70.30; 70.14; 67.95; 63.90; 63.79; 40.51; 39.37; 31.48; 29.0; 28.87; 28.01; 27.17; 25.85; 25.45; 22.16; 19.08; 16.64; 13.0; 12.93; HRMS (ESI+, m / z) calcd for (C51H87O16N3+ NH4)+requires 1015.6425, found: 1015.6437. ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((4-((2-methyl-3- (nonyloxy)-2-((nonyloxy)methyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H- pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-bis(pentyloxy)benzoate (UM-1185):

[0340] The targeted compound was prepared according to the general procedure for click chemistry (white powder; 81 mg; 78%).1H NMR (400 MHz, METHANOL-d4) d 7.96 (s, 1H), 7.15 (d, J = 2.08 Hz, 2H), 6.70 (s, 1H), 5.12 (d, J = 3.67 Hz, 1H), 4.73 - 4.85 (m, 2H), 4.55 (s, 3H), 4.36 - 4.42 (m, 1H), 4.20 - 4.29 (m, 1H), 4.09 - 4.19 (m, 1H), 4.00 (t, J = 6.36 Hz, 4H), 3.81 (d, J = 13.20 Hz, 2H), 3.40 - 3.50 (m, 3H), 3.33 (s, 14H), 3.09 - 3.23 (m, 4H), 1.75 - 1.86 (m, 4H), 1.20 - 1.58 (m, 32H), 0.85 - 1.02 (m, 15H);13C NMR (101 MHz, METHANOL-d4) d 162.09; 133.48; 109.12; 95.63; 95.27; 74.86; 74.69; 73.41; 72.89; 71.95; 71.90; 69.97; 65.60; 42.28; 33.43; 31.08; 30.95; 30.87; 30.46; 29.80; 27.75; 24.13; 23.95; 14.86; 14.81; HRMS (ESI+, m / z) calcd for (C53H91O16N3+ NH4)+requires 1026.3160, found: 1026.3169. ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((4-((2-methyl-3- (nonyloxy)-2-((nonyloxy)methyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H- pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,5-bis(pentyloxy)benzoate (UM-1186):

[0341] The targeted compound was prepared according to the general procedure for click chemistry (white powder; 76 mg; 80%).1H NMR (400 MHz, METHANOL-d4) d 7.87 - 8.30 (m, 1H), 7.15 (d, J = 1.96 Hz, 2H), 6.70 (s, 1H), 5.11 (d, J = 3.30 Hz, 1H), 4.72 - 4.85 (m, 2H), 4.58 (d, J = 10.76 Hz, 3H), 4.34 - 4.42 (m, 1H), 4.21 - 4.30 (m, 1H), 4.10 - 4.18 (m, 1H), 4.00 (t, J =6.36 Hz, 4H), 3.75 - 3.88 (m, 2H), 3.44 (d, J = 9.41 Hz, 3H), 3.29 - 3.39 (m, 14H), 3.04 - 3.25 (m, 4H), 1.70 - 1.89 (m, 4H), 1.22 - 1.55 (m, 35H), 0.82 - 1.02 (m, 15H);13C NMR (101 MHz, METHANOL-d4) d 162.09; 136.29; 133.48; 109.11; 107.66; 104.26; 96.23; 95.63; 74.67; 73.59; 72.89; 71.90; 69.71; 42.30; 33.47; 31.16; 31.08; 31.00; 30.85; 30.47; 29.80; 29.75; 27.75; 24.14; 23.95; 18.46; 18.46; 14.86; 14.81; HRMS (ESI+, m / z) calcd for (C55H95O16N3+ NH4)+requires 1071.7051, found: 1071.7043. 2. Example Syntheses − Part 2

[0342] Below is an example method for preparing example compound UM-5272.

[0343] As shown above, TMS-protected trehalose compounds of formula 1 may be subjected to esterification with carboxylic acid 6 in the presence of EDC-MeI and DMAP will give key intermediate 7. A second sequential esterification occurred with respective aryl carboxylic acid 8 to produce intermediate 9. Intermediate 9 was deprotected with acidic Dowex®resin in methanol to produce diester representative compound UM-5272.

[0344] UM-5272.1H NMR (400 MHz, MeOD) δ 7.14 (s, 2H), 6.67 (s, 1H), 5.12 (s, 2H), 4.58 (d, J = 11.0 Hz, 1H), 4.35 (s, 2H), 4.20 (d, J = 41.0 Hz, 2H), 3.98 (s, 5H), 3.81 (d, J = 5.6 Hz, 2H), 3.57 – 3.34 (m, 4H), 2.35 (s, 1H), 1.78 (s, 4H), 1.52 (d, J = 40.8 Hz, 8H), 1.41 – 1.16 (m, 36H), 0.87 (d, J = 25.6 Hz, 12H); [m / z; 958.6587; M+NH3].

[0345] Alternatively, intermediate 9 may be formed using SN2 reaction of potassiumbenzoates and triflate activated trehalose, as described below.

[0346] To a stirred solution of 7 in anhydrous DCM (10 mL / g) and pyridine (6 eq. for each OH) were added triflic anhydride (2.5 eq. for each OH) at −5 °C dropwise. The reaction mixture was allowed to warm gradually at room temperature stirred for 30 min. After this the mixture was diluted with DCM and washed with cold 1 M HCl, aq. NaHCO3and then with water. The organic layer was evaporated under reduced pressure and used it for next step without any purification. The mixture of potassium salt of an aryl acid 8” (1.2 eq.) [synthesis (1.3 mmol of acid and 0.75 mL of KOTMS were dissolved in THF and stirred for 10 min and then solvent was removed under reduced pressure and used without purification)] triflate 7-Tf (0.5 eq.) and 18-crown-6 (0.5 eq. ) were heated in toluene at 70 °C for 12 hrs. After this reaction mixture was diluted with DCM and washed the organic layer with water and then with 1 N aq. NaOH solution. The organic layer was dried over MgSO4and reduced in vacuo. The crude mixture was subjected to chromatography using the Biotage®system with a 12 g silica column and a zero to 20% ethyl acetate in heptane gradient to provide 9.

[0347] The following compounds were prepared similar to UM-5272 and then characterized. ((2R,3R,4S,5R,6R)-6-(((2R,3R,4S,5R,6R)-6-(((2-heptylnonanoyl)oxy)methyl)-3,4,5- tris((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3,4,5-tris((trimethylsilyl)oxy)tetrahydro- 2H-pyran-2-yl)methyl 3,5-di-tert-butyl-2-hydroxybenzoate (UM-5240):

[0348] 1H NMR (400 MHz, MeOD) δ 7.80 (d, J = 2.5 Hz, 1H), 7.56 (d, J = 2.5 Hz, 1H), 5.15 (dd, J = 8.8, 3.8 Hz, 2H), 4.64 (dd, J = 11.8, 2.2 Hz, 1H), 4.40 (dd, J = 11.8, 6.2 Hz, 1H), 4.34 – 4.24 (m, 2H), 4.18 (ddd, J = 10.1, 6.2, 2.2 Hz, 1H), 4.04 (ddd, J = 10.1, 5.5, 2.6 Hz, 1H), 3.83 (ddd, J = 13.9, 9.7, 8.8 Hz, 3H), 3.50 (ddd, J = 16.4, 9.7, 3.7 Hz, 2H), 3.41 (dd, J = 10.1, 8.8 Hz, 1H), 2.36 (tq, J = 9.4, 5.1 Hz, 1H), 1.65 – 1.52 (m, 2H), 1.46 (d, J = 2.7 Hz, 2H), 1.26 (d, J = 5.7 Hz, 20H), 0.87 – 0.81 (m, 6H).13C NMR (101 MHz, MeOD) δ 176.77, 171.01, 158.68, 140.43, 136.80, 130.08, 123.60, 111.20, 93.47, 93.43, 73.30, 73.18, 71.88, 70.86, 70.80, 70.05, 69.93, 64.23, 63.04, 45.80, 34.66, 33.77, 32.28, 32.23, 31.55, 30.49, 29.20, 28.87, 28.83, 28.49, 27.11, 22.26, 22.25, 13.03. Expected [M+NH3]+= 829.52. Observed [M+NH3]+=830.5155. ((2R,3S,4S,5R,6R)-6-(((2R,3R,4S,5S,6R)-6-(((2-Heptylnonanoyl)oxy)methyl)-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3,4,5-tris(octyloxy)benzoate (UM-5249):

[0349] 1H NMR (400 MHz, MeOD) δ 7.29 (s, 2H), 5.14 (dd, J = 14.4, 3.7 Hz, 2H), 4.59 (dd, J = 11.7, 2.1 Hz, 1H), 4.37 – 4.24 (m, 3H), 4.17 (ddd, J = 9.2, 6.8, 2.1 Hz, 1H), 4.01 (td, J = 6.3, 4.1 Hz, 8H), 3.83 (td, J = 9.3, 5.0 Hz, 2H), 3.55 – 3.42 (m, 2H), 3.37 (dd, J = 10.3, 9.1 Hz, 1H), 2.35 (tt, J = 9.0, 5.2 Hz, 1H), 1.87 – 1.68 (m, 6H), 1.59 – 1.15 (m, 54H), 0.95 – 0.88 (m, 9H), 0.83 (td, J = 6.9, 2.7 Hz, 6H).13C NMR (101 MHz, MeOD) δ 178.02, 167.66, 154.13, 143.34, 126.19,108.88, 94.88, 94.78, 74.66, 74.62, 74.48, 73.34, 73.25, 72.35, 72.21, 71.46, 70.11, 65.69, 64.36, 47.19, 33.72, 33.68, 33.12, 33.06, 32.98, 32.96, 31.45, 30.68, 30.64, 30.59, 30.53, 30.31, 30.27, 28.55, 28.53, 27.43, 27.29, 23.81, 23.78, 23.68, 14.51, 14.49. Expected [M+NH3]+=1086.7663. Observed [M+NH3]+=1086.7724. ((2R,3S,4S,5R,6R)-6-(((2R,3R,4S,5S,6R)-6-(((2-heptylnonanoyl)oxy)methyl)-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3,5- bis(pentyloxy)benzoate (UM-5270):

[0350] 1H NMR (400 MHz, MeOD) δ 7.14 (d, J = 2.2 Hz, 2H), 6.67 (d, J = 2.1 Hz, 1H), 5.16 - 5.06 (m, 2H), 4.58 (d, J = 9.9 Hz, 1H), 4.35 (dd, J = 11.7, 5.5 Hz, 2H), 4.25 (dd, J = 11.9, 5.6 Hz, 1H), 4.18 – 4.12 (m, 1H), 4.06 – 3.96 (m, 4H), 3.81 (td, J = 9.2, 6.0 Hz, 2H), 3.53 – 3.37 (m, 3H), 2.40 – 2.27 (m, 1H), 1.83 – 1.73 (m, 4H), 1.57 (m, 2H), 1.51 – 1.34 (m, 9H), 1.24 (s, 20H), 0.94 (dd, J = 19.4, 12.4 Hz, 6H), 0.83 (dd, J = 6.9, 3.0 Hz, 6H); [m / z; 874.55; M+NH3]. 3. Example Syntheses – Part 3 Synthesis of Intermediate Fragment 1Synthesis of 5

[0351] In 150 mL of acetone, 2-(hydroxymethyl)-2-methylpropane-1,3-diol 4 (20 g, 0.17 mol) and p-TSA (10 mg, 58 μmol) were added at room temperature under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 48 h. Completion of the reaction was monitored by TLC (30% EtOAc in Heptane; visualized with vanillin stain). The organic solvent was evaporated under reduced pressure, and the crude residue was quenched with saturated aqueous NaHCO₃. The product was extracted with EtOAc, and the organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure using a rotary evaporator, affording the crude product 5, which was purified by medium pressure chromatography on silica gel to give a colorless oil (26.8 g, 98% yield). Synthesis of 6

[0352] In a 500 mL round-bottom flask, 5 (8 g, 93.6 mmol) was dissolved in anhydrous THF (150 mL) under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and NaH (60% dispersion in mineral oil, 4.94 g, 206 mmol, 2.2 equiv.) was added slowly with stirring. After 10 min of stirring at 0°C, propargyl bromide (80% in toluene, 13.4 g, 112 mmol, 1.2 equiv.) and TBAI (346 mg, 936 μmol) were added dropwise. The reaction was allowed to warm to room temperature and stirred for 16 h. Completion of the reaction was monitored by TLC (10% EtOAc in heptane, visualized with vanillin stain). The reaction was carefully quenched by the slow addition of ice- cold water at 0 °C. The product was extracted with EtOAc (100 mL × 3), and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure using a rotary evaporator. The crude residue was purified by medium pressure chromatography on silica gel toafford the desired product 6 as a colorless oil (12.6 g, 67% yield). Synthesis of 7

[0353] In a 250 mL round-bottom flask, 6 (8.2 g, 41 mmol) was dissolved in 80 mL of anhydrous methanol under a nitrogen atmosphere at room temperature. To the reaction mixture, 20% w / w Dowex®50WX8 resin was added in one portion, and the resulting mixture was stirred at room temperature for 16 hours. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 70% ethyl acetate in heptane, visualized with a vanillin stain. After completion, the reaction mixture was filtered through a pad of Celite®, and the filtrate was dried over anhydrous sodium sulfate (Na₂SO₄). The solvent was removed under reduced pressure using a rotary evaporator, affording the crude product. The crude residue was purified by medium- pressure chromatography on silica gel, yielding 7 as a colorless oil (6.2 g, 95% yield). Synthesis of 1

[0354] In a 500 mL round-bottom flask, 2-methyl-2-((prop-2-yn-1-yloxy)methyl)propane-1, 3-diol 7 (7 g, 44 mmol) was dissolved in 100 mL of anhydrous tetrahydrofuran (THF) under a nitrogen atmosphere. The reaction mixture was cooled to 0°C, and sodium hydride (NaH, 60% dispersion in mineral oil, 2.7 g, 0.11 mol, 2.5 equiv.) was added. The mixture was stirred for 10 minutes at 0 °C, followed by the addition of 1-bromo octane (26 g, 0.13 mol, 3 equiv.) and tetrabutylammonium iodide (TBAI, 6.5 g, 18 mmol, 0.4 equiv.). The reaction mixture was allowed to warm to room temperature and stirred for 48 hours. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 15% ethyl acetate in heptane, visualized with a vanillinstain. The reaction was carefully quenched by the slow addition of ice-cold water at 0°C. The product was extracted with ethyl acetate (100 mL × 2), and the combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄). After filtration, the solvent was removed under reduced pressure using a rotary evaporator. The crude residue was purified by medium-pressure chromatography on silica gel, yielding the desired product as a colorless oil (14.4 g, 85% yield). Note: TLC analysis indicated that 85% of the starting material was converted into the desired dialkylated product (1), while the remaining 15% consisted of the mono-alkylated intermediate (1a), which can be further converted into the di-alkylated product in a subsequent reaction. Synthesis of Example Unsymmetrical Trehalose Compound UM-1189:Synthesis of 989

[0355] To a stirred mixture of trehalose dihydrate 8 (25 g, 73 mmol) in 150 mL of anhydrous DMF, trimethylsilyl (E)-N-(trimethylsilyl)acetimidate (160 mL, 0.64 mol) was added dropwise. After the addition, tetrabutylammonium fluoride (TBAF, 1.0 M in THF, 4 mL, 4 mmol) was added slowly to the reaction mixture, as the reaction was exothermic. The mixture was stirred vigorously for 5 hours at room temperature. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 3% ethyl acetate in heptane, visualized with a vanillin stain. The reaction was carefully quenched with 2-propanol (10 mL). The product was extracted with m- heptane (300 mL × 2), and the combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄). After filtration, the solvent was removed under reduced pressure using a rotary evaporator. The crude product 9 residue (isolated yield) was directly carried to the next step without further purification. Note: The crude residue was either directly carried to the next step or stored at −20 °C if not processed immediately. Synthesis of 10

[0356] In a 2 L round-bottom flask, 9 (25 g, 41 mmol) was dissolved in 1000 mL of anhydrous methanol under a nitrogen atmosphere at −15 °C. A solution of potassium carbonate (K₂CO₃, 9.1 g, 66 mmol) in methanol (1000 mL) was added slowly to the reaction mixture while maintaining the temperature. The mixture was stirred for 3-4 hours at 0 °C. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 30% ethyl acetate in heptane, visualizedwith a vanillin stain. The reaction was neutralized with acetic acid (10 mL). The product was extracted with n-heptane (500 mL × 2), and the combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄). After filtration, the solvent was removed under reduced pressure using a rotary evaporator. The crude residue was further purified by flash chromatography (EtOAc:Heptane, 1:4); to yield 10 as white solid; 14 g, 66% yield. Note: TLC initially showed clean conversion. However, upon neutralization with acetic acid and evaporation of methanol, TLC analysis indicated the formation of trehalose due to the deprotection of trimethylsilyl (TMS) groups, leading to a loss of yield. Synthesis of 2

[0357] In a 250 mL round-bottom flask, 10 (8.0 g, 10 mmol) was dissolved in 150 mL of anhydrous toluene under a nitrogen atmosphere at 0 °C. To the reaction mixture, triphenylphosphine (TPP, 22 g, 83 mmol, 8 equiv.) and diisopropyl azodicarboxylate (DIAD, 17 g, 83 mmol, 8 equiv.) were added in one portion, and the mixture was stirred at 0 °C for 30 minutes. After this, trimethylsilyl azide (9.5 g, 83 mmol, 8 equiv.) was added dropwise to the reaction mixture very carefully, as it is potentially hazardous. The reaction mixture was stirred vigorously at room temperature for 16 hours. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 3% ethyl acetate in heptane, visualized with potassium permanganate (KMnO₄) or vanillin stain (which showed a very dull spot). After completion, the reaction mixture was filtered through a pad of Celite®, and the filtrate was dried over anhydrous sodium sulfate (Na₂SO₄). The solvent was removed under reduced pressure using a rotary evaporator, affording the crude product. The crude residue was purified by medium-pressure chromatography on silica gel (compound eluted with 100% heptane-5% ethyl acetate), yielding a pale yellow solid 2 (7.7 g, 86% yield).Synthesis of 11a

[0358] In a 500 mL round-bottom flask, 2 (6 g, 7.0 mmol) and 1-(2-methyl-3-(octyloxy)-2- ((prop-2-yn-1-yloxy)methyl)propoxy)octane 1 (3.2 g, 8.4 mmol) were dissolved in 100 mL of 1,4- dioxane:water (10:2). At room temperature, sodium ascorbate (0.28 g, 1.4 mmol) was added, followed by CuSO₄·5H₂O (0.087 g, 0.35 mmol). The mixture was stirred at 55°C for 16 hours. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 30% ethyl acetate in heptane, visualized with a vanillin stain. After completion, the reaction solvent was removed under reduced pressure using a rotary evaporator. The product was extracted with ethyl acetate (100 mL × 2), and the combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄). After filtration, the solvent was removed under reduced pressure using a rotary evaporator. The crude residue was purified by medium-pressure chromatography on silica gel, yielding the desired product 11 as a colorless oil (5.3 g, 60% yield). Note: TLC analysis indicated the formation of two additional side products, 11a and 11b, along with the major product 11. Of the two side products, 11a is a useful compound for the next step.Synthesis of 11a

[0359] In a 250 mL round-bottom flask, 11 (6 g, 4.8 mmol) was dissolved in a mixture of anhydrous methanol (70 mL) and dichloromethane (DCM, 30 mL). To this reaction mixture, ammonium acetate (0.92 g, 12 mmol) was added in one portion slowly. The reaction mixture was stirred at room temperature for 48 hours. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 30% ethyl acetate in heptane, visualized with vanillin stain. After completion, the reaction solvent was removed under reduced pressure using a rotary evaporator, affording the crude product. The crude residue was purified by medium-pressure chromatography on silica gel, yielding 11a as an off white solid (3.7 g, 65% yield). Synthesis of 12

[0360] In a 100 mL round-bottom flask, 11a (3 g, 7.3 mmol) was dissolved in 30 mL of anhydrous dichloromethane (DCM) under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and pyridine (0.61 mL, 7.5 mmol) was added dropwise. After the addition, triflic anhydride (0.55 mL, 3.3 mmol) was added slowly, as the reaction is exothermic. The mixture was stirred vigorously at room temperature for 1-2 hours. Completion of the reaction was monitored by thin- layer chromatography (TLC) using 30% ethyl acetate in heptane, visualized with a vanillin stain. After completion, the reaction mixture was carefully poured into ice-cold water (50 mL). The product was extracted with DCM (20 mL × 2), and the combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄). After filtration, the solvent was removed under reduced pressure using a rotary evaporator to afford colorless oil (isolated yield). The crude product 12 was immediately used for the next step without further column purification.Synthesis of 3

[0361] In a 500 mL round-bottom flask, 3,5-di-tert-butyl-2-hydroxybenzoic acid 14 (8 g, 32 mmol) was dissolved in 200 mL of anhydrous THF under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and potassium trimethylsilanolate (KOTMS, 18 mL, 35 mmol) was added dropwise. Upon complete addition, a white precipitate was formed in the reaction mixture. The mixture was stirred at room temperature for 0.5–1 hour. The reaction mixture was filtered, and the solid was washed multiple times with n-heptane (3 X 50 mL). The collected off-white solid was dried under a high vacuum, yielding 3 as an off-white solid (isolated yield not specified). This off-white solid was used directly in the next step without further column purification. Synthesis of 13

[0362] In a 100 mL round-bottom flask, potassium 3,5-di-tert-butyl-2-hydroxybenzoate 3 (1.4 g, 4.8 mmol) and 18-crown-6 (0.5 g, 1.9 mmol) were dissolved in 20 mL of anhydrous toluene under a nitrogen atmosphere. The reaction mixture was stirred for 5 minutes at room temperature. Following this, 12 (5 g, 3.8 mmol) was dissolved in 20 mL of anhydrous toluene and added dropwise to the reaction mixture. The reaction mixture was stirred at 85 °C for 2-16 hours (reaction time dependent on scale). Completion of the reaction was monitored by thin-layer chromatography (TLC) using 30% ethyl acetate in heptane, visualized with a vanillin stain. After completion, the product was extracted with ethyl acetate (15 mL × 2), and the combined organic layers were driedover anhydrous sodium sulfate (Na₂SO₄). After filtration, the solvent was removed under reduced pressure using a rotary evaporator. The crude residue was purified by medium-pressure chromatography on silica gel, yielding the desired product 13 as an off-white solid (5.3 g, 98% yield). Synthesis of UM-1189

[0363] In a 100 mL round-bottom flask, 13 (3.7 g, 41 mmol) was dissolved in 40 mL of anhydrous methanol: DCM (1:1) under a nitrogen atmosphere at room temperature. To the reaction mixture, 20% w / w Dowex®50WX8 resin was added in one portion, and the resulting mixture was stirred at room temperature for 16 hours. Completion of the reaction was monitored by thin-layer chromatography (TLC) using 20% dichloromethane in methanol, visualized with a vanillin stain. After completion, the reaction mixture was filtered through a pad of Celite®, and the filtrate was dried over anhydrous sodium sulfate (Na₂SO₄). The solvent was removed under reduced pressure using a rotary evaporator, affording the crude product. The crude residue was purified by medium- pressure chromatography on silica gel, yielding the desired product UM-1189 as an off-white solid (3.52 g, 98% yield).

[0364] UM-1189.1H NMR (400 MHz, DMSO) δ 11.33 (s, 1H), 7.93 (d, J = 13.8 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 5.35 (s, 1H), 5.27 (t, J = 8.0 Hz, 1H), 5.02 (dd, J = 11.5, 4.4 Hz, 2H), 4.93 (dd, J = 7.2, 3.7 Hz, 3H), 4.71 – 4.56 (m, 2H), 4.51 – 4.38 (m, 4H), 4.29 (dt, J = 11.5, 5.9 Hz, 1H), 4.18 – 4.07 (m, 2H), 3.68 – 3.53 (m, 2H), 3.29 – 3.20 (m, 8H), 3.20 – 3.16 (m, 1H), 3.19 – 3.09 (m, 4H), 3.07 – 2.87 (m, 1H), 1.39 (s, 12H), 1.36 – 1.16 (m, 29H), 0.84 (t, J = 6.9 Hz, 6H), 0.79 (d, J = 23.1 Hz, 3H);13C NMR (101 MHz, DMSO) δ 171.00, 158.50, 144.4, 140.69, 136.96, 130.47, 124.39, 123.91, 111.62, 93.91, 93.82, 93.68, 73.05, 72.96, 72.88, 71.89, 71.83, 71.08, 71.01, 70.79, 70.32, 69.93, 65.31, 64.51, 51.15, 35.16, 34.3, 31.71, 31.48,29.63, 29.52, 29.47, 29.23, 29.17, 26.10, 22.55, 17.76, 14.37; [m / z; 982.766; M+H]. Synthesis of Example Unsymmetrical Trehalose Compound UM-5257:

[0365] As shown below, UM-5257 may be prepared similar to UM-1189, described above. As shown above, mono-azide derivative 12 is the critical intermediate synthesized via the novel Mitsunobu method as described above. Alkyne-azide click chemistry occurs between intermediate 12 and 13 using sodium ascorbate and copper (II) sulfate in dioxane / water to afford intermediate 14. Another critical intermediate is the potassium salt, which compound 4 is treatment with potassium trimethylsilonate in THF which affords compound 15. Esterification between potassium salt 15 and trehalose derivative 14 with 18-crown-6 in toluene produces TMS-protected intermediate 16. UM-5257 is afforded after intermediate 16 is treated with acidic Dowex® resin in methanol.

[0366] As shown above, mono-azide derivative 2 is the key intermediate synthesized via the novel Mitsunobu method as described above. Alkyne-azide click chemistry occurs between intermediate 2 and 1 using sodium ascorbate and copper (II) sulfate in dioxane / water to afford intermediate 11a. Another key intermediate is the potassium salt, which compound 4 is treatment with potassium trimethylsilonate in THF which affords compound 15. Esterification between potassium salt 15 and trehalose derivative 11a with 18-crown-6 in toluene produces TMS- protected intermediate 16. UM-5257 is afforded after intermediate 16 is treated with acidic Dowex®resin in methanol.

[0367] UM-5257.1H NMR (400 MHz, DMSO) δ 7.90 (s, 1H), 7.18 (s, 2H), 5.33 (s, 1H), 5.20 (s, 1H), 5.04 – 4.81 (m, 5H), 4.63 (d, J = 23.4 Hz, 2H), 4.44 (m, 4H), 4.14 (m, 3H), 3.95 (3, 6H), 3.60 (3, 2H), 3.21 (m, 8H), 3.04 (d, J = 36.3 Hz, 6H), 1.68 (m, J = 25.4 Hz, 6H), 1.48 – 1.09 (m, 53H), 0.80 (d, J = 41.8 Hz, 18H); [m / z; 1238.95; M+H]. 4. Structure Activity Relationship (SAR) Studies

[0368] The example unsymmetrical trehalose compounds UM-1181-1191, shown below, comprise the start of a new family of potent and biologically relevant compounds.

[0369] This new family of unsymmetrical trehalose compounds which incorporate features from both the “BRAT” and “CLICK” families may be referred to herein as the “BRICK” family. As used herein, “BRAT” refers to the family of symmetrical biaryl trehalose based Mincle ligands (e.g., as described in U.S. Patent Publication No.2021 / 0139521). As used herein, “CLICK” refers to the family of symmetrical di-ether lipid triazole trehalose based Mincle ligands (e.g., as described in U.S. Patent Publication No.2020 / 0369704).

[0370] These unsymmetrical trehalose compounds provide promise by combining substituted aryl functionality and lipid constructs, which have each independently demonstrated utility asadjuvants on a symmetrical trehalose core (e.g., see U.S. Patent Publication No. 2021 / 0139521 and U.S. Patent Publication No.2020 / 0369704 respectively).

[0371] Notably, UM-1181-1191 and UM-1091 demonstrated excellent stimulation on human PBMCs (FIGS. 1A-1B). Table 1, below, summarizes various example unsymmetrical trehalose compounds, including UM-1181-1191. Table 1. Unsymmetrical trehalose compounds (i.e., “BRICK” compounds)

[0372] Table 2 below shows previously prepared symmetrical trehalose compounds that were used as benchmarks for the unsymmetrical trehalose compounds’ activity studies. Table 2. Exemplary symmetrical trehalose compounds.Example 1: Structure-Activity Relationships of Asymmetric Mincle Ligands.

[0373] Compound screening was conducted on freshly isolated hPBMCs using two modes of exposure both solution and plate coated and an initial readout for the anticipated Th17- mediated immune response being the cytokine IL-6 by ELISA (Hu et al., Medicinal Chemistry Letters 2023, 14 (12), 1647-1655). The solution-based compounds were serially diluted in EtOH and supplemented with 1% DMSO followed by dilution in EtOH. The compounds were plate coated by serially diluting in EtOH, added to a tissue culture plate, and then solvent was evaporated. To both sample preparations, hPBMC’s were added and incubated for 18-24 h. After, supernatants were collected and evaluated for IL-6 production by ELISA. Known hMincle agonists, TDM and TDB, were used as positive controls.

[0374] Antigen specific IgG (FIG.3A), IgG1 (FIG.3B) and IgG2a (FIG.3C) titers in UM- 1098 / A-SNP were significantly higher when compared to other compound families at the samedose. Only UM-1094 / A-SNP, another BRAT compound, showed similar antigen specific IgG and IgG2a antibody titers to A-SNP adjuvanted with UM-1098 (FIG. 3A and FIG. 3C). Draining lymph nodes (dLN, inguinal and popliteal) and spleens were harvested from euthanized mice 21 days post tertiary injection. Following harvest, T cells were re-stimulated with 1 µg / mL M72 for 72 hours and supernatants collected to measure cytokine production via MSD (FIGS. 4A-4C), spleen data not shown. While M72-specific splenic T-cell responses from mice vaccinated with UM-1094, UM-1194, UM-1186, or UM-1024 adjuvanted vaccines did not show significant increase in IL-5, IFNγ or IL-17A compared to mice vaccinated with UM-1098 adjuvanted vaccine or antigen alone, data not shown. Draining lymph node responses from mice vaccinated with UM- 1194 adjuvanted vaccines exhibited a significant increase in IFNγ and IL-17A cytokine concentration compared to antigen alone. This could indicate UM-1194 may be a potential backup compound to UM-1098, since UM-1194 is from a different family of CLR compounds, and it can also bias a Th1 / Th17 response. Example 2: Additional Investigation of Backup Compounds for UM-1098 and Comparison of Human and Mouse PBMC Responses.

[0375] Further studies were performed on another CLICK family compound, UM-1089, to compare the Th1 / Th17 response seen from UM-1194. An additional structure activity response study was designed to evaluate other potential lead candidates, UM-1089 (CLICK) and UM-1189 (BRICK). Earlier testing suggested species specificity among these compounds and the potential for enhanced immunity in human versus mouse. Screening was completed in both human and mice PBMCs (wild-type and Mincle KO) prior to in vivo dosing. In contrast to previous lead selection studies, the current experiment also used the lead DSNP-50 formulation.

[0376] In addition to testing lead back-up compounds in vivo; in vitro analysis was also performed in both human and mouse PBMCs. Murine PBMCs provided direct in vitro evaluation of CLR compound activity between humans and mice. Briefly, cardiac punctures were performed on euthanized C57BL / 6 and Mincle KO mice and collected in heparin filled syringes to inhibit clotting. Following a 1:1 dilution with PBS, blood was overlaid onto Lympholyte Mammal cell separation media to allow for density separation and isolation of lymphocytes and monocytes. Cells were counted than plated at 1.0x107per well. For human PBCMs, whole blood was obtained from healthy human donors through a University of Montana Institutional Review board (IRB)approved protocol and processed as described in a previous report (Smith et al., Frontiers in Immunology 2019, 10 (338): 1-13.), cells were plated at 5.0x105cells per well. Both mice and human PBMCs were stimulated with either a starting concentration 100 µM UM-1098 / A-SNP or volume matched blank A-SNPs and diluted 1:3 down the plate and allowed to incubate at 37 °C, 5% CO2for 24 hours.IL-6 was used to screen the differences between human or wild-type (C57BL / 6) and Mincle KO PBMCs (FIGS.5A-5C). It is clear there is a noticeable species-specific difference in SAR seen between human and murine systems. Murine response to UM-1098 exhibits higher potency than that of humans versus mice. Interestingly UM-1024 (branched BRAT), UM-1089 (CLICK) and UM-1189 (BRICK) demonstrated enhanced potency versus UM- 1098 in human PBMCs. The lack of IL-6 response in Mincle knockout mice (FIG. 5C) demonstrate that all three families of compounds tested activate through the Mincle receptor.

[0377] Characterization of the lead compounds from each of the three compound families (“branched BRAT,” “CLICK,” and “BRICK”) was completed in C57BL / 6 mice. Mice were vaccinated via intramuscular administration with 1 µg M72 and indicated compounds, lead adjuvant UM-1098 at 25 nmol or vehicle alone (negative control group). Serum was collected at 28 days post primary (28dp1) injection via submandibular bleeds and again at 14 days post- secondary (14dp2) via cardiac puncture. Sera was analyzed for M72 specific IgG, IgG1 and IgG2c specific antibodies (FIGS.6A-6C). IgG (FIG.6A) and IgG1 (FIG.6B) antigen specific antibody titer compared to UM-1098 / D-SNP along with a clear dose response to changing amounts of adjuvant. Both UM-1024, a representative branched BRAT compound and UM-1089, a representative CLICK compound, had significantly lower antibody titers for all isotypes tested when compared to Mincle agonist UM-1098 (FIGS. 6A-6C). The representative BRICK compound, UM-1189, demonstrated a dose responsive increase in M72-specific IgG and IgG1 titers reaching equivalent levels to UM-1098 at 25 nmol.

[0378] Following euthanasia mouse spleens and draining lymph nodes where harvested and re-stimulated with 1 µg / mL M72 for 72 hours to analyze T-cell response. Cytokine production was measured from T-cell supernatants via MSD. While M72-specific draining lymph node T cell responses from mice vaccinated with UM-1024, UM-1089, or UM-1189, adjuvanted vaccines did not show significant increase in IL-5, IFNγ or IL-17A compared to mice vaccinated with UM- 1098 adjuvanted vaccine (FIGS.7A-7C), splenic responses from mice vaccinated with UM-1189 (BRICK) adjuvanted vaccines at both 10 nmol and 25 nmol exhibited a significant increase in IL-17A cytokine concentration compared to UM-1098 at 25 nmol (FIGS.7A-7B). Both the in vitro and in vivo response to UM-1189 could indicate this BRICK compound could be a potential backup compound to UM-1098; not only is it from a different family of CLR compounds but it can also bias a strong Th17 response at lower doses and gives a strong IL-6 response in both humans and mice with formulated on diethylenetriamine (DETA) silica nanoparticles (D-SNP).

[0379] Observations from the SAR backup compound studies found that UM-1098, the lead BRAT and UM-1189 (BRICK), had a stronger in vitro human IL-5 response and a more Th1 / Th17 biased in vivo response (FIGS.7A-7C and FIGS.8A-8C), leading to the selection of both UM- 1098 and UM-1189 as lead CLR adjuvants for this discovery contract. Example 3: Asymmetric Mincle agonists improve physicochemical properties.

[0380] Asymmetric Mincle agonists were specifically designed to enhance physicochemical properties, including reduced hydrophobicity. This is reflected in their lower molecular weights and calculated logP values compared to the lead symmetric BRAT agonist, UM-1098 (Table 3). Table 3. Molecular weight (MW) and calculated logP (clogP, using ChemDraw23.1.2) values of UM-1098 and asymmetric Mincle agonists.Example 4: Asymmetric Mincle agonists help with ease of silica nanoparticle-based formulations and improve their colloidal stability.

[0381] The asymmetric Mincle agonists were designed to improve formulation properties, while maintaining or enhancing adjuvant activity. The lead 50 nm diethylenetriamine- functionalized silica nanoparticle (DSNP-50) formulation for this class of adjuvants allows us to directly compare how structural changes in a compound affect ease of formulation, colloidal stability, and immunogenicity. In addition to differences in immunological response, modifications to the structure of the asymmetric BRAT UM-1098 achieved in the asymmetric Mincle agonist were able provide improved formulation properties as well, lending themselves to smaller particlesizes (FIG.9) while maintaining good formulation reproducibility (FIG.10). This shows that the asymmetric Mincle agonists allowed easier formulation even before any compound-specific optimization to the DSNP-50 formulations. While the initial particle size for the UM-1098 (symmetric BRAT) DSNP-50 formulation is < 200 nm, determined using dynamic light scattering (DLS) measurements, the formulation flocculates (reversible) over storage at 2-8 °C (Table 4). Table 4. DSNP-50 formulations with the asymmetric Mincle agonists (UM-1189) demonstrate formulation colloidal stability over storage at 2-8 ⁰C.

[0382] It was found that DSNP-50 formulations made with UM-1189 can be consistently made to reach a size equilibrium where the hydrodynamic size remains stable around 200 d.nm with less processing cycles than is needed for UM-1098 (Table 4). The UM-1098 formulation consistently achieved a comparable initial size, but most batches eventually flocculate to a size of ~400 d.nm over storage. Example 5: Asymmetric Mincle agonists improve compatibility with high salt content in the final vaccine formulation with antigen.

[0383] Various asymmetric Mincle agonist / DSNP-50 formulations were assessed for size changes when in their final vaccine formulation (with the addition of M72 antigen and a small amount of DPBS). Formulations were made with UM-1098 (symmetric BRAT) and UM-1189 (asymmetric compound) adsorbed to DSNP-50, then M72 antigen was added to each formulation, followed by varying amounts of 2% glycerol diluent. Z-Avg sizes of the formulations at the same dilution as the vaccine in either the 2% glycerol formulation vehicle or the vaccine vehicle composition (DPBS added), minus any antigen were compared to the vaccine formulation sizes by DLS. It was observed that, in general, when the formulation was at a higher concentration in thevaccine mixture, each formulation was more resistant to salt-induced flocculation. In these results, the particle size of the UM-1098 and UM-1194 DSNP-50 vaccine formulations were ~400 and 200 d.nm respectively. UM-1189 showed the greatest resistance to salt-induced flocculation (FIG.11). Example 6: Asymmetric Mincle agonists enhance formulation versatility

[0384] UM-1098 (symmetric BRAT) demonstrated limited formulation versatility due to its poor performance in lipid-based systems such as liposomes and emulsions, eliciting only weak antigen-specific Th17-polarized immunity. In contrast, asymmetric Mincle agonists, characterized by lower clogP values and more flexible lipid chains, are more readily incorporated into lipid- based formulations. To evaluate this improved versatility, UM-1098 with its asymmetric analog UM-1189 were compared across three cationic liposome systems varying in lipid composition, membrane rigidity, and charge localization within the lipid bilayer. These formulation parameters influence stability, adjuvant presentation, antigen association, cytotoxicity, and overall pharmacokinetics and cellular uptake. In vitro assays using human PBMCs showed that UM-1189 consistently induced IL-6 responses above vehicle controls and outperformed UM-1098 across all tested liposomes: DOEPC:Chol, DOPC:DOTAP:Chol, and DOTAP:DDA:Chol (FIG. 12). Moreover, asymmetric Mincle agonists such as UM-1189 (clogP = 9.98) can be directly dissolved in the oil phase during oil-in-water (O / W) emulsion preparation, eliminating the need for organic solvents (e.g., IPA or THF) or elevated temperatures required for UM-1098. This improved partitioning simplifies large-scale manufacturing, making it more feasible, environmentally friendly, and safer. Encapsulation of UM-1189 into Squalene / DOTAP-based emulsions also resulted in smaller particle sizes, lower polydispersity, and significantly improved colloidal stability over time compared to UM-1098 (FIG.13). These findings highlight the advantages of asymmetric design in broadening the range of compatible and effective delivery systems for Mincle agonists. Example 7: Structure Activity Relationships of Additional Asymmetric Mincle Ligands

[0385] Compound screening was conducted on freshly isolated human PBMCs an initial readout for compounds they may induce or inhibit Th17-mediated immunity by measuring the cytokine IL-6 by ELISA. Compounds were dissolved in DMSO and serially diluted in media and added to PBMCs. Alternatively, the compounds were plate coated by serially diluting in EtOH,added to a tissue culture plate, and then solvent was evaporated. Alternatively, the compounds were adsorbed to silica nanoparticles prior to serially diluting and adding to PBMCs. To all sample preparations, hPBMCs were added and incubated for 18-24 h. After, supernatants were collected and evaluated for IL-6 production by ELISA. Known Mincle agonists, TDM and TDB, or symmetric compound controls (UM-1098, UM-1052, UM-1024) were used as positive controls.

[0386] Asymmetric compounds UM 1183-1190 were evaluated for IL-6 release from human PBMCs (FIG. 14). Clear improvement in activity is noted from the parent symmetrical compounds. Notably, asymmetric structures impact compound potency.

[0387] Asymmetric compound UM-1189 was evaluated for IL-6 release from human PBMCs (FIG.15). Left shifted dose response curves indicate increased compound potency for UM-1189 vs the parent symmetrical compounds UM-1194 and UM-1098.

[0388] Asymmetric compound UM-5240 were evaluated for IL-6 release from human PBMCs (FIG. 16). Both compounds exhibited enhanced potency relative to their parent symmetric compound controls B16, UM-1024 and UM-1098. B16 is shown below and UM-1024 and UM- 1098 are shown in Table 2 above.B16

[0389] Asymmetric compounds UM-5249 and UM-1189 was evaluated for IL-6 release from human PBMCs (FIG.17). Remarkable improved potency of UM-5249 was noted.

[0390] Asymmetric compounds UM-5257 and UM-5248 were evaluated for IL-6 release from human PBMCs (FIG.18).

[0391] Mice were vaccinated two times (28-day interval) via intramuscular administration with 1 µg M72 antigen and asymmetric compound UM-1189. Symmetric adjuvants UM-1098, UM-1024, UM-1089 were used as positive controls and vehicle alone was the negative control group. Antigen alone (no adjuvant) was used as the benchmark. Serum was collected at 14 days post-secondary injection. Sera was analyzed for M72 specific IgG, IgG1 and IgG2a antibodies.Both UM-1024 (a branched BRAT) and UM-1089 (a CLICK compound), had significantly lower antibody titers for all isotypes tested when compared to Mincle agonist UM-1098 (FIGS.19A- 19C). The asymmetric BRICK compound, UM-1189 demonstrated a dose responsive increase in M72-specific IgG and IgG1 titers reaching equivalent or higher levels to UM-1098 at 25 nmol.

[0392] Following intramuscular vaccinations, mouse spleens where harvested and re- stimulated with 1 µg / mL M72 for 72 hours to analyze T-cell response. Cytokine production was measured via MSD. Splenic responses from mice vaccinated with asymmetric UM-1189 (BRICK) adjuvanted vaccines at both 10 nmol and 25 nmol exhibited a significant increase in IL-17A cytokine concentration compared to UM-1098 at 25 nmol (FIGS.20A-20C).

[0393] Mincle agonist UM-1098 (symmetric BRAT) and UM-1194 (symmetric CLICK) were compared to asymmetric compound UM-1189 for reactogenicity and injection site pain (pain and inflammation) following vaccination using a murine hind limb weigh bearing model. In a blinded measurement the mice back legs stood on a scale and the right weight and left weight (g) was recorded. The average left weight was subtracted from the right average. Hence, a positive score indicates that the mice were putting more weight on their right (not vaccinated leg) and less weight on their left (vaccinated leg). The UM-1189 and UM-1194 adjuvants in SNP formulations had significantly lower injection site reactogenicity compared to UM-1098 / SNP on days 2-3 post (FIGS.21A-21B).

[0394] To determine if asymmetric compound UM-1189 was a Mincle ligand vaccination studies were completed in wt. C57 / Bl6 mice versus Mincle KO C57 / Bl6 mice. Mice were vaccinated three times (28-day interval) via intramuscular administration with 1 µg M72 antigen and asymmetric compound UM-1189. Spleens and draining lymph node cells were collected and re-stimulated with M72 antigen to evaluation Th17 recall response by cytokine assay (FIG.22). The WT C57BL6 mice demonstrated the expected M72 antigen dependent Th17 cell-mediated immune response. The Mincle KO mice did not respond, indicating the resulting immune response was Mincle-dependent.

[0395] Mice were vaccinated three times (28-day interval) via intramuscular administration with 1 µg M72 antigen and asymmetric compounds UM-1189 and UM-5240. Symmetric adjuvant UM-1098 was used as positive controls and antigen alone (blank SNP vehicle) was used as the benchmark. Serum was collected at 21 days post-tertiary injection. Sera was analyzed for M72 specific IgG antibodies by ELISA. Compound UM-1098 induced the highest anti-M72 antibodytiters (FIG.23).

[0396] Following intramuscular vaccinations, mouse spleens where harvested and re- stimulated with 1 µg / mL M72 for 72 hours to analyze T-cell response. Cytokine production was measured via MSD. Splenic IL-17A responses were noted from mice vaccinated with asymmetric UM-1189, and UM-5240 (FIG.24).

[0397] Mincle agonist UM-1098 (symmetric BRAT) was compared to asymmetric compound UM-5240 for reactogenicity and injection site pain (pain and inflammation) following vaccination using a murine hind limb weigh bearing model. In a blinded measurement the mice back legs stood on a scale and the right weight and left weight (g) was recorded. The average left weight was subtracted from the right average. Hence, a positive score indicates that the mice were putting more weight on their right (not vaccinated leg) and less weight on their left (vaccinated leg). As shown in FIG. 25, UM-5240 SNP formulations had significantly lower injection site reactogenicity compared to UM-1098.

[0398] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

Claims

CLAIMS What is claimed is:

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, ,X1is O or NRX, where RXis hydrogen or C1-4alkyl; R1a, R1cand R1eare each independently –OH or hydrogen and R1band R1dare each independently tert-butyl or trifluoromethyl; or, alternatively, R1aand R1eare each hydrogen, R1band R1dare each –OC1-12alkyl, and R1cis hydrogen or –OC1-12alkyl; R2is a five-membered heteroaryl having 1, 2, or 3 heteroatoms independently selected from the group consisting of N, O, and S, where the five-membered heteroaryl is substituted withR4is methyl or hydrogen; Xaand Xbare each independently O, S, NH, NC1-4alkyl, or CH2;n is 2-28; m is 2-28; p is 0 or 1; and Yaand Ybare each independently methyl or –N(RY)2, where RYis hydrogen or C1-4alkyl; or, alternatively,methyl.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X1is O.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X1is NRXand RXis hydrogen.

4. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1aand R1care each independently –OH or hydrogen, R1band R1dare each tert-butyl, and R1eis hydrogen.

5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein R1ais –OH or hydrogen and R1cis hydrogen.

6. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1a, R1c, and R1eare each hydrogen and R1band R1dare each –OC1-12alkyl.

7. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1aand R1eare each hydrogen and R1b, R1c, and R1dare each –OC1-12alkyl.

8. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof,,9. The compound of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein R2is the five-membered heteroaryl.

10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein the five-membered heteroaryl is triazolyl.

11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein R2is.

12. The compound of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein R2is –R3.

13. The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein14. The compound of any one of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein Xaand Xbare each O or CH2.

15. The compound of any one of claims 1-8 or 12-14, or a pharmaceutically acceptable salt thereof, wherein.

16. The compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein n is 2-14.

17. The compound of any one of claims 1-16, or a pharmaceutically acceptable salt thereof, wherein m is 2-14.

18. The compound of any one of claims 1-17, or a pharmaceutically acceptable salt thereof, wherein Yaand Ybare each methyl.

19. The compound of any one of claims 1-17, or a pharmaceutically acceptable salt thereof, wherein Yais –N(RY)2and Ybis methyl.

20. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is:,,,,,,,,.

21. An adjuvant composition comprising an effective amount of a compound of any one of claims 1-20, or a pharmaceutically acceptable salt thereof.

22. A method for inducing an enhanced immune response in a subject, comprising administering to the subject, an effective amount of the compound of any of claims 1-20, or a pharmaceutically acceptable salt thereof, or the adjuvant composition of claim 21.

23. A vaccine composition comprising: (a) an antigen; and (b) an adjuvant composition comprising an effective amount of a compound of any one of claims 1-20, or a pharmaceutically acceptable salt thereof.

24. A method for inducing or enhancing the immunogenicity of an antigen in a subject, comprising administering to the subject an effective amount of a compound of any one of claims 1-20, or a pharmaceutically acceptable salt thereof, or the vaccine composition of claim 23.

25. An immunomodulatory composition comprising an effective amount of a compound of any one of claims 1-20, or a pharmaceutically acceptable salt thereof.

26. The immunomodulatory composition of claim 25, further comprising at least one additional adjuvant or immunostimulant.

27. A method of modulating an immune response in a subject, comprising administering to the subject, an effective amount of the compound of any of claims 1-20, or a pharmaceutically acceptable salt thereof, or the immunomodulatory composition of claim 25 or 26.

28. The method of claim 27, wherein the compound or immunomodulatory composition is administered as a monotherapy.

29. The method of claim 27 or 28, wherein the immune response in the subject is increased.

30. The method of any one of claims 27-29, wherein the subject is suffering from cancer, an autoimmune disorder, or an infectious disease.

31. A method of preparing a compound of any one of claims 1-20, or a pharmaceutically acceptable salt thereof, the method comprising: reacting a compound of formula (ii):with (CH3)3Si–N3in the presence of a phosphine and an azodicarboxylate to produce a compound of formula (iii):wherein: PG, at each occurrence, is a hydroxyl protecting group, wherein the hydroxyl protecting group is a trimethyl silyl, a benzyl, or a benzoyl group.

32. The method of claim 31, wherein reacting the compound of formula (ii) with (CH3)3Si–N3in the presence of the phosphine and an azodicarboxylate comprises: adding the azodicarboxylate to a solution comprising the compound of formula (ii), the phosphine, and an aprotic organic solvent, to produce a first reaction mixture; and adding the (CH3)3Si–N3to the first reaction mixture to produce a second reaction mixture.

33. The method of claim 31 or 32, wherein the phosphine is triphenyl phosphine or trimethyl phosphine.

34. The method of any one of claims 31-33, wherein the azodicarboxylate is diisopropyl azodicarboxylate.

35. The method of any one of claims 31-34, further comprising: reacting the compound of formula (iii) with a propargyl reagent of formula (iv):in the presence of copper (II) source and a reducing agent; or, alternatively, in the presence of a copper (I) source, to produce a compound of formula (v):removing the –Si(CH3)3group to produce a compound of formula (vi):.

36. The method of claim 35, wherein the copper (II) source is copper sulfate.

37. The method of claim 35 or 36, wherein the reducing agent is sodium ascorbate.

38. The method of any one of claims 35-37, further comprising: reacting the compound of formula (vi) with a triflating agent to provide a compound of formula (vii):.

39. The method of claim 38, wherein the triflating agent is triflic anhydride or trimethylsilyl trifluoromethanesulfonate.

40. The method of claim 38 or 39, further comprising reacting the compound of formula (vii) with a salt of formula (viii):(viii), to produce a compound of formula (ix):wherein.

41. A compound of formula (iii):wherein: PG, at each occurrence, is a hydroxyl protecting group, wherein the hydroxyl protecting group is a trimethyl silyl, a benzyl, or a benzoyl group.

42. A method of preparing the compound of claim 41, the method comprising: reacting a compound of formula (ii)with (CH3)3Si–N3in the presence of a phosphine and an azodicarboxylate.

43. The method of claim 42, wherein reacting the compound of formula (ii) with (CH3)3Si– N3in the presence of the phosphine and an azodicarboxylate comprises: adding the azodicarboxylate to a solution comprising the compound of formula (ii), the phosphine, and an aprotic organic solvent, to produce a first reaction mixture; and adding the (CH3)3Si–N3to the first reaction mixture to produce a second reaction mixture.

44. The method of claim 42 or 43, wherein the phosphine is triphenyl phosphine or trimethyl phosphine.

45. The method of any one of claims 42-44, wherein the azodicarboxylate is diisopropyl azodicarboxylate.

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