Patent Publication Number: US-9884851-B2

Title: Heterocycle-bisamide inhibitors of scavenger receptor BI

Description:
REFERENCE TO RELATED APPLICATIONS 
     This application is based upon and claims the priority of U.S. Provisional Patent Application No. 61/716,118, entitled “Small Molecule Inhibitors Of Scavenger Receptor BI” filed Oct. 19, 2012, which is hereby incorporated in its entirety by reference. 
    
    
     U.S. GOVERNMENT RIGHTS 
     This invention was made with government support under NIH Grant No. RO1 HL052212. The government has certain rights in this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to the field of small molecule inhibitors of Scavenger receptors, and in particular to inhibitors of Scavenger receptor class B, type I (SR-BI) and their use. 
     BACKGROUND 
     Scavenger receptor class B, type I (SR-BI) is a member of the CD36 superfamily. Each member contains a large extracellular domain flanked by two membrane-spanning domains with short amino and carboxy-terminal intracellular tails. CD36 family members maintain about 30% amino acid sequence identity. They can differ in subcellular localization and ligand preference. For example, CD36/SCARB3 is 29% identical to SR-BI and can bind HDL but is incapable of efficient uptake of HDL cholesterol via selective lipid uptake. There are several isoforms of SR-BI with the predominant one being isoform 1 (NP_058021). Isoform 2 (called SR-BII) differs by a 40 amino acid sequence in the C-terminus that is encoded by an alternative exon and has a reduced efficiency in selective uptake of HDL lipids. 
     SR-BI mediates selective uptake of cholesterol from high-density lipoprotein (HDL) particles through a poorly understood process that is dramatically different from classic cellular endocytic uptake of lipoproteins (e.g., the uptake of low-density lipoprotein (LDL) via LDL receptors). Among other things, SR-BI serves as a co-receptor for Hepatitis C Virus (HCV) viral entry. Thus, compounds that can interfere with the interaction of the HCV and SR-BI may block or reduce HCV infection. New tools are required to enhance our understanding of SR-BI function and mechanism of action, both in vitro and in vivo as well as new pharmaceutical agents for use in diseases or conditions involving the inhibition of SR-BI function, such as in the inhibition of pathogen (e.g., HCV) cell entry. 
     SUMMARY 
     Novel compounds are disclosed that inhibit the activity of Scavenger receptor class B, type I (SR-BI). High-density lipoprotein (HDL)-focused pharmaceutical agents and methods of treatment based on such compounds are also disclosed. 
     In one aspect, the present invention provides small-molecule compounds described by Formula (I) below or a salt or solvate thereof. These compounds can inhibit the transfer of lipids mediated by the scavenger receptor class B, type I (SR-BI). These compounds can also increase the strength of binding of high-density lipoprotein (HDL) to cells expressing SR-BI, inhibit SR-BI transport of cholesteryl ester or other lipids from HDL into cells, inhibit SR-BI transport of cholesterol or other lipids from cells into HDL. 
     Among other things, SR-BI serves as a co-receptor for Hepatitis C virus (HCV) viral entry. Thus, in some embodiments of this invention, the compounds of Formula (I) can be administered to inhibit HCV or treat and/or block HCV infection. For example, a compound of Formula (I) may be administered in a dose sufficient to reduce, inhibit or block HCV infection or in combination with other inhibitors of HCV binding, entry, replication and/or release to treat or prevent HCV-based disease. 
     These and other features of the embodiments as will be apparent are set forth and described herein. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a drawing showing the molecular representation of the probe ML279; 
         FIG. 2  is a table showing the properties of the probe ML279; 
         FIG. 3  is a drawing showing the molecular representations of the reported inhibitors of SR-BI; 
         FIG. 4  is a graph showing the recovery of undissolved probe (ML279) from PBS buffer (pH 7.4, 23° C.); 
         FIG. 5  is a table showing the summary of known probe (ML279) properties computed from structure; 
         FIG. 6  is a diagram showing the synthesis of probe 2 (ML279); 
         FIG. 7  is a drawing showing the molecular representation of the tetrazole peptide MLS000888573; 
         FIG. 8  is a drawing showing the concentration response curves for the initial hit, MLS000888573; 
         FIG. 9  is a diagram showing the critical path for probe development; 
         FIG. 10  is a drawing showing the dose response curves for the probe (ML279); 
         FIG. 11  is a table showing the SAR analysis of eastern amide; 
         FIG. 12  is a table showing the SAR analysis of heterocycle, tetrazole replacements; 
         FIG. 13  is a table showing the SAR analysis of probe 2 western arene, ortho and meta substitutions; 
         FIG. 14  is a table showing the SAR analysis of probe 2 western arene, para substitutions; 
         FIG. 15  is a table showing the SAR analysis of probe 2 central amide, n-substituent; 
         FIG. 16  is a table showing the SAR analysis of central carbon substituent; 
         FIG. 17  is a diagram showing the summary of SAR performed; 
         FIG. 18  is a table showing the comparison of the probe to project criteria; 
         FIG. 19  is a drawing showing the comparison of ML279 to prior art compounds with Dil-HDL uptake; 
         FIG. 20  is a table showing the comparison of probes to select prior art compounds; 
         FIG. 21  is a drawing showing the ML279 activity in mechanism of action assays; 
         FIG. 22  are images showing that the ML279 does not inhibit endocytosis of transferrin; 
         FIG. 23  is a graph showing the chemical characterization data for probe 2 (ML279),  1 H NMR spectrum; 
         FIG. 24  is a graph showing the chemical characterization data for probe 2 (ML279),  13 C NMR spectrum; 
         FIG. 25  is a graph showing the chemical characterization data for probe 2 (ML279), UPLC-MS chromatogram; 
         FIG. 26  is a table showing the SAR analysis of additional analogs; 
         FIG. 27  is a graph showing the chemical characterization data for analog CID 650573,  1 H NMR spectrum; 
         FIG. 28  is a drawing showing the chemical characterization data for analog CID 650573, LC-MS Chromatogram; 
         FIG. 29  is a graph showing the chemical characterization data for analog CID 53347943,  1 H NMR spectrum; 
         FIG. 30  is a graph showing the chemical characterization data for analog CID 53347943, UPLC-MS chromatogram; 
         FIG. 31  is a graph showing the chemical characterization data for analog CID 53347986,  1 H NMR spectrum; 
         FIG. 32  is a graph showing the chemical characterization data for analog CID 53347986, UPLC-MS chromatogram; 
         FIG. 33  is a graph showing the chemical characterization data for analog CID 53347994,  1 H NMR spectrum; 
         FIG. 34  is a graph showing the chemical characterization data for analog CID 53347994, UPLC-MS chromatogram; 
         FIG. 35  is a graph showing the chemical characterization data for analog CID 53393832,  1 H NMR spectrum; 
         FIG. 36  is a graph showing the chemical characterization data for analog CID 53393832, UPLC-MS chromatogram; 
         FIG. 37  is a graph showing the chemical characterization data for analog BRD-K80017325-001-01-8,  1 H NMR spectrum; 
         FIG. 38  is a graph showing the chemical characterization data for analog BRD-K80017325-001-01-8, UPLC-MS chromatogram; 
         FIG. 39  is a graph showing the chemical characterization data for analog CID 53393839,  1 H NMR spectrum; 
         FIG. 40  is a drawing showing the chemical characterization data for analog CID 53393839, UPLC-MS chromatogram; 
         FIG. 41  is a graph showing the chemical characterization data for analog CID 53377453,  1 H NMR spectrum; 
         FIG. 42  is a graph showing the chemical characterization data for analog CID 53377453, UPLC-MS chromatogram; 
         FIG. 43  is a graph showing the chemical characterization data for analog CID 53347989,  1 H NMR spectrum; 
         FIG. 44  is a graph showing the chemical characterization data for analog CID 53347989, UPLC-MS chromatogram; 
         FIG. 45  is a graph showing the chemical characterization data for analog CID 53262920,  1 H NMR spectrum; 
         FIG. 46  is a drawing showing the chemical characterization data for analog CID 53262920, UPLC-MS chromatogram; 
         FIG. 47  is a graph showing the chemical characterization data for analog CID 53347998,  1 H NMR spectrum; 
         FIG. 48  is a graph showing the chemical characterization data for analog CID 53347998, UPLC-MS chromatogram; 
         FIG. 49  is a graph showing the chemical characterization data for analog CID 53347995,  1 H NMR spectrum; 
         FIG. 50  is a graph showing the chemical characterization data for analog CID 53347995, UPLC-MS chromatogram; 
         FIG. 51  is a graph showing the chemical characterization data for analog CID 53347970,  1 H NMR spectrum; 
         FIG. 52  is a graph showing the chemical characterization data for analog CID 53347970, UPLC-MS chromatogram; 
         FIG. 53  is a graph showing the chemical characterization data for analog CID 53347956,  1 H NMR spectrum; 
         FIG. 54  is a graph showing the chemical characterization data for analog CID 53347956, UPLC-MS chromatogram; 
         FIG. 55  is a graph showing the chemical characterization data for analog CID 53347992,  1 H NMR spectrum; 
         FIG. 56  is a graph showing the chemical characterization data for analog CID 53347992, UPLC-MS chromatogram; 
         FIG. 57A  is a graph showing the chemical characterization data for analog CID 5018540,  1 H NMR spectrum; 
         FIG. 57B  is a graph showing the chemical characterization data for analog CID 5018540, UPLC-MS chromatogram; 
         FIG. 58  is a graph showing the chemical characterization data for analog CID 53347939,  1 H NMR spectrum; 
         FIG. 59  is a graph showing the chemical characterization data for analog CID 53347939, UPLC-MS chromatogram; 
         FIG. 60  is a graph showing the chemical characterization data for analog CID 53347971,  1 H NMR spectrum; 
         FIG. 61  is a graph showing the chemical characterization data for analog CID 53347971, UPLC-MS chromatogram; 
         FIG. 62  is a graph showing the chemical characterization data for analog CID 53377421,  1 H NMR spectrum; 
         FIG. 63  is a graph showing the chemical characterization data for analog CID 53377421, UPLC-MS chromatogram; 
         FIG. 64  is a graph showing the chemical characterization data for analog CID 53262912,  1 H NMR spectrum; 
         FIG. 65  is a graph showing the chemical characterization data for analog CID 53262912, UPLC-MS chromatogram; 
         FIG. 66  is a graph showing the chemical characterization data for analog CID 53377445,  1 H NMR spectrum; 
         FIG. 67  is a graph showing the chemical characterization data for analog CID 53377445, UPLC-MS chromatogram; 
         FIG. 68  is a graph showing the chemical characterization data for analog CID 53347967,  1 H NMR spectrum; 
         FIG. 69  is a graph showing the chemical characterization data for analog CID 53347967, UPLC-MS chromatogram; 
         FIG. 70  is a graph showing the chemical characterization data for analog CID 53347965,  1 H NMR spectrum; 
         FIG. 71  is a graph showing the chemical characterization data for analog CID 53347965, UPLC-MS chromatogram; 
         FIG. 72  is a graph showing the chemical characterization data for analog CID 649690,  1 H NMR spectrum; 
         FIG. 73  is a graph showing the chemical characterization data for analog CID 649690, UPLC-MS chromatogram; 
         FIG. 74  is a graph showing the chemical characterization data for analog CID 53347937,  1 H NMR spectrum; 
         FIG. 75  is a graph showing the chemical characterization data for analog CID 53347937, UPLC-MS chromatogram; 
         FIG. 76  is a graph showing the chemical characterization data for analog CID 53347936,  1 H NMR spectrum; 
         FIG. 77  is a graph showing the chemical characterization data for analog CID 53347936, UPLC-MS chromatogram; 
         FIG. 78  is a graph showing the chemical characterization data for analog CID 53377432,  1 H NMR spectrum; 
         FIG. 79  is a drawing showing the chemical characterization data for analog CID 53377432, UPLC-MS chromatogram; 
         FIG. 80  is a graph showing the chemical characterization data for analog CID 53347963,  1 H NMR spectrum; 
         FIG. 81  is a graph showing the chemical characterization data for analog CID 53347963, UPLC-MS chromatogram; 
         FIG. 82  is a graph showing the chemical characterization data for analog CID 53348004,  1 H NMR spectrum; 
         FIG. 83  is a graph showing the chemical characterization data for analog CID 53348004, UPLC-MS chromatogram; 
         FIG. 84  is a graph showing the chemical characterization data for analog CID 53347981,  1 H NMR spectrum; 
         FIG. 85  is a graph showing the chemical characterization data for analog CID 53347981, UPLC-MS chromatogram; 
         FIG. 86  is a graph showing the chemical characterization data for analog CID 53347942,  1 H NMR spectrum; 
         FIG. 87  is a graph showing the chemical characterization data for analog CID 53347942, UPLC-MS chromatogram; 
         FIG. 88  is a graph showing the chemical characterization data for analog CID 53393836,  1 H NMR spectrum; 
         FIG. 89  is a graph showing the chemical characterization data for analog CID 53393836, UPLC-MS chromatogram; 
         FIG. 90  is a histogram showing the percentage of HCV positive cells for three different infection set-ups with SR-BI compounds and controls; 
         FIG. 91  is a histogram showing the percentage of HCV positive cells for two independent experiments with SR-BI compounds and controls; 
         FIG. 92  is a histogram showing the MFI of HCV positive cells for three different infection set-ups with SR-BI compounds and controls; 
         FIG. 93  is a histogram showing the MFI of HCV positive cells for two independent experiments with SR-BI compounds and controls; 
         FIG. 94  is a graph showing the MFI of the HCV positive cells in function of the compound concentration for SRB1-2 and SRB1-8; 
         FIG. 95  is a graph showing the MFI of the HCV positive cells in function of the compound concentration for SRB1-7 and SRB1-9; 
         FIG. 96  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-1 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 97  is a graph showing the normalized to DMSO control in function of the compound SRB1-1 concentration for cell viability and viral replication; 
         FIG. 98  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-2 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 99  is a graph showing the normalized to DMSO control in function of the compound SRB1-2 concentration for cell viability and viral replication; 
         FIG. 100  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-3 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 101  is a graph showing the normalized to DMSO control in function of the compound SRB1-3 concentration for cell viability and viral replication; 
         FIG. 102  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-4 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 103  is a graph showing the normalized to DMSO control in function of the compound SRB1-4 concentration for cell viability and viral replication; 
         FIG. 104  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-5 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 105  is a graph showing the normalized to DMSO control in function of the compound SRB1-5 concentration for cell viability and viral replication; 
         FIG. 106  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-6 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 107  is a graph showing the normalized to DMSO control in function of the compound SRB1-6 concentration for cell viability and viral replication; 
         FIG. 108  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-7 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 109  is a graph showing the normalized to DMSO control in function of the compound SRB1-7 concentration for cell viability and viral replication; 
         FIG. 110  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-8 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 111  is a graph showing the normalized to DMSO control in function of the compound SRB1-8 concentration for cell viability and viral replication; 
         FIG. 112  is a graph showing the percentage of HCV positive cells in function of the compound SRB1-9 concentration for no-wash and 1× wash experimental conditions; 
         FIG. 113  is a graph showing the normalized to DMSO control in function of the compound SRB1-9 concentration for cell viability and viral replication; 
         FIG. 114  is a table showing the effective concentration of each of the tested compositions. 
     
    
    
     DETAILED DESCRIPTION 
     Small-molecule compounds have been developed that inhibit the transfer of lipids mediated by the Scavenger receptor class B, type I (SR-BI). These compounds can be described by Formula (I) or a salt or solvate thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             R 1  and R 2  are independently H, halogen, cyano, haloalkyl, haloalkyloxy or OMe; or R 1  and R 2  together are —O—CH 2 —O— or —O—CF 2 —O—; 
             R 3  is H, halogen, cyano, haloalkyl, or haloalkyloxy; 
             R 4  is C 1-6 alkyl, C 3-6 cycloalkyl, methyl-C 3-6 cycloalkyl, or C 3-6 cycloheteroalkyl, wherein the heteroatom is N or O; 
             R 5  is H or CH 3 ; 
             R 6  is a C 1-6 alkyl or a C 3-6 cycloalkyl; and 
             A, B, D, and E are each independently CH, N, O, or S, wherein at least one of A, B, D, and E is N and another of A, B, D, and E is N, O, or S, 
             wherein if R 1  and R 2  are OMe and R 4  is a cyclohexane, then R 6  is not cyclopentane. 
           
         
       
    
     In some embodiments, each of A, B, D, and E are N. In some embodiments, A is N, E is O or S, and B and D are C. 
     In some embodiments, the haloalkyl is —CF 3 . In some embodiments, haloalkyloxy is —OCF 3 . 
     In some embodiments, these compounds can be described by Formula (IA) or a salt or solvate thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             R 1  and R 2  are, independently, H, halogen, cyano, haloalkyl, haloalkyloxy or OMe; or R 1  and R 2  together are —O—CH 2 —O— or —O—CF 2 —O—; 
             R 3  is H, halogen, cyano, haloalkyl, or haloalkyloxy; 
             R 4  is C 1-6 alkyl, C 3-6 cycloalkyl, methyl-C 3-6 cycloalkyl, or C 3-6 cycloheteroalkyl, wherein the heteroatom is N or O; 
             R 5  is H or CH 3 ; 
             R 6  is a C 1-6 alkyl or a C 3-6 cycloalkyl; 
             R 7  and R 8  are, independently, H, halogen, alkyl, haloalkyl, or alkoxy; and 
             A, B, D, and E are each independently CH, N, O, or S, wherein at least one of A, B, D, and E is N and another of A, B, D, and E is N, O, or S, 
             wherein if R 1  and R 2  are OMe and R 4  is a cyclohexane, then R 6  is not cyclopentane. 
           
         
       
    
     In some embodiments, each of A, B, D, and E are N. In some embodiments, A is N, E is O or S, and B and D are C. 
     In some embodiments, R 7  and R 8  are, independently, H or halogen. In some embodiments, the halogen is fluorine or chlorine. 
     In some embodiments, R 1  and R 2  together are —O—CH 2 —O— or —O—CF 2 —O—. In other embodiments, the ring defined by A, B, D, and E is a tetrazole, oxazole, isoxazole, or thiazole. In yet other embodiments, the compound of Formula (I) has the structure: 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compounds described herein can be described by Formula (III) or a salt or solvate thereof 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             Z is an optionally substituted heteroaryl or an optionally substituted heterocycle; 
             R 4  is C 1-6 alkyl, C 3-6 cycloalkyl, methyl-C 3-6 cycloalkyl, or C 3-6 cycloheteroalkyl, wherein the heteroatom is N or O; 
             R 5  is H or CH 3 ; 
             R 6  is a C 1-6 alkyl or a C 3-6 cycloalkyl; 
             A, B, D, and E are each, independently, CH, N, O, or S, wherein at least one of A, B, D, and E is N and another of A, B, D, and E is N, O, or S, 
             wherein if R 1  and R 2  are OMe and R 4  is a cyclohexane, then R 6  is not cyclopentane. 
           
         
       
    
     In some embodiments, each of A, B, D, and E are N. In some embodiments, A is N, E is O or S, and B and D are C. 
     In some embodiments, the optionally substituted heteroaryl or optionally substituted heterocycle is optionally substituted with one or more of a halogen, cyano, haloalkyl, haloalkyloxy, or alkoxy group. In some embodiments, the alkoxy group is —OMe. 
     Other examples of compounds according to Formula (I) are provided in Table I. Each of the compounds in this table has a potency for modulating (e g, inhibiting) the uptake of lipids via SR-BI (SR-BI-mediated lipid uptake) with an IC 50  of less than 10 μM and a 24-hour cytotoxicity of IC 50 &gt;35 μM. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 101 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 102 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 103 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 104 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 105 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 106 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 107 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 108 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 109 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 110 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 111 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 112 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 113 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 114 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 115 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 116 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 117 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 118 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 119 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 120 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 121 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 122 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 123 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 124 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 125 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 126 
               
               
                   
               
            
           
         
       
     
     In some embodiments, various salts of these compounds can be used Preferably, the salt is a pharmaceutically acceptable salt. In some embodiments, various solvates of these compounds can be used. 
     In some embodiments, compounds that are substantially stereoisomerically pure are used in the methods as described herein. Thus, in some embodiments, the compound of Formula (I) and/or a pharmaceutical composition comprising the compound of Formula (I) is substantially free of other stereoisomers. For example, it contains less than about 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.5% (w/w or v/v) of other stereoisomer(s). 
     Uses 
     Scavenger receptor class B, type I (SR-BI) mediates selective uptake of lipids, such as cholesterol, from high-density lipoprotein (HDL) particles, a process that is distinct from endocytic uptake of lipoproteins, such as low-density lipoprotein (LDL). The compounds of the present invention inhibit the transfer of lipids between high-density lipoprotein (HDL) and cells that is mediated by SR-BI. These compounds may inhibit the cellular selective uptake of HDL lipids, such as cholesterol and/or its esters (e.g., cholesteryl ester), as well as the efflux of certain cellular lipids, such as cholesterol, to HDL particles. 
     At the whole organism level, SR-BI controls the structure and composition of plasma HDL, and the level and fate of HDL cholesterol, including its delivery to the liver and steroidogenic tissues. It is also required for normal endothelial cell function and controlling the structure and function of various blood cells (e.g., red blood cells and platelets). It also influences hepatitis C virus infection and deep vein thrombosis. SR-BI binds HDL and functions as a cell surface transporter to move cholesterol or its esters into or out of cells and as a signaling receptor to control cell function. SR-BI can also interact with a wide variety of other ligands and transport a variety of small molecules. 
     The compounds as defined by Formula (I) inhibit the transfer of lipids between plasma high-density lipoprotein (HDL) and cells mediated by SR-BI. As noted above, these compounds can inhibit both cellular selective lipid uptake of HDL cholesterol (particularly the esterified form of cholesterol (e.g., cholesteryl ester)) and the efflux of cellular cholesterol to HDL and can be used as a therapeutic agent as well as a probe of the molecular and cellular functions of SR-BI, across diverse areas of physiology and medicine. 
     Thus, it is an aspect of this invention that the compounds of Formula (I) are administered to inhibit or reduce the transfer of lipids between HDL and cells expressing SR-BI. It is an aspect of this invention that the compounds of Formula (I) are administered to increase the strength of binding of HDL to cells expressing SR-BI. For example, a compound of Formula (I) may be administered to a subject in need thereof in an amount effective to inhibit SR-BI transport of cholesteryl ester or other lipids from HDL into cells or from cells to HDL or other acceptors of said lipids. 
     These compounds can inhibit the transfer of lipids mediated by the scavenger receptor class B, type I (SR-BI). These compounds can also increase the strength of binding of high-density lipoprotein (HDL) to cells expressing SR-BI, inhibit SR-BI transport of cholesteryl ester or other lipids from HDL into cells, and inhibit SR-BI transport of cholesterol or other lipids from cells into HDL. 
     SR-BI influences multiple facets of lipoprotein/lipid metabolism, and in vitro and in vivo studies (e.g., transgenic and knockout mice) have established a role for SR-BI in many mammalian physiologic and pathophysiologic systems. SR-BI knockout (KO) mice display increased total plasma cholesterol levels and reduced adrenal cholesterol levels. Female KO mice are infertile due to the importance of lipoprotein metabolism in ovarian function and oocyte maturation. Lipoprotein metabolism also impacts endothelial biology, platelet function, bile secretion, steroidogenesis, and cholesterol homeostasis. SR-BI is considered to be a pattern-recognition receptor (PRR), a type of immune recognition receptor for microbial substances, such as lipopolysaccharide (LPS), and has the ability to clear LPS and to suppress stimulation of NF-kB and cytokine stimulation via Toll-like receptors. 
     As noted above, SR-BI binds HDL and functions as a cell surface transporter to move cholesterol or its esters into or out of cells and as a signaling receptor to control cell function. SR-BI can also interact with and transport a wide variety of other ligands. Thus, in addition to their usefulness as therapeutic agents, in some embodiments of the present invention, the compounds as described herein can be used to map important sites of interaction on SR-BI for these processes, to help identify possible intracellular binding partners, or to improve the understanding of other aspects of SR-BI biology. 
     SR-BI has a wide variety of functions in physiology and pathophysiology. Thus, some embodiments of the invention provide compounds that modulate (e g, inhibit or block) SR-BI activity in immune cells to modulate the immune response for example, in the case of sepsis. 
     Among other things, SR-BI serves as a co-receptor for Hepatitis C Virus (HCV) viral entry. Thus, compounds that can interfere with the interaction of the HCV virus and SR-BI may block or reduce HCV infection. ITX-5061 is a compound currently under clinical review for the treatment of HCV. ITX-5061 has been shown to inhibit HCVcc and HCVpp infection of primary human hepatocytes and/or human hepatoma cell lines (Syder et al., J. Hepatology V. 54(1) January, 2011 48-55) by modulating SR-BI activity. Similarly, the compounds of Formula (I) may also be useful in treating HCV infection. 
     Thus, in some embodiments of this invention, the compounds of Formula (I) can be administered to inhibit HCV or treat and/or block HCV infection. For example, a compound of Formula (I) may be administered in a dose sufficient to reduce, inhibit or block HCV infection, either alone or in combination with other inhibitors of the HCV lifecycle. 
     Additionally, the presence of SR-BI can also enhance sporozite invasion efficiency of hepatocytes by the malaria parasite,  Plasmodium falciparum . Blockade of SR-BI by small molecules, such as the compounds of Formula (I) can aid in the understanding of the precise mechanisms that viruses and pathogens use to enter human cells and cause disease. Thus, it is an aspect of this invention that the compounds of Formula (I) are administered to inhibit or block invasion of the malaria parasite. 
     Pharmaceutical Compositions 
     In some embodiments, pharmaceutical compositions are provided that comprise the compounds of Formula (I) and at least one pharmaceutically acceptable carrier and/or excipient. In some embodiments, the pharmaceutical composition may contain a compound which is substantially free of other isomers. 
     The compounds described herein including pharmaceutically acceptable carriers can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal, buccal, and parenteral administration, including intramuscular, subcutaneous and intravenous injections. 
     Proper formulation is dependent upon the route of administration chosen. For example, for oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained, for example, by adding asolid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added. Examples include cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof such as sodium alginate. In some embodiments, the pharmaceutical composition may be formulated as a solid dosage form (e.g., a tablet or capsule), a paste, emulsion, slurry, or ointment. 
     In other embodiments, the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injection is a preferred method of administration for the compositions of the current invention. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks&#39;s solution, Ringer&#39;s solution, or physiological saline buffer. 
     Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. 
     In some embodiments, pharmaceutical compositions are provided in which the active ingredient can be a compound of Formula (I) contained in the composition in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. Determination of an effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein. 
     For any compound described herein, the therapeutically effective amount can be initially determined from in vitro assays. As is well known in the art, in some cases, therapeutically effective amounts for use in humans can be determined from animal models. A therapeutically effective dose can also be determined from human data for compounds which are known to exhibit similar pharmacological activities. The applied dose can be adjusted based on the relative potency of the administered compound as compared with that of a known compound. 
     Patient doses for oral administration of the compounds described herein, typically range from about 1 mg/day to about 10,000 mg/day, more typically from about 10 mg/day to about 1,000 mg/day, and most typically from about 50 mg/day to about 500 mg/day. Stated in terms of patient body weight, typical dosages can range from about 0.01 to about 150 mg/kg/day, more typically from about 0.1 to about 15 mg/kg/day, and most typically from about 1 to about 10 mg/kg/day, for example 5 mg/kg/day or 3 mg/kg/day. 
     Definitions and Abbreviations 
     The definitions of terms used herein are meant to incorporate the present state-of-the-art definitions recognized for each term in the chemical and pharmaceutical fields. Where appropriate, exemplification is provided. The definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group. 
     Where stereochemistry is not specifically indicated, all stereoisomers of the compounds provided herein are included within the scope of this disclosure, as pure isomers as well as mixtures thereof. Unless otherwise indicated, individual enantiomers, diastereomers, geometrical isomers, and combinations and mixtures thereof are all encompassed by the present disclosure. 
     The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C 1-6  means one to six carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, homologs and isomers of, for example, n-pentyl, n-hexyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl.” The definition of each expression, e.g. alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure. 
     The terms “alkoxyl” or “alkoxy” refers to an alkyl group, as defined herein, having an oxygen radical attached thereto. In one embodiment, alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. The alkyl portion of an alkoxy group is sized like the alkyl groups, and can be substituted by the same groups that are suitable as substituents on alkyl groups, to the extent permitted by the available valences. 
     The term “aryl” means, unless otherwise stated, a substituted or unsubstituted polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) having 3 to 10 or alternatively 3 to 7 members which are fused together or linked covalently. 
     The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen, carbon and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, —CH 2 —CH 2 —O—CH 3 , —CH 2 —CH 2 —NH—CH 3 , —CH 2 —CH 2 —N(CH 3 )—CH 3 , —CH 2 —S—CH 2 —CH 3 , —CH 2 —CH 2 , —S(O)—CH 3 , —CH 2 —CH 2 —S(O) 2 —CH 3 , —CH═CH—O—CH 3 , —Si(CH 3 ) 3 , —CH 2 —CH═N—OCH 3 , and —CH═CH—N(CH 3 )—CH 3 . Up to two heteroatoms may be consecutive, such as, for example, —CH 2 —NH—OCH 3  and —CH 2 —O—Si(CH 3 ) 3 . The term “heteroalkyl” encompass poly(ethylene glycol) and its derivatives. 
     The term “heteroaryl” refers to aryl groups that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen, carbon and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. “Aryl” and “heteroaryl” also encompass ring systems in which one or more non-aromatic ring systems are fused, or otherwise bound, to an aryl or heteroaryl system, such as a benzodioxolyl (e.g., 1,3-benzodioxol-5-yl), benzofuran, isobenzofuran, indole, isoindole, indoxazine, indazole, benzoxazole, and anthranil. In some embodiments, the heteroaryl is a thiophene, isoxazole, tetrahydrofuran, pyridyl, benzofuran, or furanopyridine. 
     Each of the above terms (e.g., “alkyl,” “alkoxy,” “aryl” and “heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below. 
     Substituents for the alkyl, and heteroalkyl radicals are generally referred to as “alkyl substituents” and “heteroalkyl substituents,” respectively, and they can be one or more of a variety of groups selected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO 2 R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O) 2 R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O) 2 R′, —S(O) 2 NR′R″, —NRSO 2 R′, —CN and —NO 2  in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R′, R″, R′″ and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″ and R″″ groups when more than one of these groups is present. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., —CF 3  and —CH 2 CF 3 ). In some embodiments, the term “alkyl” will also include groups including acyl (e.g., —C(O)CH 3 , —C(O)CF 3 , —C(O)CH 2 OCH 3 , and the like). 
     As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). 
     The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. 
     The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention. 
     The phrase “pharmaceutically acceptable” refers to additives or compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to an animal, such as a mammal (e.g., a human) The term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington&#39;s, The Science and Practice of Pharmacy, (Gennaro, A. R., ed., 19 th  edition, 1995, Mack Pub. Co.), discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds provided herein, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc. Excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer&#39;s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. 
     The term “pharmaceutical composition” refers to a composition of the compounds of Formula (I) described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients. 
     As used herein, the term “patient” is a human or other animal, who is in need of treatment with inhibitor of SR-BI or would receive benefit from inhibition of the transfer of lipids mediated by SR-BI. For example, the patient may be a human or other animal with a bacterial infection, who has been exposed to bacteria, who is at risk of exposure to a bacteria, or who is otherwise in need of antibacterial treatment. The patient may be infected or at risk of infection with hepatitis C virus. Thus, in some embodiments the patient will be in need of the therapeutic treatment as provided herein. Preferred patients are mammals. Often, the human patients considered for the present invention are institutionalized in a primary medical care facility such as a hospital or nursing home. However, any other patient is also included within the scope of the present invention. The treatment of disease associated with the use of antibiotics or cancer chemotherapies or antiviral therapies can occur on an outpatient basis or can be prescribed by a physician for home-care as well. 
     The terms “salt” and “pharmaceutically acceptable salt” refers to a salt of one or more compounds. Suitable salts and pharmaceutically acceptable salts include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, benzoic acid, acetic acid, citric acid, tartaric acid, phosphoric acid, carbonic acid, or the like. Where the compounds carry one or more acidic moieties, pharmaceutically acceptable salts may be formed by treatment of a solution of the compound with a solution of a pharmaceutically acceptable base, such as lithium hydroxide, sodium hydroxide, potassium hydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, ammonia, alkylamines, or the like. 
     The term “solvate,” as used herein, is a molecular or ionic complex of molecules or ions of a solvent with molecules or ions of the compound of Formula (I). When water is the solvent, the molecule is referred to as a “hydrate”. 
     The term “stereoisomers” refers to compounds whose molecules have the same number and kind of atoms and the same atomic arrangement, but differ in their spatial arrangement. 
     The term “substantially free of”, when used to describe a material or compound, means that the material or compound lacks a significant or detectable amount of a designated substance. In some embodiments, the designated substance is present at a level not more than about 1%, 2%, 3%, 4% or 5% (w/w or v/v) of the material or compound. For example, a preparation of a particular stereoisomer is “substantially free of” other stereoisomers if it contains less than about 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.5% (w/w or v/v) of the other stereoisomers other than the particular stereoisomer designated. 
     The term “inhibit” or “inhibition” as used herein means to reduce, slow, or stop a process, e.g., the transfer of HDL cholesterol and/or its esters between the the extracellular fluid and the cell or an infection, such as HCV infection. The term “substantially inhibit” as used herein means reducing a process by at least 70%. In some embodiments, SR-BI is inhibited at least 80%, at least 90%, at least 95%, or at least 98%. 
     The term “substituted” refers to a chemical group, such as alkyl, cycloalkyl aryl, and the like, wherein at least one hydrogen is replaced with a substituent as described herein, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, —CF 3 , —CN, or the like. The term “substituted” is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. In many embodiments, however, any single substituent has fewer than the 100 total atoms. In many embodiments, however, any single substituent has fewer than the 10 total atoms. 
     It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. 
     The term “SR-BI,” means a mammalian scavenger receptor class B, type I as well as non-mammalian homologues. In some embodiments, SR-BI is a mammal SR-BI. In some embodiments, SR-BI is a murine or human SR-BI. 
     The phrase “therapeutically effective amount” as used herein, means an amount sufficient to elicit a desired biological or medicinal response in a cell culture, tissue system, animal, or human. In some embodiments, the response includes alleviation and/or delay of onset of one or more symptoms of the disease, condition, or disorder being treated. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects. 
     The term “treatment” or “treating” as used herein means any therapeutic intervention in a patient, preferably a human, or any other animal capable of suffering from a disease or condition associated with SR-BI mediated processes. For example, the treatment may be for abnormal lipid metabolism resulting and the treatment would restore physiologically normal metabolism by reducing SR-BI activity. As another example, the treatment may be to prevent or lessen the symptoms or duration of HCV infection. This therapeutic intervention encompasses inhibition by arresting the development of clinical symptoms, e.g., slowing the progression of the disease; and relief, by, for example, causing the regression of clinical symptoms. 
     The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined—e.g., the limitations of the measurement system, or the degree of precision required for a particular purpose. For example, “about” can mean within 1 or within 2 standard deviations, as per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, and more preferably up to 5% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” means an acceptable error range for the particular value should be assumed. 
     As used herein and in the appended claims, the singular forms “a,” “an,” and “the,” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a molecule” includes one or more of such molecules, “a resin” includes one or more of such different resins and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. 
     While the above description provides examples and specific details of various embodiments, it will be appreciated that some features and/or functions of the described embodiments admit to modification without departing from the scope of the described embodiments. The above description is intended to be illustrative of the invention, the scope of which is limited only by the language of the claims appended hereto. 
     EXAMPLES 
     Aspects of the applicant&#39;s teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the applicant&#39;s teachings in any way or be necessarily indicative to the optimal ways that the invention can be practiced. 
     The compounds as described in the Table 1 above were synthesized as provided in Scheme 1. In these reactions, the racemic mixture of the compound described by Formula (II) ( FIG. 1 ) and most other analogs were conveniently prepared via Ugi 4-component reaction. By way of example, the synthesis of the compound of Formula (II) can be prepared according to either scheme outline below, which are referred to as Scheme 1 and Scheme 2; alternatively, the enantiopure compound of Formula (II) was made using standard peptide synthesis protocols. Scheme 2, which is based upon the Ugi reaction system, which is also generally described in Ugi, I.; Meyr, R.; Fetzer, U.; Steinbriickner, C. Versuche mit Isonitrilen.  Angew. Chem.  1959, 71, 386; Dömling, A. Recent developments in isocyanide based multicomponent reactions in applied chemistry.  Chem. Rev.  2006, 106, 17-89; and Marcaccini, S.; Torroba, T. The use of the Ugi four-component condensation.  Nature Protocols  2007, 2, 632-639, each of which is hereby incorporated by reference in its entirety. The compounds can be prepared according to either method. 
     The tetrazole acetic acid building block was prepared by condensing the requisite arylnitrile with sodium azide, then alkylating the resulting tetrazole with methyl bromoacetate, and finally hydrolyzing the ester (Scheme 1). The details are provided herein. 
     
       
         
         
             
             
         
       
     
     
       
         
         
             
             
         
       
     
     Example 2—Assay of Compounds 
     The compounds as described herein were assayed in vitro for HDL activity and were found to be active inhibitors of cellular HDL lipid uptake. Details are provided herein. 
     The compound of Formula (II) was found to have an average IC 50  value of 0.016 μM in the DiI-HDL uptake assay (SR-BI-mediated uptake of the fluorescent lipid DiI (1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate, the chemical formula is C 59 H 89 ClN 2 O 4 ) that was incorporated into HDL particles). 
     In the binding assay,  125 I-HDL binding to cells (4° C. or 37° C.) was measured for 2 h in the absence (total activity) or presence (nonspecific activity) of a 40-fold excess of unlabeled HDL. Stably transfected cells were seeded in the wells of 24-well plates (50,000 cells per well) in Ham&#39;s F12 medium containing 2 mM L-glutamine, 50 units/mL penicillin per 50 μg/mL streptomycin (medium A) supplemented with 5% (vol/vol) FBS and 0.25 mg/mL G418 (medium B) on day 0, and binding assays were performed on day 2. The cells were washed twice with prewarmed (37° C.) medium A plus 0.5% (wt/vol) BSA (medium C) prior to adding  125 I-HDL. For binding assays performed at 4° C., the assay plates were removed from the incubator, precooled for 30 min on ice in a cold room (4° C.), and then washed twice with cold (4° C.) medium C supplemented with 10 mM Hepes, pH 7.4, prior to adding  125 I-HDL. Specific binding or uptake is the difference between total and nonspecific activities. In assays using small molecule inhibitors (all incubations at 37° C.), immediately prior to incubation with  125 I-HDL, the cells were washed twice with medium A, preincubated with the small molecules for 1 h, and then incubated with radiolabeled lipoproteins as described above, all in medium C plus 0.5% (vol/vol) DMSO in the presence of the indicated concentrations of small molecules. 
     The compound of Formula (II) shows no apparent cytotoxicity after 24 hours of treatment (EC 50 =&gt;35 μM). The selectivity of this compound was found to be greater than 1000. See  FIG. 2 . The compound of Formula (II) has been shown to inhibit efflux of cholesterol and it does not inhibit endocytosis of transferrin. This compound was tested to be a reversible inhibitor. 
     Experimental Results 
     Using a cell-based DiI-HDL uptake assay, we performed a high-throughput screen (HTS) of the NIH MLPCN compound library. Of the 319,533 compounds tested, 3,046 compounds (0.96%) were classified as inhibitors of DiI-HDL uptake. A bis-amide (CID650573) was identified in the primary HTS as an inhibitor. It had potent activity upon retesting in the primary assay, a low hit rate in other MLPCN assays, and possessed structural properties suitable for analog synthesis. Structure/activity relationship (SAR) studies were performed to improve potency and to minimize deleterious properties. 
     These efforts generated a probe (CID53393835/ML279), a low nanomolar inhibitor with improved potency and decreased metabolic liability. ML279 was tested for cholesterol efflux inhibition, modulation of HDL binding to SR-BI, and inhibition of endocytosis. ML279 functions by inhibiting both SR-BI-mediated uptake of cholesteryl ester from HDL and efflux of free cholesterol to HDL particles. The small-molecule probe (ML279) inhibits the transfer of lipids between high-density lipoprotein (HDL) and cells mediated by the HDL receptor Scavenger receptor class B, type I (SR-BI). ML279 inhibits both cellular selective lipid uptake of HDL cholesteryl ester and DiI and efflux of cellular cholesterol to HDL. 
     SR-BI influences multiple facets of lipoprotein/lipid metabolism, and in vitro and in vivo studies (e.g., transgenic and knock out mice) have established a role for SR-BI in many mammalian physiologic and pathophysiologic systems. SR-BI knock out (KO) mice display increased total plasma cholesterol levels and reduced adrenal cholesterol levels. Female KO mice are infertile due to the importance of lipoprotein metabolism in ovarian function and oocyte maturation. Lipoprotein metabolism also impacts endothelial biology, platelet function, bile secretion, steroidogenesis, and cholesterol homeostasis. SR-BI is considered to be a pattern-recognition receptor (PRR), a type of immune recognition receptor for microbial substances, such as lipopolysaccharide (LPS), and has the ability to clear LPS and to suppress stimulation of NF-kB and cytokine stimulation via Toll-like receptors (TLRs). Further, SR-BI serves as a co-receptor for Hepatitis C Virus (HCV) viral entry, and interference with compound or blocking antibodies can reduce cellular infection. Additionally, the presence of SR-BI enhances sporozoite invasion efficiency of hepatocytes by the malaria parasite,  Plasmodium falciparum.    
     At the molecular level, SR-BI controls the structure and composition of plasma HDL, and levels and fates of HDL cholesterol, including delivery to the liver and steroidogenic tissues. SR-BI binds HDL and functions as a cell surface transporter to move cholesterol or its esters into or out of cells and as a signaling receptor to control cell function. SR-BI can also interact with and transport a wide variety of other ligands. A new probe could be used to map important sites of interaction on SR-BI for these processes, to help identify possible intracellular binding partners or to improve our knowledge of other aspects of SR-BI biology. 
     ML279 may represent a significant improvement over prior SR-BI inhibitors. Blocker of lipid transport-1 (BLT-1) has nanomolar potency but is extremely toxic to cells, which has limited the use of the compound to short term in vitro assays. ML279 overcomes the major limitations of BLT-1: specifically it is a reversible binder of SR-BI and is not cytotoxic. Further, ML279 is more potent than ITX-5061, a previously described compound that disrupts DiI uptake from DiI-HDL. 
     Scavenger receptor class B, type I (SR-BI) is a member of the CD36 superfamily. Each member contains a large extracellular domain flanked by two internal membrane-spanning domains that are adjacent to short amino and carboxy-terminal intracellular tails. CD36 family members maintain about 30% amino acid sequence identity but differ in subcellular localization and ligand preference. For example, CD36/SCARB3 is 29% identical to SR-BI and can bind HDL but is incapable of HDL uptake. There are several isoforms of SR-BI with the predominant one being isoform 1 (NP_058021). Isoform 2 of SR-BI differs by a 40 amino acid sequence in the C-terminus that is encoded by an alternative exon and has a reduced efficiency in selective uptake of HDL. SR-BI mediates selective uptake of cholesterol from high-density lipoprotein (HDL) particles through a poorly understood process that is dramatically different from classic cellular endocytic uptake of lipoproteins (e.g., low-density lipoprotein (LDL), via LDL receptors). New tools are required to enhance our understanding of SR-BI function and mechanism of action, both in vitro and in vivo. Recent disappointments in attempts to develop HDL-focused pharmaceutical agents with other molecular targets highlight the importance of developing a deeper and broader understanding of all aspects of HDL metabolism including those mediated by SR-BI. Given the central role of SR-BI in lipid transfer and metabolism, inhibitors of SR-BI function will be useful tools to further probe the mechanisms of SR-BI, both in vitro and in vivo, and may also have clinical utility, such as in the inhibition of pathogen cell entry (e.g., HCV). 
     Several small-molecule inhibitors of SR-BI have been discovered ( FIG. 3 ). In a pilot study for this project, Krieger and coworkers identified five compounds (BLTs 1-5) that block lipid transport selectively via SR-BI, with nanomolar to micromolar potencies. Assays with purified SR-BI reconstituted into liposomes show that BLTs directly act on SR-BI. However, these probes exhibit an unacceptable level of cellular toxicity. The diabetes drug glyburide, a potent inhibitor of the sulfonylurea receptors SUR1 and SUR2, was also found to have activity at SR-BI, though it was a relatively weak inhibitor of cholesterol efflux. Researchers at Sankyo discovered that the protected piperazines R-138329 and R-154716 increased HDL-cholesterol in both mice and hamsters, presumably by inhibiting SR-BI-mediated uptake of HDL. Recently, a p38 MAP kinase inhibitor (ITX-5061) was reported to have similar in vivo effects and appears to be a moderate inhibitor of SR-BI-mediated cholesterol uptake. ITX-5061 is also an inhibitor of HCV entry into cells, and is presently in Phase II trials as a HCV antiviral treatment. This compound was prepared in our laboratories and was profiled in our assays of interest. Finally, ITX-7650 has been described as an inhibitor of cholesterol uptake and HCV infection, but the structure remains undisclosed. 
     A major goal of the invention is to identify a new SR-BI inhibitor that will provide a substantial improvement over the existing tool compounds under at least one of the following categories: 1) increased potency and reversibility; 2) reduced cellular toxicity; 3) new insights into mechanism; and 4) a unique activity profile (e.g., direct inhibition of ligand binding, differential influence on selective uptake, and free cholesterol efflux). 
     Materials and Reagents 
     
         
         
           
             DiI-HDL, custom purified HDL particles derived from human blood were prepared and labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Catalog No. D-282; Invitrogen, Carlsbad, Calif.). 
             Alexa 488 HDL, human HDL particles labeled with the Alexa Fluor® 488 Protein Labeling Kit (Catalog No. A-10235, Invitrogen; Carlsbad, Calif.) were likewise purified and labeled. 
             CellTiter-Glo® Luminescent Cell Viability Assay was purchased from Promega (Catalog No. G7573; Madison, Wis.). 
             Radiolabeled cholesterol[1,2- 3 H(N)]—, 1 mCi (37 MBq) was obtained from PerkinElmer—NEN (Catalog No. NET139001MC; Waltham, Mass.). 
             Alexa Fluor-594 conjugated human transferrin (Catalog No. T-13343) was obtained from Invitrogen.
 
Cell Lines
 
           
         
       
    
     The following cell lines were used in this study:
         ldlA[mSR-BI] is a Chinese hamster ovary (CHO) cell line that overexpresses murine SR-BI, isoform 1 (NP_058021) and was obtained from the (Krieger Laboratory). This cell line was used for the primary assay and several secondary assays. A variant of this cell line expressing mutant SR-BI, where a cysteine required for interaction with BLT-1 is mutated to serine (C384S SR-BI) was used in several secondary assays.   [ldlA7] is the parental cell line to ldlA[mSR-BI] cells, and does not overexpress SR-BI, and can be used to rule out compound activity independent of SR-BI.
 
Assays
 
DiI-HDL Uptake Assay
 
(Primary Assay AID Nos. 488896, 493194, 540354 588392, 588553, 588548, 588754, 588828, 588833)
       

     ldlA[mSR-BI] cells were plated into 384-well plates at 30 μl per well and incubated overnight. As a measurable surrogate for cholesterol uptake, human HDL particles were treated with the lipophilic fluorescent dye DiI and exposed to ldlA[mSR-BI] cells in lipoprotein-free media (Ham&#39;s F12/0.5% fatty acid-free Bovine Serum Albumin (BSA)/25 mM HEPES pH 7.4 plus 10 μg protein/ml DiI-HDL). Cells took up the DiI via SR-BI over 3 hours in the presence of compound. After significant uptake of the DiI, the cells became fluorescent. The level of fluorescence correlates with the amount of DiI uptake and can be measured with a standard plate reader (here a PerkinElmer EnVision plate reader was used). The uptake of lipid (represented by DiI) was inhibited by the positive control compound BLT-1 or with an excess of HDL untreated with DiI. Primary HTS data were analyzed in Genedata Screener Assay Analyzer, and were normalized against DMSO and the positive control (1 μM BLT-1). For the HTS, the average of two replicates was used to rank order activity and to choose compounds for retests. For dose studies, percent (%) activity was determined for each concentration and the concentration response curves (CRCs) were generated with Genedata Condeseo. 
     Fluorescence Quencher Counterscreen (Secondary Assay (SA) 2: AID Nos. 588403, 540326) 
     The primary assay for this HDL project measures a reduction in fluorescence by potential inhibitors. One possible explanation for the loss of signal in the primary is the compounds have inherent fluorescence-quenching properties and reduce signal in a dose-dependent manner without actually altering any biology. We developed an assay where compounds were pinned into assay buffer containing DiI-HDL without any cells. The compounds were incubated with DiI-HDL at 5 μg protein/ml in Ham&#39;s/25 mM HEPES/0.5% fatty acid-free BSA media for 30 minutes. Then, assay plates were measured with the identical settings for the primary assay. Compounds that quench DiI fluorescence led to a dose-dependent loss of signal in this assay. Any compound that did not alter fluorescence or had an IC 50  value of &gt;30 μM was considered for further studies. 
     Cell Cytotoxicity Assay (SA 1: AID No. 540246, 588829, 588826, 588830, 588842, 602126) 
     It is possible that a compound will cause cells to decrease HDL-mediated DiI uptake due to nonspecific consequences of cellular toxicity, representing a second class of false positive hits. The cells were treated with compounds for 3 hours or 24 hours, and then cell viability was measured using the CellTiter-Glo Assay (Promega), a luciferase-based reagent that measures cellular ATP levels. The compounds were tested at different concentrations to determine IC 50  values. Compounds that were toxic after 3 hours with an IC 50  value of less than 30 μM were excluded from additional studies. Compounds that were active in the primary assay but toxic below 30 μM at 24 hours (but nontoxic at 3 hours) were still considered but none of our preferred scaffolds showed cytotoxicity at either time point. Data were normalized against DMSO and the positive control (1 μM BLT-1) in Genedata Assay Analyzer. Curves were generated with Genedata Condeseo and showed percent (%) activity for the individual doses. 
     HDL Binding Assay (SA 6: AID Nos. 588777, 588810) 
     HDL binding was assessed using Alexa Fluor 488-labeled HDL particles. For this assay, the Alexa 488 dye was directly covalently bound to apolipoprotein components of the HDL particle via primary amines; thus, no transfer of the fluorophore to cell membranes occurs. Instead, direct binding of the HDL particles to SR-BI can be measured. As a positive control, BLT-1, which is known to increase binding, was used at 1 μM. It is possible that a compound can reduce binding of HDL to the receptor, and this would lead to a decrease in signal. This assay is used to characterize the mechanism of action of a particular compound; therefore, any outcome in the assay is acceptable. Data were normalized against DMSO and the positive control (1 μM BLT-1) in Genedata Assay Analyzer. Curves were generated with Genedata Condeseo and showed percent (%) activity for the individual doses. 
     Cholesterol Efflux Assay (SA 4: AID Nos. 588818, 602152) 
     SR-BI also mediates bidirectional flux (e.g. efflux) of unesterified or ‘free’ cholesterol (FC) between cells and HDL or other acceptors. This assay is used to determine if the compounds alter the efflux of free cholesterol to HDL. Compounds that do and do not inhibit in a dose-dependent manner will be of value. 
     On Day 0, 50,000 cells per well were plated in 24-well plates. On the following day, the media was removed and [ 3 H]cholesterol in lipoprotein-deficient media was added. On Day 1, the medium was replaced with Ham&#39;s F12 medium supplemented with 10% bovine lipoprotein deficient serum with 1 μCi/ml [1,2- 3 H]cholesterol (40-60 Ci/mmol). On Day 3, the cells were washed to remove serum and incubated in Ham&#39;s F12 plus 1% fatty acid-free BSA. On Day 4, the cells were washed and pretreated with compounds for 1 hour. Subsequently, the cells were incubated for an additional 2 hours with the same concentrations of small molecules and with unlabeled HDL (final HDL concentration of 100 μg protein/ml). The medium was collected to determine released cholesterol, and the cells were lysed. The amount of [ 3 H]cholesterol in the medium and cells was determined using liquid scintillation counting. Total cellular [ 3 H]cholesterol was calculated as the sum of the radioactivity in the efflux medium plus the radioactivity in the cells and was used to calculate the [ 3 H]cholesterol efflux (percent of total [ 3 H]cholesterol released into the medium). 
     Mutant SR-BI Cholesterol Efflux Assay (SA 10: AID Nos. 588843, 602138) 
     BLT-1 is known to interact at cysteine 384 of SR-BI (16). If the cysteine is mutated to serine, BLT-1 can no longer bind and shows no ability to inhibit cholesterol uptake. This assay is used to determine if the compounds work in a similar fashion to BLT-1 and require Cys384 to alter the efflux of FC to HDL. Compounds that do and do not inhibit efflux in a dose-dependent manner will be of value. 
     On Day 0, 50,000 cells expressing murine SR-BI with the C384S mutation per well were plated in 24-well plates. On the following day, the media was removed and [ 3 H]cholesterol in lipoprotein-deficient media was added. On Day 1, the medium was replaced with Ham&#39;s F12 medium supplemented with 10% bovine lipoprotein deficient serum, 1 μCi/ml [1,2- 3 H]cholesterol (40-60 Ci/mmol). On Day 3, the cells were washed to remove serum and incubated in Ham&#39;s F12 plus 1% fatty acid-free BSA. On Day 4, the cells were washed and pretreated with compounds for 1 hour. Subsequently, the cells were incubated for an additional 2 hours with the same concentrations of small molecules and with unlabeled HDL (final HDL concentration of 100 μg protein/ml). The medium was collected to determine released cholesterol, and the cells were lysed. The amount of [ 3 H]cholesterol in the medium and within cells was determined using liquid scintillation counting. Total cellular [ 3 H]cholesterol was calculated as the sum of the radioactivity in the efflux medium plus the radioactivity in the cells and was used to calculate the [ 3 H]cholesterol efflux (percent of total [ 3 H]cholesterol released into the medium). 
     DiI-HDL Uptake Assay in the Absence of SR-BI (SA 7: AID No. 588825) 
     In the primary assay, the cell line used to measure DiI-HDL uptake lacks the LDL receptor and overexpresses SR-BI. In this assay, the parental CHO cell line (ldlA7), which lacks the LDL receptor but does not overexpress SR-BI, was used to determine if inhibitors work via non-SR-BI-related mechanisms. As in the primary assay, 10 μg protein/ml of DiI-HDL was used to measure DiI uptake into the cells after 3 hours of incubation with different concentrations of compound. Little to no uptake of DiI-HDL was observed in these cells, and the only signal observed was minimal background staining. 
     Transferrin Endocytosis Assay (SA 3: AID No. 602134) 
     This assay measures the endocytosis of an independent ligand, transferrin, which is not taken up via SR-BI but by clathrin-mediated endocytosis; thus providing a measure of the selectivity of the inhibitors. Alexa-594-labeled transferrin is taken into the cell via endocytosis and localization of labeled transferrin can be quantitated in the various intracellular compartments. If a compound inhibits endocytosis, the labeled transferrin will not enter the cell leading to a decrease of fluorescent signal. Inhibitors of interest should act selectively at SR-BI and should have no activity in this assay. 
     Cells were pre-treated with compound for 3 hours and then treated with the Alexa-594 transferrin reagent for 30 minutes (Catalog No. T-13343; Invitrogen) in serum-free media. Plates of cells were then placed onto ice, washed with ice cold PBS, and then fixed with 4% paraformaldehyde. In addition, the nuclei were stained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) (Catalog No. 21490; Invitrogen). The cells were imaged with the Molecular Devices IXM microscope. Translocation measurements were performed using MetaExpress software and normalized for cell number. 
     DiI-HDL Uptake Assay with Washout (SA 5: AID No. 588831) 
     This is a modification of the primary assay where cells are pretreated with compound but no compound is present during the DiI-HDL treatment period. ldlA[mSR-BI] cells were plated into 384-well plates at 30 μl per well and incubated overnight. Cells were treated with compound for 3 hours, then media were removed and the cells were washed with PBS before fresh lipoprotein-free media (Ham&#39;s F12/0.5% fatty acid-free Bovine Serum Albumin [BSA]/25 mM HEPES pH 7.4) was added with 10 μg protein/ml DiI-HDL. Cells took up the DiI via SR-BI over 3 hours in the absence of compound. After significant uptake of the DiI, the cells became fluorescent. The level of fluorescence correlates with the amount of DiI uptake and can be measured with a standard plate reader. The uptake of lipid (represented by DiI) was inhibited by the compound BLT-1. 
     DiI-HDL Uptake Assay with Extended Washout (SA 5: AID No. 588831) 
     This is another modification of the primary assay where cells are pretreated with compound but no compound is present during a 4-hour washout period or the subsequent 3-hour DiI-HDL treatment period. This protocol allows us to distinguish between an irreversible inhibitor and a reversible inhibitor with a slow “off-rate”. ldlA[mSR-BI] cells were plated into 384-well plates at 30 μl per well and incubated overnight. Cells were treated with compound for 3 hours, then were washed four times with PBS, once with lipoprotein-free media (Ham&#39;s F12/0.5% fatty acid-free Bovine Serum Albumin [BSA]/25 mM HEPES pH 7.4), and then returned to the incubator with lipoprotein-free media for 4 hours. Media were then removed and cells were washed with PBS before fresh lipoprotein-free media (Ham&#39;s F12/0.5% fatty acid-free Bovine Serum Albumin [BSA]/25 mM HEPES pH 7.4) was added with 10 μg protein/ml DiI-HDL. Cells took up the DiI via SR-BI over 3 hours in the absence of compound. After significant uptake of the DiI, the cells became fluorescent. The level of fluorescence correlates with the amount of DiI uptake and can be measured with a standard plate reader. The uptake of lipid (represented by DiI) was inhibited by the compound BLT-1 or with an excess of HDL untreated with DiI. 
     Lipid Transport Assay in Liposomes (SA 11: AID No. 602155) 
     This assay provides confirmation of activity via SR-BI by measuring the uptake of [ 3 H]cholesteryl ester ([ 3 H]CE) into liposomes loaded with purified SR-BI. For the purification of C-terminally epitope-tagged murine SR-BI (mSR-BI-t1) with uniform, truncated N-linked oligosaccharide chains, we overexpressed mSR-BI-t1 in HEK293S cells. The mSR-BI-t1 was expressed in an N-acetylglucosaminyltransferase I (GnTI)-defective HEK293S derivative, HEK293S GnTI (2), which generates a glycoprotein with uniform, truncated N-linked oligosaccharide chains under the control of a tetracycline-inducible promoter. mSR-BI-t1 was immunoaffinity purified to virtual homogeneity, and the detergent-solubilized receptor was reconstituted into liposomes. Subsequently, the in vitro SR-BI-mediated selective uptake of [ 3 H]CE from [ 3 H]CE-HDL in the reconstituted liposomes was measured. The mSR-BI-t1 with truncated N-linked chains was purified and reconstituted into liposomes as described previously (15). Briefly, 20 μg of SR-BI (or an equivalent volume of protein-free buffer to generate control liposomes that are devoid of SR-BI) was reconstituted into liposomes by acetone precipitation. SR-BI liposomes were washed once by resuspension of the acetone precipitate in protein-free assay medium followed by a centrifugation step for 25 min and 48,000×g at 4° C. The pellet was first reconstituted in assay medium without protein, and then an equal volume of assay medium with 1% fatty acid-free BSA was added to yield liposomes at a nominal final concentration of 18 ng SR-BI/ml. In each reaction, 30 ml were pre-incubated together with 30 ml of assay medium containing 0.5% fatty Acid-free BSA, 1% DMSO, and the indicated compounds for 60 min at 37° C. Subsequently, 20 μl of [ 3 H]CE-HDL (five replicates per sample) were added to a final concentration of 10 μg protein/ml. Incubation was continued for 4 hours at 37° C., and then selective uptake of [ 3 H]CE into liposomes was determined using the previously described filter binding assays (15). SR-BI-selective uptake values were determined. The 100% of control value represents receptor-specific activity in SR-BI-t1-containing liposomes in the presence of 0.5% DMSO without compounds, and the 0% of control value represents background selective uptake in control liposomes devoid of SR-BI-t1. 
     Radioactive HDL Uptake Assay (Wild Type SR-BI) (SA 8: AID No. 588836) 
     The goal of this assay was to verify compounds that disrupt the lipid uptake from HDL particles to SR-BI scavenger receptor-expressing cells using an alternative means of labeling HDL-particles and avoiding any type of fluorescence measurement. To measure this binding event, HDL particles are labeled with [ 3 H]cholesteryl ester and added to mSR-BI expressing cells. Cells take up the radioactive lipids from HDL via SR-BI in 2 to 3 hours. After significant uptake of the lipids from HDL, the radiolabel can be detected by liquid scintillation counting. The level of radioactivity correlates with the amount of HDL lipid uptake. The uptake of lipid from the HDL particles can be inhibited by the compound BLT-1 or when co-treated with an excess of unlabeled HDL. The mSR-BI expressing cells utilized in the assay are a Chinese hamster ovary (CHO) cell line lacking expression of the LDL receptors and overexpressing the scavenger receptor, SR-BI. Inhibitors of SR-BI and HDL lipid uptake will have a reduction in liquid scintillation counts. 
     Radioactive HDL Uptake Assay (mutant SR-BI) (SA 9: AID No. 588837) 
     BLT-1 is known to interact at cysteine 384 of SR-BI (16). If the cysteine is mutated to serine, BLT-1 can no longer bind and shows no ability to inhibit cholesterol uptake. This assay is used to determine if the compounds work in a similar fashion to BLT-1 and require Cys384 to alter the binding of HDL particles and uptake of the lipid to the mutant SR-BI scavenger receptor, using an alternative means of labeling HDL particles and avoiding any type of fluorescence measurement. To measure this lipid uptake event, HDL particles are labeled with [ 3 H]cholesteryl ester and added to C384S SR-BI cells. Cells take up HDL lipid via the mutant SR-BI in 2 to 3 hours. After significant uptake of the HDL lipid, the radiolabel can be detected by liquid scintillation counting. The level of radioactivity correlates with the amount of HDL lipid uptake. The uptake of lipid from HDL particles can be inhibited by the compound BLT-1 or when co-treated with an excess of unlabeled HDL. The mSR-BI cells utilized in the assay are a CHO cell line lacking expression of the LDL receptors and overexpressing the mutant scavenger receptor, C384S SR-BI. 
     Probe Chemical Characterization 
     The probe (ML279) was prepared as described below, and was analyzed by UPLC,  1 H and  13 C NMR spectroscopy, and high-resolution mass spectrometry. The data obtained from NMR and mass spectrometry were consistent with the structure of the probe, and UPLC analysis showed purity of &gt;95%. Characterization data ( 1 H NMR spectra and UPLC chromatograms) of the probe are provided. 
     The solubility of Probe 2 (ML279) was determined to be 28 μM in Phosphate buffered saline (PBS; pH 7.4, 23° C.) solution with 1% DMSO. The stability of the probe in PBS (1% DMSO) was measured over 48 hours. We noticed that the concentration of the probe fluctuated through the course of the assay (data not shown). We believe that this fluctuation is due to the low kinetic solubility of the probe. Thus, we decided to determine the amount of undissolved probe present in the well after it was treated with PBS for a given length of time, relative to the amount of undissolved probe at the start of the assay. The wells were centrifuged and the supernatant was removed after various time points, then acetonitrile was added to dissolve the remaining solid in the wells, and the amount of probe was quantified. From these results, the probe seems to be stable to PBS ( FIG. 4 ). The stability of the probe was confirmed by measuring stability in human plasma, with &gt;99% remaining after a 5-hour incubation period. Plasma protein binding (PPB) studies showed that it was 76.5% bound in human plasma.  FIG. 5  summarizes known properties of the probe. 
     Probe Preparation 
     The racemic version of Probe 2 (ML279), and most of the analogs prepared for SAR analysis were conveniently prepared via Ugi 4-component reaction. The enantiopure Probe 2 (ML279) was made using standard peptide synthesis protocols, as shown in  FIG. 6 . The tetrazole acetic acid building block was prepared by condensing the requisite arylnitrile with sodium azide, then alkylating the resulting tetrazole with methyl bromoacetate, and finally hydrolyzing the ester. ML279 has an IC 50  value of 16 nM in the DiI-HDL uptake assay; shows no apparent cytotoxicity after 24 hours treatment (IC 50 =&gt;35 μM); inhibits efflux of free cholesterol; does not inhibit endocytosis of transferrin and is a reversible inhibitor. 
     To identify novel inhibitors, SR-BI-mediated lipid uptake was measured in an engineered cell line that minimized potential uptake by other mechanisms, including via the LDL receptor (1). ldlA[mSR-BI] cells overexpress murine isoform 1 and selectively take up HDL lipid at similar levels as “has been reported for cultured mouse adrenal cells, perfused organs, and liver, adrenal gland and ovary in vivo” (1). This primary cell-based assay was used in a pilot screen that identified BLTs 1 through 5. The assay was slightly modified from the original protocol of Nieland et al. to increase throughput and to ease automation procedures. 
     Briefly, the plate reader was switched to a Perkin-Elmer EnVision, several washing steps were eliminated, and the cell number per well was reduced without impacting the robustness of the assay. The automated assay was first tested with a validation set of 2,240 compounds. These compounds were tested in duplicate with in-plate neutral (DMSO) and positive (1 μM BLT-1) controls. Robustness, reproducibility, and variability parameters were analyzed before initiating the full HTS. The HTS was run over the course of several weeks. Data were normalized relative to controls, and plate patterns were corrected using a multiplicative algorithm in Genedata Screener Assay Analyzer. For each compound, the average of the two replicates was determined and used for subsequent analysis. 
     Determination of hits required several criteria: only assay plates with a Z′ greater than 0.3 were accepted for analysis, compounds needed to reach 70% inhibition relative to 1 μM BLT-1, and score in fewer than 10% of HTS assays listed in PubChem. In total, 319,533 compounds were screened. Of these, 3,046 compounds were considered active (a hit rate of 0.96%), 613 were inconclusive, and 315,055 were inactive. Compounds were clustered based upon chemical structure using a customized script in Pipeline Pilot. Clusters were rated based upon structural liabilities and ranked accordingly. Substructures were analyzed and compared to inactive compounds to identify inactive analogs. Representatives from the more desirable clusters were selected for retest. In addition, a small number of inactive analogs were chosen to provide initial structure/activity relationship (SAR) data during the dose retest studies. Of these, 573 compounds were retested over a range of concentrations to validate activity, and 186 compounds showed dose-dependent inhibition of DiI-HDL uptake. 
     Since active compounds produce a decrease in signal in the primary assay, confirmation was required that the reduction in fluorescence was not a result of quenching by the compound. Compounds were tested in the presence of DiI-HDL in a cell-free version of the primary assay. Of the compounds that were active in the primary assay, 127 were found to quench the DiI signal while 59 showed no alteration of fluorescence in the quenching assay. 
     All available dry powder samples of the remaining, non-quenching compounds were procured from commercial sources and purities were determined. The compounds obtained were then retested in the primary assay and in cytotoxicity assays. We wanted to rule out any compounds that were cytotoxic, especially in the 3-hour time span of the primary DiI-HDL uptake assay. Therefore, compounds were tested in ldl[mSR-BI] cells for cytotoxicity at 3 hours and 24 hours using the CellTiter-Glo assay (Promega) that measures cellular ATP as a marker of cell viability. Of these, 11 compounds showed an IC 50  value of less than 35 μM and were excluded from further consideration. Approximately 44 compounds had an IC 50  value below 10 μM in the DiI-HDL assay. Fifteen chemical scaffolds showed potencies below 1 μM and three of these were prioritized for follow-up studies. Compounds were screened for inhibition of endocytosis using labeled transferrin, which is known to bind the transferrin receptor, enter cells by clathrin-mediated endocytosis, and move to endosomes before being recycled to the cell surface. The BLT compounds have also been tested for potential interference with endocytosis and show no such activity (12). 
     Neither the original tetrazole-peptide hit described in this report, MLS000888573 (See  FIG. 7 ), nor ML279 affected endocytosis of transferrin, indicating that they are not grossly perturbing cellular membrane trafficking pathways independent of SR-BI. With an IC 50  value in the primary assay of 0.051 μM, the peptide scaffold of MLS000888573 was thus prioritized for further development. Analogs were designed and synthesized, eventually leading to the more potent probe, ML279. 
     MLS000888573 (SID26667991 was tested across a range of concentrations up to 35 μM in the primary assay and several secondary assays. Concentration response curves were generated with Genedata Condeseo and show normalized percent activity for the individual doses (See  FIG. 8 ). DiI-HDL Uptake (AID 540354), IC 50 =0.051 μM (A); Alexa 488 HDL binding (AID 588810), AC 50 =0.6 μM (B); 3 h CellTiter-Glo (AID 588830), IC 50 &gt;35 μM (C) and 24 h CellTiter-Glo (AID 540246), IC 50 &gt;35 μM (D). 0=replicate 1, Δ=replicate 2 
     In the primary HTS, compounds were active if they decreased DiI-HDL uptake as measured by fluorescence. The positive control, BLT-1 (1 μM), caused a decrease in uptake that led to over a 2.2-fold reduction in signal. Compounds with greater than 70% activity of the positive control were considered actives and chosen for confirmation studies. 
       FIG. 9  illustrates the critical path for probe development. To explore SAR, numerous analogs were synthesized and tested. Selected results are discussed below and shown in the figures. 
     Dose Response Curves for the Probe (ML279) are provided in  FIG. 10 . ML279 was used over a range of concentrations up to 35 μM in: the primary DiI-HDL uptake assay, IC 50 =0.0162 μM (AID 588828) ( 10 A); Alexa-488 HDL binding assay, AC 50 =0.27 μM (AID 588810) ( 10 B); 24-h CellTiter-Glo cytotoxicity, IC 50 &gt;35 μM (AID 588829) ( 10 C); DiI-HDL ldlA7 counterscreen, IC 50 &gt;35 μM (AID 588825) ( 10 D). Dose curves were generated with Genedata Condeseo and show normalized percent activity for the individual doses. ¢=replicate 1, Δ=replicate 2 
     Probe 2 (ML279) can potentially act as an electrophile under biological conditions, with its tetrazole alpha to the western amide. Currently, there is no evidence to support this possibility, as it is stable to GSH, and the racemic version of the probe showed reversible inhibition of SR-BI mediated lipid uptake. Modified versions of the DiI-HDL protocol were performed to assess whether ML279 is a reversible inhibitor. In both the immediate and extended washout protocols, the observed activity was greatly attenuated, indicating that ML279 was being washed away and was not reacting with the cells in an irreversible manner (AID Nos. 588831, 602154). 
     The biological assay data and physical properties of these analogs are presented in  FIGS. 11-16 . Characterization data ( 1 H NMR spectra and UPLC chromatograms) of these analogs are provided in  FIGS. 23-89 . 
     The hit compound showed good potency (IC 50 =0.058 μM) and excellent solubility in PBS (114 μM). In order to rapidly explore the SAR at different parts of the molecule, we generally utilized the 4-component Ugi reaction, which could often provide the desired products in a single reaction from appropriate building blocks (aldehyde, amine, carboxylic acid, and isonitrile). We investigated the eastern amide portion of the molecule by varying the isonitrile building block. The SAR at this location is very steep, as smaller or larger terminal alkyl groups largely abrogated the activity. A secondary amide is required for optimal activity, as structurally related tertiary amides had greatly reduced potencies. Both tertiary amides were prepared via a stepwise peptide synthesis; N-alkylation of the hit compound gave a complex mixture due to competing C-alkylation alpha to the tetrazole. 
     Several replacements of the tetrazole ring were investigated, using arylacetic acid building blocks in the Ugi reaction that were purchased or prepared in several steps. An oxazole showed decent potency, roughly two-fold less than that of the hit compound (IC 50 =0.13 μM). The analogous thiazole had poor activity, but an isoxazole had excellent potency in the primary assay (IC 50 =0.023 μM) and good solubility. A highly simplified arene analog without a heterocycle was basically inactive. 
     The arene SAR at the western end of the scaffold was investigated next. Several ortho-substituted arenes were poorly active. Two electron-donating groups (or possibly H-bond acceptors) proved to be optimal, as removal of the 4-methoxy group decreased the activity approximately 3-fold. Pleasingly, replacement of the 3,4-dimethoxy groups with a methylenedioxy acetal gave a slight increase in activity (IC 50 =0.037 μM), and we expect will likely also give improved metabolic stability (results pending). The difluoromethylenedioxy analog was prepared but possessed significantly less activity. 
     Variation of the primary amine utilized in the Ugi reaction provided access to compounds with various nitrogen substituents at the central amide. The SAR again proved to be fairly steep, as smaller groups such as methyl and cyclopropyl had decreased activity. Introduction of a methylene spacer between the cyclohexane group and the nitrogen largely abolished the activity. Introduction of heteroatoms to the cyclohexane ring also decreased the activity significantly. The Ugi reaction using aniline as the amine component (to provide the desired N-phenyl substituent) failed using standard conditions, though we expect that it could be prepared if desired using alternative conditions. 
     Finally, substitution at the central carbon of the peptide was investigated. Surprisingly, addition of an additional methyl group abolished the activity, as did replacement of the methyl group with a bulkier isopropyl group. The use of formaldehyde in the Ugi reaction provided an achiral and unsubstituted analog, which can be compared to the prior top compound. The potencies are similar, though the solubility of the unsubstituted analog is significantly lower (3.7 μM in PBS). To investigate the importance of the chiral center in the methyl-substituted analogs, the individual enantiomers of our racemic lead compound were prepared from the D- and L-isomers of alanine (see Section 2.3). The chirality of the molecule appears to be important, as the bulk of the activity resides in the (R)-isomer (IC 50 =0.016 μM, versus IC 50 =1.6 μM for the (S)-isomer). With optimal potency and decent solubility (27 μM in PBS), we, thus, nominated CID53393835 as the probe for this series of HDL-mediated lipid uptake inhibitors. 
     The primary assay and several of the secondary assays were cell-based experiments. The compound is active in cells and the effective IC 50  value for the probe in the primary assay (0.016 μM) is well below the measured PBS solubility (28 μM). This inhibition of uptake was confirmed with an IC 50  value of 1 μM in a nonfluorescent, radiolabeled version of the assay, AID No. 588836). Since SR-BI is a cell-surface receptor (and the compound is presumed to act on the extracellular surface), cell permeability is not an issue, though the probe is expected to have reasonable permeability. ML279 showed no cytotoxicity at 24 hours (AID No. 540326). The precise mode of action for the probe is not known but several supplemental assays suggest a direct interaction with SR-BI (see Mechanism of Action, Section 4.2). The probe shows no effect on the endocytosis of transferrin, at concentrations up to 35 μM (AID No. 602134). 
     Numerous synthetic analogs were prepared and investigated SAR at five different parts of the peptide scaffold (illustrated in  FIG. 17 ), leading to the identification of the probe (ML279). In summary, 36 analogs were prepared and screened, leading to a probe with improved potency over the original hit and a reduced metabolic liability (through replacement of the dimethoxybenzene group with a methylenedioxybenzene). 
       FIG. 18  presents a comparison of the activity and selectivity profile of the probe (ML279) with the criteria decided upon in the chemical probe development plan (CPDP). 
     We compared the performance of ML279 with BLT-1, the prior art compound ITX-5061 and another probe ML278. ML279 outperformed both BLT-1 and ITX-5061. BLT-1 is a potent inhibitor of SR-BI-mediated lipid uptake and of free cholesterol efflux. It is a nonreversible covalent modifier of SR-BI and is toxic to cells. In contrast, ML279 is a reversible inhibitor of HDL uptake (AID Nos. 588831, 602154) and shows no cytotoxicity in ldlA[mSR-BI] cells (AID 588829). As discussed earlier, ITX-5061 is an SR-BI inhibitor that is currently (as of November 2011) in Phase 1b clinical trial for HCV infection. We synthesized ITX-5061 and tested it in multiple assays. In every experiment, ML279 outperformed ITX-5061, with approximately 15-fold higher potency (AID No. 588828). In these experiments ML278 appeared to be more potent than ML279, but ML279 has significantly higher solubility, which could be advantageous. 
     In  FIG. 19 , ML279 was tested against two compounds, BLT-1 and ITX-5061 in the DiI-HDL uptake assay. Each compound was used over a range of concentrations up to 35 μM. Concentration response curves were generated using Genedata Condeseo and show normalized percent activity for the individual doses (AID 588828), ML279, IC 50 =16.2 nM (A); BLT-1, IC 50 =10.8 nM (B); ITX-5061, IC50=254 nM (C) and ML278, IC 50 =0.93 nM. Also note, that single 1 μM BLT-1 data points appear as a tight column because this dose of BLT-1 was used as the positive control and tested in dose in separate wells. 0=replicate 1, Δ=replicate 2, pale grey data points=masked wells 
     As shown in  FIGS. 20 and 21 , ML279 and selected analogs were tested in several secondary assays that explore possible mechanisms of action. In addition to uptake of HDL particles and bringing esterified cholesterol into the cell, SR-BI also plays a role in the efflux of free cholesterol from the cell to recipient HDL particles. Compounds were tested to determine if there was an impact on efflux from cells. Similar to BLT-1, ML279 reduces efflux of free cholesterol from cells by about 50% ( FIG. 21B  AID No. 602152). Unlike BLT-1, ML279 was shown to be a reversible inhibitor in the DiI uptake assay. In experiments where cells were pre-treated with compounds for 2 hours, washed extensively with PBS and then incubated with DiI-HDL without compound, ML279 showed no inhibition (AID No. 588831). In comparison, BLT-1 registers an IC 50  value of 600 pM even after a 4-hour washout period (AID No. 602154), reflecting the progression of its covalent reaction with cysteine 384 of SR-BI. 
     ML279 was tested for efflux in a cell line that only expresses a mutant form of the protein where Cys384 is converted to a serine residue. In this mutant background, BLT-1 cannot reduce uptake of HDL or efflux of cholesterol. ML279 can reduce uptake and efflux in the mutant background ( FIG. 21B ) suggesting that the compound works at a different site on the receptor and does not involve Cys384. We assessed binding of HDL to SR-BI using an Alexa-488 labeled HDL. Similar to BLT-1, ML279 increases binding of HDL to the receptor (AID No. 588810). Further studies need to be performed to determine the nature of this tertiary interaction. When comparing the activity of ML279 in the DiI-HDL and radiolabeled versions of the uptake assay there is a large difference in potency (IC 50  of 0.016 μM and 1 μM, respectively). This is distinct from BLT-1 which shows equal potency in the both uptake assays. This difference may be a result of how ML279 interacts with SR-BI. 
     ML279 was also tested with SR-BI in purified liposomes. These data suggest a direct interaction between the probe and SR-BI ( FIG. 21C , AID No. 602155). These data support results in [ldlA7] cells where SR-BI is required for compound activity (AID No. 588825). 
     In  FIG. 22  ML279 was examined for the ability to modify endocytosis of Alexa fluor-594 labeled transferrin in ldlA[mSR-BI] cells with DMSO (A) or 35 μM ML279 (B) (AID No. 602134). Note the distribution of labeled transferrin throughout the cells and punctate cytoplasmic staining indicative of endosomes. Images at 40× magnification are shown. 
     The assays discussed above have focused on SR-BI relative to cholesterol metabolism. SR-BI also has roles in immunity and acts as a co-receptor for malaria and hepatitis C virus infection. 
     All reagents and solvents were purchased from commercial vendors and used as received. NMR spectra were recorded on a Bruker 300 MHz or Varian UNITY INOVA 500 MHz spectrometer as indicated. Proton and carbon chemical shifts are reported in parts per million (ppm; δ) relative to tetramethylsilane, CDCl 3  solvent, or d 6 -DMSO ( 1 H δ 0,  13 C δ 77.16, or  13 C δ 39.5, respectively). NMR data are reported as follows: chemical shifts, multiplicity (obs=obscured, app=apparent, br=broad, s=singlet, d=doublet, t=triplet, q=quartet, p=pentet, sext=sextet, sept=septet, m=multiplet, comp=complex overlapping signals); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25° C. Flash chromatography was performed using 40-60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash R f  system. Tandem liquid chromatography/mass spectrometry (LCMS) was performed on a Waters 2795 separations module and Waters 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light and aqueous potassium permanganate (KMnO 4 ) stain followed by heating. High-resolution mass spectra were obtained at the MIT Mass Spectrometry Facility with a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer. Compound purity and identity were determined by UPLC-MS (Waters, Milford, Mass.). Purity was measured by UV absorbance at 210 nm. Identity was determined on a SQ mass spectrometer by positive electrospray ionization. Mobile Phase A consisted of either 0.1% ammonium hydroxide or 0.1% trifluoroacetic acid in water, while mobile Phase B consisted of the same additives in acetonitrile. The gradient ran from 5% to 95% mobile Phase B over 0.8 minutes at 0.45 ml/min. An Acquity BEH C18, 1.7 μm, 1.0×50 mm column was used with column temperature maintained at 65° C. Compounds were dissolved in DMSO at a nominal concentration of 1 mg/ml, and 0.25 μL of this solution was injected. 
     Synthesis Details 
     A detailed synthesis scheme for ML279 is provided below: 
     
       
         
         
             
             
         
       
     
     
       
         
         
             
             
         
       
     
     5-(Benzo[d][1,3] dioxol-5-yl)-2H-tetrazole 
     A round-bottom flask equipped with a magnetic stirbar was charged with benzo[d][1,3]dioxole-5-carbonitrile (2.50 g, 17.0 mmol) and N,N-dimethylformamide (17.0 ml). Sodium azide (1.22 g, 18.7 mmol) and ammonium chloride (1.00 g, 18.7 mmol) were then added to give a white suspension. This mixture was heated to 150° C. and stirred for 24 hours. The reaction was cooled to ˜70° C., then poured on ice (˜50 ml). The mixture was acidified to pH 1 with 1.0 M aqueous hydrochloric acid solution. The precipitated solids were collected by filtration, washed with ice-cold water (10 ml), and air-dried on the filter to give the title compound as a white solid (2.79 g, 86%).  1 H NMR (300 MHz, CD 3 OD): δ 7.55 (dd, J=8.1, 1.7 Hz, 1H), 7.46 (d, J=1.5 Hz, 1H), 7.02 (d, J=8.1 Hz, 1H), 6.08 (s, 2H); MS (ESI + ): 191 (M+H). 
     
       
         
         
             
             
         
       
     
     Methyl 2-(5-(benzo[d][1,3]dioxol-5-yl)-2H-tetrazol-2-yl)acetate 
     A round-bottom flask equipped with a magnetic stirbar was charged with 5-(benzo[d][1,3]dioxol-5-yl)-2H-tetrazole (2.79 g, 14.7 mmol) and acetone (49.0 ml). The suspension was cooled to 0° C. before adding anhydrous potassium carbonate. After stirring for 30 minutes at 0° C., methyl bromoacetate (1.5 ml, 16.2 mmol) was added to the opaque, tan mixture. The reaction was stirred for 18 hours while slowly warming to room temperature. The mixture was filtered through a pad of Celite®, and the filter was thoroughly washed with additional acetone. The clear, yellow filtrate was concentrated under reduced pressure to give an orange solid. This mixture of regioisomers (˜8:1 ratio) was separated by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 40/60) to give the title compound as a white solid (2.93 g, 76%).  1 H NMR (300 MHz, CDCl 3 ): δ 7.71 (dd, J=8.1, 1.6 Hz, 1H), 7.59 (d, J=1.5 Hz, 1H), 6.91 (d, J=8.1 Hz, 1H), 6.03 (s, 2H), 5.45 (s, 2H), 3.82 (s, 3H); MS (ESI + ): 263 (M+H). 
     
       
         
         
             
             
         
       
     
     2-(5-(Benzo[d][1,3]dioxol-5-yl)-2H-tetrazol-2-yl)acetic acid 
     Methyl 2-(5-(benzo[d][1,3]dioxol-5-yl)-2H-tetrazol-2-yl)acetate (2.93 g, 11.2 mmol) was placed in a round-bottom flask equipped with a magnetic stirbar and dissolved with dichloromethane (33.5 ml) and methanol (3.7 ml). Sodium hydroxide pellets (0.89 g, 22.3 mmol) were added to the clear, pale yellow solution. The reaction was stirred at room temperature for 1 hour and the resulting opaque, white mixture was concentrated under reduced pressure to give a white solid. This material was dissolved in water (250 ml) and washed with dichloromethane (2×100 ml). The aqueous phase was acidified to pH 1 with concentrated hydrochloric acid to give an opaque white mixture. Extraction with hot ethyl acetate (3×100 ml) produced a clear aqueous layer. The combined ethyl acetate extracts were shaken over magnesium sulfate, filtered, and concentrated under reduced pressure to give title compound as a white solid (2.64 g, 95%).  1 H NMR (300 MHz, d 6 -DMSO): δ 7.62 (d, J=8.0 Hz, 1H), 7.52 (s, 1H), 7.11 (d, J=7.9 Hz, 1H), 6.14 (s, 2H), 5.70 (s, 2H); MS (ESI + ): 249 (M+H). 
     
       
         
         
             
             
         
       
     
     (R)-tert-Butyl (1-(cyclopentylamino)-1-oxopropan-2-yl)carbamate 
     N-Boc-D-alanine (250 mg, 1.32 mmol) and HOBt (243 mg, 1.59 mmol) were added to a vial with stir bar and dissolved with dry DCM (6.6 ml). Hunig&#39;s base (254 μL, 1.45 mmol) was then added, followed by cyclopentylamine (124 mg, 1.45 mmol), and EDC (304 mg, 1.59 mmol). The reaction was then stirred for 18 h, after which time LC-MS showed complete consumption of starting material and conversion to the desired product. The reaction was diluted with 1 M aq. HCl and EtOAc, then the phases were separated, and the combined organics were washed again with aq. HCl, then twice with half-saturated aq. NaHCO 3 , then brine. The organics were dried over Na 2 SO 4 , filtered, and concentrated to yield the title compound as a white solid (311 mg, 92%).  1 H NMR (300 MHz, CDCl 3 ): δ 6.11 (br s, 1H), 5.00 (br s, 1H), 4.19 (app sext, J=6.6 Hz, 1H), 4.08 (m, 1H), 1.93 (m, 2H), 1.61 (m, 4H), 1.47 (s, 9H), 1.39 (m, 2H), 1.32 (t, J=8.0 Hz, 3H); MS (ESI + ): 257.37 (M+H). 
     
       
         
         
             
             
         
       
     
     (R)-2-(Cyclohexylamino)-N-cyclopentylpropanamide 
     (R)-tert-Butyl 1-(cyclopentylamino)-1-oxopropan-2-ylcarbamate (310 mg, 1.21 mmol), DCM (6.0 ml), triethylsilane (0.97 ml, 6.1 mmol), and TFA (0.93 mL, 12.1 mmol) were added to a flask with stir bar and stirred for 18 h. The reaction was then quenched by pipetting the reaction solution slowly into a stirred half-saturated aqueous solution of NaHCO 3  (50 ml). The mixture was diluted with DCM, the layers were separated, and the organics were washed again with aq. NaHCO 3 , then brine. The organics were dried over Na 2 SO 4 , filtered, and concentrated, yielding the intermediate primary amine as an off-white solid. This was immediately dissolved with methanol (6.1 ml) and cooled on ice. Cyclohexanone (0.38 ml, 3.6 mmol) was added, followed by sodium cyanoborohydride (760 mg, 12.1 mmol) added portionwise over 10 min. The reaction was stirred for 24 h, after which time LC-MS analysis showed complete conversion to the desired product. The reaction was concentrated to remove most of the MeOH, then it was diluted with half-saturated aq. NaHCO 3  and ether. The layers were separated, then the combined organics were washed again with NaHCO 3 . dilute aq. HCl was then added to bring the product into the aqueous layer, which was then washed twice with ether to remove the excess cyclohexanone. The aqueous layer was basified again w/ aq. NaHCO 3 , then extracted twice with Et 2 O. The ether layer was then washed with brine and dried over Na 2 SO 4 , filtered, and concentrated to provide the title compound as a white solid (239 mg, 83% over two steps). A portion of the material was converted to the hydrochloride salt by dissolving in ether and adding excess HCl in Et 2 O, then concentrating.  1 H NMR (300 MHz, CD 3 OD): δ 4.13 (app p, J=6.7 Hz, 1H), 3.95 (q, J=6.9 Hz, 1H), 2.97 (m, 1H), 2.15-1.80 (comp, 6H), 1.80-1.55 (comp, 6H), 1.48 (d, J=6.9 Hz, 3H), 1.40-1.15 (comp, 6H); MS (ESI + ): 239.68 (M+H). 
     
       
         
         
             
             
         
       
     
     (R)-2-(2-(5-(Benzo[d][1,3]dioxol-5-yl)-2H-tetrazol-2-yl)-N-cyclohexylacetamido)-N-cyclopentylpropanamide (PROBE) 
     2-(5-(Benzo[d][1,3]dioxol-5-yl)-2H-tetrazol-2-yl)acetic acid (92 mg, 0.37 mmol) was sealed under nitrogen in a vial with stir bar. Dry DCM (2.2 ml), oxalyl chloride (32 μL, 0.37 mmol), and DMF (1 drop from a 22-gauge needle) were added, and the reaction was vented to an oil bubbler. The reaction was stirred for 4 h, then (R)-2-(cyclohexylamino)-N-cyclopentylpropanamide (free base, 80 mg, 0.34 mmol) was added to the resulting solution of acid chloride, followed by Hunig&#39;s Base (129 μl, 0.74 mmol) and DMAP (4.1 mg, 0.034 mmol). The reaction was stirred for 16 h, after which time LC-MS analysis showed complete conversion to the desired product. The reaction was diluted with 1 M aq. HCl and EtOAc (˜25 ml each), the layers were separated, and the combined organics were washed again with aq. HCl, then twice with half-saturated aq. NaHCO 3 , then brine. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated to a yellow oil, then purified by column chromatography (0 to 50% EtOAc/hexanes) to yield the title product as a pale yellow solid, after concentrating from an ether solution made cloudy by slowly adding pentane (90 mg, 72%).  1 H NMR (300 MHz, CDCl 3 ) δ 7.70 (dd, J=8.1, 1.5 Hz, 1H), 7.58 (d, J=1.4 Hz, 1H), 6.91 (d, J=8.1 Hz, 1H), 6.60 (br d, J=6.8 Hz, 1H), 6.04 (s, 2H), 5.59 (d, J=15.4 Hz, 1H), 5.48 (d, J=15.4 Hz, 1H), 4.10 (m, 1H), 3.96 (br m, 1H), 3.52 (br app t, J=11.4 Hz), 2.08-0.82 (comp, 21H) (peaks are broad due to rotamers);  13 C NMR (75 MHz, CDCl 3 ) δ 170.9, 165.4, 163.8, 149.6, 148.2, 121.6, 121.1, 108.8, 107.4, 101.6, 77.6, 77.2, 76.7, 58.9, 55.7, 55.1, 51.5, 33.0, 32.9, 31.6, 31.4, 25.9, 25.8, 25.0, 23.8, 15.9 (extra peaks are due to the minor rotamer). HRMS (ESI + ): calculated for C 24 H 33 N 6 O 4  [M+H] 469.2563. found 469.2552. 
     In Vitro Testing with Cells Infected with HCV 
     A series of tests were conducted using the compound of Formula II (CID53393835/ML279), other known inhibitors of SR-BI and controls with cells infected with hepatitis C virus (HCV). The cells (Huh-7.5 cells infected with Jcl 378-1 TagRFP (MOI=0.1) 3 dpi) were treated with the following SR-BI compounds and controls: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 SRB1-1 - Enantiomer of probe 2 (negative control) 
                 10 
                 uM 
               
               
                 SRB1-2 - R-154716 
                 10 
                 uM 
               
               
                 SRB1-3 - Comparative compound 
                 1 
                 uM 
               
               
                 SRB1-4 - Comparative compound 
                 10 
                 uM 
               
               
                 SRB1-5 - Probe 2 (ML279) 
                 10 
                 uM 
               
               
                 SRB1-6 - Comparative compound 
                 10 
                 uM 
               
               
                 SRB1-7 - Comparative compound 
                 0.5 
                 uM 
               
               
                 SRB1-8 - BLT-1 (Irreversible inhibitor of SR-BI) 
                 1 
                 uM 
               
               
                 SRB1-9 - ITX-5061 
                 0.5 
                 uM 
               
               
                 anti-CD81 
                 10 
                 ug/mL 
               
               
                 anti-SR-BI 
                 10 
                 ug/mL 
               
               
                   
               
            
           
         
       
     
     Three different infection protocols were followed: (1) no wash—virus inoculum was removed 4hpi and fresh medium containing compounds added without washing cells; (2) 5× wash—virus inoculum was removed 4hpi, cells were washed 5× with PBS and fresh medium containing compounds added; and (3) no wash Opt—infection were performed in serum-free OptiMem; virus inoculum was removed 4hpi and fresh medium containing compounds added without washing cells.  FIG. 90  provides a comparison of the three different infection set-ups.  FIG. 91  provides a comparison of two independent experiments. The data indicate that the compound of Formula II (SRB1-5) inhibited viral replication along with SRB1-2, 6, 7, 8 and 9. 
       FIGS. 92 and 93  provide another measure of the compound of Formula II (SRB1-5) in terms of fluorescence intensity of HCV positive cells (normalized to a DMSO control). The data show that SRB1-5 can also inhibit virus assembly/egress. 
     The data also suggest infections performed in serum-free Optimem do not increase compounds potency.  FIGS. 94-95  provide dose dependence data for various tested compounds. 
     The experiments indicate that (1) wash steps to remove virus inoculum post infection does not affect compound&#39;s potency nor does performing infections in serum free medium (OptiMEM) enhance compound&#39;s viral inhibition and (2) SRBI compounds 2/5/6/7/8/9 were able to inhibit HCV replication. Interestingly, treatment with compounds 2 and 8 resulted in increased mean fluorescence intensity (MFI) of HCV positive cells in a dose dependent manner, which correlated to their inhibitory effect. In contrast, this increase was not seen with other compounds e.g. 7 and 9. 
       FIGS. 96-97  provide further experimental results for test compound SBR1-1;  FIGS. 98-99  provide further experimental results for test compound SBR1-2;  FIGS. 100-101  provide further experimental results for test compound SBR1-3;  FIGS. 102-103  provide further experimental results for test compound SBR1-4;  FIGS. 104-105  provide further experimental results for test compound SBR1-5;  FIGS. 106-107  provide further experimental results for test compound SBR1-6;  FIGS. 108-109  provide further experimental results for test compound SBR1-7;  FIGS. 110-111  provide further experimental results for test compound SBR1-8; and  FIGS. 112-113  provide further experimental results for test compound SBR1-9. 
       FIG. 114  provides tabular data for the effective concentrations (EC 50  and EC 90 ) of each of the tested compositions. 
     The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. 
     While the applicant&#39;s teachings are described in conjunction with various embodiments, it is not intended that the applicant&#39;s teachings be limited to such embodiments. On the contrary, the applicant&#39;s teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.