NZ612912B2 - Solid forms of gyrase inhibitor (r)-1-ethyl-3-[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 solid forms of compound of formula (I) and pharmaceutically acceptable salts thereof, particularly the hydrochloride salt and the solid amorphous mesylate salt, that 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. prising said compound of formula (I) or its salts and their use for treating bacterial infection.

Description

SOLID FORMS OF GYRASE INHIBITOR (R)— l—ETHYL—3 — [5 —[2—( l—HYDROXY— l — METHYL—ETHYL)PYRIMIDIN—5—YL]—7—(TETRAHYDROFURAN—2—YL)— lH— BENZIMIDAZOL—Z—YL]UREA CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119 of United States Provisional Patent ation serial number 61/433,161 filed January 14, 2011, the entire contents of which is incorporated herein by reference.

BACKGROUND OF THE APPLICATION Bacterial resistance 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 pment of multiple drug resistance in certain strains of bacteria, such as Streptococcus pneumoniae (SP), Mycobacterium tuberculosis, and Enterococcus. The appearance of vancomycin resistant enterococcus was particularly alarming e vancomycin was formerly the only effective otic for treating this infection, and had been considered for many infections to be the drug of "last resort". While many other drug-resistant bacteria do not cause hreatening 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 n in Anti-infective Investigational Drugs, 1999, 1, 1; Levy, "The Challenge of Antibiotic ance", ific American, March, 1998).

Another concern is how quickly otic resistance can spread. For e, until the 1960's SP was universally sensitive to penicillin, and in 1987 only 0.02% of the SP strains in the US. were ant. However, by 1995 it was reported that SP resistance to penicillin was about seven percent and as high as 30% in some parts of the US. (Lewis, FDA Consumer magazine (September, 1995); Gershman in The Medical Reporter, 1997).

Hospitals, in particular, serve as centers for the formation and transmission of drug-resistant organisms. Infections occurring in hospitals, known as nosocomial infections, are becoming an increasingly serious problem. Of the two million Americans infected in _ 1 _ hospitals each year, more than half of these infections resist at least one antibiotic. The Center for e l 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 er magazine, September 1995).

As a result of the need to combat drug—resistant bacteria and the increasing failure of the available drugs, there has been a resurgent interest in ering new antibiotics. One attractive strategy for developing new antibiotics is to inhibit DNA gyrase and/or topoisomerase IV, bacterial enzymes necessary for DNA replication, and therefore, necessary for bacterial 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 topoisomerases, a group of enzymes which ze the interconversion of topological isomers ofDNA (see generally, Kornberg and Baker, DNA ation, 2d Ed., Chapter 12, 1992, W. H. n and Co.; Drlica, Molecular Microbiology, 1992, 6, 425; Drlica and Zhao, Microbiology and Molecular Biology Reviews, 1997, 61, pp. 377—392). Gyrase itself controls DNA supercoiling and relieves gical stress that occurs when the DNA strands of a parental duplex are untwisted during the replication process. Gyrase also catalyzes the conversion of d, closed circular duplex DNA to a negatively superhelical form which is more favorable for ination. 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 strands. Such a cleavage mechanism is characteristic of a type II topoisomerase. The supercoiling reaction is driven by the binding of ATP to .

The ATP is then hydrolyzed during the reaction. This ATP binding 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 dependent on the ATP/ADP ratio. In the absence of ATP, gyrase is only capable of relaxing supercoiled DNA. ial 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 amino-terminal domain which has the ATPase activity, and a y-terminal domain which interacts with GyrA and DNA. By contrast, eukaryotic type II topoisomerases are homodimers that can relax negative and positive supercoils, but cannot introduce negative supercoils. Ideally, an antibiotic based on the inhibition of bacterial DNA gyrase and/or topoisomerase IV would be selective for these enzymes and be relatively inactive against the eukaryotic type II topoisomerases.

Topoisomerase IV primarily es linked chromosome dimers at the conclusion ofDNA 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, ciprofloxacin, 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 fluoroquinolones 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—3 92). However, drug resistance has also been recognized as a problem for this class of compounds (WHO Report, "Use of Quinolones in Food Animals and ial Impact on Human Health", 1998). With the quinolones, as with other classes of antibiotics, bacteria exposed to earlier compounds often quickly develop resistance to more potent compounds in the same class.

The associated subunits sible for supplying the energy necessary for catalytic tumover/resetting of the enzymes via ATP hydrolysis are GyrB (gyrase) and ParE (topoisomerase IV), respectively (see, Champoux, J.J., Annu. Rev. Biochem., 2001, 70, pp. 369—413). nds that target these same ATP binding sites in the GyrB and ParE subunits would be useful for ng various bacterial infections (see, Charifson et al., J.

Med. Chem., 2008, 51, pp. 5243—5263).

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

For coumarin-resistant bacteria, the most prevalent point mutation is at a e ne residue that binds to the carbonyl of the coumarin ring (Arg136 in E. coli GyrB).

While s with this mutation show lower oiling and ATPase activity, they are also less sensitive to tion by coumarin drugs (Maxwell, Mol. Microbiol., 1993, 9, 681).

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

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

As bacterial resistance to otics 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 ng increasingly prevalent.

SUMMARY OF THE APPLICATION The present application is directed to solid and amorphous forms of (R)— 1-ethyl[5-[2-(1-hydroxymethyl-ethyl)pyrimidin-5 -yl][(2R)-tetrahydrofuranyl]-1H- benzimidazolyl]urea (“the benzimidazolyl urea compound”) and methods for preparing same. In one embodiment, the present ation provides for a solid form of the benzimidazolyl urea compound. In one embodiment, the solid form is FormI solid form characterized by an by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 :: 0.2) when measured using Cu K“ radiation, selected from the group consisting of 9.3, 11.7, 12.4, 13.8, 14.6, 16.0, 16.2, 16.7, 18.6, 18.9, 19.6, 20.2, 20.5, 21.3, 1.7, 22.7, 23.9, 24.5, 24.9, 25.8, 26.7, 27.9, 28.1, 28.4, 30.4, 33.5, and 37.4, when the XPRD is collected from about 5 to about 38 degrees two theta (2 0). In certain embodiments, solid Form I may be characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 :: 0.2) when measured using Cu K0, radiation, selected from the group consisting of 9.3, 16.7, 18.6, 19.6, 21.7, and 25.8, when the XPRD is collected from about 5 to about 38 degrees 2 0. In further ments, Form I is characterized by an X-ray powder diffraction pattern, as measured using Cu K“ radiation, substantially similar to Figure 1. In yet another ment, Form I is characterized by an endothermic peak having an onset temperature of about 243°C as measured by differential scanning calorimetry in which the temperature is scanned at about 10°C per minute. The present application also es for a method for ing crystal Form I of the benzimidazolyl urea compound comprising crystallizing or precipitating the compound of formula (I) from a solvent system comprising methylene chloride, methanol, or a combination thereof.

The present application also provides for solid hydrochloride salts of the benzimidazolyl urea compound. In one embodiment, the solid hydrochloride salt is Form II solid. In one ment, Form II hydrochloride salt of the present application may be characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 :: 0.2) when measured using Cu K“ radiation, ed from the group consisting of 7.0, 9.1, 11.5, 12.3, 12.4, 13.5, 16.4, 17.2, 17.9, 18.2, 19.0, 19.5, 20.6, 20.9, 22.4, 23.0, 23.5, 24.0, 24.5, 26.0, 26.5, 27.1, 27.4, 28.5, 29.4, 30.8, 33.0, when the XPRD is ted from about 5 to about 38 degrees 2 0. In a further embodiment, Form II hloride salt of the present application may be characterized by an X-ray powder diffraction n (XPRD) comprising at least three approximate peak positions (degrees 2 0 :: 0.2) when ed using Cu K0, radiation, selected from the group consisting of 7.0, 9.1, 11.5, 12.3, 12.4, 16.4, 17.9, 19.5, 24.0, and 29.4, when the XPRD is collected from about 5 to about 38 degrees 2 0. In certain embodiments, Form 11 hydrochloride salt of the present application may be characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions es 2 0 :: 0.2) when measured using Cu K0, radiation, selected from the group consisting of 7.0, 9.1, 11.5, 19.5, and 24.0, when the XPRD is collected from about 5 to about 38 degrees 2 0. In further embodiments, Form II hydrochloride salt of the present application may be characterized by an X-ray powder diffraction pattern, as measured using Cu K“ radiation, substantially similar to Figure 4. The present application also provides for a method for preparing solid hydrochloride salt of the idazolyl urea compound comprising suspending a solid free base of the benzimidazolyl urea in an acidic solvent system comprising acetonitrile or water.

In another embodiment, the solid hydrochloride salt is Form III solid. In one embodiment, Form III hydrochloride salt of the present application may be characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions es 2 0 :: 0.2) when measured using Cu K0, radiation, selected from the group consisting of6.9, 9.1, 11.0, 11.7, 12.3, 15.8, 16.9, 18.1, 18.9, 19.8, 20.9, 22.7, 23.4, 24.1, 24.8, 25.3, 27.7, 28.5, 29.5, and 31.4, when the XPRD is collected from about 5 to about 38 degrees 2 0. In certain embodiments, Form III hydrochloride salt of the present application may be characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 :: 0.2) when measured using Cu K“ ion, selected from the group consisting of 6.9, 9.1, 11.7, 18.1, 18.9, 19.8, 23.4, and 24.8, when the XPRD is ted from about 5 to about 38 degrees 2 0. In r embodiments, Form III hydrochloride salt of the present application may be characterized by an X-ray powder diffraction pattern, as measured using Cu K“ radiation, substantially similar to Figure 7. The present application also provides a method for preparing solid Form III of the benzimidazolyl urea compound comprising suspending a solid free base of the benzimidazolyl urea in an acidic solvent system comprising one or more ethereal ts and water.

The present application also provides an amorphous Form IV of the mesylate salt of the benzimidazolyl urea compound. In one embodiment, Form IV may be characterized by an X-ray powder diffraction pattern (XPRD) using Cu K“ radiation characterized by a broad halo with no discernable diffraction peak.

PTION OF FIGURES Figure 1 shows an X-ray powder diffraction pattern of solid Form I of the benzimidazolyl urea nd (free base).

Figure 2 shows a DSC thermogram of solid Form I of the benzimidazolyl urea compound.

Figure 3 shows a TGA thermogram of solid Form I of the benzimidazolyl urea compound.

Figure 4 shows an X-ray powder diffraction pattern of solid Form II of the hydrochloride salt of the benzimidazolyl urea compound.

Figure 5 shows a DSC thermogram of solid Form II of the benzimidazolyl urea compound.

Figure 6 shows a TGA gram of solid Form II of the idazolyl urea compound Figure 7 is an X-ray powder diffraction pattern of solid Form III of the hydrochloride salt of the benzimidazolyl urea compound.

Figure 8 shows a DSC gram of solid Form III of the hydrochloride salt of the idazolyl urea compound.

Figure 9 is a TGA (thermal gravimetric analysis) thermogram of solid Form III of the benzimidazolyl urea compound.

Figure 10 is an X-ray powder diffraction pattern of amorphous Form IV of the mesylate salt of the benzimidazolyl urea compound.

Figure 11 shows a DSC thermogram of amorphous Form IV of mesylate salt of the benzimidazolyl urea compound.

Figure 12 is a 1H-NMR of the mesylate salt of the benzimidazolyl urea compound.

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

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

A substantially pure Form I (free base) crystalline solid form of the benzimidazolyl urea compound may be ed from amorphous or crystalline compound by ting the compound with a polar t such as acetonitrile, methylene chloride, methanol, ethanol, or water, or combinations thereof. The benzimidazolyl urea compound may be contacted with the solvent either by saturating a on of the benzimidazolyl urea compound in the solvent at ambient temperature and allowing the mixture to stand for an extended period of time (for example, ght). atively, the benzimidazolyl urea compound may be dissolved in the solvent at ed temperature, for example, at reflux, followed by cooling the solution to room temperature or below and isolating solid Form I.

In one ment of the process, a substantially pure crystalline solid Form I of the benzimidazolyl urea compound may be prepared from amorphous or crystalline of the compound by preparing a saturated on of the compound in a polar solvent at room temperature and isolating Form I which results. In practice this can be accomplished by dissolving a sufficient amount of the benzimidazolyl urea compound in the solvent at elevated temperature (up to reflux) such that when the solution is allowed to cool to room ature a saturated solution is ed, from which Form 1 precipitates and can be isolated. In one embodiment, a t for the preparation of Form 1 is CHzClz or MeOH or mixtures thereof. Isolation of the resulting solid es Form 1.

The solid Form 1 of the benzimidazolyl urea compound (free base) may be identified by the following characteristics: a melt endotherm with an extrapolated onset of about 243°C as determined by differential scanning calorimetry using 10°C per minute scan rate; and an X-ray powder ction pattern essentially as shown in Table 1 and Figure 1 wherein the XRPD patterns were measured using a powder ctometer ed with a Cu X—ray tube source. The sample was illuminated with Cu Km radiation and XRPD data were collected from about 0 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 ation 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 ed to be used for absolute comparisons.

Figure 1 is an X-ray powder diffraction pattern of solid Form 1 of the benzimidazolyl urea compound collected from about 5 to about 38 degrees 2 0. The peaks corresponding to X-ray powder diffraction pattern having a ve intensity greater than or equal to 5% are listed in Table 1.

Figure 2 shows a DSC thermogram of solid Form 1 of the benzimidazolyl urea compound exhibiting an endotherm with an onset transition at about 243°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 collected equilibrating a 1.8 mg sample of the solid Form 1 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 3 is a TGA (thermal gravimetric analysis) thermogram of solid Form 1 of the benzimidazolyl urea compound exhibiting an initial weight loss of about 35% percent in the 50 to 260°C temperature range. Data in Figure 3 were collected equilibrating a 0.87 mg sample of solid Form 1 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. While applicants do not wish to be held to a particular explanation of the erm 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 degradation of the material as suggested by the weight loss in the TGA.

In one embodiment, the present invention provides a solid compound of formula (I): N \ N NH O or salts thereof.

In another embodiment, the solid is a solid Form I free base.

In another ment, the solid Form I is characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 :: 0.2) when measured using Cu K, radiation, selected from the group consisting of 9.3, 11.7, 12.4, 13.8, 14.6, 16.0, 16.2, 16.7, 18.6, 18.9, 19.6, 20.2, 20.5, 21.3, 21.7, 22.7, 23.9, 24.5, 24.9, 25.8, 26.7, 27.9, 28.1, 28.4, 30.4, 33.5, and 37.4, 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 :: 0.2) when measured using Cu K, radiation, selected from the group consisting of 9.3, 16.7, 18.6, 19.6, 21.7, and 25.8, when the XPRD is ted from about 5 to about 38 degrees 2 0.

In another ment, the solid Form I is characterized by an X-ray powder diffraction pattern, as measured using Cu K“ radiation, substantially similar to Figure 1.

In another embodiment, the solid Form I is further characterized by an endothermic peak having an onset temperature of about 243°C as ed by differential scanning metry in which the temperature is scanned at about 10°C per minute.

In another ment, the t invention es a method for preparing crystal Form I of the compound of formula (I) comprising precipitating the compound of WO 97270 formula (I) from a solvent system comprising methylene chloride, methanol, ethanol, or combinations thereof.

Table 1. XRPD pattern peaks for solid Form I of the benzimidazolyl urea compound [°29] W1] 1 91 2 28 3 10 4 5 l6 6 21 7 23 8 81 n— 100 In one aspect, the t application provides crystal Form II of the hydrochloric acid addition salt of the benzimidazolyl urea compound. In one embodiment, the present application provides a process for preparing solid Form II of the benzimidazolyl urea compound. The pharmaceutically acceptable hydrochloric acid on salt of the benzimidazolyl urea compound may be ed by any method known to those skilled in the art. For example, gaseous hydrochloric acid may be bubbled through a solution of the benzimidazolyl urea nd until a mono acid addition salt of the compound is prepared.

In one embodiment, the hydrochloric acid addition salt of the benzimidazolyl urea compound may precipitate out. 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 hloride salt of the benzimidazolyl urea compound and the salt precipitate. Alternatively, adding a second solvent to the mixture may precipitate the salt.

In one embodiment, the benzimidazolyl urea compound of the t application is suspended in a polar solvent. In a further ment, the polar solvent is acetonitrile. In this embodiment, the idazolyl urea compound of the present application is suspended in acetonitrile at room temperature and a stoichoimetric amount of an aqueous HCl solution is added. The sion is kept in a closed container while ng gently until it equilibrates and the benzimidazolyl urea compound is converted to the corresponding acid addition salt. In some ments, it may take anywhere from a few minutes to a few days for the free base sion to be converted to the acid addition salt.

The salt may be recovered by filtering the suspension to obtain a white solid, which may be dried at room temperature under vacuum for several hours.

Solid Form II of the hydrochloride salt of the benzimidazolyl urea nd may be identified by an X-ray powder diffraction pattern essentially as shown in Table 2 and Figure 4 wherein the XRPD patterns were measured using a powder diffractometer equipped with a Cu X—ray tube source. The sample was illuminated with Cu Kou radiation and XRPD data were collected from about 5 to about 400 20. A person skilled in the art would recognize that relative intensities of the XPRD peaks may significantly vary depending on sample ation.

Figure 4 is an X-ray powder diffraction pattern of solid Form II of the hydrochloride salt of the benzimidazolyl urea compound collected from about 0 to about 40 degrees 2 0. The peaks ponding to X—ray powder diffraction pattern having a relative intensity greater than or equal to 5% are listed in Table 2.

In one embodiment, the present invention provides a hydrochloric acid salt of the compound of formula (I): N \ N NH 0 ) a).

In another embodiment, the hydrochloride salt is in a solid form.

In r embodiment, the hydrochloride salt is a solid Form II.

In another embodiment, the solid Form II is characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 0 :: 0.2) when measured using Cu K0, radiation, selected from the group consisting of 7.0, 9.1, 11.5, 12.3, 12.4, 13.5, 16.4, 17.2, 17.9, 18.2, 19.0, 19.5, 20.6, 20.9, 22.4, 23.0, 23.5, 24.0, 24.5, 26.0, 26.5, 27.1, 27.4, 28.5, 29.4, 30.8, 33.0, when the XPRD is ted from about 5 to about 38 degrees 2 0.

In another embodiment, the solid Form II is characterized by an X-ray powder diffraction pattern (XPRD) sing at least three approximate peak positions (degrees 2 0 :: 0.2) when measured using Cu K0, radiation, selected from the group consisting of 7.0, 9.1, 11.5, 12.3, 12.4, 16.4, 17.9, 19.5, 24.0, and 29.4, when the XPRD is collected from about 5 to about 38 degrees 2 0.

In another embodiment, the solid Form II is characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak ons (degrees 2 0 :: 0.2) when measured using Cu K0, radiation, selected from the group consisting of 7.0, 9.1, 11.5, 19.5, and 24.0, when the XPRD is collected from about 5 to about 38 degrees 2 0.

In another ment, the solid Form II is characterized by an X-ray powder diffraction n, as measured using Cu K“ radiation, substantially similar to Figure 4.

In another embodiment, the present invention es a method for preparing the solid Form II comprising suspending a solid free base of the benzimidazolyl urea in an acidic solvent system comprising acetonitrile or water.

Table 2. XRPD pattern peaks for solid Form 11 of the benzimidazolyl urea compound Peak No. Position Relative [°2 0] Intensi []% 123483 12.4346 13.4801 16.3614 17.9001 18.1737 19.0475 26. 5376 9 26 28.4887 14 27 29.3904 18 28 30.8478 6 29 32.9868 5 Solid Form 111 of the hloride salt of the benzimidazolyl urea compound may be identified by an X-ray powder diffraction pattern essentially as shown in Table 3 and Figure 7 n the XRPD patterns were measured using a powder diffractometer equipped with a Cu X—ray tube source. The sample was illuminated with Cu Kou radiation and XRPD data were collected from about 5 to about 400 20. A person skilled in the art would recognize that relative intensities of the XPRD peaks may significantly vary depending on sample orientation.

Figure 5 shows a DSC thermogram of solid Form 11 of the hydrochloride salt of the idazolyl urea compound exhibiting an endotherm with an onset transition at about 216°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 collected equilibrating a 1.26 mg sample of the solid Form II 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.

Figure 6 is a TGA (thermal gravimetric analysis) thermogram of solid Form II of the idazolyl urea compound ting an initial weight loss of about 22% percent in the 50 to 230°C temperature range. Data in Figure 6 were collected equilibrating a 1.82 mg sample of solid Form II 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. 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 tion with large peak in the DSC is due to a melting transition coupled with degradation of the material as suggested by the weight loss in the TGA.

Figure 7 is an X-ray powder diffraction pattern of solid Form III of the hydrochloride salt of the benzimidazolyl urea compound collected from about 5 to about 40 s 2 0. The peaks corresponding to X—ray powder diffraction pattern having a relative intensity greater than or equal to 5% are listed in Table 3.

In one embodiment, the present invention provides a solid compound, wherein said hydrochloric salt of nd of formula (I) is a solid Form III.

In another embodiment, the solid Form III is terized by an X-ray powder diffraction n (XPRD) comprising at least three approximate peak positions (degrees 2 0 :: 0.2) when measured using Cu K0, radiation, selected from the group consisting of6.9, 9.1, 11.0,11.7,12.3,15.8, 169,181, 18.9, 19.8, 20.9, 22.7, 23.4, 24.1, 24.8, 25.3, 27.7, 28.5, 29.5, and 31.4, when the XPRD is collected from about 5 to about 38 degrees 2 0.

In another embodiment, the solid Form III is characterized by an X-ray powder diffraction pattern (XPRD) comprising at least three imate peak positions (degrees 2 0 :: 0.2) when measured using Cu K0, radiation, selected from the group consisting of 6.9, 9.1, 11.7, 18.1, 18.9, 19.8, 23.4, and 24.8, when the XPRD is ted from about 5 to about 38 degrees 2 0.

In another ment, the solid Form III is characterized by an X-ray powder diffraction pattern, as measured using Cu K“ radiation, substantially similar to Figure In another embodiment, the present invention provides a method for preparing solid Form 111 comprising suspending a solid free base of the benzimidazolyl urea in an acidic solvent system comprising one or more ethereal solvents and water.

Table 3. XRPD pattern peaks for solid Form 111 of the benzimidazolyl urea compound Peak Relative Intensity N0. 0] [%] 6.8885 17. 17 3 9.117 4 10.9885 11.7157 6 7 9 15.7808 11 16.8739 12 18.1446 13 18.936 -14— -15— -16— -17— -18— -19— -20— -23— -24— -25— 26 31.4273 6.34 Figure 8 shows a DSC thermogram of solid Form 111 of the hydrochloride salt of the benzimidazolyl urea compound exhibiting a melting endotherm with an onset transition at about 214°C. A person skilled in the art would recognize that the peak and onset temperatures of the endotherms may vary ing on the experimental conditions. Data in Figure 8 were collected equilibrating a 1.03 mg sample of the solid form at about 35°C for about 10 minutes. During the data collection period, the ature was increased at a rate of about 10°C per .

Figure 9 is a TGA (thermal gravimetric analysis) thermogram of solid Form 111 of the benzimidazolyl urea compound exhibiting an initial weight loss of about 28% percent in the 50 to 260°C temperature range. Data in Figure 9 were ted equilibrating a 3.71 mg sample of the solid form at about 35°C for about 10 minutes. During the data collection period, the temperature was sed at a rate of about 10°C per minute. 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 degradation of the material as ted by the weight loss in the TGA.

In one embodiment, solid Form III of the hydrochloride salt of the benzimidazolyl urea compound may be ed from a mixture of an ethereal solvent and aqueous HCl. In one embodiment, the ethereal solvent is THF, -tert—butyl ether (MTBE), or mixtures thereof. In a particular embodiment, the benzimidazolyl urea compound may be suspended in an ethereal solvent followed by on of a stoichiometric amount of HCl. Additional ethereal solvent may be added and the suspension may be allowed to equilibrate for a certain period of time enough to convert the free base to the corresponding HCl addition salt. It may take less than an hour to several hours for the equilibration to be completed. In certain embodiments, the suspension may be allowed to equilibrate for up to 24 hours before collecting the white solid. The solid may be ted using any method known to those skilled in the art. The solid may be dried under vacuum for several hours.

In another aspect, the present application provides an ous Form IV of the mesylate salt of the benzimidazolyl urea compound. In one embodiment, the present application provides a process for preparing amorphous Form IV of the mesylate salt of the benzimidazolyl urea compound. A pharmaceutically acceptable esulphonic acid salt of the benzimidazolyl urea compound may be prepared by any method known to those d in the art. For example, a solution of methanesulphonic acid may be added to a solution of the benzimidazolyl urea compound until a mono acid addition salt of the compound is prepared.

The mesylate salt of the benzimidazolyl urea compound may be converted to an amorphous solid Form IV using any method known to those d in the art. The ous idazolyl 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 benzimidazolyl urea compound mesylate salt may still lack es 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 10 is an X—ray powder diffraction pattern of an amorphous Form IV of the mesylate salt of the benzimidazolyl urea compound.

In one ment, the amorphous mesylate salt of the benzimidazolyl urea compound may be prepared by spray drying a solution of the salt in appropriate t.

Spray drying is well known in the art and is often used to dry thermally-sensitive materials such as pharmaceutical drugs. Spray drying also provides consistent particle distribution that can be reproduced fairly well. Any gas may be used to dry the powder although air is ly 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 preparing the amorphous Form IV of the mesylate salt of the benzimidazolyl urea compound. For e, freeze drying, drum drying, or pulse conversion drying may be used to produce an amorphous mesylate salt of the benzimidazolyl urea compound.

In one embodiment, the t invention es a solid compound of formula (I), wherein said solid is an amorphous mesylate salt of Form IV.

In another embodiment, the solid amorphous mesylate salt Form IV 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 one embodiment, a solution of the idazolyl urea compound in a polar solvent may be spray dried using a nanospray dryer equipped a condenser.

Figure 11 shows a DSC thermogram of amorphous Form IV of the mesylate salt of the idazolyl urea compound. Data in Figure 11 were collected equilibrating a 1.6 mg sample of the amorphous material at about 35°C for about 10 minutes. During the data collection period, the ature was increased at a rate of about 10°C per minute.

It is to be understood that solid Forms I, II and III and ous solid Form IV of the idazolyl urea compound or its salts, in addition to having the XRPD, DSC, TGA and other teristics 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 detector (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 s each. The data were subsequently integrated over the range of 3°— 410 2 with a step size of 0.020 and merged into one continuous pattern.

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 a Bruker D8 Advance system equipped with a Vantec—l on sensitive detector (Bruker AXS, Madison, WI). The X-Ray generator was operating at a tension of 40 kV and a current 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 (0.25s/step).

Differential Scanning Calorimetry (DSC): DSC was performed 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 aluminum pan that was crimped using lids with either no pin-hole or pin—hole lids. The DSC samples were d from 30°C to temperatures ted 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 ted + and — 1°C every 60s with ramp rates of 2 or 3 C/min.

Data was collected by Thermal Advantage Q SeriesTM re and ed by Universal Analysis 2000 software (TA ments, New , DE).

Thermogravimetric analysis (TGA): A Model Q5000 Thermogravimetric Analyzer (TA Instruments, New Castle, DE) was used for TGA measurement. Typically, approximately 3— mg of the sampleswere scanned from 30°C to temperatures indicated on the plots at a g rate of 10°C/min. Data was collected by Thermal age Q SeriesTM software and analyzed by sal Analysis 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 ceutically acceptable salt f.

In another embodiment, the present invention provides a 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 thereof, wherein the bacterial infection is characterized by the presence of one or more of Streptococcus pneumoniae, Staphylococcus epidermidis, Enterococcusfaecalis, Staphylococcus aureus, Clostridium diflicile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ns, Chlamydophila pneumoniae, Chlamydia trachomatis, hilus influenzae, Streptococcus pyogenes or B—haemolytic streptococci.

In another embodiment, the present invention provides a method of controlling, treating or reducing the advancement, severity or effects of a nosocomial or a non- nosocomial ial infection in a patient, comprising administering to said patient a pharmaceutical ition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, n the bacterial infection is selected from one or more of the following: upper respiratory infections, lower atory infections, ear infections, pleuropulmonary and bronchial ions, 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, l infections, eye infections, or granulomatous infections, uncomplicated skin and skin structure infections (uSSSI), complicated skin and skin structure infections (cSSSI), catheter infections, pharyngitis, tis, otitis externa, otitis media, bronchitis, empyema, pneumonia, ity-acquired bacterial pneumoniae (CABP), hospital-acquired pneumonia (HAP), hospital-acquired bacterial nia, ventilator-associated pneumonia (VAP), diabetic foot infections, vancomycin ant cocci infections, cystitis and pyelonephritis, renal calculi, prostatitis, nitis, complicated intra-abdominal infections (cIAI) and other inter-abdominal infections, dialysis- associated peritonitis, visceral abscesses, endocarditis, myocarditis, rditis, transfusion— associated , meningitis, encephalitis, brain abscess, osteomyelitis, tis, genital ulcers, urethritis, vaginitis, cervicitis, gingivitis, conjunctivitis, keratitis, endophthalmitisa, an infection in cystic fibrosis patients or an infection of febrile neutropenic patients.

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

According to r embodiment, the invention provides a method of decreasing or inhibiting bacterial quantity in a biological sample. This method comprises contacting said biological sample with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

The term "biological sample", 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 f. The term gical sample" also includes living organisms, in which case "contacting a nd of this invention with a biological " is synonymous with the term "administering said compound or ition sing said compound) to a mammal".

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

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

The term osocomial infections" is also referred to as community acquired infections.

In another embodiment, the bacterial infection is characterized by the presence of one or more of Streptococcus pneumoniae, Enterococcusfaecalis, or Staphylococcus aureus.

In another embodiment, the bacterial infection is characterized by the presence of one or more of E. coli, Moraxella catarrhalis, or Haemophilus influenzae.

In another ment, the ial infection is characterized by the presence of one or more of Clostridium diflicile, ria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, cterium abscessus, Mycobacterium kansasii, cterium ulcerans, Chlamydophila pneumoniae and Chlamydia tracomatis.

In another embodiment, the bacterial infection is characterized by the presence of one or more of Streptococcus pneumoniae, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus aureus, Clostridium diflicile, Moraxella catarrhalis, ria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerans, Chlamydophila pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Streptococcus pyogenes or fl-haemolytic streptococci.

In some embodiments, the bacterial infection is characterized by the presence of one or more of Methicillin resistant Staphylococcus aureus, Fluoroquinolone resistant Staphylococcus aureus, ycin intermediate resistant Staphylococcus aureus, Linezolid resistant lococcus aureus, Penicillin resistant Streptococcus pneumoniae, Macrolide resistant ococcus pneumoniae, Fluoroquinolone resistant Streptococcus pneumoniae, Vancomycin resistant Enterococcusfaecalis, Linezolid ant Enterococcus is, Fluoroquinolone resistant Enterococcusfaecalis, Vancomycin resistant Enterococcus faecium, Linezolid resistant Enterococcusfaecium, quinolone ant Enterococcus faecium, Ampicillin resistant Enterococcusfaecium, Macrolide resistant Haemophilus nzae, B—lactam resistant Haemophilus nzae, Fluoroquinolone resistant Haemophilus influenzae, B—lactam resistant lla catarrhalis, Methicillin resistant Staphylococcus epidermidis, Methicillin resistant Staphylococcus epidermidis, Vancomycin resistant Staphylococcus epidermidis, quinolone resistant lococcus epidermidis, Macrolide resistant Mycoplasma niae, lsoniazid resistant Mycobacterium tuberculosis, Rifampin resistant Mycobacterium tuberculosis, Methicillin resistant Coagulase negative staphylococcus, Fluoroquinolone resistant Coagulase negative staphylococcus, Glycopeptide intermediate resistant Staphylococcus aureus, Vancomycin resistant Staphylococcus aureus, Hetero vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin resistant Staphylococcus , Macrolide-Lincosamide—Streptogramin resistant Staphylococcus, B—lactam resistant Enterococcus faecalis, B—lactam resistant Enterococcusfaecium, de resistant Streptococcus pneumoniae, de resistant Streptococcus pyogenes, Macrolide resistant ococcus pyogenes, Vancomycin resistant staphylococcus epidermidis, Fluoroquinolone resistant Neisseria gonorrhoeae, Multidrug Resistant Pseudomonas aeruginosa or Cephalosporin resistant Neisseria gonorrhoeae. ing to another embodiment, the Methicillin resistant Staphylococci are selected from illin resistant Staphylococcus aureus, Methicillin resistant Staphylococcus epidermidis, or Methicillin resistant Coagulase negative staphylococcus.

In some embodiments, a form of a compound of formula (I), or a pharmaceutically acceptable salt f, is used to treat community acquired MRSA (i.e., cMRSA).

In other embodiments, a form of a compound of formula (I), or a pharmaceutically able salt thereof, is used to treat daptomycin resistant organism including, but not limited to, Daptomycin resistant Enterococcusfaecium and Daptomycin resistant Staphylococcus aureus.

] According to another embodiment, the Fluoroquinolone resistant Staphylococci are selected from Fluoroquinolone resistant Staphylococcus aureus, Fluoroquinolone resistant lococcus epidermidis, or Fluoroquinolone resistant ase negative staphylococcus. ing to another embodiment, the Glycopeptide ant Staphylococci are selected from Glycopeptide intermediate resistant lococcus aureus, Vancomycin resistant Staphylococcus aureus, Vancomycin intermediate resistant Staphylococcus , Hetero vancomycin intermediate resistant Staphylococcus aureus, or Hetero vancomycin resistant 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 ant Enterococcusfaecalis, or Linezolid resistant Enterococcus faecium.

According to another ment, the Glycopeptide resistant Enterococci are selected from Vancomycin resistant Enterococcusfaecium or ycin resistant Enterococcusfaecalis.

According to another embodiment, the B-lactam resistant Enterococcus is is B-lactam resistant Enterococcusfaecium.

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

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

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

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

According to another embodiment, the am resistant hilus is [3- lactam resistant Haemophilus influenzae.

According to another embodiment, the Fluoroquinolone resistant Haemophilus is Fluoroquinolone resistant Haemophilus influenzae.

According to another embodiment, the ide resistant Haemophilus is Macrolide resistant Haemophilus influenzae.

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

] According to r embodiment, the lsoniazid resistant Mycobacterium is zid resistant Mycobacterium tuberculosis.

According to another embodiment, the Rifampin resistant Mycobacterium is Rifampin resistant Mycobacterium ulosis.

According to another embodiment, the B-lactam resistant Moraxella is [3- lactam ant Moraxella catarrhalis.

According to another embodiment, the bacterial infection is characterized by the presence of one or more of the following: Methicillin resistant 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 ant Enterococcusfaecalis, Linezolid ant Enterococcusfaecalis, Fluoroquinolone resistant coccus faecalis, Vancomycin resistant Enterococcus faecium, Linezolid resistant Enterococcusfaecium, Fluoroquinolone resistant Enterococcusfaecium, Ampicillin resistant Enterococcusfaecium, Macrolide resistant Haemophilus influenzae, B—lactam resistant Haemophilus influenzae, Fluoroquinolone ant Haemophilus influenzae, B—lactam resistant Moraxella catarrhalis, illin ant Staphylococcus epidermidis, Methicillin resistant Staphylococcus epidermidis, ycin resistant Staphylococcus epidermidis, Fluoroquinolone resistant Staphylococcus epidermidis, Macrolide resistant asma pneumoniae, Isoniazid resistant Mycobacterium tuberculosis, Rifampin resistant Mycobacterium tuberculosis, Fluoroquinolone resistant Neisseria gonorrhoeae or Cephalosporin resistant Neisseria gonorrhoeae.

According to r embodiment, the ial infection is characterized by the ce of one or more of the following: Methicillin resistant Staphylococcus aureus, Methicillin resistant Staphylococcus epidermidis, Methicillin resistant Coagulase negative staphylococcus, Fluoroquinolone resistant Staphylococcus aureus, Fluoroquinolone resistant lococcus midis, Fluoroquinolone resistant Coagulase negative staphylococcus, Vancomycin resistant Staphylococcus aureus, Glycopeptide intermediate resistant Staphylococcus aureus, Vancomycin resistant Staphylococcus aureus, Vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin resistant Staphylococcus aureus, Vancomycin resistant coccusfaecium, Vancomycin resistant Enterococcusfaecalis, Penicillin resistant Streptococcus pneumoniae, Macrolide resistant Streptococcus pneumoniae, quinolone resistant Streptococcus pneumoniae, Macrolide resistant Streptococcus pyogenes, or B—lactam resistant Haemophilus nzae.

According to another embodiment, the bacterial infection is characterized by the ce of one or more of the following: Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcusfaecium, Vancomycin resistant Enterococcus faecalis, Vancomycin resistant Staphylococcus aureus, Vancomycin intermediate ant Staphylococcus aureus, Hetero vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin resistant Staphylococcus aureus, Multidrug Resistant Pseudomonas aeruginosa, lsoniazid resistant Mycobacterium tuberculosis, and in resistant Mycobacterium ulosis.

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

Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, rate, rsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, te, glucoheptanoate, glycerophosphate, glycolate, lfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, nate, salicylate, succinate, sulfate, te, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid on salts.

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

Pharmaceutical compositions of this invention comprise a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Such compositions may ally comprise an additional therapeutic agent. Such 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 immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound.

The term "pharmaceutically acceptable carrier" 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 ceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, in, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride es of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium en phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose—based nces, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene—block polymers, wool fat and self—emulsifying drug delivery s (SEDDS) such as alpha-tocopherol, polyethyleneglycol 1000 ate, or other similar polymeric delivery matrices.

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

Depending upon the particular condition, or disease state, to be treated or prevented, additional therapeutic agents, which are normally administered to treat or prevent that condition, may be administered together with the inhibitors 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 nist, an suppressant, an anti-cancer agent, an iral agent, a ne, a growth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound.

Theconmmundsofflnsinvenfionrnaybeenufloyedhiaconvenfionminanner for controlling bacterial infections levels in vivo and for treating diseases or reducing the ement or severity of effects which are mediated by bacteria. Such s 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 compound of this invention may be combined with a mmmwwMEQWamqmmbafimmmfinwmmmummnmapMEMsflfifimflmma mamamEWmmHM%mmammmmwmmwmwmwbmwmnmdmmwmwm ive to lessen the severity of that infection or disease. atively, the compounds of this invention may be used in compositions and methods for treating or protecting individuals against bacterial infections or diseases over extended periods of time. In one 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 1—2 week period. In another ment, the compounds of this invention may be used in compositions and methods for treating or protecting duals against bacterial infections or diseases over a 4-8 week period (for e, in the treatment of patients with or at risk for ping 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 ions or es over an 8—12 week period. The compounds may be employed in such compositions either alone or together with other nds of this invention in a manner consistent with the conventional utilization of enzyme inhibitors in pharmaceutical compositions. For example, a compound of this invention may be combined with pharmaceutically acceptable adjuvants conventionally employed in vaccines and stered in prophylactically effective amounts to protect duals over an extended period of time against bacterial infections or diseases.

In some embodiments, compounds of formula (I), or a pharmaceutically able salt thereof, may be used prophylactically to prevent a bacterial infection. In some embodiments, compounds of formula (I), or a pharmaceutically able salt thereof, may be used before, during or after a dental or surgical procedure to t opportunistic infections such as those encountered in bacterial endocarditis. 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, nds of formula (I), or a pharmaceutically acceptable salt thereof, may be used prophylactically in surgical procedures including but not limited to general surgery, respiratory surgery (tonsillectomy/adenoidectomy), intestinal surgery (upper GI and elective small bowel surgery, esophageal sclerotherapy and dilation, large bowel resections, acute appendectomy), trauma surgery (penetrating abdominal surgery), -urinary tract surgery (prostatectomy, urethral on, cystoscopy, l or nal hysterectomy, cesarean section), transplant surgery (kidney, liver, pancreas or kidney transplantation), head and neck surgery (skin excisions, neck dissections, laryngectomy, head and neck cancer surgeries, mandibular fractures), orthopaedic surgery (total joint replacement, traumatic open fractures), 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 t a bacterial infection. Treatment with a gyrase and/or topoisomerase 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 prophylactic treatment could be ered is when an individual is more vulnerable to infection due to, for example, weakened immunity, y, , presence of an artificial device in the body (temporary or permanent), an ical defect, exposure to high levels of bacteria or possible exposure to a e— causing pathogen. Examples of factors that could lead to weakened immunity include chemotherapy, radiation therapy, diabetes, advanced age, HIV infection, and transplantation.

An example 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 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 pathogen 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 formula (I), or a pharmaceutically acceptable salt f, 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 sequentially or concurrently to the patient. Alternatively, pharmaceutical or lactic 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 therapeutic agent or agents is an antibiotic selected from a natural penicillin, a llinase-resistant llin, an antipseudomonal penicillin, an aminopenicillin, a first generation osporin, a second generation cephalosporin, a third tion 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 embodiments, the additional therapeutic agent or agents is an antibiotic ed from a penicillin, a cephalosporin, a quinolone, an aminoglycoside or an idinone.

In other ments, the additional therapeutic agents are selected from a natural penicillin including Benzathine penicillin G, Penicillin G and Penicillin V, from a llinase-resistant penicillin including Cloxacillin, Dicloxacillin, Nafcillin and Oxacillin, from a antipseudomonal llin including Carbenicillin, Mezlocillin, Pipercillin, Pipercillin/tazobactam, Ticaricillin and Ticaricillin/Clavulanate, from an aminopenicillin including Amoxicillin, Ampicillin and llin/Sulbactam, from a first generation cephalosporin including Cefazolin, Cefadroxil, Cephalexin and rine, from a second generation cephalosporin including Cefaclor, Cefaclor-CD, Cefamandole, Cefonacid, WO 97270 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, Ceftaroline and Ceftobiprole, from a ycin including Cefotetan and Cefoxitin, from a carbapenem including Doripenem, Imipenem and Meropenem, from a monobactam including nam, from a quinolone including Cinoxacin, Nalidixic acid, Oxolininc acid and Pipemidic acid, from a fluoroquinolone including Besifloxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, xacin, Lomefloxacin, xacin, Norfloxacin, cin and Sparfloxacin, from an aminoglycoside including Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Spectinomycin, Streptomycin and Tobramycin, from a macrolide including Azithromycin, Clarithromycin and Erythromycin, from a ketolide including romycin, from a Tetracycline including Chlortetracycline, Demeclocycline, Doxycycline, Minocycline and Tetracycline, from a eptide including Oritavancin, Dalbavancin, Telavancin, Teicoplanin and Vancomycin, from a streptogramin ing Dalfopristin/quinupristin, from an oxazolidone including Linezolid, from a Rifamycin including Rifabutin and in and from other antibiotics including bactitracin, colistin, Tygacil, ycin, chloramphenicol, clindamycin, isoniazid, metronidazole, mupirocin, polymyxin B, pyrazinamide, trimethoprim/sulfamethoxazole and sulfisoxazole.

In other embodiments, the additional eutic agents are selected from a natural penicillin including Penicillin G, from a penicillinase-resistant penicillin ing Nafcillin and Oxacillin, from an antipseudomonal penicillin including Pipercillin/tazobactam, from an aminopenicillin including Amoxicillin, from a first generation cephalosporin including exin, from a second generation osporin including Cefaclor, Cefaclor- CD and Cefuroxime, from a third generation cephalosporin including Ceftazidime and axone, from a fourth generation cephalosporin including Cefepime, from a carbapenem including Imepenem, Meropenem, Ertapenem, Doripenem, Panipenem and Biapenem,a fluoroquinolone including Ciprofloxacin, Gatifloxacin, Levofloxacin and Moxifloxacin, from an aminoglycoside including Tobramycin, from a macrolide including omycin and Clarithromycin, from a Tetracycline including Doxycycline, from a glycopeptide including ycin, from a Rifamycin including Rifampin and from other antibiotics including isoniazid, pyrazinamide, Tygacil, ycin or trimethoprim/sulfamethoxazole.

In some embodiments, a solid form of a compound 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 a (I) compound, or a pharmaceutically acceptable salt thereof, is dissolved into a liquid and administered iv) composition. In some embodiments, the composition including a formula (I) compound, or a pharmaceutically acceptable salt thereof, is administered in combination with an additional otic agent, for e, 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 fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a glycopeptide, a streptogramin, an oxazolidinone, a rifamycin, or a sulfonamide. In some embodiments, the composition including a solid form of a formula (I) compound, or a pharmaceutically acceptable salt f, is stered orally, and the additional antibiotic agent, for example, 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 lycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a glycopeptide, a streptogramin, an oxazolidinone, a cin, or a sulfonamide is administered iv.

] In some embodiments, a solid form of a a (I) compound, or a pharmaceutically acceptable salt f, can be administered for the treatment of a gram negative infection. In some embodiments, the composition is a solid, liquid (e. g., a suspension), or an iv (e. g., a form of a formula (I) compound, or a pharmaceutically acceptable salt thereof, is dissolved into a liquid and administered iv) composition. In some embodiments the composition ing a formula (I) compound, or a pharmaceutically acceptable salt thereof, is administered in combination with an additional antibiotic agent, selected from a: l penicillin, a penicillinase-resistant penicillin, an antipseudomonal llin, 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 polymyxin, tetracycline or a sulfonamide. In some ments, the composition including a solid form of a formula (I) compound, or a pharmaceutically acceptable salt thereof, is administered orally, and the additional antibiotic agent, for example, a natural penicillin, a llinase-resistant penicillin, an antipseudomonal penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation osporin, a carbapenem, a cephamycin, a monobactam, a quinolone, a fluoroquinolone, an lycoside, a macrolide, a ketolide, a polymyxin, tetracycline or a amide is administered orally. In some embodiments, the additional therapeutic agent is administered The additional therapeutic agents described above may be administered separately, as part of a multiple dosage regimen, 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, y, buccally, vaginally or via an implanted reservoir. The ceutical compositions of this invention may contain any conventional xic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically able acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.

The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, ternal, intrathecal, intralesional and intracranial injection or on techniques.

The pharmaceutical, compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to ques known in the art using le dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable on or suspension in a non- toxic parenterally-acceptable t or solvent, for example, as a solution in l,3-butanediol.

Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium de 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 tic mono— or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are l pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long—chain alcohol diluent or dispersant, such as those described in Pharmacopeia Helvetica, or a similar alcohol.

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

The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable 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 components. Such materials include, but are not d to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the ceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be ated with a le ointment containing the active components suspended or dissolved in a carrier. Carriers for topical stration of the compounds of this ion 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 ded or dissolved in a r. Suitable carriers include, but are not limited to, l oil, sorbitan monostearate, rbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-administered transdermal patches are also included in this invention.

The pharmaceutical compositions of this ion may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well— 2012/021275 known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, tion promoters to enhance bioavailability, fluorocarbons, and/or other lizing or dispersing agents known in the art.

According to another embodiment, compounds of formula (I), or a pharmaceutically 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 designed to reside either ently or temporarily in a subject. Examples of implantable and indwelling s include, but are not limited to, contact lenses, central venous ers and needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal is catheters, etic joints, such as hip and knee replacements, tympanostomy tubes, urinary catheters, voice prostheses, stents, delivery pumps, vascular filters and implantable control release compositions. Biofilms can be detrimental to the health of patients with an implantable or indwelling medical device because they uce 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 pharmaceutically acceptable salt thereof. Any implantable or indwelling device can be used to deliver a nd of formula (I), or a pharmaceutically acceptable salt thereof, provided that a) the , a nd of formula (I), or a pharmaceutically acceptable salt thereof, and any pharmaceutical composition including a compound 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 ceutically acceptable salt thereof, to confer a therapeutic effect on the treated patient.

Delivery of therapeutic agents via implantable or ling s is known in the art. See for example, “Recent Developments in Coated Stents” by Hofma et al. published in Current Interventional Cardiology s 2001, 3:28—36, the entire contents of which, including references cited therein, incorporated herein by reference. Other descriptions of implantable devices can be found in US. Patent Nos. 6,569,195 and 6,322,847; and US. Patent Application Numbers 2004/0044405, 018228, 2003/0229390, 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 include ocked meshed cables. Each cable can e metal wires for structural support and polymeric wires for delivering the therapeutic agent. The polymeric wire can be dosed by immersing the polymer in a solution of the therapeutic agent.

Alternatively, the therapeutic agent can be embedded in the ric wire during the formation of the wire from polymeric precursor solutions.

In other embodiments, implantable or indwelling devices can be coated with polymeric coatings that e the therapeutic agent. The polymeric coating can be designed to control the release rate of the therapeutic agent. Controlled release of therapeutic agents can utilize s logies. Devices are known that have a monolithic layer or coating incorporating a heterogeneous solution and/or dispersion of an active agent in a ric substance, where the diffusion of the agent is rate limiting, as the agent diffuses through the polymer 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 al, such that additional pores or channels are left after the material dissolves. A matrix device is lly diffusion limited as well, but with the channels or other internal geometry of the device also g a role in releasing the agent to the fluid. The ls can be pre-existing channels or channels left behind by ed agent or other soluble substances.

Erodible or able devices typically have the active agent physically immobilized in the polymer. The active agent can be dissolved and/or dispersed throughout the polymeric material. The polymeric material is often hydrolytically degraded over time through hydrolysis of labile bonds, allowing the polymer 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, allowing hydrolysis of labile bonds h the surface, which can lead to homogeneous or bulk erosion of polymer.

The implantable 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 include a polylactic acid/polyethylene oxide (PLA-PEO) 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 ctic acid/polycaprolactone CL) copolymer. The relative amounts and dosage rates of therapeutic agent delivered over time can be controlled by controlling the relative amounts of the faster releasing rs relative to the slower releasing polymers. For higher initial release rates the proportion of faster releasing polymer can be increased ve to the slower releasing polymer. If most of the dosage is desired to be released over a long time period, most of the polymer can be the slower releasing polymer. The device can be coated by spraying the device with a solution or sion 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 ments, 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, ably 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 nd are useful in a erapy for the prevention and treatment of bacterial infections.

Typically, the pharmaceutical compositions of this invention will be administered from about 1 to 5 times per day or alternatively, as a continuous infusion.

Alternatively, the compositions of the present invention may be stered 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 ular mode of administration. A l preparation will contain from about 5% to about 95% active compound (w/w).

Preferably, such preparations contain from about 20% to about 80% active compound.

] When the compositions of this invention comprise a combination 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 monotherapy regime.

Upon improvement of a patient's condition, a maintenance dose of a compound, composition 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 ion is retained when the symptoms 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 disease symptoms.

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

According to another ment, the invention es methods for treating or preventing a bacterial infection, or disease state, comprising the step of administering to a patient any nd, pharmaceutical composition, or combination described herein. The term "patien ", 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 activity in biochemical or cellular assays for ial 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 evident 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 definitions describe terms and abbreviations used : Ac acetyl Bu butyl Et ethyl Ph phenyl Me methyl THF ydrofuran DCM dichloromethane CHzClz dichloromethane EtOAc ethyl acetate CH3CN acetonitrile EtOH ethanol EtZO diethyl ether MeOH methanol MTBE methyl tert—butyl ether DMF N,N—dimethylformamide DMA methylacetamide DMSO dimethyl sulfoxide HOAc acetic acid TEA triethylamine TFA trifluoroacetic acid TFAA trifluoroacetic anhydride Et3N triethylamine DIPEA diisopropylethylamine DIEA diisopropylethylamine K2CO3 potassium carbonate NazCO3 sodium carbonate NaZSZO3 sodium thiosulfate CszCO3 cesium carbonate NaHCO3 sodium bicarbonate NaOH sodium hydroxide NaZSO4 sodium sulfate MgSO4, magnesium sulfate K3PO4 potassium phosphate NH4Cl um chloride 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 1M 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 ic acid N2 nitrogen gas H2 hydrogen gas n—BuLi n—butyl lithium DI de—ionized Pd(OAc)2 palladium(II)acetate PPh3 nylphosphine i-PrOH isopropyl l NBS osuccinimide Pd[(Ph3)P]4 tetrakis(triphenylphosphine)palladium(0) PTFE polytetrafluoroethylene rpm revolutions per minute SM starting material Equiv. equivalents 1H-NMR proton nuclear ic nce Synthesis of the Compounds EXAMPLES THE BENZIMIDAZOLYL UREA ND Scheme 2 provides a method for preparing the benzimidazolyl urea compound.

Scheme 2 dlleAC”WP 30 psi H2, Pd/C + DP—> Br 0 14d'°xa”ei / NEt3 MeOH, rt N02 K2003, reflux N020 N02 0 NH2 0 1 2 4 MTBE, CH3CN NBS, 2°C E :OH iOH B r Ni \N N \N Pdldppflc'z TFAA, Me—THF ' aq / 45 psi H2, Pd/C aq Nazcos NaOH 2c to rt <— ‘— <—| + OQN NEtg, MeOH, THF 1 ,4-dioxane 14-doxane NH4N03 rt to 40°C reflux remix YNH O NH2 0 HZN OZN CF3 NH2 0 6 9 8 OH OH N \N I NI \N / / Ni \ N 0 3’0 JL A JL 10 EiHN H N NHE‘ '—> chiral chrom MeSO3H —> —> pH 3.5 buffer N N\ DCM, EtOH dioxane, reflux >\’NH 0 >’NH O 2°C _ rt HN HN >=o >=o Hr HN 11 HN >20 Me803H ) > 12 HN ) 13 Example l.a Preparation of 2-(2-nitrophenyl)-2,5-dihydrofuran (3a) and 2-(2-nitrophenyl)-2,3- dihydrofuran (3b) Pd(0AC)2. dppp + O + Br 0 1,4—dioxane, \ N02 K2CO3, reflux N02 0 N02 0 1 2 3a 3b l-Bromonitro-benzene (1) (600 g, 99%, 2.941 mol, Alfa Aesar A1 1686), l,3—bis(diphenylphosphino)propane (62.50 g, 97%, 147.0 mmol, Alfa Aesar A1293 l), 1,4— dioxane (2.970 L, Sigma—Aldrich 360481), ium carbonate (812.9 g, 5.882 mol, JT— Baker 301201), and 2,3-dihydrofuran (2) (1.041 kg, 99%, 1.124 L, 14.70 mol, Aldrich 200018) were mixed in a reaction vessel. A stream of nitrogen was bubbled through the stirring mixture for 4 hrs, followed by addition of palladium (II) acetate (16.51 g, 73.52 mmol, Strem ) and continuation of deoxygenation for another 10 minutes. The reaction mixture was d at reflux under en overnight (NMR of a worked-up aliquot showed complete consumption of arylbromide). The reaction mixture was allowed to cool, diluted with hexane (l L), filtered through a short plug of Florisil® (500 g, -200 mesh), and eluted with EtOAc. The filtrate was concentrated under reduced re nitrophenyl)- 2,3-dihydrofuran (3b) is volatile under high vacuum and may be somewhat unstable at room temperature) giving a mixture of (3a) and (3b) as a dark brown oil (654.0 g). The crude material was stored in the refrigerator and carried forward t further purification.

Example l.a.l Asymmetric Preparation of 2-(2-nitrophenyl)-2,5-dihydrofuran (3 a) and 2-(2-nitrophenyl)- 2,3—dihydrofuran (3b) R)Psd(OAC)2J)-osiPhos :O()1(,4-dioxane K2C03, 105°C N02 N02 N02 N02 (R)—3a (S)—3a (R)-3b (S)-3b l—bromo—2—nitrobenzene (50.0 mg, 98%, 0.2426 mmol, h 365424), potassium carbonate (67.1 mg, 0.4852 mmol, JT—Baker 301201), (R)—(-)—l—[(S)—2— (diphenylphosphino)ferrocenyl]ethyldicyclohexylphosphine ethanol adduct ((R)—(S)— JosiPhos, 7.8 mg, 0.01213 mmol, Strem 261210), 2,3—dihydrofuran (1.0 mL, 99%, 13.08 mmol, Aldrich 200018), and l,4-dioxane (0.98 mL) were mixed in a reaction tube. A stream of nitrogen was bubbled through the stirring mixture for 20 s, and then palladium (II) acetate (1.36 mg, 65 mmol, Strem 461780) was added. The tube was sealed and the reaction e stirred at 105°C overnight. HPLC of the crude reaction mixture showed nearly complete consumption of aryl bromide and formation of a 1:1 mixture of the 2-(2- henyl)-2,5-dihydrofuran (3a) and 2-(2-nitrophenyl)-2,3-dihydrofuran (3b). The reaction mixture was allowed to cool, diluted with hexane (2 mL), filtered, and rinsed with ethyl acetate. The filtrate was concentrated on a rotary evaporator to afford a brown oil (51 mg). The material was not placed under high vacuum due to volatility and stability concerns.

The crude reaction mixture was determined to be a 1:1 mixture of (3a) and (3b) by 1H NMR amhmsTMoflw%pmmwbywkaghmmmm%mmwdmmgmm0m3W6EOAMn hexane (or 0 to 100% CHzClz in hexane) to afford pure samples of (3a) and (3b). Analytical data for these samples was as s: 2—(2—nitrophenyl)—2,5-dihydrofuran (3a) was obtained as a yellow solid (97% HPLC purity, 97.0% ee): LCMS (C18 column eluting with 10—90% MeOH / water nt from 3-5 minutes with formic acid modifier) M+1: 192.05 (3.40 min); HPLC retention time of 4.2 min (YMC ODS-AQ 150 x 3.0 mm column eluting with 10—90% CH3CN / water gradient over 8 minutes with 0.1% TFA er and 1 mL/min flow rate); analytical chiral HPLC retention time of 7.4 min (major enantiomer) and 8.1 min (minor omer) eluting with 10% iPrOH / hexane on a CEL® OJ® 4.6 x 250 mm column with 1 mL/min flow rate at 30°C; 1H NMR (300 MHz, CDCl3) 5 8.02 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.45 — 7.38 (m, 1H), 6.37 — 6.30 (m, 1H), 6.11 — 6.06 (m, 1H), 6.04 — 5.98 (m, 1H), 5.02 — 4.83 (m, 2H) ppm; 13C NMR (75 MHz, CDC13) 5 146.97, 139.11, 13395,12958,12810,12809,12678,12438,8428,76421xnn;BC])EPTVthR(75 MHz, CDC13) 5 133.95 (CH), 129.58 (CH), 128.10 (CH), 128.09 (CH), 126.78 (CH), 124.38 (CPD,8428(CTD,7642(CPh)pan 2—(2—nitrophenyl)—2,3-dihydrofuran (3b) was obtained as a yellow oil % HPLC purity, 44.0% ee): LCMS (C18 column eluting with 10-90% MeOH / water gradient from 3-5 minutes with formic acid modif1er) M+1: 192.05 (3.72 min); HPLC retention time of 4.8 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 retention time of 5.96 min (major enantiomer) and 6.35 min (minor enantiomer) eluting with 10% iPrOH / hexane on a CHIRALCEL® OJ® 4.6 x 250 mm column with 1 mL/min flow rate at 30°C; 1H NMR (300 MHz, CDCl3) 5 8.08 (d, J = 8.2 Hz, 1H), 7.73 (d, J =78lfi,HD,7650,J=761h,HD,748—739(m,HD,650@LJ=241h,HD,610 (wLJ=109,74Ph,HD,495@LJ=25PR,HD,346—335(m,HD,250—239(m,HD ppm; 13C NMR (75 MHz, CDCl3) 5 , 144.98, 139.73, 133.93, 128.07, , 124.85, 99.29, 78.45, 38.29 ppm; 13C DEPT NMR (75 MHz, CDCl3) 5 144.98 (CH), 133.93 (CH), 128.07 (CH), 127.11 (CH), 124.85 (CH), 99.29 (CH), 78.45 (CH), 38.29 (CH2) ppm. 321 and 3b were carried through the reduction step to afford 2—tetrahydrofuran— 2-yl-aniline (4) as set forth in Example 1.b (below). is of this material revealed that both 321 and 3b were formed with the same major enantiomer, with an overall 70% ee. It is unknown whether the absolute stereochemistry of the major enantiomer was (R) or (S).

Example 1.b ation of 2-tetrahydrofuranyl-aniline (4) psi H2, Pd/C NEt3, MeOH rt N02 NO2 O NH2 0 5% Palladium on carbon (16.3 g, 50% wet, 3.83 mmol, Aldrich 330116) was placed in a Parr bottle under nitrogen, followed by MeOH (100 mL, er 909333). The crude mixture of 2-(2-nitrophenyl)-2,5-dihydrofuran and 2-(2-nitrophenyl)-2,3-dihydrofuran (3a & 3b) (163 g) dissolved in MeOH (3 89 mL) was added to the Parr bottle, ed by NEt3 (237.6 mL, 1.705 mol, Sigma-Aldrich 471283). The bottle was placed on a Parr shaker and saturated with H2. 30 psi H2 was added and the bottle was shaken until starting material was completely consumed (LCMS and NMR showed complete reaction). The reaction mixture was purged with nitrogen, filtered through CeliteTM and rinsed with EtOAc. The te was concentrated on a rotary evaporator giving a brown oil. The reaction was repeated three more times on the same scale and the batches were combined for purification.

The crude product was vacuum distilled (ca. 15 torr) collecting the distillate at 108 - 129°C to give (4) as a clear faint yellow oil (427.9 g, average yield was 84%; 98% GCMS purity).

LCMS (C18 column eluting with 10—90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 163.95 (1.46 min). 1H NMR (300 MHz, CDCl3) 5 7.15 — 7.04 (m, 2H), 6.77 — 6.62 (m, 2H), 4.85 — 4.77 (m, 1H), 4.18 (s, 2H), 4.12 — 4.02 (m, 1H), 3.94 — 3.85 (m, 1H), 2.25 — 1.95 (m, 4H) ppm.

WO 97270 Example 1.c Preparation of 4-bromotetrahydrofuranyl-aniline (5).

NH O MTBE, CH3CN 2 ””2 O NBS, 2°C 4 5 To a stirring solution of 2-tetrahydrofuranyl-aniline (4) (53.45 g, 327.5 mmol) in methyl tert-butyl ether (MTBE, 641.4 mL) and acetonitrile (213.8 mL) cooled to 2°C was added N—bromosuccinimide (NBS, 58.88 g, 99%, 327.5 mmol, Aldrich B81255) in 4 portions maintaining internal temperature below about 8°C. The reaction e was stirred while cooling with an ice—water bath for 30 minutes (NMR of a worked—up aliquot showed complete consumption of starting material). Aqueous 1 N Na2S203 (330 mL) was added to the reaction mixture, removed the cold bath and d for 20 minutes. The mixture was diluted with EtOAc and the layers were separated. The organic phase was washed with saturated aqueous NaHCO3 (2x), water, brine, dried over MgSO4, filtered through a short plug of , eluted with EtOAc, and concentrated under d pressure to give (5) as a very dark amber oil (82.25 g, 77—94% HPLC purity). Carried forward without further purification. LCMS (C18 column eluting with 10—90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 242.10 (2.89 min). 1H NMR (300 MHz, CDCl3) 5 7.22 (d, J = 2.3 Hz, 1H), 7.16 (dd, J = 8.4, 2.3 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H), 4.79 — 4.73 (m, 1H), 4.15 (s, 2H), 4.10 — 4.01 (m, 1H), 3.93 — 3.85 (m, 1H), 2.26 — 2.13 (m, 1H), 2.12 — 1.97 (m, 3H) ppm.

Example 1.d Preparation of N—(4-bromonitrotetrahydrofuranyl-phenyl)-2,2,2-trifluoro-acetamide (6)- TFAA, Me-THF, 2°C to rt NH4N03, rt to 40°C 0 NH 0 NHZO Y CF3 To trifluoroacetic anhydride (455.3 mL, 3.275 mol, Sigma—Aldrich 106232) stirring at 2°C was slowly added 4-bromotetrahydrofuranyl-aniline (5) (79.29 g, 327.5 mmol) as a thick oil via on funnel over 15 minutes (reaction ature rose to 14°C).

The ing oil was rinsed into the reaction mixture with anhydrous 2-methyltetrahydrofuran (39.6 mL, Sigma—Aldrich 414247). The cold bath was removed and ammonium e (34.08 g, 425.8 mmol, Aldrich 467758) was added. The reaction temperature rose to 40°C over about 30 minutes at which time a cold water bath was used to control the exotherm and bring the reaction to room temperature. The cold bath was then removed and stirring continued for another 40 minutes (HPLC showed very little remaining un-nitrated material). The reaction mixture was slowly poured into a stirring mixture of d ice (800 g). The solid precipitate was collected by filtration, washed with water, saturated aqueous NaHC03 (to pH 8), water again, and hexane. The wet solid was dried first in a convection oven at 50°C for several hours and then under reduced pressure in an oven at 40°C overnight giving (6) as a light brown solid (77.86 g, 62% yield; 98% HPLC ).

LCMS (C18 column eluting with 10—90% CH3CN / water gradient over 5 minutes with formic acid r) M+1: 383.19 (3.27 min). 1H NMR (300 MHz, CDCl3) 5 9.81 (s, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H), 4.88 (dd, J = 9.0, 6.5 Hz, 1H), 4.17 — 4.08 (m, 1H), 4.03 — 3.95 (m, 1H), 2.45 — 2.34 (m, 1H), 2.17 — 2.06 (m, 2H), 1.96 — 1.83 (m, 1H) ppm.

Example 1.e Preparation of 4-bromonitrotetrahydrofuranyl-aniline (6a). aq NaOH OZN 1,4-dioxane reflux OZN O NH O \\l’ NH2 0 N—(4-bromonitrotetrahydrofuranyl-phenyl)-2,2,2-trifluoro-acetamide (6) (54.00 g, 140.9 mmol) was dissolved in oxane (162 mL) and added aqueous 6 M NaOH (70.45 mL, 422.7 mmol, JT-Baker 567202). The reaction mixture was stirred at reflux for 2 days (HPLC showed complete conversion). The mixture was allowed to cool, diluted with MTBE (800 mL), and washed with water (2 x 200 mL), saturated aqueous NH4Cl, water, and brine. The mixture was dried over MgSO4, filtered, and concentrated under reduced pressure to give (621) as a dark amber oil (40.96 g, 93% yield; overall 92% HPLC plus NMR purity). LCMS (C18 column g with 10-90% MeOH / water gradient from 3- minutes with formic acid modifier) M+1: 287.28 (3.44 min). 1H NMR (300 MHz, CDCl3) 5 8.24 (d, J = 2.4 Hz, 1H), 7.41 (d, J = 2.3 Hz, 1H), 6.91 (s, 2H), 4.80 (t, J = 7.2 Hz, 1H), 4.14 — 4.05 (m, 1H), 3.98 — 3.90 (m, 1H), 2.36 — 2.19 (m, 1H), 2.15 — 2.01 (m, 3H) ppm.

Example If Preparation of 2-[5-(4-aminonitrotetrahydrofuranyl-phenyl)pyrimidinyl]propan ol (8).

\ N Pd(PPh3 4) N \N N C0 I aq a2 3 / 1 4—dioxane OZN 1 /B\ reflux O O NH2 0 as NH2 0 7 8 4-Bromonitrotetrahydrofuranyl-aniline (621) (40.40 g, 92%, 129.5 mmol), 1,4-dioxane (260 mL, Sigma-Aldrich 360481), 2-[5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan—2—yl)pyrimidin—2-yl]propan—2—ol (7) (41.05 g, 155.4 mmol), and aqueous 2.7 M NazCO3 (143.9 mL, 388.5 mmol) were mixed. A stream of nitrogen was bubbled through the stirring mixture for 1 hr, followed by addition of tetrakis(triphenylphosphine)palladium (0) (7.48 g, 6.47 mmol, Strem 462150). The reaction mixture was stirred at reflux for 2 hrs (HPLC showed complete reaction), allowed to cool, and diluted with EtOAc. The mixture was washed with water, saturated aqueous NH4Cl, and brine, dried over MgSO4, and filtered h a short plug of il® eluting with EtOAc. The filtrate was concentrated under reduced pressure giving dark brown oil. The oil was dissolved in CHzClz and eluted through a short plug of silica gel with CHzClz and then EtOAc. The desired fraction was concentrated on a rotary evaporator until a precipitate formed giving a thick brown slurry, which was triturated with MTBE. The solid was ted by tion, washed with MTBE, and dried under high vacuum giving (8) as a yellow solid (35.14 g, 99+% HPLC purity). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 345.00 (2.69 min). 1H NMR (300 MHz, CDCl3) 5 8.88 (s, 2H), 8.36 (d, J = 2012/021275 2.2 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.09 (s, 2H), 4.92 (t, J = 7.2 Hz, 1H), 4.62 (s, 1H), 4.20 — 4.11 (m, 1H), 4.03 — 3.94 (m, 1H), 2.39 — 2.26 (m, 1H), 2.23 — 2.08 (m, 3H), 1.64 (s, 6H) ppm. The filtrate was concentrated and purified by ISCO silica gel chromatography eluting with 0 to 80% EtOAc / hexane giving a second crop of product (8) as an amber solid (4.46 g, 88% overall yield ; 88% HPLC purity).

Example 1.g Preparation of 2-[5-(3,4-diaminotetrahydrofuranyl-phenyl)pyrimidinyl]propanol (9). :0H fiOH psi45 H2, Pd/C NEt3, MeOH THF ] To a suspension of 2-[5-(4-amino-3 -nitrotetrahydrofuranyl- phenyl)pyrimidinyl]propanol (8) (30.10 g, 87.41 mmol) and THF (90 mL) in a Parr bottle under nitrogen was added a slurry of 5% palladium on carbon (3.01 g, 50% wet, 0.707 mmol, Aldrich 330116) in MeOH (90 mL, JT—Baker ), followed by NEt3 (24.37 mL, 174.8 mmol, Sigma-Aldrich 471283). The vessel was placed on a Parr shaker and saturated with H2. After adding 45 psi H2, the vessel was shaken until consumption was complete (HPLC showed complete conversion). The reaction mixture was purged with nitrogen, filtered through CeliteTM and rinsed with EtOAc. The e was re-filtered through a 0.5 micron glass fiber filter paper sandwiched between two P5 papers, and concentrated under reduced pressure giving (9) as a light brown foam (28.96 g, 98% yield; 93% NMR purity).

LCMS (C18 column eluting with 10—90% CH3CN / water nt over 5 minutes with formic acid modifier) M+1: 315.32 (1.54 min). 1H NMR (300 MHz, CDCl3) 5 8.83 (s, 2H), 6.92 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 1.8 Hz, 1H), 4.90 (dd, J = 7.9, 6.2 Hz, 1H), 4.72 (s, 1H), 4.18 (s, 2H), 4.17 — 4.08 (m, 1H), 3.99 — 3.89 (m, 1H), 3.46 (s, 2H), 2.34 — 2.19 (m, 1H), 2.17 — 2.05 (m, 3H), 1.63 (s, 6H) ppm.

Example 1.h ation of 1-ethyl-3 -[5-[2-(1-hydroxymethyl-ethyl)pyrimidinyl]tetrahydrofuran- 2-yl-IH—benzimidazolyl]urea (11).

N\NOI Z Z I s/ O // NWNHB pH 3. 5 buffer N\ dioxane reflux >’NH HN) 11 To a stirring solution of 2-[5-(3,4-diaminotetrahydrofuranyl- phenyl)pyrimidinyl]propanol (9) (32.10 g, 102.1 mmol) in 1,4-dioxane (160.5 mL, Sigma—Aldrich 360481) was added pH 3.5 buffer (240.8 mL), prepared by dissolving NaOAc trihydrate (34.5 g) in IN aqueous H2SO4 (240 mL). 1-Ethyl(N—(ethylcarbamoyl)-C— methylsulfanyl-carbonimidoyl)urea (10) (28.46 g, 122.5 mmol, CB Research and Development) was added to the on mixture and stirred at reflux overnight (HPLC showed 99% consumption of ng diamine). The reaction mixture was cooled to room temperature and poured portion-wise (frothing) into a stirring solution of s saturated NaHCO3 (480 mL) and water (120 mL) giving pH 8-9. This 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 (11) as an off—white solid (34.48 g, 82% yield; 99.4% HPLC purity). LCMS (C18 column eluting with 10—90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 411.41 (1.73 min). 1H NMR (300 MHz, MeOD) 5 9.02 (s, 2H), 7.62 (s, 1H), 7.37 (s, 1H), 5.31 (s, 1H), 4.23 (dd, J = 14.5, 7.3 Hz, 1H), 4.01 (dd, J = 15.0, 7.1 Hz, 1H), 3.38 — 3.28 (m, 2H), 2.58 — 2.46 (m, 1H), 2.16 — 2.05 (m, 2H), 2.02 — 1.88 (m, 1H), 1.63 (s, 6H), 1.22 (t, J = 7.2 Hz, 3H) ppm.

Example 1.i Chiral chromatographic ion of 1-ethyl-3 -[5-[2-(1-hydroxymethyl-ethyl)pyrimidin-5 - yl][(2R)—tetrahydrofuranyl]-1H-benzimidazolyl]urea (12) E :OH E :OH N \ N N \ N | I / / chiral chrom N N >\’NH O >\’NH O HN HN >20 >20 HN 11 HN > > 12 A racemic sample of 1-ethyl[5-[2-(1-hydroxymethyl-ethyl)pyrimidin tetrahydrofuran—2—yl—1H-benzimidazol—2—yl]urea (11) (24.60 g) was resolved on a CHIRALPAK® IC® column (by Chiral Technologies) eluting with CHzClz / MeOH / TBA (60/ 40/ 0.1) at 35°C giving the desired enantiomer (12) as a white solid (11.35 g, 45% yield; 99+% HPLC purity, 99+% ee). Analytical chiral HPLC retention time was 6.2 min (CHIRALPAK® IC® 4.6 x 250 mm column, 1 mL/min flow rate, 30°C).

The structure and absolute stereochemistry of 12 were confirmed by single— crystal x-ray diffraction analysis. Single crystal diffraction data was acquired on a Bruker Apex II diffractometer equipped with sealed tube Cu K-alpha source (Cu K01 radiation, y = 1.54178 A) and an Apex II CCD detector. A crystal with dimensions of 1/2x 0.05 x 0.05 mm was selected, cleaned using l oil, mounted on a MicroMount and ed on a Bruker APEXH system. Three batches of 40 frames separated in reciprocal space were obtained to e an orientation matrix and initial cell ters. 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 structure was solved and refined in acentric P21 space group.

A ction data set of reciprocal space was obtained to a resolution of 0.9 A using 050 steps using 60 s exposure for each frame. Data were collected at 100 (2) K.

Integration of intensities and refinement of cell parameters were accomplished using APEXII 2012/021275 software. Observation of the crystal after data collection showed no signs of decomposition.

As shown in Fig. 1, there are two symmetry independent molecules in the ure and both symmetry independent molecules are R isomers.

The data was collected, refined and reduced using the Apex 11 software. The structure was solved using the SHELXS97 (Sheldrick, 1990); program(s) and the structure d using the SHELXL97 (Sheldrick, 1997) program. The crystal shows monoclinic cell with P21 space group. The e parameters are a = 9.8423(4) A, b = 10.8426(3) A, c = 19.4441 (7) A, [3 = 102.966(3)°. Volume = 2022.09(12) A3.

Example 1.j Preparation of the esulfonic acid salt of 1-ethyl[5-[2-(1-hydroxymethylethyl )pyrimidinyl][(2R)-tetrahydrofuranyl]-1H-benzimidazolyl]urea (13) N \N I f” / N \N MeSO3H >’NH DCM,EtOH 2°C-rt N HN>=O HN>\’NH o HN >20 ) 12 HN Me803H A stirring suspension of 1-ethyl[5-[2-(1-hydroxymethyl-ethyl)pyrimidin- 7-[(2R)—tetrahydrofuranyl]-1H-benzimidazolyl]urea (12) (9.32 g, 22.71 mmol) in absolute ethanol (93.2 mL) was cooled with an ice-water bath. Methanesulfonic acid (1.548 mL, 23.85 mmol, Sigma—Aldrich 471356) was added, removed cold bath and stirred at room temperature for 20 minutes. It was concentrated on a rotary evaporator at 35°C to a thick slurry, diluted with EtOAc, collected the solid by filtration, washed with EtOAc, and dried under reduced re giving an initial crop of (13) as a white solid (8.10 g). The filtrate was concentrated on a rotary evaporator giving a yellowish glassy foam, which was dissolved in EtOH, concentrated to a solid slurry, triturated with EtOAc / EtZO, and ted by filtration. The solid was washed with EtOAc / EtZO, combined with the first crop, and dried under reduced pressure giving (13) as a white solid (9.89 g, 86% yield; 99+% HPLC purity, WO 97270 99+% ee). Analytical chiral HPLC shows one enantiomer with ion time of 6.3 min eluting with CHzClz / MeOH / TBA (60/ 40 / 0.1) on a CHIRALPAK® IC® 4.6 x 250 mm column with 1 mL/min flow rate at 30°C. LCMS (C18 column eluting with 10—90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 411.53 (1.74 min). 1H NMR (300 MHz, MeOD) 5 9.07 (s, 2H), 7.79 (s, 1H), 7.62 (s, 1H), 5.30 (t, J = 7.3 Hz, 1H), 4.24 (dd, J = 14.6, 7.3 Hz, 1H), 4.04 (dd, J = 15.0, 7.6 Hz, 1H), 3.40 — 3.30 (m, 2H), 2.72 (s, 3H), 2.65 — 2.54 (m, 1H), 2.20 — 2.07 (m, 2H), 2.04 — 1.90 (m, 1H), 1.64 (s, 6H), 1.23 (t, J = 7.2 Hz, 3H) ppm.

Example 1.k 1—pot deprotection/ Suzuki procedure Preparation of 2-[5-(4-aminonitrotetrahydrofuranyl-phenyl)pyrimidinyl]propan 01(8).

N \N Pd(dppf)C|2 ”I \N V / aq NaZCO3 OZN + B 1,4-dioxane OYNH O 0’ \O reflux CF3 M OZN 6 NH2 0 N—(4-Bromonitrotetrahydrofuranyl-phenyl)-2,2,2-trifluoro-acetamide (6) (19.00 g, 49.59 mmol), 2—[5—(4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl)pyrimidin yl]propan—2—ol (7) (14.41 g, 54.55 mmol), aqueous 2.7 M sodium carbonate (73.48 mL, 198.4 mmol), and 1,4-dioxane (190 mL, Sigma-Aldrich 360481) were mixed. A stream of nitrogen was bubbled through the stirring mixture for 40 minutes, followed by addition of 1,1’- bis(diphenylphosphino)ferrocene dichloropalladium dichloromethane adduct (2.025 g, 2.480 mmol, Strem 460450). The reaction mixture was stirred at reflux under N2 for 7 hrs, added another 50 mL of saturated aqueous sodium carbonate, and refluxed for another 16 hrs. The reaction e was d to cool, then diluted with EtOAc (500 mL) and water (200 mL). The layers were separated and the aqueous phase extracted with EtOAc (200 mL). The combined organic phase was washed with water (500 mL), brine (500 mL), dried over NaZSO4, filtered h a Florisil® plug, and concentrated on a rotary evaporator to give crude (8) as an orange oil. Purified by ISCO silica gel chromatography eluting with 20—90% EtOAc / hexane to give (8) as an orange solid (15.00 g, 81—88% ). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 345.35 (2.68 min). 1H NMR (300 MHz, CDC13) 5 8.88 (s, 2H), 8.36 (d, J = 2.2 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.09 (s, 2H), 4.92 (t, J = 7.2 Hz, 1H), 4.62 (s, 1H), 4.20 — 4.11 (m, 1H), 4.03 — 3.94 (m, 1H), 2.39 — 2.26 (m, 1H), 2.23 — 2.08 (m, 3H), 1.64 (s, 6H) ppm.

Example 1.k Preparation of Form 1: Chiral chromatographic isolation of 1-ethyl[5-[2-(1-hydroxy methyl-ethyl)pyrimidinyl]—7-[(2R)-tetrahydrofuranyl]—1H-benzimidazolyl]urea ] A racemic sample of 1-ethyl[5-[2-(1-hydroxymethyl-ethyl)pyrimidin yl]—7—tetrahydrofuran—2—yl—1H-benzimidazol—2—yl]urea (24.60 g) was resolved on a CHIRALPAK® IC® column (by Chiral Technologies) eluting with DCM / MeOH / TBA (60 / 40/ 0.1) at 35°C. The d fractions were ted, concentrated to dryness on a rotary evaporator, then dried overnight in a vacuum oven at about 40°C giving the desired enantiomer as a white solid (11.35 g, 99+% HPLC purity, 99+% ee) which was used for physical form characterization. LCMS (C18 column eluting with 10—90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 411.66 (1.74 min). HPLC ion time was 3.61 min (YMC ODS—AQ 150 x 3.0 mm column eluting with 10—90% CH3CN/ water gradient over 8 minutes with 0.1% TFA er and 1 mL/min flow rate). Analytical chiral HPLC shows one enantiomer with retention time of 6.2 min (CHIRALPAK® IC® 4.6 x 250 mm column, 1 mL/min flow rate, 30°C). 1H NMR (300 MHz, MeOD) 5 9.02 (s, 2H), 7.62 (d, J = 1.6 Hz, 1H), 7.37 (s, 1H), 5.32 (br.s, 1H), 4.23 (dd, J = 14.8, 6.7 Hz, 1H), 4.01 (dd, J = 15.1, 7.0 Hz,1H), 3.37 — 3.29 (m, 2H), 2.58 — 2.46 (m, 1H), 2.17 — 2.06 (m, 2H), 2.03 — 1.90 (m, 1H), 1.63 (s, 6H), 1.22 (t, J = 7.2 Hz, 3H) ppm.

Example 1.1 Preparation of Form 11 To 20 mg of the benzimidazolyl urea compound was added to an HPLC vial and 1 mL acetonitrile (CH3CN) was added to the vial with stirring at room temperature. To the resulting suspension a stoichiometric amount of 1 Molar HCl solution (0.049 mL) in water was added. The vial was crimp—capped and the mixture (suspension) was allowed to equilibrate for 6 days under gentle stirring before it was filtered and the white solid, dried under vacuum for several hours and recovered for physical form characterization.

Example 1.m Preparation of Form 111 To 20 mg of the benzimidazolyl urea compound in an HPLC vial 0.5 ml of THF was added under stirring at room temperature. Stoichiometric amount of HCl was added as 1M aqueous solution (0.049 mL). 2 mL of methyl tert—butyl ether was added and the suspension was allowed to equilibrate ght with stirring. It was then filtered, and the white solid was dried under vacuum for several hours before subjecting the compound to physical form characterization.

Example 1.n Preparation of Form IV ] A suspension of 3,4—diamino-5—[(2R)—tetrahydrofuran—2— yl]phenyl]pyrimidinyl]propanol (2 g, 6.362 mmol) and (3Z)ethyl [(ethylcarbamoylamino)-methylsulfanyl-methylene]urea (1.478 g, 6.362 mmol) in dioxane (26.00 mL) and buffer pH 3.5 (100 mL, stock solution made from 1N HZSO4 and NaOAc) was stirred for 3 h under reflux (~95 oC). Then the reaction e was quenched with approximately 50 mL water. The crude reaction mixture was transferred to a larger roundbottom flask, neutralized with NaHCO3, then filtered to give a beige solid which was washed with hot water (~200 mL). The solid (1.84 g) was dried and was salted as MeSO3H salts. 2.25 g (81%ee; 98% pure; 69.8 % yield) of pure product was obtained which was submitted for supercritical fluid chromatography (SFC) chiral tion to give peak one (S- enantiomer) and peak two (R—enantiomer).

The material (the R-enantiomer) from SFC (peak 2) was suspended in MeOH (~20 mL) and basifled with sat NaHC03 (200 mL). The mixture was d for 1h, then filtered. After flltration, the solids were collected, washed with warm water (~500 mL) and dried. 1.22 g of parent molecule (free base; peak 2) was obtained which was then salted as the mesylate. 1.43 g of pure mesylate was obtained (R; 99 % ee; 99% pure).

The te salt of the compound of the present application may be prepared using methods known to those skilled in the art. For example, the free base of the idazolyl urea compound may be mixed with a stoichiometric amount of a methanesulfonic acid and the mixture concentrated until a solid is obtained. atively, the free base of the benzimidazolyl urea compound may be suspended in an appropriate solvent containing the acid and allowing the mixture to equilibrate until the free base if converted to the corresponding acid addition salt. Figure 12 shows a 1H NMR spectrum of the mesylate salt of the benzimidazolyl urea compound.

LOGY STUDIES The enzyme tion activities of compounds of this invention may be determined in the experiments described below: ] DNA Gyrase ATPase Assay The ATP hydrolysis ty of S. aureus DNA gyrase is measured by coupling the production of ADP h pyruvate kinase/lactate dehydrogenase to the oxidation ofNADH. This method has been described previously (Tamura and Gellert, 1990, J. Biol. Chem, 265, 21342).

] ATPase assays are carried out at 30°C in buffered 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 phosphoenol pyruvate, 200 ”M nicotinamide e 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 ted by the addition of ATP to a final tration of 0.9 mM, and the rate of NADH disappearance is monitored at 340 ters over the course of 10 minutes. The Ki and IC50 values are determined from rate versus concentration profiles.

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 MgCl2, 200 mM K°Glutamate, 2.5 mM phosphoenol pyruvate, 0.2 mM NADH, 1 mM DTT, 5 ug/mL linearized DNA, 50 ug/mL BSA, 30 ug/mL pyruvate kinase, and 10 ug/mL lactate dehyrodgenase (LDH). The reaction is ted 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 IC50 are determined from plots of rate vs. concentration of selected compound fit to the Morrison Equation for tight binding inhibitors.

Example 3 Susceptibility Testing in Liquid Media WO 97270 nds 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 ces: 8 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard-- Eighth Edition (2009)". Other publications such as "Antibiotics in Laboratory Medicine" (Edited by V. Lorian, Publishers Williams and Wilkins, 1996) provide essential practical techniques in laboratory antibiotic testing. The c protocols used were as follows: ol #1: Gyrase MIC determination of nds using microdilution broth method Materials: Round bottom l microtiter plates (Costar 3788) r Hinton H agar plates (MHH; BBL premix) Mueller Hinton H 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 (U.S. Biologicals S1010—5 1) Laked horse blood (Quad Five 270—100) Resazurin 0.01% Sprague Dawley Rat serum (U.S. Biologicals 1011—90B or Valley BioMedical AS306 1 SD) Pooled Mouse serum (Valley BioMedical AS3054) Strains (media, broth and agar): 1. Staphylococcus aureus ATCC #29213 a. MHH b. MHH -- 50% human serum c. MHII -- 50% rat serum d. MHII -- 50% mouse serum 9‘95“!” Staphylococcus aureus ATCC #29213 GyrB T1731 (MHll) Staphylococcus aureus, JMI collection strains; see table 5 (MHll) Staphylococcus midis, JMI collection strains; see table 5 (MHII) Enterococcusfaecalis ATCC #29212 (MHII + 3% laked horse blood) _ 53 _ 6. Enterococcusfaecium ATCC #49624 (MHII + 3% laked horse blood) 7. Enterococusfaecalis, JMI collection strains; see table 5 (MHII -- 3% laked horse blood) 8. Enterococusfaecium, JMI collection strains; see table 5 (MHII -- 3% laked horse blood) 9. Streptococcus pneumoniae ATCC #10015 (MHII + 3% laked horse blood) . Streptococcus pneumoniae, JMI tion strains; see table 5 (MHH + 3% laked horse blood) 11. flhaemolytic streptococci, Groups A, B, C, G) JMI collection strains; see table 5 (MHII + 3% laked horse blood) 12. Bacillus cereus ATCC 10987 (MHH) 13. Bacillus cereus ATCC 14579(MHII) 14. Bacillus subtilis ATCC 6638 (MHII) . Bacillus subtilis (168) ATCC 6051 (MHII) Inoculum 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 inoculated 1 mL of sterile saline provided in the kit. 2. Vortexed the wells for ~ 30 s to provide a suspension of ~108 mL. Actual y could be confirmed by plating out dilutions of this suspension. 3. d the suspension 1/ 100 by transferring 0.15 mL of cells into 15 mL (~106 mL) sterile broth (or see below) for each plate of nds tested, then swirled to mix. If more than 1 plate of compounds (> 8 compounds), including compound 12 or 13, which may be prepared by following Examples 1.i and 1.j, respectively (above), were tested, volumes were increased accordingly. a. For E. faecalis, E. faecium and S. niae: 14.1 mL MHII + 0.9 mL laked horse blood was used. 4. Used 50 pl 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.

Diluted drug/compound stocks to 200x desired final concentration in 50 uL DMSO.

If starting concentration 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.

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 nd” DMSO well at the end of the series for l.

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

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

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

After inoculum was added, mixed each well ghly 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.

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 observed (optical y in the well).

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 of S. aureus ATCC #29213 as described above, diluted 1/200 by transferring 0.15 mL of cells into 30 mL (~5x105 cells/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 media. The usual starting concentration of nd was 8 ug/mL 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.

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 incubation, added 25 [LL of 0.01% 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 recorded. When using Resazurin, the color of the dye d 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 microdilution 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 Inoculation System (Fisher ) Test Reading Mirror (Fisher) ] Agar plates with ia streaked to single colonies, freshly prepared e DMSO Strains (MHII media for all; broth and agar): 1. Escherichia coli ATCC # 25922 899:5?!“ Escherichia coli, JMI collection strains, see table 5 Escherichia coli AG100 WT Escherichia coli AG100 tolC Acinetobacter baamannii ATCC # BAA-1710 Acinetobacter baamannii ATCC # 19606 Acinetobacter baamannii, JMI collection strains, see table 5 —56— 8. Klebsiella pneumoniae ATCC # 05 9. Klebsiella pneumoniae ATCC # 700603 . Klebsiella niae, JMI collection strains, see table 5 11. Moraxella catarrhalis ATCC# 25238 12. Moraxella catarrhalis ATCC# 49143 13.A40raxeflacxnarrhafis,Jhllcoflectknrsflains,seetable5 14. Haemophilus influenzae ATCC 49247 . Haemophilus influenzae (Rdl KW20) ATCC 51907 16. Haemophilus influenzae Rd0894 (AcrA—) 17. Haemophilus influenzae, JMI collection strains, see table 5 18. Pseudomonas aeruginosa PAOl 19. monas aeruginosa, JMI collection strains, see table 5 . Proteus mirabilis, JMI collection strains, see table 5 21. Enterobacter cloacae, JMI collection s, see table 5 22. Stenotrophomonas maltophilia ATCC BAA—84 23. Stenotrophomonas maltophilia ATCC13637 Inoculunlprep: 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 sterile saline that came with the 2. Vortexed the wells for ~ 30 s to give a suspension of ~108 cells/mL. Actual density could be confirmed by plating out ons of this suspension. 3. d the suspension 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, d to mix.

If more than 1 plate of compounds (> 8 compounds), including compound 12 or 13, was to be tested, increased volumes accordingly. 4 [Bed50ulcdb(~5x104cdb)u)moGHMemmhnfimofimrweflcmumnmg 50 ul of the drug diluted in broth (see below).

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

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

. Transferred 0.5 uL of each dilution (w/Matrix ipettor) to 50 [LL of medium/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 — compound concentrations decreased in 2x steps across the rows of a microtiter plate.

All MICs were done in duplicate. 6. All wells were inoculated with 50 pl 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 reading 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).

Protocol #3: Gyrase MIC ination of nds 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 ia streaked to single colonies, freshly prepared Sterile DMSO —58— WO 97270 GasPakTM incubation containers (BD Cat. #260672) GasPak TM EZ Anaerobe container system sachets (BD Cat. #260678) {333173}; E2 {:02 ner system sachets (BD Cat. #260679) Gasl‘ak TM EZ Campy container system sachets (BD Cat. #260680) Strains: l. Clostridium le ATCC BAA-1382; pPOSQMer’N Clostridium diflz‘cile, CMI collection strains, see table 4 Clostriudium perfringens, CMI collection strains, see table 4 Bacteroidesfragilis and oides Spp., CMI collection strains, see table 4 Fusobacterium Spp., CMI collection strains, see table 4 treptococcus, Spp., CMI collections strains, see table 4 Prevotella Spp., CMI collection strains, see table 4 N. gonorrhoeae ATCC 35541 N. gonorrhoeae ATCC 49226 . Neisseria gonorrhoeae, JMI collection strains, see table 4 ll. Neisseria meningitidis, JMI collection strains, see table 4 Media preparation and growth conditions: Growth medium recommended for each ial species was prepared ing tothe CLSI publication ‘Ml l-A7 s 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"M07—A8 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard--Eighth Edition (2009)".

Plate pouring: 1. ed 100x drug stocks of each test compound as described in Table 1A. Used a mL centrifuge tube, added 100 uL of each drug stock to 10 mL of molten agar (cooled to ~ 55°C in water bath). Mixed by inverting tube 2 —3x then pour into individually labeled 60X15 mm Petri dish. 2. Routine test concentrations were: 0.002 ug/mL — l6 ug/mL (14 plates). 3. Poured 4 drug free plates: 2 as positive control, 2 as c l. 4. 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 concentration and strain ent.

] Growth of cells requiring the nance of an anaerobic environment: 1. All work performed with anaerobic bacteria was done as rapidly as possible; work performed in biosafety cabinets (i.e., aerobic environment) was completed in less then minutes before cells were returned to bic chambers. 2. Incubation of anaerobic bacteria was achieved using GasPakTM chambers. The large box style chambers (VWR 90003—63 6) required 2 anaerobic sachets (VWR 90003— 642), while the tall cylinder style rs (VWR 90003—602) only required 1 sachet.

Plate inoculation (performed in ety cabinet): 1. Streaked each strain onto individual agar plates as described above. Incubated for required time and environmental condition (i.e. anaerobic, microaerophilic, etc). 2. Used direct colony suspension method to d loopfuls of y streaked cells into ~ 4 mL 0.9% NaClz and vortexed. 3. Adjusted suspension to O.D.600 0.05 (5x10e7 cfu/mL). ed to mix. 4. Transferred ~0.2 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 ~ lx10e5 cfu/spot. When testing C. diflz‘cile, strains swarmed when grown, however ce between multi—channel pipettor spots was far enough such that swarming cells did not impair assay results. a. Inoculated2 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 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 microaerophilic strain. Some growth was visible with N. gonorrhoeae. 6. d 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 incubated at 37°C in a5% COzenvironment for 24h.

MIC determination: Examined the test plates after the correct incubation 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 l .

Table 1A: Compound dilutions for MIC ination using the agar dilution method.

Final Volume Volume Diluent, Intermediate Conc. (uL) added Stock from stock DMSO Conc. At 1:100 to 10 mL Steo u ml Source uL uL ** u/mL u mL aar 1,600 Stock 75 75 800 8 100 1,600 Stock 75 225 400 4 100 1,600 Stock 75 525 200 2 100 200 Step 4 75 75 100 200 Step 4 75 225 50 0.5 100 200 Ste 4 75 525 25 0.25 100 n-Step 7 75 75 12.5 0.125 100 Ste 7 75 225 6.25 0.06 100 Ste 7 75 525 3.1 0.03 100 --11 3 10 75 75 . 0.016 100 --12 3 10 75 . 0.008 100 --13 3 10 75 . 0.004 100 Step 14 0.4 13 75 . 0.002 100 *1,600 ug/ml = 64 ul (10mg/ml stock) + 336 ul DMSO; 400 ul total volume to start **compound dissolved and diluted in 100% DMSO Protocol #4. MIC Determination Procedure for Mycobacterium species Materials Round bottom 96—well microtiter plates (Costar 3788) or r Film plate seals (PerkinElmer, TopSeal-A #6005250 or similar) Middlebrook 7H10 broth with 0.2% glycerol brook 7H10 agar with 0.2% glycerol Middlebrook OADC Enrichment Inoculum Preparation for M. tuberculosis: 1. Used prepared frozen M. tuberculosis stock stored at —700C. M. tuberculosis was 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 ml of the frozen stock and added it to 19 ml of 7H10 broth + 10% OADC (final concentration 2.5 x 106cfu/ml). 3. From this dilution prepared a second 1:20 dilution, removed 1 ml and added it to 19 ml of fresh broth. This was the final inoculum to add to the 96—well plates.

Inoculum ation for M. kansasii, M. avium, M. sus 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. ed a 1:20 dilution by removing 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 ml and added it to 19 ml of fresh broth (final sion).

Plate Preparation: 1. Labeled . 2. Added 50 pl 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 g solution 4x the maximum concentration tested (e.g. final concentration 32 ug/ml, highest concentration tested was 8 ug/ml). Dilutions were made from the stock solution. To start at a concentration of 1 ug/ml the drugs were prepared at 4 ug/ml, so the starting concentration was 1 ug/ml. Removed 25 ul of the 1mg/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 on to the first well of the designated row.

Continued for all compounds to be tested. Using a multichannel onic pipettor, mixed 4X and serial diluted compounds h 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 kansasii for ~7 days; Nocardia and M abcessus for ~4 days; with film seals. 2012/021275 7. Read visually and recorded the s. The MIC was ed as the lowest tration of drug where no growth was observed (optical clarity in the well).

Protocol #5. Protocol for Mycobacterium tuberculosis Serum Shift MIC Assay Materials and reagents: Costar #3 904 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 7H11 with 0.2% glycerol and OADC enrichment) with bacteria ed to single colonies Sterile 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 sterilized 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 -- 0.2% glycerol -- 2 g/L dextrose + 0.85 g/L NaCl + 0.003 g/L catalase (0% FBS). 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, picked5-10 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 culture. 2. Vortexed well, then sonicated in water bath for 30 sec providing a sion of ~108 ml. Actual density could be confirmed by plating out dilutions of this suspension.

Prepared inoculum in each of the three media formulations by ng 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 Isoniazid and Novobiocin 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— dispensed 100 pL of the stock solution into the first column of a l plate. Prepared 11-step, 2-fold serial dilutions across the row for each compound by transferring 50 pl from column 1 into 50 pl of DMSO in column 2. Continued to transfer 50 pL 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 pl of each dilution into an empty microtiter well prior to the addition of 100 pl of cells. The ng concentration of Isoniazid and Novobiocin was 100 pM after the on into medium + cells; the starting tration of Ciprofloxacin and Rifampin was 10 pM after the dilution into medium + cells. Compound concentrations decreased in 2x 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 pL volume.

Used a 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.

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, ted 2-fold, 11—point dilutions 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 transferred 1 pl of each dilution into an empty microtiter well prior to the on of 100 pl of cells as done for the control compounds.

WO 97270 .0909?” All wells were inoculated with 100 pl of diluted cell suspension (see above).

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

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

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

After 24 hours the fluorescence of each well was ed at Excitation 492 nm, Emission 595 nm.

Percent inhibition by a given compound was calculated as follows: Percent tion=100-([well fluorescence—average background fluorescence]/[DMSO control — e background fluorescence] x100). MICs were scored for all three medium conditions as the lowest compound concentration that inhibited resazurin reduction (‘%-inhibition’) signal 270% at a given medium condition.

Table 2A shows the results of the MIC assay for the mesylate salt of the benzimidazolyl urea compound of this invention.

In Table 2A and in subsequent Tables and Examples, und 13” relates to the mesylate salt of Compound 12. Compounds 12 and 13 may be prepared by following Examples 1.i and 1.j, respectively (above). These are the same numbers used to fy said compounds and salts as used in the Examples above.

Table 2A — MIC Values of Selected Compounds Strain/Special Condition Protocol Compound Staphylococcus aureus 1 0.13 ATCC 29213 lococcus aureusATCC 1 0.31 29213 with Human Serum Staphylococcus aureus 1 0.53 ATCC 29213 with Rat Serum Staphylococcus aureus 1 2 ATCC 29213 with Mouse Serum Staphylococcus aureus 1 1.29 ATCC 29213 GyrB T173I Enterococcusfaecalis ATCC 1 0.081 29212, with Laked Horse Blood Enterococcusfaecium ATCC 1 0.39 49624 with Laked Horse —65— WO 97270 Strain/Special Condition Protocol Compound Blood Enterococcusfaecium ATCC 0.25 49624 Streptococcus pneumoniae 0.022 ATCC 10015, with Laked Horse Blood Bacillus cereus ATCC 10987 Bacillus cereus ATCC 14579 Bacillus subtilis ATCC 6638 Bacillus subtilis (168) ATCC 605 1 Clostridium diflicile ATCC BAA— 1 3 82 Haemophilus nzae ATCC 49247 Haemophilus influenzae (Rdl 2.5 KW20) ATCC 51907 Haemophilus influenzae 0.14 Rd0894 (AcrA-) Moraxella halis ATCC 0.071 25238 Moraxella catarrhalis ATCC 0.04 49143 Neisseria g0n0rrh0eae 1.3 ATCC 35541 Neisseria g0n0rrh0eae 2.3 ATCC 49226 Escherichia c0li AG100 WT >16 ichia c0li AG100 tolC 0.11 Escherichia c0li ATCC [\JNN 16 25922 Escherichia c0li CHE30 Escherichia c0li CHE30 tolC Escherichia c0li MC4100 Escherichia c0li MC4100 [\JNNN tolC ella pneumoniae ATCC 700603 Klebsiella pneumoniae ATCC BAA—1705 Acinetobacter baumannii ATCC 19606 Acinetobacter baumannii ATCC BAA—1710 Pseudomonas aeruginosa PAOl Strain/Special Condition ol Compound Pseudomonas aeruginosa 0.33 PAO750 Stenotropbomonas Not done maltopbilia ATCC BAA-84 Stenotropbomonas [\J Not done maltopbilia 637 Mycobacterium avium 103 0.47 M. avium Far 0.94 M. avium 3404.4 0.94 Nocardia caviae 2497 2 N. asteroids 2039 ##-l>-l>-l>-l>-l>-I>J>J> 8 N. nova 10 8 M. kansasii 303 Not Done M. kansasii 316 Not Done M kansasii 379 Not Done M tuberculosis H37RV 0.37 ATCC 25618 M tuberculosis Erdman J> 0.25 ATCC 35801 M ulosis Erdman U1 0.045 ATCC 35801 M tuberculosis Erdman U1 2 ATCC 35801 with Mouse Serum M abscessus BB2 \0t Done M abscessus MC 6005 \ot Done M abscessus MC 5931 \ot Done M abscessus MC 5605 \ot Done M abscessus MC 6025 \ot Done M abscessus MC 5908 \ot Done M abscessus BB3 \ot Done M abscessus BB4 \ot Done M abscessus BB5 \ot Done M abscessus VIC 5922 \ot Done M abscessus VIC 5960 \ot Done M abscessus BB1 \ot Done M abscessus VIC 5812 \ot Done M abscessus VIC 5901 \ot Done M abscessus BB6 \ot Done M abscessus BB8 \ot Done M abscessus VIC 5908 \ot Done M abscessus LT 949 \ot Done M abscessus BB10 \ot Done M abscessus VIC 6142 \ot Done M abscessus VIC 6136 \ot Done M abscessus VIC 6111 ##-l>-l>-l>-l>-l>-I>J>J>J>J>J>J>-b-I>J>J>-b-I>J>J> \ot Done —67— Strain/Special Condition Protocol Compound M. sus MC 6153 4 Not Done ] Table 3A shows the results of the MIC90 assay for selected compounds of this invention.

Table 3A — MIC90 Values of Selected Compounds with Panels of Gram Positive, Gram ve and Anaerobic Pathogens Compound 13 Organism Number Protocol Range MIC90 0f (Hg/m1) (Hg/m1) Isolates Tested Aerobic Gram-positive Staphylococcus aureus 67 1 0.03— 0.25 Staphlococcus epidermidis 35 1 0.03— 0.12 0.25 Enterococcusfaecalis 34 1 0.03— 0.25 0.25 Enterococcusfaecium 33 1 0.12— 0.5 Streptococcus pneumoniae 67 1 0.015— 0.06 0.06 olytic streptococci s A, B, C 28 1 0.06— 0.25 and G) 0.5 Aerobic Gram-negative Haemophilus influenzae 55 2 0.25— 8 2 Moraxella catarrhah's 26 2 0.015— 0.12 0.12 Acinetobacter baumannii 12 2 >8— >8 >8 Pseudomonas aeruginosa 12 2 >8— >8 >8 Escherichia coli 12 2 >8— >8 >8 Klebsiella pneumoniae 12 2 >8— >8 >8 Proteus mirabilis 12 2 >8— >8 >8 Enterobacter e 12 2 >8— >8 >8 Neisseria gonorrhoeae 13 3 0.5— 1 1 Neisseria meningitidis 12 3 0.015— 0.12 0.25 Anaerobes Bacteroides and Parabacter spp. 26 3 2— >16 >16 Bacteroidesfragilis 25 3 8— >16 >16 Clostridium diflicile 16 3 0.5— 16 1 Clostridium perfringens 12 3 0.12— 0.5 nd 13 Organism Number Protocol Range MIC90 0f (Hg/m1) (Hg/m1) Isolates Tested Fusobacterium Spp. 16 3 1-4 2 Peptostreptococcus Spp. 1 1 3 0.06— >16 Prevotella Spp. 13 3 05— >16 >16 In Table 4 below, the term “CMI” stands for The Clinical Microbiology Institute located in Wilsonville, Oregon.

Table 4: Panels of bic Organism Used to Generate MIC90 Data CMI# ORGANISM A2380 B. fragilis A2381 B. fragilis A2382 B. fragilis A2486 B. fragilis A2487 B. fragilis A2489 B. is A2527 B. fragilis A2529 B. fragilis A2562 B. fragilis A2627 B. fragilis A2802 B. fragilis A2803 B. fragilis A2804 B. fragilis A2805 B. is A2806 B. fragilis A2807 B. fragilis A2808 B. fragilis A2809 B. fragilis CMI# ORGANISM A2810 B. is A2811 B. fragilis A2812 B. fragilis A2813 B. fragilis A2814 B. is A2460 B. thetaiotaomicron A2462 B. thetaiotaomicron A2463 B. thetaiotaomicron A2464 B. thetaiotaomicron A2536 B. thetaiotaomicron A2591 B. uniformis A2604 B. vulgatus A2606 B. vulgatus A2613 B. ovatus A2616 B. ovatus A2815 Bacteroides tectum A2816 B. ureolyticus A2817 Bacteroides capillosus CMI# ORGANISM A2818 B. ureolyticus A2824 Parabacter distasonis A2825 B. ovatus A2826 B. uniformis A2827 B. uniformis A2828 B. vulgatus A2829 B. vulgatus A2830 B. ovatus A2831 B. thetaiotaomicron A2832 Parabacter distasonis A2833 B. thetaiotaomicron A2767 C. diflicile A2768 C. diflicile A2769 C. diflicile A2770 C. le A2771 C. diflicile A2772 C. le A2773 C. diflicile CMI# SM A2774 C. diflicile A2775 C. diflicile A2776 C. diflicile A2777 C. diflicile A2778 C. diflicile A2779 C. le A2780 C. diflicile A2140 C. perfringens A2203 C. perfringens A2204 C. perfringens A2227 C. perfringens A2228 C. perfringens A2229 C. perfringens A2315 C. perfringens A2332 C. perfringens A2333 C. perfringens A2334 C. perfringens A2389 C. perfringens CMI# ORGANISM A2390 C. perfringens A864 F. necrophorum A871 F. tum A1667 F. necrophorum A1666 F necrophorum A2249 F tum A2716 Fusobacterium species A2717 Fusobacterium species A2719 Fusobacterium species A2721 Fusobacterium species A2722 Fusobacterium species A2710 Fusobacterium species A2711 Fusobacterium species A2712 Fusobacterium species A2713 cterium species A2714 Fusobacterium species A2715 Fusobacterium species A1594 Peptostreptococcus anaerobius CMI# ORGANISM A2 15 8 Peptostreptococcus magnus A2168 Peptostreptococcus anaerobius A2170 Peptostreptococcus magnus A2 17 1 Peptostreptococcus magnus A2575 Peptostreptococcus Spp.

A2579 Peptostreptococcus asaccharolyticus A2580 Peptostreptococcus asaccharolyticus A2614 Peptostreptococcus asaccharolyticus A2620 Peptostreptococcus asaccharolyticus A2629 Peptostreptococcus Spp.

A2739 Prevotella denticola A2752 Prevotella bivia A2753 Prevotellaintermedia A2754 Prevotellaintermedia A2756 Prevotella bivia A2759 ella bivia A2760 Prevotella denticola A2761 Prevotella intermedia CMI# ORGANISM A2762 Prevotella melaninogenica A2765 Prevotella melaninogenica A2766 Prevotella melaninogenica A2821 Prevotella bivia A2822 Prevotella bivia QCBF B. is QCBT B. thetaiotaomicron QCCD C. diflicile QCBF B. fragilis QCBT B. thetaiotaomicron QCCD C. diflicile In Table 5 below, the term “JMI” stands for The Jones Microbiology Institute located in North Liberty, Iowa.

Table 5: Panels of Gram ve and Gram Negative Organism Used to Generate MIC90 Data JMI Organism JMI Isolate # Organism 394 ACB Acinetobacter baumannii 2166 ACB Acinetobacter baumannii 3060 ACB Acinetobacter nii 3170 ACB Acinetobacter baumannii 9328 ACB Acinetobacter baumannii 9922 ACB Acinetobacter baumannii 13 618 ACB Acinetobacter baumannii 14308 ACB Acinetobacter baumannii 17086 ACB obacter baumannii 17176 ACB Acinetobacter nii 30554 ACB obacter baumannii 32007 Acinetobacter baumannii 1 192 ECL Enterobacter cloacae 3096 lfiflerobackwcfloacae 5534 Enterobacter cloacae 6487 Enterobacter e 9592 Enterobacter cloacae 11680 lfiflerobackwcfloacae 12573 ECL Enterobacter cloacae 12735 ECL Enterobacter cloacae 13057 ECL Enterobacter cloacae 18048 ECL Enterobacter cloacae 173 ECL Enterobacter cloacae 29443 ECL Enterobacter cloacae 44 iEF lfiflerococcusfbecafis 355 iEF lfiflerococcusfbecafis 886 iEF lfiflerococcusfbecafis 955 iEF lfiflerococcusfbecafis 1000 Enterococcusfaecalis 1053 Enterococcusfaecalis WO 97270 JMI Organism JMI Isolate # Code sm 1142 Enterococcusfaecalis 1325 Enterococcusfaecalis 1446 Enterococcusfaecalis 2014 Enterococcusfaecalis 2103 EF Enterococcusfaecalis 2255 EF Enterococcusfaecalis 2978 EF Enterococcusfaecalis 2986 EF Enterococcusfaecalis 5027 Enterococcusfaecalis 5270 Enterococcusfaecalis 5874 Enterococcusfaecalis 7430 Enterococcusfaecalis 7904 Enterococcusfaecalis 8092 EF Enterococcusfaecalis 8691 EF Enterococcusfaecalis 9090 EF Enterococcusfaecalis 10795 EF Enterococcusfaecalis 14104 EF Enterococcusfaecalis 16481 Enterococcusfaecalis 18217 coccusfaecalis 22442 Enterococcusfaecalis 25726 Enterococcusfaecalis 26143 Enterococcusfaecalis 2813 1 EF Enterococcusfaecalis 29765 EF Enterococcusfaecalis 30279 EF Enterococcusfaecalis 31234 EF Enterococcusfaecalis 31673 EF Enterococcusfaecalis 115 Enterococcusfaecium 227 EFM Enterococcusfaecium —78— JMI sm JMI Isolate # Code Organism 414 coccusfaecium 712 Enterococcusfaecium 870 Enterococcusfaecium 911 Enterococcusfaecium 2356 Enterococcusfaecium 2364 Enterococcusfaecium 2762 Enterococcusfaecium 3062 Enterococcusfaecium 4464 coccusfaecium 4473 Enterococcusfaecium 4653 Enterococcusfaecium 4679 Enterococcusfaecium 6803 Enterococcusfaecium 6836 Enterococcusfaecium 8280 Enterococcusfaecium 8702 Enterococcusfaecium 9855 Enterococcusfaecium 10766 Enterococcusfaecium 12799 Enterococcusfaecium 13556 Enterococcusfaecium 13783 Enterococcusfaecium 14687 Enterococcusfaecium 15268 Enterococcusfaecium 15525 Enterococcusfaecium 15538 Enterococcusfaecium 18102 Enterococcusfaecium 18306 Enterococcusfaecium 19967 Enterococcusfaecium 22428 Enterococcusfaecium 23482 Enterococcusfaecium JMI Organism JMI Isolate # Organism 29658 EFM Enterococcusfaecium 597 EC Escherichia coli 847 EC ichia coli 1451 EC Escherichia coli 8682 EC Escherichia coli 11 199 EC Escherichia coli 12583 EC Escherichia coli 12792 EC Escherichia coli 13265 Escherichia coli 14594 Escherichia coli 22148 Escherichia coli 29743 Escherichia coli 30426 Escherichia coli 470 Group A Streptococcus 2965 Group A Streptococcus 3112 Group A Streptococcus 3637 Group A Streptococcus 4393 Group A Streptococcus 4546 BSA Group A Streptococcus 4615 BSA Group A Streptococcus 5848 BSA Group A ococcus 6194 BSA Group A Streptococcus 8816 BSA Group A ococcus 1 1814 Group A Streptococcus 16977 Group A Streptococcus 18083 Group A Streptococcus 18821 Group A Streptococcus 25178 Group A Streptococcus 30704 BSA Group A Streptococcus 12 BSB Group B Streptococcus JMI Organism JMI Isolate # sm 103 66 BSB Group B Streptococcus 10611 BSB Group B Streptococcus 16786 BSB Group B Streptococcus 18833 BSB Group B Streptococcus 30225 BSB Group B Streptococcus 10422 BSC Group C Streptococcus 14209 BSC Group C Streptococcus 29732 BSC Group C Streptococcus 8544 BSG Group G Streptococcus 18086 BSG Group G Streptococcus 29815 BSG Group G Streptococcus 147 Haemophilus influenzae 180 hilus influenzae 934 HI Haemophilus influenzae 970 HI Haemophilus influenzae 1298 HI Haemophilus influenzae 1819 HI Haemophilus zae 1915 HI Haemophilus influenzae 2000 hilus influenzae 2562 Haemophilus influenzae 2821 Haemophilus influenzae 3133 Haemophilus influenzae 3140 Haemophilus influenzae 3497 HI Haemophilus influenzae 3508 HI Haemophilus influenzae 3535 HI Haemophilus influenzae 4082 HI Haemophilus influenzae 4108 HI Haemophilus zae 4422 Haemophilus influenzae 4868 Haemophilus influenzae JMI Organism JMI Isolate # Organism 4872 Haemophilus influenzae 585 8 hilus zae 625 8 Haemophilus influenzae 6875 hilus influenzae 7063 HI Haemophilus influenzae 7600 HI Haemophilus influenzae 8465 HI Haemophilus influenzae 10280 HI hilus zae 10732 Haemophilus influenzae 10850 Haemophilus influenzae 113 66 Haemophilus influenzae 11716 Haemophilus influenzae 11724 Haemophilus influenzae 11908 HI Haemophilus influenzae 12093 HI Haemophilus influenzae 12107 HI Haemophilus influenzae 13424 HI Haemophilus influenzae 13439 HI Haemophilus influenzae 13 672 Haemophilus influenzae 13 687 Haemophilus influenzae 13 792 Haemophilus influenzae 13 793 Haemophilus influenzae 14440 Haemophilus zae 153 51 HI Haemophilus influenzae 153 56 HI Haemophilus influenzae 15678 HI Haemophilus influenzae 800 HI Haemophilus influenzae 17841 HI Haemophilus influenzae 18614 Haemophilus influenzae 25195 Haemophilus influenzae JMI Organism JMI Isolate # Organism 27021 Haemophilus influenzae 28326 Haemophilus influenzae 28332 Haemophilus influenzae 29918 hilus influenzae 29923 HI Haemophilus influenzae 31911 HI Haemophilus influenzae 428 KP\ Klebsiella niae 791 KP\ Klebsiella pneumoniae 836 KP\ Klebsiella pneumoniae 1422 KP\ Klebsiella pneumoniae 1674 KP\ Klebsiella pneumoniae 1883 Klebsiella pneumoniae 6486 KP\ Klebsiella niae 8789 Klebsiella pneumoniae 10705 Klebsiella pneumoniae 1 1123 Klebsiella pneumoniae 28148 Klebsiella pneumoniae 29432 Klebsiella pneumoniae 937 Moraxella catarrhalis 1290 lla catarrhalis 1830 Moraxella catarrhalis 1903 Moraxella catarrhalis 4346 Moraxella catarrhalis 4880 Moraxella catarrhalis 6241 lla catarrhalis 6551 Moraxella catarrhalis 7074 lla catarrhalis 7259 Moraxella catarrhalis 7544 Moraxella catarrhalis 8142 Moraxella catarrhalis JMI sm JMI Isolate # Organism 8451 Moraxella halis 9246 Moraxella catarrhalis 9996 Moraxella catarrhalis 12158 Moraxella catarrhalis 13443 A40raxeflacxnarrhafis 13692 A40raxeflacxnarrhafis 13817 A40raxeflacxnarrhafis 14431 A40raxeflacxnarrhafis 14762 Moraxella catarrhalis 14842 Moraxella catarrhalis 15361 Moraxella catarrhalis 15741 Moraxella catarrhalis 17843 Moraxella catarrhalis 18639 \4CUXT A40raxeflacxflarrhal$ 241 GC Neisseria gonorrhoeae 291 GC Neisseria gonorrhoeae 293 GC ria gonorrhoeae 344 GC Neisseria gonorrhoeae 451 Neisseria hoeae 474 Neisseria gonorrhoeae 491 Neisseria gonorrhoeae 493 Neisseria gonorrhoeae 503 ria gonorrhoeae 521 GC Neisseria gonorrhoeae 552 GC Neisseria gonorrhoeae 573 GC Neisseria gonorrhoeae 592 GC ria gonorrhoeae NM Neisseria meningitidis 813 Neisseria meningitidis 1725 Neisseria meningitidis JMI Organism JMI Isolate # Code Organism 2747 \VI Neisseria meningitidis 3201 Neisseria meningitidis 3335 Neisseria meningitidis 7053 Neisseria meningitidis 9407 \VI Neisseria meningitidis 10447 \V1 Neisseria meningitidis 12685 \V1 Neisseria meningitidis 12841 \V1 Neisseria meningitidis 14038 Neisseria meningitidis 1127 s mirabilis 3049 PVI Proteus mirabilis 4471 Proteus mirabilis 8793 PVI Proteus mirabilis 10702 PVI Proteus mirabilis 1 1218 PVI Proteus mirabilis 14662 PVI Proteus mirabilis 17072 PVI Proteus mirabilis 19059 PVI Proteus mirabilis 23367 PVI Proteus mirabilis 29819 PVI s lis 31419 PVI Proteus mirabilis 1881 PSA Pseudomonas aeruginosa 5061 PSA Pseudomonas aeruginosa 7909 Pseudomonas aeruginosa 8713 Pseudomonas aeruginosa 143 18 Pseudomonas aeruginosa 14772 monas aeruginosa 155 12 Pseudomonas nosa 17093 PSA Pseudomonas aeruginosa 17802 PSA Pseudomonas aeruginosa —85— WO 97270 JMI Organism JMI Isolate # Code Organism 19661 PSA Pseudomonas aeruginosa 29967 PSA Pseudomonas aeruginosa 31539 PSA Pseudomonas aeruginosa 82 SA Staphylococcus aureus 99 SA Staphylococcus aureus 13 8 SA Staphylococcus aureus 13 9 SA Staphylococcus aureus 140 SA Staphylococcus aureus 141 Staphylococcus aureus 142 Staphylococcus aureus 272 Staphylococcus aureus 287 Staphylococcus aureus 354 Staphylococcus aureus 382 SA Staphylococcus aureus 11 12 SA Staphylococcus aureus 1687 SA Staphylococcus aureus 1848 SA Staphylococcus aureus 2031 SA Staphylococcus aureus 2159 Staphylococcus aureus 2645 Staphylococcus aureus 3256 Staphylococcus aureus 3276 lococcus aureus 4044 Staphylococcus aureus 4214 SA Staphylococcus aureus 4217 SA Staphylococcus aureus 4220 SA Staphylococcus aureus 4231 SA Staphylococcus aureus 4240 SA Staphylococcus aureus 4262 lococcus aureus 4370 Staphylococcus aureus WO 97270 JMI Organism JMI Isolate # Organism 4665 Staphylococcus aureus 4666 Staphylococcus aureus 4667 Staphylococcus aureus 5026 Staphylococcus aureus 5666 SA Staphylococcus aureus 6792 SA Staphylococcus aureus 7023 SA Staphylococcus aureus 7461 SA Staphylococcus aureus 7899 Staphylococcus aureus 7901 Staphylococcus aureus 8714 Staphylococcus aureus 9374 Staphylococcus aureus 9437 Staphylococcus aureus 10056 SA Staphylococcus aureus 101 10 SA Staphylococcus aureus 1 1379 SA Staphylococcus aureus 1 1629 SA Staphylococcus aureus 1 1659 SA Staphylococcus aureus 12788 lococcus aureus 12789 Staphylococcus aureus 13043 Staphylococcus aureus 13086 lococcus aureus 13721 Staphylococcus aureus 13742 SA Staphylococcus aureus 13932 SA Staphylococcus aureus 14210 SA Staphylococcus aureus 143 84 SA Staphylococcus aureus 15428 SA Staphylococcus aureus 15430 Staphylococcus aureus 17721 Staphylococcus aureus —87— JMI Organism JMI Isolate # Organism 18688 Staphylococcus aureus 19095 Staphylococcus aureus 20195 Staphylococcus aureus 22141 lococcus aureus 22689 SA Staphylococcus aureus 27398 SA Staphylococcus aureus 29048 SA Staphylococcus aureus 29051 SA Staphylococcus aureus 30491 Staphylococcus aureus 30538 Staphylococcus aureus Staphylococcus epidermidis 53 Staphylococcus epidermidis 385 Staphylococcus epidermidis 398 Staphylococcus epidermidis 701 Staphylococcus epidermidis 713 Staphylococcus epidermidis 13 81 Staphylococcus epidermidis 2174 Staphylococcus epidermidis 2286 Staphylococcus epidermidis 2969 Staphylococcus epidermidis 3417 Staphylococcus epidermidis 3447 Staphylococcus epidermidis 4753 lococcus epidermidis 7241 Staphylococcus epidermidis 9366 Staphylococcus epidermidis 10665 lococcus midis 1 1792 Staphylococcus epidermidis 123 1 1 Staphylococcus epidermidis 13036 lococcus epidermidis 13227 Staphylococcus epidermidis JMI Organism JMI Isolate # Code Organism 13243 Staphylococcus midis 13 621 Staphylococcus epidermidis 13 63 8 Staphylococcus epidermidis 13 800 Staphylococcus epidermidis 14078 Staphylococcus epidermidis 14392 Staphylococcus epidermidis 15007 Staphylococcus epidermidis 16733 Staphylococcus epidermidis 18871 Staphylococcus epidermidis 23285 lococcus epidermidis 27805 lococcus epidermidis 29679 Staphylococcus epidermidis 29985 Staphylococcus epidermidis 30259 Staphylococcus epidermidis 31444 Staphylococcus epidermidis 268 Streptococcus pneumoniae 1264 Streptococcus pneumoniae 2482 ococcus pneumoniae 2653 Streptococcus pneumoniae 2994 Streptococcus pneumoniae 3123 Streptococcus pneumoniae 3124 Streptococcus pneumoniae 4336 Streptococcus pneumoniae 4858 ococcus pneumoniae 5606 Streptococcus niae 5881 Streptococcus pneumoniae 5897 Streptococcus pneumoniae 5900 Streptococcus pneumoniae 605 1 Streptococcus pneumoniae 6216 Streptococcus pneumoniae JMI Organism JMI Isolate # Code sm 6556 Streptococcus pneumoniae 7270 Streptococcus pneumoniae 7584 Streptococcus pneumoniae 8479 Streptococcus niae 8501 Streptococcus pneumoniae 9256 Streptococcus niae 9257 Streptococcus niae 10246 Streptococcus pneumoniae 10467 Streptococcus niae 10886 Streptococcus pneumoniae 11217 ococcus pneumoniae 11228 Streptococcus pneumoniae 11238 Streptococcus pneumoniae 11757 Streptococcus pneumoniae 11768 Streptococcus pneumoniae 12121 Streptococcus pneumoniae 12124 Streptococcus pneumoniae 12149 Streptococcus pneumoniae 12767 Streptococcus pneumoniae 12988 Streptococcus pneumoniae 13321 Streptococcus pneumoniae 13393 Streptococcus pneumoniae 13521 Streptococcus pneumoniae 13544 Streptococcus pneumoniae 13700 Streptococcus pneumoniae 13704 Streptococcus pneumoniae 13822 Streptococcus pneumoniae 13838 Streptococcus pneumoniae 14131 Streptococcus pneumoniae 14413 Streptococcus pneumoniae WO 97270 JMI Organism JMI e # Code Organism 14744 Streptococcus pneumoniae 14808 Streptococcus pneumoniae 14827 Streptococcus pneumoniae 14835 Streptococcus pneumoniae 14836 Streptococcus pneumoniae 15832 Streptococcus pneumoniae 17336 Streptococcus pneumoniae 17343 Streptococcus pneumoniae 17349 Streptococcus pneumoniae 17735 Streptococcus niae 18060 Streptococcus pneumoniae 18567 Streptococcus pneumoniae 18595 Streptococcus pneumoniae 19082 Streptococcus pneumoniae 19826 Streptococcus pneumoniae 22174 Streptococcus pneumoniae 22175 Streptococcus pneumoniae 27003 Streptococcus pneumoniae 28310 Streptococcus pneumoniae 28312 ococcus pneumoniae 29890 Streptococcus pneumoniae 29910 Streptococcus pneumoniae

Claims (4)

WHAT IS CLAIMED IS:

1. A solid compound of formula (I): or a salt thereof.

2. The solid compound of claim 1, wherein said solid is a solid Form I free base having an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 θ ± 0.2) when measured using Cu Kα radiation, selected from the group ting of 9.3, 11.7, 12.4, 13.8, 14.6, 16.0, 16.2, 16.7, 18.6, 18.9, 19.6, 20.2, 20.5, 21.3, 21.7, 22.7, 23.9, 24.5, 24.9, 25.8, 26.7, 27.9, 28.1, 28.4, 30.4, 33.5, and 37.4, when the XPRD is collected from about 5 to about 38 s 2 θ.

3. The solid compound of claim 2, n said solid Form I has an X-ray powder diffraction pattern (XPRD) comprising at least three approximate peak positions (degrees 2 θ ± 0.2) when measured using Cu Kα radiation, selected from the group consisting of 9.3, 16.7, 18.6, 19.6, 21.7, and 25.8, when the XPRD is collected from about 5 to about 38 degrees 2 θ.

4. The solid compound of claim 2 or 3, wherein said solid Form I has an X-ray powder diffraction pattern, as measured using Cu Kα ion, substantially similar to