NZ612918B2 - Solid forms of gyrase inhibitor (r)-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl]-7-(tetrahydrofuran-2-yl)-1h-benzimidazol-2-yl]urea - Google Patents

Abstract

The disclosure relates to crystalline forms of 8(R)-1-ethyl-3-[6-fluoro-5-[2-(1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl]-7-(tetrahydrofuran-2-yl)-1H-benzimidazol-2-yl]urea represented by formula (I) and hydrochloride and mesylate salts thereof, which inhibit bacterial enzymes gyrase and/or topoisomerase IV. Also disclosed are pharmaceutical compositions comprising said compound of formula (I) or its salts and their use for treating bacterial infection caused by Mycobacterium tuberculosis, Streptococcus pneumoniae, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus aureus, Clostridium difficile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerans, Chlamydophila pneumoniae, Chlamydia trachomatis, Haemophilus injluenzae, Streptococcus pyogenes or ?-haemolytic streptococci. ase IV. Also disclosed are pharmaceutical compositions comprising said compound of formula (I) or its salts and their use for treating bacterial infection caused by Mycobacterium tuberculosis, Streptococcus pneumoniae, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus aureus, Clostridium difficile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerans, Chlamydophila pneumoniae, Chlamydia trachomatis, Haemophilus injluenzae, Streptococcus pyogenes or ?-haemolytic streptococci.

Description

PCT/U82012/021280 SOLID FORMS or GYRASE INHIBITOR (R)ETHYL[6-FLUORO[2-(1- HYDROXY-l-METHYL-ETHYL)PYRIMIDINYL](TETRAHYDROFURAN—2- YL)-lH—BENZIMIDAZOLYL]UREA CROSS REFERENCE TO D APPLICATIONS This application claims the benefit under 35 U.S.C. § 119 of United States Provisional Patent Application serial number 61/433,169 filed January 14, 2011, the entire contents of which is incorporated herein by reference.

BACKGROUND OF THE APPLICATION Bacterial ance to antibiotics has long been recognized, and it is today considered to be a serious worldwide health problem. As a result of resistance, some ial infections are either difficult to treat with antibiotics or even untreatable. This problem has become especially serious with the recent development of multiple drug resistance in n strains of bacteria, such as Streptococcus niae (SP), Mycobacrerium tuberculosis, and Enterococcus. The appearance of vancomycin resistant emerococcus was particularly ng because vancomycin was ly the only effective antibiotic for treating this infection, and had been considered for many infections to be the drug of "last ". While many other drug-resistant bacteria do not cause life—threatening disease, such as enterococci, there is the fear that the genes which induce resistance might spread to more deadly organisms such as Staphylococcus aureus, where methicillin resistance is already prevalent (De Clerq, et al., Current Opinion in Anti-infective Investigational Drugs, 1999, 1, l; Levy, "The Challenge of Antibiotic Resistance", Scientific American, March, 1 998). {0003] Another concern is how quickly antibiotic resistance can spread For example, until the 1960's SP was universally sensitive to penicillin, and in 1987 only 0.02% of the SP strains in the US. were resistant. However, by 1995 it was ed that SP resistance to penicillin was about seven percent and as high as 30% in some parts of the US. (Lewis, FDA Consumer ne (September, 1995); Gershman in The Medical Reporter, I997). als, in particular, serve as centers for the formation and transmission of drug-resistant sms. Infections occurring in hospitals, known as nosocomial ions, are becoming an increasingly serious problem. Of the two million Americans infected in hospitals each year, more than half of these ions resist at least one antibiotic. The Center for Disease Control reported that in 1992, over 13,000 hospital patients died of bacterial infections that were resistant to antibiotic treatment (Lewis, "The Rise of Antibiotic- Resistant Infections", FDA Consumer ne, September 1995).

As a result of the need to combat drug-resistant ia and the increasing failure of the available drugs, there has been a resurgent interest in discovering new antibiotics. One attractive strategy for ping new antibiotics is to inhibit DNA gyrase and/or topoisomerase IV, bacterial enzymes necessary for DNA replication, and therefore, necessary for ial cell growth and division. Gyrase and/or topoisomerase IV activity are also associated with events in DNA transcription, repair and recombination.

Gyrase is one of the omerases, a group of enzymes which catalyze the onversion of topological isomers of DNA (see generally, Kornberg and Baker, DNA Replication, 2d Ed, Chapter 12, 1992, W. H. Freeman and Co.; Drlica, Molecular Microbiology, 1992, 6, 425; Drlica and Zhao, iology and Molecular Biology Reviews, 1997, 61, pp. 377-392). Gyrase itself controls DNA supercoiling and relieves topological stress that occurs when the DNA strands of a parental duplex are untwisted during the replication process. Gyrase also catalyzes the conversion of relaxed, closed circular duplex DNA to a negatively superhelical form which is more favorable for recombination. The mechanism of the supercoiling reaction involves the wrapping of gyrase around a region of the DNA, double strand breaking in that region passing a second region of the DNA through the break, and rejoining the broken s. Such a cleavage mechanism is characteristic of a type II topoisomerase, The supercoiling reaction is driven by the binding ofATP to gyrase.

The ATP is then yzed during the reaction. This ATP g and subsequent hydrolysis cause conformational changes in the DNA-bound gyrase that are necessary for its activity. It has also been found that the level of DNA supercoiling (or relaxation) is ent on the ATP/ADP ratio. In the absence of ATP, gyrase is only capable of relaxing supercoiled DNA.

Bacterial DNA gyrase is a 400 kilodalton protein tetramer consisting of two A (GyrA) and two B subunits (GyrB). Binding and cleavage of the DNA is associated with GyrA, whereas ATP is bound and hydrolyzed by the GyrB protein. GyrB consists of an PCT/U82012/021280 terminal domain which has the ATPase activity, and a carboxy—terminal domain which interacts with GyrA and DNA. By st, eukaryotic type II topoisomerases are homodimers that can relax negative and positive supercoils, but cannot introduce ve supercoils. Ideally, an antibiotic based on the inhibition of bacterial DNA gyrase and/or topoisomerase IV would be selective for this enzyme and be relatively inactive against the eukaryotic type II topoisomerases.

Topoisomerase IV ily es linked some dimers at the conclusion of DNA replication.

The widely—used quinolone antibiotics inhibit bacterial DNA gyrase (GyrA) and/or Topoisomerase IV (ParC). Examples of the quinolones include the early compounds such as nalidixic acid and oxolinic acid, as well as the later, more potent fluoroquinolones such as norfloxacin, oxacin, and trovafloxacin. These compounds bind to GyrA and/or ParC and stabilize the cleaved complex, thus inhibiting overall gyrase function, leading to cell death. The uinolones inhibit the catalytic subunits of gyrase (GyrA) and/or Topoisomerase IV (Par C) (see Drlica and Zhao, Microbiology and Molecular Biology Reviews, 1997, 61, 377~392). However, drug resistance has also been recognized as a problem for this class of compounds (WHO Report, "Use of Quinolones in Food Animals and Potential Impact on Human Health", 1998). With the quinolones, as with other s of antibiotics, bacteria exposed to earlier compounds often quickly develop cross-resistance to more potent compounds in the same class.

The associated ts responsible for supplying the energy necessary for tic turnover/resetting of the enzymes via ATP hydrolysis are GyrB (gyrase) and ParE (topoisomerase IV), respectively (see, Champoux, II, Annu. Rev. Biochem, 2001, 70, pp. 369-413). Compounds that target these same ATP binding sites in the GyrB and ParE subunits would be useful for treating various bacterial infections (see, Charifson et al., J.

Med. Chem, 2008, 51, pp. 5243-5263).

There are fewer known inhibitors that bind to GyrB. Examples include the coumarins, novobiocin and coumermycin A1, cyclothialidine, ne, and clerocidin. The ins have been shown to bind to GyrB very tightly. For example, novobiocin makes a network ofhydrogen bonds with the protein and several hydrophobic contacts. While novobiocin and ATP do appear to bind within the ATP binding site, there is minimal overlap in the bound orientation of the two compounds. The overlapping portions are the sugar unit of novobiocin and the ATP e (Maxwell, Trends in Microbiology, 1997, 5, 102).

PCT/U52012/021280 For coumarin-resistant bacteria, the most prevalent point mutation is at a surface arginine residue that binds to the carbonyl of the coumarin ring (Arg136 in E. coli GyrB), While s with this mutation show lower supercoiling and ATPase activity, they are also less sensitive to inhibition by coumarin drugs (Maxwell, Mol. iol, 1993, 9, 681).

Despite being potent inhibitors of gyrase supercoiling, the coumarins have not been widely used as antibiotics. They are generally not suitable due to their low permeability in bacteria, eukaryotic toxicity, and poor water lity (Maxwell, Trends in Microbiology, 1997, 5, 102). It would be desirable to have a new, effective GyrB and ParEinhibitor that overcomes these drawbacks and, preferably does not rely on binding to Arg136 for activity.

Such an inhibitor would be an tive antibiotic candidate, without a y of resistance problems that plague other classes of antibiotics.

As bacterial resistance to antibiotics has become an important public health problem, there is a continuing need to develop newer and more potent antibiotics. More particularly, there is a need for antibiotics that represent a new class of compounds not previously used to treat bacterial infection. Compounds that target the ATP binding sites in both the GyrB (gyrase) and ParE (topoisomerase IV) subunits would be useful for treating various bacterial infections. Such compounds would be particularly useful in treating nosocomial infections in hospitals where the ion and transmission of resistant bacteria are becoming increasingly prevalent.

SUMMARY OF THE ATION The present application relates to solid forms of ethyl[6—fluoro—5-[2-(1- hydroxy-l -methyl-ethyl)pyrimidin—5 -y1](tetrahydrofuran—2-yl)- l H-benzimidazol—Z—yl]urea (“the 6-fluoro benzimidazolyl urea compound”). In one embodiment, the present application provides solid Form I of the 6-fluoro benzimidazolyl urea compound, which is characterized by an X—ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 :b 0.2) when measured using Cu K0L radiation, ed from the group consisting of9.3,11.7,12.1,12.4,14.5, 15.9,16.3,16.6,18.5,19.4, 21.5, 22.3, 22.8, 23.8, 24.5, 25.7, 28.1, 28.4, 30.3, and 33.4, when the XPRD is collected from about 5 to about 38 s two theta (2 0). Solid Form I may also be terized by an X-ray powder diffraction pattern, as measured using Cu KCL radiation, substantially similar to Figure 1 and an endothermic peak having an onset temperature at about 318°C as measured by differential scanning calorimetry in which the temperature is scanned at about 10°C per minute. The present application also provides a method for preparing crystal Form I of the 6—fluoro benzimidazolyl urea compound by suspending a solid al of the free base in a solvent system comprising an l and an ether and isolating the solid.

Another embodiment of the application provides solid Form 11 of the hydrochloride salt of the o benzimidazolyl urea compound, characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 9 i 0.2) when measured using Cu K, radiation, selected from the group consisting of 6.7, 9.2, 16.7, 18.6, 19.5, 20.5, 25.6, and 27.5, when the XPRD is collected from about 5 to about 38 degrees 2 9. Solid Form 11 may also be characterized by an X-ray powder diffraction pattern, as measured using Cu K, ion, substantially similar to Figure 4 and by an endothermic peak having an onset temperature at about 252°C as measured by differential scanning calorimetry in which the temperature is scanned at about 10°C per minute. Solid Form II of the hydrochloride salt of the 6-fluoro idazolyl urea nd may be prepared by suspending a free base ofthe6-fluoro benzimidazolyl urea compound in an acidic solvent mixture comprising one or more ethereal solvents and water.

A further embodiment of the present application is an amorphous Form 111 of the 6-fluoro benzimidazolyl urea compound (free base), terized by an X-ray powder diffraction pattern (XPRD) using Cu Kat radiation, characterized by a broad halo with no nable diffraction peak. A further embodiment of the present application is a method for preparing an amorphous Form 111 of the 6—fluoro benzimidazolyl urea compound (free base) comprising lyophilizing, spray drying, drum drying, or pulse conversion drying a solution of the 6—fluoro benzimidazolyl urea compound.

Yet another embodiment of the t application is an amorphous Form IV of the mesylate salt of the 6-fluoro benzimidazolyl urea compound characterized by an X-ray powder diffraction pattern (XPRD) using Cu K, radiation, characterized by a broad halo with no discernable diffraction peak.

DESCRIPTION OF FIGURES Figure 1 shows an X-ray powder diffraction pattern of solid Form I of the 6-fluoro benzimidazolyl urea compound (free base) collected from about 5 to about 38 degrees 2 6.

Figure 2 shows a DSC (DifferentialScanning Calorimetry) thermogram of solid Form 1 of the o idazolyl urea compound (free base).

Figure 3 shows a TGA (thermal gravimetric analysis) thermogram of solid Form 1 of the 6-fluoro benzimidazolyl urea compound (free base).

PCT/USZOlZ/021280 Figure 4 shows an X-ray powder diffraction pattern of solid Form II of the hydrochloride salt of the 6-fluoro benzimidazolyl urea compound.

Figure 5 shows a DSC thermogram of solid Form II of the hydrochloride salt of the 6-fluoro idazolyl urea compound.

Figure 6 shows a TGA thermogram of solid Form 11 of the 6-fluoro benzimidazolyl urea compound.

Figure 7 is an X-ray powder diffraction n of an amorphous Form III of the 6- fluoro benzimidazolyl urea nd (free base).

Figure 8 shows a DSC thermogram of amorphous Form III of 6-fluoro benzimidazolyl urea (free base) exhibiting a small exotherm followed by three larger endotherms.

Figure 9 is an X-ray powder diffraction n of an amorphous Form IV of the mesylate salt of the 6-fluoro benzimidazolyl urea compound.

Figure 10 is a 1H-NMR spectrum of the mesylate salt of the 6-fluoro benzimidazolyl urea compound.

DETAILED DESCRIPTION The present application is directed to novel, substantially pure solid forms of (R)-l -ethyl~3-[6-fluoro-5 -[2-(1 ~hydroxy- l -methyl-ethyl)pyrimidin—5 —yl]-7~(tetrahydrofuran- 2-yl)-lH—benzimidazol-Z-yl]urea (“the 6-fluoro benzimidazolyl urea compound”).

The ors have discovered a free base crystalline form of the compound (Form I), a crystalline form of a pharmaceutically acceptable salt of the 6~fluoro benzimidazolyl urea compound (Form 11, corresponding to a hloride salt), an amorphous form of the free base (Form 111) as well as an amorphous form of the mesylate salt of the compound (Form IV).

Thus, one aspect of the present application is a novel solid Form I of the o benzimidazolyl urea compound (free base). In one aspect, the present application provides a process for preparing solid Form I of the 6-fluoro benzimidazolyl urea compound.

A substantially pure solid Form I of the 6—fluoro benzimidazolyl urea nd may be ed from ous or crystalline compound by contacting the compound with a solvent system comprising an alcohol and an ether and isolating the solid, The 6-fluoro benzimidazolyl urea compound may be contacted with the solvent either by saturating a solution of the 6-fluoro benzimidazolyl urea nd in the solvent at ambient temperature PCT/U52012/021280 and ng the mixture to stand for an extended period of time (for e, overnight).

Alternatively, the 6-fluoro benzimidazolyl urea compound may be dissolved in the solvent at elevated temperature, for example, at reflux, followed by cooling the solution to room ature or below and ing solid Form I.

In one embodiment of the process, a substantially pure solid Form I of the 6- fluoro benzimidazolyl urea compound may be prepared from amorphous or lline form of the compound by preparing a saturated solution of the compound in a suitable solvent at room temperature and isolating Form I which results. In practice this can be accomplished by ving a sufficient amount of the 6-fluoro benzimidazolyl urea compound in the solvent at elevated temperature (up to reflux) such that when the solution is allowed to cool to room temperature a saturated solution is obtained, from which Form I precipitates and can be ed In other embodiments, the 6—fluoro benzimidazolyl urea compound may be isolated from a reaction mixture by modifying the lity of the compound in the solvent. For example, removing some or all of the solvent or lowering the mixture ature may reduce the solubility of the 6-fluoro benzimidazolyl urea compound and solid Form I may precipitate. Alternatively, adding a second solvent to the mixture may precipitate solid Form I of the compound.

In one embodiment, the solvent for the preparation of Form I is a mixture of ethanol and ethyl ether. Isolation of the resulting solid provides Form I.

Solid Form I of the 6-fluoro benzimidazolyl urea compound may be fied by the ing characteristics: a broad endotherm at about 250°C, a melt endotherm with an extrapolated onset of about 318°C as ined by differential scanning calorimetry using °C per minute scan rate; and an X-ray powder diffraction pattern essentially as shown in Table l and Figure 1 wherein the XRPD patterns were measured using a powder diffractometer equipped with a Cu X-ray tube source. The sample was illuminated with Cu KOtl radiation and XRPD data were collected from about 5 to about 40° 20. A person skilled in the art would recognize that relative intensities of the XPRD peaks may significantly vary depending on the orientation of the sample under test and on the type and setting of the instrument used, so that the intensities in the XPRD traces included herein are to such extent illustrative and are not intended to be used for absolute comparisons.

Figure 1 is an X-ray powder diffraction n of solid Form I of 6-fluoro benzimidazolyl urea compound (free base) collected from about 5 to about 40 degrees 2 0.

The peaks corresponding to the X—ray powder diffraction pattern having a relative intensity greater than or equal to 5% are listed in Table 1.

Figure 2 shows a DSC thermogram of solid Form 1 of the 6-fluoro benzimidazolyl urea compound exhibiting a broad endotherm with an onset transition at about 250°C and an endotherrn with an onset transition at about 318°C. A person skilled in the art would recognize that the peak and onset temperatures of the endotherms may vary depending on the experimental conditions. Data in Figure 2 were ted equilibrating a 2.5 mg sample of the solid at about 35°C for about 10 minutes. During the data collection period, the temperature was increased at a rate of about 10°C per .

Figure 3 is a TGA (thermal gravimetric analysis) thermogram of solid Form Iofthe 6—fluor0 idazolyl urea compound exhibiting an initial weight loss of about 15% t in the 50 to 300°C temperature range with additional weight loss of about 25% between 300 and 400°C.

In one embodiment, the present invention provides a solid Form I of the compound of formula (I): (I).

In another embodiment, the solid Form I is characterized by an X—ray powder diffraction n (XPRD) comprising at least three approximate peak positions (degrees 2 0 i 0.2) when ed using Cu K, radiation, selected from the group ting of 9.3,11.?,12.1,12.4, 14.5,15.9,16.3,16.6, 185,194, 21.5, 22.3, 22.8, 23.8, 24.5, 25.7, 28.1, 28.4, 30.3, and 33.4, when the XPRD is collected from about 5 to about 38 degrees 2 0.

In another embodiment, the solid Form I is characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 PCT/U52012/021280 i 0.2) when measured using Cu K“ radiation, selected from the group consisting of 9.3, 16.6, 18.5, 19.4, 21.5, and 25.7, when the XPRD is collected from about 5 to about 38 degrees 2 9.

In another embodiment, the solid Form I is terized by an X-ray powder diffraction pattern, as measured using Cu Ka radiation, substantially similar to Figure l.

In another embodiment, the solid Form 1 is r characterized by an endothermic peak having an onset temperature at about 318°C as measured by differential ng calorimetry in which the temperature is scanned at about 10°C per minute.

In another embodiment, the t invention provides a method for preparing crystal Form I of the compound of formula (1) comprising suspending a solid material of the free base in solvent system comprising an alcohol and an ether and isolating the solid.

In another embodiment, the solid Form I is stable for at least one month at 40°C with relative humidity of up to 75%.

Table 1. XRPD pattern peaks for solid Form I of the 6—fluoro benzimidazolyl urea Peak No. Position Relative Intensity °20 0/0 l 9.29 66 2 11.74 14 3 12.13 14 4 12.37 15 13.71 5 6 14.18 6 7 14.54 t 19 8 15.90 23 9 16.32 24 16.59 100 11 18.49 92 12 19.43 87 13 19.94 ‘ 9 14 20.36 6 21.53 81 16 22.34 10 17 22.80 19 18 23.50 i 8 19 23.75 13 24.45 28 21 25.09 6 22 25.67 58 23 26.39 5 24 26.69 6 W0 2012l097273 PCT/U82012/021280 Peak No. on Relative Intensity °29 % 27.52 8 26 28.05 25 27 28.43 18 28 30.04 6 29 30.31 10 31 33.40 14 32 34.07 t 6 33 35.22 5 34 37.27I 5 In another aspect, the t application provides crystal Form II of the hydrochloric acid addition salt of the 6-fluoro benzimidazolyl urea compound. In one embodiment, the present application provides a process for preparing solid Form II of the 6-- fluoro idazolyl urea compound. The pharmaceutically acceptable hydrochloric acid addition salt of the 6-fluoro benzimidazolyl urea compound may be prepared by any method known to those skilled in the art.

In some embodiments, the hydrochloric acid addition salt of the 6-fluoro benzimidazolyl urea compound may precipitate out upon ion from on of an acid to a solution of the compound. In other embodiments, the acid addition salt may be isolated from the reaction mixture by modifying the solubility of the salt in the solvent. For example, removing some or all of the solvent or lowering the mixture temperature may reduce the solubility of the hydrochloride salt of the 6-fluoro benzimidazolyl urea compound and the salt precipitate, Alternatively, adding a second solvent to the mixture may precipitate the salt.

In further embodiments, gaseous hydrochloric acid may be bubbled through a solution of the o benzimidazolyl urea compound until a mono acid addition salt of the compound is prepared. In n embodiments, stoichiometric amounts of hloric acid and the 6-fluoro benzimidazolyl urea compound may be mixed together to form a mono acid on salt of the compound. For e, a solution of the 6—fluoro benzimidazolyl urea compound in a polar solvent may be mixed with a stoichiometric amount of an aqueous solution of hydrochloric acid. Examples of polar solvents that may be suitable for preparing the solid Form 11 of hydrochloride salt of 6-fluoro benzimidazolyl urea compound include ethers such as diethyl ether and tetrahydrofuran (THF).

In a particular embodiment, stoichiometric amounts of the 6-fluoro idazolyl urea compound in TI-IF and aqueous hydrochloric acid were mixed slowly and the mixture was stirred at room temperature ght. A solid white hydrochloride salt PCT/U82012/021280 of the 6-fluoro benzimidazolyl urea compound precipitated out. The solid was isolated: washed with water and dried under vacuum.

Solid Form 11 of the 6-fluoro benzimidazolyl urea compound may be identified by the ing characteristics: a broad endotherm with a peak temperature of about 210°C, a melt erm with an extrapolated onset of about 252°C as determined by ential scanning calorimetry using 10°C per minute scan rate; and an X-ray powder diffraction pattern essentially as shown in Table 2 and Figure 4 wherein the XRPD ns were measured using a powder diffractometer equipped with a Cu X—ray tube source The sample was illuminated with Cu Kal ion and XRPD data were collected from about 5 to about 40° 20‘ A person skilled in the art would recognize that relative intensities of the XPRD peaks may significantly vary ing on sample orientation.

Figure 4 is an X-ray powder diffraction pattern of solid Form 11 of the hydrochloride salt of the 6-fluoro benzimidazolyl urea compound collected from about 5 to about 38 degrees 2 6. The peaks ponding to X—ray powder diffraction pattern having a relative intensity greater than or equal to 5% are listed in Table 2.

Figure 5 shows a DSC thermogram of solid Form 11 of the hydrochloride salt of the 6-fluoro benzimidazolyl urea compound exhibiting an endotherm at about 210°C and an endotherm at about 252°C. A person skilled in the art would recognize that the peak and onset temperatures of the endotherms may vary depending on the experimental conditions.

Data in Figure 5 were ted equilibrating a 1 mg sample of the solid at about 35°C for about 10 minutes. During the data collection period, the temperature was increased at a rate of about 10°C per minute.

Figure 6 is a TGA thermogram of solid Form 11 of the ro benzimidazolyl urea compound exhibitng an initial weight loss of about 8% percent between 100 and 220°C followed by a second weight loss of about an additional 8% at between about 240 and 270°C followed by a third weight loss of about 3% between 270 and 300°C. A person skilled in the art would recognize that the onset temperatures of the weight loss may vary depending on the experimental conditions. While applicants do not wish to be held to a particular explanation of the endotherm in the DSC and weight loss in the TGA, it appears that the transition with large peak in the DSC is due to a melting transition coupled with ation of the material as suggested by the weight loss in the TGA.

In one embodiment, the present invention provides a hydrochloric acid salt of the nd of formula (I): 2012/021280 (I).

In another embodiment, the hydrochloric acid salt is Form 11 solid form.

In another embodiment, the hydrochloric acid salt of Form 11 solid form is terized by an X—ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 i 0.2) when measured using Cu K0L radiation, selected from the group consisting of 6.7, 9.2, 16.7, 18.6, 19.5, 20.5, 25.6, and 27.5, when the XPRD is collected from about 5 to about 38 degrees 2 9.

In another embodiment, the hydrochloric acid salt of Form 11 solid form is characterized by an X-ray powder diffraction pattern, as ed using Cu Ka radiation, substantially similar to Figure 4.

In another embodiment, the hydrochloric acid salt of Form 11 solid form is r characterized by an endothermic peak having an onset temperature at about 252°C as measured by differential scanning calorimetry in which the temperature is scanned at about °C per minute.

In yet another embodiment, the present invention provides a method for preparing solid Form 11 of the hydrochloride salt of the compound of formula (1) comprising suspending a free base of the o benzimidazolyl urea compound in an acidic solvent mixture comprising one or more ethereal solvents and water.

In another embodiment, the hloric acid salt of Form II solid form is stable for at least one month at 40°C with relative humidity of up to 75%. 2012/021280 Table 2. XRPD pattern peaks for solid Form 11 of the o benzimidazolyl urea compound Peak on Relative No. [°2 9] Intensity [%] 1 6.67 13 2 9.25 33 3 11.64 7 4 13.36 7 15.90 7 6 16.69 17 7 18.59 12 8 18.81 7 9 19.51 14 20.48 100 11 22.59 7 12 24.57 5 i 13 25.61 11 14 27.54 16 Another aspect of the present application is ing a composition comprising an amorphous 6-fluoro benzimidazolyl urea compound (free base). The term "amorphous" as applied herein to 6-fluoro benzimidazolyl urea compound or its salts refers to a solid state form wherein the 6-fluoro benzimidazolyl urea les are generally present in a disordered arrangement and do not form a guishable crystal lattice or unit cell. When subjected to X-ray powder diffraction, a tely amorphous compound does not produce a diffraction pattern characteristic of a crystalline form. The X—ray powder diffraction of a partially amorphous material may still lack features characteristic of a crystal form because the diffraction peaks from the crystalline portion of the sample may be too weak to be observable over the noise. Figure 7 is an X—ray powder diffraction pattern of an amorphous form III of the o benzimidazolyl urea compound (free base).

Figure 8 shows a DSC therrnogram of amorphous Form III of 6-fluoro benzimidazolyl urea (free base) exhibiting a small exotherm followed by three larger endotherms. The small exotherm has an onset temperature of 127°C whereas the three endotherms have onset temperatures of 183°C, 226°C, and 279°C. A person skilled in the art would recognize that the peak and onset temperatures of the exotherm and the endotherms may vary depending on the experimental conditions. Data in Figure 8 were collected equilibrating a 2.9 mg sample of the amorphous 6-fluoro benzimidazolyl urea compound at about 35°C for about 10 minutes. During the data collection , the temperature was increased at a rate of about 10°C per minute. 2012/021280 In another embodiment, the t invention provides an amorphous Form III of the fluoro benzimidazolyl urea compound of formula I: (I).

In another embodiment, the amorphous Form III of the fluoro idazolyl urea compound is characterized by an X-ray powder diffraction pattern (XPRD) using Cu K, radiation, characterized by a broad halo with no discernable diffraction peak.

In yet another embodiment, the present inventionprovides a method for preparing ous Form III of the 6-fluoro benzimidazolyl urea compoundcomprising lyophilizing, spray , drum drying, or pulse conversion drying a solution of the 6—fluoro benzimidazolyl urea compound.

In another aspect, the present application provides an amorphous solid phase Form IV of the mesylate salt of the o benzimidazolyl urea nd. In one embodiment, the present application provides a process for preparing solid Form IV of the mesylate salt of the 6-fluoro benzimidazolyl urea compound. A pharmaceutically acceptable methanesulphonic acid salt of the 6—fluoro idazolyl urea compound may be prepared by any method known to those skilled in the art. For example, a solution of methanesulphonic acid may be added to a solution of the 6—fluoro benzimidazolyl urea compound until a mono acid on salt of the compound is prepared. In one ment, the mesylate salt of the 6-fluoro benzimidazolyl urea compound may precipitate out upon addition of the acid to a solution of the 6-fluoro benzimidazolyl urea compound. In other embodiments, the acid addition salt may be isolated from the reaction mixture by modifying the solubility of the salt in the solvent. For example, removing some or all of the solvent or lowering the mixture temperature may reduce the solubility of the mesylate salt of the 6- fluoro benzimidazolyl urea compound and the salt precipitate. Thus, in some embodiments, the amorphous material is collected after being precipitated from a solvent or from a solution after concentrating the solution by evaporating some of the solvent, for example, using a rotator evaporator. Altematively, adding a second t to the mixture may precipitate the salt.

The te salt of the o benzimidazolyl urea compound may be converted to an amorphous solid form using any method known to those skilled in the art.

The amorphous o benzimidazolyl urea compound mesylate salt may be characterized by the absence of a diffraction pattern characteristic of a crystalline form. The X—ray powder diffraction of a partially amorphous 6-fluoro benzimidazolyl urea compound mesylate salt may still lack features characteristic of a crystal form e the diffraction peaks from the lline portion of the sample may be too weak to be observable over the noise. Figure 9 is an X—ray powder ction pattern of an amorphous Form IV of the mesylate salt of the 6— fluoro benzimidazolyl urea compound.

In one embodiment, the ous mesylate salt of the 6—fluoro benzimidazolyl urea compound may be prepared by spray drying a solution of the salt in appropriate solvent.

Spray drying is well known in the art and is often used to dry thermally-sensitive materials such as ceutical drugs. Spray drying also provides consistent particle distribution that can be reproduced fairly well. Any gas may be used to dry the powder gh air is commonly used. If the material is sensitive to air, an inert gas, such nitrogen or argon, may be used. Any method that converts a solution, slurry, suspension or an emulsion of the salt to produce a solid powder may be suitable for ing the solid amorphous Form IV of the mesylate salt of the 6-fluoro benzimidazolyl urea compound. For example, freeze drying, drum drying, or pulse conversion drying may be used to produce an amorphous mesylate salt of the 6-fluoro benzimidazolyl urea compound.

In one embodiment, a solution of the 6-fluoro benzimidazolyl urea nd in a polar solvent may be spray dried using a nanospray dryer equipped a condenser. The inlet temperature may be kept between °C.

In another embodiment, the present invention provides an amorphous Form IV of the mesylate salt of the 6-fluoro benzimidazolyl urea compound of formula I: WO 97273 PCT/U82012/021280 (I).

In another ment, the amorphous Form IV of the mesyl ate salt of the 6- fluorobenzimidazolyl urea compound is characterized by an X—ray powder diffraction pattern (XPRD) using Cu Ka radiation, characterized by a broad halo with no discemable diffraction peak It is to be understood that solid Forms I and II and ous solid Forms 111 and IV of, respectively, free base and mesylate salt of the 6-fluoro benzimidazolyl urea compound, in addition to having the XRPD, DSC, TGA and other characteristics described herein, may also possess other characteristics not described, such as but not limited to the presence of water or one or more solvent molecules.

X—Ray Powder Diffraction (XRPD): The XRPD pattern of the crystalline forms were recorded at room temperature in reflection mode using a Bruker D8 Discover system equipped with a sealed tube source and a Hi—Star area or (Bruker AXS, Madison, WI).

The X—Ray generator was operating at a tension of 40 kV and a current of 35 mA. The powder sample was placed on a Si zero-background wafer. Two frames were registered with an exposure time of 120 5 each. The data were subsequently integrated over the range of 3°— 41° 2 with a step size of 002° and merged into one continuous n.

X—Ray Powder Diffraction (XRPD) for amorphous forms: The XRPD pattern of the amorphous solid form was recorded at room temperature in reflection mode using Bruker D8 Advance system equipped with -l position sensitive detector (Bruker AXS, n, WI). The X—Ray generator was operating at a tension of 40 kV and a t of 45 mA. The powder sample was placed on a Si zero-background holder, spinning at 15 rpm during the experiment in a continuous mode using variable slit at the detector. Data was collected from 3 to 40 degrees with 0.0144653 degree increments (0.255/step). -l6- PCT/U82012/021280 Differential Scanning Calorimetry (DSC): DSC was med on a sample of the material using a DSC Q2000 differential scanning calorimeter (TA Instruments, New Castle, DE). The instrument was calibrated with indium. A sample of approximately 1-2 mg was weighed into an um pan that was crimped using lids with either no pin-hole or pin-hole lids. The DSC samples were scanned from 30°C to temperatures indicated in the plots at a heating rate of 10°C/min with 50 mL/min nitrogen flow. The samples run under modulated DSC (MDSC) were modulated + and — 1°C every 605 with ramp rates of 2 or 3 °C/min.

Data was ted by l age Q SeriesTM software and ed by Universal Analysis 2000 software (TA Instruments, New Castle, DE).

Thermogravimetric is (TGA): A Model Q5000 Thermogravimetric Analyzer (TA Instruments, New Castle, DE) was used for TGA measurement, A sample with weight of approximately 3-5 mg was scanned from 30°C to temperatures indicated on the plots at a heating rate of 10°C/min. Data was collected by Thermal Advantage Q TM software and analyzed by Universal is 2000 software (TA Instruments, New Castle, DE).

The present invention also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

The present invention also provides a method of controlling, treating or reducing the advancement, severity or effects of a nosocomial or a non—nosocomial bacterial infection in a patient, comprising administering to said patient a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt f.

In another embodiment, the present invention provides a method of controlling, treating or ng the advancement, ty or s of a nosocomial or a non— nosocomial bacterial infection in a t, comprising administering to said patient a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein the bacterial ion is characterized by the presence of one or more of Streptococcus pneumoniae, Staphylococcus epidermidz‘s, Emerococcusfizecalis, Staphylococcus aw'eus, Clostrz'dium diflz‘cz’le, Moraxella catarrhalis, Neisserz‘a gonorrhoeae, Neisseria meningitidis, Mycobacz‘erz’um avium complex, Mlcobacterium abscessus, W0 20121’097273 PCT/USZOlZ/021280 Mycobacterium kansasz‘z', Mycobacterz’um ulcerans, Chlamydophila pneumoniae, Chlamydia D'achomatis, Haemophz‘lus influenzae, Streptococcus pyogenes or B-haemolytic streptococci.

In another embodiment, the t invention provides a method of controlling, treating or reducing the advancement, severity or effects of a mial or a non- nosocomia] ial infection in a patient, comprising administering to said patient a pharmaceutical composition comprising a nd of formula (I), or a pharmaceutically acceptable salt thereof, wherein the bacterial infectionis ed from one or more of the following: upper respiratory infections, lower respiratory infections, ear infections, pleuropulmonary and bronchial infections, complicated urinary tract infections, uncomplicated urinary tract infections, intra—abdominal infections, cardiovascular infections, a blood stream infection, sepsis, bacteremia, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital infections, eye infections, or granulomatous infections, uncomplicated skin and skin structure infections (uSSSI), complicated skin and skin structure infections (cSSSI), catheter infections, pharyngitis, sinusitis, otitis externa, otitis media, itis, empyema, pneumonia, community-acquired bacterial pneumoniae (CABP), hospital-acquired pneumonia (HAP), hospital-acquired bacterial pneumonia, ventilator-associated pneumonia (VAP), diabetic foot infections, vancomycin resistant cocci infections, cystitis and pyelonephritis, renal calculi, prostatitis, peritonitis, complicated intra—abdominal infections (cIAI) and other inter-abdominal infections, dialysis- associated peritonitis, visceral abscesses, endocarditis, myocarditis, pericarditis, transfusion- associated sepsis, meningitis, encephalitis, brain abscess, yelitis, arthritis, genital ulcers, itis, vaginitis, cervicitis, gingivitis, ctivitis, keratitis, endophthalmitisa, an infection in cystic fibrosis ts or an infection of febrile neutropenic patients.

In another ment, the bacterial infection is selected from one or more of the following: community—acquired bacterial pneumoniae (CABP), hospital-acquired pneumonia (HAP), hospital-acquired bacterial nia, ventilator-associated pneumonia (VAP), bacteremia, diabetic foot infections, catheter infections, uncomplicated skin and skin structure infections ), cated skin and skin structure infections ), vancomycin resistant enterococci infections or osteomyelitis.

According to another embodiment, the ion provides a method of decreasing or inhibiting bacterial quantity in a biological . This method comprises contacting said biological sample with a compound of formula (I) or a pharmaceutically acceptable salt thereof PCT/U82012/021280 The term "biological ", as used herein, includes cell cultures or extracts f; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof. The term "biological sample" also includes living organisms, in which case "contacting a compound of this invention with a biological sample" is synonymous with the term "administering said nd or composition comprising said compound) to a mammal".

The gyrase and/or topoisomerase IV inhibitors of this invention, or pharmaceutical salts thereof, may be formulated into pharmaceutical compositions for administration to animals or . These pharmaceutical compositions effective to treat or prevent a bacterial ion which se the gyrase and/or omerase IV inhibitor in an amount sufficient to measurably decrease bacterial quantity and a pharmaceutically acceptable carrier, are another embodiment of the present invention. The term "measurably decrease bacterial quantity", as used herein means a measurable change in the number of bacteria n a sample containing said inhibitor and a sample containing only bacteria.

According to r embodiment, the methods of the present invention are useful to treat patients in the narian field including, but not limited to, zoo, laboratory, human companion, and farm animals including primates, s, reptiles and birds. Examples of said animals include, but are not limited to, guinea pigs, hamsters, gerbils, rat, mice, rabbits, dogs, cats, , pigs, sheep, cows, goats, deer, rhesus monkeys, monkeys, tamarinds, apes, baboons, gorillas, chimpanzees, orangutans, gibbons, ostriches, chickens, turkeys, ducks, and geese.

The term "non-nosocomial infections" is also referred to as community acquired infections.

In another embodiment, the bacterial infection is characterized by the presence of one or more ofStreptococcus pneumoniae, Emerococcusfaecalz‘s, or Staphylococcus aureus.

In another embodiment, the bacterial infection is characterized by the presence of one or more ofE. coli, Moraxella catarrhalis, or Haemophz'lus influenzae.

In another embodiment, the bacterial infection is characterized by the presence of one or more of CZostrz'dz'um dzfiz‘cz'le, rz'a gonorrhoeae, Neisseria itidis, Mycobacrerium avium complex, Alycobacterz'um abscessus, cterz‘um kansasz‘z‘, Mycobacterium ulcerans, Chlamydophz‘la pneumoniae and Chlamydia tracoman‘s.

In another embodiment, the bacterial infection is characterized by the presence of one or more ptococcus pneumoniae, Staphylococcus epidermidis, Enterococcus PCT/U52012/021280 faecalis, Staphylococcus aureus, Clostridium difficile, Moraxella catarrhat‘t's, ria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterz‘um sus, Mzcobacterium kansasii, ll/chobacterium ulcerans, Chlamydopht‘la pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Streptococcus pyogenes orfl-haemolytic streptococci.

In some embodiments, the bacterial infection is characterized by the presence of one or more of Methicillin ant Staphylococcus aureus, Fluoroquinolone resistant Staphylococcus aureus, ycin intermediate resistant Staphylococcus aureus, Linezolid resistant lococcus aureus, Penicillin resistant Streptococcus pneumoniae, Macrolide resistant Streptococcus pneumoniae, Fluoroquinolone resistant Streptococcus pneumoniae, Vancomycin resistant Enterococcusfaecalis, Linezolid resistant coccusfitecalis, Fluoroquinolone resistant Enterococcusfaecalis, Vancomycin resistant Enterococcus faecium, Linezolid resistant coccusfaecium, Fluoroquinolone resistant Enterococcus faecium, Ampicillin resistant Enterococcusfaecium, Macrolide resistant hilus influenzae, B-lactam resistant Haemophilus influenzae, Fluoroquinolone resistant Haemophtlus influenzae, B-lactam ant lla catarrhalis, Methicillin antStaphylococcus epia’ermidis, Methicillin ant Staphylococcus epidermidis, Vancomycin resistant Staphylococcus epidermidis, Fluoroquinolone resistant Staphylococcus epidermidis, Macrolide resistant Mycoplasmapneumoniae, lsoniazid resistant Mycobacterium tuberculosis, Rifampin resistant Mycobacterium tuberculosis, Methicillin resistant Coagulase negative lococcus, Fluoroquinolone resistant Coagulase negative staphylococcus, Glycopeptide intermediate ant Staphylococcus aureus, Vancomycin resistant Staphylococcus aureus, Hetero vancomycin intermediate resistant Staphylococcus aureus, Hetero vancornycin resistant Staphylococcus aureus, Macrolide—Lincosamide— Streptograrnin resistant Staphylococcus, B—lactam resistant Enterococcus faecalis, am resistant Enterococcusfaecium, de ant Streptococcus niae, Ketolide resistant Streptococcus pyogenes, Macrolide resistant Streptococcus pyogenes, Vancomycin resistant staphylococcus epidermidis, Fluoroquinolone resistant Neisseria gonorrhoeae, Multidrug ant Pseudomonas aeruginosa or Cephalosporin resistant Neisseria hoeae.

According to another embodiment, the Methicillin resistant Staphylococci are selected from Methicillin resistant Staphylococcus aureus, Methicillin resistant Staphylococcus epidermiclis, or Methicillin resistant Coagulase negative staphylococcus.

W0 20121097273 PCT/U52012/021280 In some embodiments, a form of a compound of formula (I), or a pharmaceutically acceptable salt thereof, is used to treat community acquired MRSA (i.e., In other embodiments, a form of a compound of formula (I), or a pharmaceutically acceptable salt thereof, is used to treat daptomycin ant sm including, but not limited to, Daptomycin resistant Enterococcusfaecium and Daptomycin resistant Staphylococcus .

According to another embodiment, the Fluoroquinolone resistant Staphylococci are selected from Fluoroquinolone resistant Staphylococcus aureus, Fluoroquinolone resistant Staphylococcus epidermidis, or Fluoroquinolone resistant Coagulase negative staphylococcus.

According to another embodiment, the Glycopeptide resistant Staphylococci are selected from Glycopeptide intermediate resistant lococcus aureus, ycin resistant Staphylococcus , Vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin ediate resistant Staphylococcus aureus, or Hetero vancomycin ant Staphylococcus aureus.

According to another embodiment, the Macrolide-Lincosamide-Streptogramin resistant Staphylococci is Macrolide-Lincosamide-Streptogramin resistant Staphylococcus aureus.

According to another embodiment, the Linezolid resistant Enterococci are selected from Linezolid resistant Enterococcusfaecalz‘s, or Linezolid resistant Enterococcusfaecium.

According to another ment, the Glycopeptide resistant Enterococci are selected from Vancomycin resistant Enterococcusfaecz’um or Vancomycin resistant Enterococcusfizecalz‘s.

According to another embodiment, the B-lactam ant Enterococcus faecalis is B-lactam resistant Enterococcusfaectum.

According to another embodiment, the Penicillin resistant Streptococci is Penicillin ant Streptococcus pneumoniae.

According to another embodiment, the Macrolide resistant ococci is Macrolide ant Streptococcus pneumonia.

According to another embodiment, the de resistant Streptococci are selected from Macrolide resistant Streptococcus pneumoniae and Ketolide resistant Streptococcus pyogenes.

PCT/U82012/021280 ] According to another embodiment, the Fluoroquinolone resistant Streptococci is Fluoroquinolone ant Streptococcus pneumoniae.

According to another embodiment, the B-lactam resistant Haemophilus is B-lactam resistant Haemophilus tnfluenzae.

] According to another embodiment, the Fluoroquinolone ant Haemophilus is Fluoroquinolone resistant Haemophilus influenzae. ing to another embodiment, the Macrolide resistant Haemophilus is Macrolide resistant Haemophilus influenzae.

According to another embodiment, the Macrolide resistant Mycoplasma is Macrolide resistant Mycoplasma pneumoniae.

According to another ment, the Isoniazid resistant Mycobacterium is Isoniazid resistant cterium tuberculosis.

] According to another embodiment, the Rifampin resistant Mycobacterium is Rifampin resistant Mycobacterium tuberculosis.

According to another embodiment, the B-lactam resistant Moraxella is B-lactam resistant Moraxella catarrhalz’s.

According to r embodiment, the bacterial infection is characterized by the presence of one or more of the following: Methicillin ant lococcus aureus, Fluoroquinolone resistant Staphylococcus aureus, Vancomycin ediate resistant Staphylococcus aureus, Linezolid resistant Staphylococcus aureus, Penicillin resistant Streptococcus niae, Macrolide resistant Streptococcus pneumoniae, Fluoroquinolone ant Streptococcus pneumoniae, Vancomycin resistant Enterococcusfaecalis, Linezolid resistant Enterococcusfaecalt’s, Fluoroquinolone resistant coccusfitecalis, Vancomycin resistant Enterococcusfaecium, Linezolid resistant Enterococcusfizecz‘um, Fluoroquinolone resistant Enterococcus faecium, Ampicillin resistant Enterococcusfaecz’um, Macrolide resistant Haemophilus influenzae, am resistant Haemophilus z'nfluenzae, quinolone resistant Haemophilus influenzae, am resistant Moraxella catarrhalts, Methicillin resistant Staphylococcus epidermidis, Methicillin resistant Staphylococcus epidermidis, Vancomycin resistant Staphylococcus epidermidis, Fluoroquinolone resistant lococcus epidermidis, Macrolide resistant A/Iwoplasma pneumonz'ae, Isoniazid ant Mycobacterz’um tuberculosis, Rifampin resistant Mycobacterium tuberculosis, Fluoroquinolone resistant Neisserz‘a gonorrhoeae or Cephalosporin resistant Neisserz’a gonorrhoeae. 2012/021280 ] According to another embodiment, the bacterial infection is characterized by the ce of one or more of the following: Methicillin resistant lococcus aureus, Methicillin resistant Staphylococcus epidermidz‘s, Methicillin ant Coagulase negative staphylococcus, Fluoroquinolone resistant Staphylococcus aureus, Fluoroquinolone resistant Staphylococcus epidermidis, Fluoroquinolone resistant Coagulase negative lococcus, Vancomycin resistant Staphylococcus aureus, Glycopeptide intermediate resistant Staphylococcus aureus, Vancomycin resistant Staphylococcus aureus, Vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin intermediate resistant Staphylococcus , Hetero vancomycin resistant lococcus aureus, Vancomycin resistant Enterococcusfaecium, Vancomycin resistant Enterococcus faecalis, llin resistant Streptococcus pneumoniae, Macrolide ant Streptococcus pneumoniae, Fluoroquinolone resistant Streptococcus pneumoniae, Macrolide resistant Streptococcus pyogenes, or B-lactam resistant Haemophz’lus tnfluenzae. ing to another embodiment, the bacterial infection is characterized by the presence of one or more of the ing: Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcusfaecium, Vancomycin resistant coccusfizecalz‘s, Vancomycin resistant Staphylococcus aureus, Vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin intermediate ant Staphylococcus aureus, Hetero vancomycin resistant Staphylococcus aureus, Multidrug ant monas rtosa, Isoniazid resistant Mycobacterz’um tuberculosis, and Rifampin resistant Mycobactertum tuberculosis.

Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases.

Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, carnphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, oayethanesulfonate, lactate, e, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, ate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts W0 2012l097273 PCT/U82012/021280 useful as intermediates in obtaining the compounds of the ion and their pharmaceutically acceptable acid on salts.

Salts derived from appropriate bases include alkali metal (eg, sodium and potassium), alkaline earth metal (e.g, magnesium), ammonium and N+(C1-4 4 salts, This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil—soluble or dispersible products may be obtained by such quatemization.

Pharmaceutical compositions of this invention comprise a compound of formula (I) or a pharmaceutically acceptable salt thereof and a ceutically acceptable carrier.

Such compositions may optionally comprise an additional therapeutic agent. Such agents include, but are not limited to, an antibiotic, an anti-inflammatory agent, a matrix metalloprotease tor, a genase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a growth factor, an immunomodulator, a prostaglandin or an anti-vascular roliferation compound.

The term "pharmaceutically acceptable r" refers to a non-toxic carrier that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.

Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum n, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, nyl pyrrolidone, cellulose-based substances, polyethylene , sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, wool fat and mulsifying drug ry systems (SEDDS) such as alpha—tocopherol, polyethyleneglycol 1000 ate, or other similar polymeric delivery matrices.

The term "pharmaceutically effective amount" refers to an amount effective in ng or ameliorating a bacterial infection in a patient. The term "prophylactically effective amount" refers to an amount effective in preventing or ntially lessening a bacterial infection in a patient.

PCT/U52012/021280 Depending upon the particular condition, or disease state, to be treated or ted, additional therapeutic agents, which are normally administered to treat or prevent that condition, may be administered together with the tors of this invention. Such therapeutic agents include, but are not limited to, an antibiotic, an anti—inflammatory agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a growth factor, an modulator, a prostaglandin or an anti—vascular hyperproliferation compound.

] The compounds of this invention may be employed in a tional manner for controlling ial infections levels in vivo and for treating diseases or ng the ement or severity of effects which are mediated by bacteria. Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary skill in the art from available methods and techniques.

For example, a nd of this invention may be combined with a pharmaceutically acceptable adj uvant for administration to a t suffering from a bacterial infection or disease in a pharmaceutically acceptable manner and in an amount ive to lessen the severity of that infection or disease.

Alternatively, the compounds of this invention may be used in compositions and s for treating or protecting individuals against bacterial infections or diseases over extended periods of time. In one ment, the compounds of this invention may be used in compositions and methods for treating or protecting individuals against bacterial infections or diseases over a 1—2 week period. In another embodiment, the compounds of this invention may be used in compositions and methods for treating or protecting individuals against bacterial infections or diseases over a 4—8 week period (for example, in the treatment of patients with or at risk for developing endocarditis or osteomyelitis). In another embodiment, the compounds of this invention may be used in compositions and methods for treating or protecting individuals against bacterial infections or diseases over an 8-12 week period. The compounds may be employed in such compositions either alone or together with other compounds of this invention in a manner consistent with the conventional utilization of enzyme inhibitors in pharmaceutical compositions. For example, a nd of this invention may be combined with pharmaceutically acceptable adjuvants tionally employed in vaccines and administered in prophylactically effective amounts to protect individuals over an extended period of time against ial infections or diseases.

W0 2012l097273 PCT/U52012/021280 In some embodiments, nds of formula (I), or a pharmaceutically acceptable salt thereof, may be used prophylactically to prevent a bacterial infection. In some embodiments, compounds of formula (I), or a pharmaceutically acceptable salt thereof, may be used before, during or after a dental or surgical procedure to prevent opportunistic infections such as those encountered in bacterial rditis. In other embodiments, compounds of formula (I), or a pharmaceutically acceptable salt thereof, may be used prophylactically in dental procedures, including but not limited to extractions, periodontal procedures, dental implant placements and endodontic surgery. In other ments, compounds of formula (I), or a pharrnaceutically acceptable salt thereof, may be used prophylactically in surgical procedures including but not limited to general y, respiratory surgery (tonsillectomy/adenoidectomy), gastrointestinal surgery (upper GI and elective small bowel surgery, esophageal sclerotherapy and dilation, large bowel resections, acute appendectomy), trauma surgery (penetrating abdominal surgery), genito-urinary tract y (prostatectomy, urethral dilation, copy, l or abdominal ectomy, cesarean section), transplant y (kidney, liver, pancreas or kidney transplantation), head and neck surgery (skin excisions, neck dissections, laryngectomy, head and neck cancer ies, mandibular fractures), orthopaedic surgery (total joint replacement, traumatic open res), vascular surgery (peripheral vascular procedures), cardiothoracic surgery, coronary bypass surgery, pulmonary resection and neurosurgery.

The term "prevent a bacterial infection" as used herein, unless otherwise indicated, means the prophylactic use of an antibiotic, such as a gyrase and/or topoisomerase IV inhibitor of the present invention, to prevent a bacterial infection. Treatment with a gyrase and/or omerase IV inhibitor could be done prophylactically to prevent an infection caused by an organism that is susceptible to the gyrase and/or topoisomerase IV inhibitor.

One general set of conditions where lactic treatment could be considered is when an individual is more able to infection due to, for example, weakened immunity, surgery, , presence of an artificial device in the body (temporary or permanent), an anatomical defect, exposure to high levels of bacteria or possible exposure to a disease-causing pathogen.

Examples of factors that could lead to weakened immunity include chemotherapy, radiation therapy, diabetes, advanced age, HIV infection, and transplantation. An e of an anatomical defect would be a defect in the heart valve that increases the risk of bacterial endocarditis. Examples of artificial devices include artificial joints, surgical pins, catheters, etc. Another set of situations where prophylactic use of a gyrase and/or topoisomerase IV W0 97273 inhibitor might be appropriate would be to prevent the spread of a pathogen between individuals (direct or ct). A specific example of prophylactic use to prevent the spread of a en is the use of a gyrase and/or topoisomerase IV inhibitor by individuals in a healthcare institution (for example a hospital or nursing home).

] The compounds of a (I), or a pharmaceutically acceptable salt thereof, may also be co—administered with other antibiotics to increase the effect of therapy or prophylaxis against various bacterial infections. When the compounds of this invention are administered in combination therapies with other agents, they may be administered tially or concurrently to the patient. atively, pharmaceutical or prophylactic compositions according to this invention comprise a combination of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and another therapeutic or prophylactic agent.

] In some embodiments, the additional eutic agent or agents is an antibiotic selected from a natural penicillin, a penicillinase-resistant penicillin, an antipseudomonal penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a quinolone, a quinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a glycopeptide, a streptogramin, an oxazolidinone, a rifamycin, or a sulfonamide.

In some ments, the additional therapeutic agent or agents is an antibiotic ed from a penicillin, a osporin, a quinolone, an aminoglycoside or an idinone.

In other embodiments, the onal therapeutic agents are ed from a natural penicillin including Benzathine llin G, Penicillin G and Penicillin V, from a penicillinase-resistant penicillin including Cloxacillin, Dicloxacillin, Nafcillin and Oxacillin, from a antipseudomonal penicillin including Carbenicillin, Mezlocillin, Pipercillin, Pipercillin/tazobactam, Ticaricillin and Ticaricillin/Clavulanate, from an aminopenicillin including Amoxicillin, Ampicillin and Ampicillin/Sulbactam, from a first generation cephalosporin including Cefazolin, Cefadroxil, Cephalexin and Cephadrine, from a second generation cephalosporin including Cefaclor, Cefaclor-CD, Cefamandole, Cefonacid, Cefprozil, Loracarbef and Cefuroxime, from a third generation cephalosporin including Cefdinir, Cefixime, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxme and Ceftriaxone, from a fourth generation cephalosporin including Cefepime, oline and Ceftobiprole, from a Cephamycin including Cefotetan and Cefoxitin, from a WO 97273 PCT/U52012/021280 carbapenem including Doripenem, Imipenem and Meropenem, from a monobactam including Aztreonam, from a quinolone including Cinoxacin, Nalidixic acid, Oxolininc acid and Pipemidic acid, from a fluoroquinolone including Besifloxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin and Sparfloxacin, from an lycoside including Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Spectinomycin, omycin and Tobramycin, from a macrolide including Azithromycin, Clarithromycin and Erythromycin, from a ketolide including Telithromycin, from a Tetracycline including Chlortetracycline, Demeclocycline, Doxycycline, Minocycline and Tetracycline, from a glycopeptide including Oritavancin, ancin, Telavancin, Teicoplanin and Vancomycin, from a streptogramin including Dalfopristin/quinupristin, from an oxazolidone including Linezolid, from a cin including Rifabutin and Rifampin and from other antibiotics including bactitracin, colistin, Tygacil, Daptomycin, chloramphenicol, clindamycin, isoniazid, metronidazole, mupirocin, polymyxin B, pyrazinamide, trimethoprim/sulfamethoxazole and sulfisoxazole.

In other embodiments, the additional therapeutic agents are selected from a l penicillin including Penicillin G, from a penicillinase—resistant penicillin including Nafcillin and Oxacillin, from an antipseudomonal penicillin including Pipercillin/tazobactam, from an aminopenicillin including Amoxicillin, from a first generation cephalosporin including Cephalexin, from a second tion cephalosporin including or, or-CD and Cefuroxime, from a third generation cephalosporin including Cefiazidime and Ceftriaxone, from a fourth generation cephalosporin including Cefepime, from a carbapenem including em, Meropenem, Ertapenem, Doripenem, Panipenem and Biapenem,a fluoroquinolone ing Ciprofloxacin, Gatifloxacin, Levofloxacin and Moxifloxacin, from an lycoside including Tobramycin, from a macrolide ing Azithromycin and Clarithromycin, from a Tetracycline including Doxycycline, from a glycopeptide including Vancomycin, from a cin including Rifampin and from other antibiotics including isoniazid, namide, Tygacil, Daptomycin or trimethoprim/sulfamethoxazole.

] In some embodiments, a solid form of a nd of formula (I), or a pharmaceutically acceptable salt thereof, can be administered for the treatment of a gram positive infection, In some embodiments, the composition is a solid, liquid (e.g, a suspension), or an iv (e. g., a form of the formula (I) compound, or a pharmaceutically acceptable salt thereof, is dissolved into a liquid and administered iv) ition. In some embodiments, the composition including a a (1) compound, or a pharmaceutically ~28— 2012/021280 acceptable salt thereof, is administered in combination with an additional antibiotic agent, for example, a natural penicillin, a penicillinase-resistant penicillin, an eudomonal penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth tion cephalosporin, a carbapenem, a cephamycin, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a glycopeptide, a streptogramin, an idinone, a rifamycin, or a sulfonamide. In some embodiments, the composition including a solid form of a formula (1) compound, or a pharmaceutically acceptable salt thereof, is administered orally, and the onal antibiotic agent, for example, a natural penicillin, a penicillinase- resistant penicillin, an antipseudomonal penicillin, an enicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation osporin, a carbapenem, a cephamycin, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a glycopeptide, a streptogramin, an oxazolidinone, a rifamycin, or a sulfonamide is administered iv.

In some embodiments, a solid form of a a (1) compound, or a pharmaceutically acceptable salt thereof, can be administered for the treatment of a gram ve infection. In some embodiments, the composition is a solid, liquid (e. g., a suspension), or an iv (e. g., a form of a formula (1) compound, or a pharmaceutically acceptable salt f, is dissolved into a liquid and administered iv) composition. In some embodiments the composition including a formula (1) compound, or a pharmaceutically acceptable salt thereof, is administered in combination with an additional otic agent, selected from a: natural penicillin, a penicillinase-resistant penicillin, an antipseudomonal penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a monobactam, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polyrnyxin, tetracycline or a sulfonamide. In some embodiments, the composition including a solid form of a formula (1) compound, or a pharmaceutically acceptable salt thereof, is administered orally, and the additional antibiotic agent, for e, a natural penicillin, a penicillinase-resistant penicillin, an antipseudomonal penicillin, an enicillin, a first generation cephalosporin, a second tion cephalosporin, a third generation osporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a ctam, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, tetracycline or a sulfonarnide is WO 97273 administered orally. In some embodiments, the additional therapeutic agent is administered The additional therapeutic agents described above may be administered tely, as part of a multiple dosage n, from the inhibitor-containing composition.

Alternatively, these agents may be part of a single dosage form, mixed together with the inhibitor in a single composition.

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The pharmaceutical compositions of this invention may contain any conventional non—toxic ceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated nd or its delivery form.

The term eral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra—articular, intrasynovial, intrasternal, intrathecal, intralesional and ranial injection or on techniques.

The ceutical, compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This sion may be formulated according to techniques known in the art using suitable sing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile inj ectable preparation may also be a e injectable solution or suspension in a non- toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharrnaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as those described in Pharmacopeia Helvetica, or a similar alcohol.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of s for oral use, carriers which are WO 97273 PCT/U82012/021280 commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and solutions and propylene glycol are administered orally, the active ient is ed with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/0r coloring agents may be added.

The pharmaceutical compositions of this invention may also be administered in the form of itories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a le non—irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active ents. Such als include, but are not d to, cocoa butter, beeswax and polyethylene glycols.

] Topical administration of the pharmaceutical itions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by l application. For application topically t0 the skin, the pharmaceutical composition should be ated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the nds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be lly applied to the lower intestinal tract by rectal itory formulation or in a suitable enema formulation. Topically-administered transdermal patches are also included in this invention.

The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, ing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

] According to another embodiment, compounds of formula (I), or a pharrnaceutically acceptable salt thereof, may also be delivered by implantation (e.g., surgically), such as with an implantable or indwelling . An implantable or indwelling device may be ed to reside either permanently or temporarily in a subject. Examples of table and indwelling devices e, but are not limited to, contact lenses, l venous catheters and less connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, such as hip and knee replacements, tyrnpanostomy tubes, urinary ers, voice prostheses, stents, delivery pumps, vascular filters and implantable control release itions. Biofilms can be ental to the health of ts with an implantable or indwelling medical device because they introduce an artificial substratum into the body and can cause persistent infections. Thus, providing compounds of formula (I), or a pharmaceutically acceptable salt thereof, in or on the implantable or indwelling device can prevent or reduce the production of a biofilm. In addition, implantable or indwelling devices may be used as a depot or reservoir of compounds of formula (I), or a pharrnaceutically acceptable salt thereof. Any implantable or indwelling device can be used to deliver compounds of a (I), or a pharmaceutically acceptable salt thereof, provided that a) the device, compounds of formula (I), or a pharrnaceutically acceptable salt thereof, and any pharmaceutical composition including compounds of formula (I), or a pharmaceutically acceptable salt thereof, are biocompatible, and b) that the device can deliver or release an effective amount of compounds of formula (I), or a pharmaceutically acceptable salt thereof, to confer a therapeutic effect on the treated patient.

Delivery of therapeutic agents via implantable or indwelling devices is known in the art. See for example, “Recent Developments in Coated Stents” by Hofina et al. published in Current entional Cardiology Reports 2001, 3:28—3 6, the entire contents of which, including references cited therein, incorporated herein by nce. Other descriptions of table devices can be found in US. Patent Nos. 6,569,195 and 6,322,847; and US.

Patent Application Numbers 2004/0044405, 2004/0018228, 229390, 2003/0225450, 2003/0216699 and 2003/0204168, each of which is incorporated herein by reference in its entirety.

In some embodiments, the implantable device is a stent. In one specific embodiment, a stent can e interlocked meshed cables. Each cable can include metal wires for structural support and ric wires for delivering the therapeutic agent. The polymeric wire can be closed by immersing the polymer in a solution of the therapeutic agent.

PCT/U52012/021280 Alternatively, the therapeutic agent can be ed in the ric wire during the formation of the wire from polymeric sor solutions.

In other embodiments, implantable or indwelling devices can be coated with polymeric coatings that include the therapeutic agent. The polymeric coating can be designed to control the release rate of the therapeutic agent. Controlled release of therapeutic agents can e various technologies. Devices are known that have a monolithic layer or coating incorporating a heterogeneous solution and/or dispersion of an active agent in a polymeric substance, where the diffusion of the agent is rate limiting, as the agent diffuses through the r to the polymer—fluid interface and is released into the surrounding fluid. In some s, a e substance is also dissolved or dispersed in the polymeric material, such that additional pores or channels are left after the material dissolves. A matrix device is generally diffusion limited as well, but with the channels or other internal geometry of the device also playing a role in releasing the agent to the fluid. The channels can be pre-existing channels or channels left behind by released agent or other soluble substances.

Erodible or degradable devices typically have the active agent physically immobilized in the polymer. The active agent can be dissolved and/or sed throughout the ric material. The polymeric material is often hydrolytically degraded over time through hydrolysis of labile bonds, allowing the r to erode into the fluid, releasing the active agent into the fluid. Hydrophilic polymers have a generally faster rate of erosion relative to hydrophobic polymers. Hydrophobic polymers are believed to have almost purely surface diffusion of active agent, having erosion from the surface inwards. Hydrophilic polymers are believed to allow water to penetrate the surface of the polymer, ng hydrolysis of labile bonds beneath the surface, which can lead to homogeneous or bulk erosion of polymer.

The table or indwelling device coating can include a blend of polymers each having a different release rate of the therapeutic agent. For instance, the coating can e a polylactic acid/polyethylene oxide EO) copolymer and a polylactic acid/polycaprolactone (PLA-PCL) copolymer. The ctic acid/polyethylene oxide (PLA- PEO) copolymer can exhibit a higher release rate of therapeutic agent relative to the polylactic acid/polycaprolactone (PLA-PCL) copolymer. The ve amounts and dosage rates of therapeutic agent delivered over time can be controlled by controlling the ve amounts of the faster releasing polymers relative to the slower releasing polymers. For higher initial release rates the proportion of faster releasing polymer can be increased relative PCT/U82012/021280 to the slower releasing polymer. If most of the dosage is desired to be ed over a long time period, most of the polymer can be the slower releasing r. The device can be coated by spraying the device with a solution or dispersion of polymer, active agent, and solvent. The solvent can be evaporated, leaving a coating of polymer and active agent. The active agent can be dissolved and/or dispersed in the polymer. In some embodiments, the co- polymers can be extruded over the device.

Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, preferably between 0.5 and about 75 mg/kg body weight per day and most preferably between about 1 and 50 mg/kg body weight per day of the active ingredient compound are useful in a erapy for the prevention and treatment of bacterial infections.

Typically, the pharmaceutical compositions of this invention will be stered from about 1 to 5 times per day or alternatively, as a continuous infusion. Alternatively, the compositions of the present invention may be administered in a pulsatile formulation. Such administration can be used as a chronic or acute therapy The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active nd (w/w). Preferably, such preparations contain from about 20% to about 80% active compound.

When the itions of this invention comprise a ation of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 10% to 80% of the dosage normally administered in a erapy regime.

] Upon improvement of a patient's condition, a maintenance dose of a compound, ition or combination of this invention may be administered, if necessary.

Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the ms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence or e symptoms.

As the skilled artisan will iate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular t will depend upon a variety of factors, including the activity of the specific compound PCT/U52012/021280 employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the e, and the patient's disposition to the disease and the judgment of the treating ian.

According to another embodiment, the invention provides methods for treating or preventing a bacterial infection, or disease state, comprising the step of administering to a patient any compound, pharmaceutical composition, or combination described . The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

The compounds of this invention are also useful as commercial reagents which effectively bind to the gyrase B and/or topoisomerase IV enzymes. As commercial reagents, the compounds of this invention, and their derivatives, may be used to block gyrase B and/or topoisomerase IV ty in biochemical or cellular assays for bacterial gyrase B and/or topoisomerase IV or their homologs or may be derivatized to bind to a stable resin as a tethered substrate for affinity chromatography applications. These and other uses which characterize commercial gyrase B and/or topoisomerase IVinhibitors will be t to those of ordinary skill in the art.

In order that this invention be more fully understood, the following schemes and examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the ion in any way.

] The following ions describe terms and abbreviations used herein: Ac acetyl Bu butyl Et ethyl Ph phenyl Me methyl THF tetrahydrofuran DCM dichloromethane CH2C12 dichloromethane EtOAc ethyl acetate CH3CN acetonitrile EtOH l 320 diethyl ether MeOH methanol MTBE methyl tert—butyl ether DMF MN—dimethylformamide DMA NN-dimethylacetamide DMSO yl sulfoxide HOAc acetic acid TEA triethylamine TFA trifluoroacetic acid PCT/U52012/021280 TFAA trifluoroacetic anhydride Eth triethylamine DIPEA diisopropylethylamine DIEA diisopropylethylamine K2C03 potassium ate Na2C03 sodium ate 3 sodium thiosulfate C32C03 cesium carbonate NaHC03 sodium bicarbonate NaOH sodium hydroxide Na2804 sodium sulfate MgSO4, magnesium sulfate K3PO4 potassium ate NH4Cl ammonium de LC/MS liquid chromatography/mass spectra GCMS gas chromatography mass spectra HPLC high performance liquid chromatography GC gas chromatography LC liquid chromatography IC ion chromatography IM intramuscular CFU/cfu colony forming units MIC minimum inhibitory concentration Hr or h hours atm atmospheres rt or RT room temperature TLC thin layer chromatography HCl hydrochloric acid H20 water EtNCO ethyl isocyanate Pd/C palladium on carbon NaOAc sodium acetate H2804 sulfuric acid N2 nitrogen gas H2 hydrogen gas n-BuLi n—butyl lithium DI de—ionized Pd(OAc)2 palladium(II)acetate PPh3 triphenylphosphine i—PrOH isopropyl alcohol NBS N-bromosuccinimide Pd[(Ph3)P]4 tetrakis(triphenylphosphine)palladium(0) PTFE polytetrafluoroethylene rpm revolutions per minute SM starting material Equiv. equivalents 1H-NMR proton nuclear magnetic resonance PCT/U82012/021280 Synthesis of the Compounds THE 6—FLUORO BENZIMIDAZOLYL UREA ND Synthesis of (R)ethyl—3-(6-fluoro-5—(2-(2-hydroxypr0panyl)pyrimidin—S-yl)—7- (tetrahydr0furan-2—yl)—1H—benzo[d ]imidazol-Z—yl)urea Scheme 3 provides a method for preparing the 6—fluoro benzoimidazolyl urea compound.

Scheme 3 szBFz(tBU3P)2 45 pSI H2, Pd/C + m + B, O EtN(iPr)2, dioxane, reflux N33, MeOH ,1 / N02 No2 o N02 14 2 15A ISB MTBE, CHgCN' NBS-15°C OH V I B 1 N \N Pd((1113mm2 Ni? 31 aqstO4 i),TFAA THF I F 2°Ct0 Ft / aq NaH003 F 1,4-dioxane / F 14dioxane V*N reflux, 5 days ii)NH4NO3, 3010 41°C YNH O reflux fie?” NH; 0 1 “18 NH2 0 19 17 45 psi H2 PdIC NEt3 MeOH THF OH Y /OH OH OH / l / N \ N N| \ N o 30’ / EIHNJ‘NN0NHEt1 chiralchrom MeSOsH / —-—-———-—) N ————)- pH fer >’NH DOM EtOH N dioxane, reflux 2-°C rt >LNH 0 HzN HN:NHO HN NH; 0 HN>= HN>= 22 >20 M9803H 21 )0 ) 23 HN PCT/U82012/021280 Example l.a Preparation of 2-(2-fluoronitro-phenyl)-2,3-dihydrofuran (15A) and 2-(2-fluoronitro- phenyl)-2,5-dihydrofuran (15B) BusP); + 0 + Br 0 EtN(iPr)2, dioxane, reflux \ N02 N02 0 N02 0 l4 2 15A 15B 2-Bromo-1—flu0ro~3~nitro—benzene (14) (200.3 g, 98%, 892.3 mmol, Bosche F6657), 1,4-dioxane (981.5 mL, Sigma-Aldrich 360481), and 2,3-dihydrofuran (2) (341.1 mL, 99%, 4.462 mol, h 200018) were charged in a reaction flask, followed by N,N— ropylethylamine (155.4 mL, 892.3 mmol, Sigma-Aldrich ) and bromo(tri—tert— butylphosphine)palladium(l) dimer (6.936 g, 8.923 mmol, Johnson Matthey C4099). The mixture was stirred at reflux for 2 hrs (HPLC showed 98% consumption of starting omide). The reaction mixture was allowed to cool; the precipitate was removed by filtration, rinsed with EtOAc, and the filtrate concentrated in vacuo to a dark reddish brown semi-solid oil. The semi-solid oil was dissolved in CH2C12, eluted through a plug of silica with CHzClz, and concentrated in vacuo giving a mixture of 15A and 15B as a dark amber oil (291.3 g). The crude product was carried forward without r purification. The major product was 2—(2-fluoro-6—nitro—phenyl)-2,3—dihydrofuran (15A) (96%): LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+11 210.23 (3.13 min); 1H NMR (300 MHZ, CDC13) 5 7.54 (dt, J = 8.0, 1.2 Hz, 1H)= 7.43 (td, J = 8.2, 5.2 Hz, 1H), 7.32 (ddd, J = 9.7, 8.3, 1.3 Hz, 1H), 6.33 (dd, J = 4.9, 2.4 Hz= 1H), .80 (t, J = 10.9 Hz,1H), 5.06 (q, J = 2.4 Hz,1H), 3.18 — 3.07 (rm 1H), 2.94 — 2.82 (m, 1H) ppm. The minor product was 2-(2-fluoronitro-phenyl)-2,5-dihydrofuran (15B) (4%): GCMS nt HP—SMS 30 m x 250 mm x 0.25 um column heating at 60°C for 2 min to 300°C over 15 min with a 1 mL/min flow rate) M+1: 210 (11.95 min). 1H NMR (300 MHz, CDC13) 5 7.47 (d, J = 8.0 Hz= 1H), 7.43 — 7.34 (m 1H), 7.30 — 7.23 (m, 1H), 6.21 — 6.15 (m, 1H),6.11— 6.06 (m, 1H), 5.97 — 5.91 (m, 1H), 4.89 — 4.73 (m, 2H) ppm. -38..

Example l.b Preparation of 3-fluoro-2—tetrahydrofuran—2-yl-aniline (16) F F F + 45 psi H2, Pd/C N02 0 N02 0 NEt3,MeOH,rt NH2 0 15A 15B 16 5% Palladium on carbon (37.3 g, 50% wet, 8.76 mmol, Aldrich 330116) was placed in a Parr bottle under en, followed by MeOH (70 mL, JT-Baker 909333). The crude e of uoronitro-phenyl)-2,3~dihydrofuran and 2-(2-fluoro-6—nitro- )—2,5-dihydrofuran (15A&15B) (186.6 g, 892.1 mmol) dissolved in MeOH (117 mL) was added to the Parr bottle, followed by NE13 (124.3 mL, 892.] mmol, Sigma-Aldrich ). The bottle was placed on a Parr shaker and saturated with H2. After adding 45 psi H2, the reaction mixture was shaken until ption of the starting material was complete (HPLC and LCMS showed complete reaction). The reaction mixture was purged with nitrogen, filtered through CeliteTM and rinsed with EtOAc. The filtrate was concentrated on a rotary evaporator giving brown oil, which was dissolved in 320 and washed with water (2x).

The ether phase was extracted with aqueous 1 N HCl (5 X 250 mL), which was washed with EtZO (3X) and then basified with aqueous 6 N NaOH to pH 12-14. The basic aqueous phase was extracted with dichloromethane (CHzClz, 4x), and the ed organic extract was washed with saturated aqueous NH4Cl, dried over MgS O4, and filtered through a pad of silica eluting with CH2C12 to 25% EtOAc /hexane. The desired filtrate was concentrated under reduced pressure giving 16 as a light brown oil (121.8 g, 84% GCMS plus NMR purity).

GCMS (Agilent HP—SMS 30 m X 250 um X 0.25 pm column heating at 60°C for 2 min to 300°C over 15 min with a 1 mL/min flow rate) M+1: 182.0 (11.44 min). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 182.10 (2.61 min). 1H NMR (300 MHz, CDClg) 6 6.97 (td, J z 8.1, 6.3 Hz, 1H), 6.43 — 6.35 (m, 2H), 5.21 ~ 5.13 (m, 1H), 4.54 (s, 2H), 4.16 — 4.07 (m, 1H), 3.90 _ 3.81 (m, 1H), 2.23 — 2.00 (m, 4H) ppm. Additional crops were obtained as follows: the combined ether phase was washed with saturated aqueous NaI-IC03, brine, dried over Na2804, decanted, and concentrated under reduced pressure. The oil was vacuum distilled (ca. 15 torr) collecting the distillate at 101 — 108°C. To a stirring solution of the distilled oil in EtOH (1 volume) at 2°C was slowly added 5 M HCl (1 eq) in iPrOH. The resulting suspension was brought to room temperature, diluted with EtOAc (3 volumes, vol/vol), and stirred for 2 hrs.

A white solid was ted by filtration, washed with EtOAc, and dried under reduced pressure giving a second crop of product as the HCl salt. The mother liquor was concentrated to a slurry, diluted with EtOAc and the solid collected by filtration, washed with EtOAc, and dried in vacuo giving the HCl salt as a third crop of the product. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 182.10 (2.58 min). 1H NMR (300 MHz, CDClg) 5 10.73 (br.s, 3H), 7.66 (d, J = 8.1 Hz, 1H), 7.33 (td, J = 8.2, 5.9 Hz, 1H), 7.13 — 7.05 (m 1H), 5.26 (dd, J = 9.0, 6.5 Hz, 1H), 4.38 — 4.28 (m, 1H), 4.00 ~ 3.91 (m, 1H), 2.59 — 2.46 (m, 1H), 2.30 — 1.95 (m, 3H) ppm. The overall yield from the three crops was 76%.

Example 1.0 Preparation of 4-brom0fluoro-2~tetrahydrofuranyl-aniline (17).

F F MTBE, CH30N NBS, -15°C NH2 0 NH2 0 16 17 To a ng solution of 3—fluorotetrahydrofuranyl-aniline (16) (131.9 g, 92%, 669.7 mmol) in methyl terr-butyl ether (1.456 L) and acetonitrile (485 mL) cooled to - °C was added N-bromosuccinimide (120.4 g, 99%, 669.7 mmol, h B81255) in 3 portions maintaining a reaction temperature below about -15°C. After complete addition, stirring was continued at —15 to ~10°C for 30 s. 1H NMR of a worked—up aliquot showed 96% consumption of starting aniline. Another 4.82 g NBS was added to the reaction mixture and stirred at ~10°C for additional 30 minutes. Aqueous 1 N Na2S203 (670 mL) was added to the on mixture. The cold bath was removed, the mixture stirred for 20 minutes, then diluted with EtOAc. The layers were separated. The organic phase was washed with saturated s NaHC03 (2x), water, and brine, dried over , ed, and concentrated under reduced pressure giving a dark amber oil. The residue was diluted with hexane and eluted through a short plug of silica with 25% EtOAc / hexane to 50% EtOAc / hexane. The desired filtrate was concentrated in vacuo giving 17 as a dark amber oil (182.9 g, 90% yield; 86% NMR purity). LCMS (C18 column eluting with 10-90% AcN / water gradient over 5 minutes with formic acid modifier) M+1: 260.12 (3.20 min). 1H NMR (300 MHz, CDC13) 5 7.15 (dd, J = 8.6, 7.6 Hz, 1H), 6.30 (dd, J = 87,13 Hz, 1H), 5.19 — W0 20121097273 512 (Hi, 1H), 4.58 (s, 2H), 4.16 _ 4.07 (m, 1H), 3.90 — 3.81 (m, 1H), 2.23 — 1.99 (m, 4H) Example 1.d Preparation ofN—(4-bromo-3 -6—nitro-2—tetrahydrofuran-2—yl-phenyl)—2,2,2-trifluoro~ acetamide (18).

Br Br F i) TFAA, THF 2°C to rt ii) NH4N03, 30 to 41°C OQN NH2 0 OYNH o 17 CF3 To trifluoroacetic anhydride (565.3 mL, 4.067 mol, Sigma-Aldrich 106232) stirring at 2°C was slowly added neat 4-bromo—3-fluoro—2-tetrahydrofuranyl-aniline (17) (123.0 g, 86%, 406.7 mmol) as a thick 011 via addition funnel over about 20 minutes (reaction temperature rose to 13°C). The remaining oil was rinsed into the reaction mixture with anhydrous THF (35 mL). The cold bath was removed and the reaction was heated to 35°C, followed by portion-wise addition ofNH4N03 (4.88 g x 20 portions, 1.22 mol, Sigma- Aldrich A7455) over 2.5 hrs maintaining the on temperature between 30 and 41°C using an ice-water bath only as needed to control the exotherm. After complete addition the reaction mixture was stirred for r 10 minutes (HPLC showed on 99% te).

It was slowly poured into d ice (1.23 kg) and stirred for 1 hr to allow formation of a filterable solid precipitate, which was collected and washed with water, sparingly with saturated aqueous NaHC03, and water again (to pH 7). The product was dried in a tion oven overnight at 40°C and then under reduced pressure in an oven at 50°C overnight giving 18 as a beige solid (152.5 g, 90% yield; 96% HPLC purity). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 401.30 (3.41 min). 1H NMR (300 MHz, CDC13) 6 10.56 (s, 1H), 8.19 (d, J = 6.6 Hz, 1H), 5.22 (dd, J =10.3, 6.4 Hz, 1H), 4.22 (dd, J =15.8,7.2 Hz, 1H), 3.99 (dd, J = 16.1, 7.5 Hz, 1H), 2.50 — 2.38 (m, 1H), 2.22 — 2.11 (m,2H),1.86 — 1,71 (m, 1H) ppm.

W0 2012(097273 PCT/U52012/021280 Example l.e Preparation of 4-bromofluoronitrotetrahydrofuranyl-aniline (19).

Br Br F F aq H2804 1,4—dioxane 02N _._____, OZN reflux, 5 days OYNH O NH2 0 A on flask was charged with N—(4-bromo~3~fluoro~6~nitro~2~ tetrahydrofuran—Z—yl~phenyl)-2,2,2-trifluoro-acetamide (18) (242.3 g, 604.1 mmol), 1,4— dioxane (1.212 L), and aqueous 2 M ic acid (362.4 mL, 724.9 mmol), and stirred at reflux for 5 days (HPLC showed 98% sion). The on mixture was allowed to cool, diluted with EtOAc, neutralized with saturated aqueous NaHC03, separated the layers, and re-extracted the aqueous phase with EtOAc (2x). The combined c phase was washed with brine (2x), dried over MgSO4, filtered and concentrated in vacuo giving 19 as a greenish brown solid (181.7 g, 94% yield; 95% HPLC purity). The product was carried to the next step t further purification. LCMS (C18 column eluting with 10-90% CH3CN/ water gradient over 5 minutes with formic acid modifier) M+1: 305.20 (3.63 min). 1H NMR (300 MHz. CDCI3) 5 8.35 (d, J = 7.3 Hz, 1H), 7.45 (s, 2H), 5.23 — 5.16 (m. 1H), 4.23 — 4.14 (m,1H), 3.93 — 3.84 (m, 1H), 2.31 — 1.96 (m, 4H) ppm.

Example 1.f Preparation of 2—[5-(4—aminofluoro-5 -nitrotetrahydrofuran—2—yl-phenyl)pyrimidin-2— yl]propanol (20). 3 OH Br E :OH N\N F’d(dppf)Clz N \N F ' ' / w / 1,4-dioxane, reflux F OZN /B\ O O NH2 0 M NH2 0 19 7 To a stirring solution of 4-bromo—3 -fluoronitrotetrahydrofurany1- aniline (19) (525.0 g, 1.721 mol, Bridge Organics Co.) in 1,4-dioxane (4.20 L, Sigma-Aldrich 360481) was added a 1,2 M aqueous solution ofNaHC03 (4.302 L, 5.163 mol). A stream of - 42 _ W0 2012I097273 PCT/USZOIZ/021280 nitrogen was d through the ng mixture for 2 hrs, ed by addition of 2-[5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolany1)pyrimidin-2—yl]propanol (7) (545.4 g, 2.065 mol, Bridge Organics Co.) and 1,1 ’-bis(diphenylphosphino)ferrocene dichloropalladium dichloromethane adduct (42.16 g, 51.63 mrnol, Strem 460450). The reaction e was stirred at reflux overnight, allowed to cool, d with EtOAc (8.4 L), and the layers were separated. The organic phase was washed with saturated aqueous NH4C1 and then brine. The aqueous phase was re—extracted with EtOAc (4 L) and washed this organic extract with brine.

The combined organic phase was dried over MgSO4, filtered through a short plug of Florisil®, eluted with EtOAc, and the filtrate concentrated on a rotary evaporator giving a dark brown wet solid. This was dissolved in CHzClz, loaded on a pad of silica gel, eluted with hexane, then 25% EtOAc / hexane, and then 50% EtOAc / hexane. The desired filtrate was concentrated on a rotary evaporator to a thick sion, and the solid was collected by filtration, triturated with MTBE, and dried in vacuo giving 20 as a bright yellow solid (55.8% yield, 90-97% HPLC purity). The filtrate was concentrated and the above purification was repeated giving a second crop of 20 as a bright yellow solid (19.7% yield). The filtrate was again concentrated giving a dark brown oil and this was loaded on a silica column with tdmmmmmmdemOzmedmww%Emmmhmmfiman%)flwwmw fractions were concentrated to a slurry and diluted with MTBE / hexane. The solid was collected by filtration and washed with minimal MTBE giving a third crop of 20 as a bright yellow solid (4.9% yield) with an overall yield of 80% from the three crops. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 363.48 (2.95 min). 1H NMR (300 MHZ, CDC13) 6 8.84 (d, J = 1.6 Hz, 2H), 8.27 (d, J = 8.0 Hz, 1H), 7.62 (s, 2H), 5.31 — 5.24 (m, 1H), 4.63 (s, 1H), 4.27 — 4.18 (m, 1H), 3.97 — 3.87 (m, 1H), 2.33 — 2.05 (m, 4H), 1.64 (s, 6H) ppm. 2012/021280 Example 1.g Preparation of 4,5-diaminofluorotetrahydrofuran—2-yl-phenyl)pyrimidin-Z- yl]propanol (21). is is N \ N 45 psi H2, Pd/C, NEt3 N \ N I I / MeOH, THF / —___._’,.

F F 02N HQN NH2 0 NH2 0 21 5% Palladium on carbon (14.21 g, 50% wet, 3.339 mmol, Aldrich 330116) was placed in a Parr bottle under nitrogen, followed by MeOH (242 mL, JT-Baker 909333) and NEts (46.54 mL, 333.9 mmol, Sigma-Aldrich 471283). 2-[5-(4-Amino-2—fluoronitro- 3-tetrahydrofuranyl-phenyl)pyrimidin—2-yl]propan—Z—ol (20) (121.0 g, 333.9 mmol) was dissolved in hot THF (360 mL), allowed to cool, added to the reaction mixture, and rinsed the residual amount of 20 with another portion of THF (124 mL). The bottle was placed on a Parr shaker and saturated with H2. After adding 45 psi H2, the bottle was shaken until consumption of 20 was complete (HPLC and LCMS showed complete reaction). The reaction mixture was purged with nitrogen, d through CeliteTM and rinsed with EtOAc.

It was re-filtered through paper (glass microfibre) and the filtrate concentrated in vacuo. The on was repeated three more times on the same scale and the batches were combined giving 21 as a brown solid (447 g. 99% yield; 93% HPLC ). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 333.46 (1.79 min). 1H NMR (300 MHz, CDC13) 5 8.81 (d, J = 1.4 Hz, 2H), 6.69 (d, J = 7.3 Hz, 1H), 5.27 ~ 5.20 (m, 1H), 4.73 (s, 1H), 4.70 (s, 2H), 4.23 4 4.14 (m, 1H), 3.94 — 3.86 (m, 1H), 3.22 (s, 2H), 2.32 — 2.22 (m, 1H), 2.18 — 1.99 (m, 3H), 1.63 (s, 6H) ppm.

PCT/U82012/021280 Example 1.h Preparation of 1 -ethyl[6-fluoro[2-(1-hydroxymethyl-ethyl)pyrimidinyl] tetrahydrofuran-z-yl-JH-benzimidazol-Z-yl]urea (22) N \ N o s’ o / EtHNJLmkNJLNHEt 10 pH 3.5 buffer dioxane, reflux NH2 0 To a stirring suspension of 2—[5—(4,5-diarninofluoro-3 —tetrahydrofuran—2-yl- phenyl)pyrimidiny1]propan—2-ol (21) (111.3 g, 334.9 mmol) and 1,4-dioxane (556.5 mL, Sigma-Aldrich 360481) was added 1-ethyl(N-(ethylcarbamoyl)-C-methylsulfanylcarbonimidoyl )urea (10) (93.36 g, 401.9 mmol, CB Research and Development) followed by a pH 3.5 buffer (1113 L), prepared by dissolving NaOAc trihydrate ( 15 8.1 g) in IN aqueous H2304 (1.100 L). The reaction mixture was stirred at reflux overnight (HPLC showed te conversion), cooled to room temperature, and poured portion-wise (to minimize frothing) into a ng solution of s saturated NaHCO; (2.23 L) giving pH 8-9. The ing e was stirred for 30 minutes, the solid was collected by filtration, washed copiously with water to neutral pH, and then more sparingly with EtOH. The solid was dried under reduced pressure giving 22 as an off-white yellowish solid (135.2 g, 94% yield; 99% HPLC purity). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+lz 429.58 (2.03 min). 1H NMR (300 MHZ, MeOD) 5 8.95 (d, J =16 Hz, 2H), 7.45 (d, J = 6.5 Hz, 1H), 5.38 (brs, 1H), 4.27 (dd, J = 14.9, 7.1 Hz, 1H), 4.01 (dd, J = 15.1, 7.0 Hz, 1H), 3.37 — 3.29 (m, 2H), 2.55 (br.s, 1H), 2.19 — 2.07 (m, 2H), 2.02 — 1.82 (br.s, 1H), 1.63 (s, 6H), 1.21 (t, J = 7.2 Hz, 3H) ppm.

PCT/U82012/021280 Example 1.i Chiral chromatographic isolation of 1-ethyl-3 -[6-fluoro-5 -[2-(1 -hydroxymethy1-ethyl)pyrimidinyl][(2R)-tetrahydrofuran- 2-y1]—1H—benzimidazolyl]urea (23) N\N N\N chiral chrom A racernic sample of l[6-fluoro[2-(1-hydroxy—1-methyl- ethyl)pyrimidinyl]tetrahydrofuranyl-lH—benzimidazol-Z-yl]urea (22) (133.60 g) was resolved on a PAK® IC® column (by Chiral Technologies) eluting with CH2C12 / MeOH / TBA (60 / 40/ 0.1) at 25°C giving the desired enantiomer 23 as an off—white solid (66.8 g, 45% yield; 99.8% HPLC purity, 99+% ee). Analytical chiral HPLC retention time was 7.7 min (CHIRALPAK® IC® 4.6 x 250 mm column, 1 mL/min flow rate, 30°C). The solid was suspended in 2:1 EtOH / Et20 (5 s), stirred for 10 s, collected by filtration, washed with 2:1 EtOH / EtzO, and dried under reduced pressure giving a white solid (60.6 g).

The structure and absolute stereochernistry of 23 were confirmed by single-crystal x-ray diffraction analysis. Single crystal diffraction data was acquired on a Bruker Apex ll diffractometer equipped with sealed tube Cu K-alpha source (Cu K0. radiation, 7 = 1.54178 A) and an Apex II CCD detector. A crystal with dimensions of 0.15 x 0.15 x 0.10 mm was selected, cleaned using mineral oil, mounted on a MicroMount and centered on a Bruker APEXII system. Three batches of 40 frames separated in reciprocal space were obtained to provide an ation matrix and initial cell parameters. Final cell parameters were obtained and refined after data collection was completed based on the full data set. Based on systematic absences and intensities tics the ure was solved and refined in acentric P21 space group. - 46 _ PCT/U82012/021280 A diffraction data set of ocal space was obtained to a tion of 0.85 A using 05° steps using 30 s exposures for each frame. Data were collected at 100 (2) K.

Integration of intensities and refinement of cell parameters were accomplished using APEXII software. Observation of the crystal after data collection showed no signs of decomposition.

As shown in Fig. 2, there are two symmetry independent molecules in the structure and both symmetry independent molecules are R isomers.

The data was collected, refined and d using the Apex 11 software. The structure was solved using the Q7 (Sheldrick, 1990); program(s) and the structure refined using the SHELXL97 (Sheldrick, 1997) program. The crystal shows inic cell with P21 space group. The e parameters are a = 9.9016(2) A b = 10.9184(2) A, c = 19.2975(4) A, p: 102.826(1)°. Volume = 2034.190) A3.

Example 1.j Preparation of the esulfonic acid salt 1-ethyl-3 —[6-fluoro[2-( 1 -hydroxy- l ~methyl-ethyl)pyrimidin-5 -yl]-7 - [(2R)-tetrahydrofuran— 2-yl]-lH—benzimidazol—Z-yl]urea (24).

M6803H DCM, EtOH 2°C - rt To a stirring suspension of 1-ethyl[6-fluoro[2-(1—hydroxymethyl- ethyl)pyrimidin—5 ~yl][(2R)—tetrahydrofuran~2~yl]—lH—benzimidazol-Z-yl]urea (23) (15 .05 g, 35.13 mmol) in dichloromethane (60 mL, J.T. Baker 931533) and absolute ethanol (15 mL, Pharmco-AAPER 111000200) was added methanesulfonic acid (2392 mL, 36.89 mmol, Sigma-Aldrich 471356). Stirred at room temperature until a clear solution was observed.

Added heptane (300 mL) slowly over about 1 hr and collected the solid precipitate by filtration (using a Whatman ative # 3 paper on top of a Whatman GF/F glass microfibre W0 20121097273 PCT/U52012/021280 paper). Dried under reduced pressure in a vacuum oven (desiccated with calcium sulfate and potassium hydroxide) overnight at 40°C giving 24 as a white solid (13.46 g, 99+% HPLC purity, 99+% ee). Analytical chiral HPLC shows one enantiomer with retention time of 8.6 min eluting with CHzClz / MeOH / TBA (60 / 4O / 0.1) on a CHIRALPAK® IC® 4.6 x 250 mm column with 1 mL/min flow rate at 30°C. A second crop of white solid product 24 (4.36 g, 98% HPLC purity, 99+% ee) was obtained from the filtrate. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 429.58 (2.03 min). 1H NMR (300 MHZ, MeOD) 5 9.00 (d, J = 1.6 Hz, 2H), 7.67 (d, J = 6.1 HZ, 1H), 5.39 (t, J = 7.7 HZ, 1H), 4.30 (dd, J = 14.9, 6.9 Hz, 1H), 4.03 (dd, J =14.8, 7.7 Hz, 1H), 3.40 — 3.31 (m, 2H), 2.72 (s, 3H), 2.70 — 2.60 (m, 1H), 2.21 — 2.08 (m, 2H), 1.98 —1.84 (m, 1H), 1.65 (s, 6H), 1.22 (t, J = 7.2 Hz, 3H) ppm.

Example 1k Preparation of 1-ethy1—3—[6-fluoro[2—(1—hydroxy-1—methyl-ethyl)pyrimidin—5 -yl] tetrahydrofuran-Z-yl-lH—benzimidazol-Z—yl]urea To a solution of 2-[5-(4,5-diaminofluor0tetrahydrofuranyl- phenyl)pyrimidin—2-yl]propan-Z-ol (7.220 g, 21.72 mmol) and 1-ethyl(N- (ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)urea (6.054 g, 26.06 mmol, CB Research and pment) in 1,4-dioxane (36.1 mL, Aldrich 360481) was added a pH 3.5 buffer (72.2 mL), prepared by dissolving NaOAc trihydrate (5.32 g) in IN aqueous H2S04 (37 mL). The reaction mixture was stirred at reflux overnight (HPLC showed complete sion), cooled to room temperature, and poured n-wise (frothing) into a stirring solution of aqueous saturated NaHCO3 (144 mL) giving pH 8-9. This was d for 20 minutes, the solid was collected by filtration, washed copiously with water to neutral pH, and then more sparingly with EtOH. The solid was dried under reduced pressure giving a beige solid (7.90 g, 99% HPLC ). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1z 429.45 (2.03 min). HPLC retention time was 3.89 min (YMC ODS—AQ 150 x 3.0 mm column eluting with 10-90% CH3CN/ water gradient over 8 minutes with 0.1% TFA modifier and 1 mL/min flow rate).

PCT/U82012/021280 Preparation of Form I Example 1.1 Chiral chromatographic isolation of (R)ethyl[6-fluoro[2-(1-hydroxy—1 -methy1- ethyl)pyrimidinyl]—7-(tetrahydrofuran—2—y1]-1H-benzimidazol-2—yl]urea A racernic sample of 1-ethyl-3—[6-fluoro[2—(1 -hydroxymethy1— pyrimidin—5-y1]—7-tetrahydrofuranyl—1H—benzimidazol-2—yl]urea (133.60 g) was resolved on a CHIRALPAK® IC® column (by Chiral Technologies) eluting with DCM / MeOH / TBA (60 / 40/ 0.1) at 25°C giving the desired enantiomer as an off-white solid (66.8 g, 99.8% HPLC purity, 99+% ee). Analytical chiral HPLC retention time was 7.7 min (CHIRALPAK® IC® 4.6 x 250 mm column, 1 mL/min flow rate, 30°C). The solid was suspended in 2:1 EtOH/ EtzO (5 volumes), stirred for 10 minutes, collected by filtration, washed with 2:1 EtOH / EtZO, and dried under d pressure giving a white solid (60.6 g). 1H NMR (300 MHz, MeOD) 5 8.95 (d, J = 1.6 Hz, 2H), 7.45 (d, J = 6.5 Hz, 1H), 5.38 (br.s,1H), 4.27 (dd, J = 14.9, 7.1 Hz, 1H), 4.01 (dd, J = 15.1, 7.0 Hz, 1H), 3.37 — 3.29 (m, 2H), 2.55 (brs, 1H), 2.19 — 2.07 (rm 2H), 2.02 — 1.82 (br.s, 1H), 1.63 (s,6H),1.21 (t, J = 7.2 Hz, 3H) ppm.

Preparation of Form 11 Example 1m To 100 mg of the 6-fluoro benzimidazolyl urea compound 1 ml of THF was added. A stoichiometric amount of HCl was added as 21 12M aqueous solution. Then 4 mL of MTBE was added and the suspension was allowed to equilibrate ght with stirring at room temperatureIt was then filtered, and the white solid was dried under vacuum for several hours.

Preparation of Form III Example In ] 100 mg of o benzimidazolyl urea compound was weighed out and dissolved in 200 mL dichloromethane/methanol 1:1 (vzv) mixture. This solution was spray dried on the Buchi B-90 Nano spray dryer (pump m 2) with a ser attached at spray rates of 100%. Inlet temperature of 101°C was used with a nitrogen flow of 10 L/min, a nitrogen maximum pressure of 10 psi and a maximum C02 pressure of 15 psi. 55 mg of white powder was recovered.

Spray drying was performed on the Buchi B-90 Nano spray dryer with a condenser attached. A solution of the the 6-fluoro benzimidazolyl urea compound was PCT/U52012/021280 prepared in a solvent system comprised of CHgClzzMethanol (1:1) and sprayed according to the parameters listed below. ation of F01m IV Example 1.0 Preparation of the methanesulfonic acid salt (R)-l-ethy1[6-fluoro[2-(l-hydroxy methyl—ethyl)pyrimidin-5—y1]-7—(tetrahydrofuranyl)-lH—benzimidazol-Z-yl]urea A ng suspension of (R)-l-ethyl-3—[6—fluoro—5-[2-(1-hydroxy-1—methyl- ethy1)pyrimidin—5 -yl]-7—(tetrahydrofuran—2-yl)- l H-benzimidazol-Z-yl]urea(2.530 g, 5.905 mmol) in romethane (22.8 mL, Sigma-Aldrich 270997) and absolute ethanol (2.5 mL) was cooled with an ice—water bath. Methanesulfonic acid (0.402 mL, 6.20 mmol, Sigma- Aldrich 471356) was added, removed the cold bath, and stirred at room temperature for 10 minutes. The mixture was concentrated on a rotary evaporator at 30°C to a thick oil, then added slowly to stirring Eth, and rinsed the residual t with CHzClz into the ether. The gummy precipitate was stirred until it broke up into a pasty solid, which was collected by filtration, washed with EtzO, and dried under reduced pressure giving an off-white solid (2.85 g, 99% HPLC , 99+% ee). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+l: 429.51 (2.49 min). HPLC retention time was 3.86 min (YMC ODS-AQ 150 x 3.0 mm column eluting with 10-90% CH3CN/ water gradient over 8 minutes with 0.1% TFA modifier and 1 mL/min flow rate). Analytical chiral HPLC shows one omer with retention time of 7.8 min eluting with DCM/ MeOH / TEA (60 / 40 / 0.1) on a CHIRALPAK® IC® 4.6 x 250 mm column with 1 mL/min flow rate at 30°C. 1H NMR (300 MHz, MeOD) 5 8.99 (d, J = 1.6 Hz, 2H), 7.67 (d, J = 6.1 Hz, 1H), 5.38 (t, J = 7.7 Hz, 1H), 4.30 (dd, J = 15.0, 6.9 Hz,1H), 4.02 (dd, J = 14.8, 7.6 Hz, 1H), 3.38 — 3.30 (m, 2H), 2.73 (s, 3H), 2.70 — 2.60 (m, 1H), 2.20 - 2.07 (m, 2H), 1.99 — 1.84 (m, 1H), 1.64 (s, 6H), 1.22 (t, J = 7.2 Hz, 3H) ppm Example l.p STABILITY DATA The te salt of the 6-fluorobenzimidazolyl urea compound was found to be chemically and physically unstable at 25°C/60%RH at the one week time point, and chemically unstable at t=2 weeks when stored at 40°C/ambient.

The free base 6-fluoro idazolyl urea compound was chemically and physically stable under all storage conditions (25°C/60%RH, 40°C/ambient, and WO 97273 40°C/75%RH) at the 1 month timepoint. Small changes were observed in the XRPD pattern, but all wereconsidered to be the same form as at time zero (F0).

The hydrochloride salt of the 6-fluoro benzimidazolyl urea nd was chemically and physically stable under all storage conditions (25°C/60%RH, 40°C/ambient, and 40°C/75%RH) at the 1 month timepoint.

Example 2 ENZWOLOGY STUDIES The enzyme inhibition activities of compounds of this invention may be ined in the experiments bed below: DNA Gyrase ATPase Assay The ATP hydrolysis activity of S. aureus DNA gyrase is measured by coupling the production of ADP through pyruvate kinase/lactate dehydrogenase to the ion of NADH. This method has been described previously (Tamura and t, 1990, J. Biol.

Chem, 265, 21342).

ATPase assays are carried out at 30°C in ed solutions containing 100 mM TRIS pH 7.6, 1.5 mM MgC12, 150 mM KCl. The coupling system contains final concentrations of 2.5 mM oenol te, 200 uM nicotinamide adenine dinucleotide (NADH), 1 mM DTT, 30 ug/ml pyruvate kinase, and 10 ug/ml lactate dehydrogenase. The enzyme (90 nM final concentration) and a DMSO solution (3 % final concentration) of a compound is added. The reaction mixture is allowed to incubate for 10 minutes at 30°C. The reaction is initiated by the addition of ATP to a final concentration of 0.9 mM, and the rate of NADH disappearance is monitored at 340 ters over the course of 10 s. The K and ICso values are determined from rate versus concentration profiles.

Table 3. Inhibition of S. aureus DNA Gyrase Selected Compound Ki (nM) Compound 23* 9 *Compound 23 may be prepared as in Example l.i, above.

DNA Topo IV ATPase Assay The conversion ofATP to ADP by S. aureus TopoIV enzyme is coupled to the conversion ofNADH to NAD+, and the progress of the reaction is measured by the change in absorbance at 340 nm. TopoIV (64 nM) is incubated with the selected compound (3% DMSO final) in buffer for 10 minutes at 30 °C. The buffer consists of 100 mM Tris 7.5, 1,5 mM MgC12, 200 mM K-Glutamate, 2.5 mM phosphoenol pyruvate, 0.2 mM NADH, 1 mM DTT, 5 pg/mL linearized DNA, 50 ug/mL BSA, 30 ug/mL te kinase, and 10 pg/mL lactate dehyrodgenase (LDH). The reaction is initiated with ATP, and rates are monitored continuously for 20 minutes at 30°C on a Molecular Devices SpectraMAX plate reader. The inhibition constant, Ki, and the leo are determined from plots of rate vs. concentration of selected compound fit to the Morrison Equation for tight binding inhibitors.

Table 4. Inhibition of S. aureus DNA Topo IV Selected Compound Ki (nM) Compound 23 12 Example 3 Susceptibility Testing in Liquid Media ] Compounds of this invention were tested for antimicrobial activity by susceptibility testing in liquid media. Such assays can be performed within the guidelines of the latest CLSI document governing such practices: 8 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard-- Eighth Edition (2009)". Other publications such as "Antibiotics in Laboratory ne" (Edited by V. Lorian, hers Williams and Wilkins, 1996) provide essential practical ques in laboratory antibiotic testing. The specific protocols used were as follows: ol #1: Gyrase MIC determination of compounds using ilution broth method ] Materials: Round bottom 96~well microtiter plates r 3788) Mueller Hinton II agar plates (MHII; BBL premix) Mueller Hinton II liquid broth (MHII; BBL premix) BBL Prompt Inoculation System (Fisher B26306) Test Reading Mirror (Fisher) Agar plates with bacteria streaked to single colonies, freshly prepared Sterile DMSO Human serum (US. Biologicals 81010-51) Laked horse blood (Quad Five 0) Resazurin 0.01% Sprague Dawley Rat serum (U.S. Biologicals 1011-90B or Valley BioMedical AS3061 SD) PCT/U82012/021280 Pooled Mouse serum (Valley ical AS3054) Strains (media, broth and agar): 1. Staphylococcus aureus ATCC #29213 a. MHII b. MHII + 50% human serum c. MHII + 50% rat serum d. MHII + 50% mouse serum 9.0).“:99’!“ Staphylococcus aureus ATCC #29213 GyrB T1731 (MHII) Staphylococcus aureus, JMI collection strains; see table 9 (MHII) Staphylococcus epidermia’is, JMI collection strains; see table 9 (MHII) Enterococcusfaecalis ATCC #29212 (MI-111+ 3% laked horse blood) Enterococcusfaecz’um ATCC #49624 (MHII + 3% laked horse blood) Enterococusfaecalz’s, JMI collection strains; see table 9 (MHH + 3% laked horse blood) 8. Enterococusfaecz‘um, JMI collection s; see table 9 (MHII + 3% laked horse blood) 9. Streptococcus niae ATCC #10015 (MHII + 3% laked horse blood) . Streptococcus niae, JMI collection strains; see table 9 (Ml-III + 3% laked horse blood) 11. B-haemolytz‘c streptococci, Groups A, B, C, G) JMI collection s; see table 9 (MI-III + 3% laked horse blood) 12. Bacillus cereus ATCC 10987 (MHH) 13. Bacillus cereus ATCC l4579(MHH) 14. Bacillus subtilis ATCC 6638 (MHII) . Bacillus subtilis (168) ATCC 6051 (MHII) um prep (for all strains other than S. aureus + 50% sera): 1. Using the BBL Prompt kit, picked 5 big or 10 small, well separated colonies from culture grown on the appropriate agar medium as indicated above and ated 1 mL of sterile saline provided in the kit. 2. Vortexed the wells for ~ 30 s to provide a suspension of ~108 cells/mL. Actual density could be confirmed by plating out dilutions of this suspension. 3. Diluted the suspension 1/100 by transferring 0.15 mL of cells into 15 mL (~106 cells/mL) sterile broth (or see below) for each plate of compounds tested, then swirled to -53_ WO 97273 PCT/U52012/021280 mix. If more than 1 plate of compounds (> 8 compounds), including compound 23 or 24,were tested, volumes were increased accordingly. a. For E. faecalis, E. faecz’um and S. pneumoniae: 14.1 mL MHII + 0.9 mL laked horse blood was used. 4. Used 50 ul cells (~5 x 104 cells) to inoculate each microtiter well containing 50 ul of the drug diluted in broth (see below).

Drug dilutions, inoculation, MIC determination: 1. All drug/compound stocks were prepared at 12.8 mg/mL concentration, usually in 100% DMSO. d drug/compound stocks to 200K desired final concentration in 50 uL DMSO.

If ng concentration of MICs was 8 ug/mL final concentration, then required 625 uL of stock + 43.75 uL DMSO. Each 200): stock was placed in a separate row of column 1 of a new 96 well microtiter plate. p.) Added 25 uL ofDMSO to columns 2 -12 of all rows of the microtiter plate containing 200x compound stocks and serially diluted 25 uL from column 1 through column 11, changed tips after each column. i.e. 25 uL compound + 25 uL DMSO = 2x dilution.

Left “no compound” DMSO well at the end of the series for control.

For each strain tested (except S. aureus + 50% human serum), prepared two microtiter plates with 50 uL of MI-HI broth using a Matrix pipettor.

Transferred 0.5 uL of each dilution rix auto-pipettor) to 50 uL of /microtiter well prior to the addition of 50 ul of cells. The usual starting concentration of compound was 8 ug/mL after the 1/200 dilution into medium + cells ~ nd concentrations decreased in 2X steps across the rows of the microtiter plate. All MICs were done in duplicate.

All wells were inoculated with 50 ul of diluted cell suspension (see above) to a final volume of 100 pl.

Afler inoculum was added, mixed each well thoroughly with a manual hannel pipettor; same tips were used going from low to high concentration of drug in the same microtiter plate.

Plates were incubated at 37°C for at least 18 hours.

Plates were viewed with a test reading mirror after 18 hours and the MIC was recorded as the lowest concentration of drug where no growth was ed (optical clarity in the well).

PCT/U52012/021280 Preparation of S. aureus + 50% human serum, S. aureus + 50% rat serum or S. aureus + 50% mouse serum. 1. Prepared 50% serum media by combining 15 mL of MHII + 15 mL human serum — total 30 mL. Increased volume in 30 mL increments when more than 1 compound plate was tested.

Used the same BBL Prompt inoculum ofS. aureus ATCC #29213 as described above, diluted 1/200 by transferring 0.15 mL of cells into 30 mL (~5x105 mL) of the 50% human serum media prepared above and swirled to mix.

Filled all test wells of the desired number of iter plates with 100 uL cells in 50% serum media Transferred 0.5 uL of each compound dilution (w/Matrix auto-pipettor) to 100 uL of cells/media. The usual starting tration of compound was 8 ug/mL after the 1/200 on into medium + cells — compound concentrations decreased in 2x steps across the rows of a microtiter plate. All MICs were done in duplicate.

Mixed each well thoroughly with a manual multichannel pipettor; same tips were used going from low to high concentration of drug in the same microtiter plate.

Plates were incubated at 37°C for at least 18 hours After tion, added 25 iiL of 001% Resazurin to each well and continued to incubate at 37°C for at least 1 additional hour or until the Resazurin color changes.

Plates were Viewed with a test reading mirror and the MIC was ed. When using Resazurin, the color of the dye changed from a dark blue to a bright pink in wells with no growth. The lowest concentration of drug that turned the dye pink was the MIC.

Protocol 2: Gyrase MIC determination of compounds against Gram negatives using ilution broth method Materials: Round bottom 96-well microtiter plates (Costar 3788) Mueller Hinton II agar plates (MHII; BBL premix) Mueller Hinton II liquid broth (MHII; BBL premix) BBL Prompt ation System (Fisher b26306) Test Reading Mirror (Fisher) Agar plates with bacteria streaked to single colonies, freshly prepared Sterile DMSO PCT/U52012/021280 Strains (MHII media for all; broth and agar): 1. Escherichia coli ATCC # 25922 2. Escherichia 0012', M1 collection strains, see table 9 3. ichia coliAGl 00 WT 4. Escherichia coli AG100 tolC . Acinetobacter baumannii ATCC # BAA-1710 6. Acinetobacter baumanm'z' ATCC # 19606 7. Acinetobacter baumannii, JMI collection strains, see table 9 8. Klebsiella pneumoniae ATCC # BAA-1705 9. Klebsiella pneumoniae ATCC # 700603 . Klebsiella pneumoniae, JMI collection strains, see table 9 11. Moraxella catarrhalis ATCC# 25238 12. Moraxella catarrhalz‘s ATCC# 49143 13. Moraxella catarrhalis, JM1 collection strains, see table 9 14. Haemophiius z'nfluenzae ATCC 49247 . Haemophilus influenzae (Rdl KW20) ATCC 51907 16. Haemophilus influenzae Rd0894 ) l7. Haemophilus influenzae, JMI tion strains, see table 9 18. Pseudomonas aerugz’nosa PAOl l9. Pseudomonas aeruginosa, JMI collection strains, see table 9 . Proteus mirabilis, JMI tion strains, see table 9 21. Enterobacter cloacae, JMI tion strains, see table 9 22. Stenotrophomorzas maltophz‘lia ATCC BAA—84 Stenotrophomonas maltophz’h’a ATCC13637 Inoculum prep: 1. Using the BBL Prompt kit, picked 5 big or 10 small, well separated colonies from cultures grown on agar medium and inoculated 1 mL e saline that came with the kit. 2. ed the wells for ~ 30 s to give a suspension of ~108 cells/mL. Actual density could be confirmed by plating out dilutions of this suspension. 3. Diluted the sion 1/100 by transferring 0.15 mL of cells into 15 mL (~106 cells/mL) sterile broth (see below) for each plate of compounds tested, swirled to mix.

PCT/U82012/021280 If more than 1 plate of compounds (> 8 compounds), including compound 23 or 24,was to be tested, sed volumes accordingly. 4. Used 50 ul cells (~5 x 104 cells) to inoculate each microtiter well containing 50 ul of the drug diluted in broth (see below).

Drug dilutions, inoculation, MIC determination: 1. All drug/compound stocks were prepared at 12.8 mg/mL concentration, usually in 100% DMSO. 2. Diluted drug/compound stocks to 200x desired final concentration in 50 uL DMSO.

If starting tration of MICs was 8 ug/mL final concentration, then required 6.25 uL of stock + 43.75 uL DMSO. Each 200x stock was placed in a separate row of column 1 of a new 96 well microtiter plate 3. Added 25 uL ofDMSO to columns 2 -12 of all rows of the iter plate containing 200x compound stocks and serially diluted 25 uL from column 1 through column 11, d tips after each column. i.e. 25 uL nd + 25 uL DMSO = 2x dilution.

Left “no compound” DMSO well at the end of the series for control. 4. For each strain tested, prepared two microtiter plates with 50 uL of MHII broth using a Matrix pipettor. . erred 0.5 uL of each dilution (w/Matrix auto-pipettor) to 50 uL of medium/microtiter well prior to the addition of 50 ul of cells. The usual starting concentration of compound was 8 ug/InL after the 1/200 dilution into medium + cells — compound concentrations decreased in 2x steps across the rows of a microtiter plate.

All MICs were done in duplicate. 6. A11 wells were inoculated with 50 ul of diluted cell suspension (see above) to a final volume of 100 pl. 7. After inoculum was added, each well was mixed thoroughly with a manual multichannel pipettor; same tips were used going from low to high concentration of drug in the same microtiter plate. 8. Plates were incubated at 37°C for at least 18 hours. 9. Plates were viewed with a test g mirror after 18 hours and the MIC was recorded as the lowest concentration of drug where no growth was observed al clarity in the well).

W0 20121097273 PCT/U52012/021280 Protocol #3: Gyrase MIC determination of compounds using Agar dilution method Materials: Petri plates 60 x 15 mm (Thermo Scientific Cat. # 12567100) Centrifuge tubes, 15 mL (Costar) BBL Prompt Inoculation System (Fisher b26306) Agar plates with bacteria streaked to single colonies, freshly prepared Sterile DMSO GasPakTM incubation containers (BD Cat. #260672) GasPak TM EZ Anaerobe container system sachets (BD Cat. #260678) GasPakTM EZ C02 container system sachets (BD Cat. #260679) GasPak TM EZ Campy ner system sachets (BD Cat. 0) Strains: 1, Clostridium ‘le ATCC BAA-1382; 2. Ciosn‘idium difiicile, CMI collection strains, see table 8 3, Clash-indium pelfi'ingens, CMI collection strains, see table 8 4 4 Bacteroz‘desflagilis and Bacteroides 3191)., CMI collection strains, see table 8 . Fusobacterz‘um spp., CMI collection strains, see table 8 6. Peptostreptococcus, spp., CMI collections strains, see table 8 7. Prevotella , CMI collection strains, see table 8 8. N. gonorrhoeae ATCC 35541 9. N. hoeae ATCC 49226 . Neisseria gonorrhoeae, JMl tion strains, see table 8 11. Neisseria meningitidis, JMI collection strains, see table 8 Media preparation and growth conditions: Growth medium recommended for each ial s was prepared according tothe CLSI publication ‘Ml 1-A7 Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard - Seventh Edition (2007)’ with the exception of N. gonorrhoeae and N. meningitidisfor which media was prepared according to"MO7-A8 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard-Eighth Edition (2009)".

W0 2012I097273 2012/021280 Plate pouring: 1. Prepared 100x drug stocks of each test compound as described in Table 1. Used a 15 mL centrifuge tube, added 100 uL of each drug stock to 10 mL of molten agar d to ~ 55°C in water bath). Mixed by inverting tube 2 -3x then pour into individually labeled 60X15 mm Petri dish. e test concentrations were: 0.002 ug/mL — l6 ug/mL (14 plates).

Poured 4 drug free plates: 2 as positive l, 2 as aerobic control.

Allowed plates to dry. Used same day or stored overnight at RT or stored up to 3daysat 4°C.

Plates were labeled accordingly for drug tration and strain placement.

Growth of cells requiring the maintenance of an anaerobic nment: 1. All work performed with anaerobic bacteria was done as rapidly as possible; work med in biosafety cabinets (i.e., aerobic environment) was completed in less then minutes before cells were ed to anaerobic chambers.

Incubation of anaerobic bacteria was achieved using GasPakTM chambers. The large box style chambers (VWR 90003-636) required 2 anaerobic sachets (VWR 90003- 642), while the tall cylinder style chambers (VWR 90003-602) only required 1 sachet.

Plate inoculation (performed in biosafety cabinet): 1. Streaked each strain onto dual agar plates as described above. Incubated for required time and environmental condition (ie. anaerobic, microaerophilic, etc).

Used direct colony suspension method to suspend loopfuls of y streaked cells into ~ 4 mL 0.9% NaClz and vortexed.

Adjusted suspension to O.D.5oo 0.05 (5x10e7 cfu/mL). Vortexed to mix.

Transferred ~O.2 mL of adjusted, mixed cultures to a 96 well plate. When 5 5 strains were tested, all strains were lined together in a single row. When testing > 5 strains, transfered strains into plate with no more that 5 strains in a single row. This was necessary to fit on the small plates.

Used multi-channel pipettor, spotted 0.002 mL of each strain from prepared 96 well plates onto each MIC test plate. This resulted in ~ lxlOeS ot. When testing C. dzfiicile, strains swarmed when grown, however distance between channel pipettor spots was far enough such that swarming cells did not impair assay results. a. InoculatedZ drug free plates first, while the other 2 drug free plates were inoculated last after the MIC test plates. The former and latter served as W0 2012IO97273 PCT/U82012/021280 growth and inoculation controls. Incubated one plate from each set of drug- free controls under required atmospheric conditions with MIC plates and one set aerobically to test for contamination with aerobic bacteria. Aerobic culture was negative for growth when working with strict be or rnicroaerophilic strain. Some growth was Visible with N. hoeae. 6. Allowed inoculum to dry (for as short a time as necessary), then placed upside down in GasPak with appropriate number of sachets and incubate. 7. Neisseriasppwere ted at 37°C in 215% COzenvironment for 24h.

] MIC determination: Examined the test plates after the correct tion time and read the MIC endpoint at the concentration where a marked reduction occurred in the appearance of growth on the test plate as compared to that of growth on the positive control plates.

Table 5: Compound dilutions for MIC determination using the agar dilution method.

Final Volume Volume Diluent, Intermediate Conc. (uL) added from stock . At 1: 100 1,600 Stock 1,600 Stock 1,600 Stock *1,600 ug/ml= 64 ul (10mg/ml stock) + 336 111 DMSO; 400 ul total volume to start **compound dissolved and dilutedin 100% DMSO PCT/U52012/021280 Protocol #4. MIC ination Procedure for Mycobacterimn species Materials Round bottom 96-well microtiter plates (Costa: 3788) or similar Film plate seals (PerkinElmer, TopSeal-A #6005250 or similar) brook 7H10 broth with 0.2% glycerol brook 7H10 agar with 02% glycerol brook OADC Enrichment Inoculum Preparation forM ulosis: 1. Used ed M tuberculosis stock stored at -700C. M fuberculosiswas grown in 7H10 broth + 10% OADC, then frozen at a concentration of 100 Klett or 5 X 107cfu/ml, 2. Prepared a 1:20 dilution by removal of 1 m1 of the frozen stock and added it to 19 ml of 7H10 broth + 10% OADC (final concentration 2.5 x 106cfu/ml). .0.) From this dilution prepared a second 1:20 dilution, d 1 ml and added it to 19 ml of fresh broth. This was the final inoculum to add to the 96-well plates.

Inoculum Preparation forM kansasii, M avium, M abscessus and Nocardia spc.: 1. Used prepared frozen stock of culture or a fresh culture grown in 7H10 broth at a concentration of 10 Klett or 5 x 107/ml. 2. Prepared a 1:20 dilution by ng 1.0 ml of the culture stock and added it to 19 ml of 7H10 broth (final concentration 2.5 X 106cfu/ml). 3. From this dilution prepared a 1:20 dilution, removed 1 m1 and added it to 19 ml of fresh broth (final suspension).

Plate Preparation: 1. Labeled plates. 2. Added 50 ul of 7H10 broth + 10% OADC to all wells being utilized for MIC determination using a multichannel electronic pipettor. 3. Prepared stock solutions of drugs (e. g. 1 mg/ml concentration) to be tested. 4. Thawed and diluted frozen stock solutions using 7H10 broth + 10% OADC to obtain a working solution 4x the maximum concentration tested (e. g. final concentration 32 pig/ml, highest concentration tested was 8 [Lg/ml). Dilutions were made from the stock solution. To start at a concentration of 1 gig/m1, the drugs were prepared at 4 rig/ml, so PCT/U82012/021280 the starting concentration was 1 ug/ml. Removed 25 pl of the lmg/ml stock and added to 6.2 ml of broth. All dilutions of drugs were done in broth.

. Added 50 ul of the 4x working solution to the first well of the designated row.

Continued for all compounds to be tested. Using a multichannel electronic pipettor, mixed 4X and serial diluted compounds through the 11th well. Discarded remaining 50 ul. Used the 12th well as the positive control. 6. Incubated plates at 37° C M tuberculosis for ~18 days;M avium and M ii for ~7 days; Nocardia andM abcessus for ~4 days; with film seals. 7. Read visually and recorded the results. The MIC was recorded as the lowest concentration of drug where no growth was observed al clarity in the well).

Protocol #5. ol for Mycobacterium tuberculosis Serum Shift MIC Assay Materials and reagents: Costar #3904 Black-sided, flat-bottom 96—well microtiter plates Middlebrook 7H9 broth (BD271310) with 0.2% glycerol Middlebrook OADC Enrichment Fetal Bovine Serum Catalase (Sigma C1345) Dextrose NaClz BBL Prompt Inoculation System (Fisher b26306) Agar plates (Middlebrook 71-111 with 0.2% glycerol and OADC ment) with ia streaked to single colonies e DMSO Media prep: 1. For serum shifted MICs, three different media were required which all had a base of 7H9 + 0.2% glycerol. It was important that all media and supplements were ized prior to MICs. 2. Prepared all media below and inoculated as described in next section. Tested all compounds against Mtb using each media. a 7H9 + 0.2% glycerol + 10% OADC (“standard” MIC media). b.7H9+O2%ngml+2ngaume+085gLNMH+OmBgflwmflme fifléFBS) W0 2012(097273 PCT/U82012/021280 c. 2x 7H9 + 0.2% glycerol + 2 g/L dextrose + 0.85 g/L NaCl + 0.003 g/L catalase combined with equal volume Fetal Bovine Serum (50% FBS).

Inoculum prep: 1. Using BBL Prompt, 5-10 well—separated colonies and inoculated 1 ml sterile saline that came in the kit. Typically plates were two to three weeks of age when used for this assay due to the slow growth of this organism in e.

Vortexed well, then sonicated in water bath for 30 sec providing a suspension of ~108 cells/ml. Actual density could be confirmed by plating out dilutions of this suspension.

Prepared inoculum in each of the three media formulations by diluting the BBL Prompt suspension 1/200 (for example: erred 0.2 ml of cells to 40 ml of medium) to obtain a starting cell density of ~106 cells/ml.

Used 100 pl cells (~5 x 104 cells) to inoculate each microtiter well containing 1 pl of drug in DMSO (see below).

Drug dilutions, inoculation, MIC determination: 1. Control drug stocks lsoniazid and ocin were prepared at 10 mM in 100% DMSO while Ciprofloxacin and Rifampin were prepared at 1 mM in 50% DMSO and 100% DMSO, respectively. Prepared dilutions- sed 100 uL of the stock solution into the first column of a l plate. Prepared ll-step, 2-fold serial dilutions across the row for each nd by transferring 50 ill from column 1 into 50 ul of DMSO in column 2. Continued to transfer 50 uL from column 2 through column 11 while mixing and changing tips at each column. Left column 12 with DMSO only as a control.

Transferred 1 ul of each dilution into an empty microtiter well prior to the addition of 100 pl of cells. The starting tration of Isoniazid and Novobiocin was 100 uM after the dilution into medium + cells; the starting concentration of Ciprofloxacin and Rifampin was 10 uM after the dilution into medium + cells. Compound concentrations decreased in 2): steps moving across the rows of the microtiter plate.

All MICs were done in duplicate at each of the three medium conditions.

Test sets of compounds were typically at 10 mM and 50 uL volume.

Used at multichannel pipettor, removed all of the volume from each column of the master plate and transferred into the first column of a new 96-well microtiter plate.

W0 20121097273 Repeated for each column of compounds on master plate, transferring into column 1 of a new 96—well plate.

. As described above for control compounds, generated 2-fold, 11-point ons of each compound using DMSO as diluent. In all cases, left column 12 as DMSO only for a control, Once all dilutions were complete, again erred 1 ul of each dilution into an empty microtiter well prior to the addition of 100 pl of cells as done for the control compounds. 50909.0 All wells were inoculated with 100 pl of diluted cell suspension (see above).

After inoculum addition, mixed plates by gently g sides of plate.

Plates were incubated in a humidified 37°C chamber for 9 days.

At 9 days added 25 ul 0.01% sterile resazurin to each well. Measured background fluorescence at Excitation 492 nrn> Emission 595 nm and returned the plate to the tor for another 24 hours.

. After 24 hours the fluorescence of each well was measured at Excitation 492 nm, Emission 595 nm. 11‘ Percent inhibition by a given compound was calculated as follows: Percent inhibition=100-([well fluorescence-average background fluorescence]/[DMSO control -average background fluorescence] x100). MICs were scored for all three medium conditions as the lowest compound concentration that ted resazurin reduction hibition’) signal 270% at a given medium ion.

Table 6 shows the results of the MIC assay for the mesylate salt of the benzimidazolyl urea compound of this ion.

In Table 6 and in subsequent Tables and Examples, und 24” is amesylate salt of “Compound 23” and may be prepared according to Example l.j, above. Thisis the same number used to identify said compound as used in the Examples above.

Table 6 - MIC Values of Compound 24 Strain/Special Condition Protocol Compound Staphylococcus aureus 1 O. 021 ATCC 29213 Staphylococcus aureusATCC 1 0.15 29213 with Human Serum Staphylococcus aureus 1 0.18 ATCC 29213 with Rat Serum Staphylococcus aureus 1 0.5 ~64- WO 97273 ATCC 29213 with Mouse Serun1 Staphylococcus aureus 0.3 ATCC 29213 GyrB T1731 Enterococcusfaecalis ATCC 0.028 29212, with Laked Horse Blood Enterococcusfaecium ATCC 0.11 49624 with Laked Horse Blood Emerococcusfaecium ATCC 0.11 49624 Streptococcus pneumoniae 0.01 ATCC 10015, with Laked Horse Blood Bacillus cereus ATCC 10987 0.031 Bacillus cereus ATCC 14579 0.031 Bacillus subtilis ATCC 6638 Bacillus subtilis (168) ATCC 605 1 Closn'idz‘um ile ATCC 0.38 BAA-1382 Haemophilus influenzae 0.5 ATCC 49247 Haemophilus influenzae (Rdl 1.3 KW20) ATCC 51907 Haemophilus influenzae 0.041 Rd0894 (AcrA—) Moraxella halis ATCC $0.016 25238 Moraxella catarrhalis ATCC 30.016 49143 ria gonorrhoeaeATCC 0.42 35541 Neisseria gonorrhoeae ATCC 49226 Escherichia coli AG100 WT Escherichia coli AG100 tolC 0.063 Escherichia coli ATCC NNN 12 25922 Escherichia coli CHE30 Escherichia coli CHE30 tolC 0.125 Escherichia coli MC4100 >16 Escherichia coli MC4100 NNNN 0.25 tolC Klebsiella pneumoniae N 16 ATCC 700603 Klebsiella pneumoniae 12 ATCC BAA-1705 Acinetobacter baumannii ~65- ATCC 19606 Acinetobacter baumannii ATCC BAA-1710 monas aeruginosa PAOl Pseudomonas aeruginosa PAO7fl) Stenotrophomonas maltophz’lia ATCC BAA-84 Stenotrophomonas N lzz’lia ATCC13637 Mycobacterium avium 103 0.18 A4 aWuniFar 0.23 A1 aWun134044 0.23 Abcanfiacaw062497 0,125 N. asteroids 2039 ll nova 10 h4.kansasfi.303 0.03 M kansasii 316 0.06 M kansasii 379 h-b-h-b-h-b-b-b-ik-b <0.015 M tuberculosis H37RV 0.015 ATCC 25618 M tuberculosis Erdman J; 0.06 ATCC 35801 M tuberculosis Erdman 0.03 ATCC 35801 M tuberculosis Erdman 0.5 ATCC 35801 with Mouse Serunn M abscessus BB2 M abscessus MC 6005 M abscessus MC 5931 0.5 M abscessus MC 5605 1.5 M abscessus MC 6025 0.75 M abscessus MC 5908 1.5 M abscessus BB3 0.5 M abscessus BB4 M abscessus BB5 0.5 M abscessus MC 5922 0.25 M sus MC 5960 0.5 M abscessus BB1 M abscessus MC 5812 M sus MC 5901 M abscessus BB6 0.5 M abscessus BB8 0.5 M abscessus MC 5908 M abscessus LT 949 M abscessus BBIO 0.015 M abscessus MC 6142 b-bth-bbh-h-ka-b-b-b-BA-bhh-h-b 0.5 M abscessus MC 6136 0.5 —66- PCT/U52012/021280 M abscessus MC 6111 0.5 M abscessus MC 6153 A-D Table 7 shows the results of the MIC90 assay for selected compounds of this invention.

Table 7— MIC90 Values of Selected Compounds with Panels of Gram ve, Gram Negative and Anaerobic Pathogens Compound 24 Organism Number Protocol Range MIC90 of (lug/ml) (Hg/ml) Tested Aerobic Gram-positive Staphylococcus aureus 67 1 0.008— 003 0.06 Staphlococcus epidermidz‘s 35 1 0.008— 0.03 0.03 Enterococcus faecalis 34 1 0.015- 0.06 0. 1 2 Enterococcusfizecium 33 1 0.003- 0‘ 12 0.25 Streptococcus pneumoniae 67 1 0.008- 0,015 0.03 fl-haemolytic ococci s A= B, C and 28 1 0.015- 0.12 G) 0.12 Aerobic Gram-negative Haemophilus influenzae 55 2 0,06— 2 1 Moraxella cararrhalz's 26 2 - 0.03 0.03 Acinetobacter baumannii 12 2 4 - >8 >8 Pseudomonas aerugz'nosa 12 2 >8— >8 >8 Escherichia coli 12 2 2 - >8 >8 Klebsiellapneumoniae 12 2 2 - >8 >8 Proteus mirabz'lz's 12 2 4 - >8 >8 Enterobacter cloacae 12 2 >8- >8 >8 Neisseria gonorrhoeae 13 3 0.12- 0.25 0.25 rz'a meningitidz‘s 12 3 0.008- 0.03 0.06 Anaerobes Bacteroz‘des and Parabacter spp. 26 3 0.12- 16 16 Bacteroidesfiagz'lz's 25 3 1- 16 16 Clostrz'dz'um difiicz’le 16 3 0.06- 4 0.25 Clostrz'dium peiy‘i'z‘ngens 12 3 0.12- 0.5 0.5 Fusobacz‘erz’um spp. 16 3 0.015 — >16 >1 6 PCT/U52012/021280 trepz‘ococcus spp. 1 1 3 0103— >16 Prevotella spp. 13 3 0.06- 16 16 In Table 8 below, the term “CMI” stands for The Clinical Microbiology Institute located in Wilsonville, Oregon. -68— Table 8 Panels of Anaerobic Organism Used to Generate MIC90 Data _______ CMI# ORGANISM __‘________—_______.

LA2380 B. fiagilis A2381 B. fi‘agilz’s A2382 B. fi‘agilz’s A2486 B. fi'agz'lz's A2487 B. fragilis A2489 B. fi‘agz‘lz‘s A2527 B. fi‘agilz‘s A2529 B. fi'agz’lz’s A2562 B. fragilis A2627 B. fi'agz'lis A2802 B. fi‘agilis A2803 B. fi'agilis A2804 B. is A2805 B. fiagilz‘s A2806 B. fi‘agilis A2807 B. flagilis A2808 B. fi'agz‘lis 2012/021280 CMI# ORGANISM [———-:—-———-—-—————1 A2809 B. fragilis A2810 B. fi'agilz's A2811 B. fi'agz‘lz‘s A2812 B. fragilis A2813 B. fragilis A2814 B. fi‘agilis A2460 8 'otaomicron A2462 . B. thetaiotaomz‘cron A2463 B. thetaiotaomz'cron A2464 B. thetaiotaomicron A2536 B. thetaioraomz'cron A2591 B. unzfiormis A2604 B. vulgatus A2606 B. vulgatus A2613 B. ovatus A2616 B. ovatus A28 15 Bacteroz’des rectum A2816 B. ureolyticus PCT/U52012/021280 CMI# ORGANISM A2817 Bacteroz’des capillosus A2818 B. tz'cus A2824 Parabacter distasom’s A2825 B. ovatus A2826 B. uniformis A2827 B. uniformis A2828 B. vulgatus A2829 B. vulgalus A2830 B. ovatus A2831 B. thetaz‘oz‘aomicron A2832 Parabacter distasom's A2833 B. thetaiotaoml'cron A2767 C. diflicz'le A2768 C. dificz‘le A2769 C. le A2770 C. diflicz’le A2771 C. diflicz‘le '____—-_—.—_.

A2772 C. dijfzcile CMI# ORGANISM A2773 C. diflicz’le A2774 C. difi‘icz’le A2775 C. dyj‘icz’le A2776 C. dyficile A2777 C. difificile A2778 C. diflicz‘le A2779 C. dzfficile A2780 C. z‘le A2140 C. peljfrmgens A2203 C. peiy‘i'ingens A2204 C. perfi'ingens A2227 C. perfi'ingens A2228 C. perfi'z'ngens A2229 C. perfiingens A2315 C. perfi‘ingens A2332 C. perfi'ingens A2333 C. perfi‘ingens A2334 C. pezfi‘z‘ngens PCT/U52012/021280 CMI# ORGANISM A2389 C. perfringens |___ A2390 C. perfringens A864 F horum A871 F. nucleatum A1667 F. necrophorum A1666 F necrophorum A2249 F. nucleatum A27 16 Fusobacterium species A2717 Fusobacterium species A2719 Fusobacz‘erium species A2721 Fusobacterium species A2722 Fusobacterium species A27 10 Fusabacz‘erium species A2711 cterium species A2712 Fusobacterium s A2713 Fusobacterium species A2714 Fusobacterium species L—__—i A27 15 Fusobacferz'um species 2012/021280 CMI# ORGANISM l__—______ A1594 Peptostreptococcus anaerobius A2158 Peptosn'eptococcus magnus A2168 Peprostreptococcus bius A2170 Peptostreptococcus magmas _“‘—T" A2171 , Peptosn'eptococcus magnus A2575 Peptosn‘eptococcus spp‘ A2579 Peptosn'eptococcus arolyn'cus A2580 Peptosrreptococcus asaccharolyrz’cus A2614 Peptosz‘reptococcus asaccharolytz‘cus A2620 Peptostrepiococcus asaccharolytz‘cus A2629 Peptostreptococcus spp.

A2739 Prevotella denticola A2752 Prevotella bivia A2753 Prevotella intermedia A2754 Prevotellcz intermedia A2756 Prevotelia bivia A2759 Prevotella bivz'a A2760 Prevotella denrz‘cola _74_ PCT/U52012/021280 ORGANISM Prevotella intermedia Prevotella melaninogenica A2765 Prevotella melaninogem‘ca A2766 Prevotella melaninogenica A2821 Prevotella bivia A2822 Prevotella bz‘w‘a QCBF B. fi‘agilz‘s '*—“ QCBT B. thetaz‘oz‘aomicron QCCD C. difiozcile _____L_______"“______——‘W QCBF B. fiagz‘lz‘s l____ QCBT B. thetaiotaomicron QCCD (Zfimdk ] In Table 9 below, the term “JMI” stands for The Jones Microbiology Institute located in North Liberty, Iowa.

Table 9: Panels of Gram Positive and Gram Negative Organism Used to Generate MIC90 Data JMI Organism JMI Isolate # Code Organism 394 ACB Acinetobacter baumannii 21 66 ACB Acinetobacter baumannii 3060 ACB Acinetobacter baumannii 3170 ACB Acinetobacter baumanm’z’ 9328 ACB Acinetobacter baumannii 9922 ACB Acinetobacter baumannii 13618 ACB Acinetobacter baumannii 14308 ACB Acinetobacter baumannii 17086 - ACB Acinetobacter baumannii 17 176 ACB Acinetobacter baumannii 30554 ACB Acinetobacter nii 32007 ACB obacter baumannii F1 192 ECL Enterobacter cloacae 3096 ECL Enterobacter cloacae 5534 ECL bacter cloacae _______________—g_________— 6487 ECL Enterobacter cloacae 9592 ECL bacz‘er cloacae 11680 ECL Enterobacz‘er cloacae 12573 ECL bacter cloacae 12735 ECL bacter cloacae 13057 ECL Enterobacz‘er cloacae 18048 ECL Enterobacz‘er cloacae 173 ECL Enterobacter cloacae 29443 ECL Enterobacter cloacae 44 BF Enterococcusfaecalz‘s 355 EF Enterococcusfaecalz’s 886 EF Enterococcusfaecalz‘s 955 EF Enterococcusfaecalz‘s 1000 EF Enterococcusfaecalz‘s 1 142 EF Enterococcusfaecalz‘s PCT/U52012/021280 JMI Organism JMI Isolate # Code sm 1446 EF Enterococcusfaecalis 2014 EF Enterococcusfaecalz‘s 2103 EF Enterococcusfaecalis 2255 EF Enterococcusfaecalis L2978 EF jnterococcusfaecalz's 2986 EF Enterococcusfaecalis 5027 EF Wterococcusfaecalis 5270 RF Enterococcusfaecalis 5874 EF Emerococcusfaecalis 7430 EF Enterococcusfaecalz’s 7904 EF coccusfaecalis 8092 EF Enterococcusfaecalis 8691 EF Enterococcusfaecalz’s 9090 BF Enterococcusfaecalz‘s l——______—_________________ 10795 EF Enterococcusfaecalis 14104 EF Enterococcusfaecalis 16481 EF Enterococcusfaecalz’s 18217 EF Emerococcusfaecall's 22442 EF Enterococcusfaecall‘s 25726 EF Enterococcusfaecalis 26143 EF Enterococcusfaecalis 28131 BF Enlerococcusfaecalz‘s 29765 EF Enterococcusfaecalis 30279 EF Enterococcusfaecalz's 31234 BF Enterococcusfaecalis 31673 EF Enterococcusfaecalis 227 EFM Enterococcusfaecz‘um 414 EFM Enrerococcusfaecium 7 12 EFM Enterococcusfaecz‘um PCT/U52012/021280 JMI Organism JMI e # Code Organism 911 Eth Ehuerococcusjhecnun 2356 EFTA coccusjhechun 2364 IEFRA lbflerococcusjhechun 2762 EFIA lfinerococcusjbechun 3062 EF1A lbuerococcusjhecnun VZZEQF___" IEFAJ lbflerococcusjhechun 4473 EFDA lfiflerocaccusjhecnnn 4653 ZEF\4 lbuerococcusjhecnun 4679 EFBA lfiuerococcusjhecnnn 6803 EFKA lagerococcusjbecnun 6836 EFWA Eknerococcusjhecnun _8280h_*~w———-w1§ESZ_~_____——— Efinerococcusjhecnun 8702 EFBA fhnerococcusjhecnun Eknerococcusjbechun 10766 EFM Enterococcusfaecium 13783 EFM Enterococcusfaecium 14687 Eth lfiuerococcusjhecnun 15268 Eth lfinerococcusjhechun 15525 EFBA layerococcusjhecnun 15538 Eth layerococcusjhecnun 18102 A lbwerococcusjhecnun ‘18306———‘H~—_iEFh4 lbflerococcusjhecnnn 19967 .Eth lhuerococcusjhecnun 22428 Eth lfiflerococcusjhecnnn 23482 _Efifi§1—______fl__ Ehuerococcusjhecnun 29658 EFDA fihnerococcusjhecnun 597 EC Escherichia 0011' 847 EC Escherichia coli W0 2012(097273 PCT/U82012/021280 JMI Organism JMI e # Code Organism 1451 EC Escherichia coli 8682 EC Escherichia coli 1 1 199 EC Escherichia coli L 12583 BC Escherichia coli 12792 BC Escherichia coli 13265 BC Escherichia coli 14594 BC Escherichia coli 22148 EC Escherichia coli 29743 BC Escherichia coli I—30426 I EC Escherichia coli 470 BSA Group A Streptococcus 7685—— BSA Group A Streptococcus 3112 BSA Group A Streptococcus 3637 BSA Group A Streptococcus _________.____—l______— 4393 BSA Group A Streptococcus 4546 BSA Group A Streptococcus 4615 BSA Group A ococcus 5848 BSA Group A Streptococcus 6194 BSA Group A Streptococcus 8816 BSA Group A Streptococcus 1 1814 BSA Group A Streptococcus 76977 BSA Group A Streptococcus 18083 BSA Group A Streptococcus 18 821 BSA Group A Streptococcus T__—____ 25178 BSA Group A Streptococcus 30704 BSA Group A Streptococcus 12 ESE Group B Streptococcus 10366 BSB Group B Streptococcus 10611 BSB Group B Streptococcus 16786 BSB Group B Streptococcus 18833 BSB Group B Streptococcus W0 20121097273 [.____— JMI Organism JMI Isolate # Code Organism 30225 BSB Group B Streptococcus 10422 BSC Group C Streptococcus 14209 BSC Group C ococcus 29732 BSC Group C Streptococcus 8544 BSG Group G ococcus 18086 BSG Group G Streptococcus 29815 BSG Group G Streptococcus 147 lHI lflamnophflusiufluenzae 180 HI htlus tnfluenzae 934 HI Haemophilus influenzae 970 HI . Haemophilus influenzae 1298 HI Haemophz’lus influenzae 1819 HI hz’lus influenzae 1915 HI Haemophilus influenzae 2000 HI Haemophilus influenzae 2562 HI I3321 Haemophilus influenzae HI W 3133 HI W L3140 HI Haemophtlus influenzae 3497 HI Haemophz‘lus z'nfluenzae 3508 HI Haemophilus influenzae 3535 HI Haemophz’lus zae 1:082 HI Haemophz‘lus influenzae 4108 HI Haemophilus influenzae 4422 HI Haemophilus influenzae 4872 HI W 5858 m 6258 HI Haemophilus influenzae 6875 HI Haemophilus influenzae 7063 HI Haemophz’lus influenzae JMI sm JMI Isolate # Code Organism 7600 H1 Haemophilus influenzae 8465 HI Haemophilus influenzae 10280 HI Haemophilus influenzae 10732 HI Haemophilus influenzae 10850 HI Haemophilus influenzae 1 1366 HI Haemophilus influenzae 11716 HI Haemophilus influenzae 1 1724 HI hilus influenzae 1 1908 HI Haemophilus influenzae 12093 HI Haemophilus influenzae [3107 HI Haemophz’lus influenzae 13424 HI Haemaphilus influenzae 13439 HI Haemophilus influenzae 13672 HI Haemophilus influenzae 13687 HI Haemophilus influenzae 13792 HI hilus influenzae 13793 HI Haemophilus influenzae 14440 HI W 15351 HI Haemophz’lus influenzae 15356 HI Haemophilus influenzae 15678 HI Haemophilus zae 15800 HI Haemophilus influenzae 17841 HI Haemophilus influenzae 18614 HI hilus influenzae 25195 HI Haemophilus influenzae 27021 HI Haemophilus influenzae 28326 HI hz‘lus influenzae 28332 HI Haemophilus influenzae _——l________—[ 29923 HI Haemophilus influenzae 31911 HI Haemophilus influenzae PCT/U82012/021280 JMI Organism JMI Isolate # Code Organism 428 KPN Klebsiella pneumoniae 791 KPN Klebsiella pneumoniae 1422 KPN Klebsiella pneumoniae 1674 KPN W 6486 KPN Klebsiella pneumoniae 8789 KPN Klebsiella pneumoniae 10705 KPN Klebsiella pneumoniae 28148 KPN Klebsiella pneumoniae 29432 KPN Klebsiella pneumoniae 937 MCAT Moraxella catarrhalz‘s 1290 MCAT MoraerZa catarrhalz‘s WIT/13W 6241 MCAT Moraxella catarrhalz’s 6551 MCAT Moraxella catarrhalis 7074 MCAT Moraxella catarrhalis 7259 JMCAT Moraxella catarrhalis 7544 MCAT lla catarrhalz’s 9246 mm 9996 MCAT Moraxella catarrhalz's 12158 MCAT Moraxella halz‘s 13692 MCAT Moraxella halz’s 13817 MCAT Moraxella catarrhalis PCT/U52012/021280 JMI e # Code Organism 14431 MCAT Moraxella catarrhalz’s 14762 MCAT Moraxella catarrhalz‘s 14842 MCAT Moraxella catarrhalz’s 15361 MCAT Moraxella catarrhalz‘s 15741 MCAT fiMoraxella catarrhalz‘s 17843 MCAT lla catarrhalz's 18639 MCAT lla catarrhall’s 241 GO Neisseria hoeae 291 GC Neisseria gonorrhoeae 293 GC Neisseria gonorrhoeae 344 GC Neisserz'a gonorrhoeae 451 GC Neisserz’a gonorrhoeae 474 GC Nez'sserz'a gonorrhoeae 491 GC Neisseria gonorrhoeae 493 i GC Neisseria gonorrhoeae m—T—W 521 GC Neisserz'a hoeae 552 ”GT—W 573 GO Neisseria gonorrhoeae 592 GC Neisserl'a gonorrhoeae NM Neisseria meningitidis 813 NM ria meningitidis 1725 NM Neisseria meningitidis 2747 NM Nez‘sserz’a meningitidis 3201 NM Neisseria meningitidis IE35 NM Neisserz’a meningitidz‘s 7053 NM Neisseria meningitidz’s 9407 NM Neisserz’a meningitidz’s 10447 NM Neisserz'a meningitidz‘s 12685 Neisseria meningitidis 12841 Net'sserz'a meningitidz’s -83— PCT/U52012/021280 JMI Organism JMI Isolate # Code Organism 1403 8 NM Nez’sserz'a meningitia’z‘s 1 127 PM Proteus mirabilis 3049 PM Proteus mirabz'lis W1 PM Proteus mtrabz'lis 8793 PM Proteus mirabz‘lz's 10702 PM Proteus mirabilis [—1 1218 PM Proteus mirabilts 14662 PM Proteus mirabl'lz's 17072 PM Proteus mtrabz'lz‘s 19059 PM Proteus mz’rabilz's 23367 PM Proteus mirabz’lis 29819 PM Proteus mirabilis 31419 PM s mtrabtlis 1 8 81 PSA I Pseudomonas aerugz‘nosa 5061 PSA Pseudomonas aeruginosa 7909 PSA Pseudomonas aerugz‘nosa 8713 PSA PseudomonasGM 14318 PSA Pseudomonas aerugz'nosa 14772 PSA Pseudomonas aeruginosa 15512 PSA Pseudomonas aerugz'nosa 17093 PSA Pseudomonas aeruginosa 17802 PSA Pseudomonas aerugz'hosa 19661 PSA Pseudomonas aeruginosa 29967 PSA monas aeruginosa 31539 PSA Pseudomonas hosa—q 82 SA Staphylococcus aureus 99 SA Staphylococcus aureus 13 9 SA Staphylococcus aureus 140 SA lococcus aureus 141 SA Staphylococcus aureus -84— PCT/U82012/021280 JMI Organism JMI Isolate # Code Organism _E75________—3iK—____—_____§ZiJEEEEEEZ§EZE£E_____ 287 SA Staphylococcus aureus [354 SA lococcus aureus 382 SA Staphylococcus aureus 1 1 12 SA Staphylococcus aureus 1687 SA Staphylococcus aureus 1848 SA Staphylococcus aureus 2031 SA Staphylococcus aureus 2159 SA Staphylococcus aureus 2645 SA Staphylococcus aureus 3256 SA Staphylococcus aura—“4 L 3276 SA Staphylococcus aureus 4044 SA Staphylococcus aureus 4214 SA Staphylococcus aureus —#___J— 4217 SA Staphylococcus aureus 4220 SA Staphylococcus aureus 4231 SA Staphylococcus aureus 4240 SA Staphylococcus aureus 4262 SA Staphylococcus aureus 4370 SA Staphylococcus aureus 4665 SA lococcus aureus 4666 SA Staphylococcus aureus 4667 SA Staphylococcus aurcus 5026 SA Staphylococcus aureus 5666 SA Staphylococcus aureus 6792 SA W 7023 SA lococcus aureus 7461 SA Staphylococcus aureus 7899 SA Staphylococcus aureus 7901 ; SA Staphylococcus aureus _____1 JMI Organism JMI Isolate # Code Organism 8714 SIX Saufludococcusaureus 10056 SA Staphylococcus aureus 10110 SIX Sayflgflococcusaureus 11379 SA W _H_J 11629 SIX Staphgdococcus<1ureus 1 1659 SA Staphylococcus aureus 12788 SIX Sayflgdococcusaureus 12789 SA Staphylococcus aureus 13043 SIX - ococcusaureus 13086 SIX Sayfludococcusaureus 13721 SIX Sayflgdococcusaureus 13742 SIX Saufludococcusaureus ococcusaureus Saqfluflococcusaureus 14384 SIX Sauflgdococcusaureus 15428 SIX éhap¢gdococcusaureus 15430 SIX Sayfludococcusaureus 17721 SIX Sayflgdococcusaureus 18688 SA Staphylococcus aureus 19095 SIX Sayfludococcusaureus 20195 SIX Suyfludococcusaureus 22141 SIX Saqfludococcusaureus 22689 SA lococcus aureus 27398 SIX Suyfludococcusaureus 29048 SIX Sayfludococcusaureus 29051 SIX Sauflgdococcusaureus 30491 SA Staphylococcus aureus 30538 SIX éhapfludococcusaureus SEPI Staphylococcus epidermidis ~86- PCT/U82012/021280 JMI Organism JMI Isolate # Code Organism 53 SEPI Staphylococcus epidermidis 385 SEPI Staphylococcus epidermia’z’s 398 SEPI W 701 SEPI rStaphylococcus epidermia’z’s 713 SEPI Staphylococcus epidermidis 1381 SEPI Staphylococcus—6W 2174 SEPI Staphylococcus cpz’dermz‘dz's 2286 SEPI Staphylococcus epidermidz’s 2969 SEPI Staphylococcus epidermidis B3417 SEPI Staphylococcus epidermidz‘s 3447 SEPI Staphylococcus epidermidis 4753 SEPI Staphylococcus midis 7241 SEPI Staphylococcus epidermidis 9366 SEPI 1 Staphylococcus epidermia’z‘s 10665 SEPI Staphylococcus mia’z’s WEE—W 13036 SEPI Staphylococcus epidermidis 13227 W 13243 SEPI Staphylococcus epidermidis 13621 SEPI Staphylococcus rmia’z’s 13638 SEPI W 13800 SEPI W 14078 SEPI Staphylococcus epidermidis 14392 SEPI lococcus epidermidis 15007 f SEPI W 16733 SEPI Staphylococcus epidermidis Staphylococcus epidermidz‘s Staphylococcus epidermidis Staphylococcus epidermidis SEPI Staphylococcus epidermidis JMI Organism JMI Isolate # Code Organism 29985 SEPI Staphylococcus epidermidz’s 30259 SEPI Staphylococcus epidermz‘dts 31444 SEPI W 268 SPN ococcus pneumoniae ITil—64 ' SPN Streptococcus pneumoniae 2482 SPN Streptococcus pneumoniae WSPN Streptococcus pneumoniae 2994 SPN Streptococcus pneumoniae 1‘3 123 SPN Streptococcus pneumoniae 3124 SPN Streptococcus pneumoniae 4336 SPN Streptococcus pneumoniae 4858 f SPN Streptococcus pneumoniae 5606 W 5881 SPN Streptococcus pneumoniae l—_____—_____________— 5897 SPN ococcus niae 5900 SPN Streptococcus pneumoniae 6051 SPN Streptococcus pneumoniae 6216 SPN Streptococcus pneumoniae 6556 SPN Streptococcus pneumoniae 7270 SPN ‘ Streptococcus pneumoniae 7584 SPN Streptococcus pneumoniae 8479 SPN Streptococcus pneumoniae 8501 SPN Streptococcus niae 9256 SPN ococcus niae 9257 SPN Streptococcus pneumoniae 10246 SPN Streptococcus pneumoniae 10467 SPN Streptococcus pneumoniae 10886 SPN Streptococcus pneumoniae 1 1217 SPN Streptococcus pneumoniae 1 1228 SPN Streptococcus pneumoniae 11238 SPN Streptococcus pneumoniae —88- PCT/U52012/021280 JMI OrganismT JMI Isolate # Code Organism 1 1757 SPN Streptococcus pneumoniae 1 1 768 SPN Streptococcus pneumoniae l_12121 SPN Streptococcus pneumoniae 12124 SPN Streptococcus pneumoniae WSPN Streptococcus niae 12767 SPN W 12988 SPN Streptococcus pneumoniae 13321 SPN Streptococcus niae 13393 SPN Streptococcus pneumoniae 13521 SPN ococcus pneumoniae 13544 SPN Streptococcus pneumoniae 13700 SPN W 13704 SPN Streptococcus pneumoniae 13822 SPN Streptococcus pneumoniae ococcus pneumoniae 14131 SPN Streptococcus pneumoniae 14413 SPN ococcus pneumoniae 14744 SPN Streptococcus pneumoniae 14808 SPN ococcus pneumonic?‘ 14827 SPN Streptococcus pneumonia-e—J 14835 SPN Streptococcus pneumontae 14836 SPN Streptococcus pneumoniae 832 SPN Streptococcus pneumoniae 17336 SPN Streptococcus pneumoniae l7734917343 SPN Streptococcus prteumom’aew1 SPN Streptococcus pneumoniae 17735 SPN W 18060 SPN Streptococcus pneumoniae __—__________1 18567 SPN Streptococcus pneumoniae 18595 SPN Streptococcus pneumoniae 19082 SPN Streptococcus pneumoniae PCT/U82012/021280 JMI Organism JMI Isolate # Code Organism 19826 SPN ococcus pneumoniae 27003 SPN Streptococcus pnezmzorzz‘cz—e—l l2_8310 SPN Streptococcus pneumoniae 28312 SPN Streptococcus pneumoniae 29890 SPN Streptococcus pneumoniae 29910 SPN Streptococcus pneumoniae -90_

Claims (3)

WHAT IS CLAIMED IS:

1. A solid Form I of the compound of formula (I): die” (I); n the solid Form I has an X—ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 G :b 0.2) when measured using Cu Kg radiation, selected from the group ting of 9.3, 11.7, 12.1, 12.4, 14.5, 15.9, 16.3, 16.6, 18.5, 19.4, 21.5, 22.3, 22.8, 23.8, 24.5, 25.7, 28.1, 28.4, 30.3, and 33.4, when the XPRD is collected from about 5 to about 38 degrees 2 9.

2. The solid Form I of claim 1, having an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 6 i 0.2) when measured using Cu KO, radiation, selected from the group consisting of 9.3, 16.6, 18.5, 19.4, 21.5, and 25.7, when the XPRD is collected from about 5 to about 38 degrees 2 0.

3. The solid Form I of claim 1 or 2, having an X-ray powder ction pattern, as measured using Cu K, radiation, substantially similar to