Imidazopyrimidine compounds and uses thereof

Abstract

The present disclosure provides compounds of Formula (I), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof. The compounds described herein are used as enhancers and/or modifiers of an immune response (e.g., innate and/or adaptive immune response), and are useful in treating and/or preventing a disease, as adjuvants in a vaccine for the disease, (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease), or as stand alone anti-infective or immune response modifying agents. Also provided in the present disclosure are pharmaceutical compositions, kits, methods, and uses including or using a compound described herein. ##STR00001##

Claims

1. A compound of Formula (I): ##STR00154## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1A is substituted or unsubstituted phenyl or substituted or unsubstituted benzyl, or substituted or unsubstituted 6-membered heteroaryl; R.sup.1 is halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OR.sup.a, or —N(R.sup.a1).sub.2; each instance of R.sup.A is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2; each instance of R.sup.B is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2; each instance of R.sup.a is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; each instance of R.sup.a1 is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, or two instances of R.sup.a1 are joined to form a substituted or unsubstituted, heterocyclic ring, or substituted or unsubstituted, heteroaryl ring; a is 1, 2, or 3; and b is 0, 1, 2, or 3.

2. The compound of claim 1, wherein the compound is of the formula: ##STR00155## or a pharmaceutically acceptable salt thereof, wherein: each instance of R.sup.2 is independently hydrogen, halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OR.sup.a, or —N(R.sup.a1).sub.2; each instance of R.sup.C is independently halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2; and c is 0, 1, 2, 3, 4, or 5.

3. The compound of claim 1, wherein R.sup.1 is halogen or substituted or unsubstituted C.sub.1-6 alkyl.

4. The compound of claim 1, wherein R.sup.1A is of the formula: ##STR00156##

5. The compound of claim 2, wherein at least one instance of R.sup.2 is substituted or unsubstituted C.sub.1-6 alkyl.

6. The compound of claim 5, wherein at least one instance of R.sup.2 is Me or Et.

7. The compound of claim 1, wherein R.sup.A is halogen, substituted or unsubstituted C.sub.1-6 alkyl, or —O (substituted or unsubstituted C.sub.1-6 alkyl).

8. The compound of claim 7, wherein R.sup.A is —F, Me, or —OMe.

9. The compound of claim 1, wherein a is 1.

10. The compound of claim 1, wherein b is 0.

11. The compound of claim 2, wherein c is 0.

12. The compound of claim 2, wherein c is 1.

13. The compound of claim 2, wherein R.sup.C is halogen or substituted or unsubstituted C.sub.1-6 alkyl.

14. The compound of claim 1, wherein the compound is of the formula: ##STR00157## ##STR00158## ##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165## or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising a compound of claim 1, and a pharmaceutically acceptable excipient.

16. The compound of claim 1, wherein R.sup.1A is of formula: wherein: ##STR00166## each instance of R.sup.c is independently halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(=O)R.sup.a, —C(=O)OR.sup.a, —C(=O)N(R.sup.a1).sub.2, or —NO.sub.2.

17. The compound of claim 2, wherein the compound is of the formula: ##STR00167## or a pharmaceutically acceptable salt thereof.

18. The compound of claim 2, wherein the compound is of the formula: ##STR00168## or a pharmaceutically acceptable salt thereof.

19. The compound of claim 2, wherein the compound is of the formula: ##STR00169## or a pharmaceutically acceptable salt thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

(2) FIGS. 1A-1D are scatter plots showing the stimulation of adult peripheral blood mononuclear cells (PBMCs) with hit compounds from a TNF AlphaLISA screen. The PBMCs were stimulated for 18 hours. (FIGS. 1A-1C) Individual scatter plots from 3 unique adult PBMC samples/donors, run in duplicate (x and y axes). (FIG. 1D) Venn diagram comparing hits between 3 unique adult donors, with 24 hits identified that passed analysis threshold for at least 2 donors and ˜10 hits common to all with a rZ>3 for each donor.

(3) FIGS. 2A-2B demonstrate that compounds 037 and 038 induce TNF and IL-1β from adult and elderly PBMCs. (FIG. 2A) Human adult (top) and elderly (bottom) PBMCs stimulated for 18 hours with the TLR7/8 agonist R848 and 24 confirmed hit compounds from TNF AlphaLISA screen. TNF (left panels) and IL-1β (right panels) were assessed in cell-free supernatants by ELISA. Data are shown as median and interquartile range of N=5. (FIG. 2B) Compound 037 and 038, both small molecules within the same class, were identified as the most potent inducers of innate immune cytokines.

(4) FIGS. 3A-3B depict the potency and efficacy of the 037 compound as compared to R848. (FIG. 3A) TNF production measured by ELISA after stimulation of human adult peripheral blood mononuclear cells (PBMCs) with R848 or compound 037 at seven different concentrations (0.05 μM-33 μM) for 18 hours (N=8-12). (FIG. 3B) TNF production measured by ELISA after stimulation of PBMCs with compound 037 and R848 at a concentration of 10 μM. Compound R848 is a small molecule imidazoquinoline that activates human leukocytes via PRRs (TLR7/8 and inflammasome).

(5) FIG. 4 are radar plots demonstrating that compound 037 induced a broad concentration-dependent cytokine response with distinct potency, efficacy and cytokine pattern as compared with R848. Shown is a 9-plex multiplex cytokine assay after stimulation of adult peripheral blood mononuclear cells (PBMCs) with compound 037 or R848 at four different concentrations (0.1 μM-33 μM). Radar plots of immune polarizing cytokine production from human adult PBMCs stimulated for 18 hours in 10% autologous plasma, represented as fold-change over RPMI unstimulated control (N=4).

(6) FIGS. 5A-5B demonstrate that while compound 037 induces robust production of TNF-α from human primary peripheral blood mononuclear cells, it demonstrates little activity towards THP1-Lucia cells via NF-κB-driven expression of luciferase. (FIG. 5A) Human adult PBMCs were stimulated for 18 hours with compound 037 or R848 at 33 μM. Production of human TNF was assessed in cell-free supernatants by ELISA (N=5). (FIG. 5B) in comparison to the positive control compound R848, 037 does not substantially activate THP1 cells, as measured by NF-κB-driven expression of luciferase (N=4). Results are shown as the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) **p<0.01 determined by repeated measure one-way ANOVA with Dunnett's multiple comparison test on log-transformed data and comparing each compound with DMSO (control condition).

(7) FIGS. 6A-6B shows the activity of compound 037 and commercially available compound 037 analogs (imidazopyrimidines) on human adult PBMCs. Human adult PBMCs were stimulated for 18 hours with compound 037 and its analogs at 33 μM. Production of human TNF was assessed in cell-free supernatants by ELISA. Results are shown as the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) and expressed as percentage TNF production (TNF %) as compared to R848 (FIG. 6A) or TNF concentration in ng/ml (FIG. 6B) (N=3-5). **p<0.01 determined by repeated measure one-way ANOVA with Dunnett's multiple comparison test on log-transformed data and comparing each compound with DMSO (control condition).

(8) FIGS. 7A-7B shows the activity of compound 037 and de novo synthesized compound 037 analogs (imidazopyrimidines) on human adult PBMCs. Human adult PBMCs were stimulated for 18 hours with compound 037 and its analogs at 33 μM. Production of human TNF was assessed in cell-free supernatants by ELISA. Results are shown as the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) and expressed as percentage TNF production (TNF %) as compared to R848 (FIG. 7A) or TNF concentration in ng/ml (FIG. 7B) (N=5). Analogs are ranked left to right by TNF % (compound 037 on the far left). Compound 037 analogs have been classified in the following categories according to TNF %: high activity—class A: TNF %>15%; medium activity—class B: 1%<TNF %≤15%; low activity—class C: TNF %≤1%. **p<0.01 determined by repeated measure one-way ANOVA with Dunnett's multiple comparison test on log-transformed data and comparing each compound with DMSO (control condition).

(9) FIG. 8 demonstrates that the compound 037 analog 02-144-3 activates both human PBMCs and mouse splenocytes. Splenocytes isolated from 6-8 weeks old C57BL/6 mice and human adult PBMCs were stimulated for 18 hours with compound 037 and its analogs at 33 μM. Production of murine IL-6 and human TNF was assessed in cell-free supernatants by ELISA. Results are presented as scatter plot in which each dot represents median production of human TNF (N=3-5) or murine IL-6 (N=6) for each compound.

(10) FIG. 9 shows correlation between in vitro and in vivo activities of compound 037 analogs. 6-8 weeks old C57BL/6 mice were immunized on Day 0 (prime) and Day 28 (boost) with rHA alone or formulated with compound 02-184-02 (inactive analog), compound 037, compound 02-144-3 (analog that was active on both human and murine cells) or alum. Serum samples were collected at Day 28 (pre-boost) and Day 42 (14 post-boost) and anti-rHA IgG titers were measured by ELISA. Results are shown as the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) of 10 mice per group. *p<0.05 and **p<0.01 determined by Kruskal-Wallis with Dunn's multiple comparison test.

(11) FIG. 10 demonstrates that imidazopyrimidines induce TNF production in a human newborn whole blood assay. Human newborn cord blood was anticoagulated with pyrogen-free heparin and stimulated with compound 037 analog family or R848 at 11 μM for 18 hours. After centrifugation, supernatants were collected and cryopreserved prior to measurement of TNF production by ELISA. N=5. **p<0.01 determined by repeated measure one-way ANOVA with Dunn's multiple comparison test on log-transformed data and comparing each compound with DMSO (control condition).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

(12) Described herein are compounds of Formula (I), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof. The compounds described herein are enhancers and/or modifiers of an immune response (e.g., innate and/or adaptive immune response), and/or adjuvants in a vaccine for a disease, e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease, or as stand alone anti-infective or immune response modifying agents. The compounds may be useful in treating or preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease) in a subject in need thereof. Also provided are pharmaceutical compositions, kits, and uses including a compound described herein.

(13) Compounds

(14) One aspect of the present disclosure relates to the compounds described herein. The compounds described herein are enhancers and/or modifiers of an immune response (e.g., innate and/or adaptive immune response), and/or adjuvants in a vaccine for a disease, e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease, or as stand alone anti-infective or immune response modifying agents. In certain embodiments, a compound described herein is an immunomodulator. In certain embodiments, a compound described herein is a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(15) In one aspect, the present disclosure provides compounds of Formula (I):

(16) ##STR00017##
and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof, wherein:

(17) R.sup.1A is substituted or unsubstituted phenyl or substituted or unsubstituted benzyl, or substituted or unsubstituted 6-membered heteroaryl;

(18) R.sup.1 is hydrogen, halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OR.sup.a, or —N(R.sup.a1).sub.2;

(19) each instance of R.sup.A is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2;

(20) each instance of R.sup.B is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2;

(21) each instance of R.sup.a is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom;

(22) each instance of R.sup.a1 is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, or two instances of Rai are joined to form a substituted or unsubstituted, heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

(23) a is 0, 1, 2, or 3; and

(24) b is 0, 1, 2, or 3;

(25) wherein the compound is not of the formula:

(26) ##STR00018##

(27) Formula (I) includes substituent R.sup.1 on the phenyl ring. In certain embodiments, R.sup.1 is hydrogen. In certain embodiments, R.sup.A1 is halogen (e.g., F, Cl, Br, or I). In certain embodiments, R.sup.1 is substituted or unsubstituted C.sub.1-6 alkyl (e.g., substituted or unsubstituted Me or substituted or unsubstituted Et). In certain embodiments, R.sup.1 is Me. In certain embodiments, R.sup.1 is Et. In certain embodiments, R.sup.1 is —OR.sup.a (e.g., —OH, —O (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —OMe, —OEt, —OPr, —OBu, or —OBn). In certain embodiments, R.sup.1 is —N(R.sup.a1).sub.2 (e.g., —NH.sub.2, —NH (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —NHMe), or —N (substituted or unsubstituted C.sub.1-6 alkyl)-(substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —NMe.sub.2)).

(28) Formula (I) includes substituent R.sup.1A. In certain embodiments, R.sup.1A is substituted or unsubstituted phenyl. In certain embodiments, R.sup.1A is of the formula:

(29) ##STR00019##
wherein each instance of R.sup.C is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2; and c is 0, 1, 2, 3, 4, or 5. In certain embodiments, at least one instance of R.sup.C is halogen. In certain embodiments, at least one instance of R.sup.C is F. In certain embodiments, at least one instance of R.sup.C is Br. In certain embodiments, at least one instance of R.sup.C is Cl. In certain embodiments, at least one instance of R.sup.C is I. In certain embodiments, at least one instance of R.sup.C is substituted or unsubstituted alkyl (e.g., C.sub.1-6 alkyl). In certain embodiments, at least one instance of R.sup.C is substituted or unsubstituted Me. In certain embodiments, at least one instance of R.sup.C is Me. In certain embodiments, at least one instance of R.sup.C is substituted methyl (e.g., —CF.sub.3 or Bn). In certain embodiments, at least one instance of R.sup.C is —CF.sub.3. In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, at least one instance of R.sup.C is substituted or unsubstituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, at least one instance of R.sup.C is substituted or unsubstituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.C is substituted or unsubstituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, at least one instance of R.sup.C is substituted or unsubstituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.C is —OR.sup.a (e.g., —OH or —O (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —OMe)). In certain embodiments, at least one instance of R.sup.C is —OMe. In certain embodiments, at least one instance of R.sup.C is —N(R.sup.a1).sub.2 (e.g., —NH.sub.2, —NH (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —NHMe), or —NMe.sub.2). In certain embodiments, at least one instance of R.sup.C is —SR.sup.a (e.g., —SH, —S (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —SMe, —SEt, —SPr, —SBu, or —SBn), or —S (substituted or unsubstituted phenyl) (e.g., —SPh)). In certain embodiments, at least one instance of R.sup.C is —CN. In certain embodiments, at least one instance of R.sup.A is —C(═O)R.sup.a (e.g., —C(═O)(substituted or unsubstituted alkyl) or —C(═O)(substituted or unsubstituted phenyl)). In certain embodiments, at least one instance of R.sup.C is —C(═O)OR.sup.a (e.g., —C(═O)OH, —C(═O)O (substituted or unsubstituted alkyl) (e.g., —C(═O)OMe), or —C(═O)O (substituted or unsubstituted phenyl)).

(30) In certain embodiments, at least one instance of R.sup.A is —C(═O)N(R.sup.a1).sub.2 (e.g., —C(═O)NH.sub.2, —C(═O)NH (substituted or unsubstituted alkyl), —C(═O)NH (substituted or unsubstituted phenyl), —C(═O)N (substituted or unsubstituted alkyl)-(substituted or unsubstituted alkyl), or —C(═O)N (substituted or unsubstituted phenyl)-(substituted or unsubstituted alkyl)). In certain embodiments, at least one instance of R.sup.C is —NO.sub.2.

(31) In certain embodiments, R.sup.1A is of the formula:

(32) ##STR00020##
In certain embodiments, R.sup.1A is of the formula:

(33) ##STR00021##

(34) In certain embodiments, R.sup.1A is substituted or unsubstituted benzyl. In certain embodiments, R.sup.1A is of the formula:

(35) ##STR00022##
wherein each instance of R.sup.2 is independently hydrogen, halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OR.sup.a, or —N(R.sup.a1).sub.2; each instance of R.sup.C is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2; and c is 0, 1, 2, 3, 4, or 5. In certain embodiments, R.sup.1A is of the formula:

(36) ##STR00023##
wherein R.sup.2 is hydrogen, halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OR.sup.a, or —N(R.sup.a1).sub.2; each instance of R.sup.C is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2; and
c is 0, 1, 2, 3, 4, or 5. In certain embodiments, R.sup.1A is of the formula:

(37) ##STR00024##

(38) In certain embodiments, at least one instance of R.sup.2 is hydrogen. In certain embodiments, at least one instance of R.sup.2 is halogen (e.g., F, Cl, Br, or I). In certain embodiments, at least one instance of R.sup.2 is substituted or unsubstituted C.sub.1-6 alkyl (e.g., substituted or unsubstituted Me or substituted or unsubstituted Et). In certain embodiments, at least one instance of R.sup.2 is Me. In certain embodiments, at least one instance of R.sup.2 is substituted or unsubstituted Me. In certain embodiments, R.sup.2 is —CH.sub.2OH. In certain embodiments, at least one instance of R.sup.2 is Et. In certain embodiments, at least one instance of R.sup.2 is —OR.sup.a (e.g., —OH, —O (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —OMe, —OEt, —OPr, —OBu, or —OBn). In certain embodiments, at least one instance of R.sup.2 is —N(R.sup.a1).sub.2 (e.g., —NH.sub.2, —NH (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —NHMe), or —N (substituted or unsubstituted C.sub.1-6 alkyl)-(substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —NMe.sub.2)). In certain embodiments, R.sup.1A is of the formula:

(39) ##STR00025##

(40) In certain embodiments, R.sup.1A is substituted or unsubstituted 6-membered heteroaryl.

(41) In certain embodiments, R.sup.1A is substituted or unsubstituted pyridine, substituted or unsubstituted pyrimidine, or substituted or unsubstituted pyrazine.

(42) Formula (I) may include one or more instances of R.sup.A on the imidazopyrimidine ring. When Formula (I) includes two or more instances of R.sup.A, any two instances of R.sup.A may be the same or different from each other. In certain embodiments, a is 0. In certain embodiments, a is 1. In certain embodiments, a is 2. In certain embodiments, a is 3. In certain embodiments, at least one instance of R.sup.A is halogen. In certain embodiments, at least one instance of R.sup.A is F. In certain embodiments, at least one instance of R.sup.A is Br, Cl, or I. In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, at least one instance of R.sup.A is Me. In certain embodiments, at least one instance of R.sup.A is substituted methyl (e.g., —CF.sub.3 or Bn). In certain embodiments, at least one instance of R.sup.A is Et. In certain embodiments, at least one instance of R.sup.A1 is substituted ethyl. In certain embodiments, at least one instance of R.sup.A is n-Pr. In certain embodiments, at least one instance of R.sup.A is i-Pr. In certain embodiments, at least one instance of R.sup.A is substituted propyl (e.g., perfluoropropyl). In certain embodiments, at least one instance of R.sup.A is Me, Et, or n-Pr. In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, at least one instance of R.sup.A is substituted or unsubstituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.A is —OR.sup.a (e.g., —OH or —O (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —OMe)). In certain embodiments, at least one instance of R.sup.A is —OMe. In certain embodiments, at least one instance of R.sup.A is —N(R.sup.a1).sub.2 (e.g., —NH.sub.2, —NH (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —NHMe), or —NMe.sub.2). In certain embodiments, at least one instance of R.sup.A is —SR.sup.a (e.g., —SH, —S (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —SMe, —SEt, —SPr, —SBu, or —SBn), or —S (substituted or unsubstituted phenyl) (e.g., —SPh)). In certain embodiments, at least one instance of R.sup.A is —CN. In certain embodiments, at least one instance of R.sup.A is —C(═O)R.sup.a (e.g., —C(═O)(substituted or unsubstituted alkyl) or —C(═O)(substituted or unsubstituted phenyl)). In certain embodiments, at least one instance of R.sup.A is —C(═O)OR.sup.a (e.g., —C(═O)OH, —C(═O)O (substituted or unsubstituted alkyl) (e.g., —C(═O)OMe), or —C(═O)O (substituted or unsubstituted phenyl)). In certain embodiments, at least one instance of R.sup.A is —C(═O)N(R.sup.a1).sub.2 (e.g., —C(═O)NH.sub.2, —C(═O)NH (substituted or unsubstituted alkyl), —C(═O)NH (substituted or unsubstituted phenyl), —C(═O)N (substituted or unsubstituted alkyl)-(substituted or unsubstituted alkyl), or —C(═O)N (substituted or unsubstituted phenyl)-(substituted or unsubstituted alkyl)). In certain embodiments, at least one instance of R.sup.A is —NO.sub.2.

(43) Formula (I) may include one or more instances of R.sup.B on the phenyl ring. When Formula (I) includes two or more instances of R.sup.B, any two instances of R.sup.B may be the same or different from each other. In certain embodiments, b is 0. In certain embodiments, b is 1. In certain embodiments, b is 2. In certain embodiments, b is 3. In certain embodiments, at least one instance of R.sup.B is halogen. In certain embodiments, at least one instance of R.sup.B is F. In certain embodiments, at least one instance of R.sup.B is Br, Cl, or I. In certain embodiments, at least one instance of R.sup.B is substituted or unsubstituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, at least one instance of R.sup.B is Me. In certain embodiments, at least one instance of R.sup.B is substituted methyl (e.g., —CF.sub.3 or Bn). In certain embodiments, at least one instance of R.sup.B is Et. In certain embodiments, at least one instance of R.sup.B is substituted ethyl. In certain embodiments, at least one instance of R.sup.B is n-Pr. In certain embodiments, at least one instance of R.sup.B is i-Pr. In certain embodiments, at least one instance of R.sup.B is substituted propyl (e.g., perfluoropropyl). In certain embodiments, at least one instance of R.sup.B is Me, Et, or n-Pr. In certain embodiments, at least one instance of R.sup.B is substituted or unsubstituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, at least one instance of R.sup.B is substituted or unsubstituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, at least one instance of R.sup.B is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, at least one instance of R.sup.B is substituted or unsubstituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.B is substituted or unsubstituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, at least one instance of R.sup.B is substituted or unsubstituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.B is —OR.sup.a (e.g., —OH or —O (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —OMe)). In certain embodiments, at least one instance of R.sup.B is —OMe. In certain embodiments, at least one instance of R.sup.B is —N(R.sup.a1).sub.2 (e.g., —NH.sub.2, —NH (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —NHMe), or —NMe.sub.2). In certain embodiments, at least one instance of R.sup.B is —SR.sup.a (e.g., —SH, —S (substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —SMe, —SEt, —SPr, —SBu, or —SBn), or —S (substituted or unsubstituted phenyl) (e.g., —SPh)). In certain embodiments, at least one instance of R.sup.B is —CN. In certain embodiments, at least one instance of R.sup.B is —C(═O)R.sup.a (e.g., —C(═O)(substituted or unsubstituted alkyl) or —C(═O)(substituted or unsubstituted phenyl)). In certain embodiments, at least one instance of R.sup.B is —C(═O)OR.sup.a (e.g., —C(═O)OH, —C(═O)O (substituted or unsubstituted alkyl) (e.g., —C(═O)OMe), or —C(═O)O (substituted or unsubstituted phenyl)). In certain embodiments, at least one instance of R.sup.B is —C(═O)N(R.sup.a1).sub.2 (e.g., —C(═O)NH.sub.2, —C(═O)NH (substituted or unsubstituted alkyl), —C(═O)NH (substituted or unsubstituted phenyl), —C(═O)N (substituted or unsubstituted alkyl)-(substituted or unsubstituted alkyl), or —C(═O)N (substituted or unsubstituted phenyl)-(substituted or unsubstituted alkyl)). In certain embodiments, at least one instance of R.sup.B is —NO.sub.2.

(44) Formula (I) may include one or more instances of R.sup.a. In certain embodiments, at least one instance of R.sup.a is hydrogen. In certain embodiments, at least one instance of R.sup.a is substituted or unsubstituted acyl (e.g., —C(═O)Me). In certain embodiments, at least one instance of R.sup.a is substituted or unsubstituted alkyl (e.g., C.sub.1-6 alkyl). In certain embodiments, at least one instance of R.sup.a is substituted or unsubstituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, at least one instance of R.sup.a is substituted or unsubstituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, at least one instance of R.sup.a is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, at least one instance of R.sup.a is substituted or unsubstituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.a is substituted or unsubstituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, at least one instance of R.sup.a is substituted or unsubstituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.a is a nitrogen protecting group when attached to a nitrogen atom (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In certain embodiments, at least one instance of R.sup.a is an oxygen protecting group when attached to an oxygen atom (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In certain embodiments, at least one instance of R.sup.a is a sulfur protecting group when attached to a sulfur atom.

(45) Formula (I) may include one or more instances of Rai. In certain embodiments, at least one instance of Rai is hydrogen. In certain embodiments, at least one instance of Rai is substituted or unsubstituted acyl (e.g., —C(═O)Me). In certain embodiments, at least one instance of Rai is substituted or unsubstituted alkyl (e.g., C.sub.1-6 alkyl). In certain embodiments, at least one instance of Rai is substituted or unsubstituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, at least one instance of Rai is substituted or unsubstituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, at least one instance of Rai is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, at least one instance of Rai is substituted or unsubstituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of Rai is substituted or unsubstituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, at least one instance of R.sup.a1 is substituted or unsubstituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of Rai is a nitrogen protecting group when attached to a nitrogen atom (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In certain embodiments, two instances of Rai are joined to form a substituted or unsubstituted, heterocyclic ring (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, two instances of Rai are joined to form a substituted or unsubstituted, heteroaryl ring (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur).

(46) In certain embodiments, the compound of Formula (I) is of the formula:

(47) ##STR00026##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein:

(48) each instance of R.sup.2 is independently hydrogen, halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OR.sup.a, or —N(R.sup.a1).sub.2;

(49) each instance of R.sup.C is independently halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.a, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(R.sup.a1).sub.2, or —NO.sub.2; and

(50) c is 0, 1, 2, 3, 4, or 5.

(51) In certain embodiments, the compound of Formula (I) is of the formula:

(52) ##STR00027##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein:

(53) each instance of R.sup.C is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR.sup.a, —N(R.sup.a1).sub.2, —SR.sup.aa, —CN, —C(═O)R.sup.a, —C(═O)OR.sup.a, —C(═O)N(Rai).sub.2, or —NO.sub.2; and

(54) c is 0, 1, 2, 3, 4, or 5.

(55) In certain embodiments, the compound of Formula (I) is of the formula:

(56) ##STR00028##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(57) In certain embodiments, the compound of Formula (I) is of the formula:

(58) ##STR00029##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(59) In certain embodiments, the compound of Formula (I) is of the formula:

(60) ##STR00030##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(61) In certain embodiments, the compound of Formula (I) is of the formula:

(62) ##STR00031## ##STR00032##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(63) In certain embodiments, the compound of Formula (I) is not of the formula:

(64) ##STR00033## ##STR00034##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

(65) In certain embodiments, the compound of Formula (I) is of the formula:

(66) ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043##
or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.
Pharmaceutical Compositions, Kits, and Administration

(67) The present disclosure also provides pharmaceutical compositions comprising a compound described herein and optionally a pharmaceutically acceptable excipient. In certain embodiments, a compound described herein is of Formula (I).

(68) In certain embodiments, the compound described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, a therapeutically effective amount is an amount effective for enhancing an immune response (e.g., innate and/or adaptive immune response). In certain embodiments, a therapeutically effective amount is an amount effective for treating a disease (e.g., proliferative disease). In certain embodiments, a therapeutically effective amount is an amount effective for serving as an adjuvant in a vaccine for a disease, e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease, or as stand alone anti-infective or immune response modifying agents. In certain embodiments, a prophylactically effective amount is an amount effective for preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease). In certain embodiments, a prophylactically effective amount is an amount effective for enhancing an immune response (e.g., innate and/or adaptive immune response), and preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease).

(69) In certain embodiments, the effective amount is an amount effective for enhancing an immune response (e.g., innate and/or adaptive immune response) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%. In certain embodiments, the effective amount is an amount effective for enhancing an immune response (e.g., innate and/or adaptive immune response) by not more than 10%, not more than 20%, not more than 30%, not more than 40%, not more than 50%, not more than 60%, not more than 70%, not more than 80%, not more than 90%, not more than 95%, or not more than 98%.

(70) In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. The use of the compounds described herein in veterinary vaccine is also within the scope of the present disclosure. “A companion animal,” as used herein, refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal, such as a rodent (e.g., mouse, rat, guinea pig, and hamster), dog, pig, rabbit, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs). In certain embodiments, the subject is a fish or reptile.

(71) In certain embodiments, the cell is present in vitro. In certain embodiments, the cell is present in vivo.

(72) Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the compound described herein (i.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

(73) Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

(74) Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

(75) Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

(76) Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

(77) Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

(78) Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.

(79) Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

(80) Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

(81) Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

(82) Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

(83) Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

(84) Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

(85) Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

(86) Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone®, Kathon®, and Euxyl®.

(87) Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

(88) Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

(89) Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, Litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

(90) Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

(91) Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

(92) The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

(93) In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.

(94) Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

(95) Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

(96) Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

(97) The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.

(98) Dosage forms for topical and/or transdermal administration of a compound described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

(99) Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical Mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.

(100) Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

(101) A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

(102) Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

(103) Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

(104) Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

(105) Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

(106) A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.

(107) Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

(108) Compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

(109) The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject. In certain embodiments, the compound or composition is administered intradermally, intramuscularly, intravaginally, intravenously, intranasally, orally, subcutaneously, topically, and/or sublingually. In certain embodiments, the compound or composition is administered as a prophylactic. In certain embodiments, the compound or composition is administered as a combination therapy with another immunomodulatory agent, an immunomodulating antibody, an immunomodulating biologic, or an inhibitor of molecular pathways that limits immune responses. In certain embodiments, the immunomodulatory agent is a pattern recognition receptor agonist (e.g., an Alum, or a Toll-like receptor (TLR) Agonist). In certain embodiments, the immunomodulating antibody or immunomodulating biologic is a cytokine, chemokine or colony stimulating factor.

(110) The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the biological sample, tissue, or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.

(111) Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

(112) A compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in enhancing an immune response (e.g., innate and/or adaptive immune response) in a subject, biological sample, tissue, or cell), serving as an adjuvant in a vaccine for a disease, e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease, improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject, biological sample, tissue, or cell, or as stand alone anti-infective or immune response modifying agents. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional pharmaceutical agent, but not both.

(113) The compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease). Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

(114) The additional pharmaceutical agents include, but are not limited to, anti-proliferative agents, anti-cancer agents, anti-angiogenesis agents, anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, and a combination thereof. In certain embodiments, the additional pharmaceutical agent is an anti-proliferative agent (e.g., anti-cancer agent). In certain embodiments, the additional pharmaceutical agent is an anti-leukemia agent. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ADE, Adriamycin RDF (doxorubicin hydrochloride), Ambochlorin (chlorambucil), ARRANON (nelarabine), ARZERRA (ofatumumab), BOSULIF (bosutinib), BUSULFEX (busulfan), CAMPATH (alemtuzumab), CERUBIDINE (daunorubicin hydrochloride), CLAFEN (cyclophosphamide), CLOFAREX (clofarabine), CLOLAR (clofarabine), CVP, CYTOSAR-U (cytarabine), CYTOXAN (cyclophosphamide), ERWINAZE (Asparaginase Erwinia chrysanthemi), FLUDARA (fludarabine phosphate), FOLEX (methotrexate), FOLEX PFS (methotrexate), GAZYVA (obinutuzumab), GLEEVEC (imatinib mesylate), Hyper-CVAD, ICLUSIG (ponatinib hydrochloride), IMBRUVICA (ibrutinib), LEUKERAN (chlorambucil), LINFOLIZIN (chlorambucil), MARQIBO (vincristine sulfate liposome), METHOTREXATE LPF (methorexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), mitoxantrone hydrochloride, MUSTARGEN (mechlorethamine hydrochloride), MYLERAN (busulfan), NEOSAR (cyclophosphamide), ONCASPAR (Pegaspargase), PURINETHOL (mercaptopurine), PURIXAN (mercaptopurine), Rubidomycin (daunorubicin hydrochloride), SPRYCEL (dasatinib), SYNRIBO (omacetaxine mepesuccinate), TARABINE PFS (cytarabine), TASIGNA (nilotinib), TREANDA (bendamustine hydrochloride), TRISENOX (arsenic trioxide), VINCASAR PFS (vincristine sulfate), ZYDELIG (idelalisib), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is an anti-lymphoma agent. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ABVD, ABVE, ABVE-PC, ADCETRIS (brentuximab vedotin), ADRIAMYCIN PFS (doxorubicin hydrochloride), ADRIAMYCIN RDF (doxorubicin hydrochloride), AMBOCHLORIN (chlorambucil), AMBOCLORIN (chlorambucil), ARRANON (nelarabine), BEACOPP, BECENUM (carmustine), BELEODAQ (belinostat), BEXXAR (tositumomab and iodine 1131 tositumomab), BICNU (carmustine), BLENOXANE (bleomycin), CARMUBRIS (carmustine), CHOP, CLAFEN (cyclophosphamide), COPP, COPP-ABV, CVP, CYTOXAN (cyclophosphamide), DEPOCYT (liposomal cytarabine), DTIC-DOME (dacarbazine), EPOCH, FOLEX (methotrexate), FOLEX PFS (methotrexate), FOLOTYN (pralatrexate), HYPER-CVAD, ICE, IMBRUVICA (ibrutinib), INTRON A (recombinant interferon alfa-2b), ISTODAX (romidepsin), LEUKERAN (chlorambucil), LINFOLIZIN (chlorambucil), Lomustine, MATULANE (procarbazine hydrochloride), METHOTREXATE LPF (methotrexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), MOPP, MOZOBIL (plerixafor), MUSTARGEN (mechlorethamine hydrochloride), NEOSAR (cyclophosphamide), OEPA, ONTAK (denileukin diftitox), OPPA, R-CHOP, REVLIMID (lenalidomide), RITUXAN (rituximab), STANFORD V, TREANDA (bendamustine hydrochloride), VAMP, VELBAN (vinblastine sulfate), VELCADE (bortezomib), VELSAR (vinblastine sulfate), VINCASAR PFS (vincristine sulfate), ZEVALIN (ibritumomab tiuxetan), ZOLINZA (vorinostat), ZYDELIG (idelalisib), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is REVLIMID (lenalidomide), DACOGEN (decitabine), VIDAZA (azacitidine), CYTOSAR-U (cytarabine), IDAMYCIN (idarubicin), CERUBIDINE (daunorubicin), LEUKERAN (chlorambucil), NEOSAR (cyclophosphamide), FLUDARA (fludarabine), LEUSTATIN (cladribine), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ABRAXANE (paclitaxel albumin-stabilized nanoparticle formulation), AC, AC-T, ADE, ADRIAMYCIN PFS (doxorubicin hydrochloride), ADRUCIL (fluorouracil), AFINITOR (everolimus), AFINITOR DISPERZ (everolimus), ALDARA (imiquimod), ALIMTA (pemetrexed disodium), AREDIA (pamidronate disodium), ARIMIDEX (anastrozole), AROMASIN (exemestane), AVASTIN (bevacizumab), BECENUM (carmustine), BEP, BICNU (carmustine), BLENOXANE (bleomycin), CAF, CAMPTOSAR (irinotecan hydrochloride), CAPOX, CAPRELSA (vandetanib), CARBOPLATIN-TAXOL, CARMUBRIS (carmustine), CASODEX (bicalutamide), CEENU (lomustine), CERUBIDINE (daunorubicin hydrochloride), CERVARIX (recombinant HPV bivalent vaccine), CLAFEN (cyclophosphamide), CMF, COMETRIQ (cabozantinib-s-malate), COSMEGEN (dactinomycin), CYFOS (ifosfamide), CYRAMZA (ramucirumab), CYTOSAR-U (cytarabine), CYTOXAN (cyclophosphamide), DACOGEN (decitabine), DEGARELIX, DOXIL (doxorubicin hydrochloride liposome), DOXORUBICIN HYDROCHLORIDE, DOX-SL (doxorubicin hydrochloride liposome), DTIC-DOME (dacarbazine), EFUDEX (fluorouracil), ELLENCE (epirubicin hydrochloride), ELOXATIN (oxaliplatin), ERBITUX (cetuximab), ERIVEDGE (vismodegib), ETOPOPHOS (etoposide phosphate), EVACET (doxorubicin hydrochloride liposome), FARESTON (toremifene), FASLODEX (fulvestrant), FEC, FEMARA (letrozole), FLUOROPLEX (fluorouracil), FOLEX (methotrexate), FOLEX PFS (methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FU-LV, GARDASIL (recombinant human papillomavirus (HPV) quadrivalent vaccine), GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, GEMZAR (gemcitabine hydrochloride), GILOTRIF (afatinib dimaleate), GLEEVEC (imatinib mesylate), GLIADEL (carmustine implant), GLIADEL WAFER (carmustine implant), HERCEPTIN (trastuzumab), HYCAMTIN (topotecan hydrochloride), IFEX (ifosfamide), IFOSFAMIDUM (ifosfamide), INLYTA (axitinib), INTRON A (recombinant interferon alfa-2b), IRESSA (gefitinib), IXEMPRA (ixabepilone), JAKAFI (ruxolitinib phosphate), JEVTANA (cabazitaxel), KADCYLA (ado-trastuzumab emtansine), KEYTRUDA (pembrolizumab), KYPROLIS (carfilzomib), LIPODOX (doxorubicin hydrochloride liposome), LUPRON (leuprolide acetate), LUPRON DEPOT (leuprolide acetate), LUPRON DEPOT-3 MONTH (leuprolide acetate), LUPRON DEPOT-4 MONTH (leuprolide acetate), LUPRON DEPOT-PED (leuprolide acetate), MEGACE (megestrol acetate), MEKINIST (trametinib), METHAZOLASTONE (temozolomide), METHOTREXATE LPF (methotrexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), MITOXANTRONE HYDROCHLORIDE, MITOZYTREX (mitomycin c), MOZOBIL (plerixafor), MUSTARGEN (mechlorethamine hydrochloride), MUTAMYCIN (mitomycin c), MYLOSAR (azacitidine), NAVELBINE (vinorelbine tartrate), NEOSAR (cyclophosphamide), NEXAVAR (sorafenib tosylate), NOLVADEX (tamoxifen citrate), NOVALDEX (tamoxifen citrate), OFF, PAD, PARAPLAT (carboplatin), PARAPLATIN (carboplatin), PEG-INTRON (peginterferon alfa-2b), PEMETREXED DISODIUM, PERJETA (pertuzumab), PLATINOL (cisplatin), PLATINOL-AQ (cisplatin), POMALYST (pomalidomide), prednisone, PROLEUKIN (aldesleukin), PROLIA (denosumab), PROVENGE (sipuleucel-t), REVLIMID (lenalidomide), RUBIDOMYCIN (daunorubicin hydrochloride), SPRYCEL (dasatinib), STIVARGA (regorafenib), SUTENT (sunitinib malate), SYLATRON (peginterferon alfa-2b), SYLVANT (siltuximab), SYNOVIR (thalidomide), TAC, TAFINLAR (dabrafenib), TARABINE PFS (cytarabine), TARCEVA (erlotinib hydrochloride), TASIGNA (nilotinib), TAXOL (paclitaxel), TAXOTERE (docetaxel), TEMODAR (temozolomide), THALOMID (thalidomide), TOPOSAR (etoposide), TORISEL (temsirolimus), TPF, TRISENOX (arsenic trioxide), TYKERB (lapatinib ditosylate), VECTIBIX (panitumumab), VEIP, VELBAN (vinblastine sulfate), VELCADE (bortezomib), VELSAR (vinblastine sulfate), VEPESID (etoposide), VIADUR (leuprolide acetate), VIDAZA (azacitidine), VINCASAR PFS (vincristine sulfate), VOTRIENT (pazopanib hydrochloride), WELLCOVORIN (leucovorin calcium), XALKORI (crizotinib), XELODA (capecitabine), XELOX, XGEVA (denosumab), XOFIGO (radium 223 dichloride), XTANDI (enzalutamide), YERVOY (ipilimumab), ZALTRAP (ziv-aflibercept), ZELBORAF (vemurafenib), ZOLADEX (goserelin acetate), ZOMETA (zoledronic acid), ZYKADIA (ceritinib), ZYTIGA (abiraterone acetate), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TK1258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g., bortezomib (Velcade)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin, aminopterin, and hexamethyl melamine, or a combination thereof. In certain embodiments, the additional pharmaceutical agent is a binder or inhibitor of an HMT (e.g., EZH1, EZH2, DOT1). In certain embodiments, the additional pharmaceutical agent is a protein kinase inhibitor (e.g., tyrosine protein kinase inhibitor). In certain embodiments, the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation. In certain embodiments, the compounds described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.

(115) Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

(116) Thus, in one aspect, provided are kits including a first container comprising a compound or pharmaceutical composition described herein. In certain embodiments, the kits are useful for treating a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease) in a subject in need thereof. In certain embodiments, the kits are useful for preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease) in a subject in need thereof. In certain embodiments, the kits are useful as enhancers and/or modifiers of an immune response (e.g., innate and/or adaptive immune response), and/or adjuvants in a vaccine for a disease, (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease) in a subject, biological sample, tissue, or cell, or as stand alone anti-infective or immune response modifying agents.

(117) In certain embodiments, a kit described herein further includes instructions for using the compound or pharmaceutical composition included in the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease) in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease) in a subject in need thereof. In certain embodiments, the kits and instructions provide for enhancing of an immune response (e.g., innate and/or adaptive immune response) in a subject, biological sample, tissue, or cell. In certain embodiments, the kits and instructions provide for use of the compounds as adjuvants in a vaccine for a disease, (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease) in a subject, biological sample, tissue, or cell, or as stand alone anti-infective or immune response modifying agents. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

(118) Methods of Treatment and Uses

(119) The compounds described herein are capable of enhancing an immune response (e.g., innate and/or adaptive immune response). The present disclosure thus also provides methods of enhancing an immune response (e.g., innate and/or adaptive immune response) in a subject, biological sample, tissue, or cell. The present disclosure further provides methods for the compounds as adjuvants in a vaccine for treatment of a wide range of diseases, such as proliferative diseases, inflammatory diseases, autoimmune diseases, infectious diseases, and chronic diseases in a subject in need thereof, or as stand alone anti-infective or immune response modifying agents.

(120) In another aspect, the present disclosure provides methods of enhancing an immune response (e.g., innate and/or adaptive immune response), the methods comprising administering to the subject an effective amount of a compound or pharmaceutical composition described herein.

(121) In another aspect, the present disclosure provides methods of enhancing an immune response (e.g., innate and/or adaptive immune response) in a biological sample, tissue, or cell, the methods comprising contacting the biological sample, tissue, or cell with an effective amount of a compound or pharmaceutical composition described herein.

(122) In certain embodiments, the immune response (e.g., innate and/or adaptive immune response) is enhanced by a compound, pharmaceutical composition, kit, use, or method described herein by at least 1%, at least 3%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, the immune response (e.g., innate and/or adaptive immune response) in a subject, biological sample, tissue, or cell is enhanced by a compound, pharmaceutical composition, kit, use, or method described herein by not more than 1%, not more than 3%, not more than 10%, not more than 20%, not more than 30%, not more than 40%, not more than 50%, not more than 60%, not more than 70%, not more than 80%, or not more than 90%.

(123) Another aspect of the present disclosure relates to methods of using the compounds as adjuvants in a vaccine for treating a disease in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition described herein. Some aspects of the present disclosure provide the vaccine as a subunit vaccine, an attenuated vaccine, or a conjugate vaccine. In some embodiments, a vaccine stimulates the subject's immune system to recognize an antigen as foreign, and enhances the subject's immune response if the subject is later exposed to the pathogen, whether attenuated, inactivated, killed, or not. Vaccines may be prophylactic, for example, preventing or ameliorating a detrimental effect of a future exposure to a pathogen, or therapeutic, for example, activating the subject's immune response to a pathogen after the subject has been exposed to the pathogen. In some embodiments, a vaccine composition is used to protect or treat an organism against a disease (e.g., an infectious disease or cancer). In some embodiments, the vaccine is a subunit vaccine (e.g., a recombinant subunit vaccine), an attenuated vaccine (e.g., containing an attenuated pathogen such as a bacterial cell or a viral genome), a live vaccine (e.g., containing a live attenuated pathogen such as a bacterium or virus), or a conjugated vaccine (e.g., a vaccine containing an antigen that is not very immunogenic covalently attached to an antigen that is more immunogenic). One non-limiting example of a conjugated vaccine comprises a LPS attached to a strong protein antigen. Another aspect of the present disclosure relates to methods of using the compounds as stand alone anti-infective or immune response modifying agents in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition described herein.

(124) In certain embodiments, a disease described herein is a proliferative disease. In certain embodiments, a disease described herein is cancer. In certain embodiments, a disease described herein is hyperplasia (e.g., germinal center (GC) hyperplasia). In certain embodiments, a disease described herein is brain cancer, breast cancer, or prostate cancer. In certain embodiments, a disease described herein is a benign neoplasm. In certain embodiments, a disease described herein is or is associated with pathological angiogenesis. In certain embodiments, a disease described herein is an inflammatory disease. In certain embodiments, a disease described herein is an autoimmune disease. In certain embodiments, a disease described herein is an infectious disease. In certain embodiments, a disease described herein is an infection by a microbial pathogen (e.g., from a mycobacterium, bacterium, fungus, a virus, parasite, or prion). In certain embodiments, a disease described herein is a microbial infectious disease. In certain embodiments, the infectious disease is a viral infectious disease. In certain embodiments, the viral infectious disease being treated or prevented is an infection with influenza. In certain embodiments, the infectious disease is sepsis. In certain embodiments, the infectious disease is a pediatric infectious disease. In certain embodiments, the infectious disease is a disease of newborns, infants and/or school age children. In certain embodiments, the infectious disease is allergy.

(125) In certain embodiments, the infectious disease is a bacterial infectious disease. In certain embodiments, the bacterial infectious disease being treated or prevented is an infection with a Gram-positive bacteria. Exemplary Gram-positive bacteria include, but are not limited to, Staphylococcus spp., Streptococcus spp., Micrococcus spp., Peptococcus spp., Peptostreptococcus spp., Enterococcus spp., Bacillus spp., Clostridium spp., Lactobacillus spp., Listeria spp., Erysipelothrix spp., Propionibacterium spp., Eubacterium spp., Corynebacterium spp., Capnocytophaga spp., Bifidobacterium spp., and Gardnerella spp. Exemplary Gram-positive bacteria include, but are not limited to, Staphylococcus spp., Streptococcus spp., Micrococcus spp., Peptococcus spp., Peptostreptococcus spp., Enterococcus spp., Bacillus spp., Clostridium spp., Lactobacillus spp., Listeria spp., Erysipelothrix spp., Propionibacterium spp., Eubacterium spp., and Corynebacterium spp. In certain embodiments, the Gram-positive bacteria is a bacteria of the phylum Firmicutes. In certain embodiments, the Gram-positive bacteria is Streptococcus.

(126) In certain embodiments, the bacterial infection being treated or prevented is an infection with a Gram-negative bacteria. Exemplary Gram-negative bacteria species include, but are not limited to, Escherichia, Citrobacter, Enterobacter, Klebsiella, Proteus, Serratia, Shigella, Salmonella, Morganella, Providencia, Edwardsiella, Erwinia, Hafnia, Yersinia, Acinetobacter, Vibrio, Aeromonas, Pseudomonas, Haemophilus, Pasteurella, Campylobacter, Helicobacter, Branhamella, Moraxella, Neisseria, Veillonella, Fusobacterium, Bacteroides, Actinobacillus, Aggregatibacter, Agrobacterium, Porphyromonas, Prevotella, Ruminobacter, Roseburia, Caulobacter, Francisella, Borrelia, Treponema, Brucella, Bordetella, and Rickettsia. In some embodiments, the bacterial infectious agent is Bacillus anthracis (causing anthrax), Bordetella pertussis (causing whooping cough), Corynebacterium diphtheriae (causing diphtheria), Clostridium tetani (causing tetanus), Haemophilus influenzae type b, pneumococcus (causing pneumococcal infections), Staphylococci spp. (including Group A or B streptococci), Mycobacterium tuberculosis, Neiserria meningitidis (causing meningococcal disease), Salmonella typhi (causing typhoid), Vibrio cholerae (causing Cholera), or Yersinia pestis (causing plague). In some embodiments, the bacterial infectious agent is anthrax, diphtheria, tetanus, Bordetella spp., Haemophilus influenzae type b, Neiserria spp., Vibrio spp., cholera, Yersinia spp., Staphylococci spp., Streptococci spp., or Salmonella spp. In some embodiments, the bacterial infectious disease is anthrax, diphtheria, tetanus, pertussis, Haemophilus influenzae type b, pneumococcal infection, meningococcal disease, cholera, plague, Staphylococcal disease, Group A or B streptococcal or pneumococcal infection, or typhoid.

(127) In certain embodiments, the Gram-negative bacteria is Bordetella pertussis. In certain embodiments, the bacterial infectious disease is pertussis. In some embodiments, the antigen is a lipopolysaccharide endotoxin (LPS) from a Gram-negative bacterium. Non-limiting examples of Gram-negative bacterial species include: Neisseria species including Neisseria gonorrhoeae and Neisseria meningitidis, Branhamella species including Branhamella catarrhalis, Escherichia species including Escherichia coli, Enterobacter species, Proteus species including Proteus mirabilis, Pseudomonas species including Pseudomonas aeruginosa, Pseudomonas mallei, and Pseudomonas pseudomallei, Klebsiella species including Klebsiella pneumoniae,

(128) Salmonella species, Shigella species, Serratia species, Acinetobacter species; Haemophilus species including Haemophilus influenzae and Haemophilus ducreyi;

(129) Brucella species, Yersinia species including Yersinia pestis and Yersinia enterocolitica, Francisella species including Francisella tularensis, Pasteurella species including Pasteurella multocida, Vibrio cholerae, Flavobacterium species, meningosepticum, Campylobacter species including Campylobacter jejuni, Bacteroides species (oral, pharyngeal) including Bacteroides fragilis, Fusobacterium species including Fusobacterium nucleatum, Calymmatobacterium granulomatis, Streptobacillus species including Streptobacillus moniliformis, Legionella species including Legionella pneumophila.

(130) Other types of bacteria include acid-fast bacilli, spirochetes, and actinomycetes.

(131) Examples of acid-fast bacilli include Mycobacterium species including Mycobacterium tuberculosis and Mycobacterium leprae. Examples of spirochetes include Treponema species including Treponema pallidum, Treponenia pertenue, Borrelia species including Borrelia burgdorferi (Lyme disease), and Borrelia recurrentis, and Leptospira species. Examples of actinomycetes include: Actinomyces species including Actinomyces israelii, and Nocardia species including Nocardia asteroides.

(132) In certain embodiments, the bacteria is Escherichia spp., Enterobacter spp. (e.g., Enterobacter cloacae), Salmonella spp. (e.g., Salmonella enteritidis, Salmonella typhi), Shigella spp., Pseudomonas spp. (e.g., Pseudomonas aeruginosa, Pseudomonas pachastrellae, Pseudomonas stutzeri), Moraxella spp. (e.g., Moraxella catarrhalis), Neisseria spp. (e.g., Neisseria gonorrhoeae, Neisseria meningitidis), Helicobacter spp., (e.g., Helicobacter pylori) Stenotrophomonas spp., Vibrio spp. (e.g., Vibrio cholerae), Legionella spp. (Legionella pneumophila), Hemophilus spp. (e.g., Hemophilus influenzae), Klebsiella spp. (e.g., Klebsiella pneumoniae), Proteus spp. (e.g., Proteus mirabilis), Serratia spp. (Serratia marcescens), Streptococcus spp., Staphylococcus spp., Corynebacterium spp., Listeria spp., and Clostridium spp., Bacillus spp. (e.g., Bacillus anthracis) Bordetella spp. (e.g., Bordetella pertussis); Borrelia spp. (e.g., Borrelia burgdorferi); Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis); Campylobacter spp. (e.g., Campylobacter jejuni); Chlamydia spp. and Chlamydophila spp. (e.g., Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci); Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani); Corynebacterium spp. (e.g., Corynebacterium diphtheriae); Enterococcus spp. (e.g., Enterococcus faecalis, Enterococcus faecium); Escherichia spp. (e.g., Escherichia coli, Enterotoxic E. coli, enteropathogenic E. coli; E. coli O157:H7); Francisella spp. (e.g., Francisella tularensis); Haemophilus spp. (e.g., Haemophilus influenzae); Helicobacter spp. (e.g., Helicobacter pylori); Legionella spp. (e.g., Legionella pneumophila); Leptospira spp. (e.g., Leptospira interrogans); Listeria spp. (e.g., Listeria monocytogenes); Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans); Mycoplasma spp. (e.g., Mycoplasma pneumoniae); Neisseria spp. (e.g., Neisseria gonorrhoeae, Neisseria meningitidis); Pseudomonas spp. (e.g., Pseudomonas aeruginosa); Rickettsia spp. (e.g., Rickettsia rickettsii); Salmonella spp. (e.g., Salmonella typhi, Salmonella typhimurium); Shigella spp. (e.g., Shigella sonnei); Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus); Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes); Treponema spp. (e.g., Treponema pallidum); Pseudodiomarina spp. (e.g., P. maritima); Marinobacter spp. (e.g., Marinobacter hydrocarbonoclasticus, Marinobacter vinifirmus) Alcanivorax spp. (e.g., alcanivorax dieselolei); Acetinobacter spp. (e.g., A. venetianus); Halomonas spp. (e.g., H. shengliensis); Labrenzia spp.; Microbulifer spp. (e.g., M. schleiferi); Shewanella spp. (e.g., S. algae); Vibrio spp. (e.g., Vibrio cholerae, Vibrio alginolyticus, Vibrio hepatarius); and Yersinia spp. (e.g., Yersinia pestis).

(133) In certain embodiments, the infectious disease is cancer. In certain embodiments, the infectious disease is a fibrotic disease, a cardiovascular disease, a graft rejection, or graft-versus-host disease. In certain embodiments, the bacterial infectious disease is anthrax, diphtheria, tetanus, pertussis, Haemophilus influenzae type b, pneumococcal infections, meningococcal disease, cholera, plague, Staphylococcal disease, a Group A streptococcal infection, a Group A pneumococcal infection, a group B streptococcal infection, a group B pneumococcal infection, or typhoid. In certain embodiments, the infectious disease is a mycobacterial infectious disease. In certain embodiments, the mycobacterial infectious disease is a tuberculosis infection or a non-tuberculous mycobacterial infection.

(134) In certain embodiments, the infectious disease is a viral infectious disease. In certain embodiments, the viral infectious disease is an infection by Retroviruses, human immunodeficiency viruses including HIV-1, HDTV-III, LAVE, HTLV-III/LAV, HIV-III, HIV-LP, Cytomegaloviruses (CMV), Picornaviruses, polio viruses, hepatitis A virus, enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses, Calciviruses, Togaviruses, equine encephalitis viruses, rubella viruses, Flaviruses, dengue viruses, encephalitis viruses, yellow fever viruses, Coronaviruses, Rhabdoviruses, vesicular stomatitis viruses, rabies viruses, Filoviruses, ebola virus, Paramyxoviruses, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus (RSV), Orthomyxoviruses, influenza viruses, Bungaviruses, Hantaan viruses, phleboviruses and Nairo viruses, Arena viruses, hemorrhagic fever viruses, reoviruses, orbiviruses, rotaviruses, Birnaviruses, Hepadnaviruses, Hepatitis B virus, parvoviruses, Papovaviridae, papilloma viruses, polyoma viruses, Adenoviruses, Herpesviruses including herpes simplex virus 1 and 2, varicella zoster virus, Poxviruses, variola viruses, vaccinia viruses, Irido viruses, African swine fever virus, delta hepatitis virus, non-A, non-B hepatitis virus, Hepatitis C, Norwalk viruses, astroviruses, and unclassified viruses. In certain embodiments, the viral infectious disease is an infection by adenovirus, poliomyelitis, Ebola, herpes viruses (e.g., herpes simplex virus), cytomegalovirus and varicella-zoster, measles, mumps, rubella, hepatitis A, hepatitis B, hepatitis C, human papilloma virus, Influenza, rabies, Japanese encephalitis, rotavirus, human immunodeficiency virus, respiratory syncytial virus, smallpox, yellow fever, or Zika Virus. In certain embodiments, the viral infectious disease is polio, chickenpox, or shingles. In certain embodiments, the viral infectious disease is an infection by human immunodeficiency virus (HIV) or respiratory syncytial virus (RSV). In certain embodiments, the infectious disease is a parasitic infectious disease. In certain embodiments, the parasitic infectious disease is an infection by malaria, Leishmania, another protozoan, or a helminth. In certain embodiments, the parasitic infectious disease is an infection by Plasmodium species, such as Plasmodium species including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax and Toxoplasma gondii. Blood-borne and/or tissues parasites include Plasmodium species, Babesia species including Babesia microli and Babesia divergens, Leishmania species including Leishmania tropica,

(135) Leishmania species, Leishmania braziliensis, Leishmania donovani, Trypanosoma species including Trypanosoma ganibiense, Trypanosoma rhodesiense (African sleeping sickness), and Trypanosoma cruzi (Chagas' disease). In certain embodiments, the parasitic infectious disease is malaria or leishmaniasis.

(136) In certain embodiments, the infectious disease is a fungal infectious disease. In certain embodiments, the fungal infectious disease is Cryptococcus species including Cryptococcus neoformans, Histoplasma species including Histoplasma capsulatum, Coccidioides species including Coccidioides immitis, Paracoccidioides species including Paracoccidioides brasiliensis, Blastomyces species including Blastomyces dermatitidis, Chlamydia species including Chlamydia trachomatis, Candida species including Candida albicans, Sporothrix species including Sporothrix schenckii, Aspergillus species, and fungi of mucormycosis. In some embodiments, the fungus is fungus is Candida spp., Aspergillus spp., Cryptococcus spp., Mucormycete, Blastomyces dermatitidis (causing blastomycosis), or endemic mycosis causing fungus such as Histoplasma capsulatum (causing histoplasmosis), or Sporothrix schenckii (causing sporotrichosis). In certain embodiments, the vaccine provides heterologous protection against a range of pathogens. Other medically relevant microorganisms have been described extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, incorporated herein by reference. In certain embodiments, a disease described herein is a chronic disease. In certain embodiments, the chronic disease is arthritis, cardiovascular disease such as heart disease, stroke, cancer (e.g., breast cancer or colon cancer), chronic respiratory diseases, diabetes, epilepsy, seizures, obesity, or an oral health problem.

(137) In still another aspect, the present disclosure provides methods of preventing a disease to be treated with a compound or pharmaceutical composition described herein in a subject in need thereof, the methods comprising administering to the subject a prophylactically effective amount of a compound or pharmaceutical composition described herein.

(138) In another aspect, the present disclosure provides the compounds described herein for use in a method described herein (e.g., a method of enhancing an immune response (e.g., innate and/or adaptive immune response), a method of treating a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease), and a method of treating and/or preventing a disease by using the compounds as medicaments or adjuvants in a vaccine for the disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease), or as stand alone anti-infective or immune response modifying agents. In certain embodiments, the present disclosure provides the compounds described herein for use as immunomodulators. In certain embodiments, the medicament is an immunomodulator.

(139) In another aspect, the present disclosure provides the compounds described herein for use in treating and/or preventing a proliferative disease in a subject. In another aspect, the present disclosure provides the compounds described herein for use as a medicament.

(140) In still another aspect, the present disclosure provides the pharmaceutical compositions described herein for use in a method described herein (e.g., a method of enhancing an immune response (e.g., innate and/or adaptive immune response), a method of treating a disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease), and a method of treating and/or preventing a disease by using the compounds as medicaments or adjuvants in a vaccine for the disease (e.g., proliferative disease, inflammatory disease, autoimmune disease, infectious disease, or chronic disease), or as stand alone anti-infective or immune response modifying agents.

EXAMPLES

(141) In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

Preparation of the Compounds Described Herein

(142) The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. For example, compounds of Formula (I) can be prepared according to Scheme 1. Where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.

Preparation of Examples

(143) ##STR00044##

2-Bromo-1-(4-methyl-3-nitrophenyl)ethanone (1)

(144) ##STR00045##

(145) 4′-Methylacetophenone (1 g, 7.5 mmol) was dissolved in conc. H.sub.2SO.sub.4 (10 mL) and the solution was cooled to 0° C. KNO.sub.3 (1 g, 9.9 mmol) was added in portions, and the mixture was stirred at 0° C. for 2 hours, then poured onto crushed ice (100 g), and extracted with EtOAc (2×50 mL). The organic phase was washed with brine and concentrated to give 1.3 g of a yellow solid. The nitro intermediate (200 mg, 1.1 mmol) was dissolved in acetonitrile (2 mL). NBS (237 mg, 1.3 mmol) and p-TsOH (200 mg, 1.1) were added. The reaction mixture was heated at 60° C. for 4 hours, then diluted with EtOAc (20 mL) and washed with sat. Na.sub.2S.sub.2O.sub.3 solution and brine. The organic layer was concentrated to give the title compound (260 mg, yield 91%) as a yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.56 (s, 1H), 8.12 (d, 1H), 7.52 (d, 1H), 4.46 (s, 2H), 2.70 (s, 3H).

6-Methyl-2-(4-methyl-3-nitrophenyl)imidazo[1,2-a]pyrimidine (2)

(146) ##STR00046##

(147) 2-Bromo-1-(4-methyl-3-nitrophenyl)ethanone (300 mg, 1.16) and 2-amino-5-methylpyrimidine (200 mg, 1.83 mmol) were dissolved in EtOH (10 mL) and the solution refluxed overnight. The solution was diluted with DCM and washed with NaHCO.sub.3 solution. The organic layer was concentrated and the residue purified by column chromatography to give the title compound (102 mg, yield 30%) as a yellow solid. MS m/z 269.9 [M+H].sup.+.

2-Methyl-5-(6-methylimidazo[1,2-a]pyrimidin-2-yl)aniline (3)

(148) ##STR00047##

(149) 6-Methyl-2-(4-methyl-3-nitrophenyl)imidazo[1,2-a]pyrimidine (600 mg, 2.24 mmol) was dissolved in THF and methanol (3:1, 4 mL). Zinc powder (2.3 g, 35.4 mmol) and NH.sub.4C.sub.1 (2 g, 37.0 mmol) were added to the solution and the mixture was stirred for 1 hour. The solid was filtered, and the filtrate was concentrated, dissolved in EtOAc, and then washed with water. The organic phase was concentrated to give compound the title compound (400 mg, 75% yield) as a yellow solid. MS m/z 238.7 [M+H].sup.+.

2-Bromo-1-(4-chloro-3-nitrophenyl)ethan-1-one (4)

(150) ##STR00048##

(151) 2-Bromo-1-(4-chloro-3-nitrophenyl)ethan-1-one was prepared using a similar procedure to that described for 1, from 4′-chloroacetophenone. .sup.1H NMR (400 MHz, DMSO-d6): δ 8.62 (s, 1H), 8.26 (d, 1H), 8.00 (d, 1H), 5.02 (s, 2H).

(152) Anilines 5-8 were prepared by similar methods to 3, from the corresponding aminopyrimidine and either 1 or 4.

(153) TABLE-US-00001 Aniline Name .sup.1H NMR (d.sub.6-DMSO) m/z [M + 1].sup.+ Starting materials 5 (500 MHz) 8.92 (m, 1H), 8.48 (m, 1H), 8.16 (s, 1H), 7.32 (d, 1H), 7.09 (d, 1H), 7.00 (m, 2H), 4.95 (s, 2H), 2.09 (s, 3H) 224.91 0 6 (500 MHz) 9.18 (m, 1H), 8.65 (d, 1H), 8.18 (s, 1H), 7.31 (d, 1H), 7.09 (m, 1H), 7.00 (d, 1H), 4.95 (s, 2H), 2.09 (s, 3H) 242.98 7 (500 MHz) 8.66 (d, 1H), 8.36 (d, 1H), 8.05 (s, 1H), 7.27 (d, 1H), 7.05 (m, 1H), 6.98 (d, 1H), 4.92 (s, 2H), 3.85 (s, 3H), 2.08 (s, 3H) 254.68 8 259.1

Example 1 FRF-02-144-1

N-(2-Methyl-5-(6-methylimidazo[1,2-a]pyrimidin-2-yl)phenyl)-2-phenylbutanamide

(154) ##STR00057##

(155) 2-Methyl-5-(6-methylimidazo[1,2-a]pyrimidin-2-yl)aniline (75 mg, 0.32 mmol) was dissolved in DMF (5 mL). Triethylamine (0.09 mL, 0.64 mmol) and 2-phenylbutanoyl chloride (150 mg, 0.82 mmol) were added and the mixture was stirred for 1 hour. The reaction mixture was diluted with EtOAc (20 mL) and washed with sat. NaHCO.sub.3 solution (20 mL). The organic phase was concentrated and purified by flash chromatography to give the title compound (26 mg, 21% yield). .sup.1H NMR (400 MHz, DMSO-d6): δ 9.59 (s, 1H), 8.72 (s, 1H), 8.41 (d, 1H), 8.23 (s, 1H), 7.90 (s, 1H), 7.68 (d, 1H), 7.44 (m, 2H), 7.37 (m, 2H), 7.27 (m, 2H), 3.69 (m, 1H), 2.30 (s, 3H), 2.11 (s, 3H), 2.10 (m, 1H), 1.74 (m, 1H), 0.94 (t, 3H). MS m/z 385.3 [M+H].sup.+.

Example 2 FRF-02-119

N-(5-(6-Fluoroimidazo[1,2-a]pyrimidin-2-yl)-2-methylphenyl)-2-phenylbutanamide

(156) ##STR00058##

(157) 2-Phenylbutanoic acid (16 mg, 0.099 mmol), diisopropylethylamine (29 uL, 0.165 mmol) and HATU (38 mg, 0.0099 mmol) were dissolved in DMF (6 mL). After 20 minutes, 5-(6-fluoroimidazo[1,2-a]pyrimidin-2-yl)-2-methylaniline (20 mg, 0.082 mmol) was added. After stirring for 18 hours, further portions of 2-phenylbutanoic acid (8 mg), diisopropylethylamine (15 uL) and HATU (18.5 mg) were added, and the mixture stirred for a further 4 hours. The mixture was diluted with DCM, washed with brine, concentrated and the residue purified by column chromatography on silica gel (0 to 5% MeOH in DCM) to give the title compound (19 mg, 56% yield). .sup.1H NMR (500 MHz, DMSO-d6): δ 9.57 (s, 1H), 9.18 (m, 1H), 8.69 (d, 1H), 8.33 (s, 1H), 7.93 (d, 1H), 7.70 (m, 1H), 7.45 (m, 2H), 7.36 (m, 2H), 7.27 (m, 2H), 3.70 (m, 1H), 2.12 (s, 3H), 2.09 (m, 1H), 1.74 (m, 1H), 0.94 (t, 3H). MS m/z 389.16 [M+H].sup.+.

(158) Examples 3-31 shown below were prepared by similar methods to Examples 1 and 2, from the corresponding aniline and either the acid chloride or carboxylic acid.

(159) TABLE-US-00002 Example (and FRF .sup.1H NMR m/z number) Name (d.sub.6-DMSO) [M + 1].sup.+ Starting materials 3 FRF-02- 162-1 (400 MHz) 9.60 (s, 1H), 8.73 (s, 1H), 8.40 (s, 1H), 8.22 (s, 1H), 8.00 (s, 1H), 7.69 (d, 1H), 7.38 (m, 4H), 7.28 (m, 2H), 3.71 (s, 2H), 357.4 0 2.30 (s, 3H), 2.20 (s, 3H) 4 FRF-02- 162-2 (400 MHz) 9.86 (s, 1H), 8.76 (s, 1H), 8.42 (d, 1H), 8.27 (s, 1H), 8.05 (s, 1H), 7.77 (d, 1H), 7.56 (d, 1H), 7.41 (m, 1H), 7.34 (m, 3H), 2.48 (s, 3H), 2.32 (s, 3H), 357.4 3 protons masked by DMSO 5 FRF-02- 144-2 (400 MHz) 10.08 (s, 1H), 8.76 (s, 1H), 8.43 (s, 1H), 8.28 (s, 1H), 8.08 (s, 1H), 7.77 (d, 1H), 7.68 (d, 1H), 7.59 (d, 1H), 7.49 (m, 2H), 7.35 (d, 1H), 2.33 (s, 377.3 3H), 2.32 (s, 3H) 6 FRF-02- 144-3 (400 MHz) 10.13 (s, 1H), 8.77 (s, 1H), 8.46 (d, 1H), 8.27 (s, 1H), 8.06 (s, 1H), 7.88 (d, 1H), 7.77 (m, 4H), 7.35 (d, 1H), 2.31 (s, 6H) 411.2 7 FRF-02- 118 (500 MHz) 10.13 (s, 1H), 9.23 (m, 1H), 8.72 (d, 1H), 8.37 (s, 1H), 8.09 (d, 1H), 7.87 (d, 1H), 7.82 (m, 3H), 7.77 (m, 1H), 7.38 (d, 1H), 2.32 (s, 3H) 414.86 8 FRF-02- 117 (500 MHz) 9.87 (s, 1H), 9.23 (m, 1H), 8.72 (d, 1H), 8.38 (s, 1H) , 8.08 (d, 1H), 7.79 (d, 1H), 7.57 (d, 1H), 7.40 (m, 1H), 7.37 (d, 1H), 7.33 (m, 2H), 2.48 (s, 3H), 2.33 (s, 3H) 361.22 0 9 FRF-02- 116 (500 MHz) 10.07 (s, 1H), 9.23 (m, 1H), 8.72 (d, 1H), 8.38 (s, 1H), 8.12 (d, 1H), 7.79 (m, 1H), 7.75 (d, 1H), 7.65 (m, 1H), 7.53 (m, 1H), 7.44 (m, 1H), 7.37 (d, 425.3 1H), 2.36 (s, 3H) 10 FRF-02- 115 (500 MHz) 10.09 (s, 1H), 9.23 (m, 1H), 8.72 (d, 1H), 8.38 (s, 1H), 8.11 (s, 1H), 7.79 (m, 1H), 7.68 (m, 1H), 7.59 (d, 1H), 7.52 (m, 2H), 7.38 (d, 1H), 2.35 (s, 3H) 381.25 11 FRF-02- 137 (500 MHz) 9.56 (s, 1H), 8.64(d, 1H), 8.39 (d, 1H), 8.20 (s, 1H), 7.88 (s, 1H), 7.65 (m, 1H), 7.45 (m, 2H), 7.36 (m, 2H), 7.26 (m, 2H), 3.85 (s, 3H), 3.69 (m, 1H), 2.11 (s, 3H), 2.09 (m, 1H), 401.4 1.74 (m, 1H), 0.94 (t, 3H) 12 FRF-02- 135 (500 MHz) 10.11 (s, 1H), 8.68 (d, 1H), 8.42 (d, 1H), 8.23 (s, 1H), 8.04 (s, 1H), 7.87 (d, 1H), 7.81 (m, 2H), 7.74 (m, 2H), 7.35 (d, 1H), 3.86 (s, 3H), 2.31 (s, 426.9 3H) 13 FRF-02- 134 (500 MHz) 9.85 (s, 1H), 8.68 (d, 1H), 8.41 (d, 1H), 8.24 (s, 1H), 8.03 (s, 1H), 7.75 (m, 1H), 7.57 (d, 1H), 7.40 (m, 1H), 7.33 (m, 3H), 3.86 (s, 3H), 2.48 (s, 3H), 2.34 (s, 3H) 372.98 0 14 FRF-02- 132 (500 MHz) 10.05 (s, 1H), 8.68 (d, 1H), 8.42 (d, 1H), 8.24 (s, 1H), 8.07 (s, 1H), 7.75 (m, 2H), 7.65 (m, 1H), 7.53 (m, 1H), 7.44 (m, 1H), 7.34 (d, 1H), 3.86 (s, 3H), 2.34 (s, 439.09 3H) 15 FRF-02- 131 (500 MHz) 10.06 (s, 1H), 8.68 (d, 1H), 8.42 (d, 1H), 8.24 (s, 1H), 8.06 (s, 1H), 7.75 (m, 1H), 7.67 (m, 1H), 7.59 (d, 1H), 7.51 (m, 2H), 7.35 (d, 1H), 3.86 (s, 393.22 3H), 2.33 (s, 3H) 16 FRF-02- 166 (500 MHz) 10.19 (s, 1H), 9.24 (m, 1H), 8.72 (d, 1H), 8.36 (s, 1H), 8.12 (s, 1H), 7.97 (m, 1H), 7.80 (m, 2H), 7.59 (m, 1H), 7.38 (d, 1H), 2.32 (s, 3H) 433.3 17 FRF-02- 167 (500 MHz) 10.15 (s, 1H), 9.23 (m, 1H), 8.72 (d, 1H), 8.37 (s, 1H), 8.10 (s, 1H), 7.92 (m, 1H), 7.81 (m, 2H), 7.72 (m, 1H), 7.38 (d, 1H), 2.31 (s, 3H) 433.28 18 FRF-02- 169 (500 MHz) 10.17 (s, 1H), 9.23 (m, 1H), 8.72 (d, 1H), 8.36 (s, 1H), 8.10 (s, 1H), 7.98 (d, 1H), 7.94 (m, 1H), 7.87 (d, 1H), 7.80 (m, 1H), 7.37 (d, 1H), 2.31 (s, 3H) 449.17 0 19 FRF-02- 171 (500 MHz) 10.05 (s, 1H), 9.23 (m, 1H), 8.71 (d, 1H), 8.36 (s, 1H), 8.07 (s, 1H), 7.79 (m, 1H), 7.69 (m, 2H), 7.62 (d, 1H), 7.36 (d, 1H), 2.46 (s, 3H), 2.31 (s, 3H) 428.93 20 FRF-02- 174-1 (400 MHz) 9.78 (s, 1H), 8.74 (s, 1H), 8.44 (d, 1H), 8.32 (s, 1H), 8.22 (s, 1H), 7.78 (d, 1H), 7.54 (d, 1H), 7.44 (m, 2H), 7.36 (m, 2H), 7.27 (m, 1H), 3.80 (m, 1H), 2.31 (s, 3H), 405.3 2.09 (m, 1H), 1.74 (m, 1H), 0.93 (t, 3H) 21 FRF-02- 174-2 (400 MHz) 10.43 (s, 1H), 8.79 (s, 1H), 8.46 (s, 1H), 8.38 (s, 1H), 8.26 (s, 1H), 7.81 (m, 5H), 7.63 (d, 1H), 2.32 (s, 3H) 431.3 22 FRF-02- 175 (500 MHz) 10.37 (s, 1H), 9.21 (m, 1H), 8.72 (d, 1H), 8.38 (s, 1H), 8.06 (s, 1H), 7.76 (m, 4H), 7.38 (d, 1H), 2.30 (s, 3H) 432.93 23 FRF-02- 176 (500 MHz) 9.70 (s, 1H), 9.22 (m, 1H), 8.71 (d, 1H), 8.37 (s, 1H), 8.06 (s, 1H), 7.77 (m, 1H), 7.57 (d, 1H), 7.35 (d, 1H), 6.87 (m, 2H), 3.81 (s, 3H), 2.47 (s, 3H), 2.31 (s, 3H) 391.05 00 24 FRF-02- 182 01 373.3 02 25 FRF-02- 183 03 373.1 04 26 FRF-02- 187 05 (500 MHz) 9.84 (s, 1H), 9.23 (m, 1H), 8.71 (d, 1H), 8.38 (s, 1H), 8.08 (s, 1H), 7.79 (m, 1H), 7.36 (d, 1H), 7.23 (d, 1H), 7.13 (s, 1H), 6.98 (m, 1H), 3.81 (s, 3H), 391.05 06 2.39 (s, 3H), 2.32 (s, 3H) 27 FRF-02- 193 07 373.4 08 28 FRF-02- 194 09 391.26 0 29 FRF-03- 081 (500 MHz) 9.51 (s, 1H), 9.19 (m, 1H), 8.71 (d, 1H), 8.33 (s, 1H), 7.98 (s, 1H), 7.70 (m, 1H), 7.46 (m, 2H), 7.37 (m, 2H), 7.28 (m, 2H), 3.97 (q, 1H), 2.12 (s, 3H), 1.47 (d, 3H) 375.1 30 FRF-03- 082 403.4 31 FRF-03- 084-1 (500 MHz) 9.19 (m, 1H), 8.76 (s, 1H), 8.70 (d, 1H), 8.34 (s, 1H), 7.83 (s, 1H), 7.72 (d, 1H), 7.47 (m, 2H), 7.41 (m, 2H), 7.28 (m, 2H), 2.04 (s, 3H), 1.62 (s, 3H) 389.2

Biological Assays of the Compounds Described Herein

Example 1. Immunomodulatory Activity of Imidazopyrimidine Compounds

(160) Human immunity is crucial to both health and illness, playing key roles in infectious, allergic, autoimmune, oncologic and chronic diseases. In this context there is growing interest in development of approaches to modulate the human immune system to prevent and/or treat illness. Infectious diseases are the leading cause of morbidity and mortality in early life. Immunization is a key strategy for preventing infectious diseases. However, immunization of newborns and infants may result in sub-optimal responses, often requires multiple booster doses and can be limited by waning immunity. Adjuvantation is a key approach to enhance vaccine-induced immunity. Adjuvants can enhance, prolong, and modulate immune responses to vaccinal antigens to maximize protective immunity, and may potentially enable effective immunization in vulnerable populations (e.g., in the very young and the elderly or for diseases lacking effective vaccines). Vaccine adjuvants also hold great potential as cancer immunotherapeutics. Small molecules that may be used as adjuvants have been reported. Utility in redirecting immune responses away from allergy (e.g., restoring a Th1/Th2 balance) has also been demonstrated for some molecules (e.g., as described in J Immunol Mar. 15, 1998, 160 (6) 2555-2559; and Adv Drug Deliv Rev. 2009 Mar. 28; 61(3):256-62, incorporated herein by reference).

(161) Described herein is a novel molecular approach to shape human immune responses using formulations of imidazopyrimidine small molecules to induce robust activation of human leukocytes in vitro and as adjuvants in vivo. The parental compound identified to have the immune enhancing and adjuvant activity was named “Compound 037” as it was the 37.sup.th compound ordered into the Levy Lab (Boston Children's Hospital) for hit verification.

(162) To identify immunomodulators/adjuvants that robustly activate human immune cells, ˜200,000 small molecules were tested against THP1 NF-κB reporter cells in a screen. As there is evidence that cell culture lines such as THP1 cells may not model responses of natural primary human leukocytes, a smaller-scale novel high throughput screening methodology was also employed, in which ˜9,000 commercially available compounds (focusing on small molecule library plates that had relatively high # of hits form the THP1 screen) were screened using primary human blood mononuclear cells (PBMC) from three different adult human donors (TNFαLISA Assay) (FIGS. 1A-1C).

(163) TNF AlphaLISA screen was comprised of three parts: 1) human peripheral blood collection and PBMC isolation—human adult peripheral blood was collected with written consent. PBMCs were isolated from the blood using Ficoll density gradient. PBMCs were stored at 50 million cells per vial in 1 ml RPMI containing 20% autologous plasma and 10% DMSO at −80° C. until use; 2) TNFαLISA Assay—On Day 1, PBMCs were thawed in 37° C. water bath for 3 min and washed twice with PBS. Trypan blue viable cell count was obtained and cells were resuspended to viable 6.67×105 cells/ml in DMEM with 10% autologous plasma. Cells were dispensed in 30 μl per well in Corning 2712 black 384-well cell culture plates (final concentration was 20,000 cells/well). Controls were added to cells manually and the test compounds were added by robotic pin transfer. Plates were then incubated at 37° C./5% CO.sub.2 humidity-controlled incubator for 18 hours. On Day 2 of the assay, plates were centrifuged and 2 μl supernatants were collected into Perkin Elmer Alpha plates. Perkin Elmer Human TNFα kits (cat #AL208F) were used to detect the presence of TNFα in supernatants. Plates were run on the EnVision instrument (Perkin Elmer) to detect light emission at 615 nm; 3) Hit Calling Method—test compounds that resulted in a robust Z score greater than 2 in both duplicates and of at least 2 of the 3 human samples of PBMCs were considered hits.

(164) Based on antibody-coated fluorometric excitation/emission beads, the Alpha technology (PerkinElmer) enables the detection and screening of target molecules, in this case TNFα, in a no-wash, highly sensitive, quantitative assay compatible with high throughput screening.

(165) The chemical libraries screened included known bioactive and commercial libraries from various sources including commercial libraries such as ChemDiv, ChemBridge, and Asinex (see attached pdf of all libraries). All libraries were owned and provided by the Institute of Chemistry and Cell Biology (ICCB)—Longwood (Harvard Medical School). Compound 037 was identified in the PBMC TNFαLISA screen.

(166) Hits were determined based on the ability of small molecules to induce TNF production in at least two of the three human adult donors screened (FIG. 1D). From this screen, the imidazopyrimidine compounds emerged as the chemical family with greatest potency in inducing robust TNF production in vitro (FIGS. 2A-2B). These results were confirmed in conventional human PBMC assays measuring cytokine production by ELISA as well as multiplexing assay for other Th-polarizing cytokines (IL-12p70, IL-6, IL-1β etc.) (FIGS. 3A, 3B, and 4). Of note, imidazopyrimidines did not activate the human THP1 cell line in any assay formatted tested, as demonstrated by the lack of activity towards the THP1 NF-κB reporter cell line (FIGS. 5A-5B).

(167) This great discrepancy in activity of the imidazopyrimidines between primary cells and THP1 cells is noteworthy, as for practical convenience many conventional high throughput screens employ cell lines such as THP-1 cells, highlighting the novelty and importance of the PBMC screening methodology in the identification of imidazopyrimidines as immune stimulating compounds. The ability of imidazopyrimidines to activate human newborn and elderly leukocytes as well as murine leukocytes was also confirmed in vitro (FIGS. 2A, 8, and 10), expanding potential utility of this compound family (e.g., to sub-populations at high risk for infection).

(168) Additionally, in vivo studies in a murine model of influenza immunization indicate that the addition of imidazopyrimidines markedly enhances vaccine responses of adult mice (FIG. 9). Mice vaccinated with recombinant Influenza Hemagglutinin (rHA)+imidazopyrimidines demonstrated higher antibody titers compared to mice vaccinated with rHA alone and equivalent or enhanced titers as compared to mice vaccinated with rHA+Alum, the most commonly used vaccine adjuvant.

(169) Additional commercially available analogs of compound 037 were profiled in the PBMC assay (FIGS. 6A, 6B, and Table 1) with R848 as a positive control, to generate some initial structure activity relationships (SAR) within the imidazopyrimidine series. Certain structural features appeared important for activity, and a further set of imidazopyrimidines was prepared and tested, with some examples showing equivalent or superior activity to both compound 037 and R848 in this assay (e.g. FIGS. 7A and 7B and Table 1).

(170) In summary, imidazopyrimidine compounds have activity as immunomodulators/adjuvants, activating human leukocytes in vitro and demonstrating adjuvanticity in mice in vivo.

(171) The imidazopyrimidine compounds described herein may be used as stand alone agents to modify human immune responses, e.g., to be applied topically to treat infections by enhancing an immune response; given orally to enhance mucosal immunity or intranasally to treat respiratory infection or to reduce allergy (e.g., allergic rhinitis); injected locally or systemically to enhance immune responses against tumors and cancers. The compounds may also be given prophylactically to induce heightened immunity for broad protection against infection or radiation injury in high risk populations. The adjunctive therapy may also be coupled with other treatments for the conditions described herein. Further, the compounds may be used as vaccine adjuvant to be formulated with vaccinal antigen to enhance, accelerate, and/or broaden immune responses and/or to reduce the number of doses required (“dose sparing”), which is very important given the costs of vaccinal antigens and challenges of multiple clinic visits when vaccine boosting is required to achieve protective immune responses.

(172) The imidazopyrimidine compounds, compositions, and methods described herein are advantageous for the following, but are not limited to, reasons: (1) Small molecule category amenable to affordable scale up for mass production/use; (2) Molecular scaffold of the compounds appears to be relatively favorable from a medicinal chemistry perspective for production and creation of congeners/analogues; (3) The compounds are active towards human cells including those at the extremes of age—e.g., newborns and the elderly; and (4) The compounds induce a distinct pattern of cytokines relative to other established small molecule adjuvants (e.g., different from R848), suggesting distinct activity.
Table 1. Activity of Exemplary Imidazopyrimidine Compounds on Stimulating TNF Production in PBMCs as Compared to Compound R848
Table 1 provides the activity of exemplary compounds on stimulating TNF production in PBMCs as compared to the benchmark compound R848. Specifically, TNF production was measured by ELISA after stimulation of adult PBMCs with compound 037 analog family or the compound R848 at 33 μM for 18 hours. Analogs are ranked top to bottom by median percentage TNF production (TNF %) as compared to R848 (N=3-5). The compounds have been classified in the following categories according to TNF %: high activity—class A: TNF %>15%; medium activity—class B: 1%<TNF %≤15%; low activity—class C: TNF %≤1%.

(173) TABLE-US-00003 TABLE 1 Activity of Exemplary Imidazopyrimidine Compounds on Stimulating TFN Production in PBMCs as compared to Compound R848 Compound TNF TNF % Activity # Molecular ID (Median) (Median) class replicates Series weight Compound Structure FRF-02- 144-2 2.5245 104.32 A 5 6-Me Imidazo- pyrimidine 376.84 FRF-02- 144-3 5.397 99.81 A 5 6-Me Imidazo- pyrimidine 410.40 FRF-02- 193 2.997 91.73 A 5 Imidazo- pyrimidine 372.43 37.16 3.5625 90.17 A 3 Imidazo- pyrimidine 407.27 0 37.37 3.73 71.12 A 3 Imidazo- pyrimidine 396.37 Compound 37 2.403 69.79 A 5 Imidazo- pyrimidine 370.46 FRF-02- 171 2.1615 65.93 A 5 6-F Imidazo- pyrimidine 428.39 FRF-02- 144-1 3.144 55.24 A 5 6-Me Imidazo- pyrimidine 384.48 FRF-02- 166 2.0745 53.91 A 5 6-F Imidazo- pyrimidine 432.35 FRF-02- 167 1.101 51.62 A 5 6-F Imidazo- pyrimidine 432.35 FRF-02- 118 1.8795 43.53 A 5 6-F Imidazo- pyrimidin 414.36 FRF-03- 082 1.008 41.65 A 5 6-F Imidazo- pyrimidine FRF-02- 174-1 0.924 35.71 A 5 6-Me Imidazo- pyrimidine 404.90 FRF-02- 187 1.191 31.32 A 5 6-F Imidazo- pyrimidine 390.42 0 37.33 0.288 22.94 A 4 Imidazo- pyrimidine 362.82 37.17 0.865 20.64 A 3 Imidazo- pyrimidine 342.40 FRF-02- 174-2 0.1455 13.99 B 5 6-Me Imidazo- pyrimidine 430.82 FRF-02- 116 0.594 8.48 B 5 6-F Imidazo- pyrimidine 425.26 FRF-03- 081 0.3195 7.51 B 5 6-F Imidazo- pyrimidine FRF-02- 162-2 0.309 6.34 B 5 6-Me Imidazo- pyrimidine 356.43 FRF-03- 084-1 0.24 6.00 B 5 6-F Imidazo- pyrimidine FRF-02- 119 0.219 4.35 B 5 6-F Imidazo- pyrimidine 388.45 FRF-02- 176 0.087 3.81 B 5 6-F Imidazo- pyrimidine 390.42 FRF-02- 117 0.138 3.06 B 5 6-F Imidazo- pyrimidine 360.39 0 FRF-02- 115 0.0625 2.42 B 5 6-F Imidazo- pyrimidine 380.81 FRF-02- 135 0.0705 1.60 B 5 6-MeO Imidazo- pyrimidine 426.40 FRF-02- 175 0.063 1.43 B 5 6-F Imidazo- pyrimidine 432.35 FRF-02- 137 0.04 1.27 B 5 6-MeO Imidazo- pyrimidine 400.48 FRF-02- 183 0.008 0.77 C 5 Imidazo- pyrimidine 372.43 FRF-02- 162-1 0.008 0.42 C 5 6-Me Imidazo- pyrimidine 356.43 FRF-02- 182 0.008 0.39 C 5 Imidazo- pyrimidine 372.43 FRF-02- 103 0.008 0.18 C 5 Imidazo- pyrimidine 342.40 FRF-02- 169 0.008 0.18 C 5 6-F Imidazo- pyrimidine 448.81 FRF-02- 132 0.008 0.18 C 5 6-MeO Imidazo- pyrimidine 437.30 0 FRF-02- 131 0.008 0.18 C 5 6-MeO Imidazo- pyrimidine 392.84 FRF-02- 134 0.008 0.18 C 5 6-MeO Imidazo- pyrimidine 372.43 FRF-02- 194 0.008 0.18 C 5 6-F Imidazo- pyrimidine 390.42
Materials and Methods

(174) Overview. To identify immunomodulators/adjuvants that robustly activate human immune cells, screening of ˜200,000 small molecules tested against THP1 cells was conducted. As there is evidence that cell culture lines such as THP-1 cells may not model responses of natural primary human leukocytes, also employed was a smaller-scale novel high throughput screening methodology in which ˜9,000 commercially available compounds were screened, focusing on small molecule library plates that had relatively high # of hits form the THP-1 screen, using primary human blood mononuclear cells from three different adult human donors and a TNFαLISA Assay (commercially purchased from PerkinElmer). See Schildberger et al., Mediators of Inflammation, Volume 2013, Article ID 697972 (2013).

(175) THP-1 Cell Line and Culture Conditions. The THP1-Lucia™ cell line was obtained from Invivogen (San Diego, Calif.). THP1-Lucia cells, which are human monocytic cells derived from the blood of a boy with leukemia, contain an NF-kB-inducible Luc reporter construct. This allows NF-kB activation to be measured by quantifying the luminescence from the secreted luciferase enzyme. THP1-Lucia cells were cultured in RPMI 1640 supplemented with 10% non-heat inactivated fetal bovine serum (FBS), 10 mM HEPES, 1.0 mM sodium pyruvate, 50 ug/ml Pen-Strep, and 100 ug/mL Normocin™. Once cultured, the cells were stored in a 37° C. incubator with 5% CO.sub.2 and a humidified atmosphere. Cells were passaged every 2-3 days and not allowed to exceed a concentration of 2.0×10.sup.6 cells/ml media.

(176) Chemical Libraries. The chemical libraries screened included known bioactive and commercial libraries from various sources (e.g., commercial libraries such as ChemDiv, ChemBridge, and Asinex; see attached pdf of all libraries). All libraries were owned and provided by the Institute of Chemistry and Cell Biology (ICCB)—Longwood (Harvard Medical School).

(177) NF-kB-induced Luminescence Assay. THP1-Lucia cells between passage 15 and 18 and suspended in culture medium were dispensed into 384-well black clear-bottom plates (Corning 3712) at 30,000 cells/30 μl/well using a Combi liquid dispenser. To allow comparison to a benchmark small molecule with known immune stimulating activity, cells at the same concentration were stimulated with 50 μM R848, a TLR7/8 agonist, in 0.3% DMSO and added to every other well of column 24, which was left empty of cells, by multichannel pipette at the same volume. Five μl of 700 nM Phorbol myristate acetate (PMA) in THP1 culture media and 2.3% DMSO, a known peripheral blood cell mitogen, was added to every other well of column 23 by multichannel pipette (final concentration in well: 100 nM in 0.3% DMSO). Five μl of THP1 culture media with 2.3% DMSO was added to the remaining wells of column 23 by multichannel pipette (final concentration 0.3% DMSO). One hundred nl aliquots of library compounds diluted in 100% DMSO were transferred from their original 384-well plates to the assay plates using a Seiko pin transfer machine. Each library plate was pinned in duplicate, yielding two assay plates with identical conditions for comparison. Plates were then incubated for 24 hours at 37 C with 5% CO.sub.2 in humid conditions. Following incubation, 10 μL of supernatant was removed from each well and transferred to a white plate (Corning 3570) using a Vprep liquid transfer machine. 10 μL of recombinant Lucia protein (Invivogen) diluted 1:2000 in THP1 culture media was added to empty well 24P. Using a Combi liquid dispenser, 50 μL/well of Quanti-Luc substrate (Invivogen) diluted 1:3 in sterile water was added to the assay plate. Immediately after adding the substrate, the luminescence was read using a PerkinElmer Envision plate reader.

(178) THP1 Cell Adherence Assay. Following incubation and supernatant transfer described above, each black assay plate was manually washed. First, remaining suspension cells were expelled into a bath of 15% bleach by shaking the plate upside down. The plate was then submerged in a 7 L bath of 1×PBS. Once submerged, each plate was shaken vigorously side-to-side to release any air bubbles forming in the wells. The plate was then removed and shaken into the bleach bath once again. This process was repeated 3 times per plate.

(179) 30 μL/well of a mix of 2 μg/mL Hoechst 33342 in PBS with 1% Para-formaldehyde was added using a Combi liquid dispenser to the washed assay plates. Plates were left in the dark to stain and fix for 20 minutes. Washed and stained assay plates were then loaded onto an Acumen laser scanning cytometer. Total fluorescence area and number of objects (nuclei) were measured using the instrument for each well.

(180) TNFαLISA Assay. Human adult peripheral blood was collected according to approved protocols. PBMCs were isolated from blood using a Ficoll density gradient. PBMCs were stored at 5×10.sup.7 cells per vial in 1 mL RPMI containing 20% autologous plasma and 10% DMSO at −80° C. until use. On Day 1, PBMCs were thawed in 37° C. water bath for 3 min and washed twice with PBS. Viability was assessed by trypan blue staining and cells were then resuspended to 6.67×10.sup.5 viable cells/mL in DMEM with 10% autologous plasma. 30 μl of cells were dispensed per well in Corning 2712 black 384-well cell culture plates (final concentration was 20,000 cells/well). Controls were added to cells manually and the test compounds were added by robotic pin transfer. Plates were then incubated at 37° C./5% CO.sub.2 humidity-controlled incubator for 24 hours. On day 2 of the assay, plates were centrifuged and 2 μl supernatants were collected into Perkin Elmer Alpha plates. Perkin Elmer Human TNFα kits (cat #AL208F) were used to detect the presence of TNFα in supernatants. Plates were run on the EnVision (Perkin Elmer) instrument to detect light emission at 615 nm.

(181) Hit Calling Method. Test compounds that resulted in a robust Z score>2 in both duplicates and at ≥2 of the 3 human samples of PBMCs were considered hits. The see following hit calling standard operating procedures (SOPs) were used for the THP-1 and TNFαLISA Assay::

(182) All Data is log 10-transformed (log 10)—CrossTalk Corrected luminescence data from columns E, and F is log-transformed (log 10) in columns K, and L. Only experimental wells are evaluated, referencing column C with an “if” logic statement. An example of the calculation in column Q, log-transforming data from column E, is shown below:

(183) =IF(C2=“X”,LOG 10(E2), “ ”)

(184) A robust Z score is calculated for each experimental well with adjusted median absolute deviation (MAD) values. First, the plate median and MAD values are generated from the log-transformed data. The absolute deviation for each well value is calculated in columns M and N, an example of which is shown below:

(185) =IF(C.sub.2=“X”, ABS(K2−$I$3), “ ”) wherein K2 is a log transformed data point, and 13 is the median value for that plate for that readout.

(186) The MAD is then calculated as the median of each of columns U-X multiplied by 1.4286, an example of which is shown below:

(187) =MEDIAN(M2:M385)*1.4286

(188) The robust Z score is then calculated as (well_value−median_plate)/(MAD_plate*1.4286), an example calculation is shown below:

(189) =IF($C2=“X”, ((K2−$I$3)/$I$5), “ ”) wherein K2 is a log-transformed well value, 13 is the plate experimental median for that readout, and 15 is the plate experimental MAD for that readout.

(190) Any wells with robust Z score values of 2 or greater in both replicates are considered a hit. This is evaluated in column Q as shown in the example below:

(191) =IF(AND((O2>$I$7), (P2>$J$7), ($C2=“X”)),TRUE,FALSE) wherein O2 and P2 are robust Z scores for replicates of the luminescence readout, while I7 and J7 represent the luminescence robust Z score threshold (2). “TRUE” will be returned in column Q if the compound meets these hit criteria.

(192) For any well that is determined to be a hit, the Plate:Well compound ID will be displayed in column R as shown in the example below:

(193) =IF(Q2,CONCATENATE(A2, “:”,B2), “ ”) in which Q2 is the TRUE/FALSE value determining the hit status of the compound while A2 is the plate ID and B2 is the well ID.

(194) Human MoDCs arrays. Heparinized human adult peripheral blood was layered onto Ficoll-Hypaque gradients (Ficoll-Paque PREMIUM, GE Healthcare, Waukesha, Wis.) blood mononuclear cell (PBMC's) layers. Monocytes were isolated from mononuclear cell fractions by positive selection with magnetic microbeads according to the manufacturer's instructions (Miltenyi Biotec, Auburn, Calif.) using CD14 as a pan-marker. Monocyte preparations were routinely >95% pure as assessed by flow cytometry for CD14 as previously described [1-5]. Isolated monocytes were cultured in tissue culture dishes at 0.4×10.sup.6 cells/ml in RPMI 1640 medium containing fresh 10% autologous platelet-poor plasma, supplemented with recombinant human (rh) IL-4 (50 ng/ml) and rhGM-CSF (100 ng/ml) (R&D Systems, Minneapolis, Minn.) with one supplement of fresh medium and cytokines at day 3 of culture. After 5-6 days, immature MoDCs, routinely HLA-DR.sup.+, CD14.sup.−, DC-SIGN.sup.+[3], of >90% purity were harvested by gently pipetting only the loosely adherent fraction and re-plated in a 96-well format at desired cell density.

(195) ELISAs and Multiplex-Analyte Assays. Supernatants derived from human PBMCs and DC stimulations were assayed by ELISA for TNF (ThermoFisher Scientific; Waltham, Mass., USA). Cytokine and chemokine expression profiles (e.g., IFNγ, IL-9, IL-10, IL-12 (p70), IL-13, IL-1β, IL-23, IL-27, IL-28A, IL-33, IL-6, MIP-3α/CCL20, and TNF) in cell culture supernatants were measured using a customized Milliplex® Human Th17 Magnetic Bead Panel according to the manufacturer's instructions (Millipore, Chicago, Ill., USA). Assays were read and analyzed on the Luminex® 100/200™ System and xPOTENT® software (Luminex, Austin, Tex.). A minimum threshold was set at the minimum detectable concentration for each individual assay, defined as three standard deviations above the mean background.

(196) In Vitro PBMC Stimulation.

(197) PBMCs were isolated from blood using a Ficoll density gradient. PBMCs either used fresh or were stored at 5×10.sup.7 cells per vial in 1 mL RPMI containing 20% autologous plasma and 10% DMSO at −80° C. until use. Stimulation plates were prepared by transferring 0.66 μl of DMSO-dissolved compounds (10 mM) to each well of a round bottom 96-well plate. PBMCs isolated from human adult donors were resuspended at a concentration of 10.sup.5 cells/200 μl of RPMI supplemented with 10% of platelet-poor plasma. 200 μl of the cell suspension were transferred to each well resulting in a final compound concentration of 33 M. After about 18 hours of incubation (37° C., 5% CO.sub.2), plates were spun down (500×g, room temperature, 5 minutes) and supernatants were harvested for further analysis.

(198) Newborn Whole Blood Assay

(199) Human newborn cord blood was collected from term newborns (n=5) immediately after cesarean section using pyrogen-free heparin as anti-coagulant. Cord blood was diluted 1:1 (v/v) in RPMI 1640 medium. One microliter of each of the indicated compounds was added to each well containing 200 microliters of diluted blood in a 96-well round-bottomed plate at the final concentration of 11 μM. The plate was then incubated in the presence of 5% CO.sub.2 at 37° C. in a humidified incubator for 18 hours. After ˜18 hours of incubation (37° C., 5% CO.sub.2), plates were spun down (500×g, room temperature, 5 minutes) and supernatants were harvested for further analysis.

(200) Animals

(201) C57BL/6 and BALB/c mice were obtained from Taconic Biosciences or Charles River Laboratories and housed in specific pathogen-free conditions in the animal research facilities at Boston Children's Hospital.

(202) Splenocyte Isolation and In Vitro Stimulation

(203) Spleens were harvested from 6-8 weeks old C57BL/6 mice. For splenocyte isolation, spleens were mashed through a 70 μM strainer, washed with PBS, and erythrocytes were lysed with 2 min of incubation in ammonium chloride-based lysis buffer (BD Biosciences). Cells were then counted and plated 2×10.sup.6 per well (round bottom 96-well plate) in 200 μl of complete culture medium (RPMI 1640 plus 10% heat-inactivated fetal bovine serum [FBS, GE Healthcare HyClone], 50 μM 2-mercaptoethanol, 2 mM 1-glutamine, 100 U/ml penicillin/streptomycin [Gibco ThermoFisher Scientific]) with 0.66 μl of DMSO-dissolved compounds (10 mM) in order to achieve a final compound concentration of 33 μM. After 18 hours of incubation (37° C., 5% CO.sub.2), plates were spun down (500×g, room temperature, 5 minutes) and supernatants were harvested for further analysis.

(204) Antigens, Immunization and Antibody Quantification

(205) For immunization experiments, adult mice were immunized intramuscularly (i.m.) in the right posterior thigh with 50 μl of vaccine containing 0.33 μg of each of the following recombinant influenza virus hemagglutinins (rHA): A/Michigan/45/2015 (H.sub.1N.sub.1), A/Hong Kong/4801/2014 (H3N2), and B/Brisbane/60/2008, contained in the 2016-2017 formulation of the FluBlok vaccine (Protein Sciences Corp.). Mice were immunized with a prime-boost schedule (two injections four weeks apart). Vaccine in all experimental groups was formulated with 10% (v/v) DMSO (except for the groups immunized with the small molecules since they were dissolved in DMSO) and 5% (v/v) Tween-80. As indicated for specific experimental groups, vaccine was also formulated with Aluminium hydroxide (100 μg) and/or compound 037 (100 nmol, final DMSO concentration 10%). Serum was collected 28 days post-prime (pre-boost blood sample) and 14 days post-boost for antibody detection. rHA-specific IgG were quantified by ELISA. High binding flat bottom 96-well plates (Corning Life Sciences) were coated with 1 μg/ml rHA in carbonate buffer pH 9.6, incubated overnight at 4° C. and blocked with PBS+BSA 1% (Sigma-Aldrich) for 1 h at room temperature (RT). Then, sera from vaccinated mice were added with an initial dilution of 1:100 and 1:4 serial dilutions in PBS+BSA 1% and incubated for 2 h at RT. Plates were then washed and incubated for 1 h at RT with HRP-conjugated anti-mouse IgG (Southern Biotech). At the end of the incubation plates were washed again and developed with tetramethylbenzidine (BD Biosciences) for 5 minutes, then stopped with 1 N H.sub.2SO.sub.4. The optical density was read at 450 nm Versamax microplate reader with SoftMax Pro Version 5 (both from Molecular Devices) and endpoint titers were calculated using as cutoff three times the optical density of the background.

(206) Statistical analysis. Statistical significance and graphic output were generated using Prism v. 5.0b (GraphPad Software) and Microsoft Excel (Microsoft Corporation, Redmond, Wash.). Results were considered significant at p values<0.05, and indicated as follows: * p<0.05, ** p<0.01, *** p<0.001.

REFERENCES

(207) 1. Dowling, D. J., et al., The ultra-potent and selective TLR8 agonist VTX-294 activates human newborn and adult leukocytes. PLoS One, 2013. 8(3): p. e58164. 2. Palmer, C. D., et al., The effect of stable macromolecular complexes of ionic polyphosphazene on HIV Gag antigen and on activation of human dendritic cells and presentation to T-cells. Biomaterials, 2014. 35(31): p. 8876-86. 3. Philbin, V. J., et al., Imidazoquinoline Toll-like receptor 8 agonists activate human newborn monocytes and dendritic cells through adenosine-refractory and caspase-1-dependent pathways. J Allergy Clin Immunol, 2012. 130(1): p. 195-204 e9. 4. Ganapathi, L., et al., The Imidazoquinoline Toll-Like Receptor-7/8 Agonist Hybrid-2 Potently Induces Cytokine Production by Human Newborn and Adult Leukocytes. PLoS One, 2015. 10(8): p. e0134640. 5. Dowling, D. J., et al., Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses. J Allergy Clin Immunol, 2017. 6. Dowling, D. J., et al., TLR7/8 adjuvant overcomes newborn hyporesponsiveness to pneumococcal conjugate vaccine at birth, JCI Insight. 2017; 2(6).

EQUIVALENTS AND SCOPE

(208) In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

(209) Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

(210) This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

(211) Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.