Patent Application
20090131481
Most antibiotics currently used and in development to treat bacterial infections impose selective pressure on microorganisms and have led to the development of widespread antibiotic resistance. Therefore, the development of an alternative approach to treating microbial infections would be of great benefit.
Multidrug resistance in bacteria is generally attributed to the acquisition of multiple transposons and plasmids bearing genetic determinants for different mechanisms of resistance (Gold et al. 1996. N. Engl. J. Med. 335:1445). However, descriptions of intrinsic mechanisms that confer multidrug resistance have begun to emerge. The first of these was a chromosomally encoded multiple antibiotic resistance (mar) locus in Escherichia coli (George and Levy, 1983. J. Bacteriol. 155:531; George and Levy 1983 J. Bacteriol. 155:541). Mar mutants of E. coli arose at a frequency of 10−6 to 10−7 and were selected by growth on subinhibitory levels of tetracycline or chloramphenicol (George and Levy, supra). These mutants exhibited resistance to tetracyclines, chloramphenicol, penicillins, cephalosporins, puromycin, nalidixic acid, and rifampin (George and Levy, supra). Later, the resistance phenotype was extended to include fluoroquinolones (Cohen et al. 1989. Antimicrob. Agents Chemother. 33:1318), oxidative stress agents (Ariza et al. 1994. J. Bacteriol. 176:143; Greenberg et al. 1991. J. Bacteriol. 73:4433), and more recently, organic solvents (White et al. 1997. J. of Bacteriology 179:6122; Asako, et al. 1997. J. Bacteriol. 176:143) and household disinfectants, e.g., pine oil and/or TRICLOSAN® (McMurry et al. 1998. FEMS Microbiology Letters 166:305; Moken et al. 1997. Antimicrobial Agents and Chemotherapy 41:2770).
The mar locus consists of two divergently positioned transcriptional units that flank a common promoter/operator region in E. coli, Salmonella typhimurium, and other Entrobacteriacae (Alekshun and Levy. 1997, Antimicrobial Agents and Chemother. 41: 2067). One operon encodes MarC, a putative integral inner membrane protein without any yet apparent function, but which appears to contribute to the Mar phenotype in some strains. The other operon comprises marRAB, encoding the Mar repressor (MarR), which binds marO and negatively regulates expression of marRAB (Cohen et al. 1994. J. Bacteriol. 175:1484; Martin and Rosner 1995. PNAS 92:5456; Seoane and Levy. 1995 J. Bacteriol. 177:530), an activator (MarA), which controls expression of other genes on the chromosome, e.g., the mar regulon (Cohen et al. 1994 J. Bacteriol. 175:1484; Gambino et. al. 1993. J. Bacteriol. 175:2888; Seoane and Levy, 1995 J. Bacteriol. 177:530), and a putative small protein (MarB) of unknown function.
Exposure of E. coli to several chemicals, including tetracycline and chloramphenicol (Hachler et al. 1991 J Bacteriol 173(17):5532-8; Ariza, 1994, J Bacteriol; 176(1):143-8), sodium salicylate and its derivatives (Cohen, 1993, J Bacteriol; 175(24):7856-62) and oxidative stress agents (Seoane et al. 1995. J Bacteriol; 177(12):3414-9) induces the Mar phenotype. Some of these chemicals act directly at the level of MarR by interacting with the repressor and inactivating its function (Alekshun. 1999. J. Bacteriol. 181:3303-3306) while others (antibiotics such as tetracycline and chloramphenicol) appear to induce mar expression by an alternative mechanism (Alekshun. 1999. J. Bacteriol. 181:3303-3306) e.g., through a signal transduction pathway.
Once expressed, MarA activates the transcription of several genes that constitute the E. coli mar regulon (Alekshun, 1997, Antimicrob. Agents Chemother. 41:2067-2075; Alekshun, 1999, J. Bacteriol. 181:3303-3306). With respect to decreased antibiotic susceptibility, the increased expression of the AcrAB/TolC multidrug efflux system (Fralick, 1996, J. Bacteriol. 178(19):5803-5; Okusu, 1996 J Bacteriol; 178(1):306-8) and decreased synthesis of OmpF (Cohen, 1988, J. Bacteriol.; 170(12):5416-22) an outer membrane protein, play major roles. Organic solvent tolerance, however, is attributed to MarA mediating increased expression of AcrAB, TolC, OmpX, and a 77 kDa protein (Aono, 1998, Extremophiles; 2(3):239-48; Aono, 1998 J Bacteriol; 180(4):938-44) but is independent of OmpF levels (Asako, 1999, Appl Environ Microbiol; 65(1):294-6).
MarA is a member of the AraC/XylS family of transcriptional activators (Gallegos et al. 1993. Nucleic Acids Res. 21:807). There are more than 100 proteins within the AraC/XylS family and a defining characteristic of this group of proteins is the presence of two helix-turn-helix (HTH) DNA binding motifs. Proteins within this family activate many different genes, some of which produce antibiotic and oxidative stress resistance or control microbial metabolism and virulence (Gallegos et al. supra).
MarA (AraC) family proteins are present in nearly all clinically important bacteria including Pseudomonas aeruginosa, Yersinia spp., E. coli (including enteroaggregative, enterotoxigenic, and enteropathogenic strains), Klebsiella spp., Shigella spp., Salmonella spp., Vibrio cholerae, Staphylococcus aureus, and Streptococcus pneumoniae (M.-T. Gallegos et al. 1993. Nuc. Acids. Res. 21:807). Inactivation of MarA (AraC) family proteins by mutation attenuates virulence of bacteria in various animal models of infection (P. Casaz et al. 2006. Microbiol. 152:3643; G. A. Champion et al. 2003. Mol. Micro. 23:323; Y. Flashner et al. 2004. Infect. Immun. 72:908; D. S. Bieber et al. 1998. Sci. 280:2114).
MarA, Rob, and SoxS proteins are required for full E. coli virulence in a murine ascending pyelonephritis model (P. Casaz et al. 2006. Microbiol. 152:3643). Deletion of genes for marA, rob, and soxS from a clinical (intestinal fistula) E. coli isolate (KM-D), removed its ability to colonize the kidneys. Wild type virulence was restored when the deletion strain (SRM) was complemented with a single chromosomal copy of the marA, soxS, or rob genes.
The Y. pseudotuberculosis MarA (AraC) family protein LcrF (also called VirF in Y. enterocolitica) regulates expression of a major virulence determinant, the type III secretion system (TTSS) (G. R. Cornelis and H. Wolf-Watz. 1997. Mol. Microbiol. 23:861-867). The TTSS delivers toxins directly into host cells via a needle-like apparatus. Mutants that do not express the TTSS show dramatic attenuation of virulence in whole cell and animal models of infection (G. R. Cornelis and H. Wolf-Watz. 1997. Mol. Microbiol. 23:861-867; L. K. Logsdon and J. Mecsas. 2003. Infect. Immun. 71:4595-4607; J. Mecsas et al., 2001, Infect. Immun., 69:2779-2787; D. M. Monack et al. 1997. Proc. Natl. Acad. Sci. U.S.A. 94:10385-10390). Flashner et al., have recently investigated the effects of lcrF deletion on the pathogenicity of Y. pestis in a mouse model of septic infection (Y. Flashner et al. 2004. Infect. Immun. 72:908-915). The LD50 (50% lethal dose) of wild type Y. pestis in this model is approximately 1 colony forming unit (CFU). When an 1:1 mixture of wild type and lcrF mutant Y. pestis was used to infect mice, the competitive index (defined as the ratio of lcrF/wt recovered following infection vs. the ratio of lcrF/wt used for infection) was <10−7 indicating severe attenuation of the mutant organism.
The Pseudomonas aeruginosa MarA (AraC) family protein ExsA regulates expression of a well established virulence determinant, the type III secretion system (T. L. Yahr et al. 2006. Mol. Micro. 62(3):631). Mutants lacking ExsA show dramatically reduced virulence in animal models of P. aeruginosa infection (V. J. Finck-Barbancon et al. 1997. Mol. Micro. 25(3):547; A. R. Hauser et al. 1998. Mol. Micro. 27:807; I. Kudoh et al. 1994. Am. J. Physiol. 267:L551; M. A. Laskowski et al. 2004. Mol. Micro. 54(4):1090; E. J. Lee et al. 2003. Invest. Opthalmol. Vis. Sci. 44(9):3892; R. S. Smith et al. 2004. Infect. & Immun. 72(3):1677). Furthermore, expression of the type III secretion system is correlated with increased severity of disease in clinical pneumonia cases, including ventilator-associated pneumonia (A. R. Hauser et al. 2002. Crit. Care Med. 30(3):521; G. S. Schulert et al. 2003. J. Infect. Dis. 188:1695; A. Roy-Burman et al. 2001. J. Infect. Dis. 183:1767).
The present invention pertains, at least in part, to a method for reducing infectivity and/or virulence of a microbial cell by contacting the cell with a transcription factor modulating compound.
In another embodiment, the present invention pertains, at least in part, to a method for modulating transcription of genes regulated by one or more transcription factors in the MarA (AraC) family. The method includes contacting a transcription factor with a transcription factor modulating compound. Specifically, in one embodiment, the transcription factor is ExsA, LcrF (VirF) or SoxS.
The present invention also pertains, at least in part, to a method for preventing bacterial growth on a contact lens by administering a composition comprising an acceptable carrier and a transcription factor modulating compound.
The present invention also pertains, at least in part, to a method for preventing or treating an infection in a patient into which an indwelling device has been implanted (e.g., ventilator-associated pneumonia in patients receiving mechanical ventilation) by administering a composition comprising a transcription factor modulating compound.
The present invention also pertains, at least in part, to methods for treating or preventing biofilm formation in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
In another embodiment, the present invention pertains, at least in part, to a method for treating or preventing a bacterial infection in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
The present invention also pertains, at least in part, to a method for prevention or treatment of a urinary tract infection in a subject by administering to the subject an effective amount of a transcription factor compound.
In yet another embodiment, the invention pertains, at least in part, to a method for treating or preventing pneumonia in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
In a further embodiment, the invention pertains, at least in part, to a method for treating burn wounds and corneal ulcers in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
In another embodiment, the present invention pertains, at least at part, a method for treating or preventing ascending pyelonephritis or kidney infection in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
In one embodiment, the present invention pertains, at least in part, to a method for inhibiting a MarA family polypeptide by contacting a Mar family polypeptide with an effective amount of a transcription factor modulating compound.
In one embodiment, the transcription factor modulating compound is a compound of formula I:
wherein:
R2, R4 and R5 are each hydrogen;
R3 is nitro or cyano;
L is —NHCO—, —NHCOCH═CH—, —NHCOCH2CH2—, —NHCOCH2—, —CH2 NHCO—, or —C≡C—;
R6 and R10 are each hydrogen, halogen, alkyl or alkoxy;
R7 and R9 are each hydrogen, alkyl or halogen; and
R8 is hydrogen, hydroxyl, carboxy, alkylcarbonylamino, amino, aminosulfonyl, alkylsulfonyl, alkoxy, halogen, alkyl, alkylamino, acylamino, cyano, acyl, heteroaryl or heterocyclic;
and pharmaceutically acceptable salts thereof.
In another embodiment, the transcription factor modulating compound is a compound of formula II:
wherein:
R3a and R4a are each independently hydrogen, —NO2, —CN, —F, or —N(CH3)2
A is phenyl or heterocyclic;
La is —NHCO— or —NHCH═CH—; and
R8a is an electron-donating or an electron-withdrawing group and pharmaceutically acceptable salts thereof.
In yet another embodiment, the transcription factor modulating compound is a compound of formula III:
wherein:
Lb is —NHCO— or —NHCOCH═CH—; and
R8b is an electron-donating or an electron-withdrawing group and pharmaceutically acceptable salts thereof.
In one embodiment, the transcription factor modulating compound is a compound of formula IV:
wherein:
R1c is —CH2 CO2H, —OCH2CO2 Et, —OCH2CH2 CO2H, —OCH2CH2 OH, —OCH2CN, —OCH2CH2CH3, —OCH3, —OH, —OCH2CH2 NH2 or hydrogen;
R2c is aryl,
R6c is hydrogen, —NO2, H, —COCH3, —CF3, —F, —OCH3, —CO2H, —CONH2, —CN, —N(CH3)2, —C(CH3)3, —SO2 CH3, —C(CH3)NOH, or
and pharmaceutically acceptable salts thereof.
In one embodiment, the transcription factor modulating compound is a compound of formula V:
wherein:
R1* is hydroxyl, OCOCO2H; a straight or branched C1-C5 alkyloxy group; or a straight or branched C1-C5 alkyl group;
A, B, D, E, W, X, Y and Z are each independently carbon or nitrogen;
wherein: R2*, R3*, R4*, R5*, R6*, R7*, R8*, R9* are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime or halogen when A, B, D, E, W, X, Y and Z are carbon; or wherein: R2*, R3*, R4*, R5*, R6*, R7*, R8*, R9* are each independently absent or hydroxyl when A, B, D, E, W, X, Y and Z are nitrogen;
R10*, R11*, R12* and R13* are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime or halogen; and pharmaceutically acceptable salts, esters and prodrugs thereof;
provided that when A, B, C, D, E, W, X, Y and Z are each carbon, one of R6* R7*, R8*, R9* is not hydrogen.
In another embodiment, the transcription factor modulating compound is a compound of formula VI:
wherein:
R1a is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
R2a, R3a, R4a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13c, R13d and R13e are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and esters, prodrugs and pharmaceutically acceptable salts thereof;
provided that when R1a is hydroxyl, R3a is nitro, R2a, R4a, R5a, R6a, R7a, R8a, R9a R10a, R11a, R12a, R13a, R13b, R13d, and R13e are hydrogen, then R13c is not hydrogen, fluorine, dimethylamino, cyano, hydroxyl, methyl or methoxy; and
provided that when R1a is hydroxyl, R3a is nitro, R2a, R4a, R5a, R6a, R7a, R8a, R9a R10a, R11a, R12a, R13a, R13b and R13d are hydrogen, then R13c and R13e are not fluorine.
In yet another embodiment, the transcription factor modulating compound is a compound of formula VII:
wherein:
R14 is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
G, J, K, L, M, Q, T and U are each independently carbon or nitrogen;
wherein: R1, R16, R17, R18, R19, R20, R21, R22, R23 and R24 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, absent, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen when G, J, K, L, M, Q, T and U are carbon; or R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24 are each independently absent or hydroxyl when G, J, K, L, M, Q, T and U are nitrogen;
R23 and R24 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, absent, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and pharmaceutically acceptable salts, esters and prodrugs thereof;
provided that when G, J, K, L, M, Q, T and U are each carbon, one of R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24, are not hydrogen.
In a further embodiment, the transcription factor modulating compound is a compound of formula VIII:
wherein:
R14a is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
R15a, R16a, R17a, R18a, R19a, R20a, R21a, R22a, R23a and R24a, R24b, R24c, R24d and R24e are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and esters, prodrugs and pharmaceutically acceptable salts thereof;
provided that at least two of R24a, R24b, R24c, R24d and R24e are not hydrogen.
In another embodiment, the transcription factor modulating compound is a compound of formula IX:
wherein:
R25 is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
R26, R27, R28, R29, R30, R31, R32, R33, R34, R35a, R35b, R35c, R35d, and R35e are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and esters, prodrugs and pharmaceutically acceptable salts thereof;
provided that at least two of R26, R27, R28 and R29 are not hydrogen.
In another embodiment, the transcription factor modulating compound is a compound of formula X:
wherein:
R25′ is a substituted straight or branched C1-C5 alkyloxy group;
R26′, R27′ R28′, R29′, R30′, R31′, R32′, R33′, R34′, R35a′, R35b′, R35c′, R35 d′, and R35e′ are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and esters, prodrugs and pharmaceutically acceptable salts thereof.
In one embodiment, the transcription factor modulating compound is a compound of formula (XI):
wherein:
R36 is hydroxyl;
R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each independently hydrogen, alkyl alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen;
R38 is cyano, nitro, oxime, alkyloxime, aryloxime, heteroaryl, amino-oxime, or aminocarbonyl;
R46c is hydrogen, acyl, fluorine, pyrizinyl, pyridinyl, cyano, imidazolyl, dialkylaminocarbonyl or dialkylamino; and esters, prodrugs and pharmaceutically acceptable salts thereof;
provided that when R38 is nitro and R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, then R46c is not dialkylamino, acyl or hydrogen; and
provided that when R38 is cyano and R37, R39, R40, R41, R42, R43, R44, R45, R46a R46b, R46d, and R46e are each hydrogen, then R46c is not dialkylamino.
In another embodiment, the transcription factor modulating compound is a compound of formula
wherein:
R47 is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
R48, R49, R50, R51, R52 and R53 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen;
Ar is aryl; and pharmaceutically acceptable salts, esters and prodrugs thereof.
In one embodiment, the transcription factor modulating compounds is a compound of formula XIII:
wherein:
R1d is hydrogen, —OH, —OCH2-aryl, —CH2CH2 CO2H, —OCH2CO2CH2CH3, —OCH2CN, —OCH2CH2 NH2, —OCH3, —OCH2CH2N+(CH3)3, —OCH2COOH, —OCH2CH2CH3, —OCH2CH2 OH, —OCH2P(O)(OH)2 or —OCH2P(O)(OCH2CH3)2;
R2d is hydrogen or —NR2daR2db;
R2da and R2db are each independently hydrogen, alkyl or aminoalkyl;
Xd is CR3d, N or NO;
R3d is absent when Xd is N or NO—NO2, hydrogen, acyl, halogen, alkoxy, —CO2H, —CONR3daR3db; cyano, —NR3dcR3dd, alkyl, —SO2R3de, —C(R3df)NOH, heterocyclic or heteroaryl;
R3da and R3db are each independently hydrogen or alkyl;
R3dc and R3dd are each independently hydrogen, alkyl or substituted carbonyl;
R3de and R3df are each independently alkyl or amino;
R4d is hydrogen, alkoxy, —NR4daR4db, alkyl, halogen, —SO2R4d c or —CO2H;
R4da and R4db are each independently hydrogen, alkyl or aminoalkyl;
R4dc is alkyl or amino;
Zd is CH, N or NO;
Ard is
when Ld is present or
when Ld and R16d are each absent;
Yd is N or CR6d;
Wd is N or CR8d;
R6d is absent when Yd is N, or hydrogen, alkyl, amino, —CO2H, —OCH2P(O)(OH)2 or alkyl;
R8d is absent when Wd is N, or hydrogen, alkyl, amino, —CO2H, —OCH2P(O)(OH)2 or alkyl;
R7d and R9d are each independently hydrogen, alkyl, amino, —CO2H, —OCH2P(O)(OH)2 or alkyl;
Ad is O, NR10d or S;
R10d is hydrogen or alkyl;
Ld is absent, or Ld is hydrogen or unsubstituted phenyl when R16d is absent, or Ld is —O—, —SO—, —SO2—, —OCH2—, —CH2—, —NR15d,
n is an integer between 0-2;
Dd and Ed are each independently NR17d; O or S
Jd is N or CR18d;
Gd is N or CR19d;
R11d is hydrogen or alkyl;
R18d is absent when Jd is N or hydrogen or alkyl;
R19d is absent when Gd is N or hydrogen or alkyl;
R12d and R13d are each independently hydrogen, alkyl, halogen or aryl;
R15d is hydrogen or alkyl;
R16d is hydrogen, alkoxy, hydroxyl, amino, alkyl, —NO2 or halogen when Ld is absent; or R16d is
when Ld is present;
Kd is CR20d or N;
Md is CR23d or N;
R20d is absent when Kd is N or hydrogen, alkyl, halogen, alkoxy or hydroxyl;
R21d is hydrogen, halogen or alkyl;
R22d is hydrogen, heteroaryl, halogen, alkoxy, cyano, acyl, —SO2R22da heterocyclic, —COOH, hydroxyl, —CF3, alkyl, amino, CO2H, aminocarbonyl or
R22da is amino or alkyl;
R23d is absent when Md is N or hydrogen, halogen, alkyl or alkoxy; or R22d and
R23d together with the carbon atoms to which they are attached are joined to form a 5- or 6-membered ring;
R24d is hydrogen, halogen or alkoxy; and pharmaceutically acceptable salts thereof; and pharmaceutically acceptable salts thereof.
In one embodiment, the transcription factor modulating compound is a compound of formula XIV:
wherein:
R1e is —OH, —OCH2-aryl, —CH2CH2 CO2H, —OCH2CO2CH2CH3, —OCH2CN, —OCH2CH2 NH2, —OCH3, —OCH2CH2N+(CH3)3, —OCH2COOH, —OCH2CH2CH3, —OCH2CH2 OH, —OCH2P(O)(OH)2 or —OCH2P(O)(OCH2CH3)2;
R2e, R4e, R53, R11e, R12e, R13e, R21e, R22e, and R24e are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, aryloxy, carbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, acyl, amino, CO2H, cyano, nitro or halogen;
R20e is absent when Ke is N or hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, aryloxy, carbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, acyl, amino, CO2H, cyano, nitro or halogen;
R23e is absent when Me is N or hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, aryloxy, carbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, acyl, amino, CO2H, cyano, nitro or halogen;
R3e is —NO2, hydrogen, acyl, halogen, alkoxy, —CO2H, —CONR3daR3db; cyano, —NR3dcR3dd, alkyl, —SO2R3de, —C(R3df)NOH, heterocyclic or heteroaryl;
R3ea is alkyl or amino;
Ke is CR20e or N;
Me is CR23e or N; and pharmaceutically acceptable salts thereof.
The invention also pertains, at least in part, to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a transcription factor modulating compound.
The Mar proteins are members of the AraC family of bacterial transcription regulators characterized by two highly conserved helix-turn-helix (HTH) DNA-binding domains. The signaling networks regulating the activity of Mar proteins vary and, while there is high conservation within the DNA binding domains, all Mar proteins bind to distinct DNA sequences in the promoter regions of the genes which they regulate. Mar proteins are present in all clinically important bacteria whose genomes have been examined including Pseudomonas aeruginosa, Yersinia spp., E. coli (including enteroaggregative, enterotoxigenic and enteropathogenic strains), Klebsiella spp., Shigella spp., Salmonella spp., Vibrio cholerae, Staphylococcus aureus and Streptococcus pneumoniae. Mar proteins confer upon bacteria the ability to cause infections, resist antibiotics and adapt to hostile environments. Inactivation of Mar proteins by mutation attenuates the virulence of bacterial pathogens in animal models of infection, but does not affect bacterial growth.
The invention relates to anti-infective transcription factor modulating compounds that target the virulence and infectivity of a microbial cell, thus preventing infection or disease in a subject. The invention pertains, at least in part, to a method for reducing the infectivity or virulence of a microbial cell, comprising contacting said cell with a transcription factor modulating compound, e.g. a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2. The term “reducing infectivity” includes decreasing or eliminating the potential of a microbial cell to cause an infection. The term “reducing virulence” includes decreasing or eliminating the ability of a microbial cell to cause disease. Examples of microbial cells, include, but are not limited to E. coli, Y. pseudotuberculosis, Klebsiella pneumoniae, Acinetobacter baumannii and P. aeruginosa. A skilled artisan, using routine techniques, would be able to determine whether a microbial cell is infective or virulent.
In one embodiment, the method of reducing infectivity or virulence of a microbial cell includes reducing the manner in which a microbial cell causes a disease. Without being bound by theory, the methods for reducing infectivity or virulence of a microbial cell may include, for example, the inhibition of the adhesion of a microbial cell to a host cell; the inhibition of the colonization of the microbial cell in the host; the inhibition of the microbial cell from entering host cells and/or entry into the host body; the reduction or elimination of the ability of the microbial cell to produce immune response inhibitors or toxins that may cause tissue damage or damage to the host cells.
The term “microbe” includes microorganisms that cause disease. For example, in one embodiment, microbes are unicellular and include bacteria, fungi, or protozoa. In another embodiment, microbes suitable for use in the invention are multicellular, e.g., parasites or fungi. In another embodiment, microbes are pathogenic for humans, animals, or plants. In one embodiment, the microbes include prokaryotic organisms. In other embodiments, the microbes include eukaryotic organisms. In a further embodiment, the microbe is antibiotic resistant.
In one embodiment, microbes against which a transcription factor modulating compound of the invention may be used are bacteria, e.g., Gram negative or Gram positive bacteria. In one embodiment, the microbe includes any bacteria that are shown to become resistant to antibiotics, e.g., display a Mar phenotype or are infectious or potentially infectious. Exemplary bacteria that contain MarA homologs include the following: E. coli (e.g., UPEC (uropathogenic) or EPEC (enteropathogenic)), Salmonella enterica (e.g., Cholerasuis (septicemia), Enteritidis enteritis, Typhimurium enteritis, Typhimurium (multi-drug resistant)), Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Enterobacter spp., Klebsiella sp., Proteus spp., Vibrio cholerae, Shigella sp., Providencia stuartii, Neisseria meningitides, Mycobacterium tuberculosis, Mycobacterium leprae, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Bordetella pertussis and Bordetella bronchiseptica.
Examples of microbes against which a transcription faction modulating compound of the invention may be used include, but are not limited to, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter baumannii, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus saccharolyticus.
In one embodiment, microbes against which a transcription factor modulating compound of the invention may be used are bacteria from the family Enterobacteriaceae. In preferred embodiments, the compound is effective against a bacteria of a genus selected from the group consisting of: Escherichia, Proteus, Salmonella, Klebsiella, Providencia, Enterobacter, Burkholderia, Pseudomonas, Aeromonas, Haemophilus, Yersinia, Acinetobacter, Neisseria, and Mycobacteria.
In yet other embodiments, the microbes against which a transcription factor modulating compound of the invention may be used are Gram positive bacteria and are from a genus selected from the group consisting of: Lactobacillus, Azorhizobium, Streptomyces, Pediococcus, Photobacterium, Haemophilus, Bacillus, Enterococcus, Staphylococcus, Clostridium, and Streptococcus.
In other embodiments, the microbes against which a transcription factor modulating compound of the invention may be used are fungi. In one embodiment, the fungus is from the genus Mucor or Candida, e.g., Mucor racmeosus or Candida albicans.
In yet other embodiments, the microbes against which a transcription factor modulating compound of the invention may be used are protozoa. In a preferred embodiment the microbe is a malaria or cryptosporidium parasite.
The term “transcription factor” includes proteins that are involved in gene regulation in both prokaryotic and eukaryotic organisms. Preferably, a transcription factor against which a modulating compound of the invention is effective is present only in a prokaryotic organism. In one embodiment, transcription factors can have a positive effect on gene expression and, thus, may be referred to as an “activator” or a “transcriptional activation factor.” In another embodiment, a transcription factor can negatively affect gene expression and, thus, may be referred to as a “repressor” or a “transcription repression factor.” Activators and repressors are generally used terms and their functions are discerned by those skilled in the art. In one embodiment, the transcription factor is ExsA, SoxS or LcrF (VirF).
Some major families of transcription factors found in bacteria include the helix-turn-helix transcription factors (HTH) (Harrison, S. C., and A. K. Aggarwal 1990. Annual Review of Biochemistry. 59:933-969) such as AraC, MarA, Rob, SoxS and LysR; winged helix transcription factors (Gajiwala, K. S., and S. K. Burley 2000. 10:110-116), e.g., MarR, Sar/Rot family, and OmpR (Huffman, J. L., and R. G. Brennan 2002. Curr Opin Struct Biol. 12:98-106, Martinez-Hackert, E., and A. M. Stock 1997. Structure. 5:109-124); and looped-hinge helix transcription factors (Huffman, J. L., and R. G. Brennan 2002 Curr Opin Struct Biol. 12:98-106), e.g. the AbrB protein family.
MarA (AraC) family proteins are present in nearly all clinically important bacteria including Pseudomonas aeruginosa, Yersinia spp., E. coli (including enteroaggregative, enterotoxigenic, and enteropathogenic strains), Klebsiella spp., Shigella spp., Salmonella spp., Vibrio cholerae, Staphylococcus aureus, and Streptococcus pneumoniae (M.-T. Gallegos et a1993. Nuc. Acids. Res. 21:807). MarA (AraC) family proteins confer upon bacteria the ability to cause infections, resist antibiotics, and adapt to hostile environments. Inactivation of MarA (AraC) family proteins by mutation attenuates virulence of bacteria in various animal models of infection (P. Casaz et al. 2006. Microbiol. 152:3643; G. A. Champion et al. 2003. Mol. Micro. 23:323; Y. Flashner et al. 2004. Infect. Immun. 72:908; D. S. Bieber et al. 1998. Sci. 280:2114).
The term “AraC family polypeptide,” “AraC/XylS family polypeptide” or “AraC/XylS family peptide” include an art recognized group of prokaryotic transcription factors which contains more than 100 different proteins (Gallegos et al., (1997) Micro. Mol. Biol. Rev. 61: 393; Martin and Rosner, (2001) Curr. Opin. Microbiol. 4:132). AraC family polypeptides include proteins defined in the PROSITE (PS) database as profile PS01124. The AraC family polypeptides also include polypeptides described in PS0041, HTH AraC Family 1, and PS01124, and HTH AraC Family 2.
In an embodiment, the AraC family polypeptides are generally comprised of, at the level of primary sequence, by a conserved stretch of about 100 amino acids, which are believed to be responsible for the DNA binding activity of this protein (Gallegos et al., (1997) Micro. Mol. Biol. Rev. 61: 393; Martin and Rosner, (2001) Curr. Opin. Microbiol. 4: 132). AraC family polypeptides also may include two helix turn helix DNA binding motifs (Martin and Rosner, (2001) Curr. Opin. Microbiol. 4: 132; Gallegos et al., (1997) Micro. Mol. Biol. Rev. 61: 393; Kwon et al., (2000) Nat. Struct. Biol. 7: 424; Rhee et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95: 10413). The term includes MarA family polypeptides and HTH proteins.
The term “helix-turn-helix protein,” “HTH protein,” “helix-turn-helix polypeptides,” and “HTH polypeptides,” includes proteins comprising one or more helix-turn-helix domains. Helix-turn-helix domains are known in the art and have been implicated in DNA binding (Ann Rev. of Biochem. 1984. 53:293).
In one embodiment, a helix-turn-helix domain containing protein is a Mar A family polypeptide. The language “MarA family polypeptide” includes the many naturally occurring HTH proteins, such as transcription regulation proteins which have sequence similarities to MarA and which contain the MarA family signature pattern, which can also be referred to as an AraC/XylS signature pattern. MarA family polypeptides have two “helix-turn-helix” domains. This signature pattern was derived from the region that follows the first, most amino terminal, helix-turn-helix domain (HTH1) and includes the totality of the second, most carboxy terminal helix-turn-helix domain (HTH2). (See PROSITE PS00041).
The MarA family of proteins (“MarA family polypeptides”) represent one subset of AraC/XylS family polypeptides and include proteins like MarA, SoxS, Rob, RamA, AarP, PqrA, etc. The MarA family polypeptides, generally, are involved in regulating resistance to antibiotics, organic solvents, and oxidative stress agents (Alekshun and Levy, (1997) Antimicrob. Agents. Chemother. 41: 2067). Like other AraC/XylS family polypeptides, MarA-like proteins also generally contain two HTH motifs as exemplified by the MarA and Rob crystal structures (Kwon et al., (2000) Nat. Struct. Biol. 7: 424; Rhee et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95: 10413). Members of the MarA family can be identified by those skilled in the art and will generally be represented by proteins with homology to amino acids 30-76 and 77-106 of MarA. Preferably, a MarA family polypeptide or portion thereof comprises the first MarA family HTH domain (HTH1) (Brunelle, 1989, J Mol Biol; 209(4):607-22). In another embodiment, a MarA polypeptide comprises the second MarA family HTH domain (HTH2) (Caswell, 1992, Biochem J.; 287:493-509). In a preferred embodiment, a MarA polypeptide comprises both the first and second MarA family HTH domains.
MarA family polypeptide sequences are “structurally related” to one or more known MarA family members, preferably to MarA. This relatedness can be shown by sequence or structural similarity between two MarA family polypeptide sequences or between two MarA family nucleotide sequences that specify such polypeptides. Sequence similarity can be shown, e.g., by optimally aligning MarA family member sequences using an alignment program for purposes of comparison and comparing corresponding positions. To determine the degree of similarity between sequences, they will be aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for nucleic acid molecule for optimal alignment with the other protein or nucleic acid molecules). The amino acid residues or bases and corresponding amino acid positions or bases are then compared. When a position in one sequence is occupied by the same amino acid residue or by the same base as the corresponding position in the other sequence, then the molecules are identical at that position. If amino acid residues are not identical, they may be similar. As used herein, an amino acid residue is “similar” to another amino acid residue if the two amino acid residues are members of the same family of residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art (see, for example, Altschul et al. 1990. J. Mol. Biol. 215:403) including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). The degree (percentage) of similarity between sequences, therefore, is a function of the number of identical or similar positions shared by two sequences (i.e., % homology=#of identical or similar positions/total #of positions ×100). Alignment strategies are well known in the art; see, for example, Altschul et al. supra for optimal sequence alignment.
MarA family polypeptides may share some amino acid sequence similarity with MarA. The nucleic acid and amino acid sequences of MarA as well as other MarA family polypeptides are available in the art. For example, the nucleic acid and amino acid sequence of MarA can be found, e.g., on GeneBank (accession number M96235 or in Cohen et al. 1993. J. Bacteriol. 175:1484). In one embodiment, a MarA family polypeptide excludes one or more of XylS, AraC, and MelR. In another embodiment, the MarA family polypeptide is involved in antibiotic resistance. In yet another embodiment, the MarA family polypeptide is selected from the group consisting of: MarA, RamA, AarP, Rob, SoxS, and PqrA.
Exemplary MarA family polypeptides are shown in Table 1, and at Prosite (PS00041) and include: AarP, Ada, AdaA, AdiY, AfrR, AggR, AppY, AraC, CafR, CelD, CfaD, CsvR, D90812, EnvY, ExsA, FapR, HrpB, InF, InvF, LcrF, LumQ, MarA, MelR, MixE, MmsR, MsmR, OrfR, Orf_f375, PchR, PerA, PocR, PqrA, RafR, RamA, RhaR, RhaS, Rns, Rob, SoxS, S52856, TetD, TcpN, ThcR, TmbS, U73857, U34257, U21191, UreR, VirF, XylR, XylS, Xys1, 2, 3, 4, Ya52, YbbB, YfiF, YisR, YzbC, and YijO.
Escherichia coli
MarA (1)
OrfR (2, 3)
SoxS (4, 5)
Proteus vulgaris
PqrA (23)
Salmonella typhimurium
MarA (24)
Klebsiella pneumoniae
RamA (27)
Haemophilus influenzae
Yersinia spp.
Providencia stuartii
AarP (31)
Pseudomonas spp.
Synechocystis spp.
LumQ (38)
Lactobacillus helveticus
U34257 (39)
Azorhizobium caulinodans
Streptomyces spp.
U21191 (41)
Streptococcus mutans
Pediococcus pentosaceus
Photobacterium leiognathi
Bacillus subtilis
a The smaller MarA homologs, ranging in size from 87 (U34257) to 138 (OrfR) amino acid residues, are represented in boldface. References are given in parentheses and are listed below.
The term “transcription factor modulating compound” or “transcription factor modulator” includes compounds which interact with one or more transcription factors, such that the activity of the transcription factor is modulated, e.g., enhanced or inhibited. The term also includes both AraC family modulating compounds and MarA family modulating compounds (e.g., compounds that modulate transcription factors of the AraC family and compounds that modulate transcription factors of the MarA family, respectively). In another embodiment, the transcription factor modulating compound is a compound which inhibits a transcription factor, e.g., a prokaryotic transcription factor or a eukaryotic transcription activation factor. In one embodiment, the transcription factor modulating compounds modulate the activity of a transcription factor as measured by assays known in the art or LANCE assays such as those described in Example 12. In one embodiment, the transcription factor modulating compound inhibits the binding of a particular transcription factor to its cognate DNA by about 10% or greater, about 40% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, about 95% or greater, or about 100% as compared to the activity in the absence of the transcription factor modulating compound.
In another embodiment, the transcription factor modulating compound is a MarR family polypeptide inhibitor. In another embodiment, the transcription factor modulating compound is a AraC family polypeptide inhibitor.
The invention also pertains to a method for preventing bacterial growth on a contact lens. The method includes contacting the contact lenses with a solution of a transcription factor modulating compound, e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2, in an acceptable carrier. The invention also pertains to a solution comprising the compound, packaged with directions for using the solution to clean contact lenses.
In yet another embodiment, the invention pertains, at least in part, to a method for the prevention or treatment of an infection in a patient into which an indwelling device has been implanted comprising administering a composition comprising a transcription factor modulating compound, e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2. The method includes contacting at least one compound of the invention with a medical indwelling device, such as to prevent or substantially inhibit the formation of a biofilm. Examples of medical indwelling devices include catheters, orthopedic devices, devices associated with endotracheal intubation, devices associated with mechanical ventilation (e.g., a ventilator) and implants.
In one embodiment, the invention pertains, at least in part, to a method for treating or preventing biofilm formation in a subject, comprising administering to said subject an effective amount of a transcription factor modulating compound, e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2. The biofilm associated states includes disorders which are characterized by the presence or potential presence of a bacterial biofilm and can include, for example, middle ear infections, cystic fibrosis, osteomyelitis, acne, dental cavities, endocarditis, pneumonia and prostatitis. Biofilm is also implicated with, e.g., Pseudomonas aeruginosa. Furthermore, the invention also pertains to methods for preventing the formation of biofilms on surfaces or in areas by contacting the area with an effective amount of a transcription factor modulating compound, e.g., a MarA family inhibiting compound, etc. In one embodiment, the biofilm associated state is ventilator associated pneumonia. In yet another embodiment, the invention pertains, at least in part to a method for treating or preventing pneumonia in a subject where the pneumonia is associated with Pseudomonas aeruginosa.
In another embodiment, the transcription factor modulating compound inhibits biofilm formation, for example, as measured by assays known in the art or the Crystal Violet assay described in Example 11. In one embodiment, the transcription factor modulating compound of the invention inhibits the formation of a biofilm by about 25% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, 99.99% or more, or by 100%, as compared to the formation of a biofilm without the transcription factor modulating compound.
The term “biofilm” includes biological films that develop and persist at interfaces in aqueous and other environments. Biofilms are composed of microorganisms embedded in an organic gelatinous structure composed of one or more matrix polymers which are secreted by the resident microorganisms. The term “biofilm” also includes bacteria that are attached to a surface in sufficient numbers to be detected or communities of microorganisms attached to a surface (Costerton, J. W., et al. (1987) Ann. Rev. Microbiol. 41:435-464; Shapiro, J. A. (1988) Sci Am. 256:82-89; O'Toole, G. et al. (2000) Annu Rev Microbiol. 54:49-79).
In a further embodiment, the invention pertains, at least in part to a method for preventing or treating a bacterial infection in a subject, comprising administering to said subject an effective amount of a transcription factor modulating compound, e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2. The term “bacterial infection” includes states characterized by the presence of bacteria which can be prevented or treated by administering the transcription factor modulating compounds of the invention. The term includes biofilm formation and other infections or the undesirable presence of a bacteria on or in a subject. In one embodiment, the bacterial infection is associated with Y. pseudotuberculosis or P. aeruginosa. In yet another embodiment, the bacterial infection is associated with burn wounds or corneal ulcers. In another embodiment, the bacterial infection is associated with the implantation of a medical device in a subject (e.g., in the case of mechanical ventilation, endotracheal intubation, catheterization, and the like). In a further embodiment, the bacterial infection is a nosocomial infection.
In a further embodiment, the invention pertains, at least in part, to a method of treating or preventing pneumonia (e.g., ventilator-associated pneumonia) in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
In another embodiment, the invention pertains, at least in part, to a method of inhibiting a MarA family polypeptide by contacting a MarA family polypeptide with an effective amount of a transcription factor modulating compound. Suitable MarA family polypeptides include, but are not limited to, ExsA, LcrF (VirF) or Sox.
In one embodiment, the invention pertains, at least in part, to a method of treating or preventing burn wounds or corneal ulcers in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
In yet another embodiment, the invention pertains, at least in part, to a method for treatment or prevention of a urinary tract infection in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
In another embodiment, the invention pertains, at least in part, to a method for treatment or prevention of a kidney infection in a subject by administering to the subject an effective amount of a transcription factor modulating compound.
In an embodiment, the invention pertains, at least in part, to a method for treatment or prevention of acute pyelonephritis in a subject, by administering to the subject an effective amount of a transcription factor modulating compound.
In one embodiment, the invention pertains, at least in part, to a method of inhibiting bacterial infectivity and/or virulence of a bacteria comprising administering an effective amount of a transcription factor modulating compound.
In one embodiment, the invention pertains to a method of treating or preventing an infection in a subject by administering an effective amount of a transcription factor modulating compound of the invention. The aforementioned infection includes, but is not limited to, an infection by Staphylococcus aureus, Enterococcus faecium, Streptococcus pyogenes, Streptococcus pneumoniae and Streptococcus pneumoniae, Y. pseudotuberculosis or P. aeruginosa.
In another embodiment, the present invention pertains, at least in part, to a method for modulating transcription of genes regulated by transcription factors in the MarA (AraC) family, comprising contacting a transcription factor with a transcription factor modulating compound. Specifically, in one embodiment, the member of the MarA (AraC) family is ExsA or VirF. AraC family proteins contain a conserved DNA binding domain with two helix-turn-helix motifs. This conserved domain spans 100 amino acids with 17 residues showing a high degree of conservation over that span representing the consensus for the family. The overall similarity of the DNA binding domain is >20% among members of the AraC family. For example, ExsA and VirF are 56% identical, 72% similar across a 266 amino acid overlap and they show 85% identity and 97% similarity in the 100 bp DNA binding domain; VirF and MarA show 23% identity, 42% similarity across a 96 amino acid overlap; and ExsA and MarA show 23% identity, 42% similarity across a 92 amino acid overlap.
In one embodiment, the transcription factor modulating compounds of the invention are a compound of formula I:
wherein:
R2, R4 and R5 are each hydrogen;
R3 is nitro or cyano;
L is —NHCO—, —NHCOCH═CH—, —NHCOCH2CH2—, —NHCOCH2—, —CH2 NHCO—, or —C≡C—;
R6 and R10 are each hydrogen, halogen, alkyl or alkoxy;
R7 and R9 are each hydrogen, alkyl or halogen; and
R8 is hydrogen, hydroxyl, carboxy, alkylcarbonylamino, amino, aminosulfonyl, alkylsulfonyl, alkoxy, halogen, alkyl, alkylamino, acylamino, cyano, acyl, heteroaryl or heterocyclic;
and pharmaceutically acceptable salts thereof.
In one embodiment, the transcription factor modulating compound is a compound of formula I, wherein: R3 is cyano, L is —NHCO—, R6, R7, R9 and R10 are each hydrogen and R8 is acyl.
In one embodiment, R3 is nitro, L is —CH2 NHCO—, R6, R7, R9 and R10 are each hydrogen and R8 is halogen (e.g., fluorine).
In another embodiment, R3 is nitro, L is —C≡C—, R6, R7, R9 and R10 are each hydrogen and R8 is halogen (e.g., fluorine). In another embodiment, L is —NHCOCH2—, R6, R7, R9 and R10 are each hydrogen, R8 is halogen (e.g., fluorine).
In yet another embodiment, R3 is nitro, L is —NHCOCH2CH2—, R6, R7, R9 and R10 are each hydrogen and R8 is halogen (e.g., fluorine).
In a further embodiment, R3 is nitro, L is —NHCOCH═CH—, R6, R7, R9 and R10 are each hydrogen. R8 may be, for example, hydrogen, halogen (e.g., fluorine), substituted alkyl (e.g., trifluoromethyl), unsubstituted alkyl (e.g., methyl), alkoxy (e.g., methoxy), carboxy, acyl, heteroaryl (e.g., triazolyl or imidizolyl) or cyano.
In one embodiment, R3 is nitro, L is —NHCOCH═CH—, R7, R8, R9 and R10 are each hydrogen and R6 is alkoxy (e.g., methoxy).
In another embodiment, R3 is nitro, L is —NHCOCH═CH—, R6, R7 or R9 are each hydrogen and R8 and R10 are each halogen (e.g., fluorine) or alkoxy (e.g., methoxy).
In a further embodiment, R3 is nitro, L is —NHCOCH═CH—, R6, R7 and R10 are each hydrogen and R8 and R9 are each halogen (e.g., fluorine).
In yet another embodiment, R3 is nitro, L is —NHCO—, R6, R7, R9 and R10 are each hydrogen and R8 is hydrogen, alkoxy (e.g., methoxy), halogen (e.g., fluorine), alkyl (e.g., methyl), cyano, acyl, heterocyclic (e.g., imidazolyl, oxazolyl, triazolyl, morpholinyl or pyrazolyl), alkylcarbonylamino (e.g., —NHCOCH3), hydroxyl, aminosulfonyl (e.g., —SO2 NH2), alkylsulfonyl (e.g., —SO2 CH3) or amino (e.g., dialkylamino such as dimethylamino).
In one embodiment, R3 is nitro, L is —NHCO—, R6, R8, R9 and R10 are each hydrogen and R7 is halogen (e.g., fluorine) or alkyl (e.g., methyl).
In another embodiment, R3 is nitro, L is —NHCO—, R7, R8, R9 and R10 are each hydrogen and R6 is halogen (e.g., fluorine) or alkyl (e.g., methyl).
In another embodiment, R8 is an electron withdrawing or an electron donating group. In yet another embodiment, R2, R3, R4, and/or R5 comprise a lipophilic group. In another embodiment, R3 is a lipophilic group and R2, R4 and R5 are each hydrogen.
In one embodiment, the transcription factor modulating compound is a compound of formula I, wherein: R3 is cyano, L is —NHCO—, R6, R7, R9 and R10 are each hydrogen and R8 is acyl.
In one embodiment, R3 is nitro, L is —CH2 NHCO—, R6, R7, R9 and R10 are each hydrogen and R8 is halogen (e.g., fluorine).
In another embodiment, R3 is nitro, L is —C≡C—, R6, R7, R9 and R10 are each hydrogen and R8 is halogen (e.g., fluorine). In another embodiment, —NHCOCH2—, R6, R7, R9 and R10 are each hydrogen, R8 is halogen (e.g., fluorine).
In yet another embodiment, R3 is nitro, L is —NHCOCH2CH2—, R6, R7, R9 and R10 are each hydrogen and R8 is halogen (e.g., fluorine).
In a further embodiment, R3 is nitro, L is —NHCOCH═CH—, R6, R7, R9 and R10 are each hydrogen. R8 may be, for example, hydrogen, halogen (e.g., fluorine), substituted alkyl (e.g., trifluoromethyl), unsubstituted alkyl (e.g., methyl), alkoxy (e.g., methoxy), carboxy, acyl, heteroaryl (e.g., triazolyl or imidizolyl) or cyano.
In one embodiment, R3 is nitro, L is —NHCOCH═CH—, R7, R8, R9 and R10 are each hydrogen and R6 is alkoxy (e.g., methoxy).
In another embodiment, R3 is nitro, L is —NHCOCH═CH—, R6, R7 or R9 are each hydrogen and R8 and R10 are each halogen (e.g., fluorine) or alkoxy (e.g., methoxy).
In a further embodiment, R3 is nitro, L is —NHCOCH═CH—, R6, R7 and R10 are each hydrogen and R8 and R9 are each halogen (e.g., fluorine).
In yet another embodiment, R3 is nitro, L is —NHCO—, R6, R7, R9 and R10 are each hydrogen and R8 is hydrogen, alkoxy (e.g., methoxy), halogen (e.g., fluorine), alkyl (e.g., methyl), cyano, acyl, heterocyclic (e.g., imidazolyl, oxazolyl, triazolyl, morpholinyl or pyrazolyl), alkylcarbonylamino (e.g., —NHCOCH3), hydroxyl, aminosulfonyl (e.g., —SO2 NH2), alkylsulfonyl (e.g., —SO2 CH3) or amino (e.g., dialkylamino such as dimethylamino).
In one embodiment, R3 is nitro, L is —NHCO—, R6, R8, R9 and R10 are each hydrogen and R7 is halogen (e.g., fluorine) or alkyl (e.g., methyl).
In another embodiment, R3 is nitro, L is —NHCO—, R7, R8, R9 and R10 are each hydrogen and R6 is halogen (e.g., fluorine) or alkyl (e.g., methyl).
In another embodiment, R8 is an electron withdrawing or an electron donating group. In yet another embodiment, R2, R3, R4, and/or R5 comprise a lipophilic group. In another embodiment, R3 is a lipophilic group and R2, R4 and R5 are each hydrogen.
In one embodiment, the transcription factor modulating compound is a compound of formula II:
wherein:
R3a and R4a are each independently hydrogen, —NO2, —CN, —F, or —N(CH3)2
A is phenyl or heterocyclic;
La is —NHCO— or —NHCH═CH—; and
R8a is an electron-donating or an electron-withdrawing group and pharmaceutically acceptable salts thereof.
In another embodiment, the transcription factor modulating compound is a compound of formula III:
wherein:
Lb is —NHCO— or —NHCOCH═CH—; and
R8b is an electron-donating or an electron-withdrawing group and pharmaceutically acceptable salts thereof.
In one embodiment, the transcription factor modulating compounds of the invention are a compound of formula IV:
wherein:
R1c is —CH2 CO2H, —OCH2CO2 Et, —OCH2CH2 CO2H, —OCH2CH2 OH, —OCH2CN, —OCH2CH2CH3, —OCH3, —OH, —OCH2CH2 NH2 or hydrogen;
R2c is aryl,
R6c is hydrogen, —NO2, H, —COCH3, —CF3—F, —OCH3—CO2H, —CONH2, —CN, —N(CH3)2, —C(CH3)3, —SO2 CH3, —C(CH3)NOH, or
and pharmaceutically acceptable salts thereof.
In one embodiment, R6c is hydrogen, R1c is —OH or —OCH2CO2H and R2c is aryl (e.g., phenyl).
In another embodiment, R6c is —COCH3, —CF3, —F, —OCH3, —CO2H, —CONH2, —CN, —N(CH3)2, —C(CH3)3, —SO2 CH3 or —C(CH3)NOH, R1c is —OH and R2c is aryl (e.g., phenyl or furanyl).
In yet another embodiment, R6c is —NO2, R1c is —CH2CH2 OH, —OCH2CO2 Et, —OCH2CH2CH3, —OCH2CH2OH, —OCH2CN, —OCH2CH2 NH2 or —OCH3 and R2c is aryl (e.g., phenyl). Alternatively, R1c is —OH and R2c is
or aryl, such as, for example, furanyl, which may be substituted with phenyl, or phenyl which may be substituted at least one of an ortho, meta or para position. The phenyl may be substituted with alkoxy (e.g., phenoxy or methoxy), hydroxyl, amino, dialkylamino (e.g., dimethylamino), —COOH, halogen (e.g., bromine), aminoalkyl (e.g., aminomethyl), alkylcarbonylamino (e.g., methylcarbonylamino), arylcarbonylamino (e.g., furanylcarbonylamino or phenylcarbonylamino) or arylcarbonylaminoalkyl (e.g., phenylcarbonylaminomethyl). The phenylcarbonylamino substituent may be further substituted at least one of an ortho, meta or para position. Examples of suitable substituents include, for example, alkoxy (e.g. methoxy), halogen (e.g., fluorine or chlorine), dialkylamino (e.g., dimethylamino) or alkyl (e.g., t-butyl or methyl).
In one embodiment, the transcription factor modulating compounds of the invention are a compound of formula V:
wherein:
R1* is hydroxyl, OCOCO2H; a straight or branched C1-C5 alkyloxy group; or a straight or branched C1-C5 alkyl group;
A, B, D, E, W, X, Y and Z are each independently carbon or nitrogen; wherein: R2*, R3*, R4*, R5*, R6*, R7*, R8*, R9* are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime or halogen when A, B, D, E, W, X, Y and Z are carbon; or wherein: R2*, R3*, R4*, R5*, R6*, R7*, R8*, R9* are each independently absent or hydroxyl when A, B, D, E, W, X, Y and Z are nitrogen;
R10*, R11*, R12* and R13* are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime or halogen; and pharmaceutically acceptable salts, esters and prodrugs thereof;
provided that when A, B, C, D, E, W, X, Y and Z are each carbon, one of R6* R7*, R8*, R9* is not hydrogen.
In one embodiment, A, B, D, E, W, X, Y and Z are each carbon, R1* is hydroxyl, R2*, R4*, R5*, R10*, R11* and R12* are each hydrogen, R3* is nitro, R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl), R6* is halogen (e.g., fluorine) and R7*, R8* and R9* are hydrogen.
In another embodiment, A, B, D, E, W, X, Y and Z are each carbon, R1* is hydroxyl, R2*, R4*, R5*, R10*, R11* and R12* are each hydrogen, R3* is nitro, R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl), R6*, R7* and R8* are hydrogen, and R9* is halogen (e.g., fluorine).
In a further embodiment, A, B, D, E, W, X, Y and Z are each carbon, R1* is hydroxyl; R2*, R4*, R5*, R10*, R11* and R12* are each hydrogen, R3* is nitro, R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl), R6*, R8* and R9* are hydrogen, and R7* is substituted alkyl (e.g., morpholinylmethyl) or unsubstituted alkyl (e.g., methyl).
In yet another embodiment, A, B, D, E, W, X, Y and Z are each carbon, R1* is hydroxyl, R2*, R4*, R5*, R10*, R11* and R12* are each hydrogen, R3* is nitro, R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl), R6*, R7* and R9* are each hydrogen and R8* is alkoxy (e.g., methoxy).
In one embodiment, A, B, D, E, W, X, Y and Z are each carbon, R1* is hydroxyl, R2*, R4*, R5*, R10*, R11* and R12* are each hydrogen, R3* is nitro and R13* is aryl, such as alkyl substituted phenyl (e.g., 4-methylphenyl). In one embodiment, R6*, R8* and R9* are each hydrogen and R7* is alkyl (e.g., ethyl).
In another embodiment, A, B, D, W, X, Y and Z are each carbon, E is nitrogen, R1* is hydroxyl, R2*, R4*, R5*, R6*, R7*, R8*, R10*, R11* and R12* are hydrogen, R3* is nitro, R9* is absent and R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl or 2,4-fluorophenyl).
In a further embodiment, B, D, E, W, X, Y and Z are each carbon, A is nitrogen, R1* is hydroxyl, R2*, R4*, R5*, R7*, R8*, R9*, R10*, R11* and R12* are hydrogen, R6* is absent, R3* is nitro and R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl or 2,4-fluorophenyl).
In yet another embodiment, A, B, D, E, X, Y and Z are each carbon, W is nitrogen, R1* is hydroxyl, R2*, R4*, R7*, R8*, R9*, R10*, R11* and R12* are each hydrogen, R3* is nitro, R5* is absent, R6* is halogen (e.g., fluorine) and R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl).
In one embodiment, A, B, D, E, X, W, and Z are each carbon, Y is nitrogen, R1* is hydroxyl, R2*, R4*, R5*, R6*, R7*, R8*, R9*, R10*, R11* and R12* are each hydrogen, R3* is hydroxyl and R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl).
In another embodiment, A, B, D, E, X, Y and Z are each carbon, W is nitrogen, R1* is hydroxyl, R2*, R3*, R4*, R6*, R7*, R8*, R9*, R10*, R11* and R12* are each hydrogen, R5* is hydroxyl and R13* is aryl, such as halogen substituted phenyl (e.g., 4-fluorophenyl).
In a further embodiment, A, B, D, E, W, X and Z are each carbon, Y is nitrogen, R1* is hydroxyl, R2*, R4*, R5*, R6*, R7*, R8*, R9*, R10*, R11* and R12* are each hydrogen, R3* is absent and R13* is aryl (e.g., substituted phenyl, such as 4-fluorophenyl).
In one embodiment, the transcription factor modulating compounds of the invention include compounds of formula VI:
wherein:
R1a is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
R2a, R5a, R4a, R5a, R6a, R7a, R5a, R9a, R10a, R11a, R12a, R13a, R13b, R13c, R13d and R13e are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and esters, prodrugs and pharmaceutically acceptable salts thereof;
provided that when R1a is hydroxyl, R3a is nitro, R2a, R4a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13d, and R13e are hydrogen, then R13c is not hydrogen, fluorine, dimethylamino, cyano, hydroxyl, methyl or methoxy; and
provided that when R1a is hydroxyl, R3a is nitro, R2a, R4a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b and R13d are hydrogen, then R13c and R13e are not fluorine.
In one embodiment, R1a is hydroxyl and R3a is cyano and R2a, R4a, R5a, R6a, R7a, R8a, R9a R10a, R11a, R12a, R13a, R13b, R13c, R13d and R13e are each hydrogen.
In another embodiment, R1a is hydroxyl, R3a is cyano, R2a, R4a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13d and R13e are each hydrogen and R13c is halogen (e.g., fluorine), alkyl (e.g., methyl) or acyl.
In yet another embodiment, R1a is hydroxyl and R3a is nitro, R2a, R4a, R5a, R6a R7a, R5a, R9a, R11a, R12a, R13a, R13b, R13c, R13d and R13e are each hydrogen and R11a is aryl (e.g., phenyl), halogen (e.g., fluorine) or alkyl (e.g., methyl).
In another embodiment, R1a is hydroxyl, R3a is nitro, R2a, R2b, R4a, R5a, R6a, R7a R8a, R9a, R10a, R12a, R13a, R13b, R13d, and R13e are each hydrogen, R13c is halogen (e.g., fluorine) and R11a is alkyl (e.g., hydroxyethyl or piperazinylmethyl).
In a further embodiment, R1a is hydroxyl, R3a is nitro, R2a, R4a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13d and R13e are each hydrogen and R13c is alkyl (e.g., isopropyl), acyl or heteroaryl (e.g., triazole, imidazole or oxazole).
In one embodiment, R1a is hydroxyl and R3a is nitro, R2a, R4a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b and R13d are each hydrogen and R13c and R13e are each alkoxy (e.g., methoxy).
In another embodiment, R1a is hydroxyl and R3a is nitro, R2a, R4a, R5a, R6a, R7a R5a, R9a, R10a, R11a, R12a, R13a, R13d and R13e are each hydrogen and R13b is alkyl (e.g., alkyl substituted with phosphonic acid or phosphonic acid dialkyl ester) and R13e is halogen (e.g., fluorine).
In one embodiment, R1a is hydroxyl, R3a is nitro, R13c is halogen (e.g., fluorine), R2a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13d and R13e are each hydrogen and R4a is alkylamino (e.g., dimethylamino or dialkylaminoalkylamino), alkyl (e.g., methyl) or alkoxy (e.g., ethoxy, phosphonic acid substituted alkoxy, ether substituted alkoxy, alkylamino substituted alkoxy, or heterocyclic substituted alkoxy, for example, morpholine substituted alkoxy or piperazine substituted alkoxy) or halogen (e.g., fluorine).
In yet another embodiment, R1a is hydroxyl, R3a is nitro, R13c is halogen (e.g., fluorine), R4a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13d and R13e are each hydrogen and R2a is alkylamino (e.g., alkylaminoalkylamino, such as dimethylaminoethylamino).
In a further embodiment, R1a is a substituted or unsubstituted straight or branched C1-C5 alkyloxy group (e.g., phosphonic acid substituted alkoxy or phosphonic acid dialkyl ester alkoxy), R3a is nitro, R13c is halogen (e.g., fluorine), R2a, R4a, R5a, R6a R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13d and R13e are each hydrogen.
In yet another embodiment, R1a is hydroxyl, R3a is nitro, R2a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13d and R13e are hydrogen, R13c is acyl and R4a is alkoxy (e.g., piperazinyl substituted alkoxy or morpholine substituted alkoxy).
In a further embodiment, R1a is hydroxyl, R3a is heteroaryl (e.g., imidazolyl or pyrazolyl), R3a, R4a, R5a, R6a, R7a, R8a, R9a, R10a, R11a, R12a, R13a, R13b, R13d and R13e are each hydrogen, and R3c is halogen (e.g., fluorine).
In another embodiment, the transcription factor modulating compounds of the invention include compounds of formula VII:
wherein:
R14 is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
G, J, K, L, M, Q, T and U are each independently carbon or nitrogen;
wherein: R5, R16, R17, R18, R19, R20, R21, R22, R23 and R24 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, absent, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen, when G, J, K, L, M, Q, T and U are carbon; or R5, R16, R17, R18, R19, R20, R21, R22, R23 and R24 are each independently absent or hydroxyl when G, J, K, L, M, Q, T and U are nitrogen;
R23 and R24 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, absent, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and pharmaceutically acceptable salts, esters and prodrugs thereof;
provided that when G, J, K, L, M, Q, T and U are each carbon, one of R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24, are not hydrogen.
In one embodiment, G, J, K, L, M, Q, T and U are each carbon, R14 is hydroxyl, R16 is nitro, R24 is aryl (e.g., phenyl, such as acyl substituted phenyl), R15, R17, R18, R19, R20 and R21 are hydrogen and R22 is halogen (e.g., fluorine).
In another embodiment, G, J, K, L, M, Q, T and U are each carbon, R14 is hydroxyl, R16 is nitro, R24 is aryl (e.g., phenyl, such as acyl substituted phenyl), R15, R17, R18, R19, R21 and R22 are hydrogen and R20 is alkyl (e.g., methyl or ethyl).
In yet another embodiment, G, J, K, L, M, Q, T and U are each carbon, R14 is hydroxyl, R16 is nitro, R24 is aryl (e.g., phenyl, such as acyl substituted phenyl), R15, R17, R18, R19, R20 and R22 are hydrogen and R21 is alkoxy (e.g., methoxy).
In a further embodiment, G, J, K, L, M, Q, T and U are each carbon, R14 is hydroxyl, R16 is nitro, R24 is aryl (e.g., phenyl, such as halogen substituted phenyl, for example, 4-fluorophenyl), R15, R17, R18, R19, R20 and R22 are hydrogen and R21 is halogen (e.g., fluorine) or alkoxy (e.g., methoxy or phosphonic acid substituted alkoxy).
In one embodiment, G, J, K, L, M, Q, T and U are each carbon, R14 is hydroxyl, R16 is nitro, R24 is aryl (e.g., phenyl, such as halogen substituted phenyl, for example, 4-fluorophenyl), R15, R17, R18, R19, R21 and R22 are hydrogen and R20 is alkyl (e.g., ethyl).
In one embodiment, G, J, K, L, Q, T and U are each carbon, M is nitrogen, R14 is hydroxyl, R16 is nitro, R15, R17, R18, R20, R21, R22 and R23 are each hydrogen, R19 is absent and R24 is aryl, such as, for example, substituted phenyl, and in particular, halogen substituted phenyl (e.g., 4-fluorophenyl) or acyl substituted phenyl (e.g., 4-acyl substituted phenyl).
In another embodiment, G, J, K, L, M, Q and T are each carbon, U is nitrogen, R14 is hydroxyl, R16 is nitro, R15, R17, R18, R19, R20, R21, and R23 are each hydrogen, R22 is absent and R24 is aryl, such as, for example, phenyl such as halogen substituted phenyl (4-fluorophenyl).
In yet another embodiment, wherein: J, K, L, M, Q, T and U are each carbon, G is nitrogen, R14 is hydroxyl, R16 is nitro, R15, R17, R19, R20, R21, R22 and R23 are each hydrogen, R18 is absent and R24 is aryl, such as, for example, phenyl, which may be substituted with halogen (e.g., 4-fluorophenyl) or acyl (e.g., 4-acylphenyl).
In one embodiment, G, J, L, M, Q, T and U are each carbon, K is nitrogen, R14 is hydroxyl, R16 is absent, R15, R17, R18, R19, R20, R21, R22 and R23 are each hydrogen and R24 is aryl, such as, for example, phenyl, which may be substituted with halogen (e.g., 4-fluorophenyl).
In one embodiment, the transcription factor modulating compounds of the invention include compounds of formula VIII:
wherein:
R14a is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
R15a, R16a, R17a, R18a, R19a, R20a, R21a, R22a, R23a and R24a, R24b, R24c, R24d and R24e are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and esters, prodrugs and pharmaceutically acceptable salts thereof;
provided that at least two of R24a, R24b, R24c, R24d and R24e are not hydrogen.
In one embodiment, R14a is hydroxyl, R15a, R17a, R18a, R19a, R20a, R21a, R22a, R23a R24a, R24b and R24e are hydrogen, R16a is nitro and R24c and R24d are joined to form a ring (e.g., a six membered ring, such as cyclohexanone).
In another embodiment, R14a is hydroxyl, R15a, R17a, R18a, R19a, R20a, R21a, R22a R23a, R24a, R24b and R24e are hydrogen, R16a is nitro and R24c is halogen (e.g., fluorine) and R24d is halogen (e.g., fluorine), alkyl (e.g., methyl) or alkoxy (e.g., methoxy).
In yet another embodiment, R14a is hydroxyl, R15a, R17a, R18a, R19a, R20a, R21a R22a, R23a, R24a, R24b and R24d are hydrogen, R16a is nitro, R24c is halogen (e.g., fluorine) and R24e is alkoxy (e.g., methoxy).
In another embodiment, the transcription factor modulating compounds of the invention include compounds of formula IX:
wherein:
R25 is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
R26, R27, R28, R29, R30, R31, R32, R33, R34, R35a, R35b, R35c, R35d, and R35e are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and esters, prodrugs and pharmaceutically acceptable salts thereof;
provided that at least two of R26, R27, R28 and R29 are not hydrogen.
In one embodiment, R25 is hydroxyl, R26, R29, R30, R31, R32, R33, R34, R35a, R35b R35d, and R35e are each hydrogen, R27 is nitro, R28 is alkyl (e.g., methyl) and R35c is acyl or heteroaryl (e.g., oxazole).
In yet another embodiment, the transcription factor modulating compounds of the invention include compounds of formula X:
wherein:
R25′ is a substituted straight or branched C1-C5 alkyloxy group;
R26′, R27′, R28′, R29′, R30′, R31′, R32′, R33′, R34′, R35a′, R35b′, R35c′, R35d′, and R35e′ are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen; and esters, prodrugs and pharmaceutically acceptable salts thereof.
In one embodiment, R26′, R28′, R29′, R30′, R31′, R32′, R33′, R34′, R35a′, R35b′, R35d′ and R35e′ are each hydrogen, R27′ is nitro, R35c′ is halogen (e.g., fluorine) and R25′ phosphonic acid substituted alkoxy, alkyl phosphonic acid substituted alkoxy, carboxylic acid substituted alkoxy or alkylamino substituted alkoxy.
In a further embodiment, the transcription factor modulating compounds of the invention include compounds of formula XI:
wherein:
R36 is hydroxyl;
R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each independently hydrogen, alkyl alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen;
R38 is cyano, nitro, oxime, alkyloxime, aryloxime, heteroaryl, amino-oxime, or aminocarbonyl;
R46c is hydrogen, acyl, fluorine, pyrizinyl, pyridinyl, cyano, imidazolyl, dialkylaminocarbonyl or dialkylamino; and esters, prodrugs and pharmaceutically acceptable salts thereof;
provided that when R38 is nitro and R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, then R46c is not dialkylamino, acyl or hydrogen; and
provided that when R38 is cyano and R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, then R46c is not dialkylamino.
In one embodiment, R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, and R38 is cyano and R46c is acyl, fluorine, cyano or imidazolyl.
In another embodiment, R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, and R38 is amino-oxime and R46c is fluorine.
In a further embodiment, R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, and R38 is nitro and R46a is pyrizinyl, pyridinyl or dialkylaminocarbonyl (e.g., dimethylaminocarbonyl).
In another embodiment, R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, and R38 is aminocarbonyl and R46c is halogen (e.g., fluorine).
In one embodiment, R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, and R38 is oxime and R46c is dialkylamino (e.g., dimethylamino).
In another embodiment, R37, R39, R40, R41, R42, R43, R44, R45, R46b, R46c, R46d, and R46e are each hydrogen, and R38 is nitro and R46a is hydroxyl.
In another embodiment, R37, R39, R40, R41, R42, R43, R44, R45, R46a, R46b, R46d, and R46e are each hydrogen, and R38 is heteroaryl (e.g., imidazolyl or pyrazolyl) and R46c is acyl.
In one embodiment, the transcription factor modulating compounds of the invention include compounds of formula XII:
wherein:
R47 is hydroxyl, OCOCO2H, a straight or branched C1-C5 alkyloxy group, or a straight or branched C1-C5 alkyl group;
R48, R49, R50, R51, R52 and R53 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy, aryloxy, heteroaryloxy, alkoxycarbonyl, aryloxycarbonyl heteroaryloxycarbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino, amino, alkylamino, arylamino, CO2H, cyano, nitro, CONH2, heteroarylamino, oxime, alkyloxime, aryloxime, amino-oxime, or halogen;
Ar is aryl; and pharmaceutically acceptable salts, esters and prodrugs thereof.
In one embodiment, R47 is hydroxyl, R48, R50, R51 and R52 are each hydrogen, Ar is furanyl, and R53 is alkenyl, which may be substituted with phenyl, such as, for example, halogen substituted phenyl (e.g., fluorophenyl).
In one embodiment, the transcription factor modulating compounds is a compound of formula XIII:
wherein:
R1d is hydrogen, —OH, —OCH2-aryl, —CH2CH2 CO2H, —OCH2CO2CH2CH3, —OCH2CN, —OCH2CH2 NH2, —OCH3, —OCH2CH2N+(CH3)3, —OCH2COOH, —OCH2CH2CH3, —OCH2CH2 OH, —OCH2P(O)(OH)2 or —OCH2P(O)(OCH2CH3)2;
R2d is hydrogen or —NR2daR2db;
R2da and R2db are each independently hydrogen, alkyl or aminoalkyl;
Xd is CR3d, N or NO;
R3de is absent when Xd is N or NO—NO2, hydrogen, acyl, halogen, alkoxy, —CO2H, —CONR3daR3db; cyano, —NR3dcR3dd, alkyl, —SO2R3de, —C(R3df)NOH, heterocyclic or heteroaryl;
R3da and R3db are each independently hydrogen or alkyl;
R3dc and R3dd are each independently hydrogen, alkyl or substituted carbonyl;
R3de and R3df are each independently alkyl or amino;
R4d is hydrogen, alkoxy, —NR4daR4db, alkyl, halogen, —SO2R4d c or —CO2H;
R4da and R4db are each independently hydrogen, alkyl or aminoalkyl;
R4dc is alkyl or amino;
Zd is CH, N or NO;
when Ld is present or
when Ld and R16d are each absent;
Yd is N or CR6d
Wd is N or CR8d
R6d is absent when Yd is N, or hydrogen, alkyl, amino, —CO2H, —OCH2P(O)(OH)2 or alkyl;
R8d is absent when Wd is N, or hydrogen, alkyl, amino, —CO2H, —OCH2P(O)(OH)2 or alkyl;
R7d and R9d are each independently hydrogen, alkyl, amino, —CO2H, —OCH2P(O)(OH)2 or alkyl;
Ad is O, NR10d or S;
R10d is hydrogen or alkyl;
Ld is absent, or Ld is hydrogen or unsubstituted phenyl when R16d is absent, or Ld is —O—, —SO—, —SO2—, —OCH2—, —CH2—, —NR15d,
n is an integer between 0-2;
Dd and Ed are each independently NR17d; O or S
Jd is N or CR1a;
Gd is N or CR19d;
R11d is hydrogen or alkyl;
R18d is absent when Jd is N or hydrogen or alkyl;
R19d is absent when Gd is N or hydrogen or alkyl;
R12d and R13d are each independently hydrogen, alkyl, halogen or aryl;
R15d is hydrogen or alkyl;
R16d is hydrogen, alkoxy, hydroxyl, amino, alkyl, —NO2 or halogen when Ld is absent; or R16d is
when Ld is present;
Kd is CR20d or N;
Md is CR23d or N;
R20d is absent when Kd is N or hydrogen, alkyl, halogen, alkoxy or hydroxyl;
R21d is hydrogen, halogen or alkyl;
R22d is hydrogen, heteroaryl, halogen, alkoxy, cyano, acyl, —SO2R22da heterocyclic, —COOH, hydroxyl, —CF3, alkyl, amino, CO2H, aminocarbonyl or
R22da is amino or alkyl;
R23d is absent when Md is N or hydrogen, halogen, alkyl or alkoxy; or R22d and
R23d together with the carbon atoms to which they are attached are joined to form a 5- or 6-membered ring;
R24d is hydrogen, halogen or alkoxy; and pharmaceutically acceptable salts thereof; and pharmaceutically acceptable salts thereof.
In one embodiment, Ld and R16d are absent; Xd is CR3d; Zd is CH; R2d and R4d are hydrogen; R1d is —OH; R3d is —NO2 and Ard is
In another embodiment, Ard is
Ad is O; Xd is CR3d; Zd is CH and R2d and R4d are each hydrogen; R1d is —OH; Ld is hydrogen; R3d is —CONR3daR3db or —NO2; and R3da and R3db are each hydrogen.
In one embodiment, Ard is
Ad is O; Xd is CR3d; Zd is CH and R2d and R4d are each hydrogen; R1d is —OH; Ld is hydrogen; R3d is —NO2; Ld is —CH2— and R16d is hydrogen.
In one embodiment, Ard is
Ad is O; Xd is CR3d; Zd is CH and R2d and R4d are each hydrogen; R1d is —OH; Ld is hydrogen; R3d is —NO2. Alternatively, Ld is unsubstituted phenyl and R16d is hydrogen.
In one embodiment, Ard is
Ld is hydrogen; Xd is CR3d; Yd is CR6d and Wd is CR8d; R2d and R4d are each hydrogen; Zd is N; R3d is hydrogen and R1d is —OCH2-aryl (e.g., in which aryl is phenyl, such as alkyl substituted phenyl, for example 4-methylphenyl), and R6d, R7d, R8d and R9d are each hydrogen.
In one embodiment, Ard is
Ld is hydrogen; Xd is CR3d; Yd is CR6d and Wd is CR8d; R2d and R4d are each hydrogen; Zd is CH; R1d is —OH and R3d, R6d, R7a, R8d and R9d are each hydrogen.
In one embodiment, Ard is
Ld is hydrogen; Xd is CR3d; Yd is CR6d and Wd is CR8d; R2d and R4d are each hydrogen; Zd is CH, R3d is —NO2, R6d, R7d, R8d and R9d are each hydrogen and R1d is —OH, —OCH2COOCH2CH3, —OCH2CH2 COOH, —OCH2COOH, —OCH2CH2CH3, —OCH2CH2 OH, —OCH2CN, —OCH2CH2NH2 or —OCH3.
In one embodiment, Ard is
Ld is hydrogen; Xd is CR3d; Yd is CR6d and Wd is CR8d; R2d and R4d are each hydrogen; Zd is CH; R1d is —OH; R6d, R7d, R8d and R9d are each hydrogen; R3d is acyl, alkyl (e.g., t-butyl or halogen substituted alkyl such as —CF3), halogen (e.g., fluorine), alkoxy (e.g., alkoxy), —CO2H, —CONR3daR3db, —CN, —NR3dcR3dd, —NO2, —SO2R3de or —C(R3df)NOH; R3da and R3db are each hydrogen; R3dc and R3dd are each alkyl (e.g., methyl); R3de is alkyl (e.g., methyl) and R3df is alkyl (e.g., methyl).
In one embodiment, Ard is
Ld is hydrogen; Xd is CR3d; Yd is CR6d and Wd is CR8d; R2d and R4d are each hydrogen; Zd is CH; R1d is —OH; R3d is —NO2; R7d R8d and R9d are each hydrogen; R6d is amino (e.g., carbonylamino, for example, aryl substituted carbonylamino such as furanyl substituted carbonylamino or alkyl substituted carbonylamino, such as methyl substituted carbonylamino).
In one embodiment, Ard is
Ld is hydrogen; Xd is CR3d; Yd is CR6d and Wd is CR8d; R2d and R4d are each hydrogen; Zd is CH; R1d is —OH; R3d is —NO2; R6d R8d and R9d are each hydrogen; and R7d is amino (e.g., —NH2 or dialkylamino, such as dialkylamino, for example, dimethylamino; carbonylamino, such as alkyl substituted carbonylamino, for example, methyl substituted carbonylamino), —CO2H or alkyl (e.g., aminoalkyl, for example, aminomethyl).
In one embodiment, Ld is absent; Ard is
Xd is CH3d; Yd is CR6d; Wd is CR8d and Zd is CH; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R1d is —OH; R3d is —NO2; and R16d is alkoxy (e.g., methoxy), amino (e.g., —NH2, dimethylamino or carbonylamino, alkyl substituted carbonylamino, for example, methyl substituted carbonylamino) or halogen (e.g., bromine).
In one embodiment, Ld is absent; Ard is
Xd is CH3d; Yd is CR6d; Wd is CR8d and Zd is CH; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R1d is —OH; R3d is —NR3dcR3dd; R3dc is hydrogen; R16d is —NO2; and R3dd is substituted carbonyl (e.g., substituted carbonyl, such as para-fluorophenyl)
In one embodiment, Ard is
Yd is CR6d and W is CR8d; Xd is CR3d and Zd is CH; Ld is —O—; R1d is —OH; R3d is —NO2 and R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d; Md is CR23d and R20d, R21d, R22d R23d and R24d are each hydrogen.
In one embodiment Ad is
R2d, R4d, R6d, R7d, R8d, R9d and R11d are each hydrogen; R1d is —OH; R3d is
Kd is CR20d and Md is CR23d; R20d R21d, R23d and R24d are each hydrogen and R22d is hydrogen or halogen (e.g., fluorine).
In one embodiment, Ard is
n is 1; R2d, R4d, R6d, R7d, R8d, R9d and R1d are each hydrogen; R1d is —OH; R3d is —NO2; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d, R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine).
In one embodiment, Ard is
R2d, R4d, R6d, R7d, R8d, R9d and R11d and are each hydrogen; R1d is —OH; R3d is —NO2; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d, R23d and R24d are each hydrogen and R22d is halogen (e.g., fluorine).
In one embodiment Ard is
R2d, R4d, R6d, R7d, R8d, R9d and R11d are each hydrogen; R1d is —OH; R3d is —NO2; R16d is
Kd is CR20d and Md is CR23d; and R20d, R21d, R22d, R23d and R24d are each hydrogen.
In one embodiment Ard is
n is 0; R1d is —OH; R3d is —NO2; R11d is hydrogen; R2d and R4d are each hydrogen; R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d, R23d and R24d are each hydrogen and R22d is hydrogen, alkoxy (e.g., methoxy), halogen (e.g., chlorine or fluorine), amino (e.g., dialkylamino, such as dimethylamino, or carbonylamino, such as alkyl substituted carbonylamino, for example methyl substituted carbonylamino), alkyl (e.g., methyl or isopropyl), cyano, —SO2R22da, acyl, heterocyclic (e.g., morpholinyl), heteroaryl (e.g., pyrazol, isoxazolyl, imidazolyl, triazolyl, pyramidinyl or pyridinyl), —CO2H, hydroxyl,
or aminocarbonyl (e.g., dimethylaminocarbonyl); and R22da is amino or alkyl (e.g., methyl).
In one embodiment, Ard is
n is 0; R1d is —OH; R3d is —NO2; R1d is hydrogen; R2d and R4d are each hydrogen; R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R22d, R23d and R24d are each hydrogen and R2d is halogen (e.g., fluorine or chlorine), alkyl (e.g., methyl) or hydroxyl.
In one embodiment, Ard is
n is 0; R1d is —OH; R3d is —NO2; R11d is hydrogen; R2d and R4d are each hydrogen; R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d and R23d are each hydrogen; R22d and R24d are each alkoxy (e.g., methyl) or R22d is halogen (e.g., fluorine) and R24d is alkoxy (e.g., methoxy).
In one embodiment Ard is
n is 0; R1d is —OH; R3d is —NO2; R11d is hydrogen; R2d and R4d are each hydrogen; R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d and R24d are each hydrogen; R22d and R23d together with the carbon atoms to which they are attached form a 6-membered ring (e.g., a cyclohexanone ring) or R22d is halogen (e.g., fluorine) and R23d is alkyl (e.g., methyl) or alkoxy (e.g., methoxy).
In one embodiment, Ard is
n is 0; R1d is —OH; R3d is —NO2; R11d is hydrogen; R2d and R4d are each hydrogen; R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R23d and R24d are each hydrogen and R21d and R22d are each halogen (e.g., fluorine).
In one embodiment, Ard is
n is 0; R1d is —OH; R11d is hydrogen; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d R20d, R21d, R23d and R24d are each hydrogen; R3d is cyano and R22d is halogen (e.g., fluorine), acyl or cyano.
In one embodiment, Ard is
n is 0; R1d is —OH: R11d is hydrogen; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d, R23d and R24d are each hydrogen; R3d is —C(R3df)NOH; R3df is amino and R22d is halogen (e.g., fluorine).
In one embodiment Ard is
n is 0; R1d is hydrogen; R1d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d, R23d and R24d are each hydrogen; R3d is —C(R3df)NOH; R3df is alkyl (e.g., methyl) and R22d is amino (e.g., dialkylamino such as dimethylamino).
In one embodiment, Ard is
n is 0; R1d is OH; R11d is hydrogen; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d, R23d and R24d are each hydrogen; CONR3daR3db; R3da and R3db are each hydrogen and R22b is halogen (e.g., fluorine).
In one embodiment Ard is
n is 0; R3d is —NO2; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d is hydrogen; R16d is
Kd is CR20d and Md is CR23d; R20d, R21d, R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine); and R1d is —OCH2COOH, —OCH2CH2N+(CH3)3, —OCH2P(O)(OH)2 or —OCH2P(O)(OCH2CH3)2.
In one embodiment, Ard is
Kd is CR20d; Md is CR23d; R3d is —NO2; R2d, R4d, R6d, R7d, R8d, R9d and R11d are each hydrogen; Zd is N and R22d is halogen (e.g., fluorine) or acyl.
In one embodiment, Ard is
Kd is CR20d; Md is CR23d; Zd is CH; R2d, R4d, R11d, R20d, R21d, R23d and R24d are each hydrogen; R3d is —NO2; R6d, R8d and R9d are each hydrogen; R7d is alkyl (e.g., ethyl); and R22d is halogen (e.g., fluorine) or acyl.
In one embodiment, Ard is
Kd is CR20d; Md is CR23d; Zd is CH; R2d, R4d, R11d, R20d, R21d, R23d and R24d are each hydrogen; R3d is —NO2; R6d, R7d and R8d are each hydrogen; R9d is alkoxy (e.g., methoxy), halogen (e.g., fluorine) or —OCH2P(O)(OH)2 and R22d is halogen (e.g., fluorine) or acyl.
In one embodiment, Ard is
Kd is CR20d; Md is CR23d; Zd is CH; R2d, R4d, R11d, R20d, R21d, R23d and R24d are each hydrogen; R3d is —NO2; R6d, R7d and R9d are each hydrogen: R8d is halo en (e.g., fluorine) and R22d is acyl.
In one embodiment, Ard is
Xd is CR20d and Md is CR23d; R2d, R4d, R7d, R9d, R11d, R20d, R21d, R23d and R24d are each hydrogen; Wd is CR8d and R8d is hydrogen; R6d is absent; Yd is N; and R22d is halogen (e.g., fluorine) or acyl.
In one embodiment, Ard is
Kd is CR20d and Md is —CR23d; R2d, R4d, R7d, R9d, R11d, R20d, R21d, R23d and R24d are each hydrogen; Yd is CR6d and R6d is hydrogen; R8d is absent and Wd is N; R22d is halogen (e.g., fluorine).
In one embodiment, Ard is
Kd is CR20d and Md is CR23d; R2d, R6d, R7d, R9d, R11d, R20d, R21d, R23d and R24d are each hydrogen; Wd is CR8d and R8d is hydrogen; Yd is CR6d and Wd is CR8d; R4d is alkyl (e.g., methyl) and R22d is acyl or heteroaryl (e.g., isoxazolyl).
In one embodiment, Ard is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d, R9d, R11d, R20d, R21d, R23d and R24d are each hydrogen; R3d is heteroaryl (e.g., imidazolyl or pyrazolyl) and R22d is acyl.
In one embodiment, Ard is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d, R9d, R11d, R20d, R21d, R23d and R24d are each hydrogen; R3d is cyano; and R22d is heteroaryl (e.g., imidazolyl).
In one embodiment, Ard is
Kd is CR20d; Md is CR23d; R3d is absent and Xd is N; R2d, R4d, R6d, R7d, R8d, R9d, R11d, R20d, R21d, R23d and R24d are each hydrogen and R22d is halogen (e.g., fluorine)
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R20d, R21d, R23d and R24d are each hydrogen; R1d is —OH; R3d is —NO2 and R22d is heteroaryl (e.g., isoxazolyl, triazolyl, imidazolyl), hydrogen, halogen (e.g., fluorine), alkyl (e.g., methyl or halogen substituted alkyl, such as trifluoromethyl), alkoxy (e.g., methoxy), cyano, hydroxyl, acyl or —SO2R22da and R22da is alkyl (e.g., methyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R1d is —OH; R3d is —NO2; R21d, R22d, R23d and R24d are each hydrogen; and R20d is alkoxy (e.g., methoxy).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R1d is —OH; R3d is —NO2; R20d, R21d and R23d are each hydrogen and R22d and R24d are each halogen (e.g., fluorine).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R1d is —OH; R3d is —NO2; R20d, R21d and R24d are each hydrogen and R22d and R23d are each halogen (e.g., fluorine).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R1d is —OH; R22d is acyl; R20d, R21d; R23d and R24d are each hydrogen; R3d is halogen (e.g., fluorine or bromine), cyano, —SO2R3de, —CF3, hydrogen, acyl or —CO2H; and R3de is amino or alkyl (e.g., methyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R1d is —OH; R20d, R21d; R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine); R3d is heteroaryl (e.g., pyrazolyl or imidizolyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R20d, R21d; R23d and R24d are each hydrogen; R1d is —OH; R3d is cyano; and R22d is hydrogen, halogen (e.g., fluorine) or alkyl (e.g., methyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d and R13d are each hydrogen; R20d R21d, R23d and R24d are each hydrogen; R1d is —OH and R3d is NO2; R22d is hydrogen; and R12d is alkyl (e.g., methyl), halogen (e.g., fluorine) or aryl (e.g., phenyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d and R13d are each hydrogen; R20d R21d, R23d and R24d are each hydrogen; R1d is —OH and R3d is NO2; R22d is halogen (e.g., fluorine); R12d is alkyl (e.g., heterocyclic substituted alkyl, such as piperazinylmethyl, or hydroxyalkyl, such as hydroxyethyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R3d is —NO2 and R1d is —OH; R6d, R7d, R8d, R9d, R11d, R12d, R13d, R20d, R21d, R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine) or acyl; R2d is hydrogen; R4d is halogen (e.g., fluorine) alkyl, (e.g., methyl), alkoxy (e.g., ethoxy, morpholine substituted ethoxy, piperazinyl substituted ethoxy, phosphate substituted alkoxy, dimethylaminoethoxy or methoxyethoxyethoxy) or —NR4daR4db; and R4da and R4db are each alkyl (e.g., methyl or dimethylaminoethyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R3d is —NO2 and R1d is —OH; R6d, R7d, R8d, R9d, R11d, R12d, R13d, R20d, R21d, R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine) or acyl; R4d is hydrogen; R2d is —NR2daR2db and R2da and R2db are each alkyl (e.g., methyl or dimethylaminoethyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R20d, R21d, R23d and R24d are each hydrogen; R3d is —NO2; R22d is halogen (e.g., fluorine) and R1d is —OCH2P(O)(OH)2 or —OCH2P(O)(OCH2CH3)2.
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R8d and R9d are each hydrogen; R11d, R12d and R13d are each hydrogen; R1d is —OH and R3d is NO2; R20d, R21d, R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine); and R7d is alkyl (e.g., morpholinyl substituted methyl or methyl).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R11d, R12d and R13d are each hydrogen; R1d is —OH and R3d is NO2; R20d, R21d R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine); R7d, R8d and R9d are each hydrogen and R6d is halogen (e.g., fluorine).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R11d, R12d and R13d are each hydrogen; R1d is —OH and R3d is NO2; R20d, R21d R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine); R6d, R7d and R8d are each hydrogen and R9d is alkoxy (e.g., methoxy).
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R1id, R12d and R13d are each hydrogen; R1d is —OH and R3d is NO2; R20d, R21d R23d and R24d are each hydrogen; R22d is halogen (e.g., fluorine); R6d, R7d and R9d are each hydrogen and R8d is halogen (e.g., fluorine).
In one embodiment, Ld is
Kd is —CR20d and Md is CR23d; Xd is CR3d, Zd is NO or N, Yd is CR6d and Wd is CR8d; R1d is —OH; R2d, R3d, R4d, R6d, R7d, R8d, R9d, R11d, R12d, R13d, R20d, R21d R23d and R24d are each hydrogen; and R22d is halogen (e.g., fluorine).
In one embodiment, Ld is
Kd is —CR20d and Md is CR23d; R3d is absent; Xd is —NO or N, Zd is CH, Yd is CR6d and Wd is CR8d; Rd is —OH; R2d, R4d, R6d, R7d, R8d, R9d, R11d, R12d, R13d, R20d, R21d, R23d and R24d are each hydrogen; and R22d is halo en (e.g., fluorine)
In one embodiment, Ld is
Xd; is CR3d and Zd is CH; R6d is absent; Yd is N and Wd is CR8d; R2d, R4d, R7d, R8d, R9d, R11d, R12d, R13d are each hydrogen; R1d and —OH and R3d is —NO2; R20d, R21d, R23d and R24d are each hydrogen and R22d is halogen (e.g., fluorine). Alternatively, R20d and R22d are each halogen (e.g., fluorine) and R21d, R23d and R24d are each hydrogen.
In one embodiment, Ld is
Xd is CR3d and Zd is CH; Yd is —CR6d and R8d is absent; Wd is N; R1d is —OH and R3d is —NO2; R2d, R4d, R6d, R7d, R9d, R11d, R12d, R13d are each hydrogen; and R20d, R21d, R23d and R24d are each hydrogen and R22d is halogen (e.g., fluorine). Alternatively, R20d and R22d are each halogen (e.g., fluorine) and R21d, R23d and R24d are each hydrogen.
In one embodiment, Ld is
Kd is CR20d and Md is CR23d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; R12d, R13d, R20a, R21a, R23a and R24a are each hydrogen; R22d is acyl; and R11d is alkyl (e.g., carbonyl substituted alkyl, such as —CH2 COOH or aminocarbonylmethyl); R1d is —OH and R3d is —NO2 or cyano.
In one embodiment, Ld is
Kd is CR20d and Md is N; R2d, R2d, R4d, R6d, R7d, R8d, R9d, R11d, R12d, R13d R20d, R21d, R23d and R24d are each hydrogen; R1d is —OH; R3d is —NO2; R22d is acyl, heteroaryl (e.g., imidazolyl) or alkyl (e.g., halogen substituted alkyl, such as trifluoromethyl).
In one embodiment, Ld is
Kd is CR20d and Md is N; R2d, R2d, R4d, R6d, R7d, R8d, R9d, R11d, R12d, R13d R20d, R21d, R23d and R24d are each hydrogen; R1d is —OH; R3d is cyano and R22d is heteroaryl (e.g., imidazolyl).
In one embodiment, Ld is
Xd is CR3d, Z is CH; Kd is CR20d and Md is CR23d; R2d, R4d, R11d, R12d, R13d, R20d, R21d, R23d and R24d are each hydrogen; R1d is —OH, R3d is —NO2 and R22d is halogen (e.g., fluorine)
In one embodiment, Ld is
R16d is; Xd is CR3d, Z is CH; Zd is CH; Yd is CR6d Kd is —CR20d and Md is CR23d; R1d is —OH and R3d is —NO2; R2d, R4d, R6d, R7d, R8d, R8d, R9d, R11d R20d, R21d, R23d and R24d are each hydrogen and R22d is halogen (e.g., fluorine).
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH, —OCH2CO2CH2CH3, —OCH2CH2CH3, —OCH2CH2 OH, —OCH2CO2H, —OCH2CN, —OCH2CH2 NH2, —OCH2CH2 CO2H or —OCH3.
In one embodiment, when R3d is —CN; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OCH2CO2H.
In one embodiment, when R3d is F; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OCH2CO2H.
In one embodiment, when R3d is H; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH or —CH2CO2H.
In one embodiment, when R3d is Cl; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OCH2CH2CH2CH3 or —OCH2CO2H.
In one embodiment, when R3d is acyl, methoxy, —CONH2, —CO2H or t-butyl; Xd is CR3d; Wd is CH; Ard is
Yd is CR6d; Zd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OCH2CO2H.
In one embodiment, when R1d is —OH; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R3d is not acyl, —CF3, F, methoxy, —CO2H, —CONH2, —NHCOCH3, cyano, dimethylamino, t-butyl, —SO2 CH3, SO2 NHCH2 furanyl, or —C(CH3)NOH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R4d is —NH2, R2d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OCH2CH2CH2CH3 or —OCH2CO2H.
In one embodiment, when R3d is H; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is chlorine, then R1d is not —OH.
In one embodiment, when R3d is Cl; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is chlorine, then R1d is not —OCH2CO2CH2CH3 or —OCH2CO2H.
In one embodiment, when R3d is Cl; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is methoxy, then R1d is not —OCH2CO2CH2CH3 or —OCH2CO2H.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R4d is —NH2, R2d, R6d, R7d and R9d are each hydrogen; R8d is methoxy, and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is N; R8 is absent; R2d, R4d, R6d, R7d, R9d are each hydrogen; and L is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH or —OCH2CO2H.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R8d and R9d are each hydrogen; R7d is trifluoromethyl, and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is methoxy, then R1d is not —OH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d and R9d are each hydrogen; R7d and R8d are each fluorine, and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydroxy, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is —NH2, then R1d is not —OH.
In one embodiment, when R3d is cyano or NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R8d and R9d are each hydrogen; R7d is —NH2, and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
R6d is absent; Yd is N; Wd is CR8d; R2d, R4d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2, CO2H, cyano CONH2 hydrogen, acyl, fluorine or trifluoromethyl; Xd is CR3d; Zd is CH; Ard is
Ad is O; R2d and R4d are each hydrogen, and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Ad is S; R2d and R4d are each hydrogen, and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R8d and R9d are each hydrogen; R7d is methoxy; and Ld is absent and R16d is —OH, then R1d is not —OH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R7d, R8d and R9d are each hydrogen; R6d is —NH2, and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is hydrogen; Xd is CR3d; Zd is CH; Ard is
Ad is O; R2d is hydrogen and R4d is fluorine, and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Ad is O; R2d and R4d are each hydrogen, and Ld is absent and R16d is methyl, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Ad is O; R2d and R4d are each hydrogen, and Ld is unsubstituted phenyl, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Ad is S; R2d and R4d are each hydrogen, and Ld is absent and R16d is methyl or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is —N(CH3)2, then R1d is not —OH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R8d and R9d are each hydrogen; R7d is —NHCOCH2, —CO2H or —N(CH3)2 and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is bromine; Xd is CR3d; Zd is CH; Ard is
Ad is O; R2d and R4d are each hydrogen and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d and R8d are each hydrogen; R9d is methyl; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R7d, R8d and R9d are each hydrogen; R6d is NHCOCH3; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R7d, R8d and R9d are each hydrogen; R6d is —NHCOfuranyl; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R8d and R9d are each hydrogen; R7d is —NHCH2 Ph, methyl, —NHCOPh, —CH2 NH2; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent and R16d is bromine or —CH2 NHCH2 Ph, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is —CH2—; R16d is —NHCH2 Ph, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d; R9d and R15d are each hydrogen; and Ld is —NR15d; R16d is —CH2 PH, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d, R8d and R9d are each hydrogen; and Ld is absent; R16d is —NHCH2 Ph, —CH2 NH2 or —NHCOCH3, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
R2d and R4d are each hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —CONH2; Xd is CR3d; Zd is CH; Ard
is
Yd is CR6d; Wd is CR8d; R2d, R4d, R6d, R7d and R8d are each hydrogen; R9d is —NH2; and Ld is absent and R16d is hydrogen or Ld is hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH: Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R20d, R21d, R22d, R23d and R24d are each hydrogen; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R20d, R21d, R23d and R24d are each hydrogen; R22d is hydrogen, methoxy or fluorine; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d; R20d, R21d, R23d and R24d are each hydrogen; R22d is hydrogen, methoxy, chlorine, fluorine,
trifluoromethoxy, cyclohexyl, —NHCOCH3, cyano, —CF3, morpholinyl, —SO2 NH2, acyl, —SO2 CH3, pyrazolyl, diethylamino, —COOH, —CH2N(CH3)2, isoxazolyl, imidazolyl, triazolyl, t-butyl, methyl, hydroxyl, or dimethylamino; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d; R20d, R21d, R22d R23d and R24d are each hydrogen; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R14d, R20d, R21d, R23d and R24d are each hydrogen; R22d is hydrogen or dimethylamino, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is N; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R21d, R22d R23d and R24d are each hydrogen; and R20d is chlorine, methoxy, methyl or fluorine, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R20d, R21d, R23d and R24d are each hydrogen; R22d is hydrogen, fluorine, methoxy, methyl, dimethylamino, chlorine, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
n is 0-2; R2d, R4d, R6d, R7d R8d, R9d and R11d are each hydrogen; and R16d is piperidinyl; —CH2CH2-piperidine or —CH2-piperidine, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R20d, R22d, R23d and R24d are each hydrogen; R21d is chlorine, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
n is 0-2; R2d, R4d, R6d, R7d R8d, R9d and R11d are each hydrogen; and R16d is diethylamino; —CH2CH2N(CH2CH3)2 or —CH2N(CH2CH3)2, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
n is 1-2; R2d, R4d, R6d, R7d R8d, R9d and R11d are each hydrogen; and R16d is —CH2CH2 Ph or —CH2 Ph, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d; R20d, R23d R22d and R24d are each hydrogen; R21d is chlorine, methoxy or dimethylamino; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
n is 0; R2d, R4d, R6d, R7d, R8d, R9d, R11d are each hydrogen; and R16d is methyl; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; Ld is absent; R2d, R4d, R6d, R7d R8d and R9d are each hydrogen; and R16d is —NHCOCH3; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; Ld is —CH2—; R16d is R2d, R4d, R6d, R7d R8d and R9d are each hydrogen; and R16d is —NH2; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
R2d, R4d, R6d, R7d R8d; R9d, R11d and R16d are each hydrogen; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d; R21d, R22d, R23d and R24d are each hydrogen; R20d is chlorine; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d; R21d, R23d and R24d are each hydrogen; R20d and R22d is chlorine; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
n is 0; R2d, R4d, R6d, R7d, R8d, R9d and R11d are each hydrogen; and R16d is t-butyl; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
n is 0; R2d, R4d, R6d, R7d R8d, R9d, R11d are each hydrogen; and R16d is t-butylmethoxy; then R1d is not —OH.
In one embodiment, when R3d is bromine, fluorine, —CF3, cyano, —CO2H, dimethylamino, acyl or hydrogen; Xd is CR3d; Zd is CH: Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d; R20d, R21d, R23d and R24d are each hydrogen; R22d is dimethylamino; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d; R20 d, R22 d, R23d and R24d are each hydrogen; R21d is fluorine, methyl or cyano; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d; R21d, R22d, R23d and R24d are each hydrogen; R20d is fluorine or methyl; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is N; R2d, R4d, R6d, R7d R8d, R9d, R11d, R20d, R21d, R22d, R23d and R24d are each hydrogen; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R20d, R21d, R23d and R24d are each hydrogen; R22d is fluorine, dimethylamino, methyl, methoxy, cyano, —CF3, hydroxyl or hydrogen, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is N; R23d is absent; R2d, R4d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R20d, R21d, R22d and R24d are each hydrogen; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R20d, R21d, R23d and R24d are each hydrogen; R22d is fluorine or methyl, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is N; R23d is absent; R2d, R4d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R20d, R21d, R22d and R24d are each hydrogen; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R21d, R22d, R23d and R24d are each hydrogen; R20d is methoxy, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d Zd is CH: Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R20d, R21d and R23d each hydrogen; R22d and R24d are each fluorine, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d Zd is CH: Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R20d, R21d and R24d each hydrogen; R22d and R23d are each fluorine, then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R20d, R21d, R23d and R24d each hydrogen; R22d is cyano, fluorine, methoxy, dimethylamino or acyl, then R1d is not —OH.
In one embodiment, when R3d is hydrogen; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R20d R21d, R23d and R24d each hydrogen; R22d is dimethylamino; R4d is methyl or —COOH, then R1d is not —OH.
In one embodiment, when R3d is dimethylamino; Xd is CR3d; Zd is CH; Ard is
Kd is CR20d; Md is CR23d; R2d, R4d, R6d, R7d R8d, R9d, R11d, R12d, R13d, R20d, R21d, R23d and R24d each hydrogen; R22d is fluorine; then R1d is not —OH.
In one embodiment, when R3d is —NO2; Xd is CR3d; Zd is CH; Ard is
Yd is CR6d; Wd is CR8d; Ld is absent R2d, R4d, R6d, R8d R9d and R16d are each hydrogen; and R7d —NHCO-4-fluorophenyl, then R1d is not —OH.
In another embodiment, the transcription factor modulating compound is a compound of formula XIV:
wherein:
R1e is —OH, —OCH2-aryl, —CH2CH2 CO2H, —OCH2CO2CH2CH3, —OCH2CN, —OCH2CH2 NH2, —OCH3, —OCH2CH2N+(CH3)3, —OCH2COOH, —OCH2CH2CH3, —OCH2CH2OH, —OCH2P(O)(OH)2 or —OCH2P(O)(OCH2CH3)2;
R2e, R4e, R53, R11e, R12e, R13e, R21e, R22e, and R24e are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, aryloxy, carbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, acyl, amino, CO2H, cyano, nitro or halogen;
R20e is absent when Ke is N or hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, aryloxy, carbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, acyl, amino, CO2H, cyano, nitro or halogen;
R23e is absent when Me is N or hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, aryloxy, carbonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, acyl, amino, CO2H, cyano, nitro or halogen;
R3e is —NO2, hydrogen, acyl, halogen, alkoxy, —CO2H, —CONR3daR3db; cyano, —NR3dcR3dd, alkyl, —SO2R3de, —C(R3df)NOH, heterocyclic or heteroaryl;
R3ea is alkyl or amino;
Ke is CR20e or N;
Me is CR23e or N; and pharmaceutically acceptable salts thereof.
In one embodiment, R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is acyl; and R3e is CO2H, acyl, —SO2R3ea, hydrogen, —CF3 or halogen (e.g., bromine or fluorine), and R3ea is alkyl (e.g., methyl) or amino.
In one embodiment, R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R3e is nitro; and R22e is alkylsulfonyl.
In one embodiment, R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R22e is acyl; R3e is cyano or nitro and R11e is alkyl (e.g., —CH2 COOH or aminocarbonylmethyl).
In one embodiment, R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, and R24e are each hydrogen; Ke is CR20e and Me is N; R23e is absent; R11e is hydrogen; R3e is —NO2 and R22e is acyl, aryl (e.g., imidazolyl) or alkyl (e.g., halogen substituted alkyl, such as trifluoromethyl).
In one embodiment, R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, and R24e are each hydrogen; Ke is CR20e and Me is N; R23e is absent; R11e is hydrogen; R3e is cyano and R22e is aryl (e.g., imidazolyl).
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is dimethylamino, fluorine, methyl, methoxy, cyano, —CF3, hydroxyl, isopropyl, hydrogen, imidazolyl, triazolyl, acyl or oxazolyl, then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, R22e and R24e are each hydrogen; Ke is CR20e and Me is N; R23e is absent; R11e is hydrogen; then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R22e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; and R21e is methoxy, then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e and R23e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; and R22e and R24e and are each fluorine, then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; and R22e and R23e and are each fluorine, then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is hydrogen, fluorine, acyl, cyano or methyl, then R3e is not cyano.
In one embodiment, when R1e is —OH, R2e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is fluorine;
R4e is fluorine, dimethylamino, methyl, ethoxy, —OCH2CH2P(O)(OH2), —OCH2CH2OCH2CH2OCH3, —OCH2CH2 morpholinyl, —OCH2CH2-4-methylpyrazinyl, —N(CH3)CH2CH2N(CH3)2 or —OCH2CH2N(CH3)2 then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is acyl; R4e is fluorine, dimethylamino, methyl, ethoxy, —OCH2CH2 morpholinyl or —OCH2CH2-methylpyrazinyl, then R3e is not —NO2.
In one embodiment, when Re is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is fluorine, then R3e is not pyrazolyl or imidazolyl.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; and R21e is —CH2P(O)(OH)2 or —CH2P(O)(OEt)2 and R22e is fluorine, then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R12e, R13e, R20e, R21e and R23e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; and R22e and R24e are each methoxy, then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R13e, R20e, R21e, R22e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; and R12e is phenyl, fluorine or methyl, then R3e is not —NO2.
In one embodiment, when R1e is —OH, R2e, R4e, R5e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is fluorine, and R12e is —CH2-4-methylpiperazine or hydroxyethyl, then R3e is not —NO2.
In one embodiment, when R1e is —OH, R4e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is acyl or fluorine; R2e is —N(CH3)CH2CH2N(CH3)2, then R3e is not —NO2.
In one embodiment, when R1e is —OCH2P(O)(OH)2 or —OCH2P(O)(OEt)2, R2e, R4e, R5e, R12e, R13e, R20e, R21e, R23e and R24e are each hydrogen; Ke is CR20e and Me is CR23e; R11e is hydrogen; R22e is fluorine, then R3e is not —NO2.
In one embodiment, the transcription factor modulating compound is a compound of Table 2, or a pharmaceutically acceptable salt thereof:
In one embodiment, the pharmaceutically acceptable salt is sodium or potassium.
The EC50 of a transcription factor modulating compound can be measured using the assay described in Example 2. In a further embodiment, the transcription factor modulating compound has an EC50 activity against SoxS of less than about 10 μM, less than about 5 μM, or less than about 1 μM, as described in Examples 3, 14 and 15. In a further embodiment, the transcription factor modulating compound can have an EC50 activity against MarA of less than about 10 μM, less than about 5 μM, or less than about 1 μM. In yet another embodiment, the transcription factor modulating compound can have an EC50 against LcrF (VirF) of less than about 10 μM, less than about 5 μM, or less than about 1 μM, as described in Examples 5 and 15. In a further embodiment, the transcription factor modulating compound can have an EC50 against ExsA of less than about 10 jtM, less than about 5 jtM, or less than about 1 jtM, as described in Examples 8 and 15.
In one embodiment, the invention pertains, at least in part, to a method for reducing or preventing the spread of microbial cells from one or more organs (e.g., liver, kidney, lungs, brain or spleen) to another organ or organs in a subject by administering to the subject an effective amount of a transcription factor modulating compound (e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XIV or a compound of Table 2). In another embodiment, the invention pertains, at least in part, to a method for reducing the bacterial burden (e.g., the amount of bacteria) in one or more organs in the subject's body (e.g., lungs, brain, liver, spleen and kidneys) by administering an effective amount of a transcription factor modulating compound compound (e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XIV or a compound of Table 2).
In another embodiment, the transcription factor modulating compound causes a log decrease in CFU/g of a tissue in an animal compared to control tissue, for example, in lung tissue or kidney tissue. This can be measured using the assay described Example 3 and 7. In one embodiment, the transcription factor modulating compound cause a log decrease in CFU/g of tissue of greater than 1.0 CFU/g. In a further embodiment, the compound causes a log decrease in CFU/g of tissue greater than 2.5 CFU/g. In one embodiment, the transcription factor modulating compound that cause a log decrease in CFU/g is compound E, F, H, M, BQ or CG.
In another embodiment, the transcription factor modulating compound (e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIV or a compound of Table 2) induces a decrease in the cytotoxicity of a microbial agent (e.g., the ability of a microbial agent to kill a cell). In one embodiment, the transcription factor modulating compound inhibits the cytotoxicity of a microbe compared to a control, as described in Examples 6 and 9. In one embodiment, the cytotoxicity is inhibited by about 10%, by about 20%, by about 30%, about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90% or about 100%.
In another embodiment, the transcription factor modulating compound effective against Pseudomonas aeruginosa is compound A, C, D, E, F, H, I, J, K, M, S, T, U, V, W, X, Y, AB, AC, AD, AE, AF, AJ, AK, AL, AM or AN.
In a further embodiment, the transcription factor modulating compound effective against Yersinia pseudotuberculosis is compound A, B, C, D, E, F, H, I, J, K, M, S, T, U, V, W, X or Y.
In a further embodiment, the transcription factor modulating compound is not apigenin.
The term “alkyl” includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C1-C6 includes alkyl groups containing 1 to 6 carbon atoms.
Moreover, the term alkyl includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can be further substituted, e.g., with the substituents described above. An “alkylaryl” or an “arylalkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural and unnatural amino acids.
The term “aryl” includes groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, the term “aryl” includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles,” “heterocycles,” “heteroaryls” or “heteroaromatics.” The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
The term “alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.
For example, the term “alkenyl” includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. The term alkenyl further includes alkenyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 or straight chain, C3-C6 for branched chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C2-C6 includes alkenyl groups containing 2 to 6 carbon atoms.
Moreover, the term alkenyl includes both “unsubstituted alkenyls” and “substituted alkenyls,” the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term “alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond.
For example, the term “alkynyl” includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. The term alkynyl further includes alkynyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term C2-C6 includes alkynyl groups containing 2 to 6 carbon atoms.
Moreover, the term alkynyl includes both “unsubstituted alkynyls” and “substituted alkynyls,” the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure. “Lower alkenyl” and “lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.
The term “acyl” includes compounds and moieties which contain the acyl radical (CH3 CO—) or a carbonyl group. It includes substituted acyl moieties. The term “substituted acyl” includes acyl groups where one or more of the hydrogen atoms are replaced by for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term “acylamino” includes moieties wherein: an acyl moiety is bonded to an amino group. For example, the term includes alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
The term “aroyl” includes compounds and moieties with an aryl or heteroaromatic moiety bound to a carbonyl group. Examples of aroyl groups include phenylcarboxy, naphthyl carboxy, etc.
The terms “alkoxyalkyl,” “alkylaminoalkyl” and “thioalkoxyalkyl” include alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.
The term “amine” or “amino” includes compounds where a nitrogen atom is covalently bonded to at least one carbon or heteroatom. The term includes “alkyl amino” which comprises groups and compounds wherein: the nitrogen is bound to at least one additional alkyl group. The term “dialkyl amino” includes groups wherein: the nitrogen atom is bound to at least two additional alkyl groups. The term “arylamino” and “diarylamino” include groups wherein: the nitrogen is bound to at least one or two aryl groups, respectively. The term “alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl” refers to an amino group which is bound to at least one alkyl group and at least one aryl group. The term “alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.
The term “amide,” “amido” or “aminocarbonyl” includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group. The term includes “alkaminocarbonyl” or “alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl or alkynyl groups bound to an amino group bound to a carbonyl group. It includes arylaminocarbonyl and arylcarbonylamino groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group. The terms “alkylaminocarbonyl,” “alkenylaminocarbonyl,” “alkynylaminocarbonyl,” “arylaminocarbonyl,” “alkylcarbonylamino,” “alkenylcarbonylamino,” “alkynylcarbonylamino,” and “arylcarbonylamino” are included in term “amide.” Amides also include urea groups (aminocarbonylamino) and carbamates (oxycarbonylamino).
The term “carbonyl” or “carboxy” includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom. The carbonyl can be further substituted with any moiety which allows the compounds of the invention to perform its intended function. For example, carbonyl moieties may be substituted with alkyls, alkenyls, alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which contain a carbonyl include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc. The term “carboxy” further includes the structure of —COOH and —COO−
The term “oximyl” includes compounds and moieties that contain a carbon connected with a double bond to a nitrogen atom, which is, in turn connected to a hydroxyl or an alkoxyl group. The term “hydrazinyl” includes compounds and moieties that contain a carbon connected with a double bond to a nitrogen atom, which is, in turn, connected to an amino group.
The term “thiocarbonyl” or “thiocarboxy” includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom.
The term “ether” includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms. For example, the term includes “alkoxyalkyl” which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.
The term “ester” includes compounds and moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group. The term “ester” includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are as defined above.
The term “thioether” includes compounds and moieties which contain a sulfur atom bonded to two different carbon or hetero atoms. Examples of thioethers include, but are not limited to alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” include compounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bonded to an alkyl group. Similarly, the term “alkthioalkenyls” and alkthioalkynyls” refer to compounds or moieties wherein: an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkynyl group.
The term “hydroxyl” or “hydroxyl” includes groups with an —OH or —O—.
The term “halogen” includes fluorine, bromine, chlorine, iodine, etc. The term “perhalogenated” generally refers to a moiety wherein: all hydrogens are replaced by halogen atoms.
The terms “polycyclyl” or “polycyclic radical” refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings.” Rings that are joined through non-adjacent atoms are termed “bridged” rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, —COOH, alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl, arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amido, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term “heteroatom” includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
The term “electron withdrawing substituent” includes substituents which tend to withdraw electron density away from the aromatic ring. Examples of such groups include, but are not limited to, ammonium (including alkylammonium, arylammonium, and heteroarylammonium), sulfonyl (including, but not limited to, alkylsulfonyl, arylsulfonyl, and heteroarylsulfonyl), halogen, perhalogenated alkyl, cyano, oxime, carbonyl (including alkylcarbonyl, arylcarbonyl, and heteroarylcarbonyl), and nitro.
The term “electron donating substituent” includes substituents which provide additional electron density to the aromatic ring. Example of electron donating substituents include O—, amines, amides, hydroxyl, alkoxy, amides, esters, alkenyl, alkyl, and aryl groups.
The term “lipophilic groups” include groups which are substantially non-polar. Examples of lipophilic groups include alkyl, alkenyl, aryl, halogens, nitro, cyano, and alkoxy groups. The groups can further be substituted with one or more halogens.
The invention provides compositions which include a therapeutically-effective amount or dose of a transcription factor modulating compound and/or a compound identified in any of the instant assays and one or more carriers (e.g., pharmaceutically acceptable additives and/or diluents). The pharmaceutical compositions of the invention may comprise any compound described in this application as a transcription factor modulating compound, an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, a MarA family inhibiting compound, a MarA inhibiting compound, compounds of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2. Each of these compounds may be used alone or in combination as a part of a pharmaceutical composition of the invention. Furthermore, a composition can also include a second antimicrobial agent, e.g., an antibiotic.
The invention pertains to pharmaceutical compositions comprising an effective amount of a transcription factor modulating compound (e.g., a MarA family polypeptide modulating compound or an AraC family polypeptide modulating compound), and a pharmaceutically acceptable carrier. In one embodiment, the transcription factor modulating compound is of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2.
In one embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a transcription factor modulating compound, wherein: said compound is of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2. In another embodiment, the pharmaceutical composition can further comprise an antibiotic.
In one embodiment, the transcription factor modulating compound (e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2) is administered in combination with an antibiotic. The language “in combination with” an antibiotic includes co-administration of the transcription factor modulating compound and with an antibiotic, administration of the transcription factor modulating compound first, followed by administration of an antibiotic, and administration of the antibiotic first, followed by administration of the transcription factor modulating compound. The transcription factor modulating compound can be administered substantially at the same time as the antibiotic or at substantially different times as the antibiotic. Optimal administration rates for a given protocol of administration of the transcription factor modulating and/or the antibiotic can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the specific compounds being utilized, the particular compositions formulated, the mode of application, the particular site of administration and the like.
The term “antibiotic” refers to chemotherapeutic agents that inhibit or abolish the growth of microbial cells (e.g., bacteria or fungi). Suitable antibiotics include, but are not limited, aminoglycosides, ancimycins, carbacephams, cephalosporins, glycopeptides, macrolides, monobactems, penicillins, polypeptides, quinolines, sulphonamides, tetracyclines and the like. One of skill in the art using conventional medical diagnoses would be able to determine the appropriate antibiotic agent to administer in combination with the transcription factor modulating compounds of the invention.
The language “effective amount” of the compound is that amount necessary or sufficient to treat, prevent or ameliorate a bacterial infection (e.g., pneumonia, urinary tract infection, kidney infection), biofilm formation, bacterial growth (e.g., on a contact lens), corneal ulcers and burn wounds in a subject. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, etc. One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the transcription factor modulating compounds without undue experimentation.
The term “subject” includes plants and animals (e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans) which are capable of suffering from a bacterial associated disorder. The term “subject” also comprises immunocompromised subjects, who may be at a higher risk for infection.
The terms “preventing” and “prevention” include the administration of an effective amount of the transcription factor modulating compound to prevent a bacterial infection (e.g., pneumonia, urinary tract infection, kidney infection), biofilm formation, bacterial growth (e.g., on a contact lens or a medical indwelling device) from occurring.
The terms “treating” and “treatment” include the administration to a subject an effective amount of the transcription factor modulating compound to treat the subject for a bacterial infection (e.g., pneumonia, urinary tract infection), biofilm formation, bacterial growth (e.g., on a contact lens), corneal ulcers and burn wounds.
The transcription factor modulating compounds of the invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of the transcription factor modulating compounds of the invention that are basic in nature are those that form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts. Although such salts must be pharmaceutically acceptable for administration to a subject, e.g., a mammal, it is often desirable in practice to initially isolate a transcription factor modulating compound of the invention from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent and subsequently convert the latter free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the base compounds of this invention are readily prepared by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is readily obtained. The preparation of other transcription factor modulating compounds of the invention not specifically described in the foregoing experimental section can be accomplished using combinations of the reactions described above that will be apparent to those skilled in the art.
The transcription factor modulating compounds of the invention that are acidic in nature are capable of forming a wide variety of base salts. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of those transcription factor modulating compounds of the invention that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmaceutically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines. The pharmaceutically acceptable base addition salts of transcription factor modulating compounds of the invention that are acidic in nature may be formed with pharmaceutically acceptable cations by conventional methods. Thus, these salts may be readily prepared by treating the transcription factor modulating compounds of the invention with an aqueous solution of the desired pharmaceutically acceptable cation and evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, a lower alkyl alcohol solution of the transcription factor modulating compounds of the invention may be mixed with an alkoxide of the desired metal and the solution subsequently evaporated to dryness.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microbes may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Pharmaceutical compositions of the present invention may be administered to epithelial surfaces of the body orally, parenterally, topically, rectally, nasally, intravaginally, intracisternally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal or vaginal suppositories.
The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
The phrases “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a sucrose octasulfate and/or an antibacterial, drug or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
In some methods, the compositions of the invention can be topically administered to any epithelial surface. An “epithelial surface” according to this invention is defined as an area of tissue that covers external surfaces of a body, or which lines hollow structures including, but not limited to, cutaneous and mucosal surfaces. Such epithelial surfaces include oral, pharyngeal, esophageal, pulmonary, ocular, aural, nasal, buccal, lingual, vaginal, cervical, genitourinary, alimentary, and anorectal surfaces.
Compositions can be formulated in a variety of conventional forms employed for topical administration. These include, for example, semi-solid and liquid dosage forms, such as liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions, slurries, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, chewing gums, lozenges, mouthwashes, rinses.
Conventionally used carriers for topical applications include pectin, gelatin and derivatives thereof, polylactic acid or polyglycolic acid polymers or copolymers thereof, cellulose derivatives such as methyl cellulose, carboxymethyl cellulose, or oxidized cellulose, guar gum, acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer, bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum, hectorite, ispaghula husk, polyvinypyrrolidone, silica and derivatives thereof, xanthan gum, kaolin, talc, starch and derivatives thereof, paraffin, water, vegetable and animal oils, polyethylene, polyethylene oxide, polyethylene glycol, polypropylene glycol, glycerol, ethanol, propanol, propylene glycol (glycols, alcohols), fixed oils, sodium, potassium, aluminum, magnesium or calcium salts (such as chloride, carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptate or tartrate).
Such compositions can be particularly useful, for example, for treatment or prevention of an unwanted cell, e.g., vaginal Neisseria gonorrhoeae, or infections of the oral cavity, including cold sores, infections of eye, the skin, or the lower intestinal tract. Standard composition strategies for topical agents can be applied to the antiinfective compounds or a pharmaceutically acceptable salt thereof in order to enhance the persistence and residence time of the drug, and to improve the prophylactic efficacy achieved.
For topical application to be used in the lower intestinal tract or vaginally, a rectal suppository, a suitable enema, a gel, an ointment, a solution, a suspension or an insert can be used. Topical transdermal patches may also be used. Transdermal patches have the added advantage of providing controlled delivery of the compositions of the invention to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium.
Compositions of the invention can be administered in the form of suppositories for rectal or vaginal administration. These can be prepared by mixing the agent with a suitable non-irritating carrier which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum or vagina to release the drug. Such materials include cocoa butter, beeswax, polyethylene glycols, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
Compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, films, or spray compositions containing such carriers as are known in the art to be appropriate. The carrier employed in the sucrose octasulfate/contraceptive agent should be compatible with vaginal administration and/or coating of contraceptive devices. Combinations can be in solid, semi-solid and liquid dosage forms, such as diaphragm, jelly, douches, foams, films, ointments, creams, balms, gels, salves, pastes, slurries, vaginal suppositories, sexual lubricants, and coatings for devices, such as condoms, contraceptive sponges, cervical caps and diaphragms.
For ophthalmic applications, the pharmaceutical compositions can be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the compositions can be formulated in an ointment such as petrolatum. Exemplary ophthalmic compositions include eye ointments, powders, solutions and the like.
Powders and sprays can contain, in addition to sucrose octasulfate and/or antibiotic or contraceptive agent(s), carriers such as lactose, talc, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
Compositions of the invention can also be orally administered in any orally-acceptable dosage form including, but not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of sucrose octasulfate and/or antibiotic or contraceptive agent(s) as an active ingredient. A compound may also be administered as a bolus, electuary or paste. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the antiinfective agent(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Sterile injectable forms of the compositions of this invention can be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
The transcription factor modulating compound or a pharmaceutically acceptable salt thereof will represent some percentage of the total dose in other dosage forms in a material forming a combination product, including liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions slurries, soaps, shampoos, detergents, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, lozenges, mouthwashes, rinses and others. Creams and gels for example, are typically limited by the physical chemical properties of the delivery medium to concentrations less than 20% (e.g., 200 mg/gm). For special uses, far less concentrated preparations can be prepared, (e.g., lower percent formulations for pediatric applications). For example, the pharmaceutical composition of the invention can comprise sucrose octasulfate in an amount of 0.001-99%, typically 0.01-75%, more typically 0.1-20%, especially 1-10% by weight of the total preparation. In particular, a preferred concentration thereof in the preparation is 0.5-50%, especially 0.5-25%, such as 1-10%. It can be suitably applied 1-10 times a day, depending on the type and severity of the condition to be treated or prevented.
Given the low toxicity of an antiinfective agent or a pharmaceutically acceptable salt thereof over many decades of clinical use as an anti-ulcerant [W. R. Garnett, Clin. Pharm. 1:307-314 (1982); R. N. Brogden et al., Drugs 27:194-209 (1984); D. M. McCarthy, New Eng J. Med., 325:1017-1025 (1991), an upper limit for the therapeutically effective dose is not a critical issue.
In one embodiment, the transcription factor modulating compounds of the invention may be administered prophylactically. For prophylactic applications, the pharmaceutical composition of the invention can be applied prior to potential infection. The timing of application prior to potential infection can be optimized to maximize the prophylactic effectiveness of the compound. The timing of application will vary depending on the mode of administration, doses, the stability and effectiveness of composition, the frequency of the dosage, e.g., single application or multiple dosage. One skilled in the art will be able to determine the most appropriate time interval required to maximize prophylactic effectiveness of the compound.
A transcription factor modulating compound, e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2, when present in a composition will generally be present in an amount from about 0.000001% to about 100%, more preferably from about 0.001% to about 50%, and most preferably from about 0.01% to about 25%.
For compositions of the present invention comprising a carrier, the composition comprises, for example, from about 1% to about 99%, preferably from about 50% to about 99%, and most preferably from about 75% to about 99% by weight of at least one carrier.
The transcription factor modulating compounds of the invention may be formulated in a composition suitable for use in environments including industry, pharmaceutics, household, and personal care. In an embodiment, the compounds of the invention are soluble in water. The modulating compounds may be applied or delivered with an acceptable carrier system. The composition may be applied or delivered with a suitable carrier system such that the active ingredient (e.g., formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2) may be dispersed or dissolved in a stable manner so that the active ingredient, when it is administered directly or indirectly, is present in a form in which it is available in a advantageous way.
Also, the separate components of the compositions of the invention may be preblended or each component may be added separately to the same environment according to a predetermined dosage for the purpose of achieving the desired concentration level of the treatment components and so long as the components eventually come into intimate admixture with each other. Further, the present invention may be administered or delivered on a continuous or intermittent basis.
The transcription factor modulating compound (e.g., a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2) may be formulated with any suitable carrier and prepared for delivery in forms, such as, solutions, microemulsions, suspensions or aerosols. Generation of the aerosol or any other means of delivery of the present invention may be accomplished by any of the methods known in the art. For example, in the case of aerosol delivery, the compound is supplied in a finely divided form along with any suitable carrier with a propellant. Liquefied propellants are typically gases at ambient conditions and are condensed under pressure. The propellant may be any acceptable and known in the art including propane and butane, or other lower alkanes, such as those of up to 5 carbons. The composition is held within a container with an appropriate propellant and valve, and maintained at elevated pressure until released by action of the valve.
The compositions of the invention may be prepared in a conventional form suitable for, but not limited to topical or local application such as an ointment, paste, gel, spray and liquid, by including stabilizers, penetrants and the carrier or diluent with the compound according to a known technique in the art. These preparations may be prepared in a conventional form suitable for enteral, parenteral, topical or inhalational applications.
The present invention may be used in compositions suitable for household use. For example, compounds of the present invention are also useful as active antimicrobial ingredients in household products such as cleansers, detergents, disinfectants, dishwashing liquids, soaps and detergents. In an embodiment, the transcription factor modulating compound of the present invention may be delivered in an amount and form effective for the prevention of colonization, removal or death of microbes.
The compositions of the invention for household use comprise, for example, at least one transcription factor modulating compound of the invention and at least one suitable carrier. For example, the composition may comprise from about 0.00001% to about 50%, preferably from about 0.0001% to about 25%, most preferably from about 0.0005% to about 10% by weight of the modulating compound based on the weight percentage of the total composition.
The transcription factor modulating compounds of the present invention may also be used in hygiene compositions for personal care. For instance, compounds of the invention can be used as an active ingredient in personal care products such as facial cleansers, astringents, body wash, shampoos, conditioners, cosmetics and other hygiene products. The hygiene composition may comprise any carrier or vehicle known in the art to obtain the desired form (such as solid, liquid, semisolid or aerosol) as long as the effects of the compound of the present invention are not impaired. Methods of preparation of hygiene compositions are not described herein in detail, but are known in the art. For its discussion of such methods, The CTFA Cosmetic Ingredient Handbook, Second Edition, 1992, and pages 5-484 of A Formulary of Cosmetic Preparations (Vol. 2, Chapters 7-16) are incorporated herein by reference.
The hygiene composition for use in personal care comprise generally at least one modulating compound of the present application and at least one suitable carrier. For example, the composition may comprise from about 0.00001% to about 50%, preferably from about 0.0001% to about 25%, more preferably from about 0.0005% to about 10% by weight of the transcription factor modulating compound of the invention based on the weight percentage of the total composition.
The transcription factor modulating compounds of the present invention may be used in industry. In the industrial setting, the presence of microbes can be problematic, as microbes are often responsible for industrial contamination and biofouling. Compositions of the invention for industrial applications may comprise an effective amount of the compound of the present invention in a composition for industrial use with at least one acceptable carrier or vehicle known in the art to be useful in the treatment of such systems. Such carriers or vehicles may include diluents, deflocculating agents, penetrants, spreading agents, surfactants, suspending agents, wetting agents, stabilizing agents, compatibility agents, sticking agents, waxes, oils, co-solvents, coupling agents, foams, antifoaming agents, natural or synthetic polymers, elastomers and synergists. Methods of preparation, delivery systems and carriers for such compositions are not described here in detail, but are known in the art. For its discussion of such methods, U.S. Pat. No. 5,939,086 is herein incorporated by reference. Furthermore, the preferred amount of the composition to be used may vary according to the active ingredient(s) and situation in which the composition is being applied.
The transcription factor modulating compounds, e.g., compounds of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2, may be useful in nonaqueous environments. Such nonaqueous environments may include, but are not limited to, terrestrial environments, dry surfaces or semi-dry surfaces in which the compound or composition is applied in a manner and amount suitable for the situation.
The transcription factor modulating compounds, e.g., compounds of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV or a compound of Table 2, of the present invention may be used to form coatings or layers on a variety of substrates including personal care products (such as toothbrushes, contact lens cases and dental equipment), healthcare products, household products, food preparation surfaces and packaging, and laboratory and scientific equipment. Further, other substrates include medical devices such as catheters, urological devices, blood collection and transfer devices, tracheotomy devices, intraocular lenses, wound dressings, sutures, surgical staples, membranes, shunts, gloves, tissue patches, prosthetic devices (e.g., heart valves) and wound drainage tubes. Still further, other substrates include textile products such as carpets and fabrics, paints and joint cement. A further use is as an antimicrobial soil fumigant.
The transcription factor modulating compounds of the invention may also be incorporated into polymers, such as polysaccharides (cellulose, cellulose derivatives, starch, pectins, alginate, chitin, guar, carrageenan), glycol polymers, polyesters, polyurethanes, polyacrylates, polyacrylonitrile, polyamides (e.g., nylons), polyolefins, polystyrenes, vinyl polymers, polypropylene, silks or biopolymers. The modulating compounds may be conjugated to any polymeric material such as those with the following specified functionality: 1) carboxy acid, 2) amino group, 3) hydroxyl group and/or 4) haloalkyl group.
The composition for treatment of nonaqueous environments may be comprise at least one transcription factor modulating compound of the present application and at least one suitable carrier. In an embodiment, the composition comprises from about 0.001% to about 75%, advantageously from about 0.01% to about 50%, and preferably from about 0.1% to about 25% by weight of a transcription factor modulating compound of the invention based on the weight percentage of the total composition.
The transcription factor modulating compounds and compositions of the invention may also be useful in aqueous environments. “Aqueous environments” include any type of system containing water, including, but not limited to, natural bodies of water such as lakes or ponds; artificial, recreational bodies of water such as swimming pools and hot tubs; and drinking reservoirs such as wells. The compositions of the present invention may be useful in treating microbial growth in these aqueous environments and may be applied, for example, at or near the surface of water.
The compositions of the invention for treatment of aqueous environments may comprise at least one transcription factor modulating compound of the present invention and at least one suitable carrier. In an embodiment, the composition comprises from about 0.001% to about 50%, advantageously from about 0.003% to about 15%, preferably from about 0.01% to about 5% by weight of the compound of the invention based on the weight percentage of the total composition.
The present invention also provides a process for the production of an antibiofouling composition for industrial use. Such process comprises bringing at least one of any industrially acceptable carrier known in the art into intimate admixture with a transcription factor modulating compound of the present invention. The carrier may be any suitable carrier discussed above or known in the art.
The suitable antibiofouling compositions may be in any acceptable form for delivery of the composition to a site potentially having, or having at least one living microbe. The antibiofouling compositions may be delivered with at least one suitably selected carrier as hereinbefore discussed using standard formulations. The mode of delivery may be such as to have a binding inhibiting effective amount of the antibiofouling composition at a site potentially having, or having at least one living microbe. The antibiofouling compositions of the present invention are useful in treating microbial growth that contributes to biofouling, such as scum or slime formation, in these aqueous environments. Examples of industrial processes in which these compounds might be effective include cooling water systems, reverse osmosis membranes, pulp and paper systems, air washer systems and the food processing industry. The antibiofouling composition may be delivered in an amount and form effective for the prevention, removal or termination of microbes.
The antibiofouling composition of the present invention generally comprise at least one compound of the invention. The composition may comprise from about 0.001% to about 50%, more preferably from about 0.003% to about 15%, most preferably from about 0.01% to about 5% by weight of the compound of the invention based on the weight percentage of the total composition.
The amount of antibiofouling composition may be delivered in an amount of about 1 mg/l to about 1000 mg/l, advantageously from about 2 mg/l to about 500 mg/l, and preferably from about 20 mg/l to about 140 mg/l.
Antibiofouling compositions for water treatment generally comprise transcription factor modulating compounds of the invention in amounts from about 0.001% to about 50% by weight of the total composition. Other components in the antibiofouling compositions (used at 0.1% to 50%) may include, for example, 2-bromo-2-nitropropane-1,3-diol (BNPD), β-nitrostyrene (BNS), dodecylguanidine hydrochloride, 2,2-dibromo-3-nitrilopropionamide (DBNPA), glutaraldehyde, isothiazolin, methylene bis(thiocyanate), triazines, n-alkyl dimethylbenzylammonium chloride, trisodium phosphate-based, antimicrobials, tributyltin oxide, oxazolidines, tetrakis(hydroxymethyl)phosphonium sulfate (THPS), phenols, chromated copper arsenate, zinc or copper pyrithione, carbamates, sodium or calcium hypochlorite, sodium bromide, halohydantoins (Br, Cl), or mixtures thereof.
Other possible components in the compositions of the invention include biodispersants (about 0.1% to about 15% by weight of the total composition), water, glycols (about 20-30%) or Pluronic (at approximately 7% by weight of the total composition). The concentration of antibiofouling composition for continuous or semi-continuous use is about 5 to about 70 mg/l.
Antibiofouling compositions for industrial water treatment may comprise compounds of the invention in amounts from about 0.001% to about 50% based on the weight of the total composition. The amount of compound of the invention in antibiofouling compositions for aqueous water treatment may be adjusted depending on the particular environment. Shock dose ranges are generally about 20 to about 140 mg/l; the concentration for semi-continuous use is about 0.5× of these concentrations.
The invention also pertains, at least in part, to a method of regulating biofilm development. The method includes administering a composition which contains a transcription factor modulating compound of the invention. The composition can also include other components which enhance the ability of the composition to degrade biofilms.
The composition can be formulated as a cleaning product, e.g., a household or an industrial cleaner to remove, prevent, inhibit, or modulate biofilm development. Advantageously, the biofilm is adversely affected by the administration of the compound of the invention, e.g., biofilm development is diminished. These compositions may include compounds such as disinfectants, soaps, detergents, as well as other surfactants. Examples of surfactants include, for example, sodium dodecyl sulfate; quaternary ammonium compounds; alkyl pyridinium iodides; TWEEN 80, TWEEN 85, TRITON X-100; BRIJ 56; biological surfactants; rhamnolipid, surfactin, visconsin, and sulfonates. The composition of the invention may be applied in known areas and surfaces where disinfection is required, including but not limited to drains, shower curtains, grout, toilets and flooring. A particular application is on hospital surfaces and medical instruments. The disinfectant of the invention may be useful as a disinfectant for bacteria such as, but not limited to, Pseudomonadaceae, Azatobacteraceae, Rhizabiaceae, Mthylococcaceae, Halobacteriaceae, Acetobacteraceae, Legionellaceae, Neisseriaceae, and other genera.
A dentifrice or mouthwash containing the compounds of the invention may be formulated by adding the compounds of the invention to dentifrice and mouthwash formulations, e.g., as set forth in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., 1990, Chapter 109 (incorporated herein by reference in its entirety). The dentifrice may be formulated as a gel, paste, powder or slurry. The dentifrice may include binders, abrasives, flavoring agents, foaming agents and humectants. Mouthwash formulations are known in the art, and the compounds of the invention may be advantageously added to them.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, genetics, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Genetics; Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al. (Cold Spring Harbor Laboratory Press (1989)); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel, F. et al. (Wiley, NY (1995)); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1984)); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London (1987)); Handbook Of Experimental Immunology, Volumes 1-IV (D. M. Weir and C. C. Blackwell, eds. (1986)); and Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)).
The contents of all references, patent applications and patents, cited throughout this application are hereby expressly incorporated by reference. Each reference disclosed herein is incorporated by reference herein in its entirety. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety.
Preparation of N-(4-aminobenzyl)-2,4-dinitroaniline (7)
To a solution of 4-aminobenzylamine (25.5 mL, 225 mmol) and powdered NaHCO3 (94.5 g, 1125 mmol) in anhydrous DMF (300 mL) was added 2,4-dinitrofluorbenzene (1) (18.8 mL, 150 mmol) dropwise at room temperature. After 2 hours, the solution was slowly diluted with water (1000 mL) to precipitate the product, which was collected on a fritted funnel while rinsing with water until the eluent was colorless. The solid was further dried under high vacuum to afford 43.0 g as a bright orange solid in 99% yield.
To a solution of N-(4-aminobenzyl)-2,4-dinitroaniline (7) (21.6 g, 74.9 mmol) in anhydrous EtOH (300 mL) and anhydrous DMF (75 mL) was slowly added sodium methoxide (30% w/w) (69.1 g, 375 mmol) at room temperature under argon atmosphere, followed by heating to 60° C. for 2 hours. After cooling to ambient temperature, the solution was diluted with water (700 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel while rinsing with water. The crude product was recrystallized in hot EtOH to afford 18.1 g as a brown solid in 90% yield.
General procedure for the preparation of N-acyl-6-nitro-2-(4-aminophenl)-1-hydroxybenzimidazoles (9) To a solution of 6-nitro-2-(4-aminophenyl)-1-hydroxybenzimidazole (8) (270 mg, 1.00 mmol) in anhydrous pyridine (2.0 mL) was added acid chlorides (2.5 mmol) or the in situ mixed anhydrides at room temperature. (The mixed anhydride was prepared by adding trimethylacetyl chloride (2.5 mmol) dropwise to a solution of the carboxylic acid (2.55 mmol) in anhydrous pyridine at 0° C. After 1 hour, 6-nitro-2-(−4-aminophenyl)-1-hydroxybenzimidazole was added in one portion.) After stirring for 2-3 hours at room temperature, the solution was diluted with 3M NaOH (6.0 mL) and stirred for another hour. The deep amber solution was diluted with water (100 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel while rinsing with water. The crude product was further purified by either preparatory HPLC or by recrystallization in hot ethanol or a mixture of hot ethanol and chloroform.
A solution of 6-nitro-2-(4-bromophenyl)-1-hydroxybenzimidazole (10) (334 mg, 1 mmol) in DMF (2 mL) and Et3 N (1 mL) was degassed with argon for 30 minutes. Phenylacetylene (408 mg), 4 mmol), CuI (38 mg, 0.2 mmol), and Pd(PPh3)4 (116 mg, 0.1 mmol) were added. Degassing was continued for another 5 minutes and the reaction vial was placed in a sand bath preheated to 100° C. overnight. The reaction was cooled and diluted with 50 mL of water and the pH was adjusted to pH 4 with 10% aqueous HCl. The solids were filtered and triturated successively with 1,2-dichloroethane and warm methanol. The resultant yellow solid was further purified by passing through a silica gel flash column eluting with EtOAc:Hexanes (1:1). Fractions containing the product were pooled and evaporated to provide 27 mg of a yellow solid.
In a pressure reaction, 4-dimethylaminophenylamidobenzonitrile (12) (26 g, 98 mmol) was dissolved in anhydrous THF (940 mL), and the solution was purged with argon for 2-3 minutes, followed by the addition of 11 mL of uniformly suspended catalyst (Raney® nickel 2400, suspension in water). After addition of a small amount of methanol to the suspension, the reactor was pressurized at 55 psi H2 while stirring vigorously for 2.5 hours. The reaction mixture was filtered over a bed of diatomaceous earth (e.g. Celite®), and washed 3×100 mL of anhydrous THF. The combined filtrates were evaporated to dryness, and further dried under high vacuum to afford 25.1 g of white solid.
To a solution of 4-phenylamidobenzylamine (13) (225 mmol) and powdered NaHCO3 (1125 mmol) in anhydrous DMF (300 mL) was added substituted 4-nitrofluorbenzene (150 mmol) dropwise at room temperature. After 2 h, the solution was slowly diluted with water (1000 mL) to precipitate the product, which was collected on a fritted funnel while rinsing with water until the eluent was colorless. The solid was further dried under high vacuum to afford the product.
To a solution of 4-[(2-nitro-phenylamino)-methyl]-phenylbenzamide (14) (74.9 mmol) in anhydrous EtOH and anhydrous DMF (75 mL) was slowly added sodium methoxide (30% w/w) (375 mmol) at room temperature under argon atmosphere, followed by heating to 60° C. for 2 h. After cooling to ambient temperature, the solution was diluted with water (700 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel while rinsing with water. The crude product was recrystallized in hot EtOH.
To a solution of 3-aminobenzylamine (225 mmol) and powdered NaHCO3 (1125 mmol) in anhydrous DMF (300 mL) was added 2,4-dinitrofluorbenzene (1) (18.8 mL, 150 mmol) dropwise at room temperature. After 2 hours, the solution was slowly diluted with water (1000 mL) to precipitate the product, which was collected on a fritted funnel while rinsing with water until the eluent was colorless. The solid was further dried under high vacuum.
To a solution of 3-aminobenzyldinitrophenylamine (16) (74.9 mmol) in anhydrous EtOH (300 mL) and anhydrous DMF (75 mL) was slowly added sodium methoxide (30% w/w) (375 mmol) at room temperature under argon atmosphere, followed by heating to 60° C. for 2 h. After cooling to ambient temperature, the solution was diluted with water (700 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel while rinsing with water. The crude product was recrystallized in hot EtOH.
To a solution of 3-aminonitrobenzoimidazolol (17) (1.00 mmol) in anhydrous pyridine (2.0 mL) was added acid chlorides (2.5 mmol) or the in situ mixed anhydrides at room temperature. (The mixed anhydride was prepared by adding trimethylacetyl chloride (2.5 mmol) dropwise to a solution of the carboxylic acid (2.55 mmol) in anhydrous pyridine at 0° C., After 1 hour, 6-nitro-2-(−4-aminophenyl)-1-hydroxybenzimidazole was added in one portion.) After stirring for 2-3 h at room temperature, the solution was diluted with 3M NaOH (6.0 mL) and stirred for another 1 h. The deep amber solution was diluted with water (100 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel while rinsing with water. The crude product was further purified by either preparatory HPLC or by recrystallization in hot ethanol or a mixture of hot ethanol and chloroform.
The general synthesis of benzimidazole compounds are seen in Scheme 5. To a solution of 4-aminobenzylamine (35.4 mL, 313 mmol) and powdered NaHCO3 (158 g, 1875 mmol) in anhydrous DMF (500 mL) at room temperature was added a solution of 2,4-dinitrofluorobenzene (19) (46.5 g or 31.4 mL, 250 mmol) in anhydrous DMF (50 mL) dropwise via addition funnel over a 2 hours period. After another 4 hours or as determined complete by HPLC, the solution comprising compound 20 was diluted with anhydrous absolute EtOH (1000 mL), then powdered t-BuOK was added (140 g, 1250 mmol). The resulting solution was heated to 60° C. for 6 hours or as judged complete by HPLC to form compounds of formula 21. After cooling to room temperature, the EtOH was removed under reduced pressure. The resulting solution was poured into vigorously stirred water (4 L) and cooled to 0° C. by means of an ice bath. The amber solution was adjusted to a pH 6 with 4M HCl, which resulted in the precipitation of the desired product. The suspended products of formula 22 were collected on a fine fritted funnel, while rinsing with cold water until the eluent was colorless. The dark orange solid was further dried under high vacuum over P2O5 to afford the desired product in sufficient purity for further reactions. Compounds AO, AP, AQ, AR, AS, AT, AU, AV, AW, AX, AY, AZ, BI, BB, BC, BD, BE, BF, BG, BH, BI, BJ, BK, BL, BN, BO, BP, BQ, BR, BU, BV, BW, CA and CB may be prepared in this manner.
General syntheses of N-acyl-6-ntiro-2-(4-aminophenyl)-1-hydroxybenzimidazoles is seen in Scheme 2 and 3. Compounds B, C, D, AH, BM, BT, BX, BZ, CD, CE, CG, CH, and CK may be synthesized in this manner.
To a solution of 4-aminobenzylamine (25.5 mL, 225 mmol) (1) and powdered NaHCO3 (94.5 g, 1125 mmol) in anhydrous DMF (300 mL) was added 2,4-dinitrofluorbenzene (1) (18.8 mL, 150 mmol) dropwise at room temperature. After 2 hours, the solution was slowly diluted with water (1000 mL) to precipitate the product, which was collected on a fritted funnel while rinsing with water until the eluent was colorless. The solid 23 was further dried under high vacuum to afford 43.0 g as a bright orange solid in 99% yield.
To a solution of N-(4-aminobenzyl)-2,4-dinitroaniline (23) (21.6 g, 74.9 mmol) in anhydrous EtOH (300 mL) and anhydrous DMF (75 mL) was slowly added sodium methoxide (30% w/w) (69.1 g, 375 mmol) at room temperature under argon atmosphere, followed by heating to 60° C. for 2 hours. After cooling to ambient temperature, the solution was diluted with water (700 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel while rinsing with water. The crude product 24 was recrystallized in hot EtOH to afford 18.1 g as a brown solid in 90% yield.
To a solution of 6-nitro-2-(4-aminophenyl)-1-hydroxybenzimidazole (24) (270 mg, 1.00 mmol) in anhydrous pyridine (2.0 mL) was added acid chlorides (2.5 mmol) or the in situ mixed anhydrides at room temperature. (The mixed anhydride was prepared by adding trimethylacetyl chloride (2.5 mmol) dropwise to a solution of the carboxylic acid (2.55 mmol) in anhydrous pyridine at 0° C.
After 1 hour, 6-nitro-2-(−4-aminophenyl)-1-hydroxybenzimidazole was added in one portion.) After stirring for 2-3 hours at room temperature, the solution was diluted with 3M NaOH (6.0 mL) and stirred for another hour. The deep amber solution was diluted with water (100 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel while rinsing with water. The crude product was further purified by either prepatory HPLC or by recrystallization in hot ethanol or a mixture of hot ethanol and chloroform.
To a solution of 4-aminobenzyl amine derivatives (27) (225 mmol) and powdered NaHCO3 (1125 mmol) in anhydrous DMF (300 mL) at was added 2,4-dinitrofluoro benzene (26) (150 mmol) dropwise at room temperature. After 2 hours, the solution was slowly diluted with water (1000 mL) to precipitate the product, which was collected on a fritted funnel rinsing with water until the eluent was colorless. The solid was further dried under high vacuum to afford a bright orange solid.
To a solution of N-(4-aminobenzyl)-2,4-dinitroaniline derivative (28) (74.9 mmol) in anhydrous EtOH (300 mL) and anhydrous DMF (75 mL) was slowly added sodium methoxide (30% w/w, 375 mmol) at room temperature under Argon atmosphere. After the addition, the solution was warmed to 60° C. for 2 hours. After cooling to ambient temperature, the solution was transferred to an Erlenmyer flask or tall beaker through dilution with water (700 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel rinsing with water. The crude product was purified by recrystallization in hot EtOH to afford a brown solid.
To a solution of 6-nitro-2-(4-aminophenyl)-1-hydroxybenzimidazole derivative (29) (1.00 mmol) in anhydrous pyridine (2.0 mL) was added acid chlorides (2.50 mmol) or the in situ formed mixed anhydrides at room temperature. After stirring for 2-3 hours, the solution was diluted with 3M NaOH (6.0 mL) and stirred for another hour. The deep amber solution was transferred to an Erlenmeyer flask or beaker through dilution with water (100 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel rinsing with water. The crude product was further purified either by preparatory HPLC, or by recrystallization in hot ethanol or a mixture of hot ethanol and chloroform.
1H NMR (300 MHz, DMSO-d6): δ 10.60 (s, 1H), 9.38 (s, 1H), 8.36-8.32 (d, 3H), 8.28 (s, 1H), 8.15-8.11 (d, 1H), 7.99-7.93 (t, 4H), 7.87-7.82 (m, 3H), 7.73-7.68 (d, 1H), 6.96-6.91 (d, 1H). MS (M+1)=375.05.
1H NMR (300 MHz, DMSO-d6): δ 10.77 (s, 1H), 9.77 (s, 1H), 8.37-8.34 (m, 4H), 8.15-8.12 (dd, 1H), 7.98-7.92 (m, 7H), 7.85-7.82 (d, 1H), 7.76-7.71 (d, 1H), 7.08-7.03 (d, 1H). MS (M+1)=467.20.
1H NMR (300 MHz, DMSO-d6); δ 10.55 (s, 1H), 8.30 (d, 2H), 8.18-7.96 (m, 6H), 7.87 (d, 1H), 7.70 (d, 1H), 7.39 (t, 3H), 6.78 (d, 2H), 3.02 (s, 6H). MS (M+1)=391.20.
1H NMR (300 MHz, DMSO-d6); δ 10.23 (s, 1H), 8.49-8.39 (t, 1H), 8.38 (s, 1H), 8.22-8.12 (m, 3H), 7.87-7.84 (d, 1H), 7.72-7.63 (m, 3H), 7.33-7.28 (t, 2H), 7.14-7.09 (d, 1H). MS (M+1)=375.05.
1H NMR (300 MHz, DMSO-d6): δ 12.81 (br s, 1H), 10.57 (s, 1H), 8.42 (s, 1H), 8.41-8.13 (m, 7H), 7.98 (t, 1H), 7.89-7.86 (d, 1H), 2.66 (s, 3H). MS (M+1)=435.10.
1H NMR (300 MHz, DMSO-d6): δ 12.65 (s, 1H), 10.65 (s, 1H), 8.33-8.36 (m, 3H), 8.13 (dd, 1H), 8.03 (d, 2H), 7.94 (d, 2H), 7.78-7.84 (m, 3H), 7.7 (d, 1H), 7.0 (d, 1H), 2.6 (s, 3H). MS (M−1)=441.
To a solution of 4-cyanoaniline derivative (31) (225 mmol) in N-methylpyrrolidone (180 mL), was added an acid chloride (225.4 mmol) over a period of 3-5 minutes with vigorous stirring. After stirring the reaction mixture for about 5 hours (until the HPLC monitoring of the reaction indicated a complete consumption of the starting materials), it was poured into about 1400 mL of water at room temperature and the resulting suspension was stirred for about 1 hour. The precipitate was filtered, washed with 4×500 mL portions of water, and dried. A second crop of solid can be obtained from the filtrate and washings. In a pressure reactor, 4-phenylamido benzonitrile intermediate (98 mmol) was dissolved in anhydrous THF (940 mL), and the solution was purged with argon for 2-3 minutes, followed by the addition of 11 mL of the uniformly suspended catalyst (Raney® nickel 2400, suspension in water). After addition of a small amount of methanol to the suspension, the reactor was pressurized at 55 psi of H2 while stirring vigorously. LC-MS monitoring of the reaction indicated a complete conversion of the starting material to the corresponding amine within 2.5 hours. The reaction mixture was filtered over a bed of diatomaceous earth (e.g., Celite®), and washed with 3×100 mL portions of anhydrous THF. The combined filtrates were evaporated to dryness, and further dried under high vacuum to afford white colored solid.
To a solution of 4-phenylamidobenzylamine derivatives (32) (225 mmol) and powdered NaHCO3 (1125 mmol) in anhydrous DMF (300 mL) at was added 2,4-dinitrofluoro benzene (26) (150 mmol) dropwise at room temperature. After 2 hours, the solution was slowly diluted with water (1000 mL) to precipitate the product, which was collected on a fritted funnel rinsing with water until the eluent was colorless. The solid was further dried under high vacuum to afford the product as a bright orange solid.
To a solution of N-{4-[(2,4-dinitrophenylamino)-methyl]-phenyl}-benzamide derivatives (39) (74.9 mmol) in anhydrous EtOH (300 mL) and anhydrous DMF (75 mL) was slowly added sodium methoxide (30% w/w)(69.1 g, 375 mmol) at room temperature under Argon atmosphere. After the addition, the solution was warmed to 60° C. for 2 hours. After cooling to ambient temperature, the solution was transferred to an Erlenmyer flask or tall beaker through dilution with water (700 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel rinsing with water. The crude product was purified by recrystallization in hot EtOH.
1H NMR (300 MHz, DMSO-d6): δ 10.57 (s, 1H), 8.30 (s, 1H), 8.29-8.06 (m, 3H), 7.86-7.83 (d, 2H), 7.67-7.44 (t, 2H), 7.41-7.38 (t, 2H), 4.36-4.32 (d, 2H). MS (M−1)=501.
1H NMR (300 MHz, DMSO-d6): δ 10.62 (s, 1H), 8.72 (s, 1H), 8.22 (t, 3H), 8.15-8.12 (m, 4H), 7.93 (d, 1H), 7.41 (t, 2H), 4.78 (t, 2H), 3.99 (t, 2H), 3.21 (s, 9H). MS (m/z, M)=478.39.
1H NMR (300 MHz, DMSO-d6): δ 10.55 (s, 1H), 8.78 (d, 1H), 8.30 (d, 2H), 8.20-8.00 (m, 5H), 7.85 (d, 1H), 7.41 (t, 2H), 5.02 (s, 2H). MS (M+1)=451.20.
1H NMR (300 MHz, DMSO-d6): δ 10.62 (s, 1H), 8.60 (d, 1H), 8.30 (d, 2H), 8.23 (dd, 1H), 7.99 (d, 2H), 7.91 (d, 1H), 7.79˜7.67 (m, 3H), 7.34 (dd, 2H), 6.86 (d, 1H), 4.95 (d, 2H), 4.19 (q, 4H), 1.33 (t, 6H). MS (M−1)=567.
1H NMR (300 MHz, DMSO-d6): δ 10.55 (s, 1H), 8.56 (d, 1H), 8.32 (d, 2H), 8.17 (dd, 1H), 7.92 (d, 2H), 7.86 (d, 1H), 7.74˜7.61 (m, 3H), 7.30 (dd, 2H), 6.80 (d, 1H), 4.50 (d, 2H). MS (M−1)=511.
To a solution of 4-(tert-butoxycarbonyl-aminomethyl)-aniline derivative (42, 0.94 mmol) in 7 mL of NMP, an acid chloride derivative (1.0 mmol) was added and the reaction was stirred at room temperature for 40 minutes. It was then poured in 100 mL of water while stirring. The precipitate was filtered, washed with water (5×15 mL) and dried to give the boc-protected product. A solution of the Boc-protected product (0.83 mmol) in 10 mL of TFA was stirred at room temperature for 20 minutes. It was then diluted with 200 mL of diethyl ether and the suspension stirred for another 10 minutes. The precipitate was filtered, washed with 3×20 mL of diethyl ether, and dried under vacuum for 6 hours to give the product (43) as its TFA salt.
To a solution of 1,5-difluoro-2,4-dinitrobenzene (44) (2.0 mmol) in 8 mL of DMF, was added sodium bicarbonate (10.0 mmol) and the compound (43) (2.0 mmol) and the reaction mixture was refluxed for 10 hours. The reaction was poured in ice-water to give a precipitate. The precipitate was filtered, washed with water, and dried under vacuum to give the desired product.
To a solution of (E)-N-{4-[(5-Fluoro-2,4-dinitro-phenylamino)-methyl]-phenyl}-3-phenyl-acrylamide (45) (0.44 mmol) in ethanol (10 mL) and DMF (10 mL), was added sodium hydride (2.2 mmol). The reaction mixture was heated at 60° C. for 3 hours. After cooling to room temperature, it was poured into ice-water, and acidified with aqueous citric acid. The resulting precipitation was collected, washed with water, and dried in vaccuo to yield the product as a yellow solid.
1H NMR (300 MHz, DMSO-d6): δ 12.57 (s, 1H), 10.52 (s, 1H), 8.31 (d, 2H), 8.16 (s, 1H), 7.91 (d, 2H), 7.74-7.62 (m, 4H), 7.30 (dd, 2H), 6.82 (d, 1H), 2.63 (s, 3H). MS (M+1)=433.
1H NMR (300 MHz, DMSO-d6): δ 12.41 (s, 1H), 10.51 (s, 1H), 8.28 (d, 2H), 8.05 (s, 1H), 7.91 (d, 2H), 7.72 (dd, 2H), 7.64 (d, 1H), 7.49 (s, 1H), 7.30 (dd, 2H), 6.82 (d, 1H), 4.22 (q, 2H), 1.36 (t, 3H). MS (M+1)=463
1H NMR (300 MHz, DMSO-d6): δ 12.47 (s, 1H), 10.67 (s, 1H), 8.50 (s, 1H), 8.32 (d, 2H), 8.18 (s, 1H), 8.10 (d, 2H), 8.01 (d, 2H), 7.88 (d, 2H), 7.84 (s, 1H), 7.69 (s, 1H), 2.62 (s, 3H). MS (M+1)=456.
1H NMR (300 MHz, DMSO-d6): δ 12.32 (s, 1H), 10.51 (s, 1H), 8.27 (d, 2H), 7.97 (s, 1H), 7.90 (d, 2H), 7.71 (dd, 2H), 7.65 (d, 1H), 7.49 (s, 1H), 7.30 (dd, 2H), 6.82 (d, 1H), 2.75 (s, 6H). MS (M+1)=462.
1H NMR (300 MHz, DMSO-d6): δ 12.72 (s, 1H), 10.55 (s, 1H), 8.33 (d, 2H), 8.28 (d, 1H), 7.92 (d, 2H), 7.81 (d, 1H), 7.75-7.70 (m, 2H), 7.64 (d, 1H), 7.30 (dd, 2H), 6.82 (d, 1H). MS (M+1)=437
To a solution of 4-aminobenzyl amine (35.4 mL, 313 mmol) and powdered NaHCO3 (158 g, 1875 mmol) in anhydrous DMF (500 mL) at room temperature was added a solution of 4-bromo-1-fluoro-2-nitrobenzene (47) (31.4 mL, 250 mmol) in anhydrous DMF (50 mL) dropwise via addition funnel over a 1 hour period. After another 4 hours or as determined complete by HPLC, the solution was diluted with anhydrous absolute ethanol (1000 mL) and powdered potassium tert-butoxide (140 g, 1250 mmol) was added in portions. This solution was subsequently heated to 60° C. for 6 hours. After cooling to room temperature, the solution was poured into stirring solution of water (4 L), then adjusted to pH 6 with 1M HCl. The slowly stirring suspension was cooled with an ice bath to facilitate solidification. The suspended product was collected on a fine fritted funnel rinsing with water until the eluent was colorless. The orange solid was further dried under high vacuum.
A 20 mL Biotage microwave vial was charged with 6-bromo-2-(4 aminophenyl)-1-hydroxybenzimidazole (48) (1.52 g, 5.00 mmol), N,N′-dimethylethylenediamine (1.10 mL, 10.0 mmol), CuI (0.952 g, 5.00 mmol), pyrazole (1.36 g, 20.0 mmol) and potassium tert-butoxide (2.24 g, 20.0 mmol) and anhydrous DMSO (20 mL). The secured vial was placed into a Biotage microwave reactor with a temperature setting of 195° C. for 45 minutes. After cooling, the vial was opened and poured into a rapidly stirring water solution. The resulting suspension was filtered through a plug of Celite rinsing with 0.5M NaOH. The water solution was loaded onto a prepared DVB column. After loading, the product was eluted with CH3 CN. The CH3 CN was removed under reduced pressure. The resulting water solution was cooled to 0° C. by an ice bath then adjusted to pH 6 with 1M HCl to precipitate the product 49. The resulting solid was collected onto a fine fritted funnel rinsing with cold water to afford a light brown solid to afford 1.52 g in 70% yield. The product was further dried under high vacuum.
To a solution of 6-pyrazole-2-(4-aminophenyl)-1-hydroxybenzimidazole (0.78 g, 2.50 mmol) and NaHCO3 (0.84 g, 10.0 mmol) in anhydrous CH3 CN (20 mL) and DMPU (5 mL) at room temperature was added 4-fluorocinnamoyl chloride (1.15 g, 6.25 mmol). After 6 hours, the solution was diluted with 3M NaOH (25 mL) and stirred for another 2 hours. The solution was transferred to another flask through dilution with water (100 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel rinsing with water. The crude product was further purified by recrystallization in hot ethanol or a mixture of hot ethanol and chloroform. 1H NMR (DMSO-d6) δ 10.49 (s, 1H), 8.61 (s, 1H), 8.33 (m, 2H), 7.94-7.63 (m, 9H), 7.32 (m, 2H), 6.84 (m, 1H), 6.55 (s, 1H). MS (M+1)=440.
To a solution of 6-pyrazole-2-(4-aminophenyl)-1-hydroxybenzimidazole (0.78 g, 2.50 mmol) and NaHCO3 (0.84 g, 10.0 mmol) in anhydrous CH3 CN (20 mL) and DMPU (5 mL) at room temperature was added 4-acetylbenzoyl chloride (1.14 g, 6.25 mmol). After 6 hours, the solution was diluted with 3M NaOH (25 mL) and stirred for another 2 hours. The solution was transferred to another flask through dilution with water (100 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel rinsing with water. The crude product was further purified by recrystallization in hot ethanol or a mixture of hot ethanol and chloroform. 1H NMR (DMSO-d6) δ 10.61 (s, 1H), 8.69-7.77 (m, 13H), 6.60 (1, 1H), 2.63 (s, 3H). MS (M+1)=438.
To a solution of 6-imidazole-2-(4-aminophenyl)-1-hydroxybenzimidazole (0.78 g, 2.50 mmol) and NaHCO3 (0.84 g, 10.0 mmol) in anhydrous CH3 CN (20 mL) and DMPU (5 mL) at room temperature was added 4-acetylbenzoyl chloride (1.14 g, 6.25 mmol). After 6 hours, the solution was diluted with 3M NaOH (25 mL) and stirred for another 2 hours. The solution was transferred to another flask through dilution with water (100 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel rinsing with water. The crude product was further purified by recrystallization in hot ethanol or a mixture of hot ethanol and chloroform. 1H NMR (DMSO-d6) δ 10.63 (s, 1H), 8.32-7.46 (m, 13H), 7.13 (1, 1H), 2.68 (s, 3H). MS (M+1)=438.
To a solution of N-(4′-aminobenzylamine)-2,4-dinitroaniline (3) (25.0 mmol), powdered NaHCO3 (6.30 g, 75.0 mmol) in anhydrous DMF at room temperature (75 mL) was added with the alkylating agent (26.3 mmol). After 12 hours, the solution was diluted with EtOH (225 mL) then powdered potassium tert-butoxide (14.0 g, 125 mmol) was added. The solution was heated to 60° C. for 3 hours. After cooling to ambient temperature, the solution was poured into cold 10% citric acid solution to precipitate product. The product was collected on a sintered funnel rinsing with cold water. The product was further dried under high vacuum and used as is in the following reaction.
To a solution of 6-nitro-2-(4-aminophenyl)-1-hydroxybenzimidazole derivative (50) (1.00 mmol) in anhydrous pyridine (2.0 mL) was added acid chlorides (2.50 mmol) or the in situ formed mixed anhydrides at room temperature. After stirring for 2-3 hours, the solution was diluted with 3M NaOH (6.0 mL) and stirred for another hour. The deep amber solution was transferred to an Erlenmeyer flask or beaker through dilution with water (100 mL) and then acidified with saturated citric acid. The resulting precipitate was collected on a sintered funnel rinsing with water. The crude product was further purified either by preparatory HPLC, or by recrystallization in hot ethanol or a mixture of hot ethanol and chloroform.
1H NMR (300 MHz, DMSO-d6) δ 8.41-8.32 (m, 2H), 8.13 (s, 1H), 7.92-7.80 (m, 3H), 7.69-7.54 (m, 6H), 7.74-7.60 (br s, 1H), 4.52 (s, 2H), 2.50 (s, 3H). MS (M+1)=481.
1H NMR (300 MHz, DMSO-d6) δ 12.88-12.57 (br s, 1H), 8.57-7.48 (m, 12H), 7.21-7.13 (m, 1H), 6.80-6.57 (m, 1H), 4.47 (s, 2H), 2.52 (s, 3H). MS (M+1)=500.
A quantitative chemiluminescence-based assay was used to measure the DNA binding activity of various MarA (AraC) family members. With this technique, biotinylated double-stranded DNA molecules (2 nM) were incubated with a MarA (AraC) protein (20 nM) fused to 6-histidine (6-His) residues in a streptavidin coated 96-well microtiter (white) plate (Pierce Biotechnology, Rockford, Ill.). Unbound DNA and protein was removed by washing and a primary monoclonal anti-6His antibody was subsequently added. A second washing was performed and a secondary HRP-conjugated antibody was then added to the mixture. Excess antibody was removed by a third wash step and a chemiluminescence substrate (Cell Signaling Technology, Beverly, Mass.) was added to the plate. Luminescence was read immediately using a Victor V plate reader (PerkinElmer Life Sciences, Wellesley, Mass.). Compounds that inhibited the binding of the protein to the DNA resulted in a loss of protein from the plate at the first wash step and were identified by a reduced luminescence signal. The concentration of compound necessary to reduce signal by 50% (EC50/IC50) was calculated using serial dilutions of the inhibitory compounds. Also, single transcription factor modulators that affect different transcription factors were identified.
Using an animal model of ascending pyelonephritis caused by E. coli, transcription factor modulating compounds were judged for the ability to affect kidney infection. Previous studies using this urinary tract infection model have shown that E. coli mutants with a soxS gene deletion colonize the mouse kidney in numbers approximately 1-log fewer than the wild type strain. Groups of female CD1 mice (n=6) were diuresed and infected with E. coli UPEC strain C189 via intravesicular inoculation. Subsequently, mice were dosed with a transcription factor modulator (25 or 50 μg/ml), a control compound, e.g., SXT, or vehicle alone (0 mg/kg), via an oral route of administration at the time of infection and once a day for 4 days thereafter, to maintain a constant level of drug in the mice. After a 5-day period of infection and prior to sacrifice via CO2/O2 asphyxiation, a urine sample was taken by gentle compression of the abdomen. Following asphyxiation, the bladder and kidneys were removed aseptically. Urine volumes and individual organ weights are recorded, the organs were suspended in sterile PBS containing 0.025% Triton X-100, and then homogenized. Serial 10-fold dilutions of the urine samples and homogenates were plated onto McConkey agar plates to determine CFU/ml (CFU=colony forming units) of urine or CFU/gram of organ.
Efficacy in these experiments was defined as a ≧2-log decrease in CFU/g organ. The CFU/g for the compounds of the invention are summarized in Table 3 below. Compounds which exhibited a 0-1.0 log decrease in CFU/g are indicated with “***,” compounds which exhibited a 1.1-2.0 log decrease in CFU/g are indicated with “**,” and compounds which exhibited no decrease in CFU/g are indicated with “*.” This data was compared to the in vitro inhibition of SoxS activity or to the concentration necessary to inhibit DNA-protein binding by 50% (EC50). Transcription factor modulating compounds that inhibited the binding of SoxS to DNA by greater than 70% are shown by “***,” compounds that inhibited the binding of SoxS between about 30% and 69% are shown by “**,” compounds that inhibited the binding of SoxS by less than 29% are shown by “*.” Compounds that gave an IC50 of greater than 5 μM are represented by “*,” compounds that gave an IC50 of between about 1 and 4.9 μM are represented by “**,” and compounds exhibiting ICso 's of less than 0.9 μM are represented by “***.”
a Represents the log10 decrease in the CFU/g kidney tissue.
b Percent inhibition of DNA-protein binding at a compound screening concentration of 50 μg/ml.
c Concentration necessary to inhibit DNA-protein binding by 50% (EC50) as determined using a dose response analysis.
d Percent inhibition of DNA-protein binding at a compound screening concentration of 25 μg/ml.
The closely related E. coli MarA, Rob and SoxS proteins have overlapping roles in the regulation of resistance to multiple antibiotics, oxidative stress agents and organic solvents. Multi-drug resistant clinical isolates of E. coli have been identified that constitutively express these proteins. As shown in
Diuresed CD1 mice were inoculated intravesicularly with ˜107 colony forming units (CFU) of E. coli C189 (clinical cystitis isolate). The kidneys were harvested at 5 days post infection. When compound GM was administered as a single dose at the time of infection, a statistically significant difference (p<0.003) was noted between the vehicle treated dose group and the 100 mg/kg compound GM dose group. When compound GM was administered subcutaneously at 0, 6, 24, 30, 48, 54, 72 and 96 hours post-infection, a statistically significant difference (p<0.05) was noted between the untreated (vehicle) and the 1, 5 and 20 mg/kg dose groups.
The Y. pseudotuberculosis protein LcrF (also called VirF in Y. enterocolitica) regulates expression of a major virulence determinant, the type III secretion system (TTSS). The TTSS delivers toxins directly into host cells, and mutants that do not express the TTSS show dramatic attenuation of virulence in whole cell and animal models of infection. In order to determine the inhibition of LcrF-DNA binding by the transcription factor modulating compounds of the invention, the MarA (AraC) family member LcrF (VirF) was cloned, expressed and purified from Y. pseudotuberculosis. The purified protein was used in a cell-free system to monitor DNA-protein interactions in vitro, methods as in Example 2. The EC50 's for inhibition of LcrF(VirF)-DNA binding by the compounds of the invention are summarized in Table 4 below. Compounds with excellent inhibition (less than 10 μM) are indicated with “***,” very good inhibition (greater than 10.0 and less than 25.0 μM) with “**,” good inhibition (greater than 25.0 μM) with “*” and compounds that were not active are indicated with “--”
In order to demonstrate that the transcription factor modulating compounds of the invention inhibit LcrF(VirF)-dependent cytotoxicity of Y. pseudotuberculosis, select compounds were screened in a whole cell system. Type III secretion, the process whereby cytotoxic proteins (Yops) are secreted from a bacterium into a host cell, in pathogenic Yersinia spp. is regulated by LcrF. Wild type Y. pseudotuberculosis are toxic toward J774 tissue culture cells whereas bacteria bearing a mutation in either yopJ (a Yop that inhibits eukaryotic signaling pathways) or lcrF are not. The cytotoxicity of wild type Y. pseudotuberculosis was exploited in order to screen compounds for their ability to penetrate the intact bacterial cell and prevent type III secretion by binding to an inactivating LcrF function.
The CytoTox 96® assay kit from Promega was used for this assay. Briefly, J774 macrophages were plated out at 2×104 cells per well in 96-well plates on the day prior to infection. Yersinia pseudotuberculosis were grown overnight at 26° C. in 2×YT media and then diluted 1:25 or 1:40 the following morning into 2×YT supplemented with 20 mM MgCl2 and 20 mM sodium oxalate. The cultures were grown for a further 90 min at 26° C. and then shifted to 37° C. for 90 minutes. The temperature shift and the sodium oxalate, which chelates calcium, lead to induction of LcrF expression. Later experiments also included the YPIIIpIB1ΔJ (YopJ mutant) and YPIIIpIB1ΔLcrF (LcrF mutant). YPIIIpIB1ΔJ is a YopJ deletion mutant and any cytotoxicity that is unrelated to YopJ (i.e. lps-mediated) will be seen with this strain. The OD600 was measured and the culture adjusted to an OD600 of 1.0. This should correspond to approximately 1.25×109 cells/mL. Dilutions were prepared in DMEM (the J774 culture media) at different multiplicity of infections (MOIs), assuming J774 cell density of 2×104. Yersinia pseudotuberculosis were added in 10 μl aliquots and cells were incubated at 37° C. either in a chamber with a CO2 generating system, or later, in a tissue culture incubator with 5% CO2 for 2 hours. Gentamicin was then added to a final concentration of 50 μg/ml and the incubations were continued either for a further 2-3 h or overnight. Controls were included for media alone, target cell spontaneous lysis, target cell maximum lysis and effector cell spontaneous lysis. For maximum lysis, triton X-100 was added to a final concentration of 0.8% 45 minutes prior to termination of the experiment. Supernatants containing released LDH were harvested following centrifugation at 1,000 rpm for 5 minutes. Supernatants were either frozen overnight or assayed immediately. 50 μl of supernatant was mixed with 50 μl fresh assay buffer and incubated in the dark for 30 minutes 501 of stop solution was added to each well and the plates were read at 490 nm. In Table 5 below, compounds that reduced Y. pseudotubercolosis cytotoxicity to 99-75% of untreated, wild type levels at 50 μg/mL are indicated with “**.” Compounds that reduced Y. pseudotubercolosis cytotoxicty to below 75% of untreated, wild type levels at 50 μg/mL are indicated with “*.” The percent cytotoxicity was measured relative to vehicle treated cells infected with wild type Y. pseudotuberculosis. Incubation with wild type Y. pseudotuberculosis yields ≧75% toxicity.
The transcription factor modulating compounds of the invention that reduced Y. pseudotuberculosis cytotoxicity were then tested in lethal and sublethal murine Y. pseudotuberculosis murine models. Groups of 4 CD1 mice (7-8 week old males) were dosed subcutaneously with either vehicle or compound (25 mg/kg) 1 day prior to infection, at the time of infection, at 8 hours and then daily for 8 days following intranasal infection with approximately 120 CFU of wild type (WT, IP2666pIB1) or ΔLcRF (JMB155) Y. pseudotuberculosis. The percent survival of infected mice following treatment with a transcription factor modulating compound, as illustrated in
Another assay was performed in which groups of 4 CD-1 mice were treated with a single subcutaneous dose of vehicle or LcrF inhibitor (25 mg/kg) one day prior to infection, at the time of infection, at 8 hours post infection, then once daily for a further 2 days. Mice were infected intranasally with 728 CFU of wild type (IP2666pIB1) or 752 CFU ΔLcrF (JMB155) Y. pseudotuberculosis. The mice were sacrificed 3 days post infection and serial dilutions of lung tissue homogenates were plated. The results are shown in Table 6, where the decrease is relative to vehicle treated mice infected with wild type Y. pseudotuberculosis. Compounds that exhibited a 0-1.0 log decrease in the CFU/g of LcrF in lung tissue are represented by “***,” compounds that exhibited a 1.1-2.0 log decrease in the CFU/g of LcrF in lung tissue are represented by “**” and compounds that exhibited no efficacy are represented by “--.” In particular, the control ΔLcrF bacteria showed dramatically attenuated virulence with a 2-log decrease in bacterial burden in the lungs compared to wild type in sublethal infections and 100% survival in infections where comparable numbers of wild type bacteria were lethal. Treatment with select transcription factor modulating compounds markedly reduced bacterial burden in the lung and decreased mortality in these mouse models of pneumonia.
ExsA regulates the expression of a major virulence determinant, the type III secretion system (TTSS). It has been shown that mutants that lack the exsA gene do not express the TTSS and these mutants show dramatically reduced virulence in whole cell assays and animal models of P. aeruginosa infection. The vast majority of clinical P. aeruginosa strains have the TTSS and expression of the TTSS is correlated with increased severity of disease in clinical pneumonia cases, including ventilator associated pneumonia. Several transcription factor modulating compounds with high activity against LcrF also showed good inhibition of ExsA-DNA binding in vitro. The MarA (AraC) family member ExsA was cloned, expressed and purified from P. aeruginosa. The purified protein was used in a cell-free system to monitor DNA-protein interactions in vitro, methods as in Example 2. The EC50 's for inhibition of ExsA-DNA binding by the compounds of the invention are summarized in Table 7 below. Compounds with excellent inhibition (less than 10 μM) are indicated with “***,” very good inhibition (greater than 10.0 and less than 25.0 μM) with “**,” good inhibition (greater than 25.0 μM) with “*” and compounds that were not active are indicated with “--.”
Transcription factor modulating compounds that exhibited measurable inhibition of ExsA-DNA binding, as described in Example 8, were screened for inhibition of ExsA-dependent P. aeruginosa cytotoxicity to macrophages in a whole cell system. In pathogenic P. aeruginosa, type III secretion is regulated by ExsA. Type III secretion is the process in which cytotoxic proteins (ExoU, ExoT, etc.) are secreted from a bacterium into a host cell. Wild type P. aeruginosa are toxic toward J774 tissue culture cells whereas bacteria bearing a mutation in exsA are not. In this example, the cytotoxicity of wild type P. aeruginosa was exploited to screen compounds for their ability to penetrate the intact bacterial cell and prevent type III secretion by binding to an inactivating ExsA function.
The CytoTox 96® assay kit from Promega was used for this assay. Briefly, J774 macrophage-like cells were plated out at 5×104 cells per well in 96-well plates on the day prior to infection. P. aeruginosa were grown overnight at 37° C. in Luria Broth and then diluted 1:25 in MinS, a minimal salt media containing the calcium chelator trisodium nitriloacetate. Experiments also included the WT ExsA mutants, in which the entire exsA coding sequence has been deleted. Mar inhibitors were added to the MinS cultures at a concentration of 50 μg/mL and the cultures were grown for a further 3 hours at 37° C. The shift to a calcium free media leads to induction of ExsA expression. Cultures were grown to an OD600 of 1.0, approximately 1×109 cells/mL. Dilutions were prepared in DMEM (the J774 culture media) at different multiplicity of infections (MOIs), assuming J774 cell density of 5×104. Media in the J774 cell wells was replaced with DMEM containing 50 μg/mL of Mar inhibitors. P. aeruginosa were added to J774 cells in 10 μl aliquots, plates were centrifuged at 1,000 rpm for 5 minutes to synchronize infection and then incubated in a tissue culture incubator with 5% CO2 for 2 h. Controls were included for media alone, target cell spontaneous lysis, target cell maximum lysis, and Mar inhibitors with J774 cells alone. For target cell maximum lysis, 101 of the CytoTox 96® assay kit lysis solution was added to untreated J774 cells 30 minutes prior to termination of the experiment. Supernatants containing released LDH were harvested following centrifugation at 1,000 rpm for 5 minutes. Supernatants were stored frozen overnight or assayed immediately. 50 μl of supernatant was mixed with 50 μl fresh LDH substrate solution and incubated in the dark for 30 minutes. 50 μl of stop solution was added to each well and the plates were read at 490 nm. In Table 8 below, compounds that reduced P. aeruginosa cytotoxicity to 99-75% of untreated, wild type levels at 50 mg/mL are indicated with “*.” Compounds that reduced P. aeruginosa cytotoxicity below 75% of untreated, wild type levels at 50 mg/mL are indicated with “**.” The percent cytotoxicity was relative to vehicle treated cells infected with wild type P. aeruginosa. Incubation with wild type P. aeruginosa yielded ≧75% toxicity. In addition, an exsA null mutant was completely non-cytotoxic.
Pseudomonas
Transcription factor modulating compounds that substantially inhibited P. aeruginosa cytotoxicity were tested in a lethal model of murine acute pneumonia. In this model, infection with ˜1×106 CFU of wild type bacteria causes >90% mortality within 48-72 hours, whereas mice infected with the same number of an exsA null mutant bacteria survive indefinitely. The efficacy of two transcription factor modulating compounds, AJ and I, were tested in vivo for their efficacy against P. aeruginosa PA103 in a mouse model of pneumonia (106 organisms inoculated intranasally). Compound AJ was administered IP at 25 mg/kg at −18, −1,2,5,20, 26 and 44 hours post-infection and mortality was assessed at various times post infection. As shown in
Compound I was also administered IP at 25 mg/kg at −18, −1,5,20,26 and 44 hours post infection. As shown in
The biofilm assay screens test compounds for their ability to inhibit bacteria from forming a biofilm.
The M9 media (“M9”) contained M9, casamino acids, and glucose. The test compound was dissolved in 10 mg/mL DMSO stock solution.
Inoculum was started the day of the experiment by adding a colony or glycerol stock stab to 2 mL M9. The tube was placed in the 37° C. shaker incubator for approximately 4-6 hours. This inoculum was referred to as the “Starter inoculum.” The inoculum was then removed from the shaker incubator and diluted to 1×106 cells/mL in M9.
Typically, there were eight of each control, including a positive and negative control. For both the positive and negative controls, 2.5 μL of DMSO was added to 200 μL of M9. 25 μL of the diluted DMSO was added to 50 μL of M9 in the assay plate.
The test compounds were screened at 20 μg/mL. 2.5 μL of the test compound were taken from a plate containing 10 mg/mL stock and added to 200 μL of M9 and mixed. 25 μL of the diluted test compound was added to 50 μL of M9 in the assay plate. The resulting concentration of the test compound was 40 μg/mL
75 μL of the inoculum at 1×106 cells/mL was added to each well containing compound and the positive controls. 75 μL M9 was added to the negative controls. The final concentration of the test compound was 20 μg/mL and the final concentration of the inoculum was 2×105 cells/mL. The plates were then placed in a microplate reader (Wallac Victor2 V) and read OD535 (“Initial growth reading”). The plates were then placed in an incubator overnight at 35° C. In the morning, the plates were read in a microplate reader at OD535 (“Final growth reading.”)
The inoculum was then removed from the wells and the plates were washed several times with tap water. 150 μL of Crystal Violet (0.02% Crystal Violet dissolved in water) was then added to each well.
The crystal violet was then removed and the plates were washed several times with tap water. 150 μL of ethanol were then added to each well, after mixing. The plates were then placed in a microplate reader and read the OD535. This was referred to as the “Crystal Violet” reading.
To determine whether a test compound inhibited growth, the Initial growth reading was subtracted from the Final growth reading (“Subtracted Growth”). The same was done for the positive controls and averaged. The % inhibition of growth was determined by the following formula:
100-(100*Subtracted growth of sample/Average growth of Pos Controls)
To determine whether a test compound inhibited Biofilm formation, the % Inhibition of Biofilm Formation was determined using the following formula:
100-(100*Crystal Violet read of sample/Average crystal violet read of Pos Controls)
This example describes a method for the identification of test compounds that inhibit the interactions of purified transcription factor such as SoxS, ExsA and/or VirF with a target DNA sequence in an in vitro system.
The 6His-tagged SoxS, ExsA and VirF purified according to respective protocol. The N-term-biotinylated double-stranded DNA has a sequence of CCG ATT TAG CAA AAC GTG GCA TCG GTC (SEQ ID NO. 1). The antibody used was the LANCE Eu-labeled anti-6×His Antibody (Eu-αHis) (Perkin Elmer cat #AD0110) which had at least 6 Europium molecules per antibody. Streptavidin conjugated to SureLight™-Allophycocyanin (SA-APC) was obtained from Perkin Elmer (cat #CR130-100). The Assay buffer contained 20 mM Hepes pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, and 30 mM KCl, and 0.2% Tween-20.
The plates or vials of the compounds to be tested were thawed. These stocks were at a concentration of 10 mg/ml in DMSO. The solutions were allowed to thaw completely, and the plates were briefly shaken on the Titermix to redissolve any precipitated compound. Thawed aliquots of SoxS, ExsA and VirF protein from the stock stored at −80° C. and 1M stock of dithiothreitol stored at −20° C. were then placed on ice.
Dilutions at 1:100 of the compounds were made into a fresh 96-well polystyrene plate. The dilutions were prepared with 100% DMSO to give a final concentration of 100 μg/ml solutions. The dilutions were vortexed on a Titermix.
Fresh DTT was added to 25-50 mL of assay buffer to produce a 1 mM final concentration. Next, 901 of assay buffer was added to each of the 10 μl protein aliquots, and the solution was mixed by pipetting. These proteins were diluted to give the required amount of each of the diluted proteins, resulting in 201 of diluted protein per well. In preparing the solutions, 20% excess was made to allow enough for control wells. Typically, depending on the protein preps and the initial binding curves that were performed, 1000-2000 fmoles of each protein was required per well. The diluted protein solutions were the placed on ice.
Three tests plates per plate of compound (for SoxS, ExsA and VirF) were prepared. Using a multichannel pipet, 5 μl of the compound was added to each well. 5 μl of DMSO was added to the blank and control wells, and 5 μl of the control inhibitor was added to the respective wells.
Using the multichannel pipet, 201 of protein was added to all wells except those designated “blank”. To these blank wells, 201 of assay buffer was added. The plates were covered with a plate sealer and incubated at room temperature, shaking on the Titermix, for 30 minutes.
Next, the DNA solution was prepared, with enough for at least 20% more wells than were tested. 15 μl (0.4 fmoles) was added per well. Then the DNA was diluted in assay buffer, and vortexed briefly to mix. The plate sealer was removed, and 15 μl of DNA solution was added to all of the wells. the plates were then resealed, and returned to the Titermix for a further 30 minutes.
After 25 minutes, the antibody solution was prepared. 0.4 fmoles of SA-APC and 0.125 fmoles of Eu-αHis were added per well in a total volume of 10 μl. Amounts were prepared sufficient for at least 20% excess. The plate sealer was the removed and 10 μlof antibody solution was added to every well. The plates were subsequently resealed, placed on the Titermix, and covered with aluminum foil. The plates were mixed for 1 hour. The plates were then read on the Wallac Victor V, using the LANCE 615/665 protocol.
For each plate, the mean control (i.e. signal from protein and DNA without inhibition), mean blank (background signal without protein) and mean inhibitor (P001407) LANCE665 counts were determined. The percentage inhibition by each molecule (each test well) was then determined according to the following equation:
% Inhibition=100−(((test−mean blank)/(mean control−mean blank)*100)
Compounds that gave 40% or greater inhibition were identified as hits and screened again for EC50.
The protocol used was identical to that outlined above, except that only 10 compounds were assayed per plate. The testing concentrations started at 10 μg/ml and were diluted two-fold from 10 to 0.078 μg/ml.
Percent inhibition was calculated as shown above. Percent inhibition was then plotted vs. log (conc. Inhibitor) using Graph pad Prism software.
An electrophoretic mobility shift assay (EMSA) using 0.1M (33P)DNA, 5 nM SoxS and 50 ug/ml of transcription factor modulating compound was used to study activity of the compound to interrupt DNA-protein interactions in vitro. Different compounds had varying activities against SoxS in vitro in an EMSA. For example, compound AU was very active, BB was moderately active, and compound BK lacked activity.
The measurement of the ability of the transcription factor modulating compounds of the invention to intercalate DNA was performed by a qualitative agarose gel assay. The assay consisted of 100 ng uncut plasmid DNA, DMSO, which relieves DNA supercoiling and converts the plasmid DNA to a single form, and transcription factor modulating compounds AU, BP, BQ, BX and a known DNA intercalator. Unlike the known DNA intercalator, the transcription factor modulating compounds were not found to intercalate DNA.
A quantitative chemiluminescence-based assay was used to measure the DNA binding activity of various MarA (AraC) family members. With this technique, biotinylated double-stranded DNA molecule (2 nM) was incubated with a MarA (AraC) protein (20 nM) fused to 6-histidine (6-His) residues in a streptavidin coated 96-well microtiter (white) plate (Pierce Biotechnology, Rockford, Ill.). Unbound DNA and protein was removed by washing and a primary monoclonal anti-6His antibody was subsequently added. A second washing was performed and a secondary HRP-conjugated antibody was then added to the mixture. Excess antibody was removed by a third wash step and a chemiluminescence substrate (Cell Signaling Technology, Beverly, Mass.) was added to the plate. Luminescence was read immediately using a Victor V plate reader (PerkinElmer Life Sciences, Wellesley, Mass.). Compounds that inhibited the binding of the protein to the DNA resulted in a loss of protein from the plate at the first wash step and were identified by a reduced luminescence signal. The results of this assay are shown in Table 9, which shows data on the inhibition of SoxS from binding the DNA. Transcription factor modulating compounds that inhibited the binding of SoxS to DNA by greater than 70% are represented by “***,” compounds that inhibited the binding of SoxS between about 30% and 69% are represented by “**,” compounds that inhibited the binding of SoxS by less than 29% are represented by “*,” and compounds that exhibited no inhibition are represented by “--.”
aValues are means of three experiments, standard deviation is less than 15%. Compounds screened at 50 μg/mL.
The ECs50 's of several transcription factor modulating compounds for the inhibition of SoxS binding to the DNA target were also calculated and the results are given in Table 11. Compounds that gave an IC50 of greater than 5 μM are represented by “***,” compounds that gave an IC50 of between about 1 and 4.9 μM are represented by “**,” and compounds exhibiting IC50's of less than 0.9 μM are represented by “***.”
Using the chemiluminescence-based assay described above in Example 2, in vitro EC50 (μM) values for the transcription factor modulating compounds were obtained for several AraC family members: MarA, SoxS and Rob (E. coli), ExsA (P. aeruginosa), Rma (S. typhimurium), PqrA (P. mirabilis) and SlyA, which is a member of a different superfamily (the MarR protein). Although SlyA contains a helix-turn-helix DNA binding motif, it is not related to members of the MarA protein family. The results are given in Table 10. Compounds that gave an EC50 of greater than 5 μM are represented by “*,” compounds that gave an EC50 of between about 1 and 4.9 μM are represented by “**,” and compounds exhibiting EC50's of less than 0.9 μM are represented by “***,” and compounds that were not tested are represented by “—.”
aStandard error <15% for all values
Results of a subsequent assay including VirF of Y. pseudotuberculosis are shown in Table 11. Compounds that gave EC50's of less than about 5 μM are shown by “*”; compounds with EC50's of between about 5.1 and 15 μM are shown by “**”; and compounds with EC50's of greater than 15.1 μM are shown by “***”.
Y. pseudo-
P.
E. coli
E. coli
tuberculosis
aeruginosa
Approximately 30 Swiss Webster mice (females, 18-24 grams) are randomized to one of 4 groups of 5-10 mice per group. Animals are briefly anesthetized by isofluorane inhalation for 10-30 seconds in order to minimize the stress during intranasal inoculation. The mice are infected intranasally with 1×106 P. aeruginosa bacteria diluted in room temperature sterile phosphate buffered saline (PBS) in a volume of 50 μL; a control group receives intranasal PBS with no bacteria. The mice are allowed to recover in an inclined position to improved infection efficacy. The mice are dosed IP with 25 mg/kg of the test compound in a maximum volume of 10 mL/kg (or equal volume of 5% PEG400, 95% H2O vehicle alone) at −1, 2, 5, 20, 26, 44 and 50 hours post-infection. Infected mice are monitored for morbidity and survival twice daily over the course of 7 days. Any mice exhibiting signs of severe illness, e.g., 20% loss of their starting body weight, severe ataxia, shaking, labored breathing, unresponsiveness, etc., are painlessly euthanized by CO2 narcoses and cervical dislocation and marked as dead. Mice infected with this inoculum of wild type P. aeruginosa (PA103) typically succumb to the infection within 48-72 hours, whereas mice infected with an ExsA null mutant strain (PA103 ΔExsA) survive indefinitely. Compounds are also tested by IV or PO administration with dose level and schedule determined from PK evaluation by these routes.
In experiments where the determination of bacterial burden in individual organs is desired, mice are infected intranasally with ˜4×105 P. aeruginosa bacteria and receive the −1, 2, and 5 hour doses of compound or vehicle control. At 18 hours post infection, all mice are euthanized by CO2 narcoses and cervical dislocation. Blood is collected immediately via cardiac puncture, and the liver, spleen and lungs are collected and weighed aseptically. Organs are homogenized in sterile PBS, and tissue and blood are plated in serial dilutions on rich media, and incubated at 37° C. for 24 hours to determine bacterial counts. In this model, infection with wild type (PA103) P. aeruginosa results in a lung bacterial burden greater than the inoculum with detectable dissemination to the peripheral tissues. Mice are not expected to develop pronounced illness in this model, but if any animals become severely moribund, they are euthanized immediately (as described previously) and marked as dead. In this model, the bacterial counts in the lungs and peripheral organs in mice infected with ExsA null mutant bacteria (PA103ΔExsA) are typically at least 2 logs lower than for mice infected with wild type (PA103) bacteria.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific polypeptides, nucleic acids, methods, assays and reagents described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.
This application claims priority to U.S. Provisional Patent Application No. 60/920,316, filed on Mar. 27, 2007; U.S. Provisional Patent Application No. 60/931,040, filed on May 21, 2007; U.S. Provisional Patent Application No. 60/934,684, filed on Jun. 15, 2007; U.S. Provisional Patent Application No. 60/973,371, filed on Sep. 18, 2007; U.S. Provisional Patent Application No. 61/016,267, filed on Dec. 21, 2007 and U.S. Provisional Patent Application No. 61/021,136, filed on Jan. 15, 2008. The contents of each of the aforementioned applications are hereby incorporated by reference in their entirety.
This application was funded, at least in part, by grant NIH NIAID 5R43AI058627-2. The government, therefore, has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60973371 | Sep 2007 | US | |
| 60920316 | Mar 2007 | US | |
| 61016267 | Dec 2007 | US | |
| 60934684 | Jun 2007 | US | |
| 60931040 | May 2007 | US | |
| 61021136 | Jan 2008 | US |
