WO2015171951A1 - 2-(4-aryl-1h-imidazol-1-yl)aniline compounds - Google Patents

Abstract

The present invention provides compounds that are useful as vaccine adjuvants and/or antitumor agents and methods for producing and using the same. In one particular aspect of the invention, compounds of the invention are of the formula (I) where R¹, R², R³ and Ar¹ are those defined herein.

Description

2-(4-ARYL-l^IMIDAZOL-l-YL)ANILINE COMPOUNDS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No.

61/989,603, filed May 7, 2014, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grant numbers

GM 103843 and GM101279 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to compounds that are useful as vaccine adjuvants and/or antitumor agents and methods for producing and using the same. In one particular aspect of the invention, compounds of the invention are 2-(4-aryl-lH-imidazol-l- yl)aniline compounds or derivatives.

BACKGROUND OF THE INVENTION

[0004] The vertebrate host defense system is broadly classified into two groups:

innate and adaptive immunity. The innate immunity provides an immediate broad response to infection by recognizing conserved structures called pathogen associated molecular patterns (PAMPs). In contrast, the adaptive immunity provides a slow specific response to infection dependent on B cell differentiation and T cell activation by a particular antigen.

[0005] It has been shown that toll-like receptors (TLRs), which are a family of pattern recognition receptors, regulate innate immunity and subsequent adaptive immune responses. Without being bound by any theory, it is believed that TLRs control activation of adaptive immune responses by up-regulating co-stimulatory molecules on antigen-presenting cells (APCs) and producing cytokines, including interferon-a (IFN-a), IFN-γ, and interleukin-12 (IL-12), that guide T cell differentiation. The ability of TLRs to activate both immune systems has made them desirable targets for vaccine adjuvants.

[0006] Currently, a number of modifications to natural TLR agonists have been used for vaccine adjuvants. For example, the modified TLR4 agonist, monophosphoryl lipid A (MPL), is used as an adjuvant in U.S. Food and Drug Administration-approved adult hepatitis B virus (HBV) vaccines. Other TLR agonists are also being investigated for vaccine adjuvant applications, including the TLR9 agonist CpG-ODN in HBV vaccines. Literature reports have suggested that the TLR2 agonists are among the most effective adjuvants as evidenced by their uses in the HIV, HBV, and human papillomavirus (HPV) vaccines. TLRl/2 agonists are also believed to decrease infection-related morbidity and mortality and improve vaccine response in the elderly. Specific TLR agonists can be used not only for vaccine adjuvants, but also for combined cancer therapies. For example, the TLR7 agonist imiquimod is one of the well-known examples and has been approved for treating basal cell skin tumors. In addition, agonists of TLR2 have been used to induce lung tumor regression, inhibit breast cancer growth, and to treat bladder cancer and pancreatic carcinoma. Furthermore, the TLRl/2 agonists are also believed to be effective in both chronic and acute inflammatory/infectious diseases such as influenza, asthma, and age-induced obesity.

[0007] Currently, none of the vaccine adjuvants in clinical or preclinical development are small molecules. Accordingly, there is a need for a small molecule compound that can used as vaccine adjuvants.

SUMMARY OF THE INVENTION

[0008] One particular aspect of the invention rovides a compound of the formula:

I

where R¹ is H, N0₂, haloalkyl, amino, cyano or -COOR⁴, wherein R⁴ is H or alkyl; each of R² and R³ is independently H, alkyl or aralkyl; and Ar'is optionally substituted aryl. In one particular embodiment of the invention, Ar¹ is not 4-nitrophenyl. Another embodiment of the invention provides compound of Formula I provided Ar¹ is not 4-nitrophenyl; or when R¹ is H, then Ar¹ is not phenyl or 4-bromophenyl; or when R¹ is N0₂, then Ar¹ is not phenyl.

[0009] Compounds of the invention can be used as vaccine adjuvants. Accordingly, another aspect of the invention provides a composition comprising a vaccine and a compound of Formula I. In one particular embodiment, the composition comprises a compound of Formula I provided Ar¹ is not 4-nitrophenyl. Another embodiment of the invention provides a composition comprising a vaccine and a compound of Formula I provided Ar¹ is not 4- nitrophenyl; or when R¹ is H, then Ar¹ is not phenyl or 4-bromophenyl; or when R¹ is N0₂, then Ar¹ is not phenyl.

[0010] Another aspect of the invention provides a composition comprising a compound of Formula I and a vaccine. In one embodiment, the vaccine comprises hepatitis B virus (HBV) vaccine, human immunodeficiency virus (HIV) vaccine, hepatitis C virus (HCV) vaccine, human papillomavirus (HPV) vaccine, or a combination thereof.

[0011] Compounds of the invention can also be used for cancer treatment. In particular, compounds of the invention can be used to treat lung carcinoma, breast cancer, bladder cancer and/or pancreatic carcinoma. Compounds of the invention also can be used to treat chronic and/or acute inflammatory / infectious diseases ranging from influenza, asthma to age-induced obesity. Compounds of the invention are also useful in decreasing the infection-related morbidity and mortality and the improving vaccine responses in aging humans.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a graphic result showing that a compound of the invention activates secreted embryonic alkaline phosphatase (SEAP) signaling in a dose-dependent manner. HEK-Blue hTLR2 cells were incubated with Compound A or GA for 24 h, and activation was evaluated by SEAP secretion in the culture supernatants by the luminescence assay.

[0013] Figures 2A-2F show selectivity of Compound A as a TLR2 signaling agonist.

Human TLR2, TLR3, TLR4, TLR5, TLR7 and TLR8 HEK-Blue cells were incubated with Compound A (0-20 μΜ) or TLR-specific agonist for 24 h, and activation was evaluated by the luminescence assay. As positive control agonists that selectively activate a specific TLR were used. Panel A shows the results of TLR1/TLR2 selectivity experiment using

PamsCSIQ as the control; Panel B shows the result of TLR3 selectivity experiment using polyinosinic-polycytidylic acid [Poly I:C] as the control; Panel C shows the result of TLR4 selectivity experiment using LPS as the control; Panel D shows the result of TLR5 selectivity experiment using FLA-BS as the control; Panel E shows the result of TLR7 selectivity experiment using R848 as the control; and Panel F shows the result of TLR8 selectivity experiment using R848 as the control.

DETAILED DESCRIPTION OF THE INVENTION

[0014] "Alkyl" refers to a saturated linear monovalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a saturated branched monovalent hydrocarbon moiety of three to twelve, typically three to six, carbon atoms. Exemplary alkyl group include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl, and the like. [0015] "Alkoxy" refers to a moiety of the formula -OR', where R' is alkyl as defined herein.

[0016] "Alkylene" refers to a saturated linear divalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a branched saturated divalent hydrocarbon moiety of three to twelve, typically three to six, carbon atoms. Exemplary alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, and the like.

[0017] "Aryl" refers to a monovalent mono-, bi- or tricyclic aromatic hydrocarbon moiety of 6 to 15 ring atoms that are optionally substituted with one or more substituents. When substituted, an aryl group typically has one, two, or three substituents within the ring structure. When two or more substituents are present in an aryl group, each substituent is independently selected. Exemplary substituents on the aryl group include, but are not limited to, nitro (-N0₂), halo, alkyl, alkoxy, cyano, haloalkyl, phenyl, cycloalkyl, -C0₂R (where R is H or alkyl), and the like.

[0018] "Aralkyl" refers to a moiety of the formula -R^bR^c where R^b is an alkylene group and R^c is an aryl group as defined herein. Exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, and the like.

[0019] "Cycloalkyl" refers to a non-aromatic, typically saturated (although one or more unsaturated bonds within the ring system can be present), monovalent mono- or bicyclic hydrocarbon moiety of three to ten ring carbons. The cycloalkyl can be optionally substituted with one or more substituents. When substituted, cycloalkyl typically has one, two, or three, substituents within the ring structure. When two or more substituents are present in a cycloalkyl group, each substituent is independently selected.

[0020] The terms "halo," "halogen" and "halide" are used interchangeably herein and refer to fluoro, chloro, bromo, or iodo.

[0021] "Haloalkyl" refers to an alkyl group as defined herein in which one or more hydrogen atom is replaced by same or different halo atoms. The term "haloalkyl" also includes perhalogenated alkyl groups in which all alkyl hydrogen atoms are replaced by halogen atoms. Exemplary haloalkyl groups include, but are not limited to, -CH₂C1, -CF₃, - CH₂CF₃, -CH2CCI3, and the like.

[0022] "Leaving group" has the meaning conventionally associated with it in synthetic organic chemistry, i.e., an atom or a group capable of being displaced by a nucleophile and includes halo (such as chloro, bromo, and iodo), alkanesulfonyloxy, arenesulfonyloxy, alkylcarbonyloxy (e.g., acetoxy), arylcarbonyloxy, mesyloxy, tosyloxy, trifluoromethanesulfonyloxy, aryloxy (e.g., 2,4-dinitrophenoxy), methoxy, N,0- dimethylhydroxylamino, and the like.

[0023] "Pharmaceutically acceptable excipient" refers to an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.

[0024] "Pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,

benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4- toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-lcarboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[0025] The terms "pro-drug" and "prodrug" are used interchangeably herein and refer to a pharmacologically substantially inactive derivative of a parent drug molecule that requires biotransformation, either spontaneous or enzymatic, within the organism to release the active drug. Prodrugs are variations or derivatives of the compounds of this invention which have groups cleavable under metabolic conditions. Prodrugs become the compounds of the invention which are pharmaceutically active in vivo when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. Prodrug compounds of this invention may be called single, double, triple etc., depending on the number of biotransformation steps required to release the active drug within the organism, and indicating the number of functionalities present in a precursor-type form. Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier,

Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, Calif., 1992). Prodrugs commonly known in the art include acid derivatives that are well known to one skilled in the art, such as, but not limited to, esters prepared by reaction of the parent acids with a suitable alcohol, or amides prepared by reaction of the parent acid compound with an amine, or basic groups reacted to form an acylated base derivative. Moreover, the prodrug derivatives of this invention may be combined with other features herein taught to enhance bioavailability. For example, a compound of the invention having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are co valently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of compounds of the invention. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma- aminobutyric acid, citrullinehomocysteine, homoserine, ornithine and methionine sulfone. Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of a compound of the invention through the carbonyl carbon prodrugsidechain.

[0026] "Protecting group" refers to a moiety, except alkyl groups, that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3^rd edition, John Wiley & Sons, New York, 1999, and Harrison and Harrison et al, Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996), which are incorporated herein by reference in their entirety. Representative hydroxy protecting groups include acyl groups, benzyl and trityl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. Representative amino protecting groups include, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC), and the like.

[0027] "Corresponding protecting group" means an appropriate protecting group corresponding to the heteroatom (i.e., N, O, P or S) to which it is attached. [0028] "A therapeutically effective amount" means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.

[0029] "Treating" or "treatment" of a disease includes: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.

[0030] When describing a chemical reaction, the terms "treating", "contacting" and

"reacting" are used interchangeably herein, and refer to adding or mixing two or more reagents under appropriate conditions to produce the indicated and/or the desired product. It should be appreciated that the reaction which produces the indicated and/or the desired product may not necessarily result directly from the combination of two reagents which were initially added, i.e., there may be one or more intermediates which are produced in the mixture which ultimately leads to the formation of the indicated and/or the desired product.

[0031] As used herein, the terms "those defined above" and "those defined herein" when referring to a variable incorporates by reference the broad definition of the variable as well as any narrow and/or preferred, more preferred and most preferred definitions, if any.

[0032] "EC₅₀" means the effective concentration, i.e., the concentration of a compound at which 50% of the maximal response would be achieved.

[0033] General Overview: Without being bound by any theory, some aspects of the invention are based compounds that are believed to be selective modulators of TLRs. In one particular embodiment, the present invention is based on the discovery of compounds that selectively target TLR1/2 to initiate downstream signaling in human embryonic kidney 293 and macrophage cell lines. The activity was initially discovered through a high-throughput chemical screen for compounds that promote IL-8 production. Structure-activity relationship studies yielded a highly potent analog called N-methyl-4-nitro-2-(4-(4- (trifluoromethyl)phenyl)-lH-imidazol-l-yl)aniline (Compound A), which can act as a single agent to selectively induce TLR1/2 dimerization, leading to NF-κΒ, TNF-a, IL-10, and iNOS production. The present invention also provides new insights into the regulation of the TLR1/2 signaling pathway by compounds disclosed herein. In addition, compounds of the invention can also be used in a variety of therapeutic applications including, but not limited to, in cancer therapy, as vaccine adjuvant, and treatment of chronic and/or acute inflammatory/infectious diseases ranging from influenza, asthma to age-induced obesity.

[0034] As discussed in detail herein, it was discovered that compounds of the invention bind to the interface of TLR1 and TLR2, facilitating heterodimer association. The discovery of compounds of the invention not only helps in better understanding of the regulatory mechanism of the TLR1/2 signaling pathway, but also renders an immense potential for vaccine or cancer therapy developments as well as treating any clinical condition that can be treated by activation of TLR1 and/or TLR2.

[0035] Compounds of the Invention: Some aspects of the invention are based on the discovery and development of selectiveTLR2 agonists. TLR2 recognizes a wide range of ligands, many of which are from Gram-positive bacteria, and it signals as a heterodimer with either TLR1 or TLR6. It is believed that none of the TLR2 agonists in clinical or preclinical development are small molecules, and some contain mixtures with more than one active ingredient. Furthermore, the activation through TLR1/2 or TLR2/6 cannot be selectively achieved in most conventionally known TLR2 agonists. Based on the experiments discussed herein, compounds of the invention target the TLR2 heterodimer protein-protein interaction, often selectively, if not specifically, and modulate the TLR1/2 response.

[0036] One particular aspect of the invention rovides a compound of the formula:

I

where R¹ is H, N0₂, haloalkyl, amino, cyano or -COOR⁴, where R⁴ is H or alkyl; each of R² and R³ is independently H, alkyl or aralkyl; and Ar¹ is optionally substituted aryl.

[0037] In some embodiments, R¹ is selected from the group consisting of N0₂, H, trifluoromethyl, amino, cyano, and-C0₂R⁴, where R⁴ is H or C1 -C4 alkyl. Still in other embodiments, R² is H. Yet in other embodiments, R³ is selected from the group consisting of methyl, ethyl, butyl, and benzyl. In other embodiments, Ar¹ is optionally substituted phenyl or naphthyl. In one particular embodiment, Ar¹ is a substituted phenyl. In one instance, the substituted phenyl is of the formula:

where R¹³ is H, N0₂, halide, cyano, haloalkyl, aryl, alkyl (e.g., methyl, ethyl, etc.), alkoxy (e.g., methoxy, ethoxy, etc.), phenyl, cycloalkyl or -COOR⁵, wherein R⁵ is H or alkyl (e.g., methyl, ethyl, etc.); R¹⁴ is H, N0₂ or halide (often F); and R¹⁵ is H or halide (often F). In some cases, R¹³ is selected from the group consisting of H, N0₂, fluoro, cyano,

trifluoromethyl, C1-C4 alkyl, C1-C4 alkoxy, phenyl, cyclohexyl, and -COOR⁵, wherein R⁵ is H or C1-C4 alkyl. In other cases, R¹⁴ is selected from the group consisting of H, N0₂ and fluoro. Still in some cases, R¹⁵ is selected from the group consisting of H or fluoro.

[0038] It should be appreciated that combinations of the various groups described herein form other embodiments. For example, in one particularly embodiment R¹ is N0₂, R² is H, R³ is methyl, and Ar¹ is 4-trifluoromethylphenyl (Compound A). In this manner, a variety of compounds are embodied within the present invention.

[0039] Another aspect of the invention provides a composition comprising a vaccine and a compound of Formula I. In one particular embodiment, Compound of Formula I is used as a vaccine adjuvant. That is, Compound of Formula I enhances efficacy of a vaccine and/or reduces the amount of vaccine required. Generally, Compound of Formula I can be used as an adjuvant to any vaccine. In one particular embodiment, Compound of Formula I is used as adjuvant to HBV vaccine, HIV vaccine, HPV vaccine, and/or HCV vaccine.

[0040] Yet other aspects of the invention provide a method for treating a clinical condition that can be treated by selective activation of TLR2. In particular, selective activation of TLR2 refers to activation of TLR2 to form TLR1-TLR2 heterodimer selectively or specifically over formation of TLR2-TLR6 heterodimer. In some instances, compounds of the invention have at least 80%, typically at least 90%, often at least 95%, and most often at least 98% selectivity for TLR1-TLR2 heterodimer formation over TLR2-TLR6 heterodimer formation. The method comprises administering to a subject in need of such a treatment a compound of Formula I.

[0041] Still in another aspect of the invention, Compound of Formula I is used to treat cancer, a chronic inflammatory disease, an acute inflammatory disease, an infection, or obesity. In some embodiments, Compound of Formula I is used to treat breast cancer, bladder cancer, pancreatic carcinoma, influenza, asthma and/or age-induced obesity.

[0042] In one particular embodiment, Compound of Formula I is used in treatment of cancer. In such embodiments, Compound of Formula I can be administered in combination with a radiation therapy, chemotherapy, monoclonal antibody therapy, or a combination thereof. It should be noted that Compound of Formula I can be administered substantially simultaneously with other cancer treatment(s), i.e., within an hour or two, either pre- or post- treatment. Alternatively, Compound of Formula I can be administered a few hours (e.g., within 12 hours, typically within 6 hours) or a few days (e.g., within a week, typically within one, two or three days) before or after administering the other cancer treatment protocol.

[0043] Still other aspects of the invention provide a method for selectively activating

TLR2 in a subject by administering an effective amount of a compound of Formula I to the subject. It should be appreciated that selectively activating TLR2 refers to selectively activating TLR1-TLR2 heterodimer formation/production as described herein.

[0044] It should be appreciated that a prodrug and/or a pharmaceutical salt of

Compound of Formula I can also be used. Accordingly, the scope of the invention includes the use of a prodrug and/or a pharmaceutical salt of Compound of Formula I.

[0045] An improved understanding of cancer pathogenesis has given rise to new treatment options including cancer immunotherapy and targeted agents. As discussed herein, Compound of Formula I can be used as anticancer agents or co-anticancer agents with radiation, monoclonal antibodies, or cytotoxic drugs, and form long-lasting protective response against tumor re-challenge. Retrospective analysis of a clinical trial for a TLR3 agonist, poly-AU, in breast cancer patients indicated an improved 20-year survival rate for patients whose tumor cells expressed TLR3. Activation of TLR2 has been demonstrated to induce lung tumor regression, inhibit breast cancer growth, treat bladder cancer, and treat pancreaticcarcinoma. Compound of Formula I can also be used in non-cancer applications. As specific agonists for TLR1/2 have been suggested to be effective in both chronic and acute inflammatory/infectious diseases ranging from influenza, asthma, and age-induced obesity^'

[0046] A library of approximately 24,000 compounds were screen for luciferase activities using an IL-8-drivenluciferase reporter in SW620 human colonic epithelial cells, which led to the identification of N-methyl-4-nitro-2-(4-(4-nitrophenyl)-lH-imidazol-l- yl)aniline (GA), containing a 1,4-diphenyl-lH-imidazole core. GA demonstrates TLR- dependent activities in vitro; nonetheless, its direct target has not been biochemically or biophysically characterized.

[0047] The direct target for GA has been identified by the present inventor and the structure of GA was modified to achieve high efficacy and potency. The chemical synthesis utilized to produce some of the compounds of the invention is illustrated in Scheme 1 below: ^{12N HCI}' ^tr'^ethy' orthoformate

H, DMF, rt 12h, 60%

Scheme 1

More detailed representative procedures for the synthesis of Compound of Formula I are illustrated in the Examples section below.

[0048] A secreted embryonic alkaline phosphatase (SEAP) assay demonstrated GA could activate TLR2 with a half maximal effective concentration (EC₅₀) of 2.51±0.42μΜ. Structure-activity relationship (SAR) studies of compounds of the invention showed EC₅₀ of Compound A to be 52.9 ± 6.2nM. See Figure 1. Cellular study results of some of the compounds of the invention are shown in Table 1 below.

Table 1. Representative SEAP Assay Results

9 : R_a— F^"

Compound Ri R₂ R₃ P EC₅₀ (nM)

GA N0₂ CH₃ N0₂ H 2510± 420

1 H CH₃ N0₂ H ND

2 N0₂ CH₃ H H ND

3 N0₂ CH₂CH₃ N0₂ H >100000

4 N0₂ n-Bu N0₂ H ND

5 N0₂ benzyl N0₂ H ND

6 N0₂ CH₃ F H 2780± 810

7 N0₂ CH₃ CH₃ H ND

8 N0₂ CH₃ OCH₃ H ND

9 N0₂ CH₃ F F >100000

10 N0₂ CH₃ H N0₂ ND

11 N0₂ CH₃ CN H 210±30

12 (Compound A) N0₂ CH₃ CF₃ H 52.9 ± 6.2

13 N0₂ CH₃ Ph H 84.7 ± 1.2

14 N0₂ CH₃ cyclohexyl H 120± 20

15 N0₂ CH₃ - - 420± 50

16 N0₂ CH₃ t-Bu H 150± 20 17 N0₂ CH₃ n-Bu H 230± 30

18 N0₂ CH₃ COOCH₃ H 990± 190

19 N0₂ CH₃ COOH H ND

20 CF₃ CH₃ CF₃ H 240± 40

21 NH₂ CH₃ CF₃ H ND

22 COOCH3 CH₃ CF₃ H >100000

23 COOH CH₃ CF₃ H ND

24 CN CH₃ N0₂ H 10960± 212θ"

* Not detected.

** The highest SEAP signaling is at least 50% lower than the highest signaling of GA.

[0049] As can be seen in Table 1 , removing either of the nitro groups at position Ri or

R₃ decreases the potency. At the same time, the potency dropped when the aliphatic chain length was increased or a benzyl group was introduced at the amino site. The methyl group on the secondary amine was maintained, and the following studies on this series focused on two primary areas: the substitutions on ring A and B. The electronic properties at the R₃- position of ring B were investigated. A fluorine at this position (compound 6) showed similar activity to GA, suggesting that electron- withdrawing groups are tolerated. The loss of potency with electron-donating groups (e.g., compounds 7 and 8) confirmed the preference of electronegativity at this position. However, more electron- withdrawing groups in ring B were not beneficial to the potency (compound 9). No activity was observed when the R₃-nitro group was switched to the R4-position indicating that a substitution on the Reposition increases the potency. The -CN or -CF₃ replacement of -N0₂ at the Reposition increased the potency by 10 to 50 fold (compound 11 and Compound A).

[0050] It appears that a hydrophobic group at Reposition might be helpful for potency as can be seen by t-butyl (compound 16), n-butyl (compound 17) and methyl benzoate (compound 18) substituents, where each had a higher potency compared to GA. Compound 17 showed a modest decrease of potency compared with the t-butyl analog.

Compound 19 showed no noticeable activity indicating that a polar substituent at R₃ position reduces the activity. As seen with the above active analogs, the R₃ position can tolerate a wide variety of substituents. In particular, an electron withdrawing -CF₃ group at the R₃ position showed one of the highest potencies. For this reason, the -CF₃ group at the R₃- position of ring B was maintained in the following structure activity relationship (SAR) studies on ring A. Compound 20 having a -CF₃ group at the Ri -position of ring A showed ~4x decrease in potency compared to Compound A. Electron-donating and hydrophobic containing aromatic systems such as amino (compound 21), ester (compound 22), and carboxylic acid (Compound 23), gave further reduction in potency (data not shown), indicating an electron- withdrawing groups in this aromatic ring increases the activity, with - N0₂ showing highest activity. The cyano group analog (compound 24) also showed decreased activity compared to Compound A.

[0051] Similar to other TLRs, TLR2 is involved in the recognition of a wide array of microbial molecules. Selectivity of Compound A to TLR2 signaling was studied. By using HEK-Blue hTLRs cells overexpressing different TLRs, including TLR2, TLR3, TLR4, TLR5, TLR7 and TLR8, the specificity of Compound A was evaluated. See Figures 2A-F. These cell lines were obtained by co-transfection of the human TLRs and secreted embryonic alkaline phosphatase (SEAP) genes into HEK293 cells. Stimulation with a TLR ligand activates NF-κΒ and AP-1, which induce the production of SEAP. Increases in the SEAP signaling correlates to TLR activation, as monitored by the luminescence intensity. Results showed Compound A strongly activated the SEAP signaling in HEK-Blue cells

overexpressing hTLR2 cells, but not in other TLR-overexpressing cells, including TLR3, TLR4, TLR5, TLR7 and TLR8. TLR-specific agonists were used as positive controls for each HEK-Blue cell line in this SEAP assay experiment. These results showed that

Compound A can specifically activate the TLR2 signaling pathway.

[0052] Among the 10 human TLRs, most TLRs act alone. Without being bound by any theory, it is believed that only TLR2 signals as a heterodimer with either TLRl or TLR6. HEK-Blue hTLR2 cells endogenously express TLRl and TLR6. To identify whether

Compound A is a TLRl/2 agonist or a TLR2/6 agonist, an antibody inhibition experiment was performed to test its selectivity. It was observed that 60 nM Compound A can efficiently activate SEAP signaling and such activation can be reversed by either the anti-fiTLRlor anti- hTLR2 antibodies in a dose-dependent manner. Cell viability experiment confirmed that the cytotoxicity by the antibodies at the used dose is negligible. By contrast, no inhibition of the SEAP signaling activated by Compound A by an anti-hTLR6 antibody with concentrations up to 10 μg/mL was observed. This activity is similar to the established TLRl/2-specific agonist, PamsCSIQ. Compound A's inhibitory activity was further tested in the similar SEAP assay using an established TLR2/6 agonist Pam₂CSK₄. It was observed that both anti-hTLR2 and anti-hTLR6 antibodies can inhibit Pam₂CSK₄ induced SEAP signaling, while anti- hTLRl cannot. These results indicate that Compound A acts by specific activation of TLRl/2 signaling.

[0053] TLRs recruit a set of adaptor proteins through homophilic interactions with their Toll/IL-1 receptor (TIR) domains. These interactions triggers downstream signaling cascades leading to the activation of transcription factor nuclear factor-kappaB (NF-KB), which controls induction of pro-inflammatory cytokines and chemokines as well as upregulates co-stimulatory molecules on dendritic cells that are essential for T-cell activation.

[0054] In order to investigate the cellular and molecular mechanisms of Compound A with NF-KB, a TLR2-sensitive U937 human macrophage cell line were developed with a GFP-labeled NF-κΒ reporter. Flow cytometry showed that Compound A activates NF-KB signaling in a dose-dependent manner. Compound A at 5 μΜ showed comparable activation to 100 ng/mL Pani₃CSK₄. As a comparison, GA showed much lower NF-κΒ activation at 20 μΜ, even up to 100 μΜ. To investigate Compound A's synergistic effect in the NF-KB signaling pathway, a known NF-κΒ inhibitor, triptolide, was used to evaluate the SEAP signaling in HEK-Blue hTLR2 cells. As expected, the results showed that triptolide can efficiently inhibit the Compound A induced SEAP signaling, which further seems to confirm Compound A works through the NF-κΒ signaling pathway.

[0055] Another downstream product of NF-κΒ activation is nitric oxide (NO). NO play important roles in various biological processes such as neuronal communication and immunity response. As expected, Compound A efficiently triggered NO production not only in Raw 264.7 cell line, but also in primary rat macrophage cells. It was also observed that NO activation could be inhibited by the TLRl/2 specific antagonist (CU-CPT22, see Cheng et al., Angew. Chem. Int. Edit., 2012, 51, 12246-12249), but had a minimal influence by a TLR4 specific inhibitor TAK242 in Raw 264.7 macrophage cells, indicating this activation was specific.

[0056] Transcription factors turned on by TLRl/2 dimerization induce the production of pro-inflammatory cytokines and type I interferons (IFNs). One of the key outputs from this activation is tumor necrosis factor-alpha (TNF-a), which has shown to be directly relevant to inflammatory diseases and cancer. To ascertain whether the NF-κΒ activity induced by Compound A reflected upregulation of TNF-a, the ability of Compound A to activate TNF-a in Raw 264.7 cells was assessed using an ELISA experiment. The cell-based experiment showed Compound A possesses the high-efficient ability to active TNF-a signaling with an EC50 of 60.46 ± 16.99 nM, which is consistent with the SEAP activation observed in HEK- Blue hTLR2 cells. Furthermore, the highest TNF-a activation signaling was comparable to the positive control in the same experiment. Overall these results provide further support that Compound A behaves like PamsCSIQ in activating the TLRl/2 pathway by inducing NF-KB activation to trigger downstream signaling, such as SEAP, NO, and TNF-a.

[0057] Quantitative RT-PCR (qRT-PCR) was performed to investigate the effects of

Compound A and PamsCSIQ on the expression of mRNA for TLR1, TLR2 and downstream pro-inflammatory cytokines TNF, IL-10 and iNOS at 2h, 8h and 24h after drug treatment. Compound A at 1 μΜ, similar to Pam₃CSK₄, potently increased TLR1 mRNA in the macrophage cell line at 24 h. Both Pam₃CSK₄ and Compound A had the highest TLR2 mRNA expression at 2h, with a gradual decline at 8 and 24 h. Compound A and Pam₃CSK₄ also each increased TNF and iNOS mRNA expression overtime. The difference was that at 8h cells treated with both drugs showed the highest expression of TNF mRNA, while highest iNOS mRNA expression appeared at 24h. Similar to TLR2 mRNA, IL-10 mRNA was upregulated by Compound A at 2h, and also decreased with increasing time. In addition, it was observed that Compound A has dose-dependent activating effects on TLR1 mRNA at 24h, TLR2 mRNA at 2h, TNF mRNA at 8h, iNOS mRNA at 24h and IL-lOmRNA at 2h, respectively. These observations indicated that Compound A acts similarly to Pam₃CSK₄, regarding its ability to active TLRl/2 signaling and promote the upregulation of downstream pro-inflammatory cytokines.

[0058] Based on their parallel behaviors, it is believed that Compound A works similarly to Pam₃CSK₄in activating the TLRl/2 signaling. To further investigate the binding target of Compound A, and also to test whether Compound A may functionally mimic the receptor recognition by Pam₃CSK₄,a biophysical competition binding assay was performed. The TLR1 and TLR2 proteins were expressed in the baculo virus insect cell expression system, and biotin-labeled Pam₃CSK₄ was used as a probe in these experiments. Human TLRl/2 proteins were immobilized as the capturing probe in the ELISA assay. Different concentrations of biotin-labeled Pam₃CSK₄ (Biotin-Pam3) were added, and the bound Biotin- Pam3 was detected by streptavidin conjugated with HRP. Biotin-Pam3 bound to human TLRl/2 in a concentration-dependent manner. In contrast, the binding to a negative control bovine serum albumin (BSA) was negligible, indicating that the Biotin-Pam3-TLRl/2 binding is specific. In addition, Compound A was found to compete with Biotin-Pam3 for binding to TLRl/2.

[0059] Anisotropy biophysical tests were also carried out for Compound A, along with the negative control Compound 23, to demonstrate that Compound A directly binds to TLR1/TLR2. The activities of the TLR1 and TLR2 proteins were validated by the fluorescence anisotropy assay with a rhodamine labeled, synthetic triacylated lipoprotein Pam₃CSK₄ as the probe. A binding affinity (Kv) of 34.9 ± 1.9nM was obtained for

Pam₃CSK₄ binding to TLRl/2. The experiment also demonstrated that Compound A was able to compete with Pam₃CSK₄ for binding to TLRl/2with an inhibition constant (IQ) of 45.4 ± 5.2 nM, which is consistent with its potency observed in various whole cell assays. The high binding affinities of Compound A to the proteins were further confirmed by a microscale thermophoresis (MST) binding experiment of Compound A to TLR1 (K_∑, of 182 ± 27 nM) and TLR2 (¾ of 478 ± 85 nM) individually. The anisotropy of rhodamine-labeled PamsCSIQ (Rho-Pam3) showed a robust increase upon addition of TLR1/TLR2 (excitation = 549 nm; emission = 566 nm). This increase is consistent with the anisotropy changes seen with ligand-receptor pairs of comparablesizes. Increasing the concentration of Compound A to 3 μιη decreased the anisotropy to background levels, presumably because of the release of the fluorescently rhodamine labeled PamsCSIQ probe. This data was then fit to a one-site competition model with an R² value of 0.99, indicating a good fitting. By contrast,

Compound A, which was used as a negative control in the anisotropy assay, demonstrated negligible binding up to 10 μΜ. These results agree with the premise that Compound A directly competes with PamsCSIQ for binding to TLR1/TLR2 with a high affinity, perhaps to the same site of on the TLRl/2 surface.

[0060] The realization that PamsCSIQ can induce TLR1 and TLR2 dimerization, and also the observation in the competing binding experiment prompted investigation of the effect of Compound A on the dimer formation of the TLR1/TLR2 complex. A special size exclusion chromatography coupled with light scattering (SEC-LS) assay was performed by using the purified extracellular domains of hTLRl and hTLR2. The instrument was calibrated by BSA (65 kDa) and aldolase (158 kDa) first, and then the hTLRl or hTLR2 peak was verified in the SEC-LS experiment. It was observed that the majority of hTLRl existed in the homodimer configuration, and the hTLR2 existed in a predominantly monomeric state. The TLR1 and TLR2 heterodimerization was tested at various concentrations of Compound A. A substantial enhancement in the ability to form heterodimers in the presence of

Compound A was observed. With increasing concentrations of Compound A, the TLRl/2 heterodimer peak increased. Incubating the TLR1 and TLR2 protein with 40 μΜ Compound A for 4h causes the TLR1 homodimer peak to disappear and a new TLRl/2 heterodimer peak to appear. PamsCSIQ was used as a positive control in the SEC-LS experiment and also promoted the TLRl/2 heterodimerization. These data provide evidence that Compound A not only selectively activates the TLRl/2 signaling pathway, but can also induces TLRl/2 heterodimerization.

[0061] Targeting the interactions between TLR family proteins has garnered great interest, but developing drug-like compounds with high affinity and selectivity is difficult. This task is particularly challenging given the ligand-binding pocket at protein-protein interfaces are highly dynamic. Several critical issues hinder attempts to target protein-protein interactions (PPIs) in general and TLR family proteins in particular: Firstly, as PPIs faces are usually extensive there may be a requirement for large ligands to interfere effectively with their function. The currently known TLR2 agonists, such as Pam₃CSK₄ for TLRl/2 and Pam₂CSK₄ or FSL-1 for TLR2/6, are synthetic lipoproteins containing three or two 15-carbon long acyl chains with molecular weights around 1500 Da, and this makes more challenge to development of low molecular weight agonists. It is important to note that a high molecular weight can be challenging for drug development because it increases metabolic liabilities and leads to poor oral availability. Second, a major challenge to developing inhibitors to target TLRs is specificity for there are at least 13 homologous TLRs present in murine macrophages and 10 in humans, making it difficult to develop specific agonists, especially between TLRl/2 and TLR2/6, two heterodimeric protein complexes that share one common TLR2 component. Furthermore, the low potency and high toxicity of native TLR agonists often limits their broad application, making the validation of drug-like compounds with low toxicity and increased potency more critical.

[0062] The present invention provides insights on all of the above issues described.

First, Compound A (362 Da) is a small-molecule agonist that selectively activates TLRl/2 by binding to what appears to be the same PPI interface as Pam₃CSK₄. The initial hit was identified from a high-throughput chemical screening and was optimized through structure- guided medicinal chemistry to exhibit a >50-fold affinity enhancement for TLRl/2.

Compound A represents a first biochemically and biophysically characterized, specific small molecule ligand for TLRl/2.

[0063] Initial cellular studies demonstrated that Compound A only activates the NF-

KB pathway in HEK-Blue hTLR2 cells which contains both hTLRl and hTLR6 with EC50 close to 50 nM, but not other HEK-Blue hTLRs cells. This activation can be inhibited by anti-hTLRl and anti-hTLR2 antibodies, but not anti-hTLR6 antibody, which suggested that Compound A has high selectivity activation to TLRl/2, not TLR2/6. A U937 monocyte cell line was also used with a NF-Kb:GFP reporter to show efficient NF-κΒ activation by

Compound A. In addition, Compound A also invoked a TNF-a response with an EC50 of 60.46 ± 16.99 nM as measured by ELISA. Furthermore, its biological activity has a direct relationship with upregulated mRNA, such as TLR1, TLR2, TNF, IL-10 and iNOS mRNA. Finally, in all cellular assays, Compound A was found to have no significant cytotoxicity at its active dose up to 100 μΜ. The low toxicity of Compound A was also confirmed in RAW 264.7 cells and HEK-Blue hTLR2 cell line using the established MTT methodology. [0064] The anisotropy biophysical assays revealed competitive binding between

Compound A and Pam₃CSK₄ that is produced by presumably binding to the same interface of the TLRl/2heterodimer with a K; of 45 nM. The binding affinities of Compound A to the proteins were also measured by the microscale thermophoresis binding experiment. The SEC-LS assay further confirmed Compound A, like the classical TLR1/2 ligand Pam₃CSK₄, induces the dimerization of TLRl and TLR2 proteins. It is important to note that Compound A does not contain long acyl chains like Pam₃CSK₄, but can functionally mimic Pam₃CSK₄ and induce the heterodimerization. This perhaps indicates that the long acyl chains are not necessary for the dimerization.

[0065] As discussed herein, the cellular and biophysical studies indicated that

Compounds of the invention activate the NF-κΒ signaling pathway by binding to the heterodimer of TLR1/2. Thus, Compounds of the invention can be used in development of a useful tool for the study of TLR1/2 function related cells, as well as in potential biomedical applications, including cancer treatment and vaccine adjuvant development.

[0066] Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. In the Examples, procedures that are constructively reduced to practice are described in the present tense, and procedures that have been carried out in the laboratory are set forth in the past tense.

EXAMPLES

[0067] General Methods: NMR spectra were acquired on a Bruker 400 spectrometer, running at 400 MHz for 1H and 101 MHz for ¹³C, respectively. 1H NMR spectra were recorded at 400 MHz in CDC1₃, (CD₃)₂SO using residual CHC1₃ (7.28 ppm), and (CH₃)₂SO (2.50 ppm) as the internal standards. ¹³C NMR spectra were recorded at 101 MHz in CDC1₃, (CD₃)₂CO or (CD₃)₂SO using residual CHC1₃ (77.16 ppm), (CH₃)₂CO (29.84 and 206.26 ppm), (CH₃)₂SO (39.97 ppm) and CH₃CN (1.32 and 118.26 ppm), as internal references. Thin layer chromatography was performed on Merck Kieselgel 60 A F254 or Silicycle 6θΑ F254 plates eluting with the solvent indicated, visualized by a 254 nm UV lamp, and stained with an ethanolic solution of 12-molybdophosphoric acid. Compounds were purified using flash chromatography (FC) (Silica gel 60, 200-400 mesh, Sorbent Tech.) or recrystalization. Mass spectrometry was performed at the mass spectrometry facility of the Department of Chemistry at University of Colorado at Boulder on a double focusing high resolution mass spectrometer. [0068] /V^methyM-nitrobenzene-l^-diamine (Scheme 1, compound C). 2-Fluoro-

5-nitroaniline (468 mg, 3.00 mmol) and methylamine hydrochloride (486 mg, 9.00 mmol) were dissolved in 15 mL EtOH and stirred at room temperature for 10 min. An aqueous solution of potassium hydroxide (1.009 g, 18.0 mmol) in 5 mL H₂0 was introduced and the mixture was stirred at reflux at 60 °C overnight. Then poured into water (100 mL) and precipitate formed was extracted with ethyl acetate (3 >< 30 mL). The organic extracts were dried over Na₂S0₄ and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel, using ethyl acetate/hexane (2: 1) as eluent, and gave N'-methyM-nitrobenzene-l^-diamine as red solid (429 mg, 86 %); 1H NMR (400 MHz, DMSO) δ 7.56 (dd, J= 8.8, 2.7 Hz, 1H), 7.41 (d, J= 2.7 Hz, 1H), 6.43 (d, J= 8.9 Hz, 1H), 6.12 (s 1H), 5.08 (s, 2H), 2.85 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 144.09, 136.99, 134.89, 116.46, 107.42, 106.94, 30.12; LRMS (ESI): calcd for: C₇H₉N₃O₂ [M+H]⁺ = 168.2, obsd [M+H]⁺ = 168.2.

[0069] l-methyl-5-nitro-lH-benzo[i ]imidazole (Scheme 1, compound D).

Hydrochloric acid (12 N solution, 167 μί) was added to a solution of compound C (251 mg, 1.50 mmol) in triethyl orthoformate (10 mL) and N,N-dimethylformamide (added with stirring until the turbidity disappeared). The mixture was stirred at room temperature for 16 h, under a nitrogen atmosphere. The solvent was evaporated under reduced pressure and the brown oily residue was purified by flash column chromatography on silica gel, using ethyl acetate/hexane (5: 1) as eluent, and gave l-methyl-5-nitro-lH-benzo[d]imidazole as light yellow solid (187 mg, 70 %): 1H NMR (400 MHz, CDC1₃) δ 8.75 (dd, J= 2.1, 0.4 Hz, 1H), 8.30 (dd, J= 8.9, 2.1 Hz, 1H), 8.08 (s, 1H), 7.49 (dd, J= 8.9, 0.5 Hz, 1H), 3.96 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 149.23, 143.15, 142.88, 139.43, 118.32, 116.00, 111.41, 31.69; LRMS (ESI): calcd for: C₈H₇N₃O₂ [M+H]⁺ = 178.2, obsd [M+H]⁺ = 178.2.

[0070] Compound F in Scheme 1: Compound D (177 mg, 1.00 mmol) and 2-bromo- l-(4-nitrophenyl)ethanone (compound E, Scheme 1) (244 mg, 1.00 mmol) were dissolved in MeOH (10 mL) and was stirred at reflux under nitrogen for 12h (monitored by TLC) [53]. The solvent was concentrated under reduced pressure. The residue was washed by acetone (3 x 2 mL) to give the white solid product F in 92 % yield: 1H NMR (400 MHz, DMSO) δ 9.91 (s, 1H), 9.36 - 9.24 (m, 1H), 8.59 (ddd, J= 9.2, 2.0, 0.9 Hz, 1H), 8.50 (d, J= 8.2 Hz, 2H), 8.41 - 8.31 (m, 3H), 6.57 (s, 2H), 4.28 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 190.69, 151.04, 148.26, 146.46, 138.90, 135.61, 132.08, 130.37, 124.52, 122.16, 115.69, 111.90, 54.73, 34.67; LRMS (ESI): calcd for: Ci₆Hi₃BrN₄0₅ [M+H]⁺ = 422.2, obsd [M+H]⁺ = 422.2. [0071] V-methyl-4-nitro-2-(4-(4-nitrophenyl)-lH-imidazol-l-yl)aniline. A solution of compound F (211 mg, 0.500 mmol) and 308 mg (4.00 mmol) of ammonium acetate in 2 ml of glacial acetic acid was refluxed for 12 h, after which the mixture was dropped into 20 ml of water. The resulting yellow precipitate is filtered off, washed with water (3 >< 3 mL) and dried under high vacuum condition to give the product N-methyl-4-nitro-2-(4-(4- nitrophenyl)-lH-imidazol-l-yl)aniline (124 mg, 73 %); 1H NMR (400 MHz, DMSO) δ 8.24 (dt, J= 8.9, 6.9 Hz, 4H), 8.11 (d, J= 8.7 Hz, 2H), 8.07 - 7.99 (m, 2H), 6.86 (d, J= 9.3 Hz, 1H), 6.77 (s, 1H), 2.82 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 150.69, 146.06, 141.31, 139.97, 139.92, 135.51, 127.25, 125.46, 124.63, 124.28, 121.37, 121.06, 110.50, 30.26;

LRMS (ESI): calcd for: Ci6Hi₃N₅0₄ [M+H]⁺ = 340.1, obsd [M+H]⁺ = 340.1. The compound can be further purified by flash silica gel column chromatography, using ethyl acetate/hexane (1 : 1) as eluent, if necessary.

[0072] V-methyl-2-(4-(4-nitrophenyl)-lH-imidazol-l-yl)aniline. Following the general method, using 1 -methyl- lH-benzo[d]imidazole (132 mg, 1 mmol) instead of compound D, gave an final orange solid N-methyl-2-(4-(4-nitrophenyl)-lH-imidazol-l- yl)aniline with overall yield 60 %: 1H NMR (400 MHz, CDC1₃) δ 8.28 - 8.20 (m, 2H), 7.99 - 7.90 (m, 2H), 7.72 (d, J= 1.2 Hz, 1H), 7.54 (d, J= 1.2 Hz, 1H), 7.43 - 7.37 (m, 1H), 7.16 (dd, J= 8.0, 1.6 Hz, 1H), 6.85 - 6.78 (m, 2H), 2.87 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 146.48, 144.36, 140.58, 140.09, 139.04, 130.72, 126.92, 125.08, 124.24, 122.33, 118.61, 116.70, 111.25, 30.22; HRMS (ESI): calcd for: Ci₆Hi₄N₄0₂ [M+H]⁺ = 295.1190, obsd

[M+H]⁺ = 295.1188.

[0073] V-methyl-4-nitro-2-(4-phenyl-lH-imidazol-l-yl)aniline. Following the general method, using 2-bromo-l-phenylethanone (199 mg, 1 mmol) instead of compound E, gave an final orange solid N-methyl-4-nitro-2-(4-phenyl-lH-imidazol-l-yl)aniline with overall yield 62 %: 1H NMR (400 MHz, DMSO) δ 8.27 - 8.17 (m, 1H), 8.03 - 7.97 (m, 1H), 7.94 - 7.79 (m, 4H), 7.39 (dd, J= 9.3, 4.6 Hz, 2H), 7.28 - 7.18 (m, 1H), 6.87 (d, J= 9.0 Hz, 1H), 6.76 - 6.60 (m, 1H), 2.82 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 150.71, 141.92, 138.84, 135.55, 134.59, 128.95, 127.04, 127.01, 124.95, 124.07, 121.77, 117.54, 110.41, 30.29; HRMS (ESI): calcd for: Ci₆Hi₄N₄0₂ [M+H]⁺ = 295.1190, obsd [M+H]⁺ = 295.1197.

[0074] V¹-ethyl-4-nitrobenzene-l,2-diamine. Following the general method, using ethylamine (597 μί, 9 mmol) instead of methylamine hydrochloride, gave an red solid N¹- ethyl-4-nitrobenzene-l,2-diamine (451 mg, 83 %): 1H NMR (400 MHz, CDC1₃) δ 7.91 - 7.79 (m, 1H), 7.70 - 7.58 (m, 1H), 6.57 (t, J= 9.6 Hz, 1H), 4.27 (s, 2H), 3.33 - 3.21 (m, 2H), 1.27 (t, 3H); ^I3C NMR (101 MHz, CDC1₃) δ 144.96, 138.13, 131.79, 119.39, 112.44, 108.15, 38.23, 14.51.

[0075] l-ethyl-5-nitro-lH-benzo[i ]imidazole. Following the general method, using

N'-ethyM-nitrobenzene-l^-diamine (271 mg, 1.5 mmol) instead of compound C, gave an white solid l-ethyl-5-nitro-lH-benzo[ ]imidazole (218 mg, 76 %): 1H NMR (400 MHz, DMSO) δ 8.59 (s, 1H), 8.55 (d, J= 2.1 Hz, 1H), 8.21 - 8.13 (m, 1H), 7.87 (d, J= 9.0 Hz, 1H), 4.37 (m, 2H), 1.50 - 1.36 (m, 3H); ¹³C NMR (101 MHz, DMSO) δ 148.23, 143.15, 143.08, 138.50, 118.29, 116.13, 111.49, 40.21, 15.58.

[0076] V-ethyl-4-nitro-2-(4-(4-nitrophenyl)-lH-imidazol-l-yl)aniline. Following the general method, using l-ethyl-5-nitro-lH-benzo[d]imidazole (191 mg, 1 mmol) instead of compound D, gave a white solid under reduced pressure. The intermediate product was washed by acetone (3 x 2 mL) and followed by the reflux in 4 mL of glacial acetic acid with 612 mg ammonium acetate (4 mmol) for 12 h. Then the mixture was dropped into 20 ml of water. The resulting yellow precipitate is filtered off, washed with water (3 x 3 mL) and dried under high vacuum condition to give the product N-ethyl-4-nitro-2-(4-(4-nitrophenyl)-lH- imidazol-l-yl)aniline (198 mg, 56 %): 1H NMR (400 MHz, DMSO) δ 8.28 (d, J= 8.9 Hz, 2H), 8.25 - 8.17 (m, 2H), 8.11 (d, J= 8.9 Hz, 2H), 8.03 (dd, J= 3.4, 1.8 Hz, 2H), 6.96 (d, J = 9.4 Hz, 1H), 6.81 - 6.70 (m, 1H), 3.33 - 3.23 (m, 2H), 1.13 (t, 3H); ¹³C NMR (101 MHz, DMSO) δ 149.75, 146.08, 141.31, 139.97, 139.93, 135.43, 127.13, 125.48, 124.61, 124.53, 121.42, 120.99, 110.71, 37.72, 14.18; HRMS (ESI): calcd for: Ci₇Hi₅N₅0₄ [M+H]⁺ =

354.1197, obsd [M+H]⁺ = 354.1194.

[0077] TV^butyM-nitrobenzene-l^-diamine. Following the N'-ethyM- nitrobenzene-l,2-diamine synthesis method, using butylamine (889 μί, 9 mmol) instead of ethylamine, gave an red solid N¹-butyl-4-nitrobenzene-l,2-diamine (546 mg, 87 %): 1H NMR (400 MHz, CDCI3) δ 7.90 - 7.80 (m, 1H), 7.64 (d, J= 2.5 Hz, 1H), 6.55 (d, J= 8.9 Hz, 1H), 4.34 (s, 1H), 3.37 (s, 2H), 3.24 (m, 2H), 1.70 (m, 2H), 1.48 (m, 2H), 1.00 (m, 3H); ¹³C NMR (101 MHz, CDCI3) δ 145.17, 137.90, 131.75, 119.49, 112.49, 108.07, 43.34, 31.26, 20.27, 13.84.

[0078] l-butyl-5-nitro-lH-benzo[i ]imidazole. Following the general method, using

N¹-butyl-4-nitrobenzene-l,2-diamine (329 mg, 1.5 mmol) instead of compound C, gave an white solid l-butyl-5-nitro-lH-benzo[d]imidazole (266 mg, 81 %): 1H NMR (400 MHz, CDCI3) δ 8.75 - 8.70 (m, 1H), 8.25 (dd, J= 8.9, 2.2 Hz, 1H), 8.09 (s, 1H), 7.48 (m, 1H), 4.25 (t, 2H), 2.00 - 1.82 (m, 2H), 1.50 - 1.27 (m, 2H), 0.99 (t, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 146.32, 143.65, 143.12, 137.95, 118.68, 117.16, 109.68, 45.37, 31.85, 19.98, 13.49. [0079] V-butyl-4-nitro-2-(4-(4-nitrophenyl)-lH-imidazol-l-yl)aniline. Following the general synthesis method of compound 3, using l-butyl-5-nitro-lH-benzo[d]imidazole (218 mg, 1 mmol) instead of l-ethyl-5-nitro-lH-benzo[d]imidazole, gave an white solid N- butyl-4-nitro-2-(4-(4-nitrophenyl)-lH-imidazol-l-yl)aniline (252 mg, 66 %): 1H NMR (400 MHz, DMSO) δ 8.28 (d, J= 9.0 Hz, 2H), 8.19 (dd, J= 9.8, 1.9 Hz, 2H), 8.11 (d, J= 9.0 Hz, 2H), 8.02 (dd, J= 6.9, 1.9 Hz, 2H), 6.95 (d, J= 9.4 Hz, 1H), 6.75 (dd, J= 7.6, 4.2 Hz, 1H), 3.23 (m, 2H), 1.52 (m, 2H), 1.41 - 1.26 (m, 2H), 0.89 (t, 3H); ¹³C NMR (101 MHz, DMSO) δ 149.92, 146.05, 141.32, 139.97, 139.91, 135.30, 127.13, 125.44, 124.63, 124.59, 121.35, 121.04, 110.73, 42.63, 30.54, 20.01, 14.19; HRMS (ESI): calcd for: Ci₉Hi₉N₅0₄ [M+H]⁺ = 382.1510, obsd [M+H]⁺ = 382.1508.

[0080] V¹-benzyl-4-nitrobenzene-l,2-diamine. Following the general method, using benzylamine (655 μί, 9 mmol) instead of methylamine hydrochloride, gave an red solid N¹- benzyl-4-nitrobenzene-l,2-diamine (613 mg, 84 %): 1H NMR (400 MHz, CDC1₃) δ 7.82 (ddd, J= 8.9, 2.5, 0.5 Hz, 1H), 7.67 (d, J= 2.5 Hz, 1H), 7.44 - 7.32 (m, 5H), 6.60 (d, J= 8.9 Hz, 1H), 4.66 (s, 1H), 4.46 (d, 2H), 3.41 (s, 2H); ¹³C NMR (101 MHz, CDC1₃) δ 144.48, 137.59, 132.14, 128.91, 127.83, 127.54, 126.99, 119.16, 112.53, 108.87, 47.96.

[0081] l-benzyl-5-nitro-lH-benzo[i ]imidazole. Following the general method, using N'-benzyM-nitrobenzene-l^-diamine (365 mg, 1.5 mmol) instead of compound C, gave an white solid l-benzyl-5-nitro-lH-benzo[<i]imidazole (312 mg, 82 %):1H NMR (400 MHz, DMSO) δ 8.75 (s, 1H), 8.57 (d, J= 2.1 Hz, 1H), 8.16 (dd, J= 9.0, 2.2 Hz, 1H), 7.78 (d, J= 9.0 Hz, 1H), 7.43 - 7.23 (m, 5H), 5.62 (s, 2H); ¹³C NMR (101 MHz, DMSO) δ 148.83, 143.35, 143.20, 138.53, 136.71, 129.29, 128.46, 127.91, 118.59, 116.25, 111.85, 48.52.

[0082] V-benzyl-4-nitro-2-(4-(4-nitrophenyl)-lH-imidazol-l-yl)aniline. Following the general synthesis method of compound 3, using l-benzyl-5-nitro-lH-benzo[d]imidazole (253 mg, 1 mmol) instead of l-ethyl-5-nitro-lH-benzo[d]imidazole, gave an white solid N- benzyl-4-nitro-2-(4-(4-nitrophenyl)-lH-imidazol-l-yl)aniline (261mg, 63 %): 1H NMR (400 MHz, DMSO) δ 8.37 - 8.24 (m, 3H), 8.20 - 8.04 (m, 5H), 7.45 - 7.30 (m, 5H), 7.26 (ddd, J = 6.2, 5.0, 2.4 Hz, 1H), 6.75 (d, J = 9.3 Hz, 1H), 4.50 (s, 2H); ¹³C NMR (101 MHz, DMSO) δ 149.80, 146.10, 141.34, 140.07, 140.02, 138.69, 135.94, 128.97, 127.45, 127.23, 126.95, 125.51, 124.65, 124.55, 121.73, 121.12, 111.38, 46.16; HRMS (ESI): calcd for: C₂₂Hi₇N₅0₄ [M+H]⁺ = 416.1354, obsd [M+H]⁺ = 416.1349.

[0083] 2-(4-(4-fluorophenyl)-lH-imidazol-l-yl)- V-methyl-4-nitroaniline.

Following the general method, using 2-bromo-l-(4-fluorophenyl)ethanone (191 mg, 1 mmol) instead of compound E, gave a white solid under reduced pressure. The intermediate product was washed by acetone (3 x 2 mL) and followed by the reflux in 4 mL of glacial acetic acid with 612 mg ammonium acetate (4 mmol) for 12 h. Then the mixture was dropped into 20 ml of water. The resulting yellow precipitate is filtered off, washed with water (3 ^x 3 mL) and dried under high vacuum condition to give the product 2-(4-(4-fluorophenyl)-lH-imidazol-l- yl)-N-methyl-4-nitroaniline (172 mg, 55 %):1H NMR (400 MHz, DMSO) δ 8.34 - 8.12 (m, 1H), 8.10 - 7.57 (m, 5H), 7.37 - 7.08 (m, 2H), 6.94 - 6.79 (m, 1H), 6.76 - 6.58 (m, 1H), 2.81 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 162.78, 160.36, 150.70, 141.06, 138.89, 135.55, 131.20, 127.03, 126.81, 124.07, 121.71, 117.37, 115.89, 115.68, 110.40, 31.13; HRMS (ESI): calcd for: C₁6H₁3FN₄O₂ [M+H]⁺ = 313.1096, obsd [M+H]⁺ = 313.1101.

[0084] V-methyl-4-nitro-2-(4-p-tolyl-lH-imidazol-l-yl)aniline. Following the general synthesis method of compound 6, using 2-bromo-l-/?-tolylethanone (213 mg, 1 mmol) instead of compound E, gave a white solid N-methyl-4-nitro-2-(4-/?-tolyl- lH-imidazol-1- yl)aniline (188 mg, 61 %):1H NMR (400 MHz, CDC1₃) δ 8.28 (dd, J= 9.2, 2.6 Hz, 1H), 8.09 (t, J= 2.7 Hz, 1H), 7.61 (dd, J= 5.5, 2.6 Hz, 3H), 7.28 - 7.11 (m, 3H), 6.76 (dd, J= 9.2, 2.9 Hz, 1H), 5.27 (s, 1H), 3.00 (s, 3H), 2.39 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 149.81, 143.70, 137.51, 137.15, 137.01, 130.32, 129.34, 126.95, 124.83, 123.76, 121.48, 115.05, 109.42, 29.97, 21.23; HRMS (ESI): calcd for: Ci₇Hi₆N₄0₂ [M+H]⁺ = 309.1347, obsd [M+H]⁺ = 309.1347.

[0085] 2-(4-(4-methoxyphenyl)-lH-imidazol-l-yl)- V-methyl-4-nitroaniline.

Following the general synthesis method of compound 6, using 2-bromo-l-(4- methoxyphenyl)ethanone (229 mg, 1 mmol) instead of compound E, gave a white solid 2-(4- (4-methoxyphenyl)-lH-imidazol-l-yl)-N-methyl-4-nitroaniline (221 mg, 68 %): 1H NMR (400 MHz, DMSO) δ 8.21 (ddd, J= 5.3, 4.6, 1.7 Hz, 1H), 7.99 (dd, J= 3.8, 2.7 Hz, 1H), 7.91 - 7.58 (m, 4H), 7.08 - 6.90 (m, 2H), 6.89 - 6.77 (m, 1H), 6.68 (dd, J= 5.1, 2.0 Hz, 1H), 3.78 (s, 3H), 2.82 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 158.62, 150.70, 141.91, 138.55, 135.55, 127.33, 126.93, 126.24, 123.99, 121.86, 116.20, 114.38, 110.36, 55.51, 30.28; HRMS (ESI): calcd for: Ci₇Hi₆N₄0₃ [M+H]⁺ = 325.1296, obsd [M+H]⁺ = 325.1299.

[0086] V-methyl-4-nitro-2-(4-(2,4,5-trifluorophenyl)-lH-imidazol-l-yl)aniline. Following the general synthesis method of compound 6, using 2-bromo-l -(2,4,5- trifluorophenyl)ethanone (253 mg, 1 mmol) instead of compound E, gave a white solid N- methyl-4-nitro-2-(4-(2,4,5-trifluorophenyl)-lH-imidazol-l-yl)aniline (188 mg, 54 %): 1H NMR (400 MHz, DMSO) δ 8.22 (dd, J= 9.3, 2.4 Hz, 1H), 8.13 - 7.91 (m, 3H), 7.73 (d, J = 3.3 Hz, 1H), 7.70 - 7.58 (m, 1H), 6.95 - 6.78 (m, 1H), 6.70 (d, J= 4.8 Hz, 1H), 2.82 (s, 3H); 13C NMR (101 MHz, DMSO) δ 150.84, 139.12, 135.50, 133.77, 127.21, 124.44, 121.31, 121.18, 121.04, 114.69, 114.48, 110.46, 107.21, 106.93, 106.72, 30.23; HRMS (ESI): calcd for: C16H11FN4O2 [M+H]⁺ = 349.0907, obsd [M+H]⁺ = 349.0909.

[0087] V-methyl-4-nitro-2-(4-(3-nitrophenyl)-lH-imidazol-l-yl)aniline. Following the general synthesis method of compound 6, using 2-bromo-l-(3-nitrophenyl)ethanone (244 mg, 1 mmol) instead of compound E, gave a white solid N-methyl-4-nitro-2-(4-(3- nitrophenyl)-lH-imidazol-l-yl)aniline (214 mg, 63 %): 1H NMR (400 MHz, DMSO) δ 8.74 - 8.61 (m, 1H), 8.32 - 8.21 (m, 2H), 8.19 (d, J= 1.2 Hz, 1H), 8.10 (ddd, J= 8.2, 2.3, 0.9 Hz, 1H), 8.05 (d, J= 2.7 Hz, 1H), 8.01 (d, J= 1.2 Hz, 1H), 7.71 (t, J= 8.0 Hz, 1H), 6.88 (d, J = 9.4 Hz, 1H), 6.74 (d, J= 4.7 Hz, 1H), 2.82 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 150.66, 148.82, 139.75, 139.50, 136.48, 135.53, 131.13, 130.64, 127.17, 124.18, 121.56, 121.48, 119.54, 119.00, 110.49, 30.28; HRMS (ESI): calcd for: Ci6Hi₃N₅0₄ [M+H]⁺ = 340.1041, obsd [M+H]⁺ = 340.1042.

[0088] 4-(l-(2-(methylamino)-5-nitrophenyl)-lH-imidazol-4-yl)benzonitrile.

Following the general synthesis method of compound 6, using 4-(2-bromoacetyl)benzonitrile (224 mg, 1 mmol) instead of compound E, gave a white solid 4-(l-(2-(methylamino)-5- nitrophenyl)-lH-imidazol-4-yl)benzonitrile (211 mg, 66 %): 1H NMR (400 MHz, DMSO) δ 8.23 (dd, J= 9.2, 2.3 Hz, 1H), 8.15 (s, 1H), 8.03 (dd, J= 13.1, 8.0 Hz, 4H), 7.86 (d, J= 8.2 Hz, 2H), 6.87 (d, J = 9.3 Hz, 1H), 6.74 (d, J= 4.6 Hz, 1H), 2.81 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 150.69, 140.26, 139.67, 139.20, 135.55, 133.09, 127.19, 125.39, 124.23, 121.44, 120.26, 119.65, 110.48, 109.04, 30.26; HRMS (ESI): calcd for: Ci₇Hi₃N₅0₂ [M+H]⁺ = 320.1142, obsd [M+H]⁺ = 320.1147.

[0089] V-methyl-4-nitro-2-(4-(4-(trifluoromethyl)phenyl)-lH-imidazol-l- yl)aniline. Following the general synthesis method of compound 6, using 2-bromo-l-(4- (trifluoromethyl)phenyl)ethanone (267 mg, 1 mmol) instead of compound E, gave a light yellow solid N-methyl-4-nitro-2-(4-(4-(trifluoromethyl)phenyl)- lH-imidazol- 1 -yl)aniline (257 mg, 71 %): 1H NMR (400 MHz, DMSO) δ 8.24 (dd, J= 9.3, 2.3 Hz, 1H), 8.18 - 8.02 (m, 4H), 7.98 (s, 1H), 7.76 (d, J= 8.2 Hz, 2H), 6.87 (d, J= 9.2 Hz, 1H), 6.74 (s, 1H), 2.82 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 150.70, 140.46, 139.45, 138.63, 135.56, 127.14, 125.98, 125.94, 125.30, 124.19, 121.54, 119.48, 110.46, 30.26; HRMS (ESI): calcd for:

Ci₇Hi₃F₃N₄0₂ [M+H]⁺ = 363.1064, obsd [M+H]⁺ = 363.1062.

[0090] 2-(4-(biphenyl-4-yl)-lH-imidazol-l-yl)- V-methyl-4-nitroaniline. Following the general synthesis method of compound 6, using l-(biphenyl-4-yl)-2-bromoethanone (275 mg, 1 mmol) instead of compound E, gave a light yellow solid 2-(4-(biphenyl-4-yl)-lH- imidazol-l-yl)-N-methyl-4-nitroaniline (300 mg, 81 %): 1H NMR (400 MHz, DMSO) δ 8.24 (dd, J= 9.2, 2.5 Hz, 1H), 8.03 (d, J= 2.7 Hz, 1H), 7.95 (ddd, J= 6.4, 4.1, 1.6 Hz, 4H), 7.72 (dd, J= 4.9, 3.7 Hz, 4H), 7.54 - 7.43 (m, 2H), 7.40 - 7.33 (m, 1H), 6.88 (d, J= 9.4 Hz, 1H), 6.78 - 6.68 (m, 1H), 2.84 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 150.70, 141.58, 140.27, 138.98, 138.56, 135.57, 133.80, 129.38, 127.73, 127.17, 127.02, 126.96, 126.80, 125.51, 124.07, 121.76, 117.79, 110.43, 30.29; HRMS (ESI): calcd for: C₂₂Hi₈N₄0₂ [M+H]⁺ = 371.1503, obsd [M+H]⁺ = 371.1501.

[0091] 2-bromo-l-(4-cyclohexylphenyl)ethanone. 2-bromo-l-(4- cyclohexylphenyl)ethanone was synthesized. Briefly, l-(4-cyclohexylphenyl)ethanone (1.213 g, 6.0 mmol), CH₂C1₂ (30 mL), CH3OH (12 mL), and tetrabutylammonium tribromide (TBA Br₃) (2.893 g, 6.0 mmol) were added to a 100-mL flask. The mixture was stirred at room temperature for 2-12 h until the red color disappeared. Then the solvent was removed under vacuum and the residue was diluted with water (20 mL) and extracted with hexane (3 x 20 mL). The organic layer was then dried over Na₂S0₄, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel, using ethyl acetate/hexane (1 :20) as eluent, and gave 2-bromo-l-(4- cyclohexylphenyl)ethanone as a clean oil (1.130 g, 67 %): 1H NMR (400 MHz, CDC1₃) δ 7.94 (d, J = 8.4 Hz, 2H), 7.38 - 7.31 (m, 2H), 4.46 (s, 2H), 2.60 (m, 1H), 1.98 - 1.84 (m, 4H), 1.83 - 1.73 (m, 1H), 1.53 - 1.36 (m, 4H), 1.36 - 1.19 (m, 1H); ¹³C NMR (101 MHz, CDC1₃) δ 190.91, 154.81, 131.81, 129.16, 127.36, 44.77, 34.03, 30.91, 26.68, 26.00.

[0092] 2-(4-(4-cyclohexylphenyl)-lH-imidazol-l-yl)-N-methyl-4-nitroaniline. Following the general synthesis method of compound 6, using 2-bromo-l-(4- cyclohexylphenyl)ethanone (281 mg, 1 mmol) instead of compound E, gave a light yellow solid 2-(4-(4-cyclohexylphenyl)-lH-imidazol-l-yl)-N-methyl-4-nitroaniline (290 mg, 77 %): 1H NMR (400 MHz, DMSO) δ 8.22 (dd, J= 9.3, 2.6 Hz, 1H), 7.99 (d, J= 2.7 Hz, 1H), 7.88 (d, J= 1.3 Hz, 1H), 7.81 (d, J= 1.3 Hz, 1H), 7.79 - 7.72 (m, 2H), 7.24 (d, J= 8.2 Hz, 2H), 6.86 (d, J= 9.4 Hz, 1H), 6.67 (d, J= 4.9 Hz, 1H), 3.34 (s, 1H), 2.82 (s, 3H), 1.81 (m, 4H), 1.72 (m, 1H), 1.51 - 1.31 (m, 4H), 1.31 - 1.16 (m, 1H); ¹³C NMR (101 MHz, DMSO) δ 150.66, 146.40, 142.08, 138.66, 135.56, 132.25, 127.18, 126.94, 125.01, 123.96, 121.83, 116.95, 110.41, 43.96, 34.44, 30.29, 26.85, 26.10; HRMS (ESI): calcd for: C₂₂H₂₄N₄0₂

[M+H]⁺ = 377.1973, obsd [M+H]⁺ = 377.1975.

[0093] V-methyl-2-(4-(naphthalen-2-yl)-lH-imidazol-l-yl)-4-nitroaniline.

Following the general synthesis method of compound 6, using 2-bromo-l-(naphthalen-2- yl)ethanone (249 mg, 1 mmol) instead of compound E, gave a light yellow solid N-methyl-2- (4-(naphthalen-2-yl)-lH-imidazol-l-yl)-4-nitroaniline (296 mg, 86 %): 1H NMR (400 MHz, DMSO) δ 8.41 (s, 1H), 8.30 - 8.16 (m, 1H), 8.07 - 8.00 (m, 3H), 7.99 - 7.86 (m, 4H), 7.56 - 7.40 (m, 2H), 6.88 (d, J = 9.3 Hz, 1H), 6.75 (dd, J = 3.4, 1.2 Hz, 1H), 2.83 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 150.72, 141.89, 139.12, 135.55, 133.82, 132.57, 132.14, 128.45, 128.39, 128.30, 128.15, 128.08, 127.05, 126.74, 125.87, 124.16, 124.12, 122.64, 121.75, 118.23, 110.42, 30.30; HRMS (ESI): calcd for: C₂oHi₆N₄0₂ [M+H]⁺ = 345.1347, obsd [M+H]⁺ = 345.1350.

[0094] 2-bromo-l-(4-tef"f-butylphenyl)ethanone. Following the general synthesis method of 2-bromo-l-(4-cyclohexylphenyl)ethanone, gave a colorless oil 2-bromo-l-(4-tert- butylphenyl)ethanone (1.026 g, 67 %): 1H NMR (400 MHz, CDC1₃) δ 8.01 - 7.91 (m, 2H), 7.58 - 7.48 (m, 2H), 4.46 (s, 2H), 1.37 (s, 9H); ¹³C NMR (101 MHz, CDC1₃) δ 190.90, 157.90, 131.40, 128.92, 125.83, 35.25, 31.02, 30.88.

[0095] 2-(4-(4-tei-i-butylphenyl)-lH-imidazol-l-yl)- V-methyl-4-nitroaniline.

Following the general synthesis method of compound 6, using 2-bromo-l-(4-tert- butylphenyl)ethanone (350 mg, 1 mmol) instead of compound E, gave a light yellow solid 2- (4-(4-fert-butylphenyl)-lH-imidazol-l-yl)-N-methyl-4-nitroaniline (242 mg, 69 %): 1H NMR (400 MHz, DMSO) δ 8.22 (dd, J= 9.3, 2.7 Hz, 1H), 8.00 (d, J= 2.7 Hz, 1H), 7.89 (d, J= 1.3 Hz, 1H), 7.82 (d, J= 1.3 Hz, 1H), 7.80 - 7.74 (m, 2H), 7.50 - 7.31 (m, 2H), 6.87 (d, J= 9.4 Hz, 1H), 6.74 - 6.62 (m, 1H), 2.82 (s, 3H), 1.31 (s, 9H); ¹³C NMR (101 MHz, DMSO) δ 150.66, 149.43, 142.00, 138.68, 135.57, 131.83, 126.94, 125.63, 124.78, 123.98, 121.83, 117.02, 110.42, 34.71, 31.63, 30.30; HRMS (ESI): calcd for: C₂oH₂₂N₄0₂ [M+H]⁺ =

351.1816, obsd [M+H]⁺ = 351.1813.

[0096] 2-bromo-l-(4-butylphenyl)ethanone. Following the general synthesis method of 2-bromo-l-(4-cyclohexylphenyl)ethanone, gave a colorless oil 2-bromo-l-(4- butylphenyl)ethanone (964 mg, 63 %): 1H NMR (400 MHz, CDC1₃) δ 7.95 - 7.90 (m, 2H), 7.32 (dd, J= 8.0, 0.5 Hz, 2H), 4.46 (s, 2H), 2.75 - 2.65 (m, 2H), 1.64 (m, 2H), 1.45 - 1.31 (m, 2H), 0.96 (t, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 190.92, 149.90, 131.68, 129.07, 128.90, 35.76, 33.12, 30.91, 22.31, 13.87.

[0097] 2-(4-(4-butylphenyl)-lH-imidazol-l-yl)-N-methyl-4-nitroaniline.

Following the general synthesis method of compound 6, using 2-bromo-l-(4- butylphenyl)ethanone (255 mg, 1 mmol) instead of compound E, gave a light yellow solid 2- (4-(4-butylphenyl)-lH-imidazol-l-yl)-N-methyl-4-nitroaniline (256 mg, 73 %): 1H NMR (400 MHz, DMSO) δ 8.22 (dd, J= 9.3, 2.6 Hz, 1H), 7.99 (d, J= 2.7 Hz, 1H), 7.88 (d, J= 1.3 Hz, 1H), 7.82 (d, J= 1.3 Hz, 1H), 7.76 (d, J= 8.2 Hz, 2H), 7.21 (d, J= 8.3 Hz, 2H), 6.86 (d, J= 9.4 Hz, 1H), 6.68 (dt, J= 9.3, 2.4 Hz, 1H), 2.82 (s, 3H), 2.64 - 2.54 (m, 2H), 1.65 - 1.49 (m, 2H), 1.32 (m, 2H), 0.91 (t, 3H); ¹³C NMR (101 MHz, DMSO) δ 150.69, 142.06, 141.09, 138.67, 135.56, 132.09, 128.85, 126.96, 124.95, 124.00, 121.82, 116.96, 110.40, 35.03, 33.56, 30.29, 22.19, 14.26; HRMS (ESI): calcd for: C20H22N4O2 [M+H]⁺ = 351.1816, obsd [M+H]⁺ = 351.1813.

[0098] 2-bromo-l-(4-butylphenyl)ethanone. Following the general synthesis method of 2-bromo-l-(4-cyclohexylphenyl)ethanone, gave a colorless oil 2-bromo-l-(4- butylphenyl)ethanone (940 mg, 61 %): 1H NMR (400 MHz, CDC1₃) δ 8.19 - 8.13 (m, 2H), 8.08 - 8.01 (m, 2H), 4.49 (s, 2H), 3.97 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 190.79, 165.91, 137.15, 134.61, 129.99, 128.84, 52.54, 30.66.

[0099] 4-(l-(2-(methylamino)-5-nitrophenyl)-lH-imidazol-4-yl)benzoate.

Following the general synthesis method of compound 6, using 2-bromo-l-(4- butylphenyl)ethanone (257 mg, 1 mmol) instead of compound E, gave a light yellow solid methyl 4-(l-(2-(methylamino)-5-nitrophenyl)-lH-imidazol-4-yl)benzoate (250 mg, 71 %): 1H NMR (400 MHz, DMSO) δ 8.26 - 8.20 (m, 1H), 8.10 - 8.07 (m, 1H), 8.04 (dd, J= 5.0, 2.1 Hz, 1H), 8.02 - 7.94 (m, 5H), 6.91 - 6.82 (m, 1H), 6.77 - 6.68 (m, 1H), 3.86 (s, 3H), 2.81 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 166.59, 150.71, 140.82, 139.46, 139.28, 135.54, 130.08, 127.83, 127.14, 124.90, 124.18, 121.54, 119.60, 110.46, 52.44, 30.27; HRMS (ESI): calcd for: Ci₈Hi₆N₄0₄ [M+H]⁺ = 353.1245, obsd [M+H]⁺ = 353.1251.

[0100] 4-(l-(2-(methylamino)-5-nitrophenyl)-lH-imidazol-4-yl)benzoic acid. To a mixture of compound 18 (169 mg, 0.5 mmol) and LiOH (11.5 mg, 1.5 mmol) in THF (3 mL) was added MeOH (0.3 mL) and ¾0 (1 mL), stirred at room temperature for 8 h [24]. Then HC1 (1 M) was added to the reaction mixture to pH = 4, and extracted with EtOAc (3 x 15 mL), wash with H₂0 (3 x 20 mL). The organic extracts were dried over Na₂S0₄ and concentrated under reduced pressure. Purified by flash chromatography on silica gel, using ethyl acetate/hexane (4: 1) as eluent, give 4-(l-(2-(methylamino)-5-nitrophenyl)-lH-imidazol- 4-yl)benzoic acid as white powder (137 mg, 81 %): 1H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 8.24 (dd, J= 9.3, 2.7 Hz, 1H), 8.05 (dd, J= 10.4, 2.0 Hz, 2H), 7.97 (t, J= 1.1 Hz, 5H), 6.87 (d, J= 9.4 Hz, 1H), 6.73 (d, J= 4.8 Hz, 1H), 2.82 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 167.75, 150.72, 140.98, 139.38, 138.77, 135.51, 130.21, 127.14, 124.72, 124.19, 121.57, 119.34, 110.45, 99.98, 30.27; HRMS (ESI): calcd for: Ci₇Hi₄N₄0₄ [M+H]⁺ = 339.1087, obsd [M+H]⁺ = 339.1089.

[0101] V-methyl-2-nitro-4-(trifluoromethyl)aniline. Following the general method, using l-chloro-2-nitro-4-(trifluoromethyl)benzene (677 mg, 3 mmol) gave an yellow solid N- methyl-2-nitro-4-(trifluoromethyl)aniline (528 mg, 80 %): 1H NMR (400 MHz, CDC1₃) δ 8.49 (dd, J= 2.0, 0.8 Hz, 1H), 8.29 (s, 1H), 7.67 (dd, J= 9.0, 2.2 Hz, 1H), 6.96 (d, J= 9.0 Hz, 1H), 3.11 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 147.68, 132.18, 132.14, 124.87, 124.83, 114.05, 29.86.

[0102] TV^methyM-itrifluoromethylJbenzene-l^-diamine. After three vacuum/H₂ cycles to remove air from the reaction tube, the stirred mixture of the N-methyl-2-nitro-4- (trifluoromethyl)aniline (440 mg, 2 mmol) and 10 % Pd/C catalyst (44 mg) in MeOH (4 mL) was hydrogenated under ambient pressure (balloon) at room temperature for 4h. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel, using ethyl acetate as eluent, and gave N'-methyl^-^rifluoromethy benzene-l^-diamine as red solid (308 mg, 81 %); 1H NMR (400 MHz, CDC1₃) δ 7.15 (ddd, J= 8.3, 2.0, 0.9 Hz, 1H), 6.98 - 6.89 (m, 1H), 6.66 (d, J = 8.2 Hz, 1H), 3.81 - 3.10 (m, 3H), 2.92 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 141.97, 133.24, 126.29, 123.60, 118.42, 112.98, 109.42, 30.60.

[0103] l-methyl-5-(trifluoromethyl)-lH-benzo[i ]imidazole. Following the general method, using N¹-methyl-4-(trifiuoromethyl)benzene-l,2-diamine (285 mg, 1.5 mmol) gave an light yellow solid l-methyl-5-(trifluoromethyl)-lH-benzo[(i]imidazole (225 mg, 75 %): 1H NMR (400 MHz, CDC1₃) δ 8.14 - 8.08 (m, 1H), 7.99 (s, 1H), 7.59 (dd, J= 8.5, 1.5 Hz, 1H), 7.49 (d, J= 8.5 Hz, 1H), 3.91 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 145.32, 143.17, 136.44, 124.90, 119.92, 119.88, 118.09, 118.05, 31.24.

[0104] V-methyl-4-(trifluoromethyl)-2-(4-(4-(trifluoromethyl)phenyl)-lH- imidazol-l-yl)aniline. Following the general synthesis method of compound 12, using 1- methyl-5-(trifluoromethyl)-lH-benzo[(i]imidazole (200 mg, 1 mmol) instead of l-methyl-5- nitro-lH-benzo[d]imidazole, gave a light yellow solid N-methyl-4-(trifluoromethyl)-2-(4-(4- (trifluoromethyl)phenyl)-lH-imidazol-l-yl)aniline (212 mg, 55 %): 1H NMR (400 MHz, CDC1₃) δ 7.90 (dd, J= 8.7, 0.7 Hz, 2H), 7.70 - 7.59 (m, 4H), 7.46 - 7.40 (m, 2H), 6.83 (d, J = 8.7 Hz, 1H), 4.35 - 4.25 (m, 1H), 2.92 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 147.01, 141.95, 138.29, 136.77, 127.84, 127.81, 125.71, 125.68, 125.64, 124.93, 124.46, 124.42, 121.82, 116.96, 110.56, 29.97; HRMS (ESI): calcd for: Ci₈Hi₃F₆N₃ [M+H]⁺ = 386.1087, obsd [M+H]⁺ = 386.1086.

[0105] V¹-methyl-2-(4-(4-(trifluoromethyl)phenyl)- lH-imidazol- l-yl)benzene- 1 ,4- diamine. After three vacuum/H₂ cycles to remove air from the reaction tube, the stirred mixture of the compound 12 (181 mg, 0.5 mmol) and 10 % Pd/C catalyst (18 mg) in MeOH (1 mL) was hydrogenated under ambient pressure (balloon) at room temperature for 6h. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel, using MeOH/CHCl3 (1 :20) as eluent, and gave N -methyl-2-(4-(4-(trif uoromethyl)phenyl)-lH-imidazol-l-yl)benzene-l,4- diamine as white solid (99 mg, 60 %); 1H NMR (400 MHz, CDC1₃) δ 7.93 (d, J= 8.1 Hz, 2H), 7.77 - 7.61 (m, 3H), 7.48 (d, J= 1.2 Hz, 1H), 6.81 (dd, J= 8.6, 2.6 Hz, 1H), 6.69 (d, J= 8.6 Hz, 1H), 6.62 (d, J= 2.6 Hz, 1H), 3.44 (s, 3H), 2.79 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 141.23, 138.44, 137.43, 137.28, 137.20, 128.94, 128.62, 125.68, 125.64, 124.88, 123.59, 122.99, 117.66, 117.25, 114.51, 112.98, 31.06; HRMS (ESI): calcd for: Ci₇Hi₅F₃N₄ [M+H]⁺ = 333.1322, obsd [M+H]⁺ = 333.1320.

[0106] Methyl 4-fluoro-3-nitrobenzoate. 4-fluoro-3-nitrobenzoic acid (1.110 g, 6 mmol) was dissolved in MeOH (30 mL). Then, cone. H₂S0₄ (298 μί, 12 mmol) was added to the solution and was stirred at reflux for 24h (monitored by TLC) [26]. The solvent was concentrated under reduced pressure. After extraction with EtOAc (50 mL), the solution was washed with distilled water (3 x 20 mL) and saturated NaHC0₃ (20 mL), and dried over Na2S04. The solution was evaporated and purified by flash chromatography on silica gel, using ethyl acetate/hexane (1 :8) as eluent, to give methyl 4-fluoro-3-nitrobenzoate as a yellow solid (1.135 g, 95 %): 1H NMR (400 MHz, CDC1₃) δ 8.76 (dd, J= 7.2, 2.2 Hz, 1H), 8.34 (ddd, J= 8.7, 4.2, 2.2 Hz, 1H), 7.41 (dd, J= 10.2, 8.7 Hz, 1H), 4.00 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 164.07, 159.41, 156.71, 136.44, 127.83, 127.23, 118.67, 52.89.

[0107] Methyl 4-(methylamino)-3-nitrobenzoate. Following the general method , using methyl 4-fluoro-3-nitrobenzoate (587 mg, 3 mmol) gave an yellow solid methyl 4- (methylamino)-3-nitrobenzoate (561 mg, 89 %): 1H NMR (400 MHz, CDC1₃) δ 8.89 (d, J = 2.1 Hz, 1H), 8.37 (s, 1H), 8.09 (ddd, J= 9.0, 2.1, 0.7 Hz, 1H), 6.88 (d, J= 9.0 Hz, 1H), 3.91 (s, 3H), 3.10 (d, J= 5.1 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 165.62, 148.49, 136.38, 131.35, 129.41, 117.18, 113.12, 52.07, 29.90.

[0108] Methyl 3-amino-4-(methylamino)benzoate. Following the general synthesis method of N¹-methyl-4-(trifluoromethyl)benzene-l,2-diamine, using methyl 4- (methylamino)-3-nitrobenzoate (420 mg, 2 mmol) instead of N-methyl-2-nitro-4- (trifluoromethyl)aniline, gave an red solid methyl 3-amino-4-(methylamino)benzoate (292 mg, 81 %): 1H NMR (400 MHz, CDC1₃) δ 7.64 (dd, J= 8.3, 1.9 Hz, 1H), 7.42 (d, J= 1.9 Hz, 1H), 6.61 (d, J= 8.3 Hz, 1H), 3.87 (s, 3H), 3.52 (s, 2H), 2.93 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 167.55, 144.01, 132.17, 124.34, 118.81, 117.88, 108.82, 51.56, 30.44.

[0109] Methyl l-methyl-lH-benzo[i ]imidazole-5-carboxylate. Following the general method I, using methyl 3-amino-4-(methylamino)benzoate (270 mg, 1.5 mmol) gave an white solid methyl 1 -methyl- lH-benzo[d]imidazole-5-carboxylate (225 mg, 79 %): 1H NMR (400 MHz, CDC1₃) δ 8.54 (dd, J= 1.5, 0.6 Hz, 1H), 8.08 (dd, J= 8.5, 1.5 Hz, 1H), 7.99 (s, 1H), 7.44 (dd, J= 8.5, 0.6 Hz, 1H), 3.97 (s, 3H), 3.90 (s, 3H); ¹³C NMR (101 MHz, CDCI3) δ 167.56, 145.19, 143.29, 137.69, 124.57, 124.49, 122.73, 109.08, 52.08, 31.22.

[0110] Methyl 4-(methylamino)-3-(4-(4-(trifluoromethyl)phenyl)-lH-imidazol-l- yl)benzoate. Following the general synthesis method of compound 12, using 1 -methyl- 1H- benzo[d]imidazole-5-carboxylate (190 mg, 1 mmol) instead of l-methyl-5-nitro-lH- benzo[d]imidazole, gave a yellow solid methyl 4-(methylamino)-3-(4-(4- (trifluoromethyl)phenyl)-lH-imidazol-l-yl)benzoate (266 mg, 61 %): 1H NMR (400 MHz, CDCI3) δ 8.08 (ddd, J= 8.7, 2.0, 0.5 Hz, 1H), 7.97 - 7.85 (m, 3H), 7.68 (d, J= 1.2 Hz, 2H), 7.66 (s, 1H), 7.44 (d, J= 1.3 Hz, 1H), 6.78 (d, J= 8.7 Hz, 1H), 4.37 (s, 1H), 3.90 (s, 3H), 2.93 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 166.32, 147.99, 141.88, 138.43, 136.92, 132.53, 128.82, 125.76, 125.72, 125.68, 125.64, 124.96, 121.70, 118.21, 117.02, 110.02, 51.89, 29.96; HRMS (ESI): calcd for:

[M+H]⁺ = 376.1268, obsd [M+H]⁺ = 376.1270.

[0111] 4-(methylamino)-3-(4-(4-(trifluoromethyl)phenyl)-lH-imidazol-l- yl)benzoic acid. Following the synthesis method of compound 19, using compound 22 (187 mg, 0.5 mmol) instead of compound 18, gave a white solid 4-(methylamino)-3-(4-(4- (trifluoromethyl)phenyl)-lH-imidazol-l-yl)benzoic acid (105 mg, 58 %): 1H NMR (400 MHz, DMSO) δ 9.17 (s, 1H), 8.44 (s, 1H), 8.19 (d, J = 8.2 Hz, 2H), 7.96 (dd, J = 8.7, 2.0 Hz, 1H), 7.88 (dd, J = 18.1, 5.1 Hz, 3H), 6.85 (d, J = 8.9 Hz, 1H), 6.39 (s, 1H), 2.77 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 167.02, 148.52, 139.14, 133.22, 129.65, 126.50, 126.47, 126.43, 126.39, 126.22, 121.29, 120.55, 117.45, 110.93, 29.94; HRMS (ESI): calcd for: C₁₈H₁₄F₃N₃O₂

[M+H]⁺ = 362.1111 , obsd [M+H]⁺ = 362.1111.

[0112] 4-(methylamino)-3-nitrobenzonitrile. Following the synthesis method of N- methyl-2-nitro-4-(trifluoromethyl)aniline, using 4-chloro-3-nitrobenzonitrile (548 mg, 3 mmol) instead of l-chloro-2-nitro-4-(trifluoromethyl)benzene, gave an yellow solid 4- (methylamino)-3-nitrobenzonitrile (468 mg, 88 %): 1H NMR (400 MHz, CDC1₃) δ 8.52 (d, J = 2.0 Hz, 1H), 8.44 (s, 1H), 7.66 (ddd, J= 9.0, 2.0, 0.7 Hz, 1H), 6.94 (d, J= 9.0 Hz, 1H), 3.13 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 147.96, 137.72, 132.08, 131.49, 117.97, 114.43, 98.11, 29.91.

[0113] 3-amino-4-(methylamino)benzonitrile. Following the synthesis method of

N¹ -methyl-4-(trifluoromethyl)benzene- 1 ,2-diamine, using 4-(methylamino)-3 - nitrobenzonitrile (354 mg, 2 mmol) instead of N-methyl-2-nitro-4-(trifluoromethyl)aniline, gave a red solid 3 -amino- 4-(methylamino)benzonitrile (233 mg, 79 %): 1H NMR (400 MHz, CDCI₃) δ 7.19 (dd, J = 8.2, 1.9 Hz, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 3.38 (s, 2H), 2.92 (s, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 143.47, 133.05, 126.75, 120.65, 119.00, 109.45, 99.16, 30.29.

[0114] l-methyl-lH-benzo[i/]imidazole-5-carbonitrile. Following the synthesis method of l-methyl-5-(trifluoromethyl)-lH-benzo[(i]imidazole, using 3-amino-4- (methylamino)benzonitrile (220 mg, 1.5 mmol) instead of N¹-methyl-4- (trifluoromethyl)benzene-l,2-diamine, gave an light yellow solid 1 -methyl- 1H- benzo[ ]imidazole-5-carbonitrile (120 mg, 51 %): 1H NMR (400 MHz, DMSO) δ 8.43 (s, 1H), 8.21 (d, J = 0.8 Hz, 1H), 7.79 (dd, J = 8.4, 0.4 Hz, 1H), 7.67 (dd, J = 8.4, 1.5 Hz, 1H), 3.90 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 147.90, 143.24, 137.93, 126.04, 124.81, 120.38, 112.36, 104.21, 31.47.

[0115] 4-(methylamino)-3-(4-(4-nitrophenyl)-lH-imidazol-l-yl)benzonitrile.

Following the general synthesis method of compound 3, using 1-methyl-lH- benzo[d]imidazole-5-carbonitrile (157 mg, 1 mmol) instead of l-ethyl-5-nitro-lH- benzo[<i]imidazole, gave an white solid 4-(methylamino)-3-(4-(4-nitrophenyl)-lH-imidazol- l-yl)benzonitrile (172 mg, 54 %): 1H NMR (400 MHz, DMSO) δ 8.33 - 8.22 (m, 2H), 8.18 (s, 1H), 8.14 - 8.05 (m, 2H), 8.00 - 7.91 (m, 1H), 7.74 (dd, J= 8.7, 1.8 Hz, 1H), 7.68 (d, J= 2.0 Hz, 1H), 6.89 - 6.79 (m, 1H), 6.28 (s, 1H), 2.74 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 148.62, 146.03, 141.38, 139.89, 139.80, 134.92, 131.55, 125.42, 124.61, 122.45, 121.00, 119.87, 111.64, 96.20, 29.97; HRMS (ESI): calcd for: Ci₇Hi₃N₃0₂ [M+H]⁺ = 320.1142, obsd [M+H]⁺ = 320.1147.

[0116] Secreted embryonic alkaline phosphatase (SEAP) reporter gene detection assay. Materials for the SEAP assay were obtained from Applied Biosystems and used according to the manufacturer's specifications. HEK 293 cells stably transfected with human TLR2, TLR3, TLR4, TLR5, TLR7, or TLR8 and a SEAP reporter gene were obtained from InvivoGen. Cells were cultured in 200 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), lOx penicillin/ streptomycin, and 10x L- glutamine. Cells were implanted in 96-well plates (4 x 10⁴ cells per well) for 24 hours at 37°C before drug treat- ment. In the next 24 hours of treatment, medium was removed from the 96-well plate and replaced with 200 ml of supplemented Opti-MEM [0.5% FBS, penicillin (50 U/ml), streptomycin (50 mg/ml), l x nonessen- tial amino acids] containing 0 to 50.0 mM (or 0 to 100 mM) of com- pounds, or the positive control ligands for different TLRs, such as Pam3CSK4 (0 to 66 nM or 0 to 100 ng/ml) for TLR1/2, poly(LC) (0 to 10.9 mg/ml) for TLR3, LPS (0 to 36.5 ng/ml) for TLR4, FLA-BS (0 to 10 mg/ml) for TLR5, and R848 (0 to 6 mg/ml) for TLR7 and TLR8. [0117] A sample buffer (15 ml) from each well was collected and trans- ferred to an opaque white 96-well plate (Microfluor 2, Thermo Scien- tific). Each well was treated with 45 ml of l x dilution buffer, covered with microseal (MSB 1001, Bio-Rad), and incubated for 30 min at 65°C. After 30 min, plates were cooled to room temperature on ice, and 50 ml of SEAP assay buffer was added to each well. After a 5-min incubation, 50 ml of disodium 3- (4-methoxyspiro { 1 ,2-dioxetane-3,2-(5-chloro) tricyclo[3.3.1.13,7]decan} -4-yl) phenyl phosphate (CSPD) diluted 1 :20 with reaction buffer was added to each well. After 20 min, the luminescence of each well was measured using a plate reader (Beckman Coulter, DTX 880) with multimode analysis software.

[0118] QUANTI-Blue SEAP assay. Cells were cultured in 200 ml of DMEM supplemented with 10% FBS, 10x penicillin/streptomycin, and 10x L-glutamine. Cells were im- planted in 96-well plates (4 ^χ 10⁴ cells per well) for 24 hours at 37°C before drug treatment. In the next 24 hours of treatment, medium was removed from the 96-well plate and replaced with 200 ml of supplemented Opti-MEM [0.5% FBS, penicillin (50 U/ml), streptomycin (50 mg/ml), l x nonessential amino acids] containing 60 nM Compound A, 0.66 nM (1 ng/ml) Pam3CSK4, or 0.77 nM (1 ng/ml) Pam2CSK4 ac- cording to the

manufacturer's protocol (InvivoGen), as well as different antibodies (0 to 10 mg/ml), including anti-hTLRl-IgG, anti-hTLR2- IgA, or anti-hTLR6-IgA (InvivoGen). A sample buffer (20 ml) from each well of the cell culture supernatants was collected and transferred to a transparent 96-well plate (Thermo Scientific). Each well was treated with 200 ml of

QUANTI-Blue (InvivoGen) buffer and incubated at 37°C for 1 hour. A purple color can be observed, and optical density was measured using a plate reader at an absorbance of 655 nm (A655).

[0119] U937 cell transfection and NF-KB:GFP reporter assay. Human macrophage

U937 cells [American Type Culture Collection (ATCC) CRL-1593.2] were grown and maintained in RPMI 1640 medium containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml). An NF-kB-GFP reporter was stably inserted using the

commercially available pGreenFire plasmid (System Biosciences). Briefly, HEK 293T cells (ATCC CRL-3216) were transfected using a 6:1 poly- ethylenimine/DNA ratio with the pGreenFire vector (4.33 mg) and the pREV (4.33 mg), pMDL (4.33 mg), and pVSVg (2 mg) viral packaging plasmids. Viral particles were harvested from the medium 48 to 72 hours after transfection and concentrated using an 8.5% PEG-8000 (polyethylene glycol, molecular weight 8000) and 10 mM NaCl solution. The concentrated virus and polybrene (8 mg/ml) were added to U937 cells for 48 hours. Then, U937 growth medium supplemented with puromycin (1 mg/ml) was used to select for stably transfected cells. After complete selection, the cells were sorted for GFP expression using a MoFlo Cytomation (Beckman Coulter) fluorescence-activated cell sorter. After sorting for insertion, cells were treated with a TLR1/2 agonist [66 nM (100 ng/ml) Pam3CSK4, InvivoGen] and sorted for activation. The top 10% of activated cells were collected for each sort until no further peak separation was achieved between the untreated and the treated cells. The sorted cells were seeded in six-well plates at 1 x 10⁶ cells per well with 3 ml of growth medium [RPMI 1640 me- dium, supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml)] and the indicated concentrations of compound and Pam3CSK4 for 24 hours at 37 °C in a 5% C0₂ humidified incubator. After 24 hours, the cells in each well were mixed and 200 ml of cells containing medium was stained by propidium iodide for 10 min before the flow cytometry analysis.

[0120] In vitro nitric oxide activation assay for RAW 264.7 cells. Raw 264.7 cells

(mouse leukemic monocyte macrophage cell line) were grown in RPMI 1640 medium supplemented with 10%> FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml), seeded in 96- well plates at 80,000 cells per well, and grown for 24 hours at 37°C in a 5% C0₂ humidified incubator. After 24 hours, nonadherent cells and medium were removed and replaced with fresh unsupplemented RPMI 1640 medium. The adherent macrophages were treated with 66 nM (100 ng/ml) Pam3CSK4 (InvivoGen) or different concentrations of Compound A. Plates were then incubated for an additional 24 hours. After incubation, 100 ml of medium was collected and added to flat black 96-well microfluor plates (Thermo

Scientific). To each well, 10 ml of 2,3-diaminonaphthalene (0.05 mg/ml in 0.62 M aqueous HCl solution) was added and incubated for 15 min in the dark. The reaction was quenched by addition of 5 ml of a 3 M aqueous NaOH solution, and the plate was read on a Beckman Coulter DTX 880 reader with excitation at 365 nm and emission at 450 nm. The nitrite (a stable metabolite of NO) concentration was determined from a nitrite standard curve.

[0121] Primary peritoneal macrophage cells isolation and nitric oxide activation assay. Sprague-Dawley rats were anesthetized with isoflurane and then decapitated.

Peritoneal cells were removed by lavage. Cold dissection solution (30.0 ml of Hanks' balanced salt solution) was placed into the peritoneal cavity, the abdomen was briefly massaged, and the fluid was removed (20 to 25 ml). The medium was centrifuged, and the cells were then washed by red blood cell lysis buffer (160 mM NH₄C1, 12 mM NaHC0₃, 100 mM EDTA, pH 7.3). After centrifugation, the cells were resuspended to 1.0 ^x 106 cells/ml in culture medium [Iscove's medium containing 10%> FBS with penicillin (50 U/ml), streptomycin (50 mg/ml), and 2 mM L-glutamine; all medium reagents from Gibco]. Cells were seeded in a 96-well plate with a density of 40,000 cells per well. After 2 hours of incubation at 37 °C in a 5% C0₂ humidified incubator, non- adherent cells were removed by washing with phosphate-buffered saline (PBS) and 200 ml of supplemented Iscove's medium was subsequently added to each well. After overnight incubation, the medium was changed to Iscove's medium without FBS and indicated concentrations of Compound A and 66 nM (100 ng/ml) Pam3CSK4 were added. After 24 hours of treatment, medium was harvested and NO in the supernatant was measured as described previously.

[0122] TNF-aELISA. Raw 264.7 cells were seeded in six-well plates at 1 x 10⁶ cells per well with 3 ml of medium [RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml)] and grown for 24 hours at 37 °C in a 5% C0₂ humidified incubator. After 24 hours, nonadherent cells and medium were removed and replaced with fresh RPMI 1640 medium (3 ml per well). The cells were treated with indicated concentrations of Compound A and 66 nM (100 ng/ml) Pam3CSK4 (InvivoGen) as positive control. Plates were then incubated for an additional 24 hours, and the cell culture supernatants were collected and frozen at -80 °C until ready for cytokine measurement. The production of the cytokine TNF-a was quantified using cytokine-specific capture antibodies, detection antibodies, and recombinant human cytokine standards according to commercially available ELISA kits from R&D Systems. The cytokine level in each sample was determined in triplicate.

[0123] Competition binding ELISA assay. The 96-well ELISA microplate (BD

Biosciences) was coated with a mixture of TLR1 (5 mg/ml) and TLR2 (4 mg/ml) or BSA (5 mg/ml) in 0.1 M acetate buffer (pH 5.0) at 4 °C overnight. The wells were washed three times with PBS supplemented with 0.05% Tween 20 (PBST) and then blocked with a 5% BSA solution at room temperature for 1 hour. After washing with PBST three times, the indicated concentration of biotin-labeled Pam3CSK4 or biotin-labeled Pam3CSK4 and Compound A mixture was added and incubated for 1 hour at room temperature. After five washings, a streptavidin- coupled HRP conjugate was diluted at a ratio of 1 :2000 (Thermo Scientific), added into the wells, and incubated at room temperature for 1 hour. After washing with PBST seven times, 100 ml of TMB reagents (BD OptEIA) was added to each well and incubated at room temperature for 10 to 30 min. Fifty microliters of 1 M H₃PO₄ was subsequently added into each well to stop the reaction, the absorbance at 450 nm was measured on a Beckman Coulter DTX 880 microplate reader, and 620 nm was chosen as the reference wavelength. [0124] qRT-PCR. RAW 264.7 cells were seeded in 6-well plates at 1 ^χ 10⁶ cells per well with 3 mL of medium (RPMI 1640 medium, supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL)) and grown for 24 h at 37 °C in a 5% C0₂ humidified incubator. After 24 h, non-adherent cells and media were removed and replaced with fresh RPMI 1640 medium (3 mL/well). The cells were treated with the indicated concentrations of Compound A and 33 nM (50 ng/mL) PamsCSIQ as positive control. Plates were then incubated for an additional 0, 2, 8 or 24 h. At these time points the medium was removed, the cells were gently washed with cold PBS (3 x 1 mL). Then 1 mL of PBS was added to each well the cells were removed off from the plate. The mixture was transferred into corresponding 1.5 mL cryotubes and frozen at -80 °C until ready for qRT-PCR. Total RNA was extracted by an RNeasy Mini Kit (SABioscience, Frederick, MD, USA) according to Manufacture's instruction. cDNA was synthesized by RT2 Easy First Strand cDNA Synthesis Kit (SABioscience, Frederick, MD, USA) according to manufacturer's instruction. The primers for TLR1, TLR2, TNF, iNOS, IL-10 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from SABioscience (Frederick, MD, USA). qPCR was performed on a CFX96™ Real-Time PCR detection system (Bio-Rad, Hercules, CA,USA) using the SYBR Green method.

[0125] Raw 264.7 cells were seeded in six-well plates at 1 ^χ 10⁶ cells per well with 3 ml of medium [RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml)] and grown for 24 hours at 37 °C in a 5% C0₂ humidified incubator. After 24 hours, nonadherent cells and medium were removed and replaced with fresh RPMI 1640 medium (3 ml per well). The cells were treated with the indicated concentrations of Compound A and 33 nM (50 ng/ml) Pam3CSK4 as positive control. Plates were then incubated for an additional 0, 2, 8, or 24 hours. At these time points, the medium was removed and the cells were gently washed with cold PBS (3 x 1 ml). Then, 1 ml of PBS was added to each well and the cells were removed from the plate. The mixture was transferred into corresponding 1.5-ml cryotubes and frozen at -80 °C until ready for qRT- PCR. Total RNA was extracted by an RNeasy Mini Kit (SABiosciences) according to the manufacturer's instruction. Complementary DNA (cDNA) was synthesized by RT2 Easy First Strand cDNA Synthesis Kit (SABiosciences) according to the manufacturer's instruction. The primers for TLR1, TLR2, TNF, iNOS, IL-10, and GAPH were purchased from SABiosciences. qPCR was performed on a CFX96 Real-Time PCR detection system (Bio-Rad) using the SYBR Green method. The data were analyzed by AAC_t method. [0126] Fluorescence Anisotropy Assay. Fluorescence polarization experiments were performed at 25 °C using a Horiba Fluorolog-3 fluorometer. Equal molar ratio of TLRl and TLR2 were incubated at 4 °C for 30 minutes before titrated with the Rho-Pam3 solution. Indicated concentration of TLRl or TLR2 or (TLR1/2) (stock solution at 10 μΜ) were titrated into 500 PBS buffer (pH = 7.4) including 10 nM (20 ng/mL) Rho-Pam3

(Invivogen), and the fluorescence anisotropy were measured at an excitation of 549 nm and an emission of 566 nm, respectively, after the proteins were added and incubated for 3 minutes. Dissociation constant was obtained by fitting the binding curve to a one-site saturation equation.

[0127] To determine the inhibitory constant of Compound A, 10 μΐ_^ of Rho-Pam3 (1 μg/mL) was added to 490 μΐ, PBS buffer (pH = 7.4), then the fluorescence anisotropy was measured at excitation of 549 nm and emission of 566 nm, respectively, by a Horiba

Fluorolog-3 fluorometer. Then, 4 μΕ TLRl (10 μΜ) and 4 μΐ, TLR2 (10 μΜ) were titrated into the above buffer and incubated for 30 min, then the anisotropy was determined. Next, serial concentrations of CU-T12-9 (or T 12-29 as a negative control) were incubated with the complex TLRl/2-Pam3 solution for 30 min at room temperature before the anisotropy was determined. Regression analysis was carried out using Origin 9.0 (OriginLab) ligand binding macro module. Experimental data were fitted into equation (1) to determine the IC₅₀ values. Equation (1): y= min+ (max-min)/(l+10^x"logIC5°) (y= total binding, x= log concentration of and rhodamine-labeled PamsCSIQ, min= nonspecific binding, max= maximum binding in absence of ligand).

[0128] Fluorescence polarization experiments were performed at 25 °C using a

Horiba Fluorolog-3 fluorometer. Equal molar ratio of TLRl and TLR2 was incubated at 4 °C for 30 min before they were titrated with the Rho-Pam3 solution. Indicated concentrations of TLRl , TLR2, or TLR1/2 (stock solution at 10 mM) were titrated into 500 ml of PBS buffer (pH 7.4) including 10 nM (20 ng/ml) Rho-Pam3 (InvivoGen), and the fluorescence anisotropy was measured at an excitation of 549 nm and an emission of 566 nm after the proteins were added and incubated for 3 min. The dissociation constant was obtained by fitting the binding curve to a one-site saturation equation.

[0129] To determine the IC₅₀ value of Compound A, 10 ml of Rho-Pam3 (1 mg/ml) was added to 490 ml of PBS buffer (pH 7.4), and then the fluorescence anisotropy was measured at an excitation of 549 nm and an emission of 566 nm by a Horiba Fluorolog-3 fluorometer. Then, 4 ml of TLRl (10 mM) and 4 ml of TLR2 (10 mM) were titrated into the above buffer and incubated for 30 min, and the anisotropy was determined. Next, serial concentrations of Compound A (or Compound 23 as a negative control) were incubated with the complex TLRl/2-Pam3 solution for 30 min at room temperature before the anisotropy was determined. Regression analysis was carried out using the Origin 9.0 (OriginLab) ligand- binding macro module. Experimental data were fitted into the following equation to determine the IC50 values: y= min+ (max-min)/(l+10^x"logIC5°), where y is the total binding, x is the log concentration of rhodamine-labeled Pam3CSK4, min is the nonspecific binding, and max is the maximum binding in the absence of ligand.

[0130] hTLRl and hTLR2 protein expression and purification. The hTLRl and hTLR2 proteins were expressed in the baculovirus insect cell expression system. Monolayers of Spodoptera frugiperda (Sf-9) cells were cotransfected with Bright Baculovirus DNA (BD BaculoGold) and the pVL1393 plasmid vector containing cDNA for TLRl and TLR2. Viral titers were amplified to ~5 x 10⁷ to 10 x 10⁷/ml virus particles. The recombinant viruses were used to infect suspension high 5 insect cells in serum- free medium (Insect-XPRESS Protein- free Insect Cell Medium with L-glutamine, Lonza) at 27 °C, 130 rpm. After incubation of high 5 insect cells with recombinant TLR2 viruses for 3 days, the cells changed to green and the TLR2-containing medium was collected after low-speed centrifugation and dialyzed

[Slide- A-Lyzer G2 Dialysis Cassettes, 10,000 molecular weight cutoff (MWCO), Pierce] against 0.1 M tris buffer (pH 8.0) containing 0.3 M NaCl. The dialyzed medium was filtered and purified by a column of nickel nitrilotriacetic acid beads (Qiagen) according to the manufacturer's instruction. The purified protein was finally dialyzed against 5 mM tris buffer (pH 7.4) containing 0.15 M NaCl and condensed by a centrifugal concentrator (Millipore, 10,000 MWCO). Electrophoretic analysis revealed that TLR2 exhibited a single band with a molecular mass of about 80 kD. About 100 mg of TLR2 protein was obtained from 500 ml of medium. After incubation of high 5 insect cells with recombinant TLRl viruses for 2 days, the cells also changed to green and the TLRl -containing medium was collected after low- speed centrifugation. The medium was filtered and purified by a column of nProtein A Sepharose beads according to the manufacturer's instruction (GE Healthcare).

Electrophoretic analysis revealed that TLRl exhibited a single band with a molecular mass of -100 kD (fig. S10).

[0131] Microscale thermophoresis (MST). Interactions between freshly prepared

CU-T12-9 and TLRl (or TLR2) were measured using MST with a Monolith NT.115

(NanoTemper Technologies GmbH). Each protein was labeled with a fluorescent dye (NT- 647) using Monolith NT Protein Labeling Kits (amine or cysteine reactive). Proteins were labeled and purified within 45 min. The concentration of NT-647-labeled TLRl (or TLR2) was held constant at 10 nM, whereas the concentration of the nonlabeled Compound A was varied between 0.31 nM and 10 mM. The MST buffer con- tained 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 10 mM MgC12, and 0.05% Tween 20. Hydrophilic glass capillaries were used in all measurements. Apparent values were determined using NanoTemper Analysis software.

[0132] SEC-LS assay. First, 60 ml of hTLR2 in PBS buffer (pH 7.4) was incubated at room temperature for 2 hours with indicated concentrations of Compound A or Pam3CSK4. Then, 60 ml of hTLRl in PBS buffer (pH 7.4) was added to the reaction mixture, which was incubated at 37 °C for an additional 2 hours. The hTLRl and hTLR2 proteins were mixed in an equimolar ratio such that the final concentration of hTLRl was 1 mg/ml (~10 mM) and hTLR2 was 0.8 mg/ml (~10 mM). All buffers and samples were filtered through a 0.1-mm filter. Then, 100 ml of prepared samples was analyzed by injection onto a Shodex KW80X size exclusion column running in PBS buffer (pH 7.4) with a flow rate of 1 ml/min. The column was in line with a multiangle light scattering detector (DAWN EOS, Wyatt

Technologies), a refractive index detector (Optilab DSP, Wyatt Technologies), and an absorbance detector (UV 3000, Spectra System) for data collection. Data were analyzed with Astra 4.9 software (Wyatt Technologies).

[0133] MTT cell viability assay. In a 96-well plate, 40,000 HEK-Blue hTLR2 cells were seeded in 200 ml of growth medium [DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml)]. Eight wells were left empty for blank controls. The plates were incubated at 37 °C, 5% C0₂ for 24 hours. In the next 24 hours of treatment, the medium was re- moved from the 96-well plate and replaced with 200 ml of supplemented Opti-MEM [0.5% FBS, penicillin (50 U/ml), streptomycin (50 mg/ml), l x nonessential amino acids] containing 0 to 100 mM of compounds. Then, 20 ml (5 mg/ml in PBS) of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution was added to each well and incubated (37°C, 5% C0₂) for another 4 hours to allow the MTT to be metabolized. The medium was removed and the plate was dried on paper towels to remove any residue. Then, 150 ml of DMSO was added in each well and shaken continuously for 40 min. When all the MTT metabolic products were dissolved, results were read by spectra- photometer at 560 nm. Optical density should be directly correlated with cell quantity. Cytotoxicity (%) was determined using the following formula: Cytotoxicity (%) = (1 - [Compounds (A₅₆o) - Background (A₅₆₀)]/[Control (A₅₆o) - Background (A₅₆₀)]) ^x 100.

[0134] The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. All references cited herein are incorporated by reference in their entirety.

Claims

What is Claimed is:

1. A compound of the formula:

wherein

R¹ is H, N0₂, haloalkyl, amino, cyano or -COOR⁴, wherein R⁴ is H or alkyl;

each of R² and R³ is independently H, alkyl or aralkyl; and

Ar'is optionally substituted aryl,

provided Ar¹ is not 4-nitrophenyl; or when R¹ is H, then Ar¹ is not phenyl or 4-bromophenyl; or when R¹ is N0₂, then Ar¹ is not phenyl.

2. The compound of Claim 1, wherein R¹ is selected from the group consisting of NO₂, H, trifluoromethyl, amino, cyano, and-CC^R⁴, wherein R⁴ is H or C₁-C₄ alkyl.

3. The compound of Claim 1, wherein R² is H.

4. The compound of Claim 1, wherein R³ is selected from the group consisting of methyl, ethyl, butyl, and benzyl.

5. The compound of Claim 1, wherein Ar¹ is optionally substituted phenyl or naphthyl.

6. The compound of Claim 5, wherein Ar¹ is an optionally substituted phenyl.

7. The compound of Claim 6, wherein said optionally substituted phenyl is of the formula:

wherein

R¹³ is H, -NO₂, halide, alkyl, alkoxy, cyano, haloalkyl, aryl, cycloalkyl or -COOR⁵, wherein R⁵ is H or alkyl;

R¹⁴ is H, N0₂ or halide; and

R¹⁵ is H or halide.

8. The compound of Claim 7, wherein R¹³ is selected from the group consisting of H, NO₂, fluoro, cyano, trifluoromethyl, C₁-C₄ alkyl, C₁-C₄ alkoxy, phenyl, cyclohexyl, and -COOR⁵, wherein R⁵ is H or C₁-C₄ alkyl.

9. The compound of Claim 7, wherein R is selected from the group consisting of H, N0₂ and fluoro.

10. The compound of Claim 7, wherein R¹⁵ is selected from the group consisting of H or fluoro.

11. A composition com rising a vaccine and a compound of the formula:

wherein

R¹ is H, N0₂, haloalkyl, amino, cyano or -COOR⁴, wherein R⁴ is H or alkyl;

each of R² and R³ is independently H, alkyl or aralkyl; and

Ar'is optionally substituted aryl.

12. The composition of Claim 11, wherein said vaccine is hepatitis B virus vaccine, human immunodeficiency virus (HIV) vaccine, hepatitis C virus vaccine, human papillomavirus (HPV) vaccine, or a combination thereof.

13. A method for treating a clinical condition that can be treated by activation of TLR1 and/or TLR2, said method comprising administering to a subject in need of such a treatment a compound of any of Claims 1-10.

14. The method of Claim 12, wherein said clinical condition comprises cancer, a chronic inflammatory disease, an acute inflammatory disease, an infection, or obesity.

15. The method of Claim 12, wherein said clinical condition comprises breast cancer, bladder cancer, pancreatic carcinoma, influenza, asthma and age-induced obesity.

16. The method of Claim 12, wherein said clinical condition comprises cancer.

17. The method of Claim 15, wherein said compound of Claim 1 is administered in combination with a radiation therapy, chemotherapy, monoclonal antibody therapy, or a combination thereof.

18. A method for selectively activating TLR1/2 heterodimer in a subject, said method comprising administering an effective amount of a compound of any of Claims 1-10.