USOO7659399B2

(12) United States Patent (10) Patent No.: US 7.659,399 B2 Bradbury et al. (45) Date of Patent: *Feb. 9, 2010

(54) 1-THIA-24A-DIAZA-CYCLOPENTAB JP O3-209367 9, 1991 NAPTHTHALENE-3,4-DIONES AND JP 10-130 149 5, 1998 RELATED COMPOUNDSAS WO WO95/298.94 11, 1995 ANT-INFECTIVE AGENTS WO WO 2005/019228 A1 3, 2005 OTHER PUBLICATIONS (75) Inventors: Barton James Bradbury, Wallingford, CT (US); Jason Allan Wiles, Hamden, Dorwald F. A. Side Reactions in Organic Synthesis, 2005, Wiley: VCH. Weinheimp. IX of Preface.* CT (US) Li, Qun et al., “Synthesis and Structure-Activity Relationships of O O 2-Pyridones: A Novel Series of Potent DNA Gyrase Inhibitors as (73) Assignee: Achillion Pharmaceuticals, Inc., New Antibacterial Agents.” J. Med. Chem. (1996) 39: 3070-3088. Haven, CT (US) Wiles, Jason A. et al., “Isothiazolopyridones: Synthesis, Structure, and Biological Activity of a New Class of Antibacterial Agents.” J. (*) Notice: Subject to any disclaimer, the term of this Med. Chem. (2006) 49: 39-42. patent is extended or adjusted under 35 Frigola, Jordi et al., “7-AZetidinylcquinolones as Antibacterial U.S.C. 154(b) by 709 days. Agents. Synthesis and Structure-Activity Relationships.” J. Med. Chem. (1993) 36: 801-810. This patent is Subject to a terminal dis- Ishiyama, Tatsuo et al., “Palladium (0)-Catalyzed Cross-Coupling claimer Reaction of Alkoxydiboron with Haloarenes: A Direct Procedure for Arylboronic Esters,” J. Org. Chem. (1995) 60:7508-7510. (21) Appl. No.: 11/326,109 (Continued) (22) Filed: Jan. 5, 2006 Primary Examiner Rita J Desai (74) Attorney, Agent, or Firm—Cantor Colburn LLP (65) Prior Publication Data (57) ABSTRACT US 2007/0129333 A1 Jun. 7, 2007 The invention provides compounds and salts of Formula I and Related U.S. Application Data Formula II: (60) Provisional application No. 60/641,548, filed on Jan. 5, 2005 Formula I s Rs O O (51) Int. Cl. R6 CO7D 417/04 (2006.01) 21 NN N-R CO7D 53/04 (2006.01) N 1S M 2 A6 IK 3/4365 (2006.01) R As Al (52) U.S. Cl...... 546/83:546/115; 514/278: 514,293 Ro (58) Field of Classification Search ...... 546/83, Rs. O R3 Formula II 546/115; 514/278, 293 5 o1 See application file for complete search history. R6 21 NN N (56) References Cited N U.S. PATENT DOCUMENTS R7 Sn As 1S M 4,767,762 A 8, 1988 Chu R9 4,945,160 A 7/1990 Kiely et al. 5,071,848 A 12/1991 Chu et al. which possess antimicrobial activity. The invention also pro 5,387,748 A 2/1995 Demuth, Jr. et al. vides novel synthetic intermediates useful in making com 5,519,016 A 5, 1996 Kimura et al. pounds of Formula I and Formula II. The variables A. As R. 5,580,872 A 12/1996 Chu et al. R. R. R. R., and R are defined herein. 5,631,256 A 5/1997 Demuth, Jr. et al. 5,645,163 A T. 1997 Werth Certain compounds of Formula I and Formula II disclosed 5,688,791 A 11/1997 Kimura et al. herein are potent and selective inhibitors of bacterial DNA 7,199,128 B2 * 4/2007 Bradbury et al...... 514,278 synthesis and bacterial replication. The invention also pro vides antimicrobial compositions, including pharmaceutical FOREIGN PATENT DOCUMENTS compositions, containing one or more compounds of For EP O 227 O39 A1 7, 1987 mula I or Formula II and one or more carriers, excipients, or EP O 394 120 A1 4, 1990 diluents. Such compositions may contain a compound of EP O878.194 11, 1998 Formula I or Formula II as the only active agent or may JP O1-160985 6, 1989 contain a combination of a compound of Formula I or For E. 82.6 88. mula II and one or more other active agents. The invention JP o,4784 7, 1990 also provides methods for treating microbial infections in JP O2-243692 9, 1990 animals. JP O2-255687 10, 1990 JP 03-058992 3, 1991 19 Claims, No Drawings US 7.659,399 B2 Page 2

OTHER PUBLICATIONS Li, Qun et al., “Synthesis and Structure-Activity Relationships of Kiely, John S. et al., “Synthesis of 7-(Alkenyl, Cycloalkenyl, and 2-Pyridones: A Novel Series of Potent DNA Gyrase Inhibitors as 1,2,3,6-Tetrahydro-4-pyridinyl)guinolones,” J. Heterocyclic Chem. Antibacterial Agents.” J. Med. Chem. (1996) 39: 3070-3088. (1991) 28: 1581-1585. Otten, Pieter A. et al., “4-Oxo-4H-quinolizine-3-carboxylic Acids as Laborde, Edgardo et al., Novel 7-Substituted Quinolone Antibacte Mg2+ Selective, Fluorescent Indicators.” Bioconjugate Chem. rial Agents. Synthesis of 7-Alkenyl, Cycloalkenyl, and 1,2,3,6- (2001) 12:203-212. Tetrahydro-4-pyridinyl-1,8-naphthyridines 1), J. Heterocyclic Tamao, Kohei and Miyaura, Norio, “Introduction to Cross-Coupling Chem. (1991) 28:191-198. Reactions.” Topics in Current Chemistry (2002) 219:1-9. Laborde, Edgardo et al., “Quinolone Antibacterials: Synthesis and Wiles, J. A. et al., “General, Efficient, One-Step Synthesis,” J. Med. Biological Activity of Carbon Isosteres of the 1-Piperazinyl and Chem. (2006) 49: 39-42. 3-Amino-1-pyrrolidinyl Side Chains 1).” J. Med. Chem. (1992) 36: 1964-1970. * cited by examiner US 7,659,399 B2 1. 2 1-THIA-24A-DIAZA-CYCLOPENTAB Pathogenic bacteria are known to acquire resistance via NAPTHTHALENE-3,4-DIONES AND several distinct mechanisms including inactivation of the RELATED COMPOUNDSAS antibiotic by bacterial enzymes (e.g., beta-lactamases that ANT-INFECTIVE AGENTS hydrolyze penicillin and cephalosporins); removal of the anti biotic using efflux pumps; modification of the target of the CROSS REFERENCE TO RELATED antibiotic via mutation and genetic recombination (e.g., peni APPLICATION cillin-resistance in Neiserriagonorrhea); and acquisition of a readily transferable gene from an external Source to create a This application claims priority from U.S. Provisional resistant target (e.g., methicillin-resistance in Staphylococcus application 60/641,548 filed Jan. 5, 2005, which is hereby 10 aureus). There are certain gram-positive pathogens, such as incorporated by reference in its entirety. Vancomycin-resistant Enterococcus faecium, which are resis tant to virtually all commercially available antibiotics. FIELD OF THE INVENTION Resistant organisms of particular note include methicillin The present invention provides 1-thia-2.4a-diaza-cyclo 15 resistant and Vancomycin-resistant Staphylococcus aureus, pentabnaphthalene-3,4-diones and related compounds, penicillin-resistant Streptococcus pneumoniae, Vancomycin which possess antimicrobial activity. Certain compounds resistant enterococci, fluoroquinolone-resistant E. coli, provided herein possess potent antibacterial, antiprotozoal, or cephalosporin-resistant aerobic gram-negative rods and imi antifungal activity. Particular compounds provided herein are penem-resistant Pseudomonas aeruginosa. These organisms also potent and/or selective inhibitors of prokaryotic DNA are significant causes of nosocomial infections and are clearly synthesis and prokaryotic reproduction. The invention pro associated with increasing morbidity and mortality. The vides anti-microbial compositions, including pharmaceutical increasing numbers of elderly and immunocompromised compositions, containing one or more carrier, diluents, or patients are particularly at risk for infection with these patho excipients. The invention provides pharmaceutical composi gens. Therefore, there is a large unmet medical need for the tions containing a 1-thia-2.4a-diaza-cyclopentabnaphtha 25 development of new antimicrobial agents. lene-3,4-dione or related compound as the only active agent or containing a 1-thia-2.4a-diaza-cyclopentabnaphthalene SUMMARY OF THE INVENTION 3,4-dione or related compound in combination with one or more other active agent, Such as one or more other antimicro The invention provides compounds of Formula I and For bial or antifungal agent. The invention provides methods for 30 mula II (shown below) and includes 1-thia-2.4a-diaza-cyclo treating or preventing microbial infections in eukaryotes, pentabnaphthalene-3,4-dione and related compounds, preferably animals, by administering an effective amount of a which possess antimicrobial activity. The invention provides 1-thia-2.4a-diaza-cyclopentabnaphthalene-3,4-dione or compounds of Formula I and Formula II that possess potent related compound to a eukaryote suffering from or Suscep and/or selective antibacterial, antiprotozoal, or antifungal tible to microbial infection. The invention also provides 35 activity. The invention also provides anti-bacterial composi methods of inhibiting microbial growth and survival by tions containing one or more compounds of Formula I or applying an effective amount of a 1-thia-2.4a-diaza-cyclo Formula II, or a salt, Solvate, or acylated prodrug of Such a pentabnaphthalene-3,4-dione or related compound. compound, and one or more carriers, excipients, or diluents. The invention also provides novel intermediates useful for The invention further comprises methods of treating and the for the synthesis of 1-thia-2.4a-diaza-cyclopentabnaph 40 preventing microbial infections, particularly bacterial and thalene-3,4-diones and related compounds. The invention protozoal infections by administering and effective amount of also provides methods of synthesis of 1-thia-2.4a-diaza-cy a compound of Formula I or Formula II to a eukaryote suf clopentabnaphthalene-3,4-diones and related compounds. fering from or susceptible to a microbial infection. These 45 microbial infections include bacterial infections, for example BACKGROUND OF THE INVENTION E. coli infections, Staphylococcus infections, Salmonella infections and protozoal infections, for example Chlamydia Antimicrobial compounds are compounds capable of infections. The invention is particularly includes methods of destroying or Suppressing the growth or reproduction of preventing or treating microbial infections in mammals, microorganisms, such as bacteria, protozoa, mycoplasma, 50 including humans, but also encompasses methods of prevent yeast, and fungi. The mechanisms by which antimicrobial ing or treating microbial infections in other animals, includ compounds act vary. However, they are generally believed to ing fish, birds, reptiles, and amphibians. function in one or more of the following ways: by inhibiting Methods of treatment include administering a compound cell wall synthesis or repair; by altering cell wall permeabil of Formula I or Formula II alone as the single active agent or ity; by inhibiting protein synthesis; or by inhibiting synthesis 55 administering a compound of Formula I in combination with of nucleic acids. For example, beta-lactam antibacterials one or more other therapeutic agent, such as an antibacterial, inhibit the essential penicillin binding proteins (PBPs) in an antifungal, an antiviral, an interferon, an efflux-pump bacteria, which are responsible for cell wall synthesis. Qui inhibitor, a beta-lactamase inhibitor, or another compound of nolones act, at least in part, by inhibiting synthesis of DNA, Formula I or Formula II. thus preventing the cell from replicating. 60 The invention also provides methods of inhibiting micro Many attempts to produce improved antimicrobials yield bial growth and Survival by applying an effective amount of equivocal results. Indeed, few antimicrobials are produced an 1-thia-2.4a-diaza-cyclopentabnaphthalene-3,4-dione or that are truly clinically acceptable in terms of their spectrum related compound. The invention includes, for example, of antimicrobial activity, avoidance of microbial resistance, methods of inhibiting microbial growth and survival on medi and pharmacology. Thus there is a continuing need for broad 65 cal instruments or on Surfaces used for food preparation by spectrum antimicrobials, and a particular need for antimicro applying a composition containing a compound of Formula I bials effective against resistant microbes. or Formula II. US 7,659,399 B2 3 4 Thus, the invention include compounds of Formula I and group, a monocyclic orbicyclic a 5- to 10-membered bicyclic Formula II saturated, partially unsaturated, or aromatic heterocylic group bound via a carbon atom; each of which R is substi tuted with 0 to 5 substituents independently chosen from (i), Formula I 5 Rs O O (ii), and (iii); where R6 (i) is chosen from halogen, hydroxy, amino, cyano, and 21 NN nitro, N- R2 N1S X (ii) is chosen from C-Calkyl, C-Calkenyl, R As 10 C-Calkynyl, (C-Calkoxy)Co-Calkyl, mono- and di-(C- C.)alkylamino, C-Chaloalkyl, C-Chaloalkoxy, (C- R9 C-cycloalkyl)Co-Cacarbohydryl, (C-C,cycloalkyl)Co Formula II Ccarbohydryl-O , (C-C,cycloalkenyl)Co Rs O o1 R3 Ccarbohydryl, (aryl)Co-Cocarbohydryl, (aryl)C-Calkoxy, 15 R6 (C-Cheterocycloalkyl)Co-Cacarbohydryl, (heteroaryl)Co 21 N N Ccarbohydryl, C-Calkylthio. —NOR —NR —(Co N Calkyl)(C=O)Rio —(Co-Calkyl)O(C=O)Rio —(Co R7 NAs S AlM Calkyl)(C=O)NRR, —(Co-Calkyl)O(C=O) NRR, —(Co-Calkyl)(C=O)OR —(Co-Calkyl)NR R9 (C=O)R. —(Co-Calkyl)NR(C=O)OR, —(Co Calkyl)NR(C=O)NRR, (Co-Calkyl)NR(C=O) and the pharmaceutically acceptable salts thereof, wherein: (C-C alkyl)NR (C=O)C) R. —(Co-Calkyl)NR A is S. O, SO, or SO. (C=S)NRR, -(Co-Calkyl)NRNR, R2, —(Co R is hydrogen, or 25 Calkyl)N=NR —(Co-Calkyl)SO-Ro. —(Co-Calkyl) R is C-Calkyl, C-Calkenyl, C-Calkynyl, (C- (S=O)OR —(Co-Calkyl)SOR. -(Co-Calkyl) C-cycloalkyl)Co-Cacarbohydryl, (C-C cycloalkenyl) SONROR, and —(Co-Calkyl)NRoSOR: and Co-Cacarbohydryl, (aryl)Co-Cacarbohydryl, or (C- (iii) is chosen from —OR —(C=O)R. —SOR, Cheterocycloalkyl)Co-Cacarbohydryl, each of which is —SOR —NRoSOR, where R is C-C alkyl, (C- substituted with 0 to 5 substituents independently cho 30 C,cycloalkyl)Co-Calkyl, (C-Cheterocycloalkyl)Co Sen from halogen, hydroxy, amino, cyano, nitro, Calkyl, (aryl)Co-Calkyl, and (heteroaryl)Co-Calkyl, C-C alkyl, C-C alkoxy, C-Chaloalkyl, where each of (ii) and (iii) is substituted with 0 to 3 sub C-Chaloalkoxy, mono- and di-C-C-alkylamino, stituents independently chosen from halogen, hydroxy, C-C alkanoyl, C-C alkylthio. =NOR =NRo, 35 amino, cyano, nitro, oxo. —COOH, -CONH2, C-C alkyl, —O(C=O)R —(C=O)NRR, —O(C=O) C-Calkenyl, C-C alkynyl, C-C alkoxy, NRoR, —(C=O)OR, —(C=O)NROR, C-Calkoxycarbonyl, (C-C,cycloalkyl)Co-Cacarbohydryl, —NR(C=O)R, NR(C=O)OR, NR (C-C,cycloalkyl)Co-Calkoxy, mono- and di-(C-C)alky (C=O)ORR, —NR(C=S)NRR, lamino, C-Chaloalkyl, C-Chaloalkoxy, C-C alkanoyl —NRoNRR, —SO-Ro, —(S—O)OR, —SOR. -SO2NRoR, and—NRoSOR: where 40 and phenyl. Rio R, and R2 are independently hydrogen, As is N or CRs and Rs is hydrogen, halogen, hydroxy, C-C alkyl, or aryl, and R is C-C alkyl or aryl. amino, cyano, nitro, or -NHNH, or Rs is C-Clalkyl, R is hydrogen, C-Calkyl, C-Calkanoyl, mono- or C-Calkoxy, mono- or di-(C-C)alkylamino, mono-, di-, or di-C-Calkylcarbamate, or C-Calkylsulfonate; each of tri-C-C alkylhydrazinyl, C-Calkanoyl, C-Calkylester, which is substituted with 0 to 3 substituents independently 45 C-Chaloalkyl, or C-Chaloalkoxy; each of which is Sub chosen from halogen, hydroxy, amino, cyano, nitro, stituted with 0 to 3 substituents independently chosen from C-C alkoxy, OO- and di-C-C-alkylamino, hydroxy, amino, halogen, oxo, C-C alkoxy, C-Chaloalkyl, C-Chaloalkyl, and C-Chaloalkoxy. C-Chaloalkoxy, and mono- and di-C-C-alkylamino. Rs is hydrogen, halogen, hydroxy, amino, cyano, nitro, or 50 Ro is C-Csalkyl, C-Calkoxy, mono- or di-(C-C)alky —NHNH, or lamino, C-C alkanoyl, C-Chaloalkyl, C-Chaloalkoxy, Rs is C-C alkyl, C-Calkoxy, mono- or di-(C-C)alky (C-C,cycloalkyl)Co-Calkyl, or phenyl, each of which is lamino, mono-, di- or tri-C-C-alkylhydrazinyl, substituted with 0 to 3 substituents independently chosen C-C alkanoyl, C-C alkylester, C-Chaloalkyl, or from halogen, hydroxy, amino, cyano, nitro. —COOH, C-Chaloalkoxy; each of which is substituted with 0 to 3 55 Substituents independently chosen from hydroxy, amino, —CONH2, C-C alkyl, C-Calkenyl, C-C alkynyl. halogen, OXO, C-C alkoxy, C-Chaloalkyl, C-Calkoxy, (C-C,cycloalkyl)Co-Calkyl, (C- C-Chaloalkoxy, and mono- and di-C-C-alkylamino. C-cycloalkyl)Co-Calkoxy, mono- and di-(C-C)alky R is hydrogen, halogen, hydroxy, amino, cyano, lamino, C-Chaloalkyl, C-Chaloalkoxy, and C-C alkanoyl. C-C alkyl, C-Calkoxy, mono- or di-(C-C)alkylamino, 60 —SO-Ro. —SOR or —SONRoR. The invention includes novel intermediates useful for the Riis chloro, bromo, iodo, —O(SO)CF, or NBF, or synthesis of antimicrobial compounds of Formula I and For R, is XR where X is absent, —CH2—CH2—, mula II. These intermediates are compounds of Formula I and -CH=CH-, -(C=O)— —(C=O)NH , or Formula II in which R, is chloro, bromo, iodo, —O(SO)CF, —C=C , and R is C-Calkyl, C-C,cycloalkyl, 65 or NBF. The invention provides methods of synthesizing C-C,cycloalkenyl, phenyl, a bicyclic 7- to 10-membered compounds of Formula I and Formula II comprising coupling saturated, partially unsaturated, or aromatic carbocyclic an intermediate of the invention to an appropriate aryl or US 7,659,399 B2 5 6 heteroarylboronic acid, aryl or heteroarylboronic acid ester, the core structure. For example, it is to be understood that or compounds substituted with Li, Mg, B, Al, Si, Zn, Cu, Zr, when (cycloalkyl)alkyl is listed as a possible substituent the or Sn at the point of coupling. point of attachment of this substituent to the core structure is in the alkyl portion. DETAILED DESCRIPTION OF THE INVENTION A dash (“ ”) that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For Chemical Description and Terminology example, —CONH is attached through the carbon atom. Prior to setting forth the invention in detail, it may be As used herein, “alkyl is intended to include both helpful to provide definitions of certain terms to be used branched and straight-chain Saturated aliphatic hydrocarbon 10 groups, having the specified number of carbon atoms. Thus, herein. Compounds of the present invention are generally the term C-C alkyl as used herein includes alkyl groups described using standard nomenclature. having from 1 to about 6 carbon atoms. When Co-C, alkyl is In certain situations, the compounds of Formula I and used herein in conjunction with another group, for example, Formula II may contain one or more asymmetric elements (aryl)Co-Calkyl, the indicated group, in this case aryl, is Such as stereogenic centers, Stereogenic axes and the like, e.g. 15 either directly bound by a single covalent bond (C), or asymmetric carbonatoms, so that the compounds can exist in attached by an alkyl chain having the specified number of different stereoisomeric forms. These compounds can be, for carbon atoms, in this case from 1 to about 4 carbon atoms. example, racemates or optically active forms. For compounds Examples of alkyl include, but are not limited to, methyl, with two or more asymmetric elements, these compounds can ethyl, n-propyl, isopropyl. n-butyl, t-butyl, n-pentyl, and sec additionally be mixtures of diastereomers. For compounds pentyl. Preferred alkyl groups are lower alkyl groups, those having asymmetric centers, it should be understood that all of alkyl groups having from 1 to about 8 carbon atoms or 1 to the optical isomers and mixtures thereofare encompassed. In about 6 carbon atoms, e.g. C-Cs and C-Calkyl groups. addition, compounds with carbon-carbon double bonds may Alkenyl as used herein, indicates a hydrocarbon chain of occur in Z- and E-forms, with all isomeric forms of the eithera straight or branched configuration having one or more compounds being included in the present invention. In these 25 carbon-carbon double bond bonds, which may occur at any situations, the single enantiomers, i.e., optically active forms stable point along the chain. Examples of alkenyl groups can be obtained by asymmetric synthesis, synthesis from include ethenyl and propenyl. optically pure precursors, or by resolution of the racemates. Alkynyl as used herein, indicates a hydrocarbon chain of Resolution of the racemates can also be accomplished, for eithera straight or branched configuration having one or more example, by conventional methods such as crystallization in 30 triple carbon-carbon bonds that may occur in any stable point the presence of a resolving agent, or chromatography, using, along the chain, such as ethynyl and propynyl. for example a chiral HPLC column. “Carbohydryl” as used herein, includes both branched and Where a compound exists in various tautomeric forms, the straight-chain hydrocarbon groups, which are saturated or invention is not limited to any one of the specific tautomers, unsaturated, having the specified number of carbon atoms. but rather includes all tautomeric forms. 35 When Co-Cearbohydryl is used herein in conjunction with The present invention is intended to include all isotopes of another group, for example, (aryl)Co-Cacarbohydryl, the atoms occurring in the present compounds. Isotopes include indicated group, in this case aryl, is either directly bound by a those atoms having the same atomic number but different single covalent bond (Co), or attached by an carbohydryl mass numbers. By way of general example, and without chain, Such as an alkyl chain, having the specified number of limitation, isotopes of hydrogen include tritium and deute 40 carbon atoms, in this case from 1 to about 4 carbon atoms. rium and isotopes of carbon include ''C, C, and ''C. Examples include C-Calkyl, Such as methyl, or 5-butyl, Certain compounds are described herein using a general C-Calkynyl such and hexynyl, and C-C alkenyl, such as formula that includes variables, e.g. A. R. R. R. R. R-7, 1-propenyl. As and A. Unless otherwise specified, each variable within “Alkoxy' represents an alkyl group as defined above with such a formula is defined independently of other variables. 45 the indicated number of carbon atoms attached through an Thus, ifa group is said to be substituted, e.g. with 0-2 R*, then oxygen bridge. Examples of alkoxy include, but are not lim said group may be substituted with up to two R* groups and ited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, R* at each occurrence is selected independently from the 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopen definition of R*. Also, combinations of substituents and/or toxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-me variables are permissible only if such combinations result in 50 thylpentoxy. In the group “(alkoxy)alkyl the alkoxy and stable compounds. alkyl groups carry the definitions set forth above and the point The term “substituted, as used herein, means that any one of attachment to the core molecule is in the alkyl. or more hydrogens on the designated atom or group is Alkanoyl indicates an alkyl group as defined above, replaced with a selection from the indicated group, provided attached through a keto (—(C=O)—) bridge. Alkanoyl that the designated atoms normal valence is not exceeded. 55 groups have the indicated number of carbon atoms, with the When a substituent is oxo (i.e., =O), imine (e.g. —NR), or carbon of the keto group being included in the numbered Oxime (e.g. =NOR) then 2 hydrogens on the atom are carbon atoms. For example a Calkanoyl group is an acetyl replaced. An "oxo, imine, or oxime Substituent on an aro group having the formula CH (C=O)—. matic group or heteroaromatic group destroys the aromatic As used herein, the terms “mono- or di-alkylamino” or character of that group, e.g. a pyridyl Substituted with oxo is 60 "mono- and di-alkylamino' indicate secondary or tertiary pyridone. Combinations of substituents and/or variables are alkyl amino groups, wherein the alkyl groups are as defined permissible only if Such combinations result in stable com above and have the indicated number of carbon atoms. The pounds or useful synthetic intermediates. A stable compound point of attachment of the alkylamino group is on the nitro or stable structure is meant to imply a compound that is gen. Examples of mono- and di-alkylamino groups include sufficiently robust to survive isolation from a reaction mix 65 ethylamino, dimethylamino, and methyl-propyl-amino. ture, and Subsequent formulation into an effective therapeutic The term “mono- or di-alkylcarbamate' indicates 1 or 2 agent. Unless otherwise specified Substituents are named into independently chosen alkyl groups, as defined above, US 7,659,399 B2 7 8 attached through a carbamate ( O(C=O)NRR) linkage indicates a (cycloalkyl)carbohydryl group as defined above where each R represents an independently chosen alkyl attached via an oxygen bridge. group. Mono-alkylcarbamate groups have the formula (—O In the terms “(cycloalkenyl)alkyl and “(cycloalkenyl)car (C=O)NHR. bohydryl the terms cycloalkenyl, alkyl, and carbohydryl are The term “alkylester indicates and alkyl group as define as defined above, and the point of attachment is on the alkyl or above attached through an ester linkage in either orientation, carbohydryl group respectively. These terms include i.e. a group of the formula—O(C=O)alkyl or a group of the examples such as cyclobutenylmethyl, cyclohexenylmethyl, formula—(C=O)Oalkyl. and cyclohexylpropenyl. The term “mono-, di-, or tri-alkylhydrazinyl' indicates "Haloalkyl indicates both branched and straight-chain from 1 to 3 independently chosen alkyl group as defined 10 saturated aliphatic hydrocarbon groups having the specified above attached through a single-bonded nitrogen-nitrogen number of carbon atoms, Substituted with 1 or more halogen linkage. At least one of the alkyl groups is attached to the atoms, generally up to the maximum allowable number of terminal nitrogen (the nitrogen not bound to the core struc halogen atoms. Examples of haloalkyl include, but are not ture). When the term mono-alkylhydrazinyl is used only the limited to, trifluoromethyl, difluoromethyl 2-fluoroethyl, terminal nitrogen is alkyl Substituted. Examples of alkylhy 15 and penta-fluoroethyl. drazinyl groups include 2-butyl-1-hydrazinyl, 2-butyl-2-me "Haloalkoxy” indicates a haloalkyl group as defined above thyl-1-hydraaZinyl, and 1,2-dimethyl-2-propyl-1-hydrazinyl. attached through an oxygen bridge. The term “alkylthio' indicates an alkyl group as defined "Halo’ or “halogen as used herein refers to fluoro, chloro, above attached through a Sulfur linkage, i.e. a group of the bromo, or iodo. formula alkyl-S . Examples include ethylthio and pen As used herein, "heteroaryl indicates a stable 5- to tylthio. 7-membered monocyclic or 7- to 10-membered bicyclic het As used herein, the term “aryl' indicates aromatic groups erocyclic ring which contains at least 1 aromatic ring that containing only carbon in the aromatic ring or rings. Typical contains from 1 to 4, or preferably from 1 to 3, heteroatoms aryl groups contain 1 to 3 separate, fused, or pendant rings chosen from N, O, and S, with remaining ring atoms being and from 6 to about 18 ring atoms, without heteroatoms as 25 carbon. When the total number of S and O atoms in the ring members. When indicated, Such aryl groups may be heteroaryl group exceeds 1, these heteroatoms are not adja further substituted with carbon or non-carbon atoms or cent to one another. It is preferred that the total number of S groups. Such substitution may include fusion to a 5 to 7-mem and O atoms in the heteroaryl group is not more than 2. It is bered Saturated cyclic group that optionally contains 1 or 2 particularly preferred that the total number of S and O atoms heteroatoms independently chosen from N, O, and S, to form, 30 in the aromatic heterocycle is not more than 1. A nitrogen for example, a 3.4-methylenedioxy-phenyl group. Aryl atom in a heteroaryl group may optionally be quaternized. groups include, for example, phenyl, naphthyl, including When indicated, such heteroaryl groups may be further sub 1-naphthyl and 2-maphthyl, and bi-phenyl. stituted with carbon or non-carbon atoms or groups. Such In the term “(aryl)alkyl, aryl and alkyl are as defined substitution may include fusion to a 5 to 7-membered satu above, and the point of attachment is on the alkyl group. This 35 rated cyclic group that optionally contains 1 or 2 heteroatoms term encompasses, but is not limited to, benzyl, phenylethyl, independently chosen from N, O, and S, to form, for example, and piperonyl. Likewise, in the term “(aryl)carbohydryl'. a 1.3dioxolo4.5-cpyridyl group. Examples of heteroaryl aryl and carbohydryl are as defined above and the point of groups include, but are not limited to, pyridyl, indolyl, pyri attachment is on the carbohydryl group, for example a phe midinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, fura 40 nyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, nylpropen-1-yl group. quinolinyl, pyrrolyl, pyrazolyl, benzbithiophenyl, isoquino The term “carbocyclic group' indicates a 5-6 membered linyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl, and 5.6, saturated, partially unsaturated, or aromatic ring containing 7,8-tetrahydroisoquinoline. only carbon ring atoms or a 7-10 membered bicyclic Satu In the terms "heteroarylalkyl and “(heteroaryl)carbohy rated, partially unsaturated, or aromatic ring system contain 45 dryl. heteroaryl, alkyl, and carbohydryl areas defined above, ing only carbon ring atoms. Unless otherwise indicated, the and the point of attachment is on the alkyl or carbohydryl carbocyclic ring may be attached to its pendant group at any group respectively. These terms include Such examples as carbon atom that results in a stable structure. I pyridylmethyl, thiophenylmethyl, and (pyrrolyl)1-ethyl. “Cycloalkyl as used herein, indicates a saturated hydro The term "heterocycloalkyl indicates a saturated cyclic carbon ring, having the specified number of carbon atoms, 50 group containing from 1 to about 3 heteroatoms chosen from usually from 3 to about 8 ring carbon atoms, or from 3 to N, O, and S, with remaining ring atoms being carbon. Het about 7 carbonatoms. Examples of cycloalkyl groups include erocycloalkyl groups have from 3 to about 8 ring atoms, and cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexylas well as more typically have from 5 to 7 ring atoms. Examples of bridged or caged saturated ring groups such as norborane or heterocycloalkyl groups include morpholinyl, piperazinyl, adamantane. 55 piperidinyl, and pyrrolidinyl groups. A nitrogen in a hetero “Cycloalkenyl as used herein, indicates an unsaturated, cycloalkyl group may optionally be quaternized. but not aromatic, hydrocarbon ring having at least one car The term "heterocyclic group' indicates a 5-6 membered bon-carbon double bond. Cycloalkenyl groups contain from 4 saturated, partially unsaturated, or aromatic ring containing to about 8 carbon atoms, usually from 4 to about 7 carbon from 1 to about 4 heteroatoms chosen from N, O, and S, with atoms. Examples include cyclohexenyl and cyclobutenyl. 60 remaining ring atoms being carbon or a 7-10 membered bicy In the terms “(cycloalkyl)alkyl.” “(cycloalkyl)carbohy clic Saturated, partially unsaturated, or aromatic heterocylic dryl.” and “(cycloalkyl)alkoxy” the terms cycloalkyl, alkyl, ring system containing at least 1 heteroatom in the two ring carbohydryl, and alkoxy areas defined above, and the point of system chosen from N, O, and S and containing up to about 4 attachment is on the alkyl, carbohydryl, or alkoxy group heteroatoms independently chosen from N, O, and S in each respectively. These terms include examples such as cyclopro 65 ring of the two ring system. Unless otherwise indicated, the pylmethyl, cyclohexylmethyl, cyclohexylpropenyl, and heterocyclic ring may be attached to its pendant group at any cyclopentylethyoxy. The term (cycloalkyl)carbohydryl-O heteroatom or carbon atom that results in a stable structure. US 7,659,399 B2 10 When indicated the heterocyclic rings described herein may modified by making non-toxic acid or base salts thereof, and be substituted on carbon or on a nitrogenatom if the resulting further refers to pharmaceutically acceptable solvates of such compound is stable. A nitrogen atom in the heterocycle may compounds and Such salts. Examples of pharmaceutically optionally be quatemized. It is preferred that the total number acceptable salts include, but are not limited to, mineral or of heteroatoms in a heterocyclic groups is not more than 4 and that the total number of S and Oatoms in a heterocyclic group organic acid salts of basic residues such as amines; alkali or is not more than 2, more preferably not more than 1. organic salts of acidic residues such as carboxylic acids; and Examples of heterocyclic groups include, pyridyl, indolyl, the like. The pharmaceutically acceptable salts include the pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, conventional non-toxic salts and the quaternary ammonium furanyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isox 10 salts of the parent compound formed, for example, from azolyl, quinolinyl, pyrrolyl pyrazolyl, benzbithiophenyl, non-toxic inorganic or organic acids. For example, conven isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl, tional non-toxic acid salts include those derived from inor dihydroisoindolyl,5,6,7,8-tetrahydroisoquinoline, pyridinyl, ganic acids such as hydrochloric, hydrobromic, Sulfuric, Sul pyrimidinyl, furanyl, thienyl, pyrrolyl pyrazolyl pyrrolidi famic, phosphoric, nitric and the like; and the salts prepared nyl, morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl. 15 from organic acids such as acetic, propionic, Succinic, gly Additional examples heterocyclic groups include, but are not limited to, phthalazinyl, oxazolyl, indolizinyl, indazolyl, colic, Stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, benzothiazolyl, benzimidazolyl, benzofuranyl, benzoisox maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, olyl, dihydro-benzodioxinyl, oxadiazolyl, thiadiazolyl, triaz salicylic, mesylic, esylic, besylic, Sulfanilic, 2-acetoxyben olyl, tetrazolyl, oxazolopyridinyl, imidazopyridinyl, isothia Zoic, fumaric, toluenesulfonic, methanesulfonic, ethane dis Zolyl, naphthyridinyl, cinnolinyl, carbazolyl, beta-carbolinyl, ulfonic, oxalic, isethionic, HOOC-(CH)n-COOH where n isochromanyl, chromanonyl, chromanyl, tetrahydroiso is 0-4, and the like. Lists of additional suitable salts may be quinolinyl, isoindolinyl, isobenzotetrahydrofuranyl, isoben found, e.g., in Remington's Pharmaceutical Sciences, 17th Zotetrahydrothienyl, isobenzothienyl, benzoxazolyl pyri ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). dopyridinyl, benzotetrahydrofuranyl, 25 benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, The term “prodrugs includes any compounds that become phenoxazinyl, phenothiazinyl, 5 pteridinyl, benzothiazolyl, compounds of Formula I when administered to a mammalian imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, Subject, e.g., upon metabolic processing of the prodrug. benzisoxazinyl, benzoxazinyl, dihydrobenzisothiazinyl, ben Examples of prodrugs include, but are not limited to, acetate, Zopyranyl, benzothiopyranyl, coumarinyl, isocoumarinyl, 30 formate and benzoate and like derivatives of functional chromanyl, tetrahydroquinolinyl, dihydroquinolinyl, dihyd groups (such as alcohol oramine groups) in the compounds of roquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl, Formula I and Formula II. dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, ben The term “therapeutically effective amount of a com Zoxazolinonyl, pyrrolyl N-oxide, pyrimidinyl N-oxide, pound of this invention means an amount effective, when pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinyl N-oxide, 35 indolyl N-oxide, indolinyl N oxide, isoquinolyl N-oxide, administered to a human or non-human patient, to provide a quinazolinyl N-oxide, quinoxalinyl N-oxide, phthalazinyl therapeutic benefit such as an amelioration of symptoms, e.g., N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl an amount effective to decrease the symptoms of a microbial N-oxide, thiazolyl N-oxide, indolizinyl N oxide, indazolyl infection, and preferably an amount sufficient to reduce the N-oxide, benzothiazolyl N-oxide, benzimidazolyl N-oxide, 40 symptoms of a bacterial, fungal, or protozoal infection. In pyrrolyl N-oxide, oxadiazolyl N-oxide, thiadiazolyl N-oxide, certain circumstances a patient having a microbial infection tetrazolyl N-oxide, benzothiopyranyl S-oxide, and benzothi may not present symptoms of being infected. Thus a thera opyranyl S.S.-dioxide. peutically effective amount of a compound is also an amount As used herein "Active agents' are compounds that have Sufficient to prevent a significant increase or significantly pharmaceutical utility, e.g. may be used to treat a patient 45 Suffering from a disease or condition, or may be used prophy reduce the detectable level of microorganism or antibodies lacticly to prevent the onset of a disease or condition in a against the microorganism in the patient's blood, serum, other patient, or that may be used to enhance the pharmaceutical bodily fluids, or tissues. The invention also includes using activity of other compounds. compounds of Formula I and Formula II in prophylactic “Salts' of the compounds of the present invention include 50 therapies. In the context of prophylactic or preventative treat inorganic and organic acid and base addition salts. The salts ment a “therapeutically effective amount' is an amount suf of the present compounds can be synthesized from a parent ficient to significantly decrease the treated human or non compound that contains a basic or acidic moiety by conven human patient's risk of contracting a microorganism tional chemical methods. Generally, such salts can be pre infection. A significant reduction is any detectable negative pared by reacting free acid forms of these compounds with a 55 change that is statistically significant in a standard parametric Stoichiometric amount of the appropriate base (such as Na, Ca,Mg, or Khydroxide, carbonate, bicarbonate, or the like), test of Statistical significance Such as Student's T-test, where or by reacting free base forms of these compounds with a p<0.05. Stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in 60 a mixture of the two. Generally, non-aqueous media like Antimicrobial Compounds ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are For the purposes of this document, the following number preferred, where practicable. Salts of the present compounds ing system will apply to the core 1-thia-2.4a-diaza-cyclo further include solvates of the compounds and of the com pentabnaphthalene-3,4-dione (when A=Sulfur) structure pound salts. 65 or core 2H-isoxazolo 5,4-bduinolizine-3,4-dione (when “Pharmaceutically acceptable salts' includes derivatives A-oxygen) structure. The numbers 1 through 9 refer spe of the disclosed compounds wherein the parent compound is cifically to positions within the tricyclic ring system whereas US 7,659,399 B2 11 12 the letters A, B and C refer to the specific six (rings A and B) (c). A is SO; e.g. the invention includes compounds of or five (ring C) member rings as shown below. Formula VII and Formula VIII.

Formula VII Rs O O R6 21 NN N- R2 N S S 10 R As / \, Ro In addition to the compounds of Formula I and Formula II, Formula VIII described above the invention also includes compounds of Rs O o1 R3 Formula I and Formula II in which for the variables (e.g. A 15 R. R. R. etc.) one or more of the following conditions are R6 met. 21 NN N The A variable N N S N (a). A is Sulfur, e.g. the invention includes compounds of R7 As Formula III and Formula IV. / \, R9

Formula III Rs O O (d). A is O; e.g. the invention includes compounds and salts of Formula IX and Formula X. R6 25 21 N N- R2 Formula IX N 1S s/ Rs O R As O R6 Ro 30 21 NN N-R, R NAs N O

Formula IV Ro Rs O o1 R3 35 Formula X R O R 3 R6 5 o1 21 NN N R6 N 21 NN N N M R7 As N S 40 R7 NAs N O N R9 R9 (b). A is SO; e.g. the invention includes compounds of Formula V and Formula VI. 45 The R variable: (a). The invention includes compounds of Formula I in Formula V which R is hydrogen, or Rs O O R is C-Calkyl or (C-C,cycloalkyl) Co-Calkyl, each of R6 which is substituted with at least one substituent chosen 21 N from hydroxy, amino, —COOH, -(C=O)NROR, N-R and —CONH; and is substituted with 0 to 3 substituents N1S s/ independently chosen from halogen, hydroxy, amino, R7 A.8 V O cyano, nitro. —COOH, - CONH2, C-C alkyl, R9 55 C-C alkoxy, C-Chaloalkyl, C-Chaloalkoxy, and mono- and di-C-C-alkylamino, and C-C alkanoyl. (b). R is hydrogen.

Formula VI The R variable: 60 (a). R is C-Calkyl, C-Calkanoyl, mono- or di-C- Rs O o1 R3 Calkylcarbamate, or C-Calkylsulfonate; each of which is R6 substituted with 0 to 3 substituents independently chosen 21 N N N from halogen, hydroxy, amino, cyano, nitro, C-C alkoxy, N 1S s/ mono- and di-C-C alkylamino, C-Chaloalkyl, and R As V 65 C-Chaloalkoxy. O (b). R is C-Calkyl or C-Calkanoyl, each of which is Ro substituted with 0 to 3 substituents independently chosen

US 7,659,399 B2 15 16 (g). R, is XR where X is absent, —CH2, —CH2—CH2—, (1) R, is XR where X is absent and R is phenyl, pyridyl —CH=CH-, or —C=C ; and 3-yl or pyrid-4-yl, each of which is substituted with 1 or 2 R is a C-Calkyl, C-C,cycloalkyl, C-C,cycloalkenyl, Substituents independently chosen from fluoro, amino, phenyl, phenyl fused to a 5- or 6-membered heterocycloalkyl hydroxy, cyano, and methyl. ring containing 1 or 2 nitrogen or oxygen atoms, indanyl 5 (m) R, is XR where X is absent and R is tetrahydroiso naphthyl, dihydronaphthyl, tetrahydronapthyl, benzb quinolin-5-yl, tetrahydroisoquinolin-6-yl, tetrahydroiso thiophenyl, benzofuranyl, dihydroindolyl, dihydroisoin quinolin-7-yl, or tetrahydroisoquinolin-8-yl, each of which is dolyl, dihydroquinolinyl, dihydroisoquinolinyl, furanyl. substituted with 0 to 3 substituents independently chosen indolyl, imidazolyl, isoindolyl, isoquinolinyl, isoxazolyl, from C-C alkyl. 10 (n) R, is XR where X is absent and R is tetrahydroiso piperazinyl, piperidinyl, pyrazinyl, pyridyl, pyrimidinyl, pyr quinolin-6-yl optionally substituted at each of the 1, 2, and 3 rolidinyl, pyrrolyl pyrazolyl, quinolinyl, quinazolinyl, qui positions with methyl. noxalinyl, thienyl, thiazolyl, tetrahydropyridinyl, tetrahy (o) R, is XR where X is absent and R is isoindol-5-yl droisoquionolinyl, or tetrahydroquinolinyl; group; each of substituted with 0 to 3 independently chosen C-C alkyl sub which is substituted with 0 to 5 substituents independently 15 stituents. chosen from halogen, hydroxy, amino, cyano, nitro, (p) R, is XR where X is absent and R is isoindol-5-yl C-C alkyl, C-Calkenyl, C-C alkoxy, mono- and di-(C- optionally substituted at each of the 1, 2, or 3 positions with C.)alkylamino, C-Chaloalkyl, C-Chaloalkoxy, (C- methyl. C, cycloalkyl)Co-Calkyl, (C-C,cycloalkyl)Co-Calkoxy, (q) R, is XR where X is absent, —CH2, —CH2—CH2—, (phenyl)Co-Calkyl, (phenyl)Co-Calkoxy, pyrrolidinyl, pip —CH=CH-, or—C=C ; or in certain embodiments X is eridinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyridyl, absent, and R is phenyl fused to a 5- or 6-membered hetero thienyl, C-C alkylthio. —(C=O)OR —(C=O) cycloalkyl ring containing 1 or 2 nitrogen or oxygen atoms, NRoRo; and —OR, —(C=O)R. —SOR, -SOR, where R is substituted with 0 to 3 substituents independently and —NRSOR, where R is C-C alkyl, piperidinyl, chosen from (i), (ii), and (iii). phenyl, naphthyl, and pyridyl; and each Ris Substituted with 25 (r) R, is XR where X is absent, —CH2, —CH2—CH2—, 0 to 3 substituents independently chosen from halogen, —CH=CH-, or—C=C ; or in certain embodiments X is hydroxy, amino, cyano, C-C alkyl, C-Calkoxy, mono- and absent, R is phenyl fused to a 5- or 6-membered heterocy di-(C-C)alkylamino, C-Chaloalkyl, and cloalkyl ring containing 1 or 2 nitrogen or oxygen atoms, C-Chaloalkoxy. where R, is substituted with 0 to 2 substituents independently (h) R, is XR where X is absent and R carries the defini 30 chosen from halogen, hydroxy, amino, C-Calkyl, and tion set forth in (f) or (g) above. C-Calkoxy. (i) R, is XR where X is absent, —CH2, —CH2—CH2—, The As variable —CH=CH-, or—C=C ; or in certain embodiments X is (a). As is nitrogen. absent, and (b). As is CRs. 35 (c). As is CRs and Rs is hydrogen, halogen, C-C alkyl, R is phenyl or pyridyl, each of which is substituted with 0 C-Calkoxy, trifluoromethyl, or trifluoromethoxy. to 3 Substituents independently chosen from halogen, hydroxy, amino, cyano, nitro, C-C alkyl, C-Calkenyl, (d). As is CRs and Rs is hydrogen or methyl. C-C alkoxy, OO- and di-(C-C)alkylamino, The R variable: C-Chaloalkyl, C-Chaloalkoxy, (C-C,cycloalkyl)Co (a). Ro is C-Calkyl, C-C alkoxy, mono- or di-(C-C) 40 alkylamino, C-C alkanoyl. C-Chaloalkyl, Calkyl, (C-C,cycloalkyl)Co-Calkoxy, (phenyl)Co C-Chaloalkoxy, (C-C,cycloalkyl)Co-Calkyl, or phenyl, Calkyl, (phenyl)Co-Calkoxy, pyrrolidinyl, piperidinyl, pip each of which is substituted with 0 to 3 substituents indepen erazinyl, morpholinyl, thiomorpholinyl, pyridyl, thienyl, dently chosen from halogen, hydroxy, amino, C-C alkyl, C-C alkylthio. —(C=O)OR, and —(C=O)NRR: C-Calkoxy, OO- and di-(C-C)alkylamino, and substituted with 1 or 2 substituents independently chosen 45 C-Chaloalkyl, and C-Chaloalkoxy. from —OR —(C=O)R. —SOR —SOR, and (b). Ro is C-C alkyl, cyclopropyl, or phenyl, each of —NRoSOR, where R is C-C alkyl, piperidinyl, phenyl, which is substituted with 0 to 3 substituents independently naphthyl, or pyridyl; and each R, is substituted with 0 to 3 chosen from halogen, hydroxy, amino, C-C alkyl, Substituents independently chosen from halogen, hydroxy, C-Calkoxy, OO- and di-(C-C)alkylamino, amino, cyano, C-Calkyl, C-Calkoxy, mono- and di-(C- 50 C-Chaloalkyl, and C-Chaloalkoxy. C.)alkylamino, C-Chaloalkyl, and C-Chaloalkoxy. (c). Ro is C-Calkyl or cyclopropyl, or Ro is phenyl Sub () R, is XR where X is absent and R is a phenyl, pyridyl, stituted with 2 substituents chosen from halogen, hydroxy, pyrimidinyl, furanyl, isoquinolinyl, quinolinyl, indolyl, pyr amino, C-C alkyl, C-Calkoxy, mono- and di-(C-C)alky rolyl, isoindolyl, dihydroisoindolyl, tetrahydroisoquinolinyl, lamino, C-Chaloalkyl, and C-Chaloalkoxy. tetrahydroisoquinolinyl, or thienyl group, each of which is 55 (d). Ro is ethyl, t-butyl, cyclopropyl, or 2,4-difluorophenyl. substituted with 0 to 3 substituents independently chosen (e). R is cyclopropyl. from halogen, hydroxy, amino, cyano, C-C alkyl, The invention includes compounds of Formula I and For C-C alkoxy, OO- and di-(C-C)alkylamino, mula II in which the variables A. R. R. R. R. R. As and C-Chaloalkyl, and C-Chaloalkoxy. Ro carry any combination of the definitions set forth for these (k) R, is XR where X is absent and R is phenyl, pyrid 60 variables above that results in a stable compound. 3-yl pyrid-4-yl, pyrimidin-5-yl, furan-3-yl, quinolin-3-yl, Certain compounds of Formula I and Formula II exhibit quinolin-5-yl, quinolin-6-yl, isoindol-5-yl, tetrahydroiso potent antibacterial, antifungal, and/or antiprotozoal activity. quinolin-5-yl, tetrahydroisoquinolin-6-yl, tetrahydroiso Particular compounds of the invention exhibit Minimum quinolin-7-yl, tetrahydroisoquinolin-8-yl, or indol-5-yl, each Inhibitory Concentrations (MIC) of 64 g/ml or less against of which is substituted with 0 to 2 substituents independently 65 StaphylocCocus aureus and/or Eschericia coli in a standard chosen from halogen, hydroxy, amino, C-C alkyl, and assay for determining the MIC of a compound against these C-Calkoxy. bacteria, such as the assay provided in Example 9 below. US 7,659,399 B2 17 18 Preferred compounds of Formula I and II exhibit MIC values of 10 g/ml or less against Staphyloccocus aureus and/or -continued Eschericia coli. More preferred compound of the Formula I Formula XII and II exhibit MIC values of 4 lug/ml or less, or even more Rs O -*. preferably 1 ug/ml or less, against Staphyloccocus aureus R6 and/or Eschericia coli. 21 NN N Certain compounds of Formula I and Formula II are selec N N tive antimicrobial agents; having the ability to kill or inhibit Y As Al the growth or reproduction of pathogenic microbial organ 10 R9 isms, while having little or no effect on the cells of fish, amphibians, reptiles, birds, or mammals. The selectivity of compounds of Formula I and Formula II may be assessed by Wherein A. As R. R. R. R. and Ro carry the definitions determining the CCs (the concentration at which 50% of the set forth above and Y is I, Br, Cl, —O(SO)CF, or NBF. 15 Here compounds of Formula XI and Formula XII play the cells are killed) for cultured cells of a higher animal, such as role of R". Y intermediate in the coupling reaction between a fish, reptiles, amphibian, bird, or mammal. Certain com R-M and R" Y. These intermediates are coupled to com pounds of the invention exhibit a CCs of greater that 100 pounds of the Formula R-M where R' is a group R. micromolar for mammalian cells. Certain compounds of the R is XR where X is absent, —CH, CH, , invention exhibit a CC50 of greater than 100 micromolar for -CH=CH-, -(C=O)— —(C=ONH) , or cultured human hepatocytes, and also exhibit MIC values of —C=C , and R is C-Calkyl, C-C,cycloalkyl, 64 ug/ml or less, preferably 10 ug/ml or less, or more prefer C-C,cycloalkenyl, phenyl, a bicyclic 7- to 10-membered ably 4 g/ml or less, or still more preferably 1 g/ml or less saturated, partially unsaturated, or aromatic carbocyclic against StaphylocCocus aureus and/or Eschericia coli. group, a monocyclic orbicyclic a 5- to 10-membered bicyclic 25 saturated, partially unsaturated, or aromatic heterocylic Without wishing to be bound to any particular theory it is group bound via a carbon atom; each of which R is substi believed that the antimicrobial properties of compounds of tuted with 0 to 5 substituents independently chosen from (i), Formula I and Formula II are due to the ability to these (ii), and (iii); where compounds to inhibit the activity of microbial DNA gyrases (i) is chosen from halogen, hydroxy, amino, cyano, and while having little or no effect on the analogous enzyme, 30 nitro, Topoisomerase II, present in higher organisms. Certain pre (ii) is chosen from C-Calkyl, C-Calkenyl, ferred compounds of the invention are 100-fold or more C-Calkynyl, (C-Calkoxy)Co-Calkyl, mono- and di-(C- selective for bacterial DNA gyrases than for mammalian, C.)alkylamino, C-Chaloalkyl, C-Chaloalkoxy, (C- particularly human, Topoisomerase II. C,cycloalkyl)Co-Cacarbohydryl. (C-C,cycloalkyl)Co 35 Ccarbohydryl-O , (C-C,cycloalkenyl)Co Synthetic Intermediates Ccarbohydryl, (aryl)Co-Cocarbohydryl, (aryl)C-Calkoxy, The invention includes novel intermediates useful for the (C-Cheterocycloalkyl)Co-Cacarbohydryl, (heteroaryl)Co synthesis of antimicrobial compounds of Formula I and For Cocarbohydryl, C-Calkylthio. =NOR =NRio —(Co mula II. Coupling reactions occur between R-M and R"-Y in Calkyl)(C=O)R —(Co-Calkyl)O(C=O)R —(Co the presence of catalyst Q, where M is Li, Mg, B, Al, Si, Zn, 40 Calkyl)(C=O)NRR, —(Co-Calkyl)O(C=O) Cu, Zr, or Sn; where Y is I, Br, Cl, O(SO)CF, or NRoRo —(Co-Calkyl)(C=O)OR —(Co-Calkyl)NR —NBF: Q is Fe, Ni, Cu, Pd, or Rh; and RandR" are organic (C=O)R. —(Co-Calkyl)NR(C=O)OR, —(Co molecules to which MandYare bound. M may also be Boron, Calkyl)NR(C=O)NRR, —(Co-Calkyl)NR(C=O) disubstituted with OH, OG, or G, where G is an optionally (C-C alkyl)NR (C=O)O—R, —(Co-Calkyl)NRio 45 (C=S)NRR, -(Co-Calkyl)NRNR, R2, —(Co Substituted Straight, branched, or cyclic alkyl group, or other Calkyl)N=NR —(Co-Calkyl)SOR —(Co-Calkyl) suitable group;Y is Br, and where Q is Pd. A general review (S=O)OR —(Co-Calkyl)SOR. -(Co-Calkyl) of this chemistry can be found in Tamao, K and Miyaura, N. SO2NRoR, and —(Co-Calkyl)NRSOR: and Topics in Current Chemistry 219: 1-9 (2002). A review of the (iii) is chosen from —OR, —(C=O)RD, SORD, use of coupling reagents in which Mis Boron with a listing of 50 - SORD, NRSO2RD, where RD is C1-C4alkyl, potential boronates, palladium catalysts, and reaction condi (C-C,cycloalkyl)Co-Calkyl, (C2 tions can be found in Miyaura, N. Topics in Current Chem C6heterocycloalkyl)Co-Calkyl, (aryl)Co-Calkyl, and istry 219: 11-59 (2002). (heteroaryl)Co-Calkyl; Thus the invention includes intermediates of Formula XI 55 where each of (ii) and (iii) is substituted with 0 to 3 sub and Formula XII: stituents independently chosen from halogen, hydroxy, amino, cyano, nitro, oxo. —COOH, -CONH2, C1-C4alkyl, C2-C4alkenyl, C2-C4alkynyl, C1-C4alkoxy, Formula XI C1-C4alkoxycarbonyl, (C-C,cycloalkyl)Co Rs O O Ccarbohydryl, (C-C,cycloalkyl)Co-Calkoxy, mono- and 60 di-(C1-C4)alkylamino, C-Chaloalkyl, C-Chaloalkoxy, R6 21 NN C2-C4alkanoyl and phenyl. N-R, M is Li, Mg, B, Al, Si, Zn, Cu, Zr, or Sn; or M is Boron, Y N 8 Al disubstituted with OH, OG, or G, where G is an optionally 65 Substituted Straight, branched, or cyclic alkyl group, an R9 optionally substituted aryl or arylalkyl group, or other Suit able group. US 7,659,399 B2 19 20 The invention also includes intermediates of Formula XIII acid and magnesium Stearate; calcium sulfate; vegetable oils, and Formula XIV. Such as peanut oil, cottonseed oil, Sesame oil, olive oil, and corn oil; polyols such as propylene glycol, glycerine, Sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, Formula XIII 5 such as the TWEENS: wetting agents, such sodium lauryl Rs O O Sulfate; coloring agents; flavoring agents; tableting agents, R6 stabilizers; antioxidants; preservatives; pyrogen-free water; 21 NN isotonic saline; and phosphate buffer Solutions. N-R, In particular, pharmaceutically acceptable carriers for sys M N Al 10 temic administration include Sugars, starches, cellulose and 8 its derivatives, malt, gelatin, talc, calcium Sulfate, vegetable R9 oils, synthetic oils, polyols, alginic acid, phosphate buffer Formula XIV Solutions, emulsifiers, isotonic saline, and pyrogen-free Rs O -*. water. Preferred carriers for parenteral administration include 15 propylene glycol, ethyl oleate, pyrrolidone, ethanol, and R6 21 N N sesame oil. Optional active agents may be, included in a pharmaceuti cal composition, which do not substantially interfere with the M s 8 Al activity of the compound of the present invention. R9 Effective concentrations of one or more of the compounds of the invention including pharmaceutically acceptable salts, esters or other derivatives thereof are mixed with a suitable In Formula XIII and XIV A. As R. R. R. R. and R. pharmaceutical carrier, excipients, adjuvant, or vehicle. In carry the definitions set forth above and M is Li, Mg, B, Al, Si, instances in which the compounds exhibit insufficient solu Zn, Cu, Zr, or Sn; or M is Boron, disubstituted with OH, OG, 25 bility, methods for Solubilizing compounds may be used. or G, where G is an optionally substituted straight, branched, Such methods are known to those of skill in this art, and or cyclic alkyl group, an optionally Substituted aryl or aryla include, but are not limited to, using cosolvents, such as lkyl group, or other Suitable group. Here compounds of For dimethylsulfoxide (DMSO), using surfactants, such as mula XIII and Formula XIV play the role of R-M in the Tween, or dissolution in aqueous sodium bicarbonate. coupling reaction. These intermediates are coupled to com 30 Derivatives of the compounds, such as salts of the compounds pounds of the Formula R"-Y where Y is I, Br, Cl, O(SO) or prodrugs of the compounds may also be used in formulat CF, or NBF, and R" carries the definition set forth above ing effective pharmaceutical compositions. for R. Upon mixing or addition of the compound(s) of Formula I and/or Formula II, the resulting mixture may be a solution, Pharmaceutical Preparations 35 Compounds and salts of Formula I and Formula II can be Suspension, emulsion or the like. The form of the resulting administered as the neat chemical, but are preferably admin mixture depends upon a number of factors, including the istered as a pharmaceutical composition or formulation. intended mode of administration and the solubility of the Accordingly, the invention provides pharmaceutical formu compound in the chosen carrier or vehicle. The effective lations comprising a compound or pharmaceutically accept concentration Sufficient for ameliorating the symptoms of the 40 disease, disorder or condition treated and may be empirically able salt of Formula I or Formula II, together with one or more determined. pharmaceutically acceptable carrier, excipients, adjuvant, The pharmaceutical compositions containing compounds diluent, or other ingredient. of general Formula I and/or Formula II may be in a form Compounds of general Formula I and Formula II may be Suitable for oral use, for example, as tablets, troches, loZ administered orally, topically, parenterally, by inhalation or 45 enges, aqueous or oily Suspensions, dispersible powders or spray, Sublingually, transdermally, via buccal administration, granules, emulsions, hard or soft capsules, or syrups or elix rectally, as an ophthalmic solution, or by other means, in irs. Compositions intended for oral use may be prepared dosage unit formulations containing conventional non-toxic according to any method known to the art for the manufacture pharmaceutically acceptable carriers, excipients, adjuvants, of pharmaceutical compositions and Such compositions may and vehicles. 50 contain one or more agents, such as Sweetening agents, fla In addition to the Subject compound, the compositions of Voring agents, coloring agents and preserving agents, in order the invention may contain a pharmaceutically acceptable car to provide pharmaceutically elegant and palatable prepara rier, one or more compatible solid or liquid filler, diluents or tions. encapsulating Substances, which are suitable for administra Oral formulations contain between 0.1 and 99% of a com tion to an animal. Carriers must be of Sufficiently high purity 55 pound of the invention and usually at least about 5% and sufficiently low toxicity to render them suitable for (weight %) of a compound of the present invention. Some administration to the animal being treated. The carrier can be embodiments contain from about 25% to about 50% or from inert or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the com 5% to 75% of a compound of invention. pound is Sufficient to provide a practical quantity of material 60 Liquids Formulations for administration per unit dose of the compound. Compounds of the invention can be incorporated into oral Exemplary pharmaceutically acceptable carriers or com liquid preparations such as aqueous or oily Suspensions, solu ponents thereof are Sugars, such as lactose, glucose and tions, emulsions, syrups, or elixirs, for example. Moreover, Sucrose; starches, such as corn Starch and potato starch; cel formulations containing these compounds can be presented lulose and its derivatives, such as Sodium carboxymethyl 65 as a dry product for constitution with water or other suitable cellulose, ethyl cellulose, and methyl cellulose; powdered vehicle before use. Such liquid preparations can contain con tragacanth; malt, gelatin; talc.; Solid lubricants, such as Stearic ventional additives, such as Suspending agents (e.g., Sorbitol US 7,659,399 B2 21 22 syrup, methyl cellulose, glucose/Sugar, syrup, gelatin, tion products of the said partial esters with ethylene oxide, for hydroxyethyl cellulose, carboxymethyl cellulose, aluminum example polyoxyethylene Sorbitan monoleate. Stearate gel, and hydrogenated edible fats), emulsifying agents (e.g., lecithin, Sorbitan monsoleate, or acacia), non Dispersible Powders aqueous vehicles, which can include edible oils (e.g., almond Dispersible powders and granules Suitable for preparation oil, fractionated coconut oil, silyl esters, propylene glycol and of an aqueous Suspension by the addition of water provide the ethyl alcohol), and preservatives (e.g., methyl or propyl p-hy active ingredient in admixture with a dispersing or wetting droxybenzoate and Sorbic acid). agent, Suspending agent and one or more preservatives. Suit Orally administered compositions also include liquid Solu able dispersing or wetting agents and Suspending agents are tions, emulsions, Suspensions, powders, granules, elixirs, 10 exemplified by those already mentioned above. tinctures, syrups, and the like. The pharmaceutically accept Tablets and Capsules able carriers Suitable for preparation of such compositions are Tablets typically comprise conventional pharmaceutically well known in the art. Oral formulations may contain preser compatible adjuvants as inert diluents, such as calcium car Vatives, flavoring agents, Sweetening agents, such as Sucrose bonate, sodium carbonate, mannitol, lactose and cellulose; or Saccharin, taste-masking agents, and coloring agents. 15 binders such as starch, gelatin and Sucrose; disintegrants such Typical components of carriers for syrups, elixirs, emul as starch, alginic acid and croScarmelose; lubricants such as sions and Suspensions include ethanol, glycerol, propylene magnesium Stearate, Stearic acid and talc. Glidants such as glycol, polyethylene glycol, liquid Sucrose, Sorbitol and silicon dioxide can be used to improve flow characteristics of water. Syrups and elixirs may beformulated with sweetening the powder mixture. Coloring agents, such as the FD&C dyes, agents, for example glycerol, propylene glycol, Sorbitol or can be added for appearance. Sweeteners and flavoring Sucrose. Such formulations may also contain a demulcent. agents, such as aspartame, Saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Suspensions Capsules (including time release and Sustained release for For a Suspension, typical Suspending agents include meth mulations) typically comprise one or more solid diluents ylcellulose, sodium carboxymethyl cellulose, Avicel 25 disclosed above. The selection of carrier components often RC-591, tragacanth and sodium alginate; typical wetting depends on secondary considerations like taste, cost, and agents include lecithin and polysorbate 80; and typical pre shelf stability. servatives include methyl paraben and sodium benzoate. Such compositions may also be coated by conventional Aqueous Suspensions contain the active material(s) in methods, typically with pH or time-dependent coatings. Such admixture with excipients suitable for the manufacture of 30 that the Subject compound is released in the gastrointestinal aqueous Suspensions. Such excipients are Suspending agents, tract in the vicinity of the desired topical application, or at for example sodium carboxymethylcellulose, methylcellu various times to extend the desired action. Such dosage forms lose, hydropropylmethylcellulose, sodium alginate, polyvi typically include, but are not limited to, one or more of cel nylpyrrollidone, gum tragacanth and gum acacia; dispersing lulose acetate phthalate, polyvinylacetate phthalate, hydrox or wetting agents; may be a naturally-occurring phosphatide, 35 ypropyl methylcellulose phthalate, ethyl cellulose, Eudragit for example, lecithin, or condensation products of an alkylene coatings, waxes and shellac. oxide with fatty acids, for example polyoxyethylene Stearate, Formulations for oral use may also be presented as hard or condensation products of ethylene oxide with long chain gelatin capsules wherein the active ingredient is mixed with aliphatic alcohols, for example heptadecaethyleneoxycet an inert Solid diluent, for example, calcium carbonate, cal anol, or condensation products of ethylene oxide with partial 40 cium phosphate or kaolin, or as Soft gelatin capsules wherein esters derived from fatty acids and a hexitol Such as polyoxy the active ingredient is mixed with water oran oil medium, for ethylene sorbitol substitute, or condensation products of eth example peanut oil, liquid paraffin or olive oil. ylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan sub Injectable and Parenteral Formulations stitute. The aqueous Suspensions may also contain one or 45 Pharmaceutical compositions may be in the form of a ster more preservatives, for example ethyl, or n- propyl p-hy ile injectable aqueous or oleaginous Suspension. This Suspen droxybenzoate. sion may be formulated according to the known art using Oily suspensions may be formulated by Suspending the those Suitable dispersing or wetting agents and Suspending active ingredients in a vegetable oil, for example peanut oil, agents that have been mentioned above. The sterile injectable olive oil, Sesame oil or coconut oil, or in a mineral oil Such as 50 preparation may also be sterile injectable Solution or Suspen liquid paraffin. The oily Suspensions may contain a thicken sion in a non-toxic parentally acceptable diluent or solvent, ing agent, for example beeswax, hard paraffin or cetyl alco for example as a solution in 1,3-butanediol. Among the hol. Sweetening agents such as those set forth above, and acceptable vehicles and solvents that may be employed are flavoring agents may be added to provide palatable oral water, Ringer's solution, and isotonic sodium chloride solu preparations. These compositions may be preserved by the 55 tion. In addition, Sterile, fixed oils are conventionally addition of an anti-oxidant such as ascorbic acid. employed as a solvent or Suspending medium. For this pur pose any bland fixed oil may be employed including synthetic Emulsions mono- or diglycerides. In addition, fatty acids such as oleic Pharmaceutical compositions of the invention may also be acid are useful in the preparation of injectables. in the form of oil-in-water emulsions. The oily phase may be 60 Compounds of Formula I and Formula II may be adminis a vegetable oil, for example olive oil or peanut oil, or a tered parenterally in a sterile medium. Parenteral administra mineral oil, for example liquid paraffin or mixtures of these. tion includes Subcutaneous injections, intravenous, intramus Suitable emulsifying agents may be naturally-occurring cular, intrathecal injection or infusion techniques. The drug, gums, for example gum acacia or gum tragacanth, naturally depending on the vehicle and concentration used, can either occurring phosphatides, for example Soybean, lecithin, and 65 be suspended or dissolved in the vehicle. Advantageously, esters or partial esters derived from fatty acids and hexitol, adjuvants such as local anesthetics, preservatives and buffer anhydrides, for example Sorbitan monoleate, and condensa ing agents can be dissolved in the vehicle. In compositions for US 7,659,399 B2 23 24 parenteral administration the carrier comprises at least about multilamellar vesicles. Liposomes can beformed from a vari 90% by weight of the total composition. ety of phospholipids, Such as cholesterol, Stearylamine or phosphatidylcholines. Suppositories Other Formulations Compounds of Formula I and Formula II may also be Other compositions useful for attaining systemic delivery administered in the form of Suppositories for rectal adminis of the Subject compounds include Sublingual, buccal and tration of the drug. These compositions can be prepared by nasal dosage forms. Such compositions typically comprise mixing the drug with a Suitable non-irritating excipient that is one or more of Soluble filler Substances such as Sucrose, Solidat ordinary temperatures but liquid at the rectal tempera Sorbitol and mannitol, and binders such as acacia, microcrys ture and will therefore melt in the rectum to release the drug. 10 talline cellulose, carboxymethyl cellulose and hydroxypropyl Such materials are cocoa butter and polyethylene glycols. methylcellulose. Glidants, lubricants, Sweeteners, colorants, Topical Formulations antioxidants and flavoring agents disclosed above may also be included. Compounds of the invention may beformulated for local or Compositions for inhalation typically can be provided in topical application, Such as for topical application to the skin 15 and mucous membranes, such as in the eye, in the form of the form of a solution, Suspension or emulsion that can be gels, creams, and lotions and for application to the eye or for administered as a dry powder or in the form of an aerosol intracistemal or intraspinal application. Topical compositions using a conventional propellant (e.g., dichlorodifluo of the present invention may be in any form including, for romethane or trichlorofluoromethane). example, Solutions, creams, ointments, gels, lotions, milks, Additional Components cleansers, moisturizers, sprays, skin, patches, and the like. The compositions of the present invention may also option Such solutions may be formulated as 0.01%-10% isotonic ally comprise an activity enhancer. The activity enhancer can Solutions, pH about 5-7, with appropriate salts. Compounds be chosen from a wide variety of molecules that function in of the invention may also be formulated for transdermal different ways to enhance antimicrobial effects of compounds administration as a transdermal patch. 25 of the present invention. Particular classes of activity enhanc Topical compositions containing the active compound can ers include skin penetration enhancers and absorbtion be admixed with a variety of carrier materials well known in enhancers. the art, Such as, for example, water, alcohols, aloe Vera gel. Pharmaceutical compositions of the invention may also allantoin, glycerine, Vitamin A and E oils, mineral oil, propy contain additional active agents can be chosen from a wide lene glycol, PPG-2 myristyl propionate, and the like. 30 variety of molecules, which can function in different ways to enhance the antimicrobial or therapeutic effects of a com Other materials suitable for use in topical carriers include, pound of the present invention. These optional other active for example, emollients, solvents, humectants, thickeners and agents, when present, are typically employed in the compo powders. Examples of each of these types of materials, which sitions of the invention at a level ranging from about 0.01% to can be used singly or as mixtures of one or more materials, are 35 about 15%. Some embodiments contain from about 0.1% to as follows: about 10% by weight of the composition. Other embodiments Emollients, such as Stearyl alcohol, glyceryl monoricino contain from about 0.5% to about 5% by weight of the com leate, glyceryl monostearate, propane-1,2-diol, butane-1,3- position. diol, mink oil, cetyl alcohol, iso-propyl isostearate, Stearic acid, iso-butyl palmitate, isocetyl Stearate, oleyl alcohol, iso 40 Packaged Formulations propyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol. The invention includes packaged pharmaceutical formula isocetyl alcohol, cetyl palmitate, dimethylpolysiloxane, di-n- tions. Such packaged formulations include a pharmaceutical butyl sebacate, iso-propyl myristate, iso-propyl palmitate, composition containing one or more compounds or salts of iso-propyl Stearate, butyl Stearate, polyethylene glycol, trieth Formula I in a container and instructions for using the com ylene glycol, lanolin, Sesame oil, coconut oil, arachis oil, 45 position to treat an animal (typically a human patient) suffer castor oil, acetylated lanolin alcohols, petroleum, mineral oil, ing from a microorganism infection) or prevent a microor butyl myristate, isostearic acid, palmitic acid, isopropyl ganism infection in an animal. linoleate, lauryl lactate, myristyl lactate, decyl oleate, and In all of the foregoing the compounds of the invention can myristyl myristate; propellants, such as propane, butane, iso be administered alone or as mixtures, and the compositions butane, dimethyl ether, carbon dioxide, and nitrous oxide; 50 may further include additional drugs or excipients as appro Solvents, such as ethyl alcohol, methylene chloride, iso-pro priate for the indication. panol, castor oil, ethylene glycol monoethyl ether, diethylene Methods of Treatment glycol monobutyl ether, diethylene glycol monoethyl ether, The invention includes methods of preventing and treating dimethyl sulphoxide, dimethyl formamide, tetrahydrofuran: microorganism infections, particularly bacterial and proto humectants, such as glycerin, Sorbitol, Sodium 2-pyrrolidone 55 Zoal infections, by administering a therapeutically effective 5-carboxylate, Soluble collagen, dibutyl phthalate, and gela amount of one or more compounds of Formula I and of tin, and powders, such as chalk, talc, fullers earth, kaolin, Formula II to an animal at risk for a microorganism infection starch, gums, colloidal silicon dioxide, sodium polyacrylate, or having a microorganism infection. The animal may be a tetra alkyl ammonium Smectites, trialkyl aryl ammonium fish, amphibian, reptile or bird, but is preferably a mammal. Smectites, chemically modified magnesium aluminium sili 60 Methods of treating and preventing microorganism infections cate, organically modified montmorillonite clay, hydrated in livestock animals, companion animals, and human patients aluminium silicate, fumed silica, carboxyvinyl polymer, are particularly preferred. Sodium carboxymethyl cellulose, and ethylene glycol The compounds disclosed herein are useful for preventing monoStearate. and treating bacterial infections in animals. Furthermore The compounds of the invention may also be topically 65 compounds of the invention may be used to treat a variety of administered in the form of liposome delivery systems, such conditions not attributed to bacterial infections. These as Small unilamellar vesicles, large unilamellar vesicles, and include diseases and disorders caused fungal infections, US 7,659,399 B2 25 26 mycoplasma infections, protozoal infections, or other condi pneumophila, Pasteurella multocida, Salmonella enteritidis, tions involving infectious organisms. Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, In Some circumstances an effective amount of a compound Yersinia enterocolitica and H. Pylorii. of Formula I or Formula II may be an amount sufficient to Non-bacterial microorganisms: Mycoplasma, Legionella reduce the symptoms of the microorganism infection. Alter 5 and Chlamydia. natively an effective amount of a Compound of Formula I Dosage levels of the order of from about 0.1 mg to about may be an amount Sufficient to significantly reduce the 140 mg per kilogram of body weight per day are useful in the amount of microorganism orantibodies against the detectable treatment of the above-indicated conditions (about 0.5 mg to in a patient’s tissues or bodily fluids. about 7g per patient per day). The amount of active ingredient Methods of treatment also include inhibiting microorgan 10 that may be combined with the carrier materials to produce a ism replication in Vivo, in an animal at risk for a microorgan single dosage form will vary depending upon the host treated ism infection or Suffering from Such an infection, by admin and the particular mode of administration. Dosage unit forms istering a sufficient concentration of a compound of Formula will generally contain between from about 1 mg to about 500 I or Formula II to inhibit bacterial survival in vitro. By “suf mg of an active ingredient. ficient concentration of a compound administered to the 15 Frequency of dosage may also vary depending on the com patient is meant the concentration of the compound available pound used and the particular disease treated. However, for in the animal's system to prevent or combat the infection. treatment of most infectious disorders, a dosage regimen of 4 Such a concentration by be ascertained experimentally, for times daily or less is preferred and a dosage regimen of 1 or 2 example by assaying blood concentration of the compound, times daily is particularly preferred. or theoretically, by calculating bioavailability. The amount of It will be understood, however, that the specific dose level a compound sufficient to inhibit bacterial survival in vitro for any particular patient will depend upon a variety of factors may be determined with a conventional assay for bacterial including the activity of the specific compound employed, the survival such as the Minimum Inhibitory Concentration age, body weight, general health, sex, diet, time of adminis (MIC) Assay disclosed in Example 9, which follows. tration, route of administration, and rate of excretion, drug The invention also includes using compounds of Formula I 25 combination and the severity of the particular disease under and Formula II in prophylactic therapies. In the context of going therapy. prophylactic or preventative treatment an effective amount of a compound of the invention is an amount Sufficient to sig Combination Administration nificantly decrease the treated animal’s risk of contracting a The compounds of the invention may also be useful in microorganism infection. 30 combination with other pharmaceutically active agents such Compounds of the invention are particularly useful for as antibacterial agents, antiviral agents, antifungal agents, treating and preventing infectious disorders. These include anti-inflammatories, interferon, efflux-pump inhibitors, and for example: ocular infections such as conjunctivitis; urinary beta-lactamase inhibitors. Antibiotic agents include any mol tract and genital infections, such as complicated urinary tract ecule that tends to prevent, inhibit or destroy life and as such, infections, acute urinary tract and genital infections, such as 35 includes anti-bacterial agents, anti-fungicides, anti-viral pyelonephritis, cervical gonococcal infections, cystitis, ure agents, and anti-parasitic agents. thral chlamydial infections, cervical chlamydial infections, A composition comprising a compound or salt of Formula urethral gonococcal infections, and prostatitis, respiratory I or Formula II in combination with one or more other anti infections, such as lower respiratory tract infections, acute bacterial agent, antiprotozoal agent, antifungal agent, antivi sinusitis, acute exacerbations of chronic bronchitis, commu 40 ral agent, interferon, efflux-pump inhibitor, or beta-lactamase nity-acquired pneumonia, and nosocomial pneumonia, skin inhibitor is provided herein. infections, such as skin-structure infections, impetigo, folli Pharmaceutical compositions of the invention include culitis, boils, Scalded skin syndrome, and cellulites, and other single dosage forms containing of a compound of Formula I infections such as bone infections, joint infections, infectious and/or Formula II and one or more other active agent, dosage diarrhea, typhoid fever, intra-abdominal infections, gyneco 45 forms containing more than one compound of Formula I logic infections, including toxic shock syndrome, pelvic and/or Formula II, and separate. administration of a com infections, and post-Surgical infections. pound of Formula I and/or Formula II with another active The disclosed compounds are useful for treating infections agent. caused by the following microorganisms: The following active agents, which are useful in combina Aerobic Gram-positive Microorganisms: Including but not 50 tions of the invention, may be isolated from an organism that limited to Enterococcus faecalis, Enterococcus faecium, Sta produces the agent or synthesized by methods known to those phylococcus aureus, Staphylococcus epidermidis, Staphyllo of ordinary skill in the art of medicinal chemistry or pur coccus saprophyticus, Streptococcus pneumoniae, Strepto chased from a commercial source. coccus pyogenes, Staphylococcus haemolyticus, and Anti-bacterial antibiotic agents include, but are not limited Staphylococcus hominis. 55 to, penicillins, cephalosporins, carbacephems, cephamycins, Aerobic Gram-negative Microorganisms: Including but carbapenems, monobactams, aminoglycosides, glycopep not limited to Campylobacter jejuni, Citrobacter diversus, tides, quinolones, tetracyclines, macrollides, and fluoroquino Citrobacter freundii, Enterobacter cloacae, Escherichia coli, lones (see Table below). Examples of antibiotic agents Haemophilus influenzae, Haemophilus parainfluenzae, Kleb include, but are not limited to, Penicillin G (CAS Registry siella pneumoniae, Moraxella catarrhalis, Morganella mor 60 No. 61-33-6); Methicillin (CAS Registry No.: 61-32-5): ganii, Neisseria gonorrhoeae, Proteus mirabilis, Proteus vul Nafcillin (CAS Registry No.: 147-52-4): Oxacillin (CAS garis, Providencia rettgeri, Providencia Stuartii, Registry No.: 66-79-5); Cloxacillin (CAS Registry No.: Pseudomonas aeruginosa, Stenotrophomonas maltophila, 61-72-3); Dicloxacillin (CAS Registry No.: 3116-76-5): Salmonella typhi, Serratia marcescens, Shigella boydii, Shi Ampicillin (CAS Registry No.: 69-53-4); Amoxicillin (CAS gella dysenteriae, Shigella flexneri, Shigella sonnei. Acineto 65 Registry No. 26787-78-0): Ticarcillin (CAS Registry No.: bacter Iwofi, Aeromonas hydrophila, Edwardsiella tarda, 34787-01-4); Carbenicillin (CAS Registry No.: 4697-36-3); Enterobacter aerogenes, Klebsiella Oxytoca, Legionella Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin US 7,659,399 B2 27 28 (CAS Registry No.: 37091-66-0); Piperacillin (CAS Registry mide, Flucytosine, Halethazole, Hexetidine, Loflucarban, No. 61477-96-1): Imipenem (CAS Registry No.: 74431-23 Nifuratel, Potassium Iodide, Propionic Acid, Pyrithione, 5): Aztreonam (CAS Registry No.:781 10-38-0); Cephalothin Salicylanilide, Sodium Propionate, Sulbentine, Tenonitro (CAS Registry No.: 153-61-7); Cefazolin (CAS Registry Zole, Triacetin, Ujothion, Undecylenic Acid, and Zinc Propi No.: 25953-19-9); Cefaclor (CAS Registry No. 70356-03 Onate. 5); Cefamandole formate sodium (CAS Registry No.: 42540 Antiviral agents include, but are not limited to, Acyclovir, 40-9); Cefoxitin (CAS Registry No.: 35607-66-0): Cidofovir, Cytarabine, Dideoxyadenosine, Didanosine, Cefuroxime (CAS Registry No. 55268-75-2); Cefonicid Edoxudine, Famciclovir, Floxuridine, Ganciclovir, Idoxuri (CAS Registry No. 61270-58-4); Cefnmetazole (CAS Regis dine, Inosine Pranobex, Lamivudine, MADU, Penciclovir, try No. 56796-20-4); Cefotetan (CAS Registry No.: 69712 10 Sorivudine, Stavudine, Trifluridine, Valacyclovir, Vidara 56-7); Cefprozil (CAS Registry No. 92665-29-7); Loracar bine, Zalcitabine, Zidovudine, Acemannan, Acetylleucine, bef (CAS Registry No.: 121961-22-6); Cefetamet (CAS Amantadine, Amidinomycin, Delavirdine, Foscamet, Indi Registry No.: 65052-63-3); CefoperaZone (CAS Registry navir, Interferon-alpha, Interferon-beta, Interferon-gamma, No. 62893-19-0); Cefotaxime (CAS Registry No. 63527 Kethoxal, Lysozyme, Methisazone, Moroxydine, Nevirap 52-6); Ceftizoxime (CAS Registry No.: 68401-81-0); Ceftri 15 ine, Podophyllotoxin, Ribavirin, Rimantadine, Ritonavir2. axone (CAS Registry No.: 73384-59-5); Ceftazidime (CAS Saquinavir, Stailimycin, Statolon, Tromantadine, and Xena Registry No. 72558-82-8); Cefepime (CAS Registry No.: Zoic Acid. 88040-23-7); Cefixime (CAS Registry No. 79350-37-1): Antiinflammatory agents include, but are not limited to, Cefpodoxime (CAS Registry No. 80210-62-4); Cefsulodin Enfenamic Acid, Etofenamate, Flufenamic Acid, Isonixin, (CAS Registry No. 62587-73-9); Fleroxacin (CAS Registry Meclofenamic Acid, Mefenamic Acid, Niflumic Acid, Talni No. 79660-72-3); Nalidixic acid (CAS Registry No.: 389 flumate, Terofenamate, Tolfenamic Acid, Aceclofenac, 08-2); Norfloxacin (CAS Registry No. 70458-96-7); Cipro Acemetacin, Alclofenac, Amfenac, Amtolimetin Guacil, Bro floxacin (CAS Registry No. 85721-33-1); Ofloxacin (CAS mfenac, BufeXamac, Cinmetacin, Clopirac, Diclofenac, Registry No.: 82419-36-1); Enoxacin (CAS Registry No.: Etodolac, Felbinac, Fenclozic Acid, Fentiazac, Glucameta 74011-58-8); Lomefloxacin (CAS Registry No. 98.079-51 25 cin, Ibufenac, Indomethacin, IsofeZolac, ISOXepac, Lona 7); Cinoxacin (CAS Registry No. 28657-80-9); Doxycycline Zolac, Metiazinic Acid, Mofezolac, Oxametacine, Pirazolac, (CAS Registry No. 564-25-0); Minocycline (CAS Registry Proglumetacin, Sulindac, Tiaramide, Tolimetin, Tropesin, No. 10118-90-8); Tetracycline (CAS Registry No.: 60-54-8); Zomepirac, Bumadizon, Butibufen, Fenbufen, Xenbucin, Amikacin (CAS Registry No.: 37517-28-5); Gentamicin Clidanac, Ketorolac, Tinoridine, Alminoprofen, Benoxapro (CAS Registry No.: 1403-66-3); Kanamycin (CAS Registry 30 fen, Bermoprofen, Bucloxic Acid, Carprofen, Fenoprofen, No. 8063-07-8); Netilmicin (CAS Registry No. 56391-56 Flunoxaprofen, Flurbiprofen, Ibuprofen, Ibuproxam, 1); Tobramycin (CAS Registry No.:32986-56-4); Streptomy Indoprofen, Ketoprofen, Loxoprofen, Naproxen, Oxaprozin, cin (CAS Registry No.: 57-92-1): Azithromycin (CAS Reg Piketoprofen, Pirprofen, Pranoprofen, Protizinic Acid, istry No. 83905-01-5); Clarithromycin (CAS Registry No.: Suprofen, Tiaprofenic Acid, Ximoprofen, Zaltoprofen, Dife 81 103-11-9); Erythromycin (CAS Registry No.: 114-07-8); 35 namizole, Epirizole, ApaZone, BenZpiperylon, Feprazone, Erythromycin estolate (CAS Registry No. 3521-62-8); Mofebutazone, Morazone, Oxyphenbutazone, Phenylbuta Erythromycin ethyl succinate (CAS Registry No.: 41342-53 Zone, PipebuZone, Propyphenazone, Ramifenazone, Suxibu 4); Erythromycin glucoheptonate (CAS Registry No.: 23067 Zone. ThiazolinobutaZone, AcetaminoSalol, Aspirin, Benory 13-2); Erythromycin lactobionate (CAS Registry No. 3847 late, Bromosaligenin, Calcium Acetylsalicylate. Diflunisal, 29-8); Erythromycin stearate (CAS Registry No.: 643-22-1); 40 Etersalate, Fendosal, Gentisic Acid, Glycol Salicylate, Imi Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin dazole Salicylate, Lysine Acetylsalicylate, Mesalamine, Mor (CAS Registry No.: 61036-64-4); Chloramphenicol (CAS pholine Salicylate, I-Naphthyl Salicylate, Olsalazine, Registry No.: 56-75-7); Clindamycin (CAS Registry No.: Parsalmide, PhenylAcetylsalicylate, Phenyl Salicylate, Sal 18323-44-9); Trimethoprim (CAS Registry No. 738-70-5): acetamide, Salicylamide O-Acetic Acid, Salicylsulfuric Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitro 45 Acid, Salsalate, Sulfasalazine, Ampiroxicam, Droxicam, furantoin (CAS Registry No.: 67-20-9); Rifampin (CAS Reg Isoxicam, Lomoxicam, PiroXicam, Tenoxicam, epsilon-Ac istry No.: 13292-46-1); Mupirocin (CAS Registry No.: etamidocaproic Acid, S-Adenosylmethionine, 3-Amino-4- 12650-69-0); Metronidazole (CAS Registry No.: 443-48-1): hydroxybutyric Acid, Amixetrine, Bendazac, BenZydamine, Cephalexin (CAS Registry No.: 15686-71-2); Roxithromy alpha-Bisabolol, Bucolome. Difenpiramide, Ditazol, Emor cin (CAS Registry No.: 80214-83-1); Co-amoxiclavuanate: 50 faZone, Fepradinol, GuaiaZulene, Nabumetone, Nimesulide, combinations of Piperacillin and Tazobactam; and their vari Oxaceprol, Paranyline, Perisoxal, ProquaZone, Superoxide ous salts, acids, bases, and other derivatives. Dismutase, Tenidap, Zileuton, 21-Acetoxypregnenolone, Anti-fingal agents include but are not limited to Amphot Alclometasone, Algestone, Amcinonide, Beclomethasone, ericin B, Candicidin, Dermostatin, Filipin, Fungichromin, Betamethasone, Budesonide, Chloroprednisone, Clobetasol, Hachimycin, Hamycin, Lucensomycin, Mepartricin, Nata 55 Clobetasone, Clocortolone, Cloprednol, Corticosterone, Cor mycin, Nystatin, Pecilocin, Perimycin, AZaserine, Griseoful tisone, CortivaZol, Deflazacort, Desonide, DeSoximetaSone, vin, Oligomycins, Neomycin, Pyrrolnitrin, Siccanin, Tuber Dexamethasone. Diflorasone. Diflucortolone. Difluprednate, cidin, Viridin, Butenafine, Naftifine, Terbinafine, Bifonazole, Enoxolone, Fluazacort, Flucloronide, Flumethasone, Butoconazole, Chlordantoin, Chlormidazole, Cloconazole, Flunisolide, Fluocinolone Acetonide, Fluocinonide, Fluocor Clotrimazole, Econazole, Enilconazole, Fenticonazole, Flut 60 tin Butyl, Fluocortolone, Fluorometholone, Fluperolone rimazole, Isoconazole, Ketoconazole, Lanoconazole, Acetate, Fluprednidene Acetate, Fluprednisolone, Flurandre Miconazole, Omoconazole, Oxiconazole, Sertaconazole, nolide, Fluticasone Propionate. Formocortal, Halcinonide, Sulconazole, Tioconazole, Tolciclate, Tolindate, Tolnaftate, Halobetasol Propionate, Halometasone, Halopredone Fluconawle, Itraconazole, Saperconazole, Terconazole, Acetale, Hydrocortamate, Hydrocortisone, Loteprednol Eta Acrisorcin, Amorolfine, Biphenamine, Bromosalicylchlo 65 bonale, Mazipredone, Medrysone, Meprednisone, Methyl ranilide, Buclosamide, Calcium Propionate, Chlorphenesin, prednisolone, Mometasone Furoate, Paramethasone, Predni Ciclopirox, Cloxyquin, Coparaffinate, Diamthazole. Exala carbate, Prednisolone, Prednisolone 25-Diethylamino US 7,659,399 B2 29 30 acetate, Prednisolone Sodium Phosphate, Prednisone, In addition, it is recognized that one optical isomer, includ Prednival, Prednylidene, Rimexolone, Tixocortol, Triamci ing a diastereomer and enantiomer, or a stereoisomer, may nolone, Triamcinolone Acetonide, Triamcinolone Bene have favorable properties over the other. When a racemic tonide, and Triamcinolone Hexacetonide. mixture is discussed herein, it is clearly contemplated to Compounds of the invention may be combined with one or include both optical isomers, including diastereomers and more Beta lactamase inhibitor when used in combination enantiomers, or one stereoisomer Substantially free of the with a beta-lactam class antibiotic, Such as penicillin or other. cephalosporins. Beta-lactamase inhibitors include, but are The invention also includes also includes all energetically not limited to Clavulanic acid, Sulbactam, Sultamacillin, and accessible conformational and torsional isomers of the com TaZobactam. 10 pounds disclosed. Compounds of the invention may also be combined with one or more efflux pump inhibitor. Such as a quinazolinone When the substituent R, in a compound of Formula I or efflux pump inhibitors, d-ornithine-d-homophenylalanine-3- Formula II is attached via an unsaturated aliphatic group, for aminoquinoline, Phe-Arg-b-naphthylamide, propafenone, a example when R, is (phenyl)CC6alkenyl, all geometric iso phenothiazine or thioxanthene efflux pump inhibitor, 1-aza 15 mers of the compound are included. This invention is further 9-oxafluorenes, N-4-2-(3,4-dihydro-6,7-dimethoxy-2(1H)- illustrated by the following examples that should not be con isoquinolinyl)ethylphenyl-9,10-dihydro-5-methoxy-9- Strued as limiting. The contents of all references, patents and oXo-4-Acridinecarboxamide, reserpine, Milbemycin, published patent applications cited throughout this applica Cinchonine, Verapamil, L-phenylalanyl-N-2-naphthalenyl tion are incorporated herein by reference. L-Argininamide (and analogs), 5'-methoxyhydnocarpin-D. methylxanthines, FK506, a cyclosporine efflux pump inhibi EXAMPLES tor, Nocardamine and other siderophores, Amiodarone, Cyclosporin A. Ro11-2933 (DMDP), Quinidine, and the opti Abbreviations cal isomers of Propranolol, Quinine (SQ1) and Quinidine, Quinine-10, 11-epoxide, Quercetin, Amitriptyline, Taxuspine 25 The following abbreviations are used in the reaction C derivatives, Emodin, MC-002434; Agosterol A; Pheophor schemes and synthetic examples, which follow. This list in bide: pyridoquinolines such as 2,2'-(2,8,10-trimethylpyrido not meant to be an all-inclusive list of abbreviations used in 3.2-gquinoline-4,6-diyl)bis(oxy)bis N,N-dimethyl-etha the application as additional standard abbreviations, which namine, Gitonavir, and Gemfibrozil. are readily understood by those skilled in the art of organic 30 synthesis, may also be used in the synthetic schemes and Synthesis of Compounds examples: The starting materials used in preparing the compounds of DMF N,N-Dimethylformamide the invention are known, made by known methods, or are DMSO–Dimethylsulfoxide commercially available. 35 LDA—Lithium diisopropylamide It is recognized that the skilled artisan in the art of organic Pd(PPh)-Tetrakis(triphenylphosphine)palladium(0) chemistry can readily carry out standard manipulations of organic compounds without further direction. Examples of TFA Trifluoroacetic acid Such manipulations are discussed in standard texts such as J. General Methods March, Advanced Organic Chemistry, John Wiley & Sons, 40 All nonaqueous reactions are performed under an atmo 1992. Those skilled in the art of organic synthesis will recog sphere of dry argon gas (99.99%) using oven- or flame-dried nize that reactions conditions and starting materials will vary glassware. All isolated products are stored for prolonged peri on occasion from those set forth in the synthetic examples ods under an atmosphere of argon at -20° C. Microwave included hereindepending of the desired final product. assisted syntheses are conducted in a commercial microwave The skilled artisan will readily appreciate that certain reac 45 reactor (Discover System, CEM Corporation). The progress tions are best carried out when other functionalities are ofreactions is monitored using thin-layer chromatography on masked or protected in the compound, thus increasing the glass plates coated with Merck silica gel 60 (F254). Flash yield of the reaction and/or avoiding any undesirable side column chromatography is performed on Merck silica gel 60 reactions. Often, the skilled artisan utilizes protecting groups (230-400 mesh). Melting points are recorded on an Electro to accomplish Such increased yields or to avoid the undesired 50 thermal Model IA9 100 digital melting point apparatus. NMR reactions. These reactions are found in the literature and are spectra are recorded at ambient temperature using a Bruker also well within the scope of the skilled artisan. Examples of Avance 300 spectrometer (H at 300.1 MHz, C at 75.5 many such manipulations can be found in, for example, T. MHz, and 'Nat 30.4 MHz). The chemical shifts for "Hand Greene, Protecting Groups in Organic Synthesis, John Wiley 'C are reported in parts per million (8) relative to external & Sons, 1981. 55 tetramethylsilane and are referenced to signals of residual The compounds of the invention may have one or more protons in the deuterated solvent. The chemical shifts for 'N chiral center. As a result, one may selectively prepare one are reported in parts per million (8) relative to external liquid optical isomer, including diastereomers and enantiomers, ammonia. Assignment of 1H and 'C NMR data is based on over another, for example by chiral starting materials, cata extensive two-dimensional correlation experiments (H-H lysts or solvents, or may prepare both Stereoisomers or both 60 COSY, H. 'C HMQC, H C HMBC, and H. 'H optical isomers, including diastereomers and enantiomers at NOESY spectra recorded using standard pulse sequences) once (a racemic mixture). Since the compounds of the inven and the usual principles of NMR spectroscopy (the magni tion may exist as racemic mixtures, mixtures of optical iso tudes of coupling constants and chemical shifts). Analytical mers, including diastereomers and enantiomers, or stereoiso HPLC is performed using a YMC Pack Pro C1850x4.6 mm mers may be separated using known methods, such as 65 5 um column with an isocratic elution of 0.24 min at 95:5 through the use of for example, chiral salts and chiral chro HO:CHCN containing 0.1% TFA followed by a 20-min matography. linear gradient elution from 95:5 to 5:95 at a flow rate of 1.0 US 7,659,399 B2 31 32 mL/min with UV detection at 254 nm. Low-resolution mass After the addition of diethyl malonate is complete, the gray spectra are recorded on a Thermo Finnigan Surveyor MSQ reaction mixture continues to stir at 0°C. for 15 min. Carbon instrument (operating in API mode) equipped with a Gilson disulfide (14.5 mL, 0.24 mol) is then added dropwise to give liquid chromatograph. High-resolution mass spectrometric a red mixture that is allowed to warm to room temperature analyses (ESI using sodium iodide as internal standard) are with stirring for 1 h. A solution of dimethylformamide (25 performed at the W. M. Keck Foundation Biotechnology mL) containing 4-methoxybenzyl chloride (25.00 g, 0.16 Resource Laboratory (Yale University, New Haven, Conn.). mol) is added dropwise to the reaction mixture at room tem Elemental analyses are performed at Atlantic Microlab, Inc. perature. The resulting orange mixture is allowed to warm to (Norcross, Ga.) and Prevalere Life Sciences, Inc. (Whites 10 room temperature with stirring for 15 h, diluted with water boro, N.Y.). (500 mL), and extracted with ethyl acetate (4x500 mL). The combined extracts are concentrated under reduced pressure Example 1 (~1 L), washed with water (3x150 mL), washed with brine (3x150 mL), dried over magnesium sulfate, and evaporated Preparation of 10-ethyl-8-phenyl-2H-isothiazolo.54 under reduced pressure to give crude 1. The crude product is bquinolizine-3,4-dione (7) purified by flash column chromatography (eluting with ethyl

21 21 NN 21 NN EtOC COEt 1. LDA 1. LDA C N 2. Et C N 2.1 C N h S

O 2 3

O O O O

21 NN OEt 21 NN OEt TFAlanisole e C N1SN SH C N1S S o1 5 4 HNOSOH NaHCO

O O O O 21 NN DMF/NaHCOPd(PPh3)4 21 NN NH Her NH ArB{O(R)H)2 C N1S s/ Air N1S s/

6 7

Step 1. Diethyl acetate/hexanes=1:4, Rf 0.21) to give 1 as a yellow solid. mp 2-bis(4-methoxybenzylsulfanyl)methylene malonate 60 1 57-59° C. "H NMR (CDC13): & 1.27 (t, J–7.0 Hz, 3H, A solution of dimethylshamide (50 mL) containing COCHCH), 3.79 (s.3H, OCH), 4.07 (s. 2H, SCH), 4.23 diethyl malonate (12.78 g., 0.08 mol) is added slowly to an (q, J=7.0Hz, 2H, COCHCH), 6.83 (m,2H, H-3/H-5), 7.20 ice-cooled suspension of sodium hydride (60% dispersion in (m. 2H, H-2/H-6). 'C{H} NMR (CDC1): 8 14.0 mineral oil, 6.53 g, 0.16 mol) in dimethylformamide (500 65 (COCHCH), 40.3 (SCH), 55.3 (OCH), 61.4 mL). The rate of addition of diethyl malonate is such as to (COCHCH), 114.1 (aromatic C-3/C-5), 128.1 (—C— maintain the temperature of the reaction mixture below 5°C. COCHCH or C CHS ), 128.7 (—C COCHCH or US 7,659,399 B2 33 34 C CHS ), 130.4 (aromatic C-2/C-6), 155.6 ( CHS LCMS m/z calcd for CHCINOS 477 (M+). found 478 C—), 159.1 (aromatic C-4, C OCH), 163.9 (IM+H+). (COCHCH). LCMS m/z calcd for CHOS 476 (M+). found 477 (IM+H+). HRMS m/z calcd for Step 4. Ethyl 8-chloro-1-ethyl-2-(4-methoxybenzyl CH-NaOS 499.1225 (M+Na+). found 499.1230. Anal. 5 Sulfanyl)-4-oxo-4H-quinolizine-3-carboxylate (4) Calcd for CHOS: C, 60.48; H, 5.92; S, 13.46. Found: C, 60.51; H, 5.97; S, 13.19. A solution of 3 (1.93 g, 4.04 mmol) in dimethyl sulfoxide (10.0 mL) is heated at 120° C. for 5.5 h, cooled to room Step 2.4-Bromo-2-propylpyridine (2) temperature, and concentrated under reduced pressure to give 10 a yellowish brown oil that solidifies upon standing. The crude Prepared by a method analogous to the preparation of product is purified by flash column chromatography (eluting 5-Chloro-2-propylpyridine, described previously (Li, Q., et with 20% ethyl acetate in methylene chloride: Rf O.53) to give al. J. Med. Chem. 39: 3070-3088 (1996)). The crude product pure 4 as a bright yellow solid. Because 4 oxidizes slowly in is purified by flash column chromatography (eluting with Solution under ambient conditions, the purified material is ethyl acetate/hexanes=1:2: Rf 0.56) to give 2 as an amber 15 stored for prolonged periods as a Solid under an atmosphere of liquid. argon in the absence of light. "H NMR (CDC1): 8 0.97 (t, J=7.5 Hz, 3H, CHCHCH), mp 159-160° C. "H NMR (CDC1): 81.09 (t, J=7.5 Hz, 3H, 1.74 (m,2H, CHCHCH), 2.75 (m,2H, CHCHCH), 7.11 CHCH), 1.44 (t, J–7.0 Hz, 3H, COCHCH), 2.87 (q, (dd, J=5.5 Hz, 1.5 Hz, 1H, pyridyl H-5), 7.16 (d. J=1.5 Hz, J=7.5 Hz, 2H, CHCH), 3.77 (s, 3H, OCH), 4.10 (s. 2H, 1H, pyridyl H-3), 8.42 (d. J=5.5 Hz, 1H, pyridyl H-6). 'C SCH), 4.51 (q, J=7.0 Hz, 2H, COCHCH), 6.78 (m, 2H, {H} NMR (CDC1): 8 13.8 (CHCHCH.), 22.8 benzylic H-3/H-5), 7.01 (dd, J=8.0 Hz, 2.0 Hz, 1H, pyridone (CHCHCH), 40.1 (CHCHCH), 121.3 (CH, C-5), 123.0 H-7), 7.16 (m, 2H, benzylic H-2/H-6), 7.63 (d. J–2.0 Hz, 1H, (CH, C-3), 144.1 (C-4, C-C1), 150.1 (CH, C-6), 1640 (C-2). pyridone H-9), 9.16 (d. J–8.0 Hz, 1H, pyridone H-6). LCMS m/z calcd for CHCIN 155 (M+). found 156 (IM+ 'C{H} NMR (CDC1,): & 14.2 (COCHCH.), 14.9 H+). HRMS m/z calcd for C8HCIN 156.0580 (M+H+). 25 (CHCH), 22.0 (CHCH), 41.6 (SCH), 55.3 (OCH), 62.0 found 156.0578. (COCHCH), 113.9 (benzylic C-3/C-5), 116.9 (C-7, CH), 119.1 (C-1), 121.0 (C-3), 121.1 (C-9, CH), 128.8 (benzylic Step 3. (E)-diethyl 2-(1-(4-methoxybenzylthio)-2-(4- C-1), 130.0 (C-6, CH), 130.2 (benzylic C-2/C-6), 138.3 (C-8, bromopyridin-2-yl)but-1-enyl)malonate (3) C Cl), 139.2 (C-9a), 144.5 (C-2, C-SCH), 1540 (C-4, 30 NC(O)), 159.1 (benzylic C-4), 166.5 (COCHCH). LCMS Lithium diisopropylamide (LDA) is formed by dropwise m/z calcd for CHCINOS 431 (IM+). found 432 (M+ addition of n-butyllithium (1.6M in hexanes, 10.1 mL, 16.2 H+). HRMS m/z calcd for CHCINNaOS 454,0856 mmol) to a stirred solution of diisopropylamine (2.3 mL, 16.4 (IM+Na+). found 454,0853. Anal. Calcd for mmol) in tetrahydrofuran (25.0 mL) at -78°C. The resulting CHCINOS: C, 61.18; H, 5.13: N, 3.24. Found: C, 61.33: solution is stirred at -78°C. for 5 min, 0°C. for 15 min, and 35 then recooled to -78°C. A solution of 4-bromo-2-propylpy H, 5.15; N, 3.33. ridine (2, 2.94g, 14.7 mmol) in tetrahydrofuran (25.0 mL) is Step 5. Ethyl 8-bromo-1-ethyl-2-mercapto-4-oxo added dropwise to this solution over a period of 30 min. The 4H-quinolizine-3-carboxylate (5) resulting deep red suspension is stirred at -78°C. for 1 h, after which time a solution of diethyl 2-bis(4-methoxybenzylsul 40 fanyl)methylene malonate (1, 7.03 g, 14.8 mmol) in tetrahy A solution containing 4 (1.0306 g, 2.39 mmol), trifluoro acetic acid (100g), and anhydrous anisole (20.0 mL) is heated drofuran (40.0 mL) is added dropwise overa period of 15 min. at 40°C. for 23 h. The resulting amber solution is evaporated The resulting yellow solution is stirred at -78°C. for 1.5 h, under reduced pressure (~ 1 mm Hg, 40°C.) to give an oily -15°C. for 1.5 h, and room temperature for 30 minto give a residue containing ethyl 8-bromo-1-ethyl-2-mercapto-4- dark orange solution. The reaction mixture is quenched with 45 a saturated aqueous solution of ammonium chloride (100 mL) oxo-4H-quinolizine-3-carboxylate (5) that is used directly in and extracted with diethyl ether (2x100 mL). The combined the next step of the synthesis. organic extracts are dried over magnesium Sulfate and evapo "H NMR (CDC1): 81.22(br,3H, CHCH.), 1.44 (t, J–7.0 rated under reduced pressure to give 3 an orange oil. The Hz, 3H, COCHCH), 2.88 (br, 2H, CHCH), 4.48 (br, 2H, product is purified by flash column chromatography (eluting 50 COCHCH), 5.85 (br. 1H, SH), 6.92 (dd, J=8.0 Hz, 2.0 Hz, with 5% ethyl acetate in methylene chloride. 1H, H-7), 7.53 (br. 1H, H-9), 9.12 (d. J=8.0 Hz, 1H, H-6). H NMR (DMSO-d6): 8 0.76 (t, J–7.5 Hz, 3H, CHCH), LCMS m/z calcd for CHCINOS 311 (M+). found 312 1.17 (t, J–7.0 Hz, 6H, 2xCOCHCH), 2.81 (q, J–7.5 Hz, (IM+H+). 2H, CHCH), 3.74 (s.3H, OCH), 4.01 (s. 2H, SCH), 4.10 (q, J–7.0 Hz, 4H, 2xCOCH2CH), 4.41 (s, 1H, 55 Step 6.7-Chloro-9-ethyl-1-thia-2.4a-diazacyclopenta CH(COCHCH)), 6.90 (m, 2H, benzylic H-3/H-5), 7.28 bnaphthalene-3,4-dione (6) (m. 2H, benzylic H-2/H-6), 7.44 (d, J-2.0 HZ, 1 H. pyridyl H-3), 7.52 (dd, J=5.5 Hz, 2.0 Hz, 1H, pyridyl H-5), 8.56 (d. A solution of sodium bicarbonate (2.00g, 23.8 mmol) in J=5.5 Hz, 1H, pyridyl H-6). 'C{H} NMR (DMSO-d6): 8 water (60 mL) is added to a solution of 5 (from above, ~2.39 12.4 (CHCH), 13.9 (CH(COCHCH)), 28.3 (CHCH), 60 mmol) in tetrahydrofuran (60 mL) at room temperature. 39.5 (SCH), 55.0 (OCH), 58.5 (CH(COCHCH.),), 61.1 Hydroxylamine-O-sulfonic acid (1.08 g, 9.55 mmol) is added (CH(COCHCH)), 113.9 (benzylic C-3/C-5), 122.9 (py to this mixture as a Solid and in one portion. The resulting ridyl C-5), 123.0 (pyridyl C-3), 125.7 (=C(SAr)(CH yellow mixture is stirred at room temperature for 3.5 h and (COCHCH))), 129.3 (benzylic C-1), 130.2 (benzylic quenched by addition of an aqueous solution of 5% hydro C-2/C-6), 143.4 (pyridyl C-4, C. Cl), 150.6 (pyridyl C-6). 65 chloric acid (500 mL). The precipitate that forms is collected 153.3 (—C(CHCH)(pyridyl)), 158.4 (benzylic C-4, by filtration, washed with distilled water (3x50 mL), washed C OCH), 159.1 (pyridyl C-1), 167.2 (CH(COCHCH)). exhaustively with diethyl ether, and dried in vacuo to give US 7,659,399 B2 35 36 pure 6 as a yellow solid. Because 6 decomposes slowly in Example 2 Solution under ambient conditions, the purified material is stored for prolonged periods as a Solid under an atmosphere of Preparative HPLC Conditions argon in the absence of light. 5 Preparative HPLC was performed using a YMC Pack Pro Rf 0.25 (methylene chloride/methanol/acetic acid-200/10/ C18 150x20.0 mm 5um column with an isocratic elution of 1). mp 222-223° C. "H NMR (DMSO-d6): 8 1.14 (t, J=7.5 Hz, 0.35 min at 90:10 water:acetonitrile containing 0.1% trifluo 3H, CHCH), 2.75 (q, J–7.5 Hz, 2H, CHCH), 7.05 (dd, roacetic acid followed by a 23.6-min linear gradient elution J=8.0Hz, J-2.0Hz, 1H, pyridone H-6), 7.87 (d. J=2.0Hz, 1H, from 90:10 to 10:90 at a flow rate of 18.9 mL/min with UV pyridone H-8), 8.89 (d. J–8.0 Hz, 1H, pyridone H-5). 10 detection at 254 nm. Analytical HPLC was performed using a 'C{H} NMR (DMSO-d6): 8 13.3 (CHCH), 21.6 YMC Pack Pro C18 50x4.6 mm 5um column with an iso (CHCH), 104.0 (br, C-3a), 106.1 (C-9), 1144 (C-6, CH), cratic elution of 0.24 min at 95:5 water:acetonitrile contain 119.3 (C-8, CH), 129.7 (C-5, CH), 137.2 (C-7, C C1), 137.5 ing 0.1% trifluoroacetic acid followed by a 20-min linear (C-8a), 152.9 (C-4, NC(O)), 160.9 (br, C-9a, C S), 166.8 gradient elution from 95:5 to 5:95 at a flow rate of 1.0 mL/min (C-3, CONS). LCMS m/z calcd for CHCINOS 280 15 with UV detection at 254 nm. (M+). found 281 (IM+H+). HRMS m/z calcd for Example 3 CHCINNaOS 302.9971 (M+Na+). found 302.9969. Anal. Calc’d for CHCIN.O.S.H.O: C, 48.24; H, 3.71; N, 9-Ethyl-7-pyridin-4-yl-1-thia-2.4a-diaza-cyclopenta 9.38. Found: C, 48.06; H, 3.40; N, 9.16. Ibnaphthalene-3,4-dione hydrochloride (8) Step 6a, 2-N-7-Chloro-9-ethyl-1-thia-2,4a-diaza

cyclopentablnaphthalene-3,4-dione (2-N-7) This compound is prepared as outlined above for 6 using 25 'Nhydroxylamine-O-sulfonic acid (98+ atom%). The 'C NMR spectrum of 2-N-6 is identical to that reported above for 6 with one exception that follows. 'C{H} (DMSO-d6): 8 166.8 (d. 1JN–C–3.5 Hz). Because exchange 30 of the N-H group of 2-N-6 is rapid (the corresponding broad 1H resonance showed rapid H-D exchange with DO at Tetrakis(triphenylphosphine)palladium(0) (26.1 mg, room temperature), it is necessary to cool samples to obtain 0.023 mmol), 4-pyridinylboronic acid (89.4 mg. 0.727 'N NMR spectroscopic data. 'N NMR (DMF-d7, -50° C): 35 mmol), and a 1 M aqueous solution of sodium bicarbonate (2.2 mL, 2.2 mmol) are added sequentially under an atmo & 104.6 (s). In accord with the 'N data recorded at low sphere of argon, to a solution containing 7-chloro-9-ethyl-1- temperature, no 1JN-H coupling was observed in 1H NMR thia-2.4a-diazacyclopentabnaphthalene-3,4-dione (40.9 spectra of 2-N-7 recorded in DMF-d7 at temperatures mg, 0.145 mmol) and dimethylformamide (4.0 mL). The ranging from +60 to -55° C. LCMS m/z calcd for 40 resulting orange mixture is irradiated in a microwave for 15 CHCINNOS (IM+) 281. found 282 (M+H+). min (110° C.: 300 W) to give a red mixture. The solvent is evaporated under reduced pressure (~10 mm Hg, 50° C.), and Step 7. 10-ethyl-8-phenyl-2H-isothiazolo.5,4-b the remaining solid washed with diethyl ether (3x15 mL). The quinolizine-3,4-dione (7) product is extracted from this solid using hot dimethyl Sul 45 foxide (5x2 mL) and purified by preparative HPLC. The purified product is isolated as the trifluoroacetate salt and Pd(PPh) (3.1 mg, 6% mole), boronic acid or ester (2 converted to the hydrochloride salt by addition of a solution of equiv., 0.088 mmole) and NaHCO solution (1M, 0.2 mL, 4.5 hydrogen chloride (~1.25M in methanol) followed by evapo equiv.) under argon at room temperature is added to a stirred ration to give a deep red Solid; this process is repeated twice. solution of 7-Bromo-9-ethyl-1-thia-2.4a-diazacyclopentab 50 Purity by HPLC: 98.3%; tR=4.98 min. mp 289-290° C. naphthalene-3,4-dione 0.044 mmole) in DMF (1 mL). The dec. "H NMR (DMSO-d6): 8 1.22 (t, J–7.5 Hz, 3H, reaction mixture is degassed by bubbling argon through for CHCH), 2.97 (q, J=7.5 Hz, 2H, CHCH), 7.52 (dd, J-8.0 10 minutes at room temperature. The reaction tube is sealed HZ, J=1.5 Hz, 1H, pyridone H-6), 8.25 (d. J=1.5 Hz, 1H, and then heated in a microwave (100 W. 130°C.) until the pyridone H-8), 8.40 (d. J=6.0 Hz, AA" part of AAXX' spin reaction reaches completion (10-20 minutes). The reaction 55 system, 2H, pyridinyl H-3/H-5), 8.96 (d. J=6.0 Hz, XX' part mixture is cooled to room temperature and then filtered. The of AAXX' spin system, 2H. pyridinyl H-2/H-6), 9.02 (d. filtrate is concentrated in vacuo. The residue is dissolved in a J=8.0 Hz, 1H, pyridone H-5). C{1H NMR (DMSO-d6): 8 mixture of DMF:CHC1:MeOH (0.5:3:0.5) (4 mL) and pre 13.7 (CHCH), 21.8 (CHCH), 105.3 (C-3a), 109.5 (C-9), cipitated with diethyl ether. This dissolution and precipitation 110.8 (C-6, CH), 121.4 (C-8, CH), 123.9 (pyridinyl C-3/C-5), 60 128.8 (C-5, CH), 136.5 (C-7), 136.7 (C-8a), 143.7 (pyridinyl step is repeated 5 times. The resulting light yellow solid is C-2/C-6), 149.9 (pyridinyl C-4), 152.9 (C-4, NC(O)), 160.9 washed with water (3 mL) and dried to afford the title com (C-9a, C S), 166.8 (C-3, CONS). LCMS m/z calcd for pound. CHNOS 323 (M++). found 324 (M+H+). HRMS General information concerning the preparation of C C m/z calcd for C2HNOS 324.0807 (M--H+). found bond at C-7 of a pyridone core via Suzuki cross-coupling may 65 324.0807. Anal. Calcd for CHNOS.HC1.2.2H2O: C, be found in Otten, P. A. et al. Bioconjugate Chem. (2001) 12: 51.11; H, 4.64; N, 10.52. Found: C, 51.03; H, 4.54: N, 10.67. 2O3-212. Analytical HPLC: th=4.98 min. US 7,659,399 B2 37 38 Example 4 Example 5 7-(2,6-Dimethylpyridin-4-yl)-9-ethyl-1-thia-2.4a- 7-(4-Aminomethylphenyl)-9-ethyl-1-thia-2.4a-diaza diaza-cyclopentabnaphthalene-3,4-dione hydro- cyclopentablnaphthalene-3,4-dione (10) chloride (9) 5

10 21 N

HN 15 2

Under an atmosphere of argon, tetrakis(triphenylphos phine)palladium(0) (20.4 mg., 0.018 mmol), 4-aminometh Tetrakis(triphenylphosphine)palladium(0) (25.9 mg, ylphenylboronic acid (110.0 mg. 0.587 mmol), and a 1 M 0.022 mmol), 2,6-dimethyl-4-pyridinylboronic acid (112.8 aqueous solution of sodium bicarbonate (1.75 mL, 1.75 mg, 0.747 mmol), and a 1 M aqueous solution of sodium mmol) were added sequentially to a solution containing bicarbonate (2.3 mL, 2.3 mmol) are added sequentially under 25 7-chloro-9-ethyl-1-thia-2.4a-diazacyclopentabnaphtha an atmosphere of argon, to a solution containing 7-chloro-9- lene-3,4-dione (33.0 mg 0.118 mmol) and dimethylforma ethyl-1-thia-24a-diazacyclopentablnaphthalene-3,4-dione mide (3.0 mL). The resulting orange mixture was irradiated in (42.0 mg 0.150 mmol) and dimethylformamide- (4.0 mL). a microwave for 15 min (110° C.; 300 W) to give a red The resulting Orange mixture is irradiated 181COWaWe for mixture. The solvent is evaporated under reduced pressure 15 min (110° C.; 300 W) to give a red mixture. The solventO is (~10 mm Hg, 50° C.), and the remaining orange Solid is evaporated under reduced pressure (~10 mm Hg, 50° C.), and 30 washed with diethyl ether (3x15 mL). the remaining solid washed with diethyl ether (3x15 mL). The product is extracted from this solid using hot dimethyl Sul- f MSN als S.N.O.S 351 (M+). foxide (5x2 mL) and purified by preparative HPLC. The ou (M+ +, X), ( 6). purified product is isolated as the trifluoroacetate salt and H NMR (acetic acid-da): & 1.32 (t, J–7.5 Hz, 3H, converted to the hydrochloride salt by addition of a solution of 35 CHCH), 2.90 (q, J–7.5 Hz, 2H, CHCH), 436 (s. 2H, hydrogen chloride (~1.25M in methanol) followed by evapo- CHN), 7.49 (dd, J=8.0 Hz, J=1.5 Hz, 1H, pyridone H-6), ration to give a deep red Solid; this process is repeated twice. 7.69 (d. J–8.5 Hz, AA part of AAXX' spin system, 2H, Purity by HPLC. 99.6%; tR-5.80 min. mp 279-280 C. phenyl H-3 and H-5), 7.91 (d. J.8.5 Hz, XX part of AAXX dec. H NMR (DMSO-d6): 8 1.21 (t, J=7.5 Hz, 3H, spin system, 2H, phenyl H-2 and H-6), 7.94 (d. J=1.5 Hz, 1H, CHCH), 2.80 (s, GH, pyridinylCH.), 2.96 (d. J–7.5 Hz, 2H, pyridone H-8),924 (d. J-8-0 Hz, 1H. pyridone H-5). CHCH), 7.45 (dd, J=8.0 Hz, J=1.5 Hz, 1H, pyridone H-6), Analytical HPLC: th=6.86 min. 8.20 (brs, 1H, pyridone H-8), 8.28 (s. 2H, pyridinyl H-3/H- 5), 8.96 (d. J=8.0Hz, 1H, pyridone H-5). LCMS m/z calcd for Example 6 C19HNOS 351 (M+). found 352 (M+H+). HRMS m/z calcd for C19H18NOS 352.1120 (M+H+). found 45 Preparation of 1-Thia-2.4a,8-triaza-cyclopentab 352.1120. naphthalene-diones

ONa F NH2 Fn 2 21 NN NŠs N 1.HC EtOH HN EtO O HO N F 2. NH3 F F

oHCI F F F

1. n-Buli 2.1 US 7,659,399 B2 39 40 -continued O O

F F EtOC 21 NN OEt 2n - 2 COEt 1. A h C N N N S 2. POCl, DMF HO N N S F 1. F

O F F /TFAfanisole O O O O O O F 21 NN of H2NOSO3H F 21 NN Pd(PPh3)4 F 21 NN NaHCO NH, DMF/NaHCOs NH C Sa 1s SH C N 1s S/ AB (or H Ar N 1s

F F

F F F

30 Example 7 may also be prepared by the method disclosed by Otten, P. A. et al. (Bioconjugate Chem., 12: 203-212 (2001) which is Additional 1-thia-2.4a-diaza-cyclopentabnaphtha- hereby incorporated by reference for its teachings regarding lene-3,4-diones the preparation of C–C bond at C-7 of the pyridone via 35 Suzuki cross-coupling. Additional compounds of Formula I are prepared by the methods illustrated in Examples 1. Compounds of Formula I Additional compounds of Formula I are listed in Table I.

TABLE 1

Cpd. # Structure Name

11 9-Cyclopropyl-6-fluoro-7-pyridin-4- yl-1-thia-24a-diaza cyclopentabnaphthalene-3,4-dione

12 9-Cyclopropyl-6-fluoro-7-pyridin-3- yl-1-thia-24a-diaza cyclopentabnaphthalene-3,4-dione

US 7,659,399 B2 57 58 Example 8 Minimum Inhibitory Concentration (MIC) Assay Whole-cell antibacterial activity is determined by broth X-ray Crystal Structure of 7-Chloro-9-ethyl-1-thia-2, microdilution using conditions recommended by the 4a-diazacyclopentabnaphthalene-3,4-dione (6) National Committee for Clinical Laboratory Standards (NC CLS). The minimum inhibitory concentration (MIC) was Data Collection defined as the lowest concentration of antimicrobial agent to A yellow blade crystal of CHCINOS having approxi completely inhibit visible growth after 18-24 h at 37°C. mate dimensions of 0.25x0.10x0.10 mm was mounted with Test compounds are dissolved in DMSO and diluted 1:50 epoxy cement on the tip of a fine glass fiber. All measure 10 in Mueller-Hinton II broth (Becton-Dickinson) to produce a ments were made on a Nonius KappaCCD diffractometer 256 ug/ml stock solution. In a 96-well microtiter plate, the with graphite monochromated Mo KC. radiation. compound solution is serially two-fold diluted in Mueller Cell constants and an orientation matrix for data collection Hinton II broth. After the compounds are diluted, a 50 ug corresponded to a primitive monoclinic cell with dimensions: aliquot of the test organism (~1x10 cfu/mL) is added to each a=7.0574(14) A 15 well of the microtiter plate. The final test concentrations b=9.950(2) A range from 0.125-128ug/mL. Inoculated plates are incubated c=16.661(3) A in ambient air at 37° C. for 18 to 24 hours. The organisms V=1165.6(4) A selected for testing included laboratory strains S. aureus ATCC 29213 and E. coli ATCC 25922 (strains purchased B=94.96(3) from AmericanType Culture Collection, Manassas, Va.). The minimum inhibitory concentration (MIC) was determined as For Z=4 and F.W.-280.72, the calculated density is 1.600 the lowest concentration of compound that inhibited visible g/cm. Based on a statistical analysis of intensity distribution, growth of the test organism. and the Successful solution and refinement of the structure, the space group was determined to be P2/c (#14). 25 Topoisomerase IV Activity The data were collected at a temperature of 173(2) K to a Enzyme activity is measured by a decatenation assay that maximum 20 value of 56.54. Five omega Scans consisting of 37, 29, 34, 14, and 17 data frames, respectively, were col monitors the ATP-dependent unlinking of DNA minicircles lected with a frame width of 2.0° and a detector-to-crystal from kinetoplast DNA. Analysis is done by gel electrophore distance, Dx, of 35.0 mm. Each frame was exposed twice (for 30 sis through 1% agarose/TBE gel. DNA is visualized with an the purpose of de-Zingering) for a total of 40s. The data Alpha Imager 2200 Analysis System and the ICso determined frames were processed and scaled using the DENZO software by nonlinear regression analysis with Graphpad Prism soft package. ware. S. aureus topoismerase IV enzyme subunits, grlA and grlB, are purified to homogeneity from over-expression plas Data Reduction 35 mids in E. coli. A total of 4725 reflections were collected of which 2872 were unique and observed (R-0.0321). The linear absorp DNA Gyrase Activity tion coefficient, u, for Mo KC radiation is 5.00 cm', and no Enzyme activity is measured by a Supercoiling assay that absorption correction was applied. The data were corrected monitors the ATP-dependent conversion of relaxed pBR for Lorentz and polarization effects. 40 DNA to the supercoiled form. Analysis is done by gel elec Structure Solution and Refinement trophoresis through 1% agarose/TBE gel. DNA is visualized The structure was solved by direct methods and expanded with an Alpha Imager 2200 Analysis System and the ICso using Fourier techniques. The non-hydrogen atoms were determined by nonlinear regression analysis with Graphpad refined anisotropically, and hydrogen atoms, with exceptions Prism software. E. coli DNA gyrase enzyme subunits, gyra noted, were treated as idealized contributions. The final cycle 45 and gyrB, are purified to homogeneity from over-expression of full-matrix least-squares refinement on F was based on constructs in E. coli. 2872 observed reflections (D2.00O(I)) and 167 variable parameters and converged with unweighted and weighted Human Topoisomerase II Activity agreement factors of Enzyme activity is measured by a DNA cleavage assay that 50 monitors generation of linear DNA from Supercoiled pBR DNA. Human p170 topoisomerase II is purchased from Topo Gen. Analysis is done by electrophoresis through 1% agarose/ TAE gel with 0.5 ug/mL ethidium bromide, followed by The maximum and minimum peaks on the final difference visualization and quantification with Alpha Imager 2200. The Fourier map corresponded to 0.324 and -0.347e/A respec 55 tively. EC value is defined as the effective concentration of drug required to enhance enzyme-mediated cleavage of double Example 9 Stranded DNA twofold. Compounds 8 and 9 exhibit an MIC of 10 g/ml against S. Antimicrobial Activity of Compounds of the 60 aureaus and E. coli when tested in the above assay for mini Invention mal inhibitory activity. These compounds also exhibit inhibi tion of e. coli DNA gyrase Supercoiling (ICso<1 uM) and The antimicrobial activity of the compounds of the inven inhibition of S. aureus topoisomerase IV activity (ICso tion may be evaluated by a number of methods, including the <10 uM). Compounds 8 and 9 do not inhibit S. aureus DNA following visual minimum inhibitory concentration (MIC) 65 gyrase Supercoiling activity (ICs >200 LM) and do not dis assay. This assay determines the minimum concentration of play activity against human topoisomerase II (EC2>150 uM). compound required to inhibit growth of a bacterial strain. EC2 is US 7,659,399 B2 59 60 the effective concentration of drug required to enhance enzyme-mediated cleavage of double-stranded DNA. TABLE II CELL PLATING POSITIVE Example 10 LINE MEDIA DENSITY CONTROL CHO 1. F-12 Nutrient 7,000 cells well Terfenadine Cell Viability Staining with Alamar Blue (Chinese Mixture (Gibco CCso = hamster #11765-054) 4.3-6.5 L.M ovary) containing 10% To determine whether the microcidal effect observed FBS, 1% Pen Strep, against S. aureus and E. coli is specific to bacterial cells, 10 1.5 g/L Sodium Bicarbonate compounds are screened for cell viability effects on several 2. McCoy's 5a human cell types. medium, 10% FBS and PSGIn Optimal cell density is first determined by plating cells in a HEP 2 Minimum Essential 7,000 cells well Terfenadine 96-well plate standard sterile tissue culture plates in 100 ul 15 (laryngeal Medium - Alpha CCso = media, 10% FBS at six cell densities from 500 cells/well to carcinoma) Medium (Gibco 3-5 M # 12571-063) 15,000 cells/well. A cell free well containing only media is containing 10% used as a control. Cells are incubated at 37°C. in a 5% CO, FBS, 1% Pen Strep, incubator for 24 hours. 10% culture volume (10 microliters) 1.5 g/L Sodium of Alamar Blue (Biosource, DAL 1100, 100 mL) is then Bicarbonate added. Cells are incubated at 37° C. in a 5% CO incubator and read in a Victor V plate reader, 544 nm excitation, 590 nm Example 11 emission, at 3, 4, and 24 hours after the addition of Alamar Blue. The cell number vs. change in fluorescence is plotted to Pharmmaceutical Formulations determine linearity of signal vs. cell number. The optimal 25 density varies between 500-15,000 cells/well depending on Examples 11A through 11D are examples of pharmaceu the specific cell type. The optimal density is selected based on tical compositions containing the compounds of Formula I. the highest number of cells that is still in the linear response The abbreviation A.M. stands for an antimicrobial com range. 30 pound of the present invention. Determination of Compound Cytotoxicity Example 1 1A Cells are plated at optimal cell density in a standard sterile tissue culture 96 well plate, and incubated at 37°C. O/N in a Capsules 5% CO2 incubator. 12 to 48 hours post-plating media is 35 20 grams of the A.M., 6 grams sodium lauryl sulfate, 56 removed. The cells are washed 1 or 2 times with 1xPBS and grams starch, 56 grams lactose, 0.8 grams colloidal silicon replaced with fresh media containing the test compound in dioxide, and 1.2 grams magnesium Stearate are vigorously 1% DMSO. 24 to 72 hours after addition of compound, the stirred together. The resulting mixture is subsequently filled media is removed, and the cells washed 1 to 2 times with into 1000 Suitable hardened gelatin capsules, comprising 1xPBS. Fresh media containing/10 volume of Alamar Blue is 40 each 20 mg of the active ingredient. then added. Plates are incubated 4 hours at 37° C. in a 5% CO2 incubator and read in a Victor V plate reader, 544 nm Example 11B excitation, 590 nm emission. Compounds are diluted to 20 micromolar in 1% DMSO Film-Coated Tablets 45 and media and screened in duplicate to obtain single concen Preparation of tablet core: A mixture of 10 grams of the tration cytotoxicity data. Eight concentration points from A.M., 57 grams lactose and 20 grams starch is mixed well and 0.78 micromolar to 100 micromolar, run in duplicate, are used thereafter humidified with a solution of 0.5 grams sodium to determine cyctotoxicity CC50 values. Cells with 1% dodecyl sulfate, and 1.0 grams polyvinylpyrrolidone (KOL DMSO and media are used as a negative control, compounds 50 LIDON-K 90) in about 20 ml of water. The wet powder having a known CC50 against a particular cell type are used as mixture is sieved, dried, and sieved again. Then 100 grams positive controls. microcrystalline cellulose (AVICEL) and 15 grams hydroge The change in fluorescence vs. concentration of test com nated vegetable oil (STEROTEX) are added. The whole is pound is plotted to determine the cytotoxicity of the com mixed well and compressed into tablets, giving 1000 tablets, pound. 55 each containing 10 mg of the active ingredient. Sample media conditions, optimal plating densities, and Coating: Ethyl cellulose (0.5 grams, ETHOCEL 22 CPS) positive control compounds for two cell types screened are in 15 ml of dichloromethane is added to a solution of 1.0 grams methyl cellulose (Methocel 60 HG.RTM.) in 7.5 ml of presented in Table II. denatured ethanol. Then 7.5 ml of dichloromethane and 0.25 Preferred compounds disclosed in Example 1 and 3 to 6 60 ml 1,2,3-propanetriol are added. Polyethylene glycol (1.0 exhibit CC50 values greater than 10 uM against each of the grams) is melted and dissolved in 7.5 ml of dichloromethane cell lines listed below. Other cell types that may be used and added to the cellulose-containing Solution. Magnesium include but are not limited to Balb/3TC, CEM-SS, HeLa, Octadecanoate (0.25 grams), 0.5 grams polyvinylpyrroli HepG2, HT-29, MRC-5, SK-N SH, U-87 MG, 293T, and done, and 3.0 ml of concentrated color Suspension Huh-7. More preferred are compounds with a CCs value 65 (OPASPRAY K-1-2109) are added and the whole mixture greater than 50 uM. Most preferred are compounds with a homogenized. The tablet cores are coated with this mixture in CCso value greater than 100 uM. a coating apparatus. US 7,659,399 B2 61 62 Example 11C mono- and di-(C-C)alkylamino, =NOR, where Ro is C-C alkyl, and —OR, where R, is (C- Injectible Solutions Cheterocycloalkyl)Co-Calkyl; As is CRs: (i) 1.8 grams methyl 4-hydroxybenzoate and 0.2 grams 5 Rs is hydrogen, C-C alkyl, C-C alkoxy; and propyl 4-hydroxybenzoate are dissolved in about 0.5 L of Ro is (C-C,cycloalkyl)Co-Calkyl or phenyl, each of boiling water. After cooling to about 50° C., 4 grams lactic which is substituted with 0 to 3 substituents indepen acid, 0.05 grams propylene glycol, and 4 grams of the A.Mare dently chosen from halogen. added while stirring. The solution is cooled to room tempera 2. A compound of Formula II ture and Supplemented with water for injection q. S. giving a 10 Solution containing 4 mg/ml of A.M. The solution is sterilized by filtration and filled in sterile containers. Formula II (ii) 100.0 g of an acid salt of an A.M. of the invention is Rs O o1 R3 dissolved in boiling water. After cooling to about 50° C., 37.5 R6 grams lactic acid (90% by weight) are added while stirring. 15 21 NN N The solution is cooled to room temperature and water is added | N to 1 L. The solution is sterilized by filtration and filled in N1SN X sterile containers. R (iii) 5.00 g of an acid salt of an A.M. of the invention is Rs R9 dissolved in boiling water. After cooling to about 50° C., 2.20 grams lactic acid (90% by weight) are added while stirring. The solution is cooled to room temperature and water is added or a pharmaceutically acceptable salt thereof, wherein to 100 ml. A is S. O, SO, or SO; R is hydrogen, C-Calkyl, C-Calkanoyl, mono- or Example 1 1D 25 di-C-Calkylcarbamate, or C-Calkylsulfonate; each of which is substituted with 0 to 3 substituents indepen Cream dently chosen from halogen, hydroxy, amino, cyano, nitro, C-C alkoxy, mono- and di-C-C alkylamino, Phase I contains Sorbitan monostearate (2.0 g), Polyoxy C-Chaloalkyl, and C-Chaloalkoxy: ethylene (20) sorbitan monostearate (1.5 g), Synthetic sper 30 Rs is hydrogen, halogen or amino; maceti (3.0 g) Cetyl stearyl alcohol (10.0 g) and 2-Octyldode R is hydrogen, halogen, or amino; canol (13.5g). The phase I mixture is heated to 75° C., stirred R7 is XR where and mixed. Phase II contains A.M. (1.0 g). Phase II is added X is absent or —C=C , and to phase I, stirred and suspended. Phase III contains Benzyl R is phenyl or a monocyclic or bicyclic 5- to 10-mem alcohol (1.0 g) and demineralized water (q. s. 100 g). Phase III 35 bered bicyclic Saturated, partially unsaturated, or aro is heated to 75° C. and added to phase II. The cream is mixed matic heterocylic group containing at least one nitro intensively and cooled slowly to room temperature, with fur genatom and bound via a carbon atom; each of which ther stirring. After cooling to room temperature the cream is R is substituted with 0 to 5 substituents indepen homogenized. dently chosen from halogen, hydroxy, amino, cyano, C-Calkyl optionally Substituted with amino, (C- What is claimed is: 40 Calkoxy)Co-Calkyl, mono- and di-(C-C)alky 1. A compound of Formula I lamino, =NOR, where Rio is C-C alkyl, and —OR, where R, is (C-Cheterocycloalkyl)Co Formula I Calkyl; Rs O Rs is hydrogen, C-C alkyl, or C-C alkoxy; and O 45 R is (C-C,cycloalkyl)Co-Calkyl, or phenyl, each of R6 21 NN which is substituted with 0 to 3 substituents indepen NH dently chosen from halogen. N N X 3. A compound or salt of claim 1, wherein A is S. R7 50 4. A compound or salt of claim 3 wherein Rs is hydrogen. 5. A compound or salt of claim 3 wherein: R is hydrogen, Rs Ro fluoro, or amino. 6. A compound or salt of claim 5 wherein R is fluoro or or a pharmaceutically acceptable salt thereof, wherein hydrogen. A is S, O, SO, or SO; 55 7. A compound or salt of claim3 wherein Rs is hydrogen or Rs is hydrogen, halogen or amino methoxy. Ro is hydrogen, halogen, or amino, cyano; 8. A compound or salt of claim 3 wherein R is R is XR where C-C cycloalkyl. X is absent or —C=C , and 9. A compound or salt of claim 8 wherein R is cyclopropyl R is phenyl, or a monocyclic or bicyclic 5- to 10 60 or 2,4-difluorophenyl. membered bicyclic Saturated, partially unsaturated, 10. A compound or salt of claim 1 wherein or aromatic heterocylic group containing at least one R, is XR where nitrogen atom and bound via a carbon atom; each of X is absent or —C=C and which R is substituted with 0 to 5 substituents inde R is phenyl, dihydroindolyl, dihydroisoindolyl, dihydro pendently chosen from 65 quinolinyl, dihydroisoquinolinyl, indolyl, imidazolyl, halogen, hydroxy, amino, cyano, C-Calkyl optionally isoindolyl, isoquinolinyl, isoxazolyl, piperazinyl, pip Substituted with amino, (C-Calkoxy)Co-Calkyl, eridinyl, pyrazinyl, pyridyl, pyririnidinyl, pyrrolidinyl, US 7,659,399 B2 63 64 pyrrolyl pyrazolyl, quinolinyl, quinazolinyl, quinoxali 9-Cyclopropyl-6-fluoro-7-quinolin-6-yl-1-thia-2.4a nyl, thiazolyl, tetrahydropyridinyl, tetrahydroisoqui diaza-cyclopentabnaphthalene-3,4-dione; onolinyl, or tetrahydroquinolinyl; group; each of which 9-Cyclopropyl-6-fluoro-7-4-(2-piperazin-1-yl-ethoxy)- is substituted with 0 to 5 substituents independently phenyl-1-thia-2.4a-diaza-cyclopentabnaphthalene-3, chosen from halogen, hydroxy, amino, cyano, 4-dione; (missing cmp 25) C-Calkyl optionally substituted with amino, (C- 7-(4-Aminomethyl-phenyl)-9-cyclopropyl-6-fluoro-1- Calkoxy)Co-Calkyl, mono- and di-(C-C)alky thia-2.4a-diaza-cyclopentabnaphthalene-3,4-dione; lamino, =NOR, where Rio is C-C alkyl, and 9-Cyclopropyl-6-fluoro-7-pyridin-3-ylethynyl-1-thia-2, —OR, where R, is C-Cheterocycloalkyl)Co 4a-diaza-cyclopentabnaphthalene-3,4-dione; Calkyl. 10 4-7-(4-Aminomethyl-phenyl)-9-cyclopropyl-6-fluoro-3, 11. A compound or salt of claim 10 wherein 4-dioxo-3H.4H-1-thia-2.4a-diaza-cyclopentabnaph R is a phenyl, dihydroindolyl, dihydroisoindolyl, dihyd thalen-2-yl)-butyric acid; roquinolinyl, dihydroisoquinolinyl, indolyl, isoindolyl, Acetic acid 9-cyclopropyl-6-fluoro-7-(6-methyl-pyridin isoquinolinyl, pyridyl, pyririnidinyl, quinolinyl, tetrahy 3-yl)-4-oxo-4H-1-thia-2.4a-diaza-cyclopentabnaph droisoquionolinyl, or tetrahydroquinolinyl; group; each 15 thalen-3-yl ester; of which is substituted with 0 to 5 substituents indepen 5-Amino-9-cyclopropyl-6-fluoro-7-pyrimidin-5-yl-1- dently chosen from halogen, hydroxy, amino, cyano, thia-2.4a-diaza-cyclopentabnaphthalene-3,4-dione; C-C alkyl, C-C alkoxy, and mono- and di-(C-C) 6-Amino-9-cyclopropyl-7-pyridin-3-yl-1-thia-2.4a-diaza alkylamino. cyclopentabnaphthalene-3,4-dione; 12. A compound or salt of claim 11 in which X is absent. 9-Cyclopropyl-6-fluoro-8-methoxy-7-(1-methyl-2,3-di 13. A compound or salt of claim 12 in which R is phenyl hydro-1H-isoindol-5-yl)-1-thia-2.4a-diaza-cyclopenta or pyridyl, each of which is substituted with 0 to 3 substituents bnaphthalene-3,4-dione: independently chosen from halogen, hydroxy, amino, cyano, 9-Cyclopropyl-8-methoxy-7-(1-methyl-2,3-dihydro -1H C-C alkyl, C-C alkoxy, and mono- and di-(C-C)alky isoindol-5-yl)-1-thia-2.4a-diaza-cyclopentabnaphtha lamino. 25 lene-3,4-dione; 14. A pharmaceutical composition comprising a com 3-6-Fluoro-9-(2-fluoro-cyclopropyl)-3,4-dioxo-2,3-di pound or salt of claim 1 together with a pharmaceutically hydro-4H-1-thia-2.4a-diaza-cyclopentabnaphthalen acceptable carrier, diluent, or excipient. 7-yl)-benzonitrile; and 15. A compound or salt thereof of claim 1, wherein the 9-(2,4-Difluoro-phenyl)-6-fluoro-7-(1,2,3,4-tetrahydro compound is 30 isoquinolin-6-yl)-1-thia-2.4a-diaza-cyclopentabnaph 9-Cyclopropyl-6-fluoro-7-pyridin-4-yl-1-thia-2.4a-diaza thalene-3,4-dione. cyclopentabnaphthalene-3,4-dione: 16. A compound or salt of claim 13 in which R is a phenyl, 9-Cyclopropyl-6-fluoro-7-pyridin-3-yl-1-thia-2.4a-diaza pyridyl, pyrimidinyl, isoquinolinyl, quinolinyl, indolyl, pyr cyclopentabnaphthalene-3,4-dione; rolyl, isoindolyl, dihydroisoindolyl, tetrahydroisoquinolinyl, 9-Cyclopropyl-6-fluoro-7-(6-methyl-pyridin-3-yl)-1- 35 or tetrahydroisoquinolinyl group, each of which is Substituted thia-2.4a-diaza-cyclopentabnaphthalene-3,4-dione; with 0 to 3 substituents independently chosen from 9-Cyclopropyl-6-fluoro-7-(1,2,3,4-tetrahydro-isoquino halogen, hydroxy, amino, cyano, C-C alkyl, lin-6-yl)-1-thia-2.4a-diaza-cyclopentabnaphthalene C-Calkoxy, mono- and di-(C1-C4)alkylamino, 3,4-dione; C-Chaloalkyl, and C-Chaloalkoxy. 9-Cyclopropyl-7-(2,6-dimethyl-pyridin-4-yl)-6-fluoro-1- 40 17. A compound or salt of claim 16 in which thia-2.4a-diaza-cyclopentabnaphthalene-3,4-dione; R is phenyl, pyridyl-3-yl or pyrid-4-yl, each of which is 9-Cyclopropyl-6-fluoro-7-(1-methyl-2,3-dihydro -1H substituted with 1 or 2 substituents independently cho isoindol-5-yl)-1-thia-2.4a-diaza-cyclopentabnaphtha Sen from fluoro, amino, hydroxy, cyano, and methyl. lene-3,4-dione; 18. A compound or salt of claim 13 in which 9-Cyclopropyl-6-fluoro-7-pyrimidin-5-yl-1-thia-2.4a 45 R is isoindol-5-yl, tetrahydroisoquinolin-5-yl, tetrahy diaza-cyclopentabnaphthalene-3,4-dione; droisoquinolin-6-yl, tetrahydroisoquinolin-7-yl, or tet 3-(9-Cyclopropyl-6-fluoro-3,4-dioxo-2,3-dihydro-4H-1- rahydroisoquinolin-8-yl, each of which is substituted thia-2.4a-diaza-cyclopentabnaphthalen-7-yl)-ben with 0 to 3 substituents independently chosen from Zonitrile; C-C alkyl. 9-Cyclopropyl-6-fluoro-7-(1H-indol-2-yl)-1-thia-2.4a 50 19. A compound or salt of claim 10 in which R is dihy diaza-cyclopentabnaphthalene-3,4-dione; droindolyl, dihydroisoindolyl, tetrahydroisoquionolinyl, or 9-Cyclopropyl-6-fluoro-7-(4-hydroxy-3-methoxy-phe tetrahydroquinolinyl, where R is substituted with 0 to 2 nyl)-1-thia-2.4a-diaza -cyclopentabnaphthalene-3,4- Substituents independently chosen from halogen, hydroxy, dione; amino, C-C alkyl, and C-Calkoxy. 7-(3-Amino-4-fluoro-phenyl)-9-cyclopropyl-6-fluoro-1- 55 thia-2.4a-diaza-cyclopentabnaphthalene-3,4-dione; k k k k k