HK1189876A - 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

Description

Solid forms of the gyrase inhibitor (R) -1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- (tetrahydrofuran-2-yl) -1 h-benzimidazol-2-yl ] urea

CROSS-REFERENCE TO RELATED APPLICATIONS

The benefit of U.S. provisional patent application serial No. 61/433,161, filed 2011, 1, 14, was claimed in this application under 35u.s.c. § 119, the entire contents of which are incorporated herein by reference.

Application background

The resistance of bacteria to antibiotics has long been recognized and is today considered a serious worldwide health problem. Some bacterial infections are either difficult to treat with antibiotics or even untreatable due to resistance. This problem has become particularly serious with the recent development of multidrug resistance in specific bacterial strains, such as Streptococcus Pneumoniae (SP), Mycobacterium tuberculosis (Mycobacterium tuberculosis), and Enterococcus (Enterococcus). The emergence of vancomycin resistant enterococci is of particular concern because vancomycin has previously been the only effective antibiotic for treating this infection, and has been considered as a "last resort" drug for many infections. Although many other drug-resistant bacteria, such as enterococci, do not cause life-threatening diseases, there is a concern that genes inducing Resistance may spread to more lethal organisms, such as Staphylococcus aureus (Staphyloccocusareureus), for which methicillin Resistance has become prevalent (De Clerq et al, Current opinion in Anti-infectious Investigational Drugs, 1999, 1, 1; Levy, "The challenge of antimicrobial Resistance", Scientific American, March, 1998).

Another concern is how rapidly resistance to antibiotics can spread. For example, until the sixties of the twentieth century, SPs were universally susceptible to penicillin, and in 1987, only 0.02% of SP strains were resistant in the united states. However, by 1995, SP resistance to penicillin was reported to be about seven percent and as high as 30% in some parts of The United states (Lewis, FDAConsumer magazine (9 months 1995); Gershman in The Medical Reporter, 1997).

Hospitals in particular serve as centers for the formation and dissemination of drug resistant organisms. Infections occurring in hospitals, known as nosocomial infections, are becoming an increasingly serious problem. Of the two million americans infected annually in hospitals, more than half of these infections are resistant to at least one antibiotic. The center for disease control reported that more than 13,000 hospitalized patients died from bacterial Infections Resistant to Antibiotic treatment in 1992 (Lewis, "The Rise of Antibiotic-Resistant Infections," FDAConsumer magazine, month 9 1995).

Interest in the development of resuscitation of new antibiotics has emerged due to the need to combat drug-resistant bacteria and the increasing failure of available drugs. An attractive strategy for the development of new antibiotics is the inhibition of DNA gyrase and/or topoisomerase IV, bacterial enzymes that are essential for DNA replication and thus for bacterial cell growth and division. Gyrase and/or topoisomerase IV are also associated with events in DNA transcription, repair and recombination.

Gyrases are one of the topoisomerases, a group of enzymes that catalyze the interconversion of topoisomers of DNA (see generally, Kornberg and Baker, DNA Replication, 2 nd edition, Chapter 12, 1992, W.H.Freeman and Co.; Drica, molecular Microbiology, 1992, 6, 425; Drica and Zhao, Microbiology and molecular Biology Reviews, 1997, 61, p 377-392). Gyrase itself controls DNA supercoiling and relieves the topological stress that occurs when the DNA strands of the parent duplex unwind during the replication process. Gyrase also catalyzes the conversion of relaxed closed circular duplex DNA into a negative supercoiled form that is more favorable for recombination. The mechanism of the supercoiling reaction involves the wrapping of gyrase around one region of DNA, double-strand breaks in that region, crossing the break by a second region of DNA, and rejoining the broken strands. Such cleavage mechanisms are characteristic of type II topoisomerases. The supercoiling reaction is driven by the binding of ATP to gyrase. ATP is subsequently hydrolyzed during the reaction. This ATP binding and subsequent hydrolysis causes a conformational change in the DNA-bound gyrase, which is essential 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 able to relax supercoiled DNA.

Bacterial DNA gyrase is a 400 kilodalton protein tetramer composed of two a subunits (GyrA) and two B subunits (GyrB). Binding and cleavage of DNA is associated with GyrA, while ATP is bound and hydrolyzed by GyrB protein. GyrB consists of an amino-terminal domain with atpase activity and a carboxy-terminal domain that interacts with GyrA and DNA. In contrast, eukaryotic type II topoisomerases are homodimers that can relax negative and positive supercoils but cannot introduce negative supercoils. Ideally, antibiotics based on inhibition of bacterial DNA gyrase and/or topoisomerase IV are selective for these enzymes and relatively inactive against eukaryotic type II topoisomerases.

Topoisomerase IV primarily eliminates chromosome dimers that are linked at the end of DNA replication.

The widely used quinolone antibiotics inhibit bacterial DNA gyrase (GyrA) and/or topoisomerase iv (parc). Examples of quinolones include early compounds such as nalidixic acid and oxolinic acid, and later, more potent fluoroquinolones such as norfloxacin, ciprofloxacin and trovafloxacin. These compounds bind to GyrA and/or ParC and stabilize the cleaved complex, thereby inhibiting overall gyrase function, resulting in cell death. Fluoroquinolones inhibit the catalytic subunit of gyrase (gyrA) and/or topoisomerase IV (ParC) (see Drlic and Zhao, Microbiology and molecular Biology Reviews, 1997, 61, 377-392). However, resistance has also been recognized as a problem with this class of compounds (WHO Report, "Use of Quinolonesen Food Animals and Potential Impact on Human Health", 1998). For quinolones, as with other classes of antibiotics, bacteria exposed to earlier compounds often rapidly develop cross-resistance to more potent compounds in the same class.

The relevant subunits responsible for supplying the energy required for the catalytic turnover/resetting of the enzyme by ATP hydrolysis are GyrB (gyrase) and ParE (topoisomerase IV), respectively (see Champoux, j.j., annu.rev.biochem., 2001, 70, p. 369-413). Compounds that target these same ATP binding sites in the GyrB and ParE subunits would be useful for treating a variety of bacterial infections (see Charifson et al, j.med.chem., 2008, 51, pages 5243-5263).

There are fewer known inhibitors that bind to GyrB. Examples include coumarin, novobiocin and coumaromycin A1, cyclothialidine, cinodin and clooxetine. Coumarin has been shown to bind very tightly to GyrB. For example, novobiocin forms a hydrogen bonding network with the protein and several hydrophobic contacts. While novobiocin and ATP do appear to bind within the ATP binding site, there is minimal overlap in the direction of binding of the two compounds. The overlapping parts are the sugar unit of novobiocin and adenine of ATP (Maxwell, Trends in microbiology, 1997, 5, 102).

For coumarin-resistant bacteria, the most common point mutation is on the surface arginine residue, which binds to the carbonyl of the coumarin loop (Arg 136 in escherichia coli (e.coli) GyrB). Although enzymes with such mutations show lower supercoiled and atpase activities, they are also less sensitive to inhibition by coumarin drugs (Maxwell, mol. microbe. 1993, 9, 681).

Despite being effective inhibitors of gyrase supercoils, coumarin has not been widely used as an antibiotic. They are generally unsuitable due to their low penetration in bacteria, eukaryotic toxicity and poor water solubility (Maxwell, Trends in Microbiology, 1997, 5, 102). It would be desirable to have new potent GyrB and ParE inhibitors that overcome these disadvantages and preferably do not rely on binding to Arg136 for activity. Such inhibitors would be attractive antibiotic candidates without a history of resistance problems that plague other classes of antibiotics.

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

Summary of the application

The present application relates to solid and amorphous forms of (R) -1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]Ureas ("benzimidazolyl urea compounds") and processes for preparing them. In one embodiment, the present application provides a benzimidazolyl urea compound in solid form. In one embodiment, the solid form is a form I solid form characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αAt least 3 approximate peak positions (2 theta angles ± 0.2) are included when measured radiatively, 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 XPRD is collected from dicyta (2 theta) of about 5 degrees to about 38 degrees. In a particular embodiment, solid form I is characterized by an X-ray powder diffraction Pattern (XPRD) when using Cu K_αRadiation measuring time bagAt least 3 approximate peak positions (2 θ angles ± 0.2) are included, which are selected from 9.3, 16.7, 18.6, 19.6, 21.7, and 25.8 when XPRD is collected from 2 θ of about 5 degrees to about 38 degrees. In a further embodiment, form I is characterized, e.g., by Cu K_αThe X-ray powder diffraction pattern measured by the radiation is substantially similar to that of figure 1. And in yet another embodiment, form I is characterized by an endothermic peak having an onset temperature of about 243 ℃ as measured by differential scanning calorimetry in which the temperature is scanned at about 10 ℃/minute. The present application also provides a process for preparing crystalline form I of the benzimidazolyl urea compound, comprising crystallizing or precipitating the compound of formula (I) from a solvent system comprising dichloromethane, methanol, or a combination thereof.

The present application also provides solid hydrochloride salts of the benzimidazolyl urea compounds. In one embodiment, the solid hydrochloride salt is a form II solid. In one embodiment, form II hydrochloride of the present application is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 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 XPRD is collected from about 5 degrees to about 38 degrees 2 theta. In a further embodiment, form II hydrochloride of the present application is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 7.0, 9.1, 11.5, 12.3, 12.4, 16.4, 17.9, 19.5, 24.0, and 29.4 when XPRD is collected from 2 theta of about 5 degrees to about 38 degrees. In a particular embodiment, form II hydrochloride herein is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 7.0, 9.1, 11.5, 19.5, and 24.0 when XPRD is collected from 2 theta of about 5 degrees to about 38 degrees. In a further embodiment, the form II hydrochloride salt of the present application can be characterized asE.g. with Cu K_αThe X-ray powder diffraction pattern measured by the radiation is substantially similar to that of figure 4. The present application also provides a process for preparing a solid hydrochloride salt of a benzimidazolyl urea compound comprising suspending a solid free base of benzimidazolyl urea in an acidic solvent system comprising acetonitrile or water.

In another embodiment, the solid hydrochloride salt is a form III solid. In one embodiment, form III hydrochloride of the present application is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 6.9, 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 XPRD is collected from 2 theta of about 5 degrees to about 38 degrees. In a particular embodiment, form III hydrochloride of the present application is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 6.9, 9.1, 11.7, 18.1, 18.9, 19.8, 23.4, and 24.8 when XPRD is collected from 2 theta of about 5 degrees to about 38 degrees. In a further embodiment, the form III hydrochloride salt of the present application is characterized, e.g., by Cu K_αThe X-ray powder diffraction pattern measured by the radiation is substantially similar to that of fig. 7. The present application also provides a process for preparing solid form III of a benzimidazolyl urea compound comprising suspending a solid free base of benzimidazolyl urea in an acidic solvent system comprising one or more ether solvents 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 is characterized by the use of Cu K characterized by an extended halo with an unrecognizable diffraction peak_αX-ray powder diffraction pattern (XPRD) of the radiation.

Brief Description of Drawings

Figure 1 shows the X-ray powder diffraction pattern of solid form I of the benzimidazolyl urea compound (free base).

Figure 2 shows the 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 the 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 thermogram of solid form II of the benzimidazolyl urea compound.

FIG. 7 is an X-ray powder diffraction pattern of solid form III of the hydrochloride salt of a benzimidazolyl urea compound.

Figure 8 shows a DSC thermogram of solid form III of the hydrochloride salt of the benzimidazolyl urea compound.

Figure 9 is a TGA (thermogravimetric analysis) thermogram of solid form III of the benzimidazolyl urea compound.

FIG. 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 the mesylate salt of the benzimidazolyl urea compound.

FIG. 12 is a depiction of the mesylate salt of a benzimidazolyl urea compound¹H-NMR。

Detailed description of the invention

The present application relates to novel substantially pure solid forms of (R) -1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1 h-benzimidazol-2-yl ] urea ("benzimidazolyl urea compounds").

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 a benzimidazolyl urea compound.

A crystalline solid of form I (free base) of a substantially pure benzimidazolyl urea compound can be prepared from an amorphous or crystalline compound by contacting the compound with a polar solvent such as acetonitrile, dichloromethane, methanol, ethanol, or water, or a combination thereof. The benzimidazolyl urea compound may be contacted with the solvent by saturating a solution of the benzimidazolyl urea compound in the solvent at ambient temperature and allowing the mixture to stand for an extended period of time (e.g., overnight). Alternatively, the benzimidazolyl urea compound may be dissolved in the solvent at elevated temperature, e.g. under reflux, followed by cooling the solution to room temperature or lower and isolating the solid form I.

In one embodiment of the process, a crystalline solid form I of a substantially pure benzimidazolyl urea compound may be prepared from the amorphous or crystalline compound by preparing a saturated solution of the compound in a polar solvent at room temperature and isolating the resulting form I. 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 temperature, a saturated solution is obtained from which form I precipitates and can be isolated. In one embodiment, the solvent used to prepare form I is CH₂Cl₂Or MeOH or mixtures thereof. Isolation of the resulting solid provided form I.

The solid form I of the benzimidazolyl urea compound (free base) can be identified by the following features: a melting endotherm having an extrapolated onset point of about 243 ℃ as determined by differential scanning calorimetry using a scan rate of 10 ℃/minute; and an X-ray powder diffraction pattern substantially as shown in table 1 and figure 1, wherein the XRPD pattern was measured using a powder diffractometer equipped with a Cu X-ray tube source. With Cu Ka₁The sample is irradiated and XRPD data is collected from about 0to about 40 ° 2 θ. The state of the artOne will appreciate that the relative intensities of XRPD peaks may vary significantly, depending on the orientation of the sample being examined and the type and arrangement of instrument used, and thus the intensities in the XPRD traces included herein are to some extent illustrative and not intended for absolute comparison.

Fig. 1 is an X-ray powder diffraction pattern of solid form I of the benzimidazolyl urea compound collected from about 5 degrees to about 38 degrees 2 Θ. The peaks corresponding to the X-ray powder diffraction pattern having a relative intensity greater than or equal to 5% are listed in table 1.

Figure 2 shows a DSC thermogram of solid form I of the benzimidazolyl urea compound, which exhibits an endotherm with an initial transition at about 243 ℃. One skilled in the art will recognize that the peak and onset temperature of the endotherm may vary depending on the experimental conditions. A1.8 mg sample of solid form I was equilibrated at about 35 deg.C for about 10 minutes and the data in FIG. 2 was collected. During the sample collection period, the temperature was increased at a rate of about 10 deg.C/minute.

Figure 3 is a TGA (thermogravimetric analysis) thermogram of solid form I of the benzimidazolyl urea compound showing an initial weight loss of about 35% in the temperature range of 50 to 260 ℃. The data in fig. 3 were collected by equilibrating 0.87mg of the solid form I sample at about 35 ℃ for about 10 minutes. During the sample collection period, the temperature was increased at a rate of about 10 deg.C/minute. While applicants do not wish to be bound by a specific explanation of the endotherm in DSC and the weight loss in TGA, it appears that the transition with the large peak in DSC is due to the melting transition associated with material degradation implied by the weight loss in TGA.

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

or a salt thereof.

In another embodiment, the solid is solid form I free base.

In another embodiment, solid form I is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 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 XPRD is collected from about 5 degrees to about 38 degrees 2 theta.

In another embodiment, solid form I is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 9.3, 16.7, 18.6, 19.6, 21.7, and 25.8 when XPRD is collected from 2 theta of about 5 degrees to about 38 degrees.

In another embodiment, solid form I is characterized, e.g., by the use of Cu K_αRadiometric, substantially similar to the X-ray powder diffraction pattern of fig. 1.

In another embodiment, solid form I is further characterized by having an endothermic peak with an onset temperature at about 243 ℃ as measured by differential scanning calorimetry in which the temperature is scanned at about 10 ℃/minute.

In another embodiment, the present invention provides a crystalline form I process for preparing a compound of formula (I) comprising precipitating a compound of formula (I) from a solvent system comprising dichloromethane, methanol, ethanol, or a combination thereof.

TABLE 1 XRPD pattern peaks for solid form I of benzimidazolyl urea compounds

In one aspect, the present application provides a crystalline form II of a 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 acid addition salts of pharmaceutically acceptable benzimidazolyl urea compounds may be prepared 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 compound until the mono-acid addition salt of the compound is prepared. In one embodiment, a hydrochloric acid addition salt of a benzimidazolyl urea compound may be precipitated. 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 temperature of the mixture may reduce the solubility of the hydrochloride salt of the benzimidazolyl urea compound and the salt precipitates out. Alternatively, adding a second solvent to the mixture may precipitate the salt.

In one embodiment, the benzimidazolyl urea compound of the present application is suspended in a polar solvent. In a further embodiment, the polar solvent is acetonitrile. In this embodiment, the benzimidazolyl urea compound of the present application is suspended in acetonitrile at room temperature and a stoichiometric amount of aqueous HCl is added. The suspension was maintained in a closed vessel while gently stirring until it equilibrated and the benzimidazolyl urea compound was converted to the corresponding acid addition salt. In some embodiments, the conversion of the free base suspension to an acid addition salt may take from minutes to days. The salt can be recovered by filtering the suspension to obtain a white solid which can be dried under vacuum at room temperature for several hours.

The solid form II of the hydrochloride salt of the benzimidazolyl urea compound may be identified by an X-ray powder diffraction pattern substantially as shown in table 2 and figure 4, wherein the XRPD pattern is measured using a powder diffractometer equipped with a Cu X-ray tube source. With Cu Ka₁The sample is irradiated and XRPD data is collected from about 5 to about 40 ° 2 θ. In the field ofOne skilled in the art will recognize that the relative intensities of XRPD peaks may vary significantly depending on the sample orientation.

FIG. 4 is an X-ray powder diffraction pattern of solid form II of the hydrochloride salt of the benzimidazolyl urea compound collected from about 0to about 40 degrees 2 θ. The peaks corresponding to the 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 hydrochloride salt of a compound of formula (I):

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

In another embodiment, the hydrochloride salt is solid form II.

In another embodiment, solid form II is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 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 XPRD is collected from about 5 degrees to about 38 degrees 2 theta.

In another embodiment, solid form II is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 7.0, 9.1, 11.5, 12.3, 12.4, 16.4, 17.9, 19.5, 24.0, and 29.4 when XPRD is collected from 2 theta of about 5 degrees to about 38 degrees.

In another embodiment, solid form II is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αWhen measuring radiation, includes at least3 approximate peak positions (2 theta angles ± 0.2) selected from 7.0, 9.1, 11.5, 19.5, and 24.0 when XPRD is collected from 2 theta of about 5 degrees to about 38 degrees.

In another embodiment, solid form II is characterized, e.g., by the use of Cu K_αRadiometric, substantially similar to the X-ray powder diffraction pattern of fig. 4.

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

TABLE 2 XRPD pattern peaks for solid form II of benzimidazolyl urea compounds

The solid form III of the hydrochloride salt of the benzimidazolyl urea compound may be identified by an X-ray powder diffraction pattern substantially as shown in table 3 and figure 7, wherein the XRPD pattern is measured using a powder diffractometer equipped with a Cu X-ray tube source. With Cu Ka₁The sample is irradiated and XRPD data is collected from about 5 to about 40 ° 2 θ. Those skilled in the art will recognize that the relative intensities of the XPRD peaks may vary significantly depending on the sample orientation.

Figure 5 shows a DSC thermogram of solid form II of the hydrochloride salt of the benzimidazolyl urea compound which exhibits an endotherm with an initial transition at about 216 ℃. One skilled in the art will recognize that the peak and onset temperature of the endotherm may vary depending on the experimental conditions. A 1.26mg sample of solid form II was equilibrated at about 35 ℃ for about 10 minutes and the data in fig. 5 was collected. During the sample collection period, the temperature was increased at a rate of about 10 deg.C/minute.

Figure 6 is a TGA (thermogravimetric analysis) thermogram of solid form II of the benzimidazolyl urea compound showing an initial weight loss of about 22% in the temperature range of 50-230 ℃. A 1.82mg sample of solid form II was equilibrated at about 35 ℃ for about 10 minutes and the data in fig. 6 was collected. During the sample collection period, the temperature was increased at a rate of about 10 deg.C/minute. While applicants do not wish to be bound by a specific explanation of the endothermic curve in DSC and weight loss in TGA, it appears that the transition with the large peak in DSC is due to the melting transition associated with material degradation implied by the weight loss in TGA.

FIG. 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 degrees 2 θ. The peaks corresponding to the 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 the hydrochloride salt of the compound of formula (I) is solid form III.

In another embodiment, solid form III is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 6.9, 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 XPRD is collected from 2 theta of about 5 degrees to about 38 degrees.

In another embodiment, solid form III is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αThe radiation measurement includes at least 3 approximate peak positions (2 theta angles ± 0.2) selected from 6.9, 9.1, 11.7, 18.1, 18.9, 19.8, 23.4, and 24.8 when XPRD is collected from 2 theta of about 5 degrees to about 38 degrees.

In another embodiment, solid form III is characterized, for example, by Cu K_αThe X-ray powder diffraction pattern measured by the radiation is substantially similar to that of fig. 7.

In another embodiment, the present application also provides a process for preparing solid form III comprising suspending a solid free base of benzimidazolyl urea in an acidic solvent system comprising one or more ethereal solvents and water.

TABLE 3 XRPD pattern peaks for solid form III of benzimidazolyl urea compounds

Figure 8 shows a DSC thermogram of hydrochloride solid form III of the benzimidazolyl urea compound showing a melting endotherm with an initial transition at about 214 ℃. One skilled in the art will recognize that the peak and onset temperature of the endotherm may vary depending on the experimental conditions. The data in fig. 8 was collected by equilibrating a 1.03mg sample in solid form at about 35 ℃ for about 10 minutes. During the sample collection period, the temperature was increased at a rate of about 10 deg.C/minute.

Figure 9 is a TGA (thermogravimetric analysis) thermogram of solid form III of the benzimidazolyl urea compound showing an initial weight loss of about 28% in the temperature range of 50-260 ℃. The data in fig. 9 was collected by equilibrating a 3.71mg sample of the solid form at about 35 ℃ for about 10 minutes. During the sample collection period, the temperature was increased at a rate of about 10 deg.C/minute. While applicants do not wish to be bound by a specific explanation of the endothermic curve in DSC and weight loss in TGA, it appears that the transition with the large peak in DSC is due to the melting transition associated with material degradation implied by the weight loss in TGA.

In one embodiment, the solid form III of the hydrochloride salt of the benzimidazolyl urea compound may be prepared from a mixture of an ethereal solvent and water-soluble HCl. In one embodiment, the ethereal solvent is THF, methyl tert-butyl ether (MTBE), or a mixture thereof. In certain embodiments, the benzimidazolyl urea compound may be suspended in the ethereal solvent, followed by the addition of a stoichiometric amount of HCl. Additional ethereal solvent may be added and the suspension may be allowed to equilibrate for a time sufficient to convert the free base to the corresponding HCl addition salt. Equilibration may take less than an hour to several hours to complete. In particular embodiments, the suspension may be allowed to equilibrate for up to 24 hours before collecting the white solid. The solids can be collected using any method known to those skilled in the art. The solid can be dried under vacuum for several hours.

In another aspect, the present application provides an amorphous 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. The mesylate salt of a pharmaceutically acceptable benzimidazolyl urea compound may be prepared by any method known to those skilled in the art. For example, a solution of methanesulfonic acid can 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 the amorphous solid form IV using any method known to those skilled in the art. The amorphous mesylate salt of the benzimidazolyl urea compound is characterized by the absence of a diffraction pattern of the crystalline form. X-ray powder diffraction of the partially amorphous 6-fluorobenzimidazolyl urea compound mesylate salt may still lack the characteristic attributes of the crystalline form because the derived peak from the crystalline portion of the sample is too weak to be observed more than noise. FIG. 10 is an X-ray powder diffraction pattern of amorphous form IV of the mesylate salt of the benzimidazolyl urea compound.

In one embodiment, amorphous mesylate salts of benzimidazolyl urea compounds may be prepared by spray drying a salt solution in a suitable solvent. Spray drying is well known in the art and is commonly used to dry heat sensitive materials such as pharmaceutical drugs. Spray drying also provides a consistent particle distribution that can be reproduced quite well. Any gas may be used to dry the powder, although air is typically used. If the material is sensitive to air, an inert gas such as nitrogen or argon may be used. Any method of converting a solution, slurry, suspension or emulsion of a salt to produce a solid powder may be suitable for preparing amorphous form IV of the mesylate salt of the benzimidazolyl urea compound. For example, freeze drying, drum drying or pulse transfer drying may be used to produce amorphous mesylate salt of the benzimidazolyl urea compound.

In one embodiment, the present invention provides a solid compound of formula (I), wherein the solid is form IV amorphous mesylate.

In another embodiment, the solid amorphous mesylate salt form IV is characterized by the use of a Cu K characterized by an extended halo with no discernible diffraction peak_αX-ray powder diffraction pattern (XPRD) of the radiation.

In one embodiment, the solution of the benzimidazolyl urea compound in the polar solvent may be spray-dried using a nano spray dryer equipped with a condenser.

Figure 11 shows a DSC thermogram of amorphous form IV of the mesylate salt of the benzimidazolyl urea compound. A 1.6mg sample of amorphous material was equilibrated at about 35 ℃ for about 10 minutes and the data in fig. 11 was collected. During the sample collection period, the temperature was increased at a rate of about 10 deg.C/minute.

It is to be understood that solid forms I, II and III and amorphous solid form IV of the benzimidazolyl urea compound or salt thereof, in addition to having XRPD, DSC, TGA, and other features described herein, may also have other features not described, such as, but not limited to, the presence of water or one or more solvent molecules.

X-ray powder diffraction pattern (XRPD): XRPD patterns of the crystalline form were recorded in reflection mode at room temperature using a Bruker D8Discover system (Bruker AXS, Madison, WI) equipped with a closed tube wave source and a Hi-Star plane detector. The X-ray generator was operated at 40kV tension and 35mA current. Powder samples were placed on Si zero background wafers. Two frames were registered with 120 seconds exposure times each. The data were then integrated in 0.02 steps over 2 of 3-41 and merged into one continuous pattern.

X-ray powder diffraction pattern (XRPD) for amorphous form: XRPD patterns were recorded as amorphous solids in reflectance mode at room temperature using a Bruker D8Advance system (Bruker AXS, Madison, WI) equipped with a Vantec-1 position sensitive detector. The X-ray generator was operated at 40kV tension and 45mA current. The powder sample was placed on a Si zero background holder and rotated at 15rpm during the experiment in a continuous mode using a variable slit on the detector. Data was collected from 3 to 40 degrees in 0.0144653 degree increments (0.25 sec/step).

Differential Scanning Calorimetry (DSC): DSC was performed on samples of the material using a DSC Q2000 differential scanning calorimeter (taiinstruments, New Castle, DE). The instrument was calibrated with indium. About 1-2mg of the sample was weighed into an aluminum pan crimped with a pinhole-free cap or a pinhole cap. The DSC sample was scanned at a heating rate of 10 ℃/min and with a 50 mL/min nitrogen flow from 30 ℃ to the temperature shown in the plot. Samples run under adjusted dsc (mdsc) were adjusted at + and-1 ℃ every 60 seconds with ramp rates (ramp rates) of 2 or 3 ℃/min.

Data were collected by Thermal Advantage Q series (TM) software and analyzed by Universal Analysis2000 software (TA Instruments, New Castle, DE).

Thermogravimetric analysis (TGA): a model Q5000 thermogravimetric analyzer (TA Instruments, New Castle, DE) was used for TGA measurements. Typically, about 3-5mg of sample is scanned from 30 ℃ to the temperature shown in the plot at a heating rate of 10 ℃/minute. Data were collected by ThermalAdvantage Q series (tm) software and analyzed by Universal Analysis2000 software (TA Instruments, New Castle, DE).

The 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 progression, severity or effect of a nosocomial or 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.

In another embodiment, the present invention also provides a method of controlling, treating, or reducing the progression, severity, or effect of a nosocomial or 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 said bacterial infection is characterized by the presence of one or more of: streptococcus pneumoniae, Staphylococcus epidermidis (Staphylococcus epidermidis), enterococcus faecalis (Enterococcus faecalis), Staphylococcus aureus (Staphylococcus aureus), Clostridium difficile (Clostridium difficile), Moraxella catarrhalis (Moraxella catarrhalis), Neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), Mycobacterium avium complex (Mycobacterium complex), Mycobacterium abscessus (Mycobacterium vaccae), Mycobacterium vaccae (Mycobacterium vaccaria), Mycobacterium kansasii (Mycobacterium kansasii), Mycobacterium ulcerous (Mycobacterium ulcerocens), Chlamydia pneumoniae (Mycobacterium pneoniae), Chlamydia trachomatis (Chlamydia trachomatis), Haemophilus Haemophilus (Streptococcus influenzae), Streptococcus pyogenes (Streptococcus pyogenes) or Streptococcus pyogenes (Streptococcus pyogenes).

In another embodiment, the invention also provides a method of controlling, treating or reducing the progression, severity or effect of a nosocomial or 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 said bacterial infection is selected from one or more of the following: upper respiratory tract infections, lower respiratory tract infections, ear infections, pleuropneumoniae and bronchial infections, complicated urinary tract infections, non-complicated urinary tract infections, intra-abdominal infections, cardiovascular infections, bloodstream infections, sepsis, bacteremia, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital infections, eye infections or granuloma infections, non-complicated skin and skin structure infections (uSSSI), complicated skin and skin structure infections (cSSSI), catheter infections, pharyngitis, sinusitis, otitis externa, otitis media, bronchitis, empyema, pneumonia, community-acquired bacterial pneumonia (CABP), hospital-acquired pneumonia (HAP), hospital-acquired bacterial pneumonia, respirator-associated pneumonia (VAP), diabetic foot infections, vancomycin-resistant enterococcus infections, cystitis and pyelonephritis, kidney stones, kidney-derived pneumonia (cabs), and liver inflammation, Prostatitis, peritonitis, complicated intra-abdominal infections (cIAI) and other intra-abdominal infections, dialysis-related peritonitis, visceral abscesses, endocarditis, myocarditis, pericarditis, transfusion-related septicemia, meningitis, encephalitis, brain abscesses, osteomyelitis, arthritis, genital ulcers, urethritis, vaginitis, cervicitis, gingivitis, conjunctivitis, keratitis, endophthalmitis, infections in cystic fibrosis patients, or infections in febrile neutropenia patients.

In another embodiment, the bacterial infection is selected from one or more of the following: community-acquired bacterial pneumonia (CABP), hospital-acquired pneumonia (HAP), hospital-acquired bacterial pneumonia, ventilator-associated pneumonia (VAP), bacteremia, diabetic foot infection, catheter infection, non-complicated skin and skin structure infections (uSSSI), complicated skin and skin structure infections (cSSSI), vancomycin-resistant enterococcal infections, or osteomyelitis.

According to another embodiment, the present invention provides a method of reducing or inhibiting the number of bacteria in a biological sample. Such methods comprise contacting the 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 thereof; biopsy material or extract thereof from a mammal; and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof. The term "biological sample" also includes living organisms, in which case "contacting a compound of the invention with a biological sample" is synonymous with the term "administering said compound or a composition comprising said compound to a mammal".

The gyrase and/or topoisomerase IV inhibitors or pharmaceutical salts thereof of the present invention may be formulated into pharmaceutical compositions for administration to a mammal or human. Another embodiment of the invention is the pharmaceutical composition effective for treating or preventing a bacterial infection comprising an amount of a gyrase and/or topoisomerase IV inhibitor sufficient to measurably reduce the number of bacteria and a pharmaceutically acceptable carrier. As used herein, the term "measurably reduce the number of bacteria" means a measurable change in the number of bacteria between a sample containing the inhibitor and a sample containing only bacteria.

According to another embodiment, the methods of the invention are useful for treating patients in the veterinary field, including but not limited to zoos, laboratories, human partners and farm animals, including primates, rodents, reptiles and birds. Examples of such animals include, but are not limited to, guinea pigs, hamsters, gerbils, rats, mice, rabbits, dogs, cats, horses, pigs, sheep, cows, goats, deer, rhesus monkeys, tamarinds, apes, baboons, gorillas, chimpanzees, orangutans, gibbons, ostriches, chickens, turkeys, ducks, and geese.

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

In another embodiment, the bacterial infection is characterized by the presence of one or more of streptococcus pneumoniae, enterococcus faecalis, or staphylococcus aureus.

In another embodiment, the bacterial infection is characterized by the presence of one or more of escherichia coli, moraxella catarrhalis, or haemophilus influenzae.

In another embodiment, the bacterial infection is characterized by the presence of one or more of the following: clostridium difficile, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerosa, Chlamydophila pneumoniae and Chlamydia trachomatis.

In another embodiment, the bacterial infection is characterized by the presence of one or more of the following: streptococcus pneumoniae, Staphylococcus epidermidis, enterococcus faecalis, Staphylococcus aureus, Clostridium difficile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerous, Chlamydia pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Streptococcus pyogenes, or beta-hemolytic streptococci.

In some embodiments, the bacterial infection is characterized by the presence of one or more of the following: methicillin-resistant Staphylococcus aureus, fluoroquinolone-resistant Staphylococcus aureus, vancomycin intermediate-resistant Staphylococcus aureus, linezolid-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, macrolide-resistant Streptococcus pneumoniae, fluoroquinolone-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis, linezolid-resistant Enterococcus faecalis, fluoroquinolone-resistant Enterococcus faecalis, vancomycin-resistant Enterococcus faecium (Enterococcus faecium), linezolid-resistant Enterococcus faecium, fluoroquinolone-resistant Enterococcus faecium, ampicillin-resistant Enterococcus faecium, macrolide-resistant Haemophilus influenzae, beta-lactam-resistant Haemophilus influenzae, fluoroquinolone-resistant Haemophilus influenzae, beta-lactam-resistant Moraxella catarrhalis, methicillin-resistant Staphylococcus epidermidis, vancomycin-resistant Staphylococcus epidermidis, fluoroquinolone-resistant Staphylococcus epidermidis, macrolide-resistant mycoplasma pneumoniae, isoniazid-resistant mycobacterium tuberculosis, rifampin-resistant mycobacterium tuberculosis, methicillin-resistant Coagulase-negative Staphylococcus (Coagulase negative Staphylococcus), fluoroquinolone-resistant Coagulase-negative Staphylococcus, glycopeptide intermediate-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, heterogeneous vancomycin intermediate-resistant Staphylococcus aureus, heterogeneous vancomycin-resistant Staphylococcus aureus, macrolide-lincosamide-streptogramin-resistant Staphylococcus (staphyloccus), beta-lactam-resistant enterococcus faecalis, beta-lactam-resistant enterococcus faecium, ketolide-resistant streptococcus pneumoniae, ketolide-resistant streptococcus pyogenes, macrolide-resistant streptococcus pyogenes, Vancomycin-resistant Staphylococcus epidermidis, fluoroquinolone-resistant Neisseria gonorrhoeae, multidrug-resistant Pseudomonas aeruginosa (Pseudomonas aeruginosa) or cephalosporin-resistant Neisseria gonorrhoeae.

According to another embodiment, the methicillin-resistant staphylococcus is selected from the group consisting of methicillin-resistant staphylococcus aureus, methicillin-resistant staphylococcus epidermidis or methicillin-resistant coagulase-negative staphylococcus.

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

In other embodiments, a form of the compound of formula (I) or a pharmaceutically acceptable salt thereof is used to treat daptomycin resistant organisms, including but not limited to daptomycin resistant enterococcus faecium and daptomycin resistant staphylococcus aureus.

According to another embodiment, the fluoroquinolone resistant staphylococcus is selected from the group consisting of fluoroquinolone resistant staphylococcus aureus, fluoroquinolone resistant staphylococcus epidermidis, and fluoroquinolone resistant coagulase negative staphylococcus.

According to another embodiment, the glycopeptide resistant staphylococcus is selected from the group consisting of glycopeptide intermediate resistant staphylococcus aureus, vancomycin intermediate resistant staphylococcus aureus, heterogeneous vancomycin intermediate resistant staphylococcus aureus, and heterogeneous vancomycin resistant staphylococcus aureus.

According to another embodiment, the macrolide-lincosamide-streptogramin resistant staphylococcus is macrolide-lincosamide-streptogramin resistant staphylococcus aureus.

According to another embodiment, the linezolid-resistant enterococcus is selected from linezolid-resistant enterococcus faecalis or linezolid-resistant enterococcus faecium.

According to another embodiment, the glycopeptide resistant enterococcus is selected from vancomycin resistant enterococcus faecium or vancomycin resistant enterococcus faecalis.

According to another embodiment, the β -lactam resistant enterococcus faecalis is a β -lactam resistant enterococcus faecium.

According to another embodiment, the penicillin-resistant streptococcus is penicillin-resistant streptococcus pneumoniae.

According to another embodiment, the macrolide-resistant streptococcus is macrolide-resistant streptococcus pneumoniae

According to another embodiment, the ketolide resistant streptococcus is selected from the group consisting of macrolide resistant streptococcus pneumoniae and ketolide resistant streptococcus pyogenes.

According to another embodiment, the fluoroquinolone resistant streptococcus is a fluoroquinolone resistant streptococcus pneumoniae.

According to another embodiment, the β -lactam resistant haemophilus is β -lactam resistant haemophilus influenzae.

According to another embodiment, the fluoroquinolone resistant haemophilus is fluoroquinolone resistant haemophilus influenzae.

According to another embodiment, the macrolide-resistant haemophilus is macrolide-resistant haemophilus influenzae.

According to another embodiment, the macrolide-resistant mycoplasma is macrolide-resistant mycoplasma pneumoniae.

According to another embodiment, the isoniazid resistant mycobacterium is an isoniazid resistant mycobacterium tuberculosis.

According to another embodiment, the rifampicin resistant mycobacterium is rifampicin resistant mycobacterium tuberculosis.

According to another embodiment, the beta-lactam resistant moraxella is beta-lactam resistant 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, vancomycin intermediate-resistant Staphylococcus aureus, linezolid-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, macrolide-resistant Streptococcus pneumoniae, fluoroquinolone-resistant Streptococcus pneumoniae, vancomycin-resistant enterococcus faecalis, linezolid-resistant enterococcus faecalis, fluoroquinolone-resistant enterococcus faecalis, vancomycin-resistant enterococcus faecium, linezolid-resistant enterococcus faecium, fluoroquinolone-resistant enterococcus faecium, ampicillin-resistant enterococcus faecium, macrolide-resistant Haemophilus influenzae, beta-lactam-resistant Haemophilus influenzae, fluoroquinolone-resistant Haemophilus influenzae, beta-lactam-resistant Moraxella catarrhalis, methicillin-resistant Staphylococcus epidermidis, vancomycin-resistant Staphylococcus epidermidis, fluoroquinolone-resistant staphylococcus epidermidis, macrolide-resistant mycoplasma pneumoniae, isoniazid-resistant mycobacterium tuberculosis, rifampin-resistant mycobacterium tuberculosis, fluoroquinolone-resistant neisseria gonorrhoeae or cephalosporin-resistant neisseria gonorrhoeae.

According to another embodiment, the bacterial infection is characterized by the presence of one or more of the following: methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, methicillin-resistant coagulase-negative Staphylococcus aureus, fluoroquinolone-resistant Staphylococcus epidermidis, fluoroquinolone-resistant coagulase-negative Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, glycopeptide intermediate-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, vancomycin intermediate resistant staphylococcus aureus, heterogeneous vancomycin resistant staphylococcus aureus, vancomycin resistant enterococcus faecium, vancomycin resistant enterococcus faecalis, penicillin resistant streptococcus pneumoniae, macrolide resistant streptococcus pneumoniae, fluoroquinolone resistant streptococcus pneumoniae, macrolide resistant streptococcus pyogenes, or beta-lactam resistant haemophilus influenzae.

According to another embodiment, the bacterial infection is characterized by the presence of one or more of the following: methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococcus faecium, vancomycin-resistant enterococcus faecalis, vancomycin-resistant staphylococcus aureus, vancomycin intermediate-resistant staphylococcus aureus, heterogeneous vancomycin-resistant staphylococcus aureus, multidrug-resistant pseudomonas aeruginosa, isoniazid-resistant mycobacterium tuberculosis, and rifampicin-resistant mycobacterium tuberculosis.

Pharmaceutically acceptable salts of the compounds of the present invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, picrate, tartrate, dihydrogensulfate, butyrate, citrate, camphorate, glycolate, camphorate, nicotinate, picrate, pivalate, salicylate, succinate, sulfate, tartrate, etc, Thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not themselves pharmaceutically acceptable, may be used to prepare salts which are useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

Salts derived from suitable bases include alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., magnesium), ammonium, and N⁺（C_1-4Alkyl radical)₄And (3) salt. The present invention also contemplates the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products can be obtained by such quaternization.

The pharmaceutical compositions of the present invention comprise a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Such compositions may optionally comprise additional therapeutic agents. Such agents include, but are not limited to, antibiotics, anti-inflammatory agents, matrix metalloproteinase inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressive agents, anti-cancer agents, antiviral agents, cytokines, growth factors, immunomodulators, prostaglandins, or anti-vascular hyperproliferative compounds.

The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that can be administered to a patient along with a compound of the present invention without destroying its pharmacological activity.

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

The term "pharmaceutically effective amount" 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 reducing a bacterial infection in a patient.

Depending on the particular condition or disease state to be treated or prevented, additional therapeutic agents typically administered to treat or prevent the condition may be administered with the inhibitors of the invention. Such therapeutic agents include, but are not limited to, antibiotics, anti-inflammatory agents, matrix metalloproteinase inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressive agents, anti-cancer agents, antiviral agents, cytokines, growth factors, immunomodulatory agents, prostaglandins, or anti-vascular hyperproliferative compounds.

The compounds of the invention may be used in a conventional manner to control the level of bacterial infection in vivo and to treat diseases or reduce the progression or severity of bacterially-mediated effects. Such methods of treatment, their dosage levels and requirements can be selected by one of ordinary skill in the art based on available methods and techniques.

For example, a compound of the invention can be used in combination with a pharmaceutically acceptable adjuvant for administration to a patient suffering from a bacterial infection or disease in a pharmaceutically acceptable manner and in an amount effective to reduce the severity of that infection or disease.

Alternatively, the compounds of the present invention may be used in compositions and methods for treating or protecting an individual against a bacterial infection or disease for an extended period of time. In one embodiment, the compounds of the invention may be used in compositions and methods for treating or protecting an individual against bacterial infection or disease during 1-2 cycles. In another embodiment, the compounds of the invention may be used in compositions and methods for treating or protecting an individual against a bacterial infection or disease during 4-8 cycles (e.g., in treating a patient suffering from or at risk of developing endocarditis or osteomyelitis). In another embodiment, the compounds of the invention may be used in compositions and methods for treating or protecting an individual against bacterial infection or disease during 8-12 cycles. The compounds may be used in such compositions, alone or in conjunction with other compounds of the invention, in a manner consistent with the conventional use of enzyme inhibitors in pharmaceutical compositions. For example, the compounds of the present invention may be combined with pharmaceutically acceptable adjuvants conventionally used in vaccines and administered in prophylactically effective amounts to protect individuals against bacterial infection or disease for extended periods of time.

In some embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof may be used prophylactically to prevent a bacterial infection. In some embodiments, the compounds of formula (I) or pharmaceutically acceptable salts thereof may be used to prevent opportunistic infections such as those encountered in bacterial endocarditis before, during or after dental or surgical procedures. In other embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be used prophylactically in dental procedures, including but not limited to tooth extraction, periodontal surgery, dental implant placement, and endodontic procedures. In other embodiments, compounds of formula (I) or pharmaceutically acceptable salts thereof may be used prophylactically in surgical procedures, including, but not limited to, general surgery, respiratory surgery (tonsillectomy/adenoidectomy), gastrointestinal surgery (upper GI and selective small bowel surgery, esophageal sclerotherapy and dilation, large bowel resection, acute appendectomy), trauma surgery (penetrating abdominal surgery), genitourinary tract surgery (prostatectomy, urethral dilation, cystoscopy, vaginal or abdominal hysterectomy, cesarean section), transplantation surgery (kidney, liver, pancreas, or kidney transplantation), head and neck surgery (dermectomy, cervical lymph node sweeping, laryngectomy, head and neck cancer surgery, mandibular fracture), orthopedic surgery (total joint replacement, traumatic open fracture), vascular surgery (peripheral vascular surgery), Chest and heart surgery, coronary bypass surgery, pneumonectomy, and neurosurgery.

As used herein, unless otherwise indicated, the term "preventing bacterial infection" means the prophylactic use of an antibiotic, such as a gyrase and/or topoisomerase IV inhibitor of the present invention, to prevent bacterial infection. Treatment with gyrase and/or topoisomerase IV inhibitors may be performed prophylactically to prevent infection by organisms sensitive to gyrase and/or topoisomerase IV inhibitors. One general group of conditions in which prophylactic treatment may be considered is when an individual is more susceptible to infection for reasons such as reduced immunity, surgery, trauma, the presence of artificial devices in the body (temporary or permanent), anatomical defects, exposure to high levels of bacteria or possible exposure to disease-causing pathogens. Examples of factors that can lead to reduced immunity include chemotherapy, radiation therapy, diabetes, aging, HIV infection, and transplantation. An example of an anatomical defect would be a defect in a heart valve, which increases the risk of bacterial endocarditis. Examples of artificial devices include artificial joints, surgical needles, catheters, and the like. Another group of situations where prophylactic use of gyrase and/or topoisomerase IV inhibitors may be appropriate would be to prevent transmission of pathogens (directly or indirectly) between individuals. A particular example of a prophylactic use to prevent pathogen transmission is the use of gyrase and/or topoisomerase IV inhibitors by individuals in a healthcare facility (e.g. a hospital or nursing home).

The compounds of formula (I) or pharmaceutically acceptable salts thereof may also be co-administered with other antibiotics to increase the effectiveness of the treatment or prevention against a variety of bacterial infections. When the compounds of the present invention are administered in combination therapy with other agents, they may be administered to the patient sequentially or simultaneously. Alternatively, a pharmaceutical or prophylactic composition according to the invention comprises 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 one or more therapeutic agents is an antibiotic selected from the group consisting of: natural penicillins, penicillinase-resistant penicillins, pseudomonad-resistant penicillins, aminopenicillins, first-generation cephalosporins, second-generation cephalosporins, third-generation cephalosporins, fourth-generation cephalosporins, carbapenems, cephamycins, quinolones, fluoroquinolones, aminoglycosides, macrolides, ketolides, polymyxins, tetracyclines, glycopeptides, streptogramins, oxazolidinones, rifamycins, or sulfonamides.

In some embodiments, the additional one or more therapeutic agents is an antibiotic selected from a penicillin, a cephalosporin, a quinolone, an aminoglycoside, or an oxazolidinone.

In other embodiments, the additional therapeutic agent is selected from the group consisting of natural penicillins including benzathine penicillin G, and penicillin V, penicillinase resistant penicillins including cloxacillin, dicloxacillin, nafcillin, and oxacillin, anti-pseudomonas penicillins including carbenicillin, mezlocillin, piperacillin/tazobactam, ticarcillin, and ticarcillin/clavulanic acid, aminopenicillin including amoxicillin, ampicillin, and ampicillin/sulbactam, cephalosporins including cefazolin, cefadroxil, cephalexin, and cefradine from the first generation, cephalosporins including cefaclor, cefaclor-CD, cefamandole, cefnici, cefprozil, chlorocefuroxime, and cefuroxime from the third generation including cefdinir, Cefixime, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime and ceftriaxone, selected from the fourth generation cephalosporins including cefepime, cefaclor (Ceftaroline) and ceftriaxone, selected from the cephalosporins including cefotetan and cefoxitin, selected from the carbapenems including doripenem, imipenem and meropenem, selected from the monoamidomycins including aztreonam, selected from the quinolones including cinoxacin, nalidixic acid, oxolinic acid and pipemidic acid, selected from the fluoroquinolones including besifloxacin, ciprofloxacin, enoxacin, gatifloxacin, glafloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, oxpocetine and sparfloxacin, selected from the aminoglycosides including amikacin, gentamycin, kanamycin, neomycin, netilmicin, spectinomycin, streptomycin and tobramycin, selected from macrolides including azithromycin, clarithromycin and erythromycin, from ketolides including telithromycin, from tetracyclines including chlortetracycline, demeclocycline, doxycycline, minocycline and tetracycline, from glycopeptides including oritavancin, dabetencin, telavancin, teicoplanin and vancomycin, from streptogramins including dalfopristin/quinupristin, from oxazolidinones including linezolid, from rifamycins including rifabutin and rifampin, and from other antibiotics including bacitracin, colistin, tigecycline, daptomycin, chloramphenicol, clindamycin, isoniazid, metronidazole, mupirocin, polymyxin B, pyrazinamide, trimethoprim/sulfamethoxazole and sulfisoxazole.

In other embodiments, the additional therapeutic agent is selected from the group consisting of natural penicillins including penicillin G, penicillinase-resistant penicillins including nafcillin and oxacillin, anti-pseudomonas penicillins including piperacillin/tazobactam, aminopenicillin including amoxicillins, first generation cephalosporins including cephalexin, second generation cephalosporins including cefaclor, cefaclor-CD, and cefuroxime, third generation cephalosporins including ceftazidime and ceftriaxone, fourth generation cephalosporins including cefepime, carbapenems including imipenem, meropenem, ertapenem, doripenem, panipenem and biapenem, fluoroquinolones including ciprofloxacin, gatifloxacin, levofloxacin and moxifloxacin, aminoglycosides including tobramycin, macrolides including azithromycin and clarithromycin, tetracyclins including doxycycline, glycopeptides include vancomycin, rifamycins include rifampicin, and other antibiotics include isoniazid, pyrazinamide, tigecycline, daptomycin, or trimethoprim/sulfamethoxazole.

In some embodiments, a solid form of a compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered to treat a gram-positive infection. In some embodiments, the composition is a solid, liquid (e.g., a suspension), or iv (e.g., a form of a compound of formula (I) or a pharmaceutically acceptable salt thereof dissolved as a liquid and administered iv) composition. In some embodiments, the composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an additional antibiotic agent, such as a natural penicillin, a penicillinase-resistant penicillin, an anti-pseudomonas 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, a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in solid form is administered orally, and iv. an additional antibiotic agent such as a natural penicillin, a penicillinase resistant penicillin, an anti-pseudomonas 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 is administered.

In some embodiments, a solid form of a compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered to treat gram-negative infections. In some embodiments, the composition is a solid, liquid (e.g., a suspension), or iv (e.g., a form of a compound of formula (I) or a pharmaceutically acceptable salt thereof dissolved as a liquid and administered iv) composition. In some embodiments, a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an additional antibiotic agent selected from the group consisting of: natural penicillins, penicillinase-resistant penicillins, anti-pseudomonas penicillins, aminopenicillins, first-generation cephalosporins, second-generation cephalosporins, third-generation cephalosporins, fourth-generation cephalosporins, carbapenems, cephamycins, monobactams, quinolones, fluoroquinolones, aminoglycosides, macrolides, ketolides, polymyxins, tetracyclines or sulfonamides. In some embodiments, a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in solid form is administered orally, and an additional antibiotic agent such as a natural penicillin, a penicillinase resistant penicillin, an anti-pseudomonas penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a monoamide, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, or an amide is administered orally. In some embodiments, the additional therapeutic agent is administered iv.

The additional therapeutic agents described above may be administered separately from the inhibitor-containing composition as part of a multiple dose regimen. Alternatively, these agents may be part of a single dosage form mixed together with the inhibitor in a single composition.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, externally, rectally, nasally, buccally, vaginally or via an implanted reservoir. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of sterile injectable preparations, for example, as sterile injectable aqueous or oleaginous suspensions. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural 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 similar orally-administered alcohols.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, and aqueous suspensions 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 capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and solutions and polyethylene glycols are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration. These compositions may be prepared by mixing a compound of the invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the active ingredients. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

External administration of the pharmaceutical compositions of the present invention is particularly useful when the desired treatment involves externally applying an easily accessible area or organ. For external application to the skin, the pharmaceutical compositions should be formulated in a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for external application of the compounds of the present invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable emulsion or cream containing the active ingredient suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitol monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the invention may also be applied externally to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Externally applied transdermal patches are also included in the present invention.

The pharmaceutical compositions of the present invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other stabilizers or dispersants known in the art.

According to another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof may also be delivered by implantation (e.g. surgery), for example with an implantable or indwelling device. Implantable or indwelling devices may be designed to reside permanently or temporarily in a subject. Examples of implantable and indwelling devices include, but are not limited to, contact lenses, central venous catheters and needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints such as hip and knee replacements, tympanostomy tubes, urinary catheters, voice prostheses, stents, delivery pumps, vascular filters, and implantable controlled release compositions. Biofilms can be detrimental to patient health with implantable or indwelling devices because they introduce artificial substrates into the body and can cause persistent infections. Thus, providing a compound of formula (I) or a pharmaceutically acceptable salt thereof in or on an implantable or indwelling device may prevent or reduce biofilm production. In addition, implantable or indwelling devices may be used as a reservoir or reservoir for a compound of formula (I) or a pharmaceutically acceptable salt thereof. Any implantable or indwelling device may be used to deliver a compound of formula (I) or a pharmaceutically acceptable salt thereof, provided that a) the device, a compound of formula (I) or a pharmaceutically acceptable salt thereof, and any pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof are biocompatible, and b) the device may deliver or release an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to confer therapeutic efficacy to a patient being treated.

The delivery of therapeutic agents from implantable or indwelling devices is known in the art. See, e.g., "Recent Developments in Coated Steps" published by Hofma et al in Current Interactive graphics Reports2001, 3:28-36, the entire contents of which, including the references cited therein, are incorporated herein by reference. Other descriptions of implantable devices can be found in U.S. patent nos. 6,569,195 and 6,322,847; and U.S. patent application nos. 2004/0044405, 2004/0018228, 2003/0229390, 2003/0225450, 2003/0216699 and 2003/0204168, each of which is incorporated by reference herein in its entirety.

In some embodiments, the implantable device is a stent. In a particular embodiment, the stent may comprise interlocking mesh cables. Each cable may include a metal wire for structural support and a polymeric wire for delivery of a therapeutic agent. The polymeric thread may be administered by dipping the polymer into a solution of the therapeutic agent. Alternatively, the therapeutic agent may be embedded in the polymeric wire during formation of the wire from the polymeric precursor solution.

In other embodiments, the implantable or indwelling device may be coated with a polymeric coating that includes a therapeutic agent. The polymeric coating can be designed to control the release rate of the therapeutic agent. Controlled release of therapeutic agents can utilize a variety of techniques. Devices are known having integral layers or coatings that integrate heterogeneous solutions and/or dispersions of active agents in polymeric substances, wherein diffusion of the agent is rate limiting as the agent diffuses through the polymer to the polymer-liquid interface and is released into the surrounding liquid. In some devices, the soluble substance is also dissolved or dispersed in the polymeric material, such that additional pores or channels are left behind after the material is dissolved. Matrix devices are also generally diffusion limited, but channels or other internal geometries of the device also play a role in the release of reagents into the liquid. The channel may also be a pre-existing channel or a channel left by the release of a reagent or other soluble substance.

Erodible or degradable devices typically physically immobilize the active agent in the polymer. The active agent may be dissolved and/or dispersed throughout the polymeric material. The polymeric material degrades hydrolytically, typically through hydrolysis of the labile bond, allowing the polymer to erode into the liquid, releasing the active agent into the liquid. Hydrophilic polymers have generally faster rates of erosion relative to hydrophobic polymers. Hydrophobic polymers are believed to have almost pure surface diffusion of the active agent, with erosion from the surface inward. Hydrophilic polymers are believed to allow water to penetrate the surface of the polymer, allowing hydrolysis of labile bonds below the surface, which can result in uniform or extensive erosion of the polymer.

The coating of the implantable or indwelling device may comprise a blend of polymers, each having a different rate of release of the therapeutic agent. For example, the coating may comprise a polylactic acid/polyethylene oxide (PLA-PEO) copolymer or a polylactic acid/polycaprolactone (PLA-PCL) copolymer. The polylactic acid/polyethylene oxide (PLA-PEO) copolymer may exhibit a higher therapeutic agent release rate relative to the polylactic acid/polycaprolactone (PLA-PCL) copolymer. The relative amount of therapeutic agent delivered over time and the rate of dosage can be controlled by controlling the relative amount of faster-releasing polymer relative to slower-releasing polymer. For higher initial release rates, the proportion of faster releasing polymer may be increased relative to slower releasing polymer. If most of the dose is to be released over a long period of time, most of the polymer may be a slower releasing polymer. Device coating the device may be by spraying the device with a solution or dispersion of the polymer, active agent and solvent. The solvent may evaporate leaving a coating of the polymer and active agent. The active agent may be dissolved and/or dispersed in the polymer. In some embodiments, the copolymer may be extruded onto a device.

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

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

When the compositions of the present 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, the compound and the additional agent should be present at dosage levels ranging from about 10% to 80% of the dosage normally administered in a monotherapy regimen.

Upon improvement of the condition of the patient, a maintenance dose of a compound, composition or combination of the invention may be administered, if desired. Subsequently, depending on the symptoms, the dose or frequency of administration or both, as a function of the symptoms, may be reduced to a level that maintains an improved condition, and when the symptoms have been alleviated to a desired level, treatment should be discontinued. However, patients may require interstitial therapy on a long-term basis based on any recurring or disease symptoms.

As the skilled person will appreciate, lower or higher doses than those described above may be required. The particular dose and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the particular compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, and the psychological disposition of the patient to the disease and the judgment of the treating physician.

According to another embodiment, the present invention provides a method for treating or preventing a bacterial infection or disease state comprising the step of administering to a patient any of the compounds, pharmaceutical compositions or combinations described herein. As used herein, the term "patient" means an animal, preferably a mammal, and most preferably a human.

The compounds of the invention are also useful as commercial reagents in effective combination with gyrase B and/or topoisomerase IV enzymes. As commercial reagents, the compounds of the invention and their derivatives can be used to block the activity of gyrase B and/or topoisomerase IV in biochemical or cellular assays for bacterial gyrase B and/or topoisomerase IV or homologues thereof, or can be derivatized to bind to a stable resin as a tethered substrate for affinity chromatography applications. These and other uses for characterizing commercial gyrase B and/or topoisomerase IV inhibitors will be apparent to those of ordinary skill in the art.

In order that the invention may be more fully understood, the following schemes and examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

The following definitions describe the terms and abbreviations described herein:

ac acetyl group

Bu butyl

Et Ethyl group

Ph phenyl

Me methyl group

THF tetrahydrofuran

DCM dichloromethane

CH₂Cl₂Methylene dichloride

EtOAc ethyl acetate

CH₃CN acetonitrile

EtOH ethanol

Et₂O diethyl ether

MeOH methanol

MTBE methyl tert-butyl ether

DMF N, N-dimethylformamide

DMA N, N-dimethylacetamide

DMSO dimethyl sulfoxide

HOAc acetic acid

TEA Triethylamine

TFA trifluoroacetic acid

TFAA trifluoroacetic anhydride

Et₃N-Triethylamine

DIPEA diisopropylethylamine

DIEA diisopropylethylamine

K₂CO₃Potassium carbonate

Na₂CO₃Sodium carbonate

Na₂S₂O₃Sodium thiosulfate

Cs₂CO₃Cesium carbonate

NaHCO₃Sodium bicarbonate

NaOH sodium hydroxide

Na₂SO₄Sodium sulfate

MgSO₄Magnesium sulfate

K₃PO₄Potassium phosphate

NH₄Cl ammonium chloride

LC/MS liquid chromatography/Mass Spectrometry

GCMS gas chromatography mass spectrum

HPLC high performance liquid chromatography

GC gas chromatography

LC liquid chromatography

IC ion chromatography

IM muscle

CFU/CFU colony forming units

MIC minimum inhibitory concentration

Hr or h hours

atm atmospheric pressure

RT or RT Room temperature

TLC thin layer chromatography

HCl hydrochloric acid

H₂O water

EtNCO Ethyl isocyanate

Pd/C palladium on carbon

NaOAc sodium acetate

H₂SO₄Sulfuric acid

N₂Nitrogen gas

H₂Hydrogen gas

n-BuLi n-butyllithium

DI deionized

Pd(OAc)₂Palladium acetate (II)

PPh₃Triphenylphosphine

i-PrOH Isopropanol

NBS N-bromosuccinimide

Pd[(Ph₃)P]₄Tetrakis (triphenylphosphine) palladium (0)

PTFE Polytetrafluoroethylene

rpm revolutions per minute

SM raw material

Equivalent weight of Equiv

¹H-NMR proton nuclear magnetic resonance

Synthesis of Compounds

Examples

Benzimidazolyurea compounds

Scheme 2 provides a process for preparing benzimidazolyl urea compounds.

Scheme 2

Example 1.a

Preparation of 2- (2-nitrophenyl) -2, 5-dihydrofuran (3 a) and 2- (2-nitrophenyl) -2, 3-dihydrofuran (3 b)

1-bromo-2-nitrobenzene (1) (600g, 99%, 2.941mol, AlfaAesar A11686), 1, 3-bis (diphenylphosphino) propane (62.50g, 97%, 147.0 mmol, AlfaAesar A12931), 1, 4-dioxane (2.970L, Sigma-Aldrich360481), potassium carbonate (812.9 g, 5.882mol, JT-Baker 301201), and 2, 3-dihydrofuran (2) (1.041 kg, 99%, 1.124L, 14.70mol, Aldrich 018) were mixed in a reaction vessel. The nitrogen stream was bubbled through the stirred mixture for 4 hours, then palladium (II) acetate (16.51 g, 73.52mmol, Strem 461780) was added and deoxygenation continued for another 10 minutes. The reaction mixture was stirred under reflux under nitrogen overnight (NMR of a working-up aliquot showed complete consumption of aryl bromide). The reaction mixture was allowed to cool, diluted with hexane (1L), and passed throughWas filtered (500 g, -200 mesh) and eluted with EtOAc. The filtrate was concentrated under reduced pressure (2- (2-nitrophenyl) -2, 3-dihydrofuran (3 b) volatile under high vacuum, and may be slightly unstable at room temperature) to giveA mixture of (3 a) and (3 b) was obtained as a dark brown oil (654.0 g). The crude material was stored in a refrigerator and advanced without further purification.

Example 1.a.1

Asymmetric preparation of 2- (2-nitrophenyl) -2, 5-dihydrofuran (3 a) and 2- (2-nitrophenyl) -2, 3-dihydrofuran (3 b)

1-bromo-2-nitrobenzene (50.0 mg, 98%, 0.2426mmol, Aldrich 365424), potassium carbonate (67.1 mg, 0.4852mmol, JT-Baker 301201), (R) - (-) -1- [ (S) -2- (diphenylphosphino) ferrocenyl]Ethyldicyclohexylphosphine ethanol adduct ((R) - (S) -JosiPhos, 7.8mg, 0.01213mmol, Strem 261210), 2, 3-dihydrofuran (1.0 mL, 99%, 13.08mmol, Aldrich 200018) and 1, 4-dioxane (0.98 mL) were combined in a reaction tube. The nitrogen stream was bubbled through the stirred mixture for 20 min, then palladium (II) acetate (1.36 mg, 0.006065mmol, Strem 461780) was added. The tube was sealed and the reaction mixture was stirred at 105 ℃ overnight. HPLC of the crude reaction mixture shows almost complete consumption of aryl bromide and formation of a 1:1 mixture of 2- (2-nitrophenyl) -2, 5-dihydrofuran (3 a) and 2- (2-nitrophenyl) -2, 3-dihydrofuran (3 b). The reaction mixture was allowed to cool, diluted with hexane (2 mL), filtered and washed with ethyl acetate. The filtrate was concentrated on a rotary evaporator to give a brown oil (51 mg). Due to volatility and stability concerns, the material was not placed under high vacuum. Crude reaction mixture is passed through¹H NMR analysis determined a 1:1 mixture of (3 a) and (3 b). The oil was purified by treatment with 0-38% EtOAc in hexane (or 0-100% CH in hexane)₂Cl₂) Eluted silica gel chromatography to provide pure samples of (3 a) and (3 b). The analytical data for these samples are as follows:

2- (2-Nitrophenyl)) 2, 5-dihydrofuran (3 a) was obtained as a yellow solid (97% HPLC purity, 97.0% ee): LCMS (C18 column eluted with 10-90% MeOH/water gradient from 3-5 min using formic acid modifier) M + 1: 192.05 (3.40 minutes); HPLC retention time of 4.2 min (10-90% CH over 8 min)₃CN/Water gradient elution YMC ODS-AQ150x3.0mm column, using 0.1% TFA modifier and 1 mL/min flow rate); in thatAnalytical chiral HPLC retention time on a 4.6x250mm column eluting with 10% iPrOH in hexanes for 7.4 minutes (major enantiomer) and 8.1 minutes (minor enantiomer) using a flow rate of 1 mL/min at 30 ℃;¹H NMR（300MHz，CDCl₃）δ8.02（d，J=8.2Hz，1H），7.73（d，J=7.9Hz，1H），7.64（t，J=7.6Hz，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（75MHz，CDCl₃）δ146.97，139.11，133.95，129.58，128.10，128.09，126.78，124.38，84.28，76.42ppm；¹³C DEPT NMR（75MHz，CDCl₃）δ133.95（CH），129.58（CH），128.10（CH），128.09（CH），126.78（CH），124.38（CH），84.28（CH），76.42（CH₂）ppm。

2- (2-Nitrophenyl) -2, 3-dihydrofuran (3 b) was obtained as a yellow oil (79-90% HPLC purity, 44.0% ee): LCMS (C18 column eluted with 10-90% MeOH/water gradient from 3-5 min using formic acid modifier) M + 1: 192.05 (3.72 minutes); HPLC retention time of 4.8 min (10-90% CH over 8 min)₃CN/Water gradient elution YMCODS-AQ150x3.0mm column, using 0.1% TFA modifier and 1 mL/min flow rate); in thatAnalytical chiral HPLC retention time on a 4.6x250mm column eluting with 10% iPrOH in hexanes for 5.96 minutes (major enantiomer) and 6.35 minutes (minor enantiomer) using a flow rate of 1 mL/min at 30 ℃;¹H NMR（300MHz，CDCl₃）δ8.08（d，J=8.2Hz，1H），7.73（d，J=7.8Hz，1H），7.65（t，J=7.6Hz，1H），7.48–7.39（m，1H），6.50（q，J=2.4Hz，1H），6.10（dd，J=10.9，7.4Hz，1H），4.95（q，J=2.5Hz，1H），3.46–3.35（m，1H），2.50–2.39（m，1H）ppm；¹³C NMR（75MHz，CDCl₃）δ146.60，144.98，139.73，133.93，128.07，127.11，124.85，99.29，78.45，38.29ppm；¹³C DEPT NMR（75MHz，CDCl₃）δ144.98（CH），133.93（CH），128.07（CH），127.11（CH），124.85（CH），99.29（CH），78.45（CH），38.29（CH₂）ppm。

3a and 3b are subjected to a reduction step to provide 2-tetrahydrofuran-2-yl-aniline (4) as shown in example 1.b (below). Analysis of this material revealed that 3a and 3b were formed from the same major enantiomer, with an overall 70% ee. The absolute stereochemistry of the major enantiomer is not known as (R) or (S).

Example 1.b

Preparation of 2-tetrahydrofuran-2-yl-aniline (4)

5% Palladium on carbon (16.3g, 50% wet, 3.83mmol, Aldrich330116) was placed in a Parr bottle under nitrogen followed by MeOH (100 mL, JT-Baker 909333). 2- (2-Nitrophenyl) -2, 5-dihydrofuran and 2- (2-Nitrophenyl) -2, 3-dihydrofuran (3 a) dissolved in MeOH (389 mL)&3b) (163 g) of the crude mixture was added to a Parr bottle followed by NEt₃(237.6 mL, 1.705mol, Sigma-Aldrich 471283). Place the vial on a Parr shaker and use H₂And (4) saturation. Addition of 30psi H₂And the vial shaken until the starting material was completely consumed (LCMS and NMR showed complete reaction). The reaction mixture was purged with nitrogen and passed through Celite^TMFiltered and washed with EtOAc. The filtrate is filtered in a cycloneConcentrate on the evaporator to give 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 (about 15 torr) and the distillate was collected at 108 ℃ and 129 ℃ to give (4) (427.9 g, average yield 84%; 98% GCMS purity) as a clear pale yellow oil. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 163.95 (1.46 min).¹H NMR（300MHz，CDCl₃）δ7.15–7.04（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。

Example 1.c

Preparation of 4-bromo-2-tetrahydrofuran-2-yl-aniline (5)

To a stirred solution of 2-tetrahydrofuran-2-yl-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 ℃ was added 4 parts of N-bromosuccinimide (NBS, 58.88g, 99%, 327.5mmol, Aldrich B81255), maintaining the internal temperature below about 8 ℃. The reaction mixture was stirred while cooling with an ice water bath for 30 minutes (NMR of working aliquot showed complete consumption of starting material). Dissolving 1N Na in water₂S₂O₃(330 mL) was added to the reaction mixture, the cold water bath removed and stirred for 20 minutes. The mixture was diluted with EtOAc and the layers were separated. The organic phase was dissolved with saturated aqueous NaHCO₃(2X), water, brine wash, over MgSO₄Dry above, filter through a short plug of silica, elute with EtOAc, and concentrate under reduced pressure to give (5) (82.25 g, 77-94% HPLC purity) as a very dark amber oil. Proceed without further purification. LCMS (10-90% CH over 5 min)₃CN/Water gradient elution C18 column with formic acid modifier) M + 1: 242.10 (2.89 minutes).¹H NMR（300MHz，CDCl₃）δ7.22（d，J=2.3Hz，1H），7.16（dd，J=8.4，2.3Hz，1H），6.54（d，J=8.4Hz，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-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl) -2,2, 2-trifluoroacetamide (6)

To trifluoroacetic anhydride (455.3 mL, 3.275mol, Sigma-Aldrich 106232) stirred at 2 ℃ was slowly added 4-bromo-2-tetrahydrofuran-2-yl-aniline (5) (79.29 g, 327.5 mmol) as a viscous oil over 15 minutes (reaction temperature increased to 14 ℃) via an addition funnel. The remaining oil was washed into the reaction mixture with anhydrous 2-methyltetrahydrofuran (39.6 mL, Sigma-Aldrich 414247). The cold bath was removed and ammonium nitrate (34.08 g, 425.8mmol, Aldrich 467758) was added. The reaction temperature was raised to 40 ℃ over about 30 minutes, at which time a cold water bath was used to control the exothermicity and bring the reaction to room temperature. The cold bath was then removed and stirring continued for an additional 40 minutes (HPLC showed very little non-nitrated material remaining). The reaction mixture was slowly poured into a stirred mixture of crushed ice (800 g). The solid precipitate was collected by filtration and taken up with water, saturated aqueous NaHCO₃(to pH 8), water and hexane washes again. The wet solid was first dried in a convection oven at 50 ℃ for several hours and then under reduced pressure in an oven at 40 ℃ overnight to give (6) as a pale brown solid (77.86 g, 62% yield; 98% HPLC purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 383.19 (3.27 min).¹H NMR（300MHz，CDCl₃）δ9.81（s，1H），8.08（d，J=2.2Hz，1H），7.73（d，J=2.2Hz，1H），4.88（dd，J=9.0，6.5Hz，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-bromo-2-nitro-6-tetrahydrofuran-2-yl-aniline (6a)

N- (4-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl) -2,2, 2-trifluoroethylphthalamide (6) (54.0Og)140. gmol) was dissolved in 1, 4-dioxane (162mL) and water soluble 6M NaOH (70.45 mL, 422.7mmol, JT-Baker 567202) was added. The reaction mixture was stirred at reflux for 2 days (HPLC showed complete consumption). The mixture was allowed to cool, diluted with MTBE (800 mL), and dissolved in water (2 × 200 mL), saturated water, NH₄Cl, water and brine. The mixture was stirred over MgSO₄Dried, filtered and concentrated under reduced pressure to give (6a) (40.96 g, 93% yield; overall 92% HPLC plus NMR purity) as dark amber oil. LCMS (C18 column eluted with 10-90% MeOH/water gradient from 3-5 min using formic acid modifier) M + 1: 287.28 (3.44 min).¹H NMR（300MHz，CDCl₃）δ8.24（d，J=2.4Hz，1H），7.41（d，J=2.3Hz，1H），6.91（s，2H），4.80（t，J=7.2Hz，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 1.f

Preparation of 2- [5- (4-amino-3-nitro-5-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl ] propan-2-ol (8).

4-bromo-2-nitro-6-tetrahydrofuran-2-yl-aniline (6a) (40.409, 92%0, 129.5mmol), 1, 4-dioxane (26OmL, Sigma-Aldrich360481), 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 water-soluble 2.7M Na₂CO₃(143.9 mL, 388.5 mmol) were mixed. A nitrogen stream was bubbled through the stirred mixture for 1 hour, followed by the addition of tetrakis (triphenylphosphine) palladium (0) (7.48 g, 6.47mmol, Strem 462150). The reaction mixture was stirred at reflux for 2 hours (HPLC showed complete reaction), allowed to cool and diluted with EtOAc. The mixture is dissolved in water and saturated water₄Cl and brine, over MgSO₄Is dried and passed throughWas filtered through a short plug of EtOAc and eluted. The filtrate was concentrated under reduced pressure to give a dark brown oil. Dissolving oil in CH₂Cl₂In, and CH for short plug through silica gel₂Cl₂Followed by EtOAc elution. The desired fractions were concentrated on a rotary evaporator until a precipitate formed, giving a thick brown paste which was triturated with MTBE. The solid was collected by filtration, washed with MTBE, and dried under high vacuum to give (8) (35.14 g, 99+% HPLC purity) as a yellow solid. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 345.00 (2.69 min).¹H NMR（300MHz，CDCl₃) δ 8.88 (s, 2H), 8.36 (d, J =2.2Hz, 1H), 7.56 (d, J =2.1Hz, 1H), 7.09 (s, 2H), 4.92 (t, J =7.2Hz, 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 0-80% EtOAc/hexanes to give the product (8) as a second crop of amber solid (4.46 g, 88% overall yield; 88% HPLC purity).

Example 1.g

Preparation of 2- [5- (3, 4-diamino-5-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl ] propan-2-ol (9).

To 2- [5- (4-amino-3-nitro-5-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl under nitrogen]To the suspension of propan-2-ol (8) (30.10g, 87.41mmol) and THF (90mL) in a Parr bottle was added a slurry of 5% palladium on charcoal (3.01 g, 50% wet, 0.707mmol, Aldrich330116) in MeOH (90mL, JT-Baker 909333), followed by NEt₃(24.37 mL, 174.8mmol, Sigma-Aldrich 471283). Place the container on a Parr shaker and use H₂And (4) saturation. After addition of 45psi H₂After this time, the vessel was shaken until consumption was complete (HPLC showed complete conversion). The reaction mixture was purged with nitrogen and passed through Celite^TMFiltered and washed with EtOAc. The filtrate was refiltered through a 0.5 micron glass fiber filter paper sandwiched between two sheets of P5 paper and concentrated under reduced pressure to give (9) as a light brown foam (28.96 g, 98% yield; 93% NMR purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 315.32 (1.54 min).¹H NMR（300MHz，CDCl₃）δ8.83（s，2H），6.92（d，J=1.8Hz，1H），6.88（d，J=1.8Hz，1H），4.90（dd，J=7.9，6.2Hz，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

Preparation of 1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl ] urea (11).

To 2- [5- (3, 4-diamino-5-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl]To a stirred solution of propan-2 ol (9) (32.10 g, 102.1 mmol) in 1, 4-dioxane (160.5 mL, Sigma-Aldrich360481) was added ph3.5 buffer (240.8 mL) prepared by dissolving NaOAc trihydrate (34.5 g) in 1N water-soluble H₂SO₄Prepared (240 mL). 1-Ethyl-3- (N- (ethylcarbamoyl) -C-methylthio-carboimino) urea (10) (28.46 g, 122.5mmol, CB Research and Development) was added to the reaction mixture and stirred at reflux overnight (HPLC showed 99% consumption of the starting diamine). The reaction mixture was cooled to room temperature and poured (bubbled) with aqueous saturated NaHCO₃(480 mL) and water (120 mL) to give a pH of 8-9. This was stirred for 30 minutes, the solid was collected by filtration, washed well with water to neutral pH, and then washed more sparingly with EtOH. The solid was dried under reduced pressure to give (11) (34.48 g, 82% yield; 99.4% HPLC purity) as an off-white solid. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 411.41 (1.73 minutes).¹HNMR（300MHz，MeOD）δ9.02（s，2H），7.62（s，1H），7.37（s，1H），5.31（s，1H），4.23（dd，J=14.5，7.3Hz，1H），4.01（dd，J=15.0，7.1Hz，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=7.2Hz，3H）ppm。

Example 1.i

Chiral chromatographic separation of 1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea (12)

At 35 ℃ in CH₂C1₂MeOH/TEA (60/40/0.1) elutedResolution of 1-Ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl on a column (by Chiral Technologies)]-7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl]A racemic sample of urea (11) (24.60 g) gave the desired enantiomer (12) (11.35 g, 45% yield; 99+% HPLC purity, 99+% ee) as a white solid. Analytical chiral HPLC retention time is 6.2 min: (4.6X250mm column, 1 mL/min flow rate, 30 ℃).

The structure and absolute stereochemistry of 12 was confirmed by single crystal x-ray diffraction analysis. In a sealed tube equipped with a source of Cu K-alpha (Cu K alpha emission,) And single crystal diffraction data were obtained on a Bruker Apex II diffractometer with Apex II CCD detector. Crystals with dimensions of 1/2x0.05x0.05mm were selected, cleaned using mineral oil, fixed on MicroMount and concentrated on a bruker apexii system. Three batches of 40 frames separated in reciprocal space were obtained to provide the orientation matrix and initial cell parameters. The final cell parameters were obtained and refined based on the full dataset after data collection was complete. P2 for structural and centerless based system extinction and intensity statistics₁Refining in space group.

Diffraction data sets of reciprocal space were obtained using 0.5 ° steps for an exposure of 60 seconds per frame toThe resolution of (2). Data was collected at 100 (2) K. Integration of intensity and refinement of lattice parameters were done using APEXII software. Observation of the crystals after data collection showed no decomposition signsMillion. As shown in fig. 1, there are two symmetrically independent molecules in the structure and both symmetrically independent molecules are R isomers.

Data were collected, refined and simplified using Apex II software. Using SHELXS97 (Sheldrick, 1990); the program solved the structure and refined it using the SHELXL97 (Sheldrick, 1997) program. The crystal exhibits a P2₁A space group of monoclinic cells. The lattice parameter isβ=102.966（3）°。

Example 1.j

Preparation of the mesylate salt of 1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea (13).

Cooling of 1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] with an ice water bath]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]A stirred suspension of urea (12) (9.329, 22.71 mmol) in absolute ethanol (93.2 mL). Methanesulfonic acid (1.548 mL, 23.85mmol, Sigma-Aldrich 471356) was added, the cold bath removed and stirred at room temperature for 20 minutes. It was concentrated on a rotary evaporator at 35 ℃ to a thick slurry, diluted with EtOAc, the solid collected by filtration, washed with EtOAc, and dried under reduced pressure to give the initial harvest (13) (8.10 g) as a white solid.The filtrate was concentrated on a rotary evaporator to give a yellowish glassy foam, which was dissolved in EtOH, concentrated to a solid slurry and concentrated with EtOAc/Et₂Triturate and collect by filtration. The solid was washed with EtOAc/Et₂O washes, combined with the first harvest, and dried under reduced pressure gave (13) as a white solid (9.89 g, 86% yield; 99+% HPLC purity, 99+% ee). Analytical chiral HPLC is shown in4.6X250mm column on CH₂Cl₂One enantiomer, having a retention time of 6.3 minutes, was eluted with MeOH/TEA (60/40/0.1) using a flow rate of 1 mL/min at 30 ℃. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 411.53 (1.74 min).¹H NMR（300MHz，MeOD）δ9.07（s，2H），7.79（s，1H），7.62（s，1H），5.30（t，J=7.3Hz，1H），4.24（dd，J=14.6，7.3Hz，1H），4.04（dd，J=15.0，7.6Hz，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=7.2Hz，3H）ppm。

Example 1.k

1 pot deprotection/Suzuki procedure

Preparation of 2- [5- (4-amino-3-nitro-5-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl ] propan-2-ol (8).

N- (4-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl) -2,2, 2-trifluoroacetamide (6) (19.00g, 49.59mmol), 2- [5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrimidin-2-yl]Propan-2-ol (7) (14.41 g, 54.55 mmol), water-soluble 2.7M sodium carbonate (73.48 mL, 198.4 mmol) and 1, 4-dioxane (190 mL, Sig)ma-Aldrich 360481). A nitrogen stream was bubbled through the stirred mixture for 40 minutes, followed by addition of 1, 1' -bis (diphenylphosphino) ferrocene palladium dichloride dichloromethane adduct (2.025 g, 2.480mmol, Strem 460450). The reaction mixture was refluxed under N₂Stirring was continued for 7 hours, an additional 50mL of saturated water soluble sodium carbonate was added and heating at reflux for an additional 16 hours. The reaction mixture was allowed to cool, then diluted with EtOAc (500 mL) and water (200 mL). The layers were separated and the aqueous phase was extracted with EtOAc (200 mL). The combined organic phases were washed with water (500 mL), brine (500 mL) and washed with Na₂SO₄Is dried byThe plug was filtered and concentrated on a rotary evaporator to give the crude product (8) as an orange oil. Purification by ISCO silica gel chromatography eluting with 20-90% EtOAc/hexanes gave (8) (15.00 g, 81-88% purity) as an orange solid. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 345.35 (2.68 min).¹H NMR（300MHz，CDCl₃）δ8.88（s，2H），8.36（d，J=2.2Hz，1H），7.56（d，J=2.1Hz，1H），7.09（s，2H），4.92（t，J=7.2Hz，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 I: chiral chromatographic separation of 1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea

Eluted with DCM/MeOH/TEA (60/40/0.1) at 35 deg.CResolution of 1-Ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl on a column (by Chiral Technologies)]-7-tetrahydrofuran-2-yl-1H-Benzimidazol-2-yl]Racemic sample of urea (24.60 g). The desired fraction was collected, concentrated to dryness on a rotary evaporator, followed by drying in a vacuum oven at about 40 ℃ overnight to give the desired enantiomer as a white solid (11.35 g, 99+% HPLC purity, 99+% ee) which was used for physical form characterization. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 411.66 (1.74 min). HPLC retention time was 3.61 min (10-90% CH over 8 min)₃CN/Water gradient elution YMC ODS-AQ150x3.0mm column, using 0.1% TFA modifier and 1 mL/min flow rate). Analytical chiral HPLC showed one enantiomer with a retention time of 6.2 min: (4.6X250mm column, 1 mL/min flow rate, 30 ℃).¹H NMR（300MHz，MeOD）δ9.02（s，2H），7.62（d，J=1.6Hz，1H），7.37（s，1H），5.32（br.s，1H），4.23（dd，J=14.8，6.7Hz，1H），4.01（dd，J=15.1，7.0Hz，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=7.2Hz，3H）ppm。

Example 1.l

Preparation of form II

Add 20mg of benzimidazolyl urea compound to the HPLC vial and add 1mL of acetonitrile (CH)₃CN) was added to the vial with stirring at room temperature. To the resulting suspension was added a stoichiometric amount of 1 molar HCl solution in water (0.049 mL). The vial was pressed on the lid and the mixture (suspension) was allowed to equilibrate under gentle stirring for 6 days before it was filtered and the white solid was dried under vacuum for several hours and recovered for physical form characterization.

Example 1.m

Preparation of form III

To 20mg of the benzimidazolyl urea compound in the HPLC vial was added 0.5ml of THF at room temperature with stirring. A stoichiometric amount of HCl (0.049 mL) was added as a 1M aqueous solution. 2mL of methyl-t-butyl ether was added and the suspension was allowed to equilibrate overnight with stirring. It was then filtered and the white solid was dried under vacuum for several hours before physical form characterization of the mixture was performed.

Example 1.n

Preparation of form IV

2- [5- [3, 4-diamino-5- [ (2R) -tetrahydrofuran-2-yl]Phenyl radical]Pyrimidin-2-yl]Propan-2-ol (2 g, 6.362 mmol) and (3Z) -1-ethyl-3- [ (ethylcarbamoylamino) -methylthio-methylene]Urea (1.478 g, 6.362 mmol) in dioxane (26.00 mL) and buffer pH3.5 (100 mL, from 1N H)₂SO₄And NaOAc) was stirred at reflux (-95 ℃) for 3 hours. The reaction mixture was then quenched with about 50mL of water. The crude reaction mixture was transferred to a larger round bottom flask with NaHCO₃Neutralization followed by filtration gave a beige solid which was washed with hot water (-200 mL). The solid (1.84 g) was dried and salified to MeSO₃And (4) H salt. 2.25g (81% ee; 98% pure; 69.8% yield) of pure product were obtained, which was submitted for chiral separation by Supercritical Fluid Chromatography (SFC) to give peak one (S-enantiomer) and peak two (R-enantiomer).

The material (R-enantiomer) from SFC (Peak 2) was suspended in MeOH (. about.20 mL) and saturated NaHCO was used₃Basified (200 mL). The mixture was stirred for 1 hour and then filtered. After filtration, the solid was collected, washed with warm water (-500 mL) and dried. 1.22g of the parent molecule (free base; peak 2) was obtained, which was subsequently salified to the mesylate salt. 1.43g of pure methanesulfonate salt were obtained. (R; 99% ee; 99% pure).

The mesylate salt of the compound of the present application can be prepared using methods known to those skilled in the art. For example, the free base of the benzimidazolyl urea compound may be mixed with a stoichiometric amount of methanesulfonic acid, and the mixture is mixedConcentrate until a solid is obtained. Alternatively, the free base of the benzimidazolyl urea compound may be suspended in a suitable solvent containing the acid and the mixture allowed to equilibrate until the free base is converted to the corresponding acid addition salt. FIG. 12 shows the mesylate salt of a benzimidazolyl urea compound¹H NMR spectrum.

Example 2

Enzymology study

The enzyme inhibitory activity of the compounds of the present application can be determined in the experiments described below:

DNA gyrase ATPase assay

The ATP hydrolysis activity of S.aureus DNA gyrase was measured by coupling ADP production by pyruvate kinase/lactate dehydrogenase with oxidation of NADH. This method has been previously described (Tamura and Gellert, 1990, j.biol.chem., 265, 21342).

At 30 ℃ in the presence of 100mM TRIS pH7.6, 1.5mM MgCl₂ATPase assay was performed in 150mM KCl buffer. The conjugate system contained final concentrations of 2.5mM phosphoenolpyruvate, 200. mu.M Nicotinamide Adenine Dinucleotide (NADH), 1mM DTT, 30ug/ml pyruvate kinase, and 10ug/ml lactate dehydrogenase. The enzyme (90 nM final concentration) and DMSO solution of compound (3% final concentration) were added. The reaction mixture was allowed to incubate at 30 ℃ for 10 minutes. The reaction was initiated by adding ATP to a final concentration of 0.9mM and the rate of NADH disappearance was monitored at 340nm over the course of 10 minutes. Determination of K from a velocity vs. concentration profile_iAnd IC₅₀The value is obtained.

DNA Topo IV ATPase assay

The ATP to ADP conversion of the staphylococcus aureus TopoIV enzyme was coupled with the NADH to NAD + conversion, and the progress of the reaction was measured by the change in absorbance at 340 nm. TopoIV (64 nM) was incubated with the selected compound (final 3% DMSO) in buffer for 10 min at 30 ℃. The buffer solution consists of 100mM Tris7.5, 1.5mM MgCl2, 200mMK glutamate, 2.5mM phosphoenolpyruvate, 0.2mM NADH, 1mM DTT, 5. mu.g/mL linearized DNA, 50. mu.g/mL BSA, 30. mu.g/mL pyruvate kinase, and 10. mu.g/mL Lactate Dehydrogenase (LDH). The reaction was initiated with ATP and the rate was monitored continuously on a Molecular devices SpectraMAX plate reader for 20 minutes at 30 ℃. Determination of inhibition constants (Ki) and IC from rate vs. concentration plots of selected compounds₅₀The Morrison equation was fitted for tight binding inhibitors.

Example 3

Sensitivity testing in liquid media

The compounds of the invention were tested for antimicrobial activity by sensitivity testing in liquid media. Such assays may be performed within the guidelines of the latest CLSI documents that govern such practices: "M07-A8Methods for Dilution of Antimicrobial scientific substrates for bacteriosis that Grow Aerobically; approval standard-eighth edition (2009) ". Other publications such as "Antibiotics in Laboratory Medicine" (edited by v. lorian, publishers Williams and Wilkins, 1996) provide basic practical techniques in Laboratory antibiotic testing. The specific protocol used was as follows:

scheme # 1: determination of gyrase MIC of Compounds Using the Trace dilution Broth method

Materials:

round bottom 96-well microtiter plate (Costar 3788)

Mueller Hinton II agar plates (MHII; BBL premix)

Mueller Hinton II liquid broth (MHII; BBL premix)

BBL Prompt Inoculation System（Fisher B26306）

Test reading mirror (Fisher)

Agar plates of bacteria streaked to single colonies, freshly prepared

Sterile DMSO

Human serum (U.S.biologicals S1010-51)

Cracking horse blood (Quad Five 270-100)

Resazurin 0.01%

Sprague Dawley rat serum (U.S. biologicals1011-90B or Valley biomedical AS3061 SD)

Pooled mouse serum (Valley Biomedical AS 3054)

Strain (medium, broth and agar):

1. staphylococcus aureus ATCC #29213

a.MHII

MHII +50% human serum

MHII +50% rat serum

MHII +50% mouse serum

2. Staphylococcus aureus ATCC #29213GyrB T173I (MHII)

3. Staphylococcus aureus, JMI collection strain; see Table 5 (MHII)

4. Staphylococcus epidermidis, JMI collection strain; see Table 5 (MHII)

5. Enterococcus faecalis ATCC #29212 (MHII +3% lysed horse blood)

6. Enterococcus faecium ATCC #49624 (MHII +3% lysed horse blood)

7. Enterococcus faecalis, JMI collection strain; see Table 5 (MHII +3% lysed horse blood)

8. Enterococcus faecium, JMI collected strain; see Table 5 (MHII +3% lysed horse blood)

9. Streptococcus pneumoniae ATCC #10015 (MHII +3% lysed horse blood)

10. Streptococcus pneumoniae, JMI collection strain; see Table 5 (MHII +3% lysed horse blood)

11. Beta-hemolytic streptococci, group A, B, C, G) JMI collection strain; see Table 5 (MHII +3% lysed horse blood)

12. Bacillus cereus ATCC10987 (MHII)

13. Bacillus cereus ATCC14579 (MHII)

14. Bacillus subtilis ATCC6638 (MHII)

15. Bacillus subtilis (168) ATCC6051 (MHII)

Inoculum preparation (for all strains except staphylococcus aureus +50% serum):

1. using the BBL Prompt kit, 5 large or 10 small well-separated colonies were picked from cultures grown on the appropriate agar medium as indicated above and inoculated with 1mL of sterile saline provided in the kit.

2. Vortex the hole for-30 seconds to provide-10⁸cells/mL of suspension. The actual density can be confirmed by plating out a dilution of this suspension.

3. For each plate of compound tested, 0.15mL of cells were transferred to 15mL (. about.10)⁶cells/mL) sterile broth (or see below), the suspension 1/100 was diluted and then vortex mixed. If more than 1 plate of compound is to be tested (>8 compounds) including compound 12 or 13, which can be prepared by following examples 1.i and 1.j (above), then the volume can be increased accordingly.

a. For enterococcus faecalis, enterococcus faecium and streptococcus pneumoniae: horse blood was lysed using 14.1mL MHII +0.9 mL.

4. 50 μ l cells (. about.5X 10) were used⁴Cells) to inoculate each microtiter containing 50 μ l of drug diluted in brothWells (see below).

Drug dilution, inoculation, MIC determination:

1. all drug/compound stocks were prepared at a concentration of 12.8mg/mL, typically in 100% DMSO.

2. Drug/compound stock was diluted to 200x desired final concentration in 50 μ L DMSO. If the MICs starting concentration is 8. mu.g/mL final concentration, then 6.25. mu.L of stock solution + 43.75. mu.L of DMSO is required. Each 200x stock was placed in a separate row of column 1 of a new 96 well microtiter plate.

3. 25 μ L of DMSO was added to all rows of microtiter plates containing 200 Xstock of compound in columns 2-12 and serial dilutions of 25 μ L were made from column 1 through column 11, with the tip replaced after each column. I.e., 25 μ L compound +25 μ L DMSO =2x dilution. A "no compound" DMSO well was left at the end of the series for control.

4. For each strain tested (except staphylococcus aureus +50% human serum), two microtiter plates were prepared with 50 μ L MHII broth using Matrix pipettes.

5. 0.5. mu.L of each dilution (w/Matrix automated pipettor) was transferred to 50. mu.L of medium/microtiter wells before 50. mu.L of cells were added. After dilution 1/200 into media + cells, the typical starting concentration of compound was 8 μ g/mL-the compound concentration decreased in 2X steps across the rows of the microtiter plate. All MICs were done in duplicate.

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

7. After addition of the inoculum, each well was mixed thoroughly with a manual multi-channel pipettor; the same tip was used from low to high drug concentration on the same microtiter plate.

8. The plates were incubated at 37 ℃ for at least 18 hours.

9. Plates were viewed with a test reading scope after 18 hours and when no growth was observed (optical clarity in wells), MIC was recorded as the lowest concentration of drug.

Preparation of staphylococcus aureus +50% human serum, staphylococcus aureus +50% rat serum or staphylococcus aureus +50% mouse serum.

1. 50% serum medium was prepared by combining 15mL MHII +15mL human serum-30 mL total. When more than 1 compound plate was tested, the volume was increased in 30mL increments.

2. Using the same BBLPrompt inoculum of Staphylococcus aureus ATCC #29213 as described above, 0.15mL of cells were transferred to 30mL (. about.5X 10)⁵cells/mL) 1/200 in 50% human serum medium prepared above, and vortex mixed.

3. All test wells of the desired number of microtiter plates were loaded with 100. mu.L of cells in 50% serum medium.

4. 0.5. mu.L of each compound dilution (w/Matrix robotic pipettor) was transferred to 100. mu.L of cells/medium. After dilution 1/200 into media + cells, the typical starting concentration of compound was 8 μ g/mL-compound concentration decreased in 2x steps across the rows of the microtiter plate. All MICs were done in duplicate.

5. Thoroughly mixing each well with a manual multi-channel pipettor; the same tip was used from low to high drug concentration on the same microtiter plate.

6. The plates were incubated at 37 ℃ for at least 18 hours. After incubation, 25 μ L of 0.01% resazurin was added to each well and incubation was continued at 37 ℃ for at least another 1 hour or until the resazurin was discolored.

7. The plates were viewed with a test reading mirror and the MIC recorded. When resazurin is used, the color of the dye changes from dark blue to light pink in the wells without growth. The lowest concentration of drug that turns the dye to pink is the MIC.

Scheme # 2: gram-negative gyrase MIC assay using microdilution broth method

Materials:

round bottom 96-well microtiter plate (Costar 3788)

Mueller Hinton II agar plates (MHII; BBL premix)

Mueller Hinton II liquid broth (MHII; BBL premix)

BBL Prompt Inoculation System（Fisher b26306）

Test reading mirror (Fisher)

Agar plates of bacteria streaked to single colonies, freshly prepared

Sterile DMSO

Strain (MHII medium for all; broth and agar):

1. escherichia coli ATCC #25922

2. Coli, JMI Collection Strain, see Table 5

3. Escherichia coli AG100WT

4. Escherichia coli AG100tolC

5. Acinetobacter baumannii (Acinetobacter baumannii) ATCC # BAA-1710

6. Acinetobacter baumannii ATCC #19606

7. Acinetobacter baumannii, JMI Collection Strain, see Table 5

8. Klebsiella pneumoniae (Klebsiella pneumoniae) ATCC # BAA-1705

9. Klebsiella pneumoniae ATCC #700603

10. Klebsiella pneumoniae, JMI Collection Strain, see Table 5

11. Moraxella catarrhalis ATCC #25238

12. Moraxella catarrhalis ATCC #49143

13. Moraxella catarrhalis, JMI Collection Strain, see Table 5

14. Haemophilus influenzae ATCC49247

15. Haemophilus influenzae (Rd 1KW 20) ATCC51907

16. Haemophilus influenzae Rd0894 (AcrA-)

17. Haemophilus influenzae, JMI Collection Strain, see Table 5

18. Pseudomonas aeruginosa PAO1

19. Pseudomonas aeruginosa, JMI Collection Strain, see Table 5

20. Proteus mirabilis (Proteus mirabilis), JMI Collection Strain, see Table 5

21. Enterobacter cloacae (Enterobacter cloacae), JMI Collection Strain, see Table 5

22. Stenotrophomonas maltophilia ATCCBA-84

23. Stenotrophomonas maltophilia ATCC13637

Preparation of inoculum:

1. using the BBL Prompt kit, 5 large or 10 small well-separated colonies were picked from cultures grown on agar medium and inoculated into 1mL of sterile saline with the kit.

2. Vortex the hole for-30 seconds to give-10⁸cells/mL of suspension. The actual density can be confirmed by plating out a dilution of this suspension.

3. For each plate of compound tested, 0.15mL of cells were transferred to 15mL (. about.10)⁶cells/mL) sterile broth (see below), the suspension 1/100 was diluted and vortex mixed. If more than 1 plate of compound is tested (>8 compounds), including compounds 12 or 13, are increased accordinglyAdding volume.

4. 50 μ l cells (. about.5X 10) were used⁴Cells) to seed each microtiter well containing 50 μ l of drug diluted in broth (see below).

Drug dilution, inoculation, MIC determination:

1. all drug/compound stocks were prepared at a concentration of 12.8mg/mL, typically in 100% DMSO.

2. Drug/compound stock was diluted to 200x desired final concentration in 50 μ L DMSO. If the MICs starting concentration is 8. mu.g/mL final concentration, then 6.25. mu.L of stock solution + 43.75. mu.L of DMSO is required. Each 200x stock was placed in a separate row of column 1 of a new 96 well microtiter plate.

3. 25 μ L of DMSO was added to all rows of microtiter plates containing 200 Xstock of compound in columns 2-12 and serial dilutions of 25 μ L were made from column 1 through column 11, with the tip replaced after each column. I.e., 25 μ L compound +25 μ L DMSO =2x dilution. A "no compound" DMSO well was left at the end of the series for control.

4. For each strain tested, two microtiter plates were prepared with 50 μ L MHII broth using Matrix pipettes.

5. 0.5. mu.L of each dilution (w/Matrix automated pipettor) was transferred to 50. mu.L of medium/microtiter wells before 50. mu.L of cells were added. After dilution 1/200 into media + cells, the typical starting concentration of compound was 8 μ g/mL-the compound concentration decreased in 2X steps across the rows of the microtiter plate. All MICs were done in duplicate.

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

7. After addition of the inoculum, each well was mixed thoroughly with a manual multi-channel pipettor; the same tips were used from low to high drug concentration in the same microtiter plate.

8. The plates were incubated at 37 ℃ for at least 18 hours.

9. The plates were viewed with a test reading mirror after 18 hours and when no growth was observed (optical clarity in the wells), the MIC was recorded as the lowest concentration of drug.

Scheme # 3: gyrase MIC assay of compounds using agar dilution method

Materials:

petri dish 60x15mm (Thermo Scientific catalog # 12567100)

Centrifuge tube, 15mL (Costar)

BBL Prompt Inoculation System（Fisher b26306）

Agar plates of bacteria streaked to single colonies, freshly prepared

Sterile DMSO

GasPak^TMIncubation container (BD catalog # 260672)

GasPak^TMEZ Anaerobe Container System pouch (BD Cat # 260678)

GasPak^TMEZ C02 Container System pouch (BD Cat # 260679)

GasPak^TMEZ Campy Container System pouch (BD Cat # 260680)

The strain is as follows:

1. clostridium difficile ATCC BAA-1382;

2. clostridium difficile, CMI Collection Strain, see Table 4

3. Clostridium perfringens (Clostridium perfringens), CMI Collection Strain, see Table 4

4. Bacteroides fragilis (Bacteroides fragilis) and Bacteroides species (Bacteroides spp.), CMI Collection of strains, see Table 4

5. Clostridium species (Fusobacterium spp.), CMI Collection Strain, see Table 4

6. Streptococcus species (spp), CMI Collection strains, see Table 4

7. Prevotella species (Prevotella spp.), CMI Collection strains, see Table 4

8. Neisseria gonorrhoeae ATCC35541

9. Neisseria gonorrhoeae ATCC49226

10. Neisseria gonorrhoeae, JMI Collection Strain, see Table 4

11. Neisseria meningitidis, JMI Collection Strain, see Table 4

Media preparation and growth conditions:

according to CLSI publication "M11-A7 Methods for antimicrobial surgery Testing of antimicrobial Bacteria; applied Standard-derived Edition (2007) "preparation of growth media recommended for each microorganism species, the media of which is in accordance with" M07-A8Methods for Dilution of Antimicrobial biological assays for bacterial Grow Aerobically, in addition to Neisseria gonorrhoeae and Neisseria meningitidis; an Approved Standard- -height Edition (2009) "was prepared.

And (3) flattening:

1.a 100x stock of drug was prepared for each test compound as described in table 1A. Using a 15mL centrifuge tube, 100uL of each drug stock was added to 10mL of agar melt (cooled to 55 ℃ in a water bath). The tubes were mixed 2-3X upside down and then poured into individually labeled 60X15mm petri dishes.

2. The conventional test concentrations were: 0.002ug/mL to 16ug/mL (14 plates).

3. Pour 4 drug-free plates: 2 pieces served as positive controls and 2 pieces as aerobic controls.

4. The plate was allowed to dry. Used on the same day or stored overnight at RT or at 4 ℃ for up to 3 days.

5. Plates were labeled accordingly for drug concentration and strain placement.

Growth of cells requiring maintenance of an anaerobic environment:

1. all work performed with anaerobes is done as quickly as possible; the work performed in the biosafety cabinet (i.e., anaerobic environment) is completed in less than 30 minutes before the cells are returned to the anaerobic culture chamber.

2. Using GasPak^TMThe culture chamber realizes the incubation of anaerobic bacteria. The large box culture chamber (VWR 90003-636) requires 2 anaerobic sachets (VWR 90003-642), while the high cylinder culture chamber (VWR 90003-602) requires only 1 sachet.

Plating (performed in biosafety cabinet):

1. each strain was streaked onto agar plates as described above. Incubation for the required time and under ambient conditions (i.e. anaerobic, microaerophilic, etc.).

2. The colony suspension method was used directly to suspend the cell rings of fresh streaked culture to-4 ml0.9% NaCl₂Inner and swirl.

3. The suspension was adjusted to o.d.₆₀₀0.05 (5 × 10e7 cfu/mL). Vortex and mix.

4. Transfer-0.2 mL of the adjusted mixed culture to a 96-well plate. When < 5 strains were tested, all strains were ranked together in a single row. When >5 strains were tested, the strains were transferred into plates with no more than 5 strains in a single row. This is necessary to fit the small plate.

5. 0.002mL of each strain from the prepared 96-well plate was spotted on each MIC test plate using a multi-channel pipette. This results in 1 × 10e5 cfu/point. When tested for clostridium difficile, the strains migrated in colonies while growing, however, the distance between the multi-channel pipette spots was far enough that the migrated cells did not compromise the assay results.

a. The 2 drug-free plates were inoculated first, while the other 2 drug-free plates were inoculated last after the MIC test plate. The former and the latter served as growth and inoculation controls. One plate in each set of no-drug control inoculations was incubated with the MIC plate under the required atmospheric conditions and set up in the presence of oxygen to test for aerobic contamination. Aerobic cultures are negative for growth when operated with strictly anaerobic or microaerophilic strains. Some growth was seen with neisseria gonorrhoeae.

6. The inoculum was allowed to dry (as short a time as possible) and then placed upside down for incubation in GasPak with the appropriate number of sachets.

7. Neisseria species at 37 ℃ 5% CO₂Incubate for 24 hours in ambient.

MIC determination:

the tested plates were examined after the correct incubation time and the MIC end-point was read at a concentration where growth on the test plate appeared significantly reduced in appearance compared to growth on the positive control plate.

Table 1A: compound dilution for MIC assay using agar dilution method

1,600ug/ml =64ul (10 mg/ml stock) +336ul DMSO; 400ul total volume to start

Compounds were dissolved and diluted in 100% DMSO

Scheme #4. MIC determination procedure for Mycobacterium species

Material

Round-bottom 96-well microtiter plates (Costar 3788) or the like

Thin film plate seal (PerkinElmer, TopSeal-A #6005250 or the like)

Middlebrook7H10 broth with 0.2% glycerol

Middlebrook7H10 agar with 0.2% glycerol

Middlebrook OADC Enrichment

Inoculum preparation for mycobacterium tuberculosis:

1. the used prepared frozen Mycobacterium tuberculosis stock was stored at-70 ℃. Mycobacterium tuberculosis grown in 7H10 broth +10% OADC, followed by 100Klett or 5X10⁷The concentration of cfu/ml is frozen,

2. by taking 1ml of frozen stock and adding it to 19ml of 7H10 broth +10% OADC (final concentration 2.5X 10)⁶cfu/ml), a 1:20 dilution was prepared.

3. From this dilution a second 1:20 dilution was prepared, 1ml was taken and 19ml of fresh broth was added. This was the final inoculum added to the 96-well plate.

Inoculum preparation for mycobacterium kansasii, mycobacterium avium, mycobacterium abscessus and nocardia species:

1. used prepared frozen stock culture or cultured with 10Klett or 5X10⁷Fresh cultures grown at a concentration of 7H10 broth/ml.

2. By taking 1.0ml of the culture stock and adding it to 19ml of 7H10 broth (final concentration 2.5X 10)⁶cfu/ml), a 1:20 dilution was prepared.

3. From this dilution a 1:20 dilution was prepared, 1ml was taken and 19ml of fresh broth (final suspension) was added.

Plate preparation:

1. the plate is labeled.

2. Using a multi-channel electric pipette, 50 μ Ι of 7H10 broth +10% OADC was added to all wells used for MIC determination.

3. A stock solution (e.g., 1mg/ml concentration) of the drug to be tested is prepared.

4. The frozen stock solution was thawed and diluted with 7H10 broth +10% OADC to obtain the maximum concentration for the working solution 4x test (e.g., a final concentration of 32 μ g/ml, with the highest concentration tested being 8 μ g/ml). Dilutions were prepared from stock solutions. To start at a concentration of 1. mu.g/ml, the drug was prepared at 4. mu.g/ml, so the starting concentration was 1. mu.g/ml. Mu.l of the stock solution 1mg/ml was removed and 6.2ml of broth was added. All dilutions of the drug were done in broth.

5. Add 50. mu.l of 4 Xworking solution to the first well of the indicated row. Continue for all compounds tested. Using a multi-channel electric pipettor, mix 4X and serial dilution compounds until the 11 th well. The remaining 50. mu.l were discarded. The 12 th well was used as a positive control.

6. Incubating the plate at 37 ℃ for 18 days with Mycobacterium tuberculosis; m. avium and M.kansasii for-7 days; nocardia and Mycobacterium abscessus for-4 days; a film seal is used.

7. The readings were visually observed and the results recorded. MIC was recorded as the lowest concentration of drug where no growth was observed (optical clarity in wells).

Scheme #5 protocol for M.tuberculosis seroconversion MIC assay

Materials and reagents:

costar #3904 Black-sided, flat-bottomed 96-well microtiter plate

Middlebrook7H9 broth with 0.2% glycerol (BD 271310)

Middlebrook OADC Enrichment

Fetal bovine serum

Catalase (Sigma C1345)

Dextrose

NaCl₂

BBL Prompt Inoculation System（Fisher b26306）

Bactero agar plates with streaking to a single colony (Middlebrook 7H11 with 0.2% glycerol and OADC enrichment)

Sterile DMSO

Preparing a culture medium:

1. for seroconverted MICs, three different media were required, all with a basis of 7H9+0.2% glycerol. It is important that all media and supplements be sterilized prior to MICs.

2. All media below were prepared and inoculated as described in the next section. All compounds were tested against Mtb using each medium.

a.7H9+0.2% Glycerol +10% OADC ("Standard" MIC Medium)

b.7H9+0.2% Glycerol +2g/L dextrose +0.85g/L NaCl +0.003g/L Catalase (0% FBS).

c. 2X7H9+0.2% glycerol +2g/L dextrose +0.85g/L NaCl +0.003g/L catalase in combination with an equal volume of fetal bovine serum (50% FBS).

Preparation of inoculum:

1. using BBL Prompt, 5-10 well-isolated colonies were picked and inoculated in 1ml of sterile saline from the kit. Typically, plates have two to three weeks of age when used in such assays due to slow growth of such organisms in culture.

2. Vortex well and then sonicate in a water bath for 30 seconds, providing-10⁸Cell/ml suspension. The actual density can be confirmed by plating out a dilution of this suspension.

3. BBL Prompt suspension was diluted by 1/200 (e.g., 0.2ml cells were transferred to 40ml medium) Inoculum was prepared in each of the three medium formulations to obtain-10⁶Initial cell density of cells/ml.

4. 100 μ l of cells (. about.5X 10) were used⁴Cells) to inoculate each microtiter well containing 1 μ Ι of drug in DMSO (see below).

Drug dilution, inoculation, MIC determination:

1. control drug stock solutions isoniazid and novobiocin were prepared at 10mM in 100% DMSO, while ciprofloxacin and rifampin were prepared at 1mM in 50% DMSO and 100% DMSO, respectively. Prepared dilutions-100 μ Ι _ stock was dispensed into the first column of a 96 well plate. An 11-step, 2-fold serial dilution was prepared across rows for each compound by transferring 50 μ Ι from column 1 into 50 μ Ι DMSO in column 2. Transfer of 50 μ l from column 2 to column 11 was continued while mixing and changing the tips at each column. Column 12 with DMSO alone was left as a control.

2.1 μ l of each dilution was transferred to an empty microtiter well before adding 100 μ l of cells. The initial concentration of isoniazid and novobiocin after dilution into medium + cells was 100 μ M; after dilution into medium + cells, the initial concentration of ciprofloxacin and rifampicin was 10 μ M. Compound concentration decreased in 2x steps moving across the rows of the microtiter plate. All MICs were done in duplicate under each of the three media conditions.

3. Test groups of compounds are typically at 10mM and 50 μ L volumes.

4. Using a multi-channel pipette, all volumes from each column of the master plate are removed and transferred into the first column of a new 96-well microtiter plate. This was repeated for each column of compounds on the master plate and transferred to column 1 of a new 96 well plate.

5. For control compounds, as described above, DMSO was used as a diluent to generate a 2-fold, 11-point dilution of each compound. In all cases, column 12 with DMSO alone was left for control. Once all dilutions were completed, as was done for the control compound, 1 μ Ι of each dilution was transferred into an empty microtiter well before 100 μ Ι of cells were added.

6. All wells were inoculated with 100. mu.l of diluted cell suspension (see above).

7. After addition of inoculum, the plates were mixed by gently tapping the sides of the plates.

8. The plates were incubated in a humidified 37 ℃ culture chamber for 9 days.

9. At day 9, 25 μ l of 0.01% sterile resazurin was added to each well. Background fluorescence was measured at excitation 492nm, emission 595nm, and the plates were returned to the incubator for another 24 hours.

After 24 hours, the fluorescence of each well was measured at excitation 492nm, emission 595 nm.

The percent inhibition for a given compound was calculated as follows: percent inhibition =100- ([ pore fluorescence-mean background fluorescence ]/[ DMSO control-mean background fluorescence ] x 100). MICs scores were performed for all three media conditions, which were the lowest compound concentrations that inhibited Resazurin reduction ("% -inhibition") signal ≧ 70% under the given media conditions.

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

In table 2A and subsequent tables and examples, "compound 13" relates to the mesylate salt of compound 12. Compounds 12 and 13 can be prepared by following examples 1.i and 1.j (above), respectively. These are the same numbers used in the examples above to identify the compounds and salts.

TABLE 2A MIC values for selected Compounds

Strains/specific conditions	Scheme(s)	Compound 13
			Staphylococcus aureus ATCC29213	1	0.13
Staphylococcus aureus ATCC29213 human serum	1	0.31
			Staphylococcus aureus ATCC29213 rat serum was used	1	0.53
Staphylococcus aureus ATCC29213 mouse serum was used	1	2
			Staphylococcus aureus ATCC29213GyrB T173I	1	1.29
Enterococcus faecalis ATCC29212, obtained by cracking horse blood	1	0.081
			Enterococcus faecium ATCC49624 lysed horse blood was used	1	0.39
Enterococcus faecium ATCC49624	1	0.25
			Streptococcus pneumoniae ATCC10015, using lysed horse blood	1	0.022
Bacillus cereus ATCC10987	1	0.5
			Bacillus cereus ATCC14579	1	0.5
Bacillus subtilis ATCC6638	1	>8
			Bacillus subtilis (168) ATCC6051	1	>8
Clostridium difficile ATCC BAA-1382	3	1
			Haemophilus influenzae ATCC49247	2	1
Haemophilus influenzae (Rd 1KW 20) ATCC51907	2	2.5
			Haemophilus influenzae Rd0894 (AcrA-)	2	0.14
Moraxella catarrhalis ATCC25238	2	0.071

Strains/specific conditions	Scheme(s)	Compound 13
			Moraxella catarrhalis ATCC49143	2	0.04
Neisseria gonorrhoeae ATCC35541	3	1.3
			Neisseria gonorrhoeae ATCC49226	3	2.3
Escherichia coli AG100WT	2	>16
			Escherichia coli AG100tolC	2	0.11
Escherichia coli ATCC25922	2	16
			Escherichia coli CHE30	2	>16
Escherichia coli CHE30tolC	2	0.5
			Escherichia coli MC4100	2	>16
Escherichia coli MC4100tolC	2	1
			Klebsiella pneumoniae ATCC700603	2	>16
Klebsiella pneumoniae ATCC BAA-1705	2	>16
			Acinetobacter baumannii ATCC19606	2	>16
Bob shi buAcinetobacter ATCC BAA-1710	2	>16
			Pseudomonas aeruginosa PAO1	2	>16
Pseudomonas aeruginosa PAO750	2	0.33
			Stenotrophomonas maltophilia ATCC BAA-84 stenotrophomonas maltophilia ATCC13637 Mycobacterium avium 103 Mycobacterium avium Far	2244	Incomplete 0.470.94
Mycobacterium avium 3404.4	4	0.94
			Nocardia caviae (Nocardia caviae) 2497	4	2
Nocardia asteroides (N. asteroids) 2039	4	8
			Nocardia neoformans (N.nova) 10	4	8
M. kansasii 303	4	Unfinished
			M. kansasii 316	4	Unfinished
Mycobacterium kansasii 379	4	Unfinished
			Mycobacterium tuberculosis H37Rv ATCC25618	4	0.37
Mycobacterium tuberculosis Erdman ATCC35801	4	0.25
			Mycobacterium tuberculosis Erdman ATCC35801	5	0.045
Mycobacterium tuberculosis Erdman ATCC35801 mouse serum	5	2
			Mycobacterium abscessus BB2	4	Unfinished
Mycobacterium abscessus MC6005	4	Unfinished
			M. abscessus MC5931	4	Unfinished
Mycobacterium abscessus MC5605	4	Unfinished
			Mycobacterium abscessus MC6025	4	Unfinished

Strains/specific conditions	Scheme(s)	Compound 13
			Mycobacterium abscessus MC5908	4	Unfinished
Mycobacterium abscessus BB3	4	Unfinished
			Mycobacterium abscessus BB4	4	Unfinished
Mycobacterium abscessus BB5	4	Unfinished
			M. abscessus MC5922	4	Unfinished
Mycobacterium abscessus MC5960	4	Unfinished
			Mycobacterium abscessus BB1	4	Unfinished
Mycobacterium abscessus MC5812	4	Unfinished
			M. abscessus MC5901	4	Unfinished
Mycobacterium abscessus BB6	4	Unfinished
			Mycobacterium abscessus BB8	4	Unfinished
Mycobacterium abscessus MC5908	4	Unfinished
			Mycobacterium abscessus LT949	4	Unfinished
Mycobacterium abscessus BB10	4	Unfinished
			Mycobacterium abscessus MC6142	4	Unfinished
Mycobacterium abscessus MC6136	4	Unfinished
			Mycobacterium abscessus MC6111	4	Unfinished
Mycobacterium abscessus MC6153	4	Unfinished

Table 3A shows the results of the MIC90 assay for selected compounds of the invention.

TABLE 3A MIC90 values for a panel of selected compounds for gram-positive, gram-negative and anaerobic pathogens

In Table 4 below, the term "CMI" represents the clinical microbiology Institute, located in Wilsonville, Oregon.

Table 4: panel of anaerobic organisms for generating MIC90 data

CMI#	Biological body
		A2380	Bacteroides fragilis
A2381	Bacteroides fragilis
		A2382	Bacteroides fragilis
A2486	Bacteroides fragilis
		A2487	Bacteroides fragilis
A2489	Bacteroides fragilis
		A2527	Bacteroides fragilis
A2529	Bacteroides fragilis
		A2562	Bacteroides fragilis
A2627	Bacteroides fragilis
		A2802	Bacteroides fragilis
A2803	Bacteroides fragilis
		A2804	Bacteroides fragilis

CMI#	Biological body
		A2805	Bacteroides fragilis
A2806	Bacteroides fragilis
		A2807	Bacteroides fragilis
A2808	Bacteroides fragilis
		A2809	Bacteroides fragilis
A2810	Bacteroides fragilis
		A2811	Bacteroides fragilis
A2812	Bacteroides fragilis
		A2813	Bacteroides fragilis
A2814	Bacteroides fragilis
		A2460	Bacteroides thetaiotaomicron (B. Thetaioomicron)
A2462	Bacteroides thetaiotaomicron
		A2463	Bacteroides thetaiotaomicron
A2464	Bacteroides thetaiotaomicron
		A2536	Bacteroides thetaiotaomicron
A2591	Bacteroides simplex (B.uniformis)
		A2604	Bacteroides vulgare (B.vulgatus)
A2606	Bacteroides vulgaris
		A2613	Oval bacterium (B.ovatus)
A2616	Leptobacterium ovatus
		A2815	Bacteroides camouflagus (Bacteroides tectom)
A2816	Ureolyticus (B. ureolyticus)
		A2817	Flavobacterium sp. (Bacteroides capitosus)
A2818	Ureaplasma urealyticum
		A2824	Parabacter distasonis
A2825	Leptobacterium ovatus
		A2826	Bacteroides simplex
A2827	Bacteroides simplex
		A2828	Bacteroides vulgaris
A2829	Bacteroides vulgaris
		A2830	Leptobacterium ovatus
A2831	Bacteroides thetaiotaomicron
		A2832	Parabacter distasonis
A2833	Bacteroides thetaiotaomicron
		A2767	Clostridium difficile
A2768	Clostridium difficile
		A2769	Clostridium difficile

CMI#	Biological body
		A2770	Clostridium difficile
A2771	Clostridium difficile
		A2772	Clostridium difficile
A2773	Clostridium difficile
		A2774	Clostridium difficile
A2775	Clostridium difficile
		A2776	Clostridium difficile
A2777	Clostridium difficile
		A2778	Clostridium difficile
A2779	Clostridium difficile
		A2780	Clostridium difficile
A2140	Clostridium perfringens
		A2203	Clostridium perfringens
A2204	Clostridium perfringens
		A2227	Clostridium perfringens
A2228	Clostridium perfringens
		A2229	Clostridium perfringens
A2315	Clostridium perfringens
		A2332	Clostridium perfringens
A2333	Clostridium perfringens
		A2334	Clostridium perfringens
A2389	Clostridium perfringens
		A2390	Clostridium perfringens
A864	Necrotic shuttle rodFungus (F. necrophorum)
		A871	Fusobacterium nucleatum (F.nucleolus)
A1667	Fusobacterium necrophorum
		A1666	Fusobacterium necrophorum
A2249	Fusobacterium nucleatum
		A2716	Fusobacterium species
A2717	Fusobacterium species
		A2719	Fusobacterium species
A2721	Fusobacterium species
		A2722	Fusobacterium species
A2710	Fusobacterium species
		A2711	Fusobacterium species
A2712	Shuttle rodGenus species of the genus
		A2713	Fusobacterium species

CMI#	Biological body
		A2714	Fusobacterium species
A2715	Fusobacterium species
		A1594	Anaerobic digestion of Streptococcus (Peptostreptococcus anaerobius)
A2158	Streptococcus magnus (Peptostreptococcus magnus)
		A2168	Anaerobic digestion of streptococci
A2170	Streptococcus macrodigestive system
		A2171	Streptococcus macrodigestive system
A2575	Digestion of Streptococcus species
		A2579	Streptococcus undenatured (Peptostreptococcus asaccharolyticus)
A2580	Streptococcus undenatured
		A2614	Streptococcus undenatured
A2620	Streptococcus undenatured
		A2629	Digestion of Streptococcus species
A2739	Prevotella denticola (Prevotella denticola)
		A2752	Two-way Prevotella (Prevotella bivia)
A2753	Prevotella intermedia (Prevotella intermedia)
		A2754	Prevotella intermedia
A2756	Prevotella bifidus
		A2759	Prevotella bifidus
A2760	Prevotella denticola
		A2761	Prevotella intermedia
A2762	Prevotella nigricans (Prevotella melaninogenica)
		A2765	Prevotella nigricans producing bacterium
A2766	Prevotella nigricans producing bacterium
		A2821	Prevotella bifidus
A2822	Prevotella bifidus
		QCBF	Bacteroides fragilis
QCBT	Bacteroides thetaiotaomicron
		QCCD	Clostridium difficile
QCBF	Bacteroides fragilis
		QCBT	Bacteroides thetaiotaomicron
QCCD	Clostridium difficile

In Table 5 below, the term "JMI" represents the Jones microbiology Institute at North Liberty, Iowa.

Table 5: panel of gram positive and gram negative organisms for generating MIC90 data

JMI isolate #	JMI biological coding	Biological body
			394	ACB	Acinetobacter baumannii
2166	ACB	Acinetobacter baumannii
			3060	ACB	Acinetobacter baumannii
3170	ACB	Acinetobacter baumannii
			9328	ACB	Acinetobacter baumannii
9922	ACB	Acinetobacter baumannii
			13618	ACB	Acinetobacter baumannii
14308	ACB	Acinetobacter baumannii
			17086	ACB	Acinetobacter baumannii
17176	ACB	Acinetobacter baumannii
			30554	ACB	Acinetobacter baumannii
32007	ACB	Acinetobacter baumannii
			1192	ECL	Drain trenchEnterobacter
3096	ECL	Enterobacter cloacae
			5534	ECL	Enterobacter cloacae
6487	ECL	Enterobacter cloacae
			9592	ECL	Enterobacter cloacae
11680	ECL	Enterobacter cloacae
			12573	ECL	Enterobacter cloacae
12735	ECL	Enterobacter cloacae
			13057	ECL	Enterobacter cloacae
18048	ECL	Enterobacter cloacae
			25173	ECL	Enterobacter cloacae
29443	ECL	Enterobacter cloacae
			44	EF	Enterococcus faecalis
355	EF	Enterococcus faecalis
			886	EF	Enterococcus faecalis
955	EF	Enterococcus faecalis
			1000	EF	Enterococcus faecalis
1053	EF	Enterococcus faecalis
			1142	EF	Enterococcus faecalis
1325	EF	Enterococcus faecalis
			1446	EF	Enterococcus faecalis
2014	EF	Enterococcus faecalis
			2103	EF	Enterococcus faecalis
2255	EF	Enterococcus faecalis
			2978	EF	Enterococcus faecalis

JMI isolate #	JMI biological coding	Biological body
			2986	EF	Enterococcus faecalis
5027	EF	Enterococcus faecalis
			5270	EF	Enterococcus faecalis
5874	EF	Enterococcus faecalis
			7430	EF	Enterococcus faecalis
7904	EF	Enterococcus faecalis
			8092	EF	Enterococcus faecalis
8691	EF	Enterococcus faecalis
			9090	EF	Enterococcus faecalis
10795	EF	Enterococcus faecalis
			14104	EF	Enterococcus faecalis
16481	EF	Enterococcus faecalis
			18217	EF	Enterococcus faecalis
22442	EF	Enterococcus faecalis
			25726	EF	Enterococcus faecalis
26143	EF	Enterococcus faecalis
			28131	EF	Enterococcus faecalis
29765	EF	Enterococcus faecalis
			30279	EF	Enterococcus faecalis
31234	EF	Enterococcus faecalis
			31673	EF	Enterococcus faecalis
115	EFM	Enterococcus faecium
			227	EFM	Enterococcus faecium
414	EFM	Enterococcus faecium
			712	EFM	Enterococcus faecium
870	EFM	Enterococcus faecium
			911	EFM	Enterococcus faecium
2356	EFM	Enterococcus faecium
			2364	EFM	Enterococcus faecium
2762	EFM	Enterococcus faecium
			3062	EFM	Enterococcus faecium
4464	EFM	Enterococcus faecium
			4473	EFM	Enterococcus faecium
4653	EFM	Enterococcus faecium
			4679	EFM	Enterococcus faecium
6803	EFM	Enterococcus faecium
			6836	EFM	Enterococcus faecium

JMI isolate #	JMI biological coding	Biological body
			8280	EFM	Enterococcus faecium
8702	EFM	Enterococcus faecium
			9855	EFM	Enterococcus faecium
10766	EFM	Enterococcus faecium
			12799	EFM	Enterococcus faecium
13556	EFM	Enterococcus faecium
			13783	EFM	Enterococcus faecium
14687	EFM	Enterococcus faecium
			15268	EFM	Enterococcus faecium
15525	EFM	Enterococcus faecium
			15538	EFM	Enterococcus faecium
18102	EFM	Enterococcus faecium
			18306	EFM	Enterococcus faecium
19967	EFM	Enterococcus faecium
			22428	EFM	Enterococcus faecium
23482	EFM	Enterococcus faecium
			29658	EFM	Enterococcus faecium
597	EC	Escherichia coli
			847	EC	Escherichia coli
1451	EC	Escherichia coli
			8682	EC	Escherichia coli
11199	EC	Escherichia coli
			12583	EC	Escherichia coli
12792	EC	Escherichia coli
			13265	EC	Escherichia coli
14594	EC	Escherichia coli
			22148	EC	Escherichia coli
29743	EC	Escherichia coli
			30426	EC	Escherichia coli
470	BSA	Group A streptococcus
			2965	BSA	Group A streptococcus
3112	BSA	Group A streptococcus
			3637	BSA	Group A streptococcus
4393	BSA	Group A streptococcus
			4546	BSA	Group A streptococcus
4615	BSA	Group A streptococcus
			5848	BSA	Group A streptococcus

JMI isolate #	JMI biological coding	Biological body
			6194	BSA	Group A streptococcus
8816	BSA	Group A streptococcus
			11814	BSA	Group A streptococcus
16977	BSA	Group A streptococcus
			18083	BSA	Group A streptococcus
18821	BSA	Group A streptococcus
			25178	BSA	Group A streptococcus
30704	BSA	Group A streptococcus
			12	BSB	Group B streptococcus
10366	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 streptococci
			18086	BSG	Group G streptococci
29815	BSG	Group G streptococci
			147	HI	Haemophilus influenzae
180	HI	Haemophilus influenzae
			934	HI	Haemophilus influenzae
970	HI	Haemophilus influenzae
			1298	HI	Haemophilus influenzae
1819	HI	Haemophilus influenzae
			1915	HI	Haemophilus influenzae
2000	HI	Haemophilus influenzae
			2562	HI	Haemophilus influenzae
2821	HI	Haemophilus influenzae
			3133	HI	Haemophilus influenzae
3140	HI	Haemophilus influenzae
			3497	HI	Haemophilus influenzae
3508	HI	Haemophilus influenzae
			3535	HI	Haemophilus influenzae
4082	HI	Haemophilus influenzae
			4108	HI	Haemophilus influenzae

JMI isolate #	JMI biological coding	Biological body
			4422	HI	Haemophilus influenzae
4868	HI	Haemophilus influenzae
			4872	HI	Haemophilus influenzae
5858	HI	Haemophilus influenzae
			6258	HI	Haemophilus influenzae
6875	HI	Haemophilus influenzae
			7063	HI	Haemophilus influenzae
7600	HI	Haemophilus influenzae
			8465	HI	Haemophilus influenzae
10280	HI	Haemophilus influenzae
			10732	HI	Haemophilus influenzae
10850	HI	Haemophilus influenzae
			11366	HI	Haemophilus influenzae
11716	HI	Haemophilus influenzae
			11724	HI	Haemophilus influenzae
11908	HI	Haemophilus influenzae
			12093	HI	Haemophilus influenzae
12107	HI	Haemophilus influenzae
			13424	HI	Haemophilus influenzae
13439	HI	Haemophilus influenzae
			13672	HI	Haemophilus influenzae
13687	HI	Haemophilus influenzae
			13792	HI	Haemophilus influenzae
13793	HI	Haemophilus influenzae
			14440	HI	Haemophilus influenzae
15351	HI	Haemophilus influenzae
			15356	HI	Haemophilus influenzae
15678	HI	Haemophilus influenzae
			15800	HI	Haemophilus influenzae
17841	HI	Haemophilus influenzae
			18614	HI	Haemophilus influenzae
25195	HI	Haemophilus influenzae
			27021	HI	Haemophilus influenzae
28326	HI	Haemophilus influenzae
			28332	HI	Haemophilus influenzae
29918	HI	Haemophilus influenzae
			29923	HI	Haemophilus influenzae

JMI isolate #	JMI biological coding	Biological body
			31911	HI	Haemophilus influenzae
428	KPN	Klebsiella pneumoniae
			791	KPN	Klebsiella pneumoniae
836	KPN	Klebsiella pneumoniae
			1422	KPN	Klebsiella pneumoniae
1674	KPN	Klebsiella pneumoniae
			1883	KPN	Klebsiella pneumoniae
6486	KPN	Klebsiella pneumoniae
			8789	KPN	Klebsiella pneumoniae
10705	KPN	Klebsiella pneumoniae
			11123	KPN	Klebsiella pneumoniae
28148	KPN	Klebsiella pneumoniae
			29432	KPN	Klebsiella pneumoniae
937	MCAT	Moraxella catarrhalis
			1290	MCAT	Moraxella catarrhalis
1830	MCAT	Moraxella catarrhalis
			1903	MCAT	Moraxella catarrhalis
4346	MCAT	Moraxella catarrhalis
			4880	MCAT	Moraxella catarrhalis
6241	MCAT	Moraxella catarrhalis
			6551	MCAT	Moraxella catarrhalis
7074	MCAT	Moraxella catarrhalis
			7259	MCAT	Moraxella catarrhalis
7544	MCAT	Moraxella catarrhalis
			8142	MCAT	Moraxella catarrhalis
8451	MCAT	Moraxella catarrhalis
			9246	MCAT	Moraxella catarrhalis
9996	MCAT	Moraxella catarrhalis
			12158	MCAT	Moraxella catarrhalis
13443	MCAT	Moraxella catarrhalis
			13692	MCAT	Moraxella catarrhalis
13817	MCAT	Moraxella catarrhalis
			14431	MCAT	Moraxella catarrhalis
14762	MCAT	Moraxella catarrhalis
			14842	MCAT	Moraxella catarrhalis
15361	MCAT	Moraxella catarrhalis
			15741	MCAT	Moraxella catarrhalis

JMI isolate #	JMI biological coding	Biological body
			17843	MCAT	Moraxella catarrhalis
18639	MCAT	Moraxella catarrhalis
			241	GC	Neisseria gonorrhoeae
291	GC	Neisseria gonorrhoeae
			293	GC	Neisseria gonorrhoeae
344	GC	Neisseria gonorrhoeae
			451	GC	Neisseria gonorrhoeae
474	GC	Neisseria gonorrhoeae
			491	GC	Neisseria gonorrhoeae
493	GC	Neisseria gonorrhoeae
			503	GC	Neisseria gonorrhoeae
521	GC	Neisseria gonorrhoeae
			552	GC	Neisseria gonorrhoeae
573	GC	Neisseria gonorrhoeae
			592	GC	Neisseria gonorrhoeae
25	NM	Neisseria meningitidis
			813	NM	Neisseria meningitidis
1725	NM	Neisseria meningitidis
			2747	NM	Neisseria meningitidis
3201	NM	Neisseria meningitidis
			3335	NM	Neisseria meningitidis
7053	NM	Neisseria meningitidis
			9407	NM	Neisseria meningitidis
10447	NM	Neisseria meningitidis
			12685	NM	Neisseria meningitidis
12841	NM	Neisseria meningitidis
			14038	NM	Neisseria meningitidis
1127	PM	Proteus mirabilis
			3049	PM	Proteus mirabilis
4471	PM	Proteus mirabilis
			8793	PM	Proteus mirabilis
10702	PM	Proteus mirabilis
			11218	PM	Proteus mirabilis
14662	PM	Proteus mirabilis
			17072	PM	Proteus mirabilis
19059	PM	Proteus mirabilis
			23367	PM	Proteus mirabilis

JMI isolate #	JMI biological coding	Biological body
			29819	PM	Proteus mirabilis
31419	PM	Proteus mirabilis
			1881	PSA	Pseudomonas aeruginosa
5061	PSA	Pseudomonas aeruginosa
			7909	PSA	Pseudomonas aeruginosa
8713	PSA	Pseudomonas aeruginosa
			14318	PSA	Pseudomonas aeruginosa
14772	PSA	Pseudomonas aeruginosa
			15512	PSA	Pseudomonas aeruginosa
17093	PSA	Pseudomonas aeruginosa
			17802	PSA	Pseudomonas aeruginosa
19661	PSA	Pseudomonas aeruginosa
			29967	PSA	Pseudomonas aeruginosa
31539	PSA	Pseudomonas aeruginosa
			82	SA	Staphylococcus aureus
99	SA	Staphylococcus aureus
			138	SA	Staphylococcus aureus
139	SA	Staphylococcus aureus
			140	SA	Staphylococcus aureus
141	SA	Staphylococcus aureus
			142	SA	Staphylococcus aureus
272	SA	Staphylococcus aureus
			287	SA	Staphylococcus aureus
354	SA	Staphylococcus aureus
			382	SA	Staphylococcus aureus
1112	SA	Staphylococcus aureus
			1687	SA	Staphylococcus aureus
1848	SA	Staphylococcus aureus
			2031	SA	Staphylococcus aureus
2159	SA	Staphylococcus aureus
			2645	SA	Staphylococcus aureus
3256	SA	Staphylococcus aureus
			3276	SA	Staphylococcus aureus
4044	SA	Staphylococcus aureus
			4214	SA	Staphylococcus aureus
4217	SA	Staphylococcus aureus
			4220	SA	Staphylococcus aureus

JMI isolate #	JMI biological coding	Biological body
			4231	SA	Staphylococcus aureus
4240	SA	Staphylococcus aureus
			4262	SA	Staphylococcus aureus
4370	SA	Staphylococcus aureus
			4665	SA	Staphylococcus aureus
4666	SA	Staphylococcus aureus
			4667	SA	Staphylococcus aureus
5026	SA	Staphylococcus aureus
			5666	SA	Staphylococcus aureus
6792	SA	Staphylococcus aureus
			7023	SA	Staphylococcus aureus
7461	SA	Staphylococcus aureus
			7899	SA	Staphylococcus aureus
7901	SA	Staphylococcus aureus
			8714	SA	Staphylococcus aureus
9374	SA	Staphylococcus aureus
			9437	SA	Staphylococcus aureus
10056	SA	Staphylococcus aureus
			10110	SA	Staphylococcus aureus
11379	SA	Staphylococcus aureus
			11629	SA	Staphylococcus aureus
11659	SA	Staphylococcus aureus
			12788	SA	Staphylococcus aureus
12789	SA	Staphylococcus aureus
			13043	SA	Staphylococcus aureus
13086	SA	Staphylococcus aureus
			13721	SA	Staphylococcus aureus
13742	SA	Staphylococcus aureus
			13932	SA	Staphylococcus aureus
14210	SA	Staphylococcus aureus
			14384	SA	Staphylococcus aureus
15428	SA	Staphylococcus aureus
			15430	SA	Staphylococcus aureus
17721	SA	Staphylococcus aureus
			18688	SA	Staphylococcus aureus
19095	SA	Staphylococcus aureus
			20195	SA	Staphylococcus aureus

JMI isolate #	JMI biological coding	Biological body
			22141	SA	Staphylococcus aureus
22689	SA	Staphylococcus aureus
			27398	SA	Staphylococcus aureus
29048	SA	Staphylococcus aureus
			29051	SA	Staphylococcus aureus
30491	SA	Staphylococcus aureus
			30538	SA	Staphylococcus aureus
25	SEPI	Staphylococcus epidermidis
			53	SEPI	Staphylococcus epidermidis
385	SEPI	Staphylococcus epidermidis
			398	SEPI	Staphylococcus epidermidis
701	SEPI	Staphylococcus epidermidis
			713	SEPI	Staphylococcus epidermidis
1381	SEPI	Staphylococcus epidermidis
			2174	SEPI	Staphylococcus epidermidis
2286	SEPI	Staphylococcus epidermidis
			2969	SEPI	Staphylococcus epidermidis
3417	SEPI	Staphylococcus epidermidis
			3447	SEPI	Staphylococcus epidermidis
4753	SEPI	Staphylococcus epidermidis
			7241	SEPI	Staphylococcus epidermidis
9366	SEPI	Staphylococcus epidermidis
			10665	SEPI	Staphylococcus epidermidis
11792	SEPI	Staphylococcus epidermidis
			12311	SEPI	Staphylococcus epidermidis
13036	SEPI	Staphylococcus epidermidis
			13227	SEPI	Staphylococcus epidermidis
13243	SEPI	Staphylococcus epidermidis
			13621	SEPI	Staphylococcus epidermidis
13638	SEPI	Staphylococcus epidermidis
			13800	SEPI	Staphylococcus epidermidis
14078	SEPI	Staphylococcus epidermidis
			14392	SEPI	Staphylococcus epidermidis
15007	SEPI	Staphylococcus epidermidis
			16733	SEPI	Staphylococcus epidermidis
18871	SEPI	Staphylococcus epidermidis
			23285	SEPI	Staphylococcus epidermidis

JMI isolate #	JMI biological coding	Biological body
			27805	SEPI	Staphylococcus epidermidis
29679	SEPI	Staphylococcus epidermidis
			29985	SEPI	Staphylococcus epidermidis
30259	SEPI	Staphylococcus epidermidis
			31444	SEPI	Staphylococcus epidermidis
268	SPN	Streptococcus pneumoniae
			1264	SPN	Streptococcus pneumoniae
2482	SPN	Streptococcus pneumoniae
			2653	SPN	Streptococcus pneumoniae
2994	SPN	Streptococcus pneumoniae
			3123	SPN	Streptococcus pneumoniae
3124	SPN	Streptococcus pneumoniae
			4336	SPN	Streptococcus pneumoniae
4858	SPN	Streptococcus pneumoniae
			5606	SPN	Streptococcus pneumoniae
5881	SPN	Streptococcus pneumoniae
			5897	SPN	Streptococcus pneumoniae
5900	SPN	Streptococcus pneumoniae
			6051	SPN	Streptococcus pneumoniae
6216	SPN	Streptococcus pneumoniae
			6556	SPN	Streptococcus pneumoniae
7270	SPN	Streptococcus pneumoniae
			7584	SPN	Streptococcus pneumoniae
8479	SPN	Streptococcus pneumoniae
			8501	SPN	Streptococcus pneumoniae
9256	SPN	Streptococcus pneumoniae
			9257	SPN	Streptococcus pneumoniae
10246	SPN	Streptococcus pneumoniae
			10467	SPN	Streptococcus pneumoniae
10886	SPN	Streptococcus pneumoniae
			11217	SPN	Streptococcus pneumoniae
11228	SPN	Streptococcus pneumoniae
			11238	SPN	Streptococcus pneumoniae
11757	SPN	Streptococcus pneumoniae
			11768	SPN	Streptococcus pneumoniae
12121	SPN	Streptococcus pneumoniae
			12124	SPN	Streptococcus pneumoniae

JMI isolate #	JMI biological coding	Biological body
			12149	SPN	Streptococcus pneumoniae
12767	SPN	Streptococcus pneumoniae
			12988	SPN	Streptococcus pneumoniae
13321	SPN	Streptococcus pneumoniae
			13393	SPN	Streptococcus pneumoniae
13521	SPN	Streptococcus pneumoniae
			13544	SPN	Streptococcus pneumoniae
13700	SPN	Streptococcus pneumoniae
			13704	SPN	Streptococcus pneumoniae
13822	SPN	Streptococcus pneumoniae
			13838	SPN	Streptococcus pneumoniae
14131	SPN	Streptococcus pneumoniae
			14413	SPN	Streptococcus pneumoniae
14744	SPN	Streptococcus pneumoniae
			14808	SPN	Streptococcus pneumoniae
14827	SPN	Streptococcus pneumoniae
			14835	SPN	Streptococcus pneumoniae
14836	SPN	Streptococcus pneumoniae
			15832	SPN	Streptococcus pneumoniae
17336	SPN	Streptococcus pneumoniae
			17343	SPN	Streptococcus pneumoniae
17349	SPN	Streptococcus pneumoniae
			17735	SPN	Streptococcus pneumoniae
18060	SPN	Streptococcus pneumoniae
			18567	SPN	Streptococcus pneumoniae
18595	SPN	Streptococcus pneumoniae
			19082	SPN	Streptococcus pneumoniae
19826	SPN	Streptococcus pneumoniae
			22174	SPN	Streptococcus pneumoniae
22175	SPN	Streptococcus pneumoniae
			27003	SPN	Streptococcus pneumoniae
28310	SPN	Streptococcus pneumoniae
			28312	SPN	Streptococcus pneumoniae
29890	SPN	Streptococcus pneumoniae
			29910	SPN	Streptococcus pneumoniae

Claims (22)

1.A solid compound of formula (I):

or a salt thereof.

2. The solid compound of claim 1, wherein the solid is solid form I free base.

3. The solid compound of claim 2, wherein said solid form I is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αAt least three approximate peak positions (degrees 2 θ ± 0.2) are included when measured radiometrically, which are selected from 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 XPRD is collected from about 5 to about 38 degrees 2 θ.

4. The solid compound of claim 2, wherein said solid form I is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αAt least three approximate peak positions (degrees 2 θ ± 0.2) are included in the radiometric measurements, which are selected from 9.3, 16.7, 18.6, 19.6, 21.7, and 25.8 when XPRD is collected from about 5 to about 38 degrees 2 θ.

5. The solid compound of claim 2, wherein said solid form I is characterized as using Cu K_αRadiometric, substantially similar to the X-ray powder diffraction pattern of figure 1.

6. The solid compound of claim 2, wherein said solid form I is further characterized by an endothermic peak having an onset temperature of about 243 ℃ as measured by differential scanning calorimetry in which the temperature is scanned at about 10 ℃/minute.

7. A process for preparing crystalline form I of the compound of formula (I) according to claim 1, comprising precipitating the compound of formula (I) from a solvent system comprising dichloromethane, methanol, ethanol, or a combination thereof.

8. A hydrochloride salt of a compound of formula (I):

9. the hydrochloride salt of claim 8 in solid form.

10. The hydrochloride salt of claim 9, wherein the salt is solid form II.

11. The solid form II of claim 10, wherein said solid form II is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αAt least three approximate peak positions (degrees 2 theta + -0.2) are included when measured radiometrically, which are selected from 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 XPRD is collected from about 5 to about 38 degrees 2 theta.

12. The solid form II of claim 10, wherein said solid form II is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αAt least three approximate peak positions (degrees 2 θ ± 0.2) are included in the radiometric measurements, which when XPRD is collected from about 5 to about 38 degrees 2 θ is selected from 7.0, 9.1, 11.5, 12.3, 12.4, 16.4, 17.9, 19.5, 24.0, and 29.4.

13. The solid form II of claim 10 characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αAt least three approximate peak positions (degrees 2 θ ± 0.2) are included in the radiometric measurements, which when XPRD is collected from about 5 to about 38 degrees 2 θ is selected from 7.0, 9.1, 11.5, 19.5, and 24.0.

14. The solid form II of claim 10, characterized by the use of Cu K_αRadiometric, substantially similar to the X-ray powder diffraction pattern of fig. 4.

15. A process for preparing solid form II of claim 10 comprising suspending a solid free base of a benzimidazolyl urea in an acidic solvent system comprising acetonitrile or water.

16. The solid hydrochloride salt compound of claim 9, wherein the hydrochloride salt of the compound of formula (I) is solid form III.

17. The solid form III of claim 16, wherein the solid form III is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αAt least three approximate peak positions (degrees 2 θ ± 0.2) are included when measured radiometrically, which are selected from the group consisting of 6.9, 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 XPRD is collected from about 5 to about 38 degrees 2 θ.

18. The solid form III of claim 16, wherein the solid form III is characterized by an X-ray powder diffraction pattern (XPRD) when using Cu K_αAt least three approximate peak positions (degrees 2 θ ± 0.2) are included in the radiometric measurements, which are selected from 6.9, 9.1, 11.7, 18.1, 18.9, 19.8, 23.4, and 24.8 when XPRD is collected from about 5 to about 38 degrees 2 θ.

19. The solid form III of claim 16, characterized by the use of Cu K_αRadiometric, substantially similar to the X-ray powder diffraction pattern of fig. 7.

20. A process for preparing solid form III according to claim 16, comprising suspending a solid free base of benzimidazolyl urea in an acidic solvent system comprising one or more ethereal solvents and water.

21. The solid compound of claim 1, wherein the solid is form IV amorphous mesylate.

22. The solid amorphous mesylate salt form IV of claim 21, which is characterized by an X-ray powder diffraction pattern (XPRD) using Cu ka radiation characterized by an extended halo with no discernible diffraction peak.