Vaccine adjuvants

Abstract

Provided herein are compositions comprising a vaccine composition and an agent that induces innate immune responses in primary human monocyte derived dendritic cells (MDDCs) and methods of using the agent that induces innate immune responses in and activation of MDDCs to increase effectiveness of the vaccine.

Description

96SB-705001-WO / 24-014-02PCTVACCINE ADJUVANTSRELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 761,717, filed February 21, 2025. The content of this related application is incorporated herein by reference in its entirety for all purposes.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under 75N93019C00046 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUNDField

[0003] Described herein are compounds, and methods of using such compounds as adjuvants of a vaccine such as an inactivated influenza virus vaccine.Description of the Related Technology

[0004] Dendritic cells (DCs) are pivotal antigen-presenting cells that bridge the innate and adaptive immune systems. Activation of dendritic cells (DCs) is a critical property for effective vaccine adjuvants whose ability to drive effective T cell response to the antigens determines the immunogenicity of vaccines. Adjuvants amplify the immunogenicity, enabling stronger and longer-lasting protection against pathogens. Current vaccines for influenza are only effective for short durations, particularly in elderly individuals and patients with chronic diseases with weakened immune responses. Thus, there is a substantial demand for the development of new adjuvants to improve the immunogenicity and durability of the existing influenza vaccines. The present technology provides a new vaccine adjuvant to improve vaccine durability and cross-protection by exploiting the innate immune pathway activation in dendritic cells. Potential innate immune and proinflammatory pathways that the adjuvant candidate targets include but are not limited to: cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING), Toll-like receptor (TLR), RIG-I like receptors (RLR), NOD-like receptors (NLR), AIM2, STAT, NFKB, MAPK.SUMMARY

[0005] The present disclosure relates to compounds that activate innate immune pathways to dendritic cell activation and increase immunity in a subject. One aspect of the disclosure includes a method of increasing the effectiveness of a vaccine in a subject, which comprises administering a compound of Formula (I), or a pharmaceutically acceptable salt thereof,R3(I),wherein: R1is hydrogen, or C1-C3 alkyl; R2is hydrogen, or halo; and R3is C16-C19 alkyl, or C16-C19 alkenyl, to the subject in a dose and schedule configured to increase the effectiveness of the vaccine, wherein the subject is administered with the vaccine.

[0006] Some embodiments provide a compound of Formula (I),R3(I)or a pharmaceutically acceptable salt thereof, wherein:R1is hydrogen, or C1-C3 alkyl; R2is hydrogen, or halo; and R3is C16-C19 alkyl, or C16-C19 alkenyl.

[0007] Some embodiments provide a compound of Formula (II),R3(II)or a pharmaceutically acceptable salt thereof, wherein:R3is C16-C19 alkyl, or C16-C19 alkenyl; i) R4is C1-C6alkoxyl, or halo; and R5and R6are hydrogen, or ii) R4is hydrogen; and R5and R6are halo.

[0008] Some embodiments provide a compound of Formula (III),(Ill)or a pharmaceutically acceptable salt thereof, wherein:R1is hydrogen, or C1-C3 alkyl; and R2is hydrogen, or halo.

[0009] In some embodiments, the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, is administered concurrently with the vaccine. In such embodiments, it is contemplated that the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof can be an immune enhancer for the vaccine. In some embodiments, the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, is administered at least a day after administering the vaccine. In some embodiments, the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, is administered at least 2 days after administering the vaccine. In some embodiments, the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, is administered a plurality of times in a regular interval after administering the vaccine.

[0010] In some embodiments, the effectiveness of the vaccine is increased by activating the innate immune pathway. In some embodiments, the effectiveness of the vaccine is increased by increasing memory B cell population in the subject. In some embodiments, the effectiveness of the vaccine is increased by increasing memory B cell precursor population in the subject. In some embodiments, the effectiveness of the vaccine is increased by increasing replenishment of memory B cell population in the subject after rechallenge. In some embodiments, memory B cell population, memory B cell precursor population, and / or the replenishment of memory B cell population is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or a value or range between any two of these values. In some embodiment, the memory B cell population, memory B cell precursor population, and / or the replenishment of memory B cell population is increased by at least 10%.

[0011] In some embodiments, the dose and schedule are sufficient to increase antigen-specific antibody titers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or a valueor range between any two of these values in the subject compared to a subject not receiving the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, after administering the vaccine. In some embodiments, the dose and schedule are sufficient to increase antigen-specific antibody titers at least 50% in the subject compared to a subject not receiving the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, after administering the vaccine. In some embodiments, the dose and schedule are sufficient to increase antigen-specific antibody titers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or a value or range between any two of these values in the subject compared to a subject not receiving the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, after administering rechallenge. In some embodiments, the dose and schedule are sufficient to increase antigen-specific antibody titers at least 50% in the subject compared to a subject not receiving the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, after rechallenge.

[0012] Another aspect of the disclosure includes a method of increasing immunity against an antigen in a subject having an immune response against the antigen, which comprises administering a compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, to the subject in a dose and schedule effective to increase a secondary immune response upon re-exposure to the antigen compared to a subject not being administered the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof. In some embodiments, the dose of the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof is between 1.0 - 5.0 mg / kg. In some embodiments, the dose is about 2.5 mg / kg.

[0013] In some embodiments, the compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, is administered during the immune response. In some embodiments, the schedule comprises administration at least a day after the immune response. In some embodiments, the schedule comprises administration at least 2 days after the immune response. In some embodiments, the schedule comprises administration at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or a value or range between any two of these values after the immune response. In some embodiments, the schedule comprises administration a plurality of times in a regular interval after the immune response.

[0014] In some embodiments, the dose and schedule are sufficient to activate the innate immune pathway in the subject.

[0015] Some embodiments provide a pharmaceutical composition comprising a compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises a vaccine composition, wherein the compound is present in the pharmaceutical composition in a dose effective to increase effectiveness of the vaccine.

[0016] Some embodiments provide a method of increasing the effectiveness of a vaccine in a subject, comprising: administering a compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, to the subject in a dose and schedule configured to increase the effectiveness of the vaccine, wherein the subject is administered with the vaccine.

[0017] Some embodiments provide a compound of Formula (I) or Formula (II) or Formula (III) for use in increasing the effectiveness of a vaccine in a subject. The use can include administering the compound and the vaccine to the subject

[0018] Some embodiments provide a method of increasing immunity against an antigen in a subject having an immune response against the antigen, comprising, comprising: administering a compound of Formula (I) or Formula (II) or Formula (III), or a pharmaceutically acceptable salt thereof, to the subject in a dose and schedule configured to induce innate immune responses in primary human monocyte derived dendritic cells (MDDCs) compared to a subject not being administered the compound, or a pharmaceutically acceptable salt thereof.

[0019] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figures 1 A-1D show Compound 11 significantly enhances total IgG and neutralizing HAI titers to QIV vaccination in NHP. (Figures 1 A-1C) Longitudinal assessment of total anti -Influenza IgG binding titers comparing Compound 11, formulated with alum, to no adjuvant and control adjuvants of alum and Addavax. (Figure ID) Neutralizing HAI titers at Day 180 Statistics performed by unpaired t-test. *=p<0.05, **=p<0.01, ****=p<0.0001

[0021] Figures 2A-2B show Compound 11 induces a unique cytokine profile of balanced inflammatory and anti-inflammatory cytokines that correlate with higher Ab titers. (Figure 2A)Polar plots of categories of cytokines demonstrating a balanced inflammatory and antiinflammatory environment in alum-11 (red). Pro-inflammatory= IL- lb, IL-6, TNFa; Anti-Inflammatory = IL-10, TGFB1-3; Thl= IFNg, IL-2, IL-12p70, TNFa; Th2= IL-4, IL-5, IL-13; Thl7= IL-17A, IL-21, IL-22. (Figure 2B) Spearman correlations for all 24 NHP across 4 treatment groups showing a significant and positive correlation between cytokines in plasma and Day 180 total IgG ant-influenza Ab titers. *=p<0.05INCORPORATION BY REFERENCE

[0022] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.DETAILED DESCRIPTIONDefinitions

[0023] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0024] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0025] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.

[0026] The terms “individual,” “patient,” or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e g. constant or intermittent) of ahealth care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker).

[0027] “ Treatment” of a state, disorder or condition e.g., cancer) includes: (1) preventing or delaying the appearance of clinical or sub-clinical symptoms of the disorder developing in a human that is afflicted with or pre-disposed to the disorder but does not yet experience or display clinical or subclinical symptoms of the disorder; and / or (2) inhibiting the disorder, including arresting, reducing or delaying the clinical manifestation of the disorder or at least one clinical or sub-clinical symptom thereof; and / or (3) relieving the disorder, e.g., causing regression of the disorder or at least one of its clinical or sub-clinical symptoms; and / or (4) causing a decrease in the severity of one or more symptoms of the disorder. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0028] As used herein, the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited feature but not the exclusion of any other features. Thus, as used herein, the term “comprising” is inclusive and does not exclude additional, unrecited features. In some embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of.” The phrase “consisting essentially of’ is used herein to require the specified feature(s) as well as those which do not materially affect the character or function of the claimed disclosure. As used herein, the term “consisting" is used to indicate the presence of the recited feature alone.

[0029] Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well of any dividual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well of any dividual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention, unless the context clearly dictates otherwise.

[0030] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0031] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0032] The term “measuring,” as used herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations.

[0033] As used herein, “treatment of’ or “treating,” ‘applying”, or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and / or a prophylactic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient is still afflicted with the underlying disorder. For prophylactic benefit, the compositions are, in some embodiments, administered to a patient at risk of developing a particular disease or condition, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease has not been made.

[0034] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0035] It is understood that in the present description, combinations of substituents and / or variables of the depicted formulae are permissible only if such contributions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc at room temperature (e.g. 21 °C).

[0036] The terms below, as used herein, have the following meanings, unless indicated otherwise:

[0037] “Alkyl” refers to a straight-chain or branched-chain fully saturated hydrocarbon radical having from 1 to 30 carbon atoms (C1-C30 alkyl), 1 to 6 carbon atoms (Ci-Ce alkyl), 7 to 30 carbon atoms (C7-C30 alkyl), 11 to 19 carbon atoms (C11-C19 alkyl), 16 to 19 carbon atoms (C16-C19 alkyl). Examples include, but are not limited to methyl, ethyl, n-propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3 -methyl- 1 -butyl, 2-methyl-3 -butyl, 2,2-dimethyl- 1 -propyl, 2-methyl-1 -pentyl, 3 -methyl- 1 -pentyl, 4-methyl-l -pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- 1 -butyl, 3,3-dimethyl-l-butyl, 2-ethyl-l -butyl, n-butyl, isobutyl, secbutyl, t-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl and hexyl, and longer alkyl groups, such as heptyl, octyl, and the like. Whenever it appears herein, a numerical range such as “C1-6 alkyl”, means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated. In some embodiments, the alkyl is a Ci-10 alkyl. In some embodiments, the alkyl is a C1-6 alkyl. In some embodiments, the alkyl is a C1-5 alkyl. In some embodiments, the alkyl is a Ci-4 alkyl. In some embodiments, the alkyl is a C1-3 alkyl. In some embodiments, the alkyl is a C1-2 alkyl. In some embodiments, the alkyl is a C16-19 alkyl. In some embodiments, the alkyl is a C16-18 alkyl. In some embodiments, the alkyl is a C16-17 alkyl. In some embodiments, the alkyl is a Ci6 alkyl. In some embodiments, the alkyl is a C17 alkyl. In some embodiments, the alkyl is a Cis alkyl. In some embodiments, the alkyl is a C19 alkyl. Unless stated specifically in the specification, an alkyl group is not substituted. In some embodiments, the alkyl is substituted with one or more halogen, -CN, -OH, or -OCH3. In some embodiments, the alkyl is substituted with one or more halogen atoms.

[0038] “Alkenyl” refers to a straight-chain or branched-chain hydrocarbon radical having one or more carbon-carbon double-bonds and having from 2 to 30 carbon atoms (C2-C30 alkenyl), 2 to 6 carbon atoms (C2-C6 alkenyl), 7 to 30 carbon atoms (C7-C30 alkenyl), 11 to 19 carbon atoms (C11-C19 alkenyl), 16 to 19 carbon atoms (C16-C19 alkenyl). The group may be in either the cis or trans or Z or E conformation about the double bond(s). Examples include, but are not limited to ethenyl (-CH=CH2), 1-propenyl (-CH2CH=CH2), isopropenyl [-C(CH3)=CH2], butenyl, 1,3-butadienyl and the like. Whenever it appears herein, a numerical range such as “C2-C6 alkenyl”, means that thealkenyl group may consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkenyl” where no numerical range is designated. Unless stated specifically in the specification, an alkenyl group is not substituted. In some embodiments, the alkenyl is substituted with one or more halogen, -CN, -COOH, -COOCH3, -OH, -OCH3, -NH2, or -NO2. In some embodiments, the alkenyl is substituted with one or more halogen, -CN, -OH, or -OCH3. In some embodiments, the alkenyl is substituted with one or more halogen. When more than one carbon-carbon double bonds are present in the alkenyl, two or more carbon-carbon double bonds may be conjugated or unconjugated.

[0039] “Alkoxy” refers to a radical of the formula -Oalkyl where alkyl is defined as above. In some embodiments, the alkoxy is substituted with one or more halogen, -CN, -COOH, -COOCH3, -OH, -OCH3, -NH2, or -NO2. In some embodiments, the alkoxy is substituted with one or more halogen, -CN, -OH, or -OCH3. In some embodiments, the alkoxy is substituted with one or more halogen.

[0040] “Halogen” or “halo” refers to fluorine, chlorine, bromine, or iodine. In some embodiments, halogen is fluorine, chlorine, or bromine. In some embodiments, halogen is fluorine or chlorine. In some embodiments, halogen is fluorine or bromine. In some embodiments, halogen is chlorine or bromine.

[0041] “Haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 2-fluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.

[0042] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0043] While more and more vaccines against various infectious disease have been developed, the efficacy of such vaccines are not often satisfactory. The present disclosure relates to compounds that boost the immunity against an antigen or boost the effect of a vaccine against the antigen by inducing innate immune responses. In one aspect, a method of increasing the effectiveness of a vaccine in a subject is disclosed.

[0044] In another aspect, a method of increasing immunity against an antigen in a subject having an immune response against the antigen is disclosed.Compounds

[0045] Some embodiments provide a compound of Formula (I),(I)or a pharmaceutically acceptable salt thereof, wherein:R1is hydrogen, or C1-C3 alkyl; R2is hydrogen, or halo; and R3is C16-C19 alkyl, or C16-C19 alkenyl.

[0046] In some embodiments, R1is hydrogen. In some embodiments, R1is -CH3. In some embodiments, R2is hydrogen or chloro. In some embodiments, R2is hydrogen. In some embodiments, R2is chloro.

[0047] Some embodiments provide a compound of Formula (I),(I)or a pharmaceutically acceptable salt thereof, wherein:

[0048] R1is hydrogen, or substituted or unsubstituted C1-C3 alkyl; R2is hydrogen, halo, -CN, haloalkyl, or C1-C6 alkoxy; and R3is substituted or unsubstituted C16-C19 alkyl, or substituted or unsubstituted C16-C19 alkenyl. In some embodiments, R3is C16-C19 alkyl. In some embodiments, R3is C17 alkyl. In some embodiments, R3is C16-C19 alkenyl. In some embodiments, C17alkenyl. In some embodiments, R1is C1-C3 alkyl substituted with one or more substituents each independently selected from halogen, -CN, -OH, and -OCH3. In some embodiments, R2is halo, -CN, haloalkyl, or Ci-Ce alkoxy. In some embodiments, R2is halo, haloalkyl, or Ci-Ce alkoxy. In some embodiments, R3is C16-C19 alkyl substituted with one or more substituents each independently selected from halogen, -CN, -OH, and -OCH3. In some embodiments, R3is C17 alkyl substituted with one or more substituents each independently selected from halogen, -CN, -OH, and -OCH3. In some embodiments, R3is C16-C19 alkenyl substituted with one or more substituents each independently selected from halogen, -CN, -OH, and -OCH3. In some embodiments, R3is C17 alkenyl substituted with one or more substituents each independently selected from halogen, -CN, -OH, and -OCH3.

[0049] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is:pharmaceutically acceptable salt thereof.

[0050] Some embodiments provide a compound of Formula (II),(II)or a pharmaceutically acceptable salt thereof, wherein:R3is C16-C19 alkyl, or C16-C19 alkenyl; i) R4is C1-C6alkoxyl, or halo; and R5and R6are hydrogen, or ii) R4is hydrogen; and R5and R6are halo.

[0051] Some embodiments provide a compound of Formula (II),(II)or a pharmaceutically acceptable salt thereof, wherein:R3is a substituted or unsubstituted C16-C19 alkyl, or a substituted or unsubstituted C16-C19 alkenyl. In some embodiments, R3is a C16-C19 alkyl substituted with one or more substituents each independently selected from fluoro, chloro, bromo, -CN, -OH, and -OCH3, or a C16-C19 alkenyl substituted with one or more substituents each independently selected from fluoro, chloro, bromo, -CN, -OH, and -OCH3; and R4, R5and R6are independently hydrogen, Ci-Ce alkoxyl, halo, or nitro. In some embodiments, R3is a linear C16-C19 alkyl, or an linear C16-C19 alkenyl. In some embodiments, R4is Ci-Ce alkoxyl, or halo; and R5and R6are hydrogen. In some embodiments, R4is hydrogen; and R5and R6are halo.

[0052] In some embodiments, R4is Ci-Ce alkoxyl. In some embodiments, R4is halo. In some embodiments, R5and R6are each independently fluoro, chloro, or bromo. In some embodiments, R5and R6are each chloro. In some embodiments, R3is C16-C19 alkyl. In some embodiments, R3is a linear C16-C19 alkyl. In some embodiments, R3is C17 alkyl. In some embodiments, R3is C16-C19 alkenyl. In some embodiments, R3is alinear C16-C19 alkenyl. In some embodiments, R3is C17 alkenyl. In some embodiments, the compound of Formula (II), or a pharmaceutically acceptable salt thereof, is:

[0053] Some embodiments provide a compound of Formula (III),oO SII I H H^R1(III)or a pharmaceutically acceptable salt thereof, wherein:

[0054] R1is hydrogen, or substituted or unsubstituted C1-C3 alkyl; and R2is hydrogen, halo, -CN, haloalkyl, or Ci-Ce alkoxy. In some embodiments, R1is C1-C3 alkyl substituted with one or more substituents each independently selected from halogen, -CN, -OH, and -OCH3. In some embodiments, R2is halo, -CN, haloalkyl, or Ci-Ce alkoxy. In some embodiments, R2is halo, haloalkyl, or Ci-Ce alkoxy. In some embodiments, R1is hydrogen, or C1-C3 alkyl; and R2is hydrogen, or halo.

[0055] In some embodiments, the compound of Formula (III), or a pharmaceutically acceptable saltOH N Nthereof, is:, orN H Hor a pharmaceutically acceptable salt thereof.Pharmaceutical Composition

[0056] Some embodiments provide a pharmaceutical composition comprising a compound of Formula (I) or a compound of Formula (II) or a compound of Formula (III), or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some instances, the pharmaceutical composition describe herein is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrathecal, intracerebral, intracerebroventricular, or intracranial) administration. In other instances, the pharmaceutical composition describe herein is formulated for oral administration. In still other instances, the pharmaceutical composition describe herein is formulated for intranasal administration.

[0057] In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous dosage forms, or solid dosage forms. In some embodiments, solid dosage forms are in tablets, or capsules.

[0058] In some instances, the pharmaceutical formulation further includes pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0059] In some instances, the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include thosehaving sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0060] In some instances, the pharmaceutical formulation further includes diluent which are used to solubilize and / or stabilize compounds because they provide a more stable environment. Salts dissolved in buffered solutions (which also provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as Di-Pac® (Amstar); mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner’s sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0061] In some instances, the pharmaceutical formulation includes filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0062] Solubilizers include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like. Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.Therapeutic Regimens

[0063] In some embodiments, the compounds or the pharmaceutical compositions described herein are administered for therapeutic applications. The compounds or the pharmaceutical compositions can be used to (or for use to) increase the effectiveness of the vaccine in the subject. The compounds or the pharmaceutical compositions can be administered to the subject to increase the effectiveness of the vaccine. The compounds or the pharmaceutical compositions can beadministered to the subject in a dose and schedule effective for the vaccine. Thus, a dose and schedule of administering the agent can vary depending on the type of vaccines, for example, live-attenuated vaccines (e.g., measles vaccine, rotavirus vaccine, smallpox vaccine, chickenpox vaccine, yellow fever vaccine, etc.), inactivated vaccines (e.g., flu vaccine, polio vaccine, Hepatitis A vaccine, rabies vaccine, etc.), subunit, recombinant, polysaccharide, and conjugate vaccines (e.g., Hib disease vaccine, Hepatitis B vaccine, whooping cough vaccine, pneumococcal disease vaccine, meningococcal disease vaccine, shingles vaccine, etc ), or toxoid vaccine (e.g., diphtheria vaccine, tetanus vaccine, etc.). In addition, a dose and schedule of administering the pharmaceutical compositions can vary depending on the age, health condition, or biology of the subject.

[0064] For example, in some embodiments, the dose of compound for administering to a subject (e.g., human) can be 0.1 - 20 mg / kg, 0.2 - 15 mg / kg, 0.5 - 15 mg / kg, 0.5-10 mg / kg, 0.8- 10 mg / kg, 1.0 - 10 mg / kg, 1.0 - 9.0 mg / kg, 1.0 - 8.0 mg / kg, 1.0 - 7.0 mg / kg, 1.0 - 6.0 mg / kg, 1.0 - 5.0 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, or 5.0 mg / kg, 0.1 - 20 mg / kg, between 0.2 - 15 mg / kg, between 0.5 - 15 mg / kg, between 0.5-10 mg / kg, between 0.8- 10 mg / kg, between 1.0 - 10 mg / kg, between 1.0 - 9.0 mg / kg, between 1.0 -8.0 mg / kg, between 1.0 - 7.0 mg / kg, between 1.0 - 6.0 mg / kg, or between 1.0 - 5.0 mg / kg. In some embodiments, the dose of compound for administering to a subject (e.g., human) can be 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 7.0 mg / kg, 8.0 mg / kg, 9.0 mg / kg, 10.0 mg / kg, 11.0 mg / kg, 12.0 mg / kg, 13.0 mg / kg, 14.0 mg / kg, 15.0 mg / kg, 16.0 mg / kg, 17.0 mg / kg, 18.0 mg / kg, 19.0 mg / kg, 20.0 mg / kg, or the dose of compound for administering to a subject (e.g., human) can be within a range defined by any two of the preceding values. As used herein, the term “mg / kg” or “milligrams per kilogram” means amount of a compound of Formula (I) or a compound of Formula (II) or a compound of Formula (III), or a pharmaceutically acceptable salt thereof, measured in mg for each kilogram of a subject’s body mass. For example, 1.5 mg / kg means administration of 1.5 mg of a compound of Formula (I) or a compound of Formula (II) or a compound of Formula (III), or a pharmaceutically acceptable salt thereof, per kilogram of subject’s body mass such that a 60 kg subject would be administered 90 mg of the compound of Formula (I) or a compound of Formula (II) or a compound of Formula (III), or a pharmaceutically acceptable salt thereof, in the dose.

[0065] In some embodiments, the compounds can be administered concurrently with the vaccine. In some embodiments, the compounds can be administered at least within 10 min, within 30 min, within 1 hour, within 2 hours, within 3 hours, within 6 hours, within 12 hours after the vaccineadministration. In some embodiments, the agent can be administered at least a day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 12 days, at least 14 days, or at least 30 days after administering the vaccine. In some embodiments, where the vaccine administration schedule comprises a prime administration and a booster administration, the compounds can be administered between the prime administration and the booster administration. In some embodiments, the compounds can be administered a plurality of times in a regular interval after administering the vaccine. For example, once a day, once every two days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 10 days, once every 14 days from day 0, day 2, day 3, day 4, day 5, day 6, day 7, day 10, day 14, day 28 after the administration of the vaccine.

[0066] In certain embodiments, the compound is present in the pharmaceutical composition in a dose effective to increase effectiveness of the vaccine. In some embodiments, the dose is effective to increase antigen-specific antibody titers at least 50% in a subject when administered, compared to a subject not receiving the composition. In some embodiments, the dose is sufficient to increase the effectiveness of the vaccine by activating cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. In some embodiments, the effectiveness of the vaccine is increased by increasing memory B cell population in the subject. In some embodiments, the effectiveness of the vaccine is increased by increasing memory B cell precursor population in the subject.

[0067] Thus, in certain instances, administration of the compound can be customized in a dose and / or a schedule to increase the effectiveness of the vaccine. The effectiveness of the vaccine can be determined, assessed, or predicted in various methods. For example, in some embodiments, the effectiveness of the vaccine can be determined by measuring a parameter compared to the control (e.g. a patient that has not been treated with the compound).

[0068] Some embodiments provide a method of increasing the effectiveness of a vaccine in a subject, comprising:administering a compound of Formula (I) or a compound of Formula (II) or a compound of Formula (III), or a pharmaceutically acceptable salt thereof, to the subject in a dose and schedule configured to increase the effectiveness of the vaccine, wherein the subject is administered with the vaccine.

[0069] Some embodiments provide a method of increasing immunity against an antigen in a subject having an immune response against the antigen, comprising:administering a compound of Formula (I) or a compound of Formula (II) or a compound of Formula (III), or a pharmaceutically acceptable salt thereof, to the subject in a dose and schedule configuredto induce innate immune responses in primary human monocyte derived dendritic cells (MDDCs) compared to a subject not being administered the compound.

[0070] In some embodiments, the dose is between 1.0 - 5.0 mg / kg. In some embodiments, the dose the dose is about 2.5 mg / kg. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered concurrently with the vaccine. In some embodiments, the compound is an immune enhancer for the vaccine. In some embodiments, the compound is an immune enhancer for the vaccine. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered before or during the immune response. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered at least a day after administering the vaccine. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or a value or range between any two of these values after administering the vaccine. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered at least 2 days after administering the vaccine. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered a plurality of times (e.g., in a regular interval) after administering the vaccine. In some embodiments, the compound increases effectiveness of the vaccine by inducing innate immune responses in primary human monocyte derived dendritic cells (MDDCs).

[0071] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or a value or range between any two of these values after the immune response. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered at least a day after the immune response. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered at least 2 days after the immune response. In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered a plurality of times (e.g., in a regular interval) after the immune response.

[0072] In some embodiments, the dose and schedule are sufficient to increase antigen-specific antibody titers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or a value or range between any two of these values in the subject compared to a subject not receiving the compound after administering the vaccine. For example, the dose and schedule are sufficient to increase antigen-specific antibody titers at least 50% in the subject compared to a subject not receiving the compound after administering the vaccine. In some embodiments, the dose and schedule are sufficient to increase antigen-specific antibody titers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or a value or range between any two of these values inthe subject compared to a subject not receiving the compound after rechallenge. For example, the dose and schedule are sufficient to increase antigen-specific antibody titers at least 50% in the subject compared to a subject not receiving the compound after rechallenge.

[0073] In some embodiments, the effectiveness of the vaccine is increased by increasing memory B cell population in the subject. The memory B cell population can be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or a value or range between any two of these values. In some embodiments, the effectiveness of the vaccine is increased by increasing memory B cell precursor population in the subject. The memory B cell precursor population can be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or a value or range between any two of these values.

[0074] In some embodiments, the effectiveness of the vaccine is increased by increasing replenishment of memory B cell population in the subject after rechallenge. In some embodiments, the replenishment of memory B cell population is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or a value or range between any two of these values after rechallenge.Kits / Article of Manufacture

[0075] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more of the compositions and methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[0076] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[0077] For example, the container(s) include target nucleic acid molecule described herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

[0078] A kit typically includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0079] In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.

[0080] In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U. S. Food and Drug Administration for prescription drugs, or the approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.EXAMPLESExample 1A: Murine Model

[0081] Female BALB / c mice were vaccinated with 1.5 pg of HA-equi valent of quadrivalent inactivated influenza vaccine (QIV) in the hamstring muscles. Compound was used at a stock solution of 10 pM concentration and admixed in equal volumes with the QIV, resulting in 5 pM final concentration for injection. Animals were given a prime and booster vaccination, spaced three weeks apart. Antibody responses to adjuvanted vaccination were measured by ELISA using recombinant trimeric soluble hemagglutinin as a coating antigen or by hemagglutination inhibition (HAI) assays. Serum was collected 2 weeks after prime or booster vaccination and probed for ELISA- binding titers. Groups that received PBS, DMSO, unadjuvanted QIV or QIV adjuvanted with benchmarks IMDQ-PC or AddaVax were included as controls. Compound 11 showed robust activity in comparison to controls.Example IB: Induction of interferon stimulatory gene 54 and CD86 and MHC-II surface cell activation marker expressions in human monocyte derived dendritic cells (MDDCs) MDDC isolation:

[0082] Primary monocyte-derived dendritic cells (MDDCs) are prepared from fresh, healthy donor blood from the San Diego Blood Bank. Peripheral blood mononuclear cells (PBMCs) from the blood are isolated by Ficoll-Paque (GE Healthcare) density centrifugation and incubated with CD14+ Micro beads per manufacturer’s instructions (Miltenyi Biotec). The isolated CD14+ monocytes are plated at 1.5 x 106cells / mL in RPMI 1640 (Life Technology) supplemented with 10% fetal bovine serum (FBS), 1000 U / mL of human IL-4 (Peprotech) and 500 U / mL of human GM-CSF (Peprotech), and then allowed to differentiate for 5 days with a supplement of another volume of media with cytokines on the 3rd day. 2-3 donors are pooled and seeded at 20,000 cells per well of 96 well plate in 0.1 mL media.qRT-PCR-based analysis of ISG54 expression.

[0083] MDDCs are treated with 0.1, 1 and 10 pM of each compound in media with 0.1% DMSO final for 8 hrs, followed by lysing of cells. The cell lysates are subjected to cDNA synthesis (Applied Biosystem), followed by qPCR reaction with primers specific ISG 54 transcript. Values are normalized against the DMSO control and expressed as fold induction. The highest fold induction of ISG 54 at any concentration is defined as Emax of each compound.MDDC maturation and activation assay.

[0084] MDDCs, pooled from multiple donors, were treated at 1 to 10 pM of compounds for 24 hours and fixed with paraformaldehyde, followed by immunostaining against MHC-II (HLA-DR) and CD86 DC activation markers. The cells were subjected to high throughput image analysis with Celigo. Mean Fluorescence Intensity (MFI) per cell was measured and the median of MFIs was calculated for each marker for each compound. For each compound, fold inductions over DMSO and the Comparison Compound were calculated. Score of either 0 or 1 was assigned depending on whether the fold induction is lower or higher than 1 respectively.Chemical Synthesis

[0085] In some instances, reactions were performed in oven-dried glassware under an atmosphere of argon with magnetic stirring. In some instances, solvents and chemicals used were purchased from Sigma-Aldrich or Acros, and were used as received without further purification. Purity and characterization of compounds were established by a combination of liquid chromatography-massspectroscopy (LC-MS) and NMR analytical techniques. Silica gel column chromatography was carried out using prepacked silica cartridges from RediSep (ISCO Ltd.) and eluted using an Isco Companion system.1H- and13C-NMR spectra were obtained on a Jeol 400 spectrometer at 400 MHz and 100 MHz, respectively. Chemical shifts are reported in δ (ppm) relative to residual solvent peaks or TMS as internal standards. Coupling constants are reported in Hz. HPLC-MS analyses were performed on a Shimadzu 2010EV LCMS using the following conditions: Kromisil C18 column (reverse phase, 4.6 mm × 50 mm); a linear gradient from 10% acetonitrile and 90% water to 95% acetonitrile and 5% water.

[0086] Compounds listed in Table 1 can be prepared using the procedure described in Scheme 1 and according to the procedure of Example 2.Scheme 1DCM-DMF, rt, 12hExample 2. Preparation of octadecyl 2-chloro-4-(3-(2-methoxybenzoyl)thioureido)benzoate (Compound 5)O

[0087] To a solution of 2-chloro-4-(3-(4-methoxy-3-nitrobenzoyl)thioureido)benzoic acid (425 mg, 50% Wt, 1 eq, 583 pmol) in DMF (5 mL) and DCM (10 mL) was added DMAP (7 mg, 0.1 eq, 58 pmol) at ice bath temperature. The resulting mixture was stirred 12 min and then treated with octadecan- l-ol (315 mg, 2 eq, 1.17 mmol). The resulting mixture was stirred 12 h at RT. The solvent was removed under reduced pressure and the crude material was first purified by normal phase HPLC (100% hexane to 70:30 hexane / ethyl acetate) on a 40 min run. Solvent was removed and then the material was purified by reverse phase prep HPLC (CH3OH-water system with 40 min run with 80 mL / min flow). The solvent was removed to afford the octadecyl 2-chloro-4-(3-(2-methoxybenzoyl)thioureido)benzoate (Compound 5, 180 mg, 293 pmol, 50.2 %).1H NMR (400MHz, DMSO-D6) 5 12.70 (s, 1H), 11.37 (s, 1H), 8.17 (s, 1H), 7.87 (d, J= 8.4 Hz, 2H), 7.78 (m, 1H), 7.31 (m, 1H), 7.17 (m, 1H), 4.28 (s, 1H), 4.01 (s, 3H), 1.70 (m, 2H), 1.39 (s, 9H), 1.23 (m, J= 3.8 Hz, 13H), 1.10 - 0.46 (m, 12H). M+H = 617.52.TABLE 1ID StructureCompound O1J. JL J\N N ^^ CIII 1 H HCompound 02O?hE A Jk JL Jx^i YNN ^^ CIII 1 H HCompound 03 0 SII 1 H HCompound 04II 1 H HCompound 051 1 A JIffV^N Nz^ / ^CI[1 1 H H1Compound 06r<^J^ N N '^^Cl|| 1 H HStructure

[0088] Compounds listed in Table 2 can be prepared according to the procedure of Example 3 A or Example 3B.Example 3A. Synthesis of 2-chloro-4-(3-(4-ethoxy-3-nitrobenzoyl)thioureido)benzoic acidStep 1: Synthesis of 4-ethoxy-3-nitrobenzoyl chloride

[0089] A stirring mixture of 4-ethoxy-3 -nitrobenzoic acid (1A, 150 mg, 710 pmol, leq), and thionyl chloride (311 pL, 4.26 mmol, 6 eq) was heated at 80 °C for 2 h. Excess thionyl chloride was removed under reduced pressure to afford 4-ethoxy-3-nitrobenzoyl chloride (Compound 2A).Compound 2A was used without further purification to the next step.Step 2: Synthesis of 4-ethoxy-3-nitrobenzoyl isothiocyanate

[0090] A stirring cold solution of Compound 2A in acetone (3 mL) maintained with an ice-bath was treated with potassium thiocyanate (138 mg, 1.42 mmol, 2 eq). The resulting mixture was stirred for 30 min. The mixture was allowed to warm to RT and stirred for 1 h. The solvent was removed under reduced pressure and 4-ethoxy-3-nitrobenzoyl isothiocyanate (Compound 3A) was used in the next step without further purification.Step 3: Synthesis of 2-chloro-4-(3-(4-ethoxy-3-nitrobenzoyl)thioureido)benzoic acid

[0091] A solution of Compound 3A in ethyl acetate (5 mL) was treated with 4-amino-2-chlorobenzoic acid (Compound 4A, 122 mg, 710 pL, 1 eq). The resulting mixture was heated toreflux for 2 h. The mixture was then allowed to cool to RT and the solvent was removed under reduced pressure. The mixture was processed using reverse phase HPLC (water: acetonitrile;40: 60) to afford 2-chloro-4-(3-(4-ethoxy-3-nitrobenzoyl)thioureido)benzoic acid (Compound 11) as an off-white solid (200 mg, 67%). LC-MS (ESI): M / Z = 422 (M-l)’.Example 3B. Synthesis of 2-chloro-4-(3-(2-methoxybenzoyl)thioureido)benzoic acidStep 1: Synthesis of 2-methoxybenzoyl isothiocyanate

[0092] To a solution of thionyl chloride (7.15 g, 4.38 mL, 10 eq, 60.1 mmol) was added 2-methoxybenzoic acid (914 mg, 1 eq, 6.01 mmol) and the reaction mixture was refluxed for 1 hours. The excess thionyl chloride was removed from the mixture using CHCh trituration. The crude acid chloride material was used in the next step without any purification. To the acid chloride was added acetone and then potassium thiocyanate (1.17 g, 2 eq, 12.0 mmol) at ice bath temperature. The resulting mixture was stirred for 30 min at 56 °C. The solvent was removed and used for the next step without any further purification.Step 2: Synthesis of 2-chloro-4-(3-(2-methoxybenzoyl)thioureido)benzoic acid

[0093] To a solution of 2-methoxybenzoyl isothiocyanate (1.16 g, 1 eq, 6 mmol) in ethyl acetate (5 mL) was added the 4-amino-2-chlorobenzoic acid (1.03 g, 1 eq, 6 mmol) and the resulting mixture was refluxed for 2 hours. The title compound was isolated after removal of solvent.TABLE 2ID Structure Compound 11 Cl 0 0 SII 1 H HNO2Compound 12 0 0 S AV tlH rW AV1 II J H HCompound 13 0ClCompound 14 0 0 SCompound 15 0o sCompound 16 "xo 00 S r<= |A^'OH N N H I H H 1'“ZOHID StructureCompound 17 Cl 0ClCompound 18xo 00 S C I OHH J H H 1o oNO2Compound 19IZ TZCompound 207' o Q— oO S 2°=oI LJH HaCl

[0094] Compounds listed in Table 3 can be prepared according to the procedure of Example 4.Example 4. Synthesis of 4-(3-(4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoyl)thioureido)-2-chlorobenzoic acidOStep 1: Synthesis of 2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethyl 4-methylbenzenesulfonate

[0095] A solution of 2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethan-l-ol (6A, 285 mg, 2.06 mmol, 1 eq) in dry DCM (3 mL) was cooled to 0 °C using ice bath and then treated with DMAP (8 mg, 62 pmol, 3 mol %), and pyridine (667 pL, 8.25 mmol, 4 eq). Subsequently, 4-methylbenzenesulfonyl chloride (786 mg, 4.13 mmol, 2 eq) in dry DCM (5 mL) was added dropwise and the resulting mixture stirred at rt for 16 h. Solvent was removed under reduced pressure and the remainder was purified by silica column (hexanes: EtO AC; 60:40) to afford 2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethyl 4-methylbenzenesulfonate (7A) as a colorless oil (216 mg, 36%).Step 2: Synthesis of methyl 4-(2-)3-(but-3-yn-l-yl)-3H-diazirine-3-yl)ethoxy)benzoate

[0096] A solution of methyl 4-hydroxybenzoate (8A, 121 mg, 794 pmol, 1.1 eq) in dry DMF (5 mL) was cooled to 0 °C using ice bath and then treated with K2CO3 (299 mg, 2.17 mol, 3 eq). The resulting mixture was stirred at 0 °C for 20 min. Subsequently, 2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethyl 4-methylbenzenesulfonate (7A, 211 mg, 722 pmol, 1 eq) was added to the mixture, and stirred at 50 °C for 16 h under nitrogen atmosphere. Solvent was removed under reduced pressure and the remainder was purified by reverse phase HPLC (water: acetonitrile; 20:80) to obtain methyl 4-(2-)3-(but-3-yn-l-yl)-3H-diazirine-3-yl)ethoxy)benzoate (9A) as a clear oil (136 mg, 69%).Step 3: Synthesis of 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoic acid

[0097] A solution of methyl 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoate (9A, 136 mg, 499 pmol, 1 eq) in THF and MeOH (6 mL, 1:1) in a 4-dram vial was treated with a solution of LiOH H2O (126 mg, 3.0 mmol, 6 eq) dissolved in DI water (3 mL) dropwise addition. The resulting mixture was stirred at rt for 20 h. Solvent was removed under reduced pressure and the remainder was combined with water. IN HC1 was added to adjust the pH to 4. Material precipitated out which was extracted in DCM. Solvent was removed under reduced pressure to afford 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoic acid (10A) as a white powder (110 mg, 85%).Step 4: Synthesis of 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoyl chloride

[0098] A mixture of 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoic acid (10A, 43.5 mg, 168 pmol, leq), and thionyl chloride (111 pL, 1.52 mmol, 9 eq) was stirred at 80 °C for 2 h. Excess thionyl chloride was removed under reduced pressure to afford 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoyl chloride (Compound HA). Compound HA was used without further purification to the next step.Step 5: Synthesis of 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoyl isothiocyanate

[0099] A solution of Compound 11A in acetone (3 mL) was cooled to 0 °C using ice bath and treated with potassium thiocyanate (32.7 mg, 337 pmol, 2 eq). The resulting mixture was stirred for 30 min. Then the mixture was allowed to warm to rt and stirred for another 1 h. Subsequently, solvent was removed under reduced pressure to afford 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoyl isothiocyanate (Compound 12A). The product was used in the next step without further purification.Step 6: Synthesis of 4-(3-(4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoyl)thioureido)-2-chlorobenzoic acid

[0100] A solution of 4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoyl isothiocyanate (12A) in ethyl acetate (3 mL) was treated with 4-amino-2-chlorobenzoic acid (28.9 mg, 168 pmol, 1 eq). The resulting mixture was refluxed for 16 h and then allowed to cool to RT. Solvent was removed under reduced pressure and the resultant was purified by reverse phase HPLC (water: acetonitrile; 30:70) to afford 4-(3-(4-(2-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)ethoxy)benzoyl)thioureido)-2-chlorobenzoic acid (101) as an off-white solid (43 mg, 55%). LC-MS (ESI): M / Z = 469 (M-l)’.TABLE 3ID StructureCompound 102NGNH H V0Y Y Y |0 s / ° °\ Compound 103CIN=NO S X^yOHOCompound 104N.=N IjYH H?'o s / O OH Compound 105NVNA H H0Y Y Y 1o s \A / OH0Example 5A. Enhanced total IgG and HAI neutralizing titers to Influenza vaccination in nonhuman primate (NHP)

[0101] To demonstrate pre-clinical efficacy, Compound 11 was formulated with alhydrogel (aluminum hydroxide, “alum”) and combined with the QIV influenza vaccination (the formulation of compound 11 with alum combined with QIV influenza vaccination is referred to herein as QIV+alum-11 or QIV+alhydrogel-11). QIV+alum-11 was compared to QIV alone along with controls of QlV+alum (alum combined with QIV influenza vaccination) and QIV+Addavax (Addavax combined with QIV influenza vaccination). Addavax is a research analog for the adjuvant MF59 which is currently licensed for use with QIV. N=6 NHP per group were given a prime dose followed 28 days later by a boost dose. Plasma / serum was harvested longitudinally (baseline, 2- / 7- / 14- / 21-days post-prime, 2- / 7- / 14- / -21 days post-boost, and 180 days post-prime). Total antiinfluenza IgG binding titers and neutralizing HAI titers were quantified. QIV+alum-11 induced significantly higher IgG binding titers compared to QIV alone (Figure 1A), QIV+Addavax (FigureIB), and QlV+alum (Figure 1C). Neutralizing HAI titers were also significantly elevated at Day 180 in QIV-alum-11 compared to QIV alone and QIV+Addavax (Figure ID).Example 5B. Alum-11 induces a balanced inflammatory and anti-inflammatory cytokine profiles that is correlated with increased Ab titers

[0102] Profiling of cytokines in the plasma of NHP revealed that QIV+alum-11 induced a balanced inflammatory and anti-inflammatory state, which was distinct from the profiles induced by alum and Addavax (Figure 2A). Importantly, this balanced cytokine profile remains elevated in alum- 11 at the pre-boost timepoint, where levels of these cytokines are significant positive correlates of antiinfluenza Ab titers at Day 180 (Figure 2B). These data demonstrate a unique adjuvant effect of Compound 11 compared to alum alone and Addavax, that is mechanistically linked to heightened vaccine efficacy.

[0103] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A compound of Formula (I),or a pharmaceutically acceptable salt thereof, wherein:R1is hydrogen, or C1-C3 alkyl;R2is hydrogen, or halo; andR3is C16-C19 alkyl, or C 16-C19 alkenyl.

2. The compound of claim 1, wherein R1is hydrogen.

3. The compound of claim 1, wherein R1is -CH3.

4. The compound of any one of claims 1 to 3, wherein R2is hydrogen or chloro.

5. The compound of claim 4, wherein R2is hydrogen.

6. The compound of claim 4, wherein R2is chloro.

7. The compound of any one of claims 1 to 6, wherein R3is C16-C19 alkyl.

8. The compound of claim 7, wherein R3is C17 alkyl.

9. The compound of any one of claims 1 to 6, wherein R3is C16-C19 alkenyl.

10. The compound of claim 9, wherein R3is C17 alkenyl.

11. The compound of claim 1, wherein the compound is:Opharmaceutically acceptable salt thereof.

12. A compound of Formula (II),(II)or a pharmaceutically acceptable salt thereof, wherein:R3is C16-C19 alkyl, or C16-C19 alkenyl;i) R4is Ci-Ce alkoxyl, or halo; and R5and R6are hydrogen, orii) R4is hydrogen; and R5and R6are halo.

13. The compound of claim 12, wherein R4is Ci-Ce alkoxyl.

14. The compound of claim 12, wherein R4is halo.

15. The compound of claim 12, wherein R5and R6are each independently fluoro, chloro, or bromo.

16. The compound of claim 12, wherein R5and R6are each chloro.

17. The compound of any one of claims 12 to 16, wherein R3is C16-C19 alkyl.

18. The compound of claim 17, wherein R3is C17 alkyl.

19. The compound of any one of claims 12 to 16, wherein R3is C16-C19 alkenyl.

20. The compound of claim 19, wherein R3is C17 alkenyl.

21. The compound of claim 12, wherein the compound is:or a pharmaceutically acceptable salt thereof.

22. A compound of Formula (III),(Ill)or a pharmaceutically acceptable salt thereof, wherein:R1is hydrogen, or C1-C3 alkyl; andR2is hydrogen, or halo.

23. The compound of claim 22, wherein R1is hydrogen.

24. The compound of claim 22, wherein R1is -CH3.

25. The compound of any one of claims 22 to 24, wherein R2is hydrogen or chloro.

26. The compound of claim 25, wherein R2is hydrogen.

27. The compound of claim 25, wherein R2is chloro.

29. A pharmaceutical composition comprising a compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient.

30. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition further comprises water.

31. A method of increasing the effectiveness of a vaccine in a subject, comprising:administering a compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, to the subject in a dose and schedule configured to increase the effectiveness of the vaccine, wherein the subject is administered with the vaccine.

32. The method of claim 31, wherein the dose is between 1.0 - 5.0 mg / kg.

33. The method of claim 32, wherein the dose is about 2.5 mg / kg.

34. The method of any one of claims 31 to 33, wherein the compound, or a pharmaceutically acceptable salt thereof, is administered concurrently with the vaccine.

35. The method of claim 34, wherein the compound is an immune enhancer for the vaccine.

36. The method of any one of claims 31 to 35, wherein the compound is administered at least a day after administering the vaccine.

37. The method of claim 36, wherein the compound is administered at least 2 days after administering the vaccine.

38. The method of any one of claims 31 to 37, wherein the compound is administered a plurality of times in a regular interval after administering the vaccine.

39. The method of any one of claims 31 to 38, wherein the compound increases effectiveness of the vaccine by inducing innate immune responses in primary human monocyte derived dendritic cells (MDDCs).

40. The method of any one of claims 31 to 39, wherein the dose and schedule are sufficient to increase antigen-specific antibody titers at least 50% in the subject compared to a subject not receiving the compound after rechallenge.

41. The method of any one of claims 31 to 40, wherein the dose and schedule are sufficient to increase the effectiveness of the vaccine by activating cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) pathway.

42. The method of any one of claims 31 to 41, wherein the effectiveness of the vaccine is increased by increasing memory B cell population in the subject, optionally the memory B cell population is increased by at least 10%.

43. The method of any one of claims 31 to 42, wherein the effectiveness of the vaccine is increased by increasing memory B cell precursor population in the subject, optionally the memory B cell precursor population is increased by at least 10%.

44. A method of increasing immunity against an antigen in a subject having an immune response against the antigen, comprising:administering a compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, to the subject in a dose and schedule configured to induce innate immune responses in primary human monocyte derived dendritic cells (MDDCs) compared to a subject not being administered a compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof.

45. The method of claim 44, wherein the dose is between 1.0 - 5.0 mg / kg.

46. The method of claim 44 or 45, wherein the dose is about 2.5 mg / kg.

47. The method of any one of claims 44 to 46, wherein the compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, is administered before or during the immune response.

48. The method of any one of claims 44 to 47, wherein the schedule comprises administration at least a day before or after the immune response.

49. The method of any one of claims 44 to 48, wherein the schedule comprises administration at least 2 days before or after the immune response.

50. The method of any one of claims 44 to 49, wherein the dose and schedule are sufficient to wherein the dose and schedule are sufficient to increase antigen-specific antibody titers at least 50% in the subject compared to a subject not receiving the compound after re-exposure to the antigen.

51. The method of any one of claims 44 to 50, wherein the dose and schedule are sufficient to increase the effectiveness of the vaccine by activating cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) pathway.

52. The method of any one of claims 44 to 51, wherein the effectiveness of the vaccine is increased by increasing memory B cell population in the subject, optionally the memory B cell population is increased by at least 10%.

53. The method of any one of claims 44 to 52, wherein the effectiveness of the vaccine is increased by increasing memory B cell precursor population in the subject, optionally the memory B cell precursor population is increased by at least 10%.

54. A pharmaceutical composition, comprising: a vaccine composition and a compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, wherein the compound is present in the composition in a dose effective to increase effectiveness of the vaccine.

55. The pharmaceutical composition of claim 54, wherein the dose is between 1.0 - 5.0 mg / kg.

56. The pharmaceutical composition of claim 54 or 55, wherein the dose is about 2.5 mg / kg.

57. The pharmaceutical composition of any one of claims 54 to 56, wherein the dose is effective to induce innate immune responses in primary human monocyte derived dendritic cells (MDDCs) in a subject when administered.

58. The pharmaceutical composition of any one of claims 54 to 57, wherein the dose is effective to increase antigen-specific antibody titers at least 50% in a subject when administered, compared to a subject not receiving the composition.

59. The pharmaceutical composition of any one of claims 54 to 58, wherein the dose is sufficient to increase the effectiveness of the vaccine by activating cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) pathway.

60. The pharmaceutical composition of any one of claims 54 to 59, wherein the effectiveness of the vaccine is increased by increasing memory B cell population in the subject, optionally the memory B cell precursor population is increased by at least 10%.

61. The pharmaceutical composition of any one of claims 54 to 60, wherein the effectiveness of the vaccine is increased by increasing memory B cell precursor population in the subject, optionally the memory B cell precursor population is increased by at least 10%.

62. The compound of any one of claims 1 to 28 for use in increasing the effectiveness of a vaccine in a subject.

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