Patent Publication Number: US-11040949-B2

Title: Compounds affecting pigment production and methods for treatment of bacterial diseases

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application is a continuation-in-part of U.S. non-provisional patent application Ser. No. 16/867,540 filed May 5, 2020, which is a continuation-in-part of U.S. non-provisional patent application Ser. No. 16/041,838 filed Jul. 23, 2018, which claims priority from a U.S. provisional patent application Ser. No. 62/535,540 filed Jul. 21, 2017, and the disclosures of which are incorporated by reference in their entireties. 
    
    
     TECHNICAL FIELD 
     The present invention relates to compounds and derivatives thereof, composition comprising said compounds and/or derivatives, and methods for treating microbial infections and/or related diseases or conditions. More specifically, the present compounds, derivatives, composition comprising thereof, and methods are for bacterial infections. 
     BACKGROUND OF THE INVENTION 
       Staphylococcus aureus  is a major human pathogen in communities and hospitals, causing a variety of infections that ranges from harmless infections to life threatening conditions [18]. With the wide-spread dissemination of methicillin-resistant  S. aureus  (MRSA) in hospitals and in communities, treating  S. aureus  associated infections has become increasingly difficult [19]. Staphyloxanthin has been proven to be an important factor in promoting bacterial invasion [1]. Five genes, crtOPQMN, located in an operon are responsible for the biosynthesis of the pigment. The transcription of the operon is driven by a σ B -dependent promoter upstream of crtO, and ends with a terminator downstream of crtN [2]. The pigments that endow  S. aureus  with a golden color also make it resistant to attack from reactive oxygen species (ROS) and neutrophils [3]. Pigmented bacteria have increased resistance to the host&#39;s immune defenses [4]. 
     In a mouse subcutaneous model of infection, animals infected with a wild-type strain of  S. aureus  had higher bacterial loads and larger visible lesions than those infected with non-pigmented bacteria [4]. The reduced virulence of bacterial strains with defective carotenoid synthesis was also shown in a mouse systemic  S. aureus  infection model [3]. In vitro and in vivo data suggest that blocking pigment synthesis may reduce pathogenicity. 
     Dehydrosqualene synthase (CrtM) catalyses the first step of the biosynthetic pathway, was shown to be a target for anti-infective therapy based on virulence factor neutralization. Diphenylamine was found to be an inhibitor of 4,4-diapophytoene desaturase (CrtN) at high micromolar level [5]. Another potential inhibitor of CrtN, naftifine, a FDA approved antifungal compound was shown to reduce bacterial load in different mice infection models [6]. However, there remains a need for new compounds and methods of treatment for staphylococcal infections. 
     SUMMARY OF THE INVENTION 
     Provided herein are compounds and methods for prevention and/or treatment of microbial infections and/or related disease or conditions. In a first aspect, the present invention provides compounds and/or their derivatives which can be represented by Formula (II): 
                         
wherein R1 is selected from:
 
                         
or
 
any four-, five-, six-, seven-, eight-, nine-, ten-, eleven-, or twelve-membered heterocyclyl, cycloalkenyl, or cycloalkyl,
 
where R3 and R4 can be independently or jointly selected from the group: H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR11 and —OC(═O)R11);
 
or R3 and R4 can be bonded together to form a four-, five-, or six-membered heterocyclyl, cycloalkenyl, or cycloalkyl;
 
R5 can be selected from the group: H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR11 and —OC(═O)R11); and
 
where R10 and R11 can be independently or jointly selected from the group consisting of: a (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers; and alkynyl;
 
X is selected from N or C,
 
A is single bond or double bond;
 
Q is selected from N or C,
 
M is selected from O or C, and
 
wherein R2 is selected from:
 
                         
where R6 and R7 can be independently or jointly selected from O or absent;
 
R8 and R9 can be independently or jointly selected from H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR1 and —OC(═O)R11), or R8 and R9 can be bonded together to form a four-, five-, or six-membered heterocyclyl, cycloalkenyl, or cycloalkyl, and
 
where R10 and R11 can be independently or jointly selected from the group consisting of: a (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers; and alkynyl, and
 
Z is selected from C or S.
 
     In one embodiment, the present compounds and/or the derivatives thereof can be an anti-virulent agent for bacteria. 
     In another embodiment, the present compounds and/or the derivatives thereof are effective in reducing virulence of bacteria. 
     In other embodiment, the bacteria that the present compounds and/or the derivatives thereof are effective in reducing their virulence comprise  Staphylococci  sp. 
     In yet another embodiment, the bacteria that the present compounds and/or the derivatives thereof are effective in reducing their virulence comprise  Staphylococcus aureus  ( S. aureus ). 
     In still another embodiment, the bacteria that the present compounds and/or the derivatives thereof are effective in reducing their virulence comprise methicillin-resistant  S. aureus  (MRSA). 
     In other embodiment, said reducing the virulence of bacteria by the compounds and/or derivatives thereof comprises inhibiting biosynthesis of staphyloxanthin in said bacteria and/or inhibiting or reducing production of pigments that are resistant to the bacterial host&#39;s immune defenses. 
     A composition for preventing and/or treating the microbial infections and/or related diseases or conditions comprising an effective amount of the compounds and/or the derivatives thereof in the first aspect is also provided herein. 
     In one embodiment, said microbial infections are bacterial infections. 
     In another embodiment, said microbial infections comprise staphylococcal infections. 
     In other embodiment, the composition further comprises a pharmaceutically acceptable carrier, salt, ester, excipient, vehicle, prodrug, solvent, and diluent, or any combination thereof. 
     In a second aspect, the present invention provides methods for preventing and/or treating the microbial infections and/or related diseases or conditions including administering to a subject a composition comprising an effective amount of one or more compounds of Formula (II): 
                         
wherein R1 is selected from:
 
                         
or
 
any four-, five-, six-, seven-, eight-, nine-, ten-, eleven-, or twelve-membered heterocyclyl, cycloalkenyl, or cycloalkyl,
 
where R3 and R4 can be independently or jointly selected from the group: H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR11 and —OC(═O)R11);
 
or R3 and R4 can be bonded together to form a four-, five-, or six-membered heterocyclyl, cycloalkenyl, or cycloalkyl;
 
R5 can be selected from the group: H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR11 and —OC(═O)R11); and
 
where R10 and R11 can be independently or jointly selected from the group consisting of: a (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers; and
 
alkynyl;
 
X is selected from N or C,
 
A is single bond or double bond;
 
Q is selected from N or C,
 
M is selected from O or C, and
 
wherein R2 is selected from:
 
                         
where R6 and R7 can be independently or jointly selected from O or absent;
 
R8 and R9 can be independently or jointly selected from H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR11 and —OC(═O)R11),
 
or R8 and R9 can be bonded together to form a four-, five-, or six-membered heterocyclyl, cycloalkenyl, or cycloalkyl, and
 
where R10 and R11 can be independently or jointly selected from the group consisting of: a (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers; and alkynyl, and
 
Z is selected from C or S.
 
     In an exemplary embodiment, R3 and R4 are independently or jointly selected from Br. 
     In one embodiment, the microbial infections are bacterial infection. 
     In another embodiment, the microbial infections comprise Staphylococcal infections. 
     In other embodiment, the microbial infections and/or related diseases or conditions are caused by  Staphylococci  sp. 
     In yet another embodiment, the  Staphylococci  sp. comprise  Staphylococcus aureus  ( S. aureus ). 
     In still another embodiment,  S. aureus  comprise methicillin-resistant  S. aureus  (MRSA). 
     In other embodiment, the microbial infections and/or related diseases or conditions comprise infections of the skin and soft tissue, bone and joint, surgical wound, indwelling devices, lung and heart valves. 
     In certain embodiments, the present method further comprises reducing virulence of bacteria causing the microbial infections and/or related disease or conditions. 
     In some other embodiments, the present method further comprises inhibiting biosynthesis of staphyloxanthin in said bacteria and/or inhibiting or reducing production of pigments that are resistant to the bacterial host&#39;s immune defenses. 
     In another embodiment, said subject or bacterial host is a mammal. 
     In other embodiment, said subject or bacterial host is human. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent of application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee. 
       In the following detailed description, reference is made to the accompanying figures, depicting exemplary, non-limiting and non-exhaustive embodiments of the invention. So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, can be had by reference to the embodiments, some of which are illustrated in the appended figures. It should be noted, however, that the figures illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention can admit to other equally effective embodiments. 
         FIGS. 1A-1D  show the in vitro pigment inhibition by compound NP16:  FIG. 1A  shows the inhibition of wild-type (WT)  S. aureus  pigmentation using increasing concentrations of NP16;  FIG. 1B  shows the pigment inhibition by NP16; the IC 50  for pigment formation is ˜300 nM;  FIG. 1C  depicts the chemical structure of compound NP16;  FIG. 1D  shows the growth curve of  S. aureus  COL in the presence of different concentrations of NP16. All data represent mean values±SD. 
         FIGS. 2A-2D  show that NP16 treatment leads to increased sensitivity to oxidation and neutrophil killing:  FIG. 2A  depicts the cytotoxic activity of compound NP16 on MDCK cells; 
         FIG. 2B  shows the increased susceptibility of the NP16-treated  S. aureus  COL strain to killing by hydrogen peroxide;  FIG. 2C  shows the increased susceptibility of the NP16-treated  S. aureus  COL to killing by neutrophils;  FIG. 2D  is the UV spectrum of carotenoids extracted from different strains, with or without NP16 treatment. All data represent mean values±SD (***P&lt;0.001; ****P&lt;0.0001). P values were determined using GraphPad Prism using an unpaired parametric t test with Welch&#39;s correction. 
         FIGS. 3A-3F  show the in vivo effect of CrtN and its inhibition by NP16.  FIGS. 3A and 3B  show the bacteria recovered from the livers and spleens, respectively, of mice infected with the wild-type COL or COL-ΔcrtN strains;  FIGS. 3C and 3D  show the bacteria recovered from the livers and spleens, respectively, of mice infected with the COL strain, with or without compound NP16 treatment;  FIG. 3E  shows the bacteria recovered from the kidneys of mice infected with clinical isolate strain AE052 or AE052-ΔcrtN;  FIG. 3F  shows the bacteria recovered from the kidneys of mice infected with strain AE052, with or without compound NP16 treatment. All data represent mean values±SEM (*P&lt;0.05; **P&lt;0.01; ***P&lt;0.001). P values were determined using GraphPad Prism using an unpaired parametric t test with Welch&#39;s correction. 
         FIG. 4  shows in vivo efficacy of staphyloxanthin inhibitors from selected NP-16 analogues. 
         FIG. 5  shows the X-ray powder diffraction pattern of the compounds NP16-XL-016 (“IM032”) and NP16-XL-061 (“IM032-C”) in terms of the intensity (cps) against two-theta (degree). 
         FIG. 6A  shows the melting temperature and enthalpy of IM032. 
         FIG. 6B  shows the melting temperature and enthalpy of IM032-Cl. 
         FIG. 7A  illustrates relative pigment production of 5 different  S. aureus  strains inhibited by IM032. Data is presented as mean±SD. 
         FIG. 7B  illustrates relative pigment production of another 5 different  S. aureus  strains inhibited by IM032. Data is presented as mean±SD. 
         FIG. 8  shows the change in pigment production in different strains of  S. aureus  by different concentrations of IM032 (nM, log 10  scale). 
         FIG. 9  shows the effects of IM032 and vancomycin in the bacteria counts in various organs of a mouse bacteraemia model by IV inoculation of MRSA USA 300 in immune competent BALB/c mice. 
         FIG. 10  shows the effects of IM032 and vancomycin in the bacteria counts in various organs of a mouse bacteraemia model by IV inoculation of MRSA USA 300 ATCC BAA-1717 intravenous infection model within immune competent BALB/c mice. 
         FIG. 11A  shows the healing effect of IM032 (30 mg/kg, via oral, twice a day, 12 hours each interval) on skin infection caused by methicillin-resistant  S. aureus  (MRSA) as compared to mupirocin (2%, via topical, twice a day, 12 hours each interval) and linezolid (100 mg/kg, via oral, twice a day, 12 hours each interval) in terms of the percentage of wound closure. 
         FIG. 11B  are photos from the top view of mice with skin infection caused by MRSA before (at Day 0) and after different treatments (at Day 7): top left received 2% mupirocin via topical BID for 7 days; top right received 100 mg/kg linezolid via oral BID for 7 days; bottom received 30 mg/kg IM032 via oral BID for 7 days. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Following an established screening method for finding agents that reduce  Staphyloccous aureus  pigmentation [7], it is identified that the present compounds, termed NP16 and its derivatives, have block pigment production in  S. aureus  by targeting the 4,4-diapophytoene desaturase (CrtN). CrtN is proposed as a novel target for anti-virulence treatments in  S. aureus. S. aureus  staphyloxanthin contributes substantially to pathogenesis by interfering with host immune clearance mechanisms, but has little impact on ex vivo survival of the bacteria. Without wanting to be bound by theory, it is provided that agents blocking staphyloxanthin production may discourage the establishment and maintenance of bacterial infection without exerting selective pressure for antimicrobial resistance. 
     NP16 and its derivatives can be represented by Formula (II): 
                         
wherein R1 is selected from:
 
                         
or
 
any four-, five-, six-, seven-, eight-, nine-, ten-, eleven-, or twelve-membered heterocyclyl, cycloalkenyl, or cycloalkyl,
 
where R3 and R4 can independently or jointly be selected from the group: H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR11 and —OC(═O)R11);
 
or R3 and R4 can be bonded together to form a four-, five-, or six-membered heterocyclyl, cycloalkenyl, or cycloalkyl;
 
R5 can be selected from the group: H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR11 and —OC(═O)R11); and
 
where R10 and R11 can be independently or jointly selected from the group consisting of: a (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers; and
 
alkynyl;
 
X is selected from N or C,
 
A is single bond or double bond;
 
Q is selected from N or C,
 
M is selected from O or C, and
 
wherein R2 is selected from:
 
                         
where R6 and R7 can be independently or jointly selected from O or absent;
 
R8 and R9 can be independently or jointly selected from H; F; Cl; Br; I; OH; CN; (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers thereof; alkynyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl; thiol; thioalkyl; alkoxy; alkylthio; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy; phosphonic acid; phosphate ester; sulfonic acid (—SO 3 H); sulfonate ester; sulfonamide; alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino; alkylthio; heteroalkyl; alkyltriphenylphosphonium; heterocyclyl; ketone (═O); ether (—OR10); and ester (—COOR11 and —OC(═O)R11), or R8 and R9 can be bonded together to form a four-, five-, or six-membered heterocyclyl, cycloalkenyl, or cycloalkyl, and
 
where R10 and R11 can be independently or jointly selected from the group consisting of: a (C 1-4 )alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; (C 2-4 )alkenyl, such as ethenyl, propenyl, butenyl, where the double bond can be located at any position in the alkenyl carbon chain, and including any alkenyl conformational isomers; and
 
alkynyl, and
 
Z is selected from C or S.
 
     Preferably, R3 and R4 are independently or jointly selected from Br. 
     The compounds of Formula (II) can include, but are not limited to, those compounds listed in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Compounds Blocking Staphyloxanthin Production 
               
            
           
           
               
               
               
            
               
                 Sample  
                   
                   
               
               
                 name 
                 Chemical Name 
                 Structure 
               
               
                   
               
               
                 NP16 
                 3-phenyl-N-[4-(1- pyrrolidinylsulfonyl)- phenyl]acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-010 
                 3-phenyl-N-[4-(1- piperidine-1-sulfonyl)- phenyl]acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-011 
                 3-(4-acetoxyl- phenyl)-N-[4-(1- piperidine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-012 
                 3-(5-acetoxyl- phenyl)-N-[4-(1- piperidine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-013 
                 3-(6-acetoxyl- phenyl)-N-[4-(1- piperidine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-014 
                 3-(4-bromophenyl)- N-[4-(1-piperidine- 1-sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-015 
                 3-(5-bromophenyl)- N-[4-(1-piperidine- 1-sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-016 
                 3-(6-bromophenyl)- N-[4-(1-piperidine- 1-sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-017 
                 3-(4-methylphenyl)- N-[4-(1-piperidine- 1-sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-018 
                 3-(6-methylphenyl)- N-[4-(1-piperidine- 1-sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-019 
                 3-phenyl-N-[4-(1- indole-1-sulfonyl)- phenyl]acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-020 
                 3-(4-bromophenyl)- N-(4-(1-indole-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-021 
                 3-(5-bromophenyl)- N-(4-(1-indole-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-022 
                 3-phenyl-N-[4- (1-pyrrolidinyl- sulfonyl)phenyl]- propanamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-023 
                 3-(5-acetoxyl- phenyl)-N-[4-(1- indole-1-sulfonyl)- phenyl]acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-024 
                 3-(6-acetoxyl- phenyl)-N-[4-(1- indole-1-sulfonyl)- phenyl]acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-025 
                 3-(4-acetoxyl- phenyl)-N-[4-(1- indole-1-sulfonyl)- phenyl]acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-026 
                 3-(6-bromophenyl)- N-[4-(1-indole-1- sulfonyl)phenyl]- acrylainide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-027 
                 3-(4-methylphenyl)- N-[4-(1-indole-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-028 
                 3-(6-methylphenyl)- N-[4-(1-indole-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-029 
                 3-(4-bromophenyl)- N-[4-(1,2,3,4--tetra- hydroquinoline-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-030 
                 3-phenyl-N-[4- (1,2,3,4--tetra- hydroquinoline-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-031 
                 3-(4-bromophenyl)- N-[4-(3,4-dihydro- 1H-isoquinoline-2- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-032 
                 3-phenyl-N-[4- (3,4-dihydro-1H- isoquinoline-2- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-035 
                 3-(4-phenylphenyl)- N-[4-(1-indole-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-036 
                 3-phenyl-N-{[4- (N-phenyl-3- phenylprop-2- enamido)sulfonyl]- phenyl}-acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-037 
                 3-phenyl-N-[(4- phenylsulfamoyl)- phenyl]acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-038 
                 3-(6-bromophenyl)- N-[(4-phenylsul- famoyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-039 
                 3-(6-bromophenyl)- N-{[4-(N-phenyl- 3-6-bromophenyl- prop-2-enamido)- sulfonyl]phenyl}- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-040 
                 3-(2,6-difluoro- phenyl)-N-[(4- phenylsulfamoyl)- phenyl]acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-041 
                 3-(6-fluorophenyl)- N-[(4-phenyl- sulfamoyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-042 
                 3-(pyridin-3-yl)- N-[(4-phenyl- sulfamoyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-043 
                 3-(6-cyanophenyl)- N-[(4-phenyl- sulfamoyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-044 
                 3-(pyridin-2-yl)- N-[(4-phenyl- sulfamoyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-045 
                 3-(2,6-difluoro- phenyl)-N-[4-(1- piperidine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-046 
                 3-(pyridin-3-yl)-N- [4-(1-piperidine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-047 
                 3-(6-cyanophenyl)- N-[4-(1-piperidine- 1-sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-048 
                 3-(6-bromphenyl)- N-[(4-phenylamine- carbonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-049 
                 3-(6-cyanophenyl)- N-[(4-phenylamine- carbonyl)phenyl]- acryiamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-050 
                 3-(2,6-difluorophenyl)- N-[(4-phenylamine- carbonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-051 
                 3-(6-fluorophenyl)- N-[(4-phenylamine- carbonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-052 
                 3-(6-bromophenyl)- N-[4-(4-methyl- 1,4-piperazine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-053 
                 3-(2,6-difluoro- phenyl)-N-[4-(4- methyl-1,4- piperazine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-054 
                 3-(6-fluorophenyl)- N-[4-(4-methyl- 1,4-piperazine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-055 
                 3-(6-cyanophenyl)- N-[4-(4-methyl- 1,4-piperazine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-056 
                 3-(pyridin-3-yl)- N-[4-(4-methyl- 1,4-piperazine-1- sulfonyl)phenyl]- acrylainide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-057 
                 4-(5-phenyl-1,3- oxazole)-N-[4- (1-piperidine-1- sulfonyl)phenyl]- amide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-058 
                 3-(2,6-dibromo- phenyl)-N-[4-(1- piperidine-1- sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-059 
                 4-[2-(4-cyano- phenyl)-1,5- oxazole]-N-[4- (1-piperidine-1- sulfonyl)phenyl]- amide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-060 
                 4-[2-(thiophen- 5-yl)-1,5-oxazole]- N-[4-(1-piperidine- 1-sulfonyl)phenyl]- amide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 NP16- XL-061 
                 3-(6-chlorophenyl)- N-[4-(1-piperidine- 1-sulfonyl)phenyl]- acrylamide 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     One or more compounds of Formula (II) can be combined and/or mixed with one or more of a pharmaceutically acceptable carrier, salt, ester, excipient, vehicle, prodrug, solvent, and diluent to make a composition. 
     As used herein, the phrase “pharmaceutically acceptable” can mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and/or in humans. 
     As used herein, the term “carrier” can refer to a diluent, adjuvant, excipient, and/or vehicle with which the compound and/or antibiotic are administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. 
     As used herein, the phrase “pharmaceutically acceptable salt” can refer to derivatives of the compounds defined herein, wherein the parent compound is modified by making acid or base salts thereof. 
     The method of treating and/or preventing a microbial infection in a subject can include, but is not limited to, administering to a subject an effective amount of one or more compounds of Formula (I). 
     As used herein, the terms “treatment” or “treating” can refer to arresting or inhibiting, or attempting to arrest or inhibit, the development or progression of an infection and/or causing, or attempting to cause, the reduction, suppression, regression, or remission of an infection and/or a symptom thereof. As would be understood by those skilled in the art, various clinical and scientific methodologies and assays may be used to assess the development or progression of an infection, and similarly, various clinical and scientific methodologies and assays may be used to assess the reduction, regression, or remission of an infection or its symptoms. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the infection as well as those prone to have the infection or those in whom the infection is to be prevented. In at least some forms, the infection being treated can include, but is not limited to,  Staphylococcus aureus  infection. In other forms, the infection being treated is a microbial infection. 
     The administration can include, but is not limited to: administration though oral or oral cavity pathways, which administration includes administration in capsule, tablet, liquid, film, granule, spray, syrup, or other such forms; administration through non-oral pathways, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like; administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or the like; as well as administration topically; and administration via controlled released formulations, depot formulations, and infusion pump delivery. 
     For intravenous administration, the compounds can be packaged in solutions of sterile isotonic aqueous buffer, emulsions, or nanosuspensions to make the composition. When necessary, the composition can also include a solubilizing agent. The composition of the compounds can be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrated solution in a hermetically sealed container such as an ampoule or sachette indicating the amount of active agent. If the compound is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the compound is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to injection. 
     As used herein, the term “subject” can refer to an animal. Typically, the terms “subject” and “patient” may be used interchangeably herein in reference to a subject. As such, a “subject” can include a human that is being treated for a microbial infection as a patient. 
     As used herein, the term “animal” can refer to a mouse, rat, dog, cat, rabbit, pig, monkey, chimpanzee, and human. 
     As used herein, the terms “effective amount” and “therapeutically effective amount,” can be used interchangeably, as applied to the compounds, antibiotics, and pharmaceutical compositions described can mean the quantity necessary to render the desired therapeutic result. For example, an effective amount is a level effective to treat, cure, or alleviate the symptoms of an infection for which the composition and/or antibiotic, or pharmaceutical composition, is/are being administered. Amounts effective for the particular therapeutic goal sought will depend upon a variety of factors including the infection being treated and its severity and/or stage of development/progression; the bioavailability and activity of the specific compound and/or antibiotic, or pharmaceutical composition, used; the route or method of administration and introduction site on the subject; the rate of clearance of the specific composition and other pharmacokinetic properties; the duration of treatment; inoculation regimen; drugs used in combination or coincident with the specific composition; the age, body weight, sex, diet, physiology and general health of the subject being treated; and like factors well known to one of skill in the relevant scientific art. Some variation in dosage will necessarily occur depending upon the condition of the subject being treated, and the physician or other individual administering treatment will, in any event, determine the appropriate dosage for an individual patient. Furthermore, the therapeutic methods described would not only apply to treatment in a subject, but could be applied to cell cultures, organs, tissues, or individual cells in vivo, ex vivo or in vitro. 
     The term “hydrocarbyl” as used herein includes reference to a moiety consisting exclusively of hydrogen and carbon atoms; such a moiety may comprise an aliphatic and/or an aromatic moiety. The moiety may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of hydrocarbyl groups include C 1-6  alkyl (e.g. C 1 , C 2 , C 3  or C 4  alkyl, for example methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl); C 1-6  alkyl substituted by aryl (e.g. benzyl) or by cycloalkyl (e.g. cyclopropylmethyl); cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl); aryl (e.g. phenyl, naphthyl or fluorenyl) and the like. 
     The term “alkyl” as used herein includes reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of alkyl groups include “C 1-6  alkyl” and “C 2-10  alkyl”. The term “C 1-6  alkyl” as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms. The term “C 2-10  alkyl” as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. This term includes reference to groups such as methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl or tert-butyl), pentyl, hexyl and the like. In particular, the alkyl moiety may have 1, 2, 3, 4, 5 or 6 carbon atoms. 
     The terms “alkenyl” and “C 2-6  alkenyl” as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one double bond, of either E or Z stereochemistry where applicable. This term includes reference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl and the like. 
     The terms “alkynyl” and “C 2-6  alkynyl” as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one triple bond. This term includes reference to groups such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl and 3-hexynyl and the like. 
     The terms “alkoxy” and “C 1-6  alkoxy” as used herein include reference to —O-alkyl, wherein alkyl is straight or branched chain and comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1, 2, 3 or 4 carbon atoms. This term includes reference to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like. 
     The term “cycloalkyl” as used herein includes reference to an alicyclic moiety having 3, 4, 5, 6, 7 or 8 carbon atoms. The group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl and the like. 
     The term “aryl” as used herein includes reference to an aromatic ring system comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms. Aryl is often phenyl but may be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like. 
     “Cyclic group” means a ring or ring system, which may be unsaturated or partially unsaturated but is usually saturated, typically containing 5 to 13 ring-forming atoms, for example a 5- or 6-membered ring. The ring or ring system may be substituted with one or more hydrocarbyl groups. Cyclic group includes carbocyclyl and heterocyclyl moeities. 
     The term “carbocyclyl” as used herein includes reference to a saturated (e.g. cycloalkyl) or unsaturated (e.g. aryl) ring moiety having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon ring atoms. In particular, carbocyclyl includes a 3- to 10-membered ring or ring system and, in particular, 5- or 6-membered rings, which may be saturated or unsaturated. The ring or ring system may be substituted with one or more hydrocarbyl groups. A carbocyclic moiety is, for example, selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like. 
     The term “heterocyclyl” as used herein includes reference to a saturated (e.g. heterocycloalkyl) or unsaturated (e.g. heteroaryl) heterocyclic ring moiety having from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon and sulphur. In particular, heterocyclyl includes a 3- to 10-membered ring or ring system and more particularly a 5- or 6-membered ring, which may be saturated or unsaturated. The ring or ring system may be substituted with one or more hydrocarbyl groups. 
     A heterocyclic moiety is, for example, selected from oxiranyl, azirinyl, 1, 2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, especially thiomorpholino, indolizinyl, isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, cumaryl, indazolyl, triazolyl, tetrazolyl, purinyl, 4/V-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazoiyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl, chromanyl and the like. 
     The term “heterocycloalkyl” as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus and sulphur. The group may be a polycyclic ring system but more often is monocyclic. This term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolizidinyl and the like. The ring or ring system may be substituted with one or more hydrocarbyl groups. 
     The term “heteroaryl” as used herein includes reference to an aromatic heterocyclic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen and sulphur. The group may be a polycyclic ring system, having two or more rings, at least one of which is aromatic, but is more often monocyclic. The ring or ring system may be substituted with one or more hydrocarbyl groups. This term includes reference to groups such as pyrimidinyl, furanyl, benzo[b]thiophenyl, thiophenyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridinyl, benzo[b]furanyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl and the like. 
     The term “halogen” as used herein includes reference to F, Cl, Br or I. 
     The expression “halogen containing moiety” as used herein includes reference to a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulphur which moiety includes at least one halogen. The moiety may be hydrocarbyl for example C 1-6  alkyl or C 1-6  alkoxy, or carbocyclyl for example aryl. 
     The term “substituted” as used herein in reference to a moiety means that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described substituents. The term “optionally substituted” as used herein means substituted or un-substituted. It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible. 
     The term “enantiomer” as used herein means one of two stereoisomers that have mirror images of one another. 
     The term “racemate” as used herein means a mixture of equal amounts of enantiomers of a chiral molecule. 
     The term “diastereomer” as used herein means one of a class of stereoisomers that are not enantiomers, but that have different configurations at one or more of the equivalent chiral centers. Example of diasteromers are epimers that differ in configuration of only one chiral center. 
     The term “stereoisomer” as used herein means one of a class of isomeric molecules that have the same molecular formula and sequence of bonded atoms, but different three-dimensional orientations of their atoms in space. 
     The term “prodrug” as used herein refers to a medication that is administered as an inactive (or less than fully active) chemical derivative that is subsequently converted to an active pharmacological agent in the body, often through normal metabolic processes. 
     The term “independently” used herein refers to two or more moieties each selected from a list of atoms or groups, which means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties. 
     The term “jointly” used herein refers to two or more moieties are identical selected from a list of atoms or groups. In other words, the identity of each moiety is therefore dependent of the identities of the one or more other moieties being referred to be “jointly” selected from the list of atoms or groups. 
     Examples 
     The examples and embodiments described herein are for illustrative purposes only and various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto. 
     Bacteria, Mice, and Chemical Reagents 
     The strains of  S. aureus  and  E. coli  are listed in Table 2. BALB/c mice were purchased from Charles River Laboratories.  S. aureus  was propagated in Terrific broth (TB) or on TB agar (Life Technologies; or in Brain Heart Infusion broth (BHI) or on BHI agar (Oxoid). Unless otherwise indicated, all experiments were performed with bacteria derived from light-protected  S. aureus  36-48 h stationary phase cultures, the point at which pigmentation phenotypes were readily apparent. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Strains 
               
            
           
           
               
               
               
            
               
                 Strains 
                 Description 
                 Source 
               
               
                   
               
               
                 
                   E. coli 
                 
                   
                   
               
               
                 Rosetta (DE3) 
                 Host strain for gene expression 
                 Lab source 
               
               
                 
                   S. aureus 
                 
                   
                   
               
               
                 RN4220 
                 Intermediate cloning host 
                 Lab source 
               
               
                 COL 
                 Laboratory strain 
                 Lab source 
               
               
                 AE052 
                 Clinical isolate 
                 [8] 
               
               
                 COL-ΔcrtN 
                 COL with crtN gene replaced ermC cassette 
                 This study 
               
               
                 AE052-ΔcrtN 
                 AE052 with crtN gene replaced with ermC 
                 This study 
               
               
                   
                 cassette 
                   
               
               
                 USA300 
                 CA-MRSA, USA300 FPR3757, ATCC BAA- 
                 ATCC 
               
               
                   
                 1556 
               
               
                   
               
            
           
         
       
     
     Minimum Inhibitory Concentration (MIC) Tests 
     MIC was determined by inoculating 5×10 4    S. aureus  cells in 100 μl BHI medium in 96-well plates with a serial dilution of antibiotics. The MIC was defined as the minimum concentration resulting in a cell density less than 0.05 OD at 620 nm, which corresponded to no visible growth, after incubating for 18 h at 37° C. 
     Evaluation of NP-16 analogues in Staphyloxanthin production 
     The in vitro pigment inhibition studies were performed by  S. aureus  USA300 cultured in BHI with or without the presence of inhibitor compounds at 37° C. and 250 rpm for 36-48 hours. The bacteria were washed twice with PBS prior to the staphyloxanthin purification with methanol. The OD of the extracts were monitor at 450 nm using DTX880 multi-plate reader spectrophotometer (Beckman). The concentration range tested for the compounds were between 300 nM to 700 nM, and control groups were added with equal volume of DMSO. 
     Cytotoxicity Evaluation of Other NP-16 Analogues in Raw 264.7 Cells 
     The cytotoxicity of NP-16 and some of it analogues in Raw 264.7 cells was also evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay according to manufacturer&#39;s instructions. A toxic control (1%) SDS was included to ensure the MTT assay was working properly. The highest concentration of NP-16 analogues used was 500 μM due to solubility limitations. SigmaPlot 11.0 (SPSS, IL) was used for graph plotting. Experiments were carried out in triplicate and repeated twice. 
     The cytotoxicity of NP-16 and some of its analogues was tested against Raw 264.7 cells, and the cell tolerance of each compounds were documented in Table 3. Along with the in vitro staphyloxanthin production of the NP-16 analogues were being tested, the compounds can exert staphyloxanthin production inhibition. The staphyloxanthin from the overnight-cultured bacteria were extracted with methanol and quantified in via spectrophotometry. The results were presented in relative ratio to NP-16 in both the inhibition ratio as well as the TC50 (concentration for decreasing cell viability by 50%) in Table 3 (sample names correspond to those listed in Table 1). 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                 TC50 to Raw  
                 Relative ratio to NP16  
               
               
                 Sample Name 
                 264.7 cells 
                 based on inhibition ratio 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 NP16 
                 &gt;200 
                 1 
               
               
                 NP16-XL-010 
                 &gt;200 
                 1.43 
               
               
                 NP16-XL-011 
                 75 
                 1.31 
               
               
                 NP16-XL-012 
                 50 
                 1.16 
               
               
                 NP16-XL-013 
                 37.5 
                 0.28 
               
               
                 NP16-XL-014 
                 &gt;200 
                 0.74 
               
               
                 NP16-XL-015 
                 &gt;200 
                 1.18 
               
               
                 NP16-XL-016 
                 &gt;200 
                 2.71 
               
               
                 NP16-XL-017 
                 &gt;200 
                 0.09 
               
               
                 NP16-XL-018 
                 &gt;200 
                 2.48 
               
               
                 NP16-XL-019 
                 &gt;200 
                 1.7 
               
               
                 NP16-XL-020 
                 &gt;200 
                 0.17 
               
               
                 NP16-XL-021 
                 &gt;200 
                 0.76 
               
               
                 NP16-XL-022 
                 &gt;200 
                 −0.08 
               
               
                 NP16-XL-023 
                 18.7 
                 1.34 
               
               
                 NP16-XL-024 
                 170 
                 0.43 
               
               
                 NP16-XL-025 
                 170 
                 1.92 
               
               
                 NP16-XL-026 
                 &gt;200 
                 2.67 
               
               
                 NP16-XL-027 
                 &gt;200 
                 0.79 
               
               
                 NP16-XL-028 
                 &gt;200 
                 2.31 
               
               
                 NP16-XL-029 
                 &gt;200 
                 0.53 
               
               
                 NP16-XL-030 
                 &gt;200 
                 1.26 
               
               
                 NP16-XL-031 
                 37.5 
                 0.23 
               
               
                 NP16-XL-032 
                 190 
                 1.17 
               
               
                 NP16-XL-035 
                 50 
                 −0.38 
               
               
                 NP16-XL-036 
                 &gt;200 
                 3.04 
               
               
                 NP16-XL-037 
                 &gt;200 
                 3.02 
               
               
                 NP16-XL-038 
                 &gt;200 
                 3.23 
               
               
                 NP16-XL-039 
                 &gt;200 
                 3.23 
               
               
                 NP16-XL-040 
                 &gt;200 
                 2.51 
               
               
                 NP16-XL-041 
                 &gt;200 
                 3.17 
               
               
                 NP16-XL-042 
                 &gt;200 
                 1.44 
               
               
                 NP16-XL-043 
                 &gt;200 
                 3.23 
               
               
                 NP16-XL-044 
                 200 
                 2.47 
               
               
                 NP16-XL-045 
                 200 
                 0.88 
               
               
                 NP16-XL-046 
                 &gt;200 
                 −0.05 
               
               
                 NP16-XL-047 
                 &gt;200 
                 3.18 
               
               
                 NP16-XL-048 
                 &gt;200 
                 0.23 
               
               
                 NP16-XL-049 
                 50 
                 0.21 
               
               
                 NP16-XL-050 
                 100 
                 −0.03 
               
               
                 NP16-XL-051 
                 150 
                 −0.16 
               
               
                 NP16-XL-052 
                 &gt;200 
                 3.18 
               
               
                 NP16-XL-053 
                 &gt;200 
                 1.26 
               
               
                 NP16-XL-054 
                 &gt;200 
                 2.86 
               
               
                 NP16-XL-055 
                 &gt;200 
                 3.19 
               
               
                 NP16-XL-056 
                 &gt;200 
                 0 
               
               
                 NP16-XL-057 
                   
                 0.14 
               
               
                 NP16-XL-058 
                   
                 0.16 
               
               
                 NP16-XL-059 
                   
                 0.06 
               
               
                 NP16-XL-060 
                   
                 0.06 
               
               
                   
               
            
           
         
       
     
     crtN Expression, Purification and Enzymatic Assay 
     CrtN with a histidine-maltose binding protein (MBP) tag was overexpressed in  E. coli  Rosetta (DE3) cells. A 10 ml overnight culture was transferred into 1 L of LB medium supplemented with 100 μg/ml ampicillin. Induction was carried out with 1 mM IPTG for 12 hours at 16° C. at an OD of 0.6 at 600 nm. The cell lysate was loaded onto a Ni-NTA column, and CrtN was eluted using a 75-ml linear gradient of 0-0.4 M imidazole in 50 mM sodium phosphate buffer, with 400 mM sodium chloride, pH 6.6. The collected fractions were analysed by SDS-PAGE to confirm the peak for MBP-CrtN. The target peak fractions were concentrated and the buffer was exchanged to loading buffer without imidazole using a PD-10 column (GE Healthcare). The collected solution was treated with TEV protease at 4° C. overnight. The protein sample was applied to a maltose column, and the flow-through was collected as native CrtN protein. For enzyme assay, 10 μg of purified CrtN was incubated with 100 μl of 4,4′-diapophytoene liposomes (containing 5 nmol of 4,4′-diapophytoene), 150 μM FAD and buffer II (20 mM phosphate buffer pH 8.0, 100 mM NaCl) in a total volume of 660 μl at 37° C. for 2 h (standard assay). The reaction was stopped by adding 1 volume of CHCl 3 : MeOH (2:1, v/v). Followed by mixing, the sample was centrifuged at 16,000 g for 10 min. The organic phase was dried for LC/MS analysis. 
     Isolation of Carotenoids 
     The substrate (4,4′-diapophytoene) and product (4,4′-diaponeurosporene) were extracted from strains COL-ΔcrtN and COL-ΔcrtOP. Carotenoids were extracted from cell pellets using 300 ml of methanol per liter of cultured bacteria pellet until all visible pigments were removed. After centrifugation (4° C. and 8,000 g), colored supernatants were pooled and concentrated to 50 ml using an EZ-2 Plus centrifugal evaporator (Genevac Inc., Gardiner, N.Y., USA). A sample was mixed with 100 ml of EtOAc and 200 ml of NaCl (2.5 M). The extract sample in the upper organic phase was collected, washed with same volume of distilled water, and dried using the EZ-2 Plus evaporator. Dried samples were ready for silica gel isolation or stored at −70° C. prior to analysis. For structural elucidation, carotenoids were identified using a combination of HPLC retention times, UV-visible absorption spectra, and mass fragmentation spectra. Mass fragmentation spectra were monitored using both negative and positive ion modes in a mass range of m/z 200-1000 on the Varian 1200L LC/MS system equipped with an atmospheric pressure chemical ionization interface. 
     Hydrogen Peroxide Susceptibility Assay 
       S. aureus  was grown in BHI with or without NP16 (40 μM). After 2 days, bacteria were washed twice in PBS, diluted to a concentration of 1×10 7  CFUs per 100 μl reaction mixture in a 96-well plate. Hydrogen peroxide (H 2 O 2 ) in PBS was added to a 440 mM final concentration, and the plate was incubated for 1 hr at 37° C. with shaking. The reaction was stopped by the addition of 1,000 U/ml of exogenous catalase (Sigma-Aldrich, St. Louis, Mo.), and bacterial viability was assessed by plating dilutions on BHI agar plates. 
     Bactericidal Activity of Polymorphonuclear Leukocytes 
     The killing of  S. aureus  by human polymorphonuclear leukocytes (PMNs) was determined as previously described [9], with some modifications. Briefly, PMNs (10 6 ) were mixed with ˜10 7  opsonized  S. aureus  bacteria MOI=10 in 24-well tissue culture plates. After centrifuged at 380 g for 8 min, plates were incubated at 37° C. for up to 1.5 h. PMNs were lysed with saponin (20 min on ice) and plated on BHIA plates. The percent survival was calculated by normalized with time zero. Statistics were performed with the Student&#39;s t-test (GraphPad Prism). 
     Murine Model of Intraperitoneal Infection 
     Six- to eight-week-old female Balb/c mice were injected intravenous (i.v) with 1×10 7  CFUs of early stationary phase  S. aureus  USA300 or isogenic  S. aureus  mutant USA300-ΔcrtN. For the treatment study, mice were randomized into groups at the start of the experiment and administered, i.p. either 17.25 mg/kg of the selected NP-16 analogues or vehicle (5% DMSO with 5% Tween-80) as a control, twice per day. The NP16, NP16-XL-026 and NP16-XL-043 were administered via intraperitoneal route at 17.25 mg/kg. The kidney bacterial recovery was compared against vehicle control 7 days after drug treatment. 
     With the use of intravenous infection of  S. aureus  USA300 complemented with isogenic knockouts of −ΔcrtN, mice euthanized on day 7 post infection with bacterial counts of kidney being quantified, NP16-XL-026 and NP16-XL-043 exhibit similar inhibitory effect as NP-16 ( FIG. 4 ) 
     For other  S. aureus  sub-type, eight- to ten-week-old female BALB/c mice were injected intraperitoneally (i.p) with 4×10 8  CFUs of early stationary phase  S. aureus  COL. After 3 d, animals were euthanized, the liver and spleen were isolated, homogenized in PBS, and plated on to obtain viable counts. For the treatment study, mice were randomized into two groups at the start of the experiment and administered, i.p., either 0.35 mg of NP16 or PBS with 5% Tween-80 as a control, twice per day, starting on d −1 to d 2 (a total of eight doses for each). Intraperitoneal challenge with 4×10 8  CFUs of early stationary phases  S. aureus  COL was performed on d 0. The mice were sacrificed on d 3 for enumeration of bacterial CFUs in liver and spleen homogenates. 
     For the clinical isolate  S. aureus  strain AE052, all operations were similar to those used for the COL strain, except 10 8  CFUs of early stationary phase bacteria were used in the infection model, and kidneys were collected for monitoring bacterial loads. Statistics were performed using the Student&#39;s t-test (GraphPad Prism). 
     Compound NP16 Reduces Pigment Production 
     Compound NP16 (structure shown in  FIG. 1C ) had potent activity against  S. aureus  pigment formation in vitro, as shown in  FIG. 1A , with IC 50  values ranging from 100 to 300 nM ( FIG. 1B ). In the biosynthesis of staphyloxanthin, the product of CrtN, 4,4′-diaponeurosporene, is a yellowish compound while products prior to CrtM catalysis are colorless. Thus, NP16 treatment is proposed to target CrtM or CrtN or other regulators that affect the expression of the crtOPQMN cluster, such as sigB or ispA [10]. The MIC of NP16 for USA300 was greater than 500 μM ( FIG. 1D ). 
     The functions of the five encoded enzymes were characterized by product analysis of gene deletion mutants. Firstly, in staphyloxanthin biosynthesis, two molecules of farnesyl diphosphate are condensed head-to-head to form dehydrosqualene (4,4′-diapophytoene), catalyzed by the dehydrosqualene synthase CrtM. Secondly, dehydrosqualene is dehydrogenated by the dehydrosqualene desaturase CrtN to form the yellow intermediate 4,4′-diaponeurosporene. Thirdly, oxidation of the terminal methyl group of 4,4′-diaponeurosporene is catalyzed by a mixed function oxidase CrtP, to form 4,4′-diaponeurosporenic acid. Then, glycosyl 4,4′-diaponeurosporenoate is formed by esterification of glucose at the C 1 ″ position of 4,4′-diaponeurosporenic acid with CrtQ, a glycosyltransferase involved. Finally, glucose at the C 6 ″ position is esterified with the carboxyl group of 12-methyltetradecanoic acid by the acyltransferase CrtO to yield staphyloxanthin. Staphyloxanthin was identified as β-D-glucopyranosyl 1-O-(4,4′-diaponeurosporen-4-oate)-6-O-(12-methyltetradecanoate). 
     Inhibition of CrtN by NP16 Results in H 2 O 2  and Neutrophil Killing 
     To probe the biological activities of CrtN, an isogenic crtN mutant in the COL strain via allelic replacement was generated. The mutation resulted in loss of yellow pigment. Compound NP16 had no effect on the growth of MDCK cells ( FIG. 2A ). A decrease in pigment production was found in  S. aureus  grown in the presence of this NP16 ( FIG. 1A ). Blocking  S. aureus  pigment formation has led to an increase in the susceptibility of the pathogen to hydrogen peroxide killing. For the non-pigmented strain RN6390, the susceptibility was similar irrespective of NP16 treatment ( FIG. 2B ). Additionally, as a carotenoid producing strain ( FIG. 2D ), COL survived significantly better than RN6390 and NP16-treated COL in human neutrophils ( FIG. 2C ). 
     Animal Studies 
     Using a systemic  S. aureus  infection model, the enzyme CrtM from  S. aureus  was identified to be a target for anti-infective therapy, based on virulence factor neutralization [3]. A similar model was applied to determine if crtN is also essential for infections in mice. The loss of staphyloxanthin reduced invasive disease potential, as mice inoculated with the isogenic  S. aureus  mutant COL-ΔcrtN showed lower bacterial population from the liver and spleen, compared with the 4×10 8  CFUs of wild-type  S. aureus  (by intraperitoneal injection), which led to a sustained infection ( FIGS. 3 a  and 3 b   ). Because the COL strain is a low virulence strain, no bacteria were detected in the kidneys from day 1 to day 3. 
     Another highly virulent clinical isolate, AE052, and its isogenic  S. aureus  mutant lacking the CrtN enzyme were also examined by these tests. Compared to wildtype strain, mutant strain in kidney was cleared by host after 72 hours post infection ( FIG. 3E ). 
     With the same intraperitoneal challenge used in  FIGS. 3 a , 3 b  and 3 e   , one group of mice (n=14) was treated with 0.35 mg of NP16 twice per day (days −1, 0, 1 and 2), and a second group (n=12) with a vehicle control. Upon sacrificing the mice at 72 hours,  S. aureus  COL bacterial counts in the livers and spleens of mice treated with compound NP16 were significantly lower than those of the control group (P&lt;0.01) ( FIGS. 3 c  and 3 d   ). In the case of AE052 infections, bacterial counts in the kidneys of the mice (n=10 for both groups) treated with NP16 were significantly lower than those of the control group (P&lt;0.001), with 6 of 10 below the detection threshold, versus only 2 of 10 in the control group ( FIG. 3F ). This result indicates a 98% decrease in surviving bacteria in the treatment groups infected with COL or AE052. 
     Discussion 
     It is identified that NP-16 is an inhibitor for CrtN and can exhibit anti-virulence effect on  S. aureus . CrtM and CrtN are key enzymes in staphyloxanthin biosynthesis [11]. While staphyloxanthin plays a major role in  S. aureus  tolerance to host defence, it provides a basis for potential target for rational drug design for the use against  S. aureus . It is proposed that a novel anti-infective drug without direct bactericidal properties, only targeting mechanisms that renders the pathogens susceptible to normal host innate immune clearance, is provided. As there is 30% sequence identity between the human SQS and the bacterial CrtM, and they share significant structural features. The presence of such homologue discouraged the employment of CrtM as druggable target this is further supported by a study focusing on the improvement of the specificity of BPH652 against CrtM was published recently [12]. Compared with CrtM, CrtN has no homologous enzyme in the human cholesterol biosynthesis pathway, making it an attractive drug target. A recently proposed CrtN inhibitor, nafitifine, is a topically administered antifungal compound [13], which has been shown to suppress chemotaxis, chemokinesis, chemiluminescence, and superoxide anion production of polymorphonuclear leukocytes at high concentrations [14]. The effects of naftifine are not stable in different organ (from no effect to reduced bacterial load for nearly 4 log) and inconsistency with CrtN mutant (always reduced bacterial load from 0.2 to 2 log at most). It is believed that this indicates that CrtN should not be the primary target of naftifine [6]. 
     ROS are employed by phagocytic cells to eliminate bacteria. They are generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [15]. The bacterial carotenoids expressed by  S. aureus  may have a protective function against these defensive molecules [4, 16]. Evidence supported that a pigment-deficient  S. aureus  strain was more sensitive to oxidants, hydrogen peroxide and singlet oxygen, in vitro, as compared to a wild-type  S. aureus  strain [1]. Using intra-bacterial inhibition assay system, showed that the isogenic crtN mutant, which exhibited interrupted carotenoid synthesis, was more sensitive to purified human neutrophils. This confirmed the importance of CrtN in the intracellular survival of  S. aureus.    
     CrtN inhibitors without direct bactericidal properties should possess theoretical advantages of not exerting a direct selective pressure on the pathogen or normal flora to develop drug resistance. Our approach, as well as other virulence factor-based concepts [3, 17] for highly specific anti-staphylococcal therapy relies mainly on the host normal innate immune response for pathogen clearance. Such strategies are much more ideal for clinical treatment and prophylactic applications with limited risk of developing drug resistant pathogen unlike the case observed with antibiotics. 
     Turning to  FIG. 5 , the X-ray powder diffraction (XRPD) pattern of compound NP16-XL-016 (IM032) and compound NP16-XL-061 (IM032-Cl) are put together, and the result shows that the two patterns are not superimposed, indicating their difference in crystal structure. The difference in crystal structure between these two compounds may explain why NP16-XL-061 has a lower melting point (Tm) and higher enthalpy ( FIG. 6B ) than those of NP16-XL-016 ( FIG. 6A ), even though they differ only in the substituted group at C 6  position on the 6-membered ring structure, and the two different substituents both belong to halogen. 
     The following table (Table 4) illustrates the solubility of two different compounds, NP16-XL-016 (hereinafter as “IM032”) and NP16-XL-061 (hereinafter as “IM032-C”), in two solutions with different pH values simulating the pH of gastric juice and human intestinal fluid using HCl and phosphate buffer respectively. 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                 Solubility in pH 2 (0.1N HCl + 
                 Solubility in pH 7.4 (KH2PO4 + 
               
               
                   
                 0.1% Tween 80)-simulating 
                 0.1% Tween 80)-simulating 
               
               
                   
                 human stomach 
                 human intestine 
               
               
                   
               
             
            
               
                 IM032 
                 2.03 μg/mL 
                   1.74 μg/mL 
               
               
                 IM032-Cl 
                 &lt;LOQ 
                 0.08564 μg/mL 
               
               
                   
               
            
           
         
       
     
     From Table 4, IM032 exhibits a higher solubility in both simulated gastric and intestinal fluids than IM032-Cl. It is understood that after taken orally, a compound or molecule, especially an active pharmaceutical ingredient, has to be dissolved in the gastrointestinal tract before absorption. Solubility plays pivotal role in the drug absorption process (Amidon et al., 1995). The solubility of IM032 in two biorelevant media (pH 2 simulates gastric fluid and pH 7.4 simulates intestinal fluid) is remarkably (&gt;20×) higher than that of IM032-Cl, implying a significantly better absorbability of IM032. The significantly higher solubility in simulated gastric and intestinal fluids may also imply a higher availability in our systemic circulation (a higher bioavailability), e.g., a higher plasma level, of a compound or molecule than that with a lower solubility in simulated gastric and intestinal fluids. In contrast, the solubility of IM032-Cl in both stomach and intestines is so poor that it will not be absorbed orally. As a result, as shown in previous therapeutic compounds with low solubility below, regardless of the efficacy of the compound in vitro, compounds with poor solubility seldomly have sufficient bioavailability to made into a drug. 
     Indeed, solubility plays a pivotal role in determining the success of pharmaceutical development, and there are occasions where drugs have to be withdrawn upon discovery of poor solubility. Taking Paclitaxel as an example, its nanoparticulate formulation, Abraxane, was provided to overcome the issues of Taxol, which uses a high organic content medium, which causes a number of major side-effects such as hypersensitivity, to solubilize paclitaxel that is extremely poorly soluble. Another example is ritonavir, which was completely withdrawn by Abbvie upon discovery that crystalline ritonavir exhibited polymorphism, where the more stable polymorph was less soluble. Due to the significantly lower bioavailability resulted from poor solubility, ritonavir had to be completely withdrawn for redevelopment. 
     As the solubility of a drug candidate can be partially determined by the crystallinity of the solute and its interaction with a solvent/solvents, it is possible to change the nature of the solvent or select a suitable solvent system that can dissolve the solute and interact therewith. 
     Table 5 further demonstrates the difference in solubility of IM032 and IM032-Cl in some commonly used solvents for oral administration: 
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                 IM032-Cl solubility (mg/mL) 
                 IM032 solubility (mg/mL) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Labrasol ALF 
                 6.08 
                 11.84 
               
               
                 PEG300 
                 7.15 
                 &gt;20 
               
               
                 PEG400 
                 6.94 
                 &gt;20 
               
               
                 1:1 Labrasol 
                 6.34 
                 16.07 
               
               
                 ALF/PEG300 
                   
                   
               
               
                 1:1 Labrasol 
                 7.60 
                 &gt;15 
               
               
                 ALF/PEG400 
               
               
                   
               
            
           
         
       
     
     From Table 5, it shows that IM032 is more soluble in most of the solvents used for oral formulation than IM032-Cl, revealing that IM032 is more “drug-like” and a better candidate for pharmaceutical development into oral dosage forms compared with IM032-Cl. Finding an acceptable oral formulation is not just for human consumption. Indeed, an oral formulation that can solubilize a compound well is equally important for animal studies such as efficacy, toxicology, which are prerequisite for human clinical trials. All these reveal the importance of a drug to dissolve in different orally acceptable solvents. As such, although the structural difference between IM032 and IM032-Cl is only in one substituted group at the same position on a 6-membered cyclic group, it greatly impacts on the solubility thereof in different physiologically relevant media and different solvents for formulating into a potential formulation to be orally administered to a subject in need thereof. 
     Effect of IM032 on Staphyloxanthin Production of Different Strains of  Staphylococcus aureus:    
     This study aimed to evaluate IM032 for its in vitro efficacy to inhibit the production of staphyloxanthin in different strains of  S. aureus.    
     Compound IM032 was evaluated for its in vitro efficacy to inhibit staphyloxanthin production in 10 different  Staphylococcus aureus  ( S. aureus ) strains, SH1000, HG003, ATCC29213, ATCC700698, COL, JE2, LAC, USA300-3, Newman and ST239III. IM032 showed inhibition of staphyloxanthin production in all 10 tested strains with IC50 ranging from 1.2 to 70 nM. 
     FPR3757, ATCC29213, Mu3 and Newman were purchased from ATCC; COL and SH1000 were gifts from Professor Ambrose L. Cheung, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, HG003 was a gift from Professor Suzanne Walker, Department of Chemistry and Chemical Biology, Harvard University; JE2 was a gift from Professor Chia Lee, Department of Microbiology and Immunology, Kansas State University; LAC was a gift from Professor Anthony R. Richardson, Department of Microbiology &amp; Molecular Genetics University of Pittsburgh. USA300-3 was a gift from Professor Daniel Lopez, National Centre for Biotechnology, Spanish National Research Council; ST239III was a clinical isolate from Dr. P L Ho, The University of Hong Kong. They were cryopreserved as single-use frozen working stock cultures which were stored at −80° C. until use. 
     The negative control was dimethyl sulfoxide (DMSO) which was used to prepare stock solutions and dilutions; positive control in this test was  S. aureus  strain FPR3757 treated with IM032. 
     The test item was dissolved at 44.9 mg/mL in DMSO at 37° C., aliquoted into several tubes, and stored at −20° C. until use. On the day of testing, a stock solution was serially diluted to testing concentrations with DMSO. All solutions were vortexed and mixed with a pipette to achieve homogeneity immediately before dilution. A correction factor for purity was not applied to the test item preparations. 
     Each test item solution (6 μL) was combined with 0.6 mL of  S. aureus  culture in brain heart infusion broth (CFU per well=1×10 7 ). 12-wells repeats were performed for each concentration in 96-well 2-mL plates. The plates were then incubated in a shaker at 37° C., 250 rpm for 24 hours. The bacteria were centrifuged at 4,000 rpm for 10 min. Next, the bacteria from 4-wells were combined and were washed twice with PBS. Staphyloxanthin was extracted with 300 μL of methanol in a water bath at 60° C. for 1 hour. After centrifugation, 100 μL of supernatant was transferred to a 96-well cell culture plate and OD 450  was measured. The relative pigment production at each tested concentration was calculated as follows: 
     
       
         
           
             
               Relative 
               ⁢ 
               
                   
               
               ⁢ 
               pigment 
               ⁢ 
               
                   
               
               ⁢ 
               production 
             
             = 
             
               
                 
                   OD 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   450 
                   ⁢ 
                   
                     ( 
                     
                       treatment 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       group 
                     
                     ) 
                   
                 
                 - 
                 
                   OD 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   450 
                   ⁢ 
                   
                     ( 
                     blank 
                     ) 
                   
                 
               
               
                 
                   OD 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   450 
                   ⁢ 
                   
                     ( 
                     
                       negative 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       control 
                     
                     ) 
                   
                 
                 - 
                 
                   OD 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   450 
                   ⁢ 
                   
                     ( 
                     blank 
                     ) 
                   
                 
               
             
           
         
       
     
     The IC 50  for each test was determined based on the calculated inhibition ratio from above with Prism 6.0 by employing a non-linear regression (four parameters) fitting method with assigned bottom and top at 0.05 and 0.95 respectively. The IC 50  presented is the best-fit value. 
       S. aureus  strains (ATCC29213, HG003, Mu3, SH1000, and COL) were treated with IM032 at final concentrations of 4,000, 1,000, 250, 63, 16, 4 and 1 nM ( FIG. 7A ).  S. aureus  strains (JE2, USA300-3, LAC, ST239III and Newman) were treated with IM032 at final concentrations of 250, 63, 16, 4, 1, 0.2 and 0.06 nM ( FIG. 7B ). A clear bactericidal effect as evidenced by a reduction in optical density was not observed at any concentration of IM032. The determined IC 50  values of the tested strains range from 1.2 to 70 nM (Tables 6 and 7). 
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                   
                   
                 Drug  
                 Staphyloxanthin 
                 IC 50    
               
               
                   
                 No. 
                 Strains 
                 resistance 
                 production 
                 (nM) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 1 
                 FPR3757 
                 MRSA (CA) 
                 Moderate 
                 20 
               
               
                   
                 2 
                 ATCC29213 
                 MSSA 
                 Moderate 
                 18 
               
               
                   
                 3 
                 Mu3 
                 hVISA (HA) 
                 Weak 
                 1.8 
               
               
                   
                 4 
                 SH1000 
                 MSSA 
                 High 
                 70 
               
               
                   
                 5 
                 HG003 
                 MSSA 
                 High 
                 46 
               
               
                   
                 6 
                 COL 
                 MRSA (HA) 
                 Weak 
                 1.2 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                   
                   
                 Drug  
                 Staphyloxanthin 
                 IC 50    
               
               
                   
                 No. 
                 Strains 
                 resistance 
                 production 
                 (nM) 
               
               
                   
                   
               
             
            
               
                   
                 1 
                 FPR3757 
                 MRSA (CA) 
                 Moderate 
                 28 
               
               
                   
                 2 
                 JE2 
                 MRSA 
                 Moderate 
                 26 
               
               
                   
                 3 
                 USA300-3 
                 MRSA (HA) 
                 Moderate 
                 27 
               
               
                   
                 4 
                 LAC 
                 MRSA 
                 Moderate 
                 27 
               
               
                   
                 5 
                 ST239III 
                 MRSA (HA) 
                 Weak 
                 19 
               
               
                   
                 6 
                 Newman 
                 MSSA 
                 Weak 
                 21 
               
               
                   
                   
               
            
           
         
       
     
     The negative control (DMSO) group showed orange colour and the mean OD 450  reading was the highest, indicating the presence of staphyloxanthin production. The positive control group with IM032 treated FPR3757 had an IC 50  of 20 nM in the first experiment and 28 nM in the second, in agreement with the previous study (IC 50 =17 nM) and therefore the results are considered valid. 
     Effect of IM032 on Staphyloxanthin Production of 10 Strains of  Staphylococcus aureus    
       FIG. 8  shows the change in pigment production in different strains of  S. aureus  by different concentrations of IM032 (nM, log 10  scale). 
     It is observed from the result in  FIG. 8  and the following table (Table 8) that IM032 is able to inhibit the production of staphyloxanthin in 11 strains of  S. aureus  in vitro: 
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Strain 
                 Type 
                 IC 50  (nM) 
               
               
                   
               
             
            
               
                 SH1000 
                 MSSA 
                 70.5 ± 6 
               
               
                 HG003 
                 MSSA 
                 54.4 ± 4 
               
               
                 USA300-JE2 
                 MSSA 
                 37.7 ± 4 
               
               
                 USA300 (FPR-3757) 
                 CA-MRSA 
                 30.8 ± 5 
               
               
                 USA300-3 
                 HA-MRSA 
                 42.8 ± 6 
               
               
                 Newman 
                 MSSA 
                 23.7 ± 1 
               
               
                 USA300-LAC 
                 MRSA 
                 43.6 ± 5 
               
               
                 ATCC29213 
                 MSSA 
                 30.0 ± 5 
               
               
                 Clinical isolate ST239III 
                 HA-MRSA 
                 16.3 ± 8 
               
               
                 Mu3 
                 VISA 
                  2.6 ± 1 
               
               
                 COL 
                 HA-MRSA 
                  0.9 ± 1 
               
               
                   
               
               
                 Keys: 
               
               
                 MSSA: methicillin-suspectible  S. aureus ; 
               
               
                 CA-MRSA: community-acquired MRSA; 
               
               
                 HA-MRSA: hospital-acquired MRSA; 
               
               
                 VISA: vancomycin-immediate  S. aureus   
               
            
           
         
       
     
     Efficacy of IM032 in a Mouse (LD0-20) Bacteremia Model Infected with Methicillin-Resistant  Staphylococcus  (MRSA USA300) (BAA-1717) 
     A bacterial strain, USA 300 MRSA BAA-1717, was used to infect BALB/c (female). Test animals were intravenously (IV) inoculated with MRSA ATCC BAA-1717 at a target density of 1×10 6  CFU/mouse. Test substance, IM032 at 0, 0.3, 1, 3, 10, and 30 mg/kg, was administered orally (PO) twice daily (BID) for a total of 7 dosing days. 
     The reference agents, vancomycin at 3 mg/kg was administered IV once (QD) at 1 h after infection for 7 consecutive days. 
     With reference to  FIG. 9 , the animals were IV inoculated with MRSA USA 300 ATCC BAA-1717 at 1.02×10 6  CFU/mouse. IM032 at 0.3, 1, 3, 10, and 30 mg/kg were orally (PO) administrated twice daily (BID) at 1 and 7 h after the infection on Day 1, and then twice daily at 6-hour intervals in the next 6 days for a total of 7 days of dosing. Vancomycin, at 3 mg/kg, was administered intravenously (IV) at 1 h after the infection on Day 1 and then once a day in the next 6 days for a total of 7 days of dosing. All the test animals in the IM032 treatment, reference vancomycin and the vehicle control groups were sacrificed at 168 hours (Day 7) after the infection. Tissues of lung and liver were excised for bacterial enumeration and represented as CFU/gram. Statistical significance compared to the respective vehicle control was determined by unpaired student t-tests. Statistical significance is represented as* p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001. 
     With reference to  FIG. 10 , bacterial density in lung and liver were determined. The animals were IV inoculated with MRSA USA 300 ATCC BAA-1717 at 1.02×10 6  CFU/mouse. IM032 at 0.3, 1, 3, 10, and 30 mg/kg were orally (PO) administrated orally (PO) twice daily (BID) at 1 and 7 h after the infection on Day 1, and then twice daily at 6-hour intervals in the next following 6 days for a total of 7 days of dosing. Days. Vancomycin, at 3 mg/kg, was administered intravenously (IV) once (QD) at 1 h after the infection on Day 1 and then once a day in the next 6 days for a total of 7 days of dosing. For 7 consecutive days. All the test animals in the IM032 treatment, reference vancomycin and the vehicle control groups were sacrificed at 168 hours (Day 7) after the infection. Blood samples were collected by cardiac puncture and organ tissues were excised for bacterial enumeration which are represented as CFU/mL for the blood or CFU/gram for organs. Statistical significance compared to the respective vehicle control was determined by unpaired student t-tests. Statistical significance is represented as *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001. 
     All test animals in the IM032 treatment, vancomycin and the vehicle control groups were sacrificed at 168 h (Day 7) after infection. Tissues of kidney, lung, liver and spleen were excised for bacterial enumeration, CFU/gram (tissues). Unpaired student t-test was performed to assess statistical significance (p&lt;0.05) in the bacterial counts of the treated animals compared to the carrier control group. 
     This test aimed to evaluate the efficacy of IM032 in a mouse bacteremia model by IV infecting of immune competent BALB/c mice with MRSA USA 300 (ATCC BAA-1717). At 1×10 6  CFU/mouse. 
     Therefore, compared with vancomycin, the in vivo experiment shows that IM032 achieved a statistically significant reduction in bacteria count across major organs. 
     The Efficacy of IM032 in a Mouse (LD0-20) Bacteremia Model Infected with Methicillin-Resistant  Staphylococcus  (MRSA USA300) (BAA-1717) 
       FIG. 11A  shows the healing effect of IM032 (30 mg/kg, via oral, twice a day, 12 hours each interval) on skin infection caused by methicillin-resistant  S. aureus  (MRSA) as compared to mupirocin (2%, via topical, twice a day, 12 hours each interval) and linezolid (100 mg/kg, via oral, twice a day, 12 hours each interval) in terms of the percentage of wound closure. Mice were challenged with MRSA skin infection Compared with topical dosing of 2% Mupirocin and oral dosing of Linezolid at 100 mg/kg twice a day, oral dosing of ALS-4 at 30 mg/kg twice a day showed statistically significant improvement in wound healing. Specifically, at the end of the study on Day 7, ALS-4 exhibited 63.8% of wound closure compared with 48.4% for oral Linezolid and 43.2% for topical Mupirocin 2%. Visual wound healing/closure in different treatment groups of mice can be observed in  FIG. 11B . Among the three groups, the mice treated with IM032 via oral administration at BID for 7 days appears to have the best visual wound healing from the observed size of the closure. 
     It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. 
     All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. 
     It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or practiced with other methods, protocols, reagents, cell lines and animals. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts, steps or events are required to implement a methodology in accordance with the present invention. Many of the techniques and procedures described, or referenced herein, are well understood and commonly employed using conventional methodology by those skilled in the art. 
     Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and/or as otherwise defined herein. 
     REFERENCE 
     The following references are incorporated herein by reference in their entirety:
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