NZ612961B2 - Pyrimidine gyrase and topoisomerase iv inhibitors - Google Patents

Abstract

612961 The disclosure relates to solid forms of compound of formula (I) and pharmaceutically acceptable salts thereof, wherein the variables X and R are as defined herein, particularly the methanesulfonic acid salt, which inhibit bacterial enzymes gyrase and/or topoisomerase IV. The compounds of formula (I) either possess a broad range of anti bacterial activity and advantageous toxicological properties or are prodrugs of compounds having said activity. Also disclosed are pharmaceutical compositions comprising said compound of formula (I) or its salts and their use for treating nosocomial or a non-nosocomial bacterial infections which are characterized by the presence of one or more of Streptococcus pneumoniae, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus aureus, Clostridium difJicile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, ycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerans, Chlamydophila pneumoniae, Chlamydia trachomatis, Haemophilus injluenzae, Streptococcus pyogenes or ?-haemolytic streptococci. of formula (I) either possess a broad range of anti bacterial activity and advantageous toxicological properties or are prodrugs of compounds having said activity. Also disclosed are pharmaceutical compositions comprising said compound of formula (I) or its salts and their use for treating nosocomial or a non-nosocomial bacterial infections which are characterized by the presence of one or more of Streptococcus pneumoniae, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus aureus, Clostridium difJicile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, ycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerans, Chlamydophila pneumoniae, Chlamydia trachomatis, Haemophilus injluenzae, Streptococcus pyogenes or ?-haemolytic streptococci.

Description

PYRIMIDINE GYRASE AND TOPOISOMERASE IV INHIBITORS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119 of United States Provisional Patent Application serial number ,965 filed y 14, 201 1,of United States Provisional Patent Application serial number ,174 filed August 4, 2011, of United States Provisional Patent ation serial number 61/515,249 filed August 4, 2011, and of United States Provisional Patent Application serial number 61/499,134 filed June 20, 2011, the entire contents of each application hereby incorporated by reference.

BACKGROUND OF THE INVENTION Bacterial resistance to antibiotics has long been recognized, and it is today considered to be a serious worldwide health problem. As a result of resistance, some bacterial infections are either lt to treat with antibiotics or even untreatable. This problem has become especially serious with the recent development of multiple drug resistance in certain strains of bacteria, such as Streptococcus pneumoniae (SP), cterium tuberculosis, and Enterococcus. The appearance of Vancomycin resistant enterococcus was particularly alarming because vancomycin was formerly the only effective antibiotic for treating this infection, and had been considered for many infections to be the drug of "last resort". While many other drug-resistant bacteria do not cause life-threatening disease, such as enterococci, there is the fear that the genes which induce resistance might spread to more deadly organisms such as Staphylococcus aureus, where methicillin resistance is already prevalent (De Clerq, et al., Current Opinion in Anti-infective Investigational Drugs, 1999, 1, 1, Levy, "The nge of Antibiotic ance", Scientific American, March, 1998) Another concern is how quickly antibiotic resistance can . For example, until the 1960's SP was universally ive to penicillin, and in 1987 only 0.02% of the SP strains in the US. were resistant. However, by 1995 it was reported that SP resistance to penicillin was about seven percent and as high as 30% in some parts of the US. (Lewis, FDA er magazine (September, 1995), Gershman in The Medical Reporter, 1997).

Hospitals, in ular, serve as centers for the formation and transmission of drug-resistant organisms. Infections occurring in hospitals, known as nosocomial infections, are becoming an increasingly serious problem. Of the two n Americans infected in hospitals each year, more than half of these infections resist at least one antibiotic. The Center for Disease Control reported that in 1992, over 13,000 hospital patients died of bacterial infections that were resistant to antibiotic treatment (Lewis, "The Rise of Antibiotic- ant Infections", FDA Consumer magazine, ber 1995).

As a result of the need to combat drug-resistant bacteria and the increasing e of the available drugs, there has been a resurgent st in discovering new antibiotics. One attractive strategy for developing new antibiotics is to inhibit DNA gyrase and/or topoisomerase IV, bacterial s ary for DNA replication, and therefore, necessary for bacterial cell grth and division. Gyrase and/or topoisomerase IV activity are also associated with events in DNA transcription, repair and recombination.

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

The ATP is then hydrolyzed during the reaction. This ATP binding and subsequent ysis cause conformational changes in the DNA-bound gyrase that are ary for its activity. It has also been found that the level of DNA supercoiling (or relaxation) is dependent on the ATP/ADP ratio. In the absence of ATP, gyrase is only capable of relaxing oiled DNA.

Bacterial DNA gyrase is a 400 kilodalton protein tetramer consisting of two A (GyrA) and two B subunits . g and cleavage of the DNA is associated with GyrA, whereas ATP is bound and hydrolyzed by the GyrB protein. GyrB consists of an terminal domain which has the ATPase activity, and a carboxy-terminal domain which interacts with GyrA and DNA. By contrast, eukaryotic type II omerases are homodimers that can relax negative and positive oils, but cannot introduce negative 2012/021270 oils. Ideally, an antibiotic based on the inhibition of bacterial DNA gyrase and/or topoisomerase IV would be selective for these enzymes and be vely inactive against the eukaryotic type II topoisomerases.

Topoisomerase IV primarily resolves linked chromosome dimers at the conclusion of DNA replication.

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

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

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

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

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

While enzymes with this mutation show lower supercoiling and ATPase activity, they are also less sensitive to inhibition by coumarin drugs ll, Mol. Microbiol., 1993, 9, 681).

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

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

As bacterial resistance to antibiotics has become an important public health problem, there is a continuing need to develop newer and more potent antibiotics. More particularly, there is a need for antibiotics that represent a new class of compounds not previously used to treat bacterial infection. Compounds that target the ATP binding sites in both the GyrB (gyrase) and ParE somerase IV) ts would be useful for treating various bacterial infections. Such compounds would be particularly useful in treating nosocomial infections in hospitals where the formation and transmission of ant bacteria are becoming singly prevalent. Furthermore, there is a need for new antibiotics having a broad spectrum of activity with advantageous toxicological properties.

SUMMARY OF THE ION The present invention is directed to compounds and pharmaceutically acceptable salts thereof, useful as gyrase and/or topoisomerase IV inhibitors. The gyrase and/or topoisomerase IV inhibitors of the present invention may be represented by formula (I) or salts thereof: wherein R is hydrogen or fluorine; X is hydrogen, —PO(OH)2, —PO(OH)O'M+, —PO(O')2-2M+, or —PO(O')2-D2+, M+ is a pharmaceutically acceptable monovalent cation; and D2+ is a ceutically acceptable divalent cation. The compounds of formula (I) either possess a broad range of anti-bacterial activity and advantageous toxicological properties or are prodrugs of compounds having said activity.

The present invention also relates to compounds of formula (IA), or ceutically acceptable salts thereof, useful as gyrase and/or topoisomerase IV inhibitors. The compounds of formula (IA) are assed by formula (I). The compounds of formula (IA) may be represented as: (1A) wherein R is hydrogen or e. The nds of formula (IA) possess a broad range of anti-bacterial activity and advantageous toxicological properties.

The present invention also relates to compounds of formula (IE), or pharmaceutically acceptable salts thereof, useful as gs for gyrase and/or omerase IV inhibitors. The compounds of formula (IB) are encompassed by formula (I). The compounds of formula (IB) may be represented as: WO 97269 N \N \NH o ) (1B) wherein X is —PO(OH)2, —PO(OH)O'M+, —PO(O')2-2M+, or —PO(O')2-D2+, M+ is a pharmaceutically acceptable monovalent cation; and D2+ is a pharmaceutically acceptable divalent cation. The nds of formula (IB) are ate ester gs of the compound (R)- l -ethyl-3 -(6-fluoro-5 -(2-(2-hydroxypropanyl)pyrimidin-5 - (tetrahydrofuranyl)-lH-benzo[d]imidazolyl)urea, which possesses a broad range of anti- bacterial activity and advantageous logical properties. In addition to the compounds ed herein, the present invention further provides a pharmaceutical ition comprising a compound of formula (I) (which includes other formulae encompassed by formula (I) such as formulae (IA), (IB), (IC) and (ID)) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another embodiment, the present invention relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a ceutically acceptable carrier, and an additional therapeutic agent selected from an antibiotic, an anti-inflammatory agent, a matrix metalloproteinase tor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a grth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound.

In another ment, the present invention relates to a method of treating a bacterial infection in a mammal in need thereof, comprising administering to said mammal a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof In a further embodiment, the t invention relates to a method of treating a bacterial infection in a mammal in need thereof, comprising administering to said mammal a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an antibiotic, an anti-inflammatory agent, a matrix metalloproteinase inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a grth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound, either as part of a multiple dosage form together with said compound or as a separate dosage form.

BRIEF DESCRIPTION OF THE DRAWINGS is a thermal ellipsoid plot of two symmetry independent molecules of compound 12. is a l ellipsoid plot of two ry independent molecules of compound 23.

DETAILED DESCRIPTION As used herein, the term “halogen” means F, Cl, Br, or I.

Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure, i.e., the R and S configurations for each asymmetric center.Therefore, single stereochemical isomers as well as omeric and diastereomeric mixtures of the t compounds are within the scope of the invention. ically-labeled forms of compounds of formula (I) wherein one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature are also included herein. Examples of es that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, en, , and fluorine, such as 2H, 3H, 13C, 14C, 15N, 18O, and 170.

Such radio-labeled and stable-isotopically labeled compounds are useful, for example, as research or diagnostic tools or gyrase and/or topoisomerase IV inhibitors with improved therapeutic profile. The structures also encompass zwitterionic forms of the compounds or salts, where appropriate.

In one embodiment, compounds of a (I) e compounds of formula (IC) (1C) wherein R is as defined above.

In another embodiment, nds of formula (I) include compounds of formulae (ID) and (IE) as set forth below: N>\’NH o ) (1D) (R)-l -ethyl-3 -(5-(2-(2-hydroxypropanyl)pyrimidin—5 -yl)-7 -(tetrahydrofuran—2 - yl)-lH-benzo[d]imidazolyl)urea, or a pharmaceutically acceptable salt thereof; and (1E) (R)-l -ethyl-3 -(6-fluoro-5 -(2-(2-hydroxypropanyl)pyrimidin-5 -yl)-7 - hydrofuran—2-yl)-lH-benzo[d]imidazolyl)urea, or a pharmaceutically acceptable salt thereof Unless otherwise stated, the phrase “compounds of a (1)” is intended to include other formulae set forth herein that are encompassed by formula (I) including formulae (IA), (IB), (IC), (ID), and (IE).

The compounds of formula (IB) are prodrugs of their parent compound, l [6-fluoro-5 -[2-(l -hydroxy- l -methyl-ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - lH- benzimidazolyl]urea. Thus, the activity exhibited upon stration of the prodrug is principally due to the presence of the parent compound that results from cleavage of the prodrug.

The term “prodrug” refers to compounds which are drug precursors which, following stration and absorption, release the drug in vivo via some metabolic process.

In l, a prodrug possesses less biological activity than its parent drug. A prodrug may also improve the physical properties of the parent drug and/or it may also improve overall drug efficacy, for example through the reduction of toxicity and unwanted effects of a drug by controlling its absorption, blood levels, metabolic distribution and cellular uptake.

The term “parent compound” or “parent drug” refers to the biologically active entity that is released via enzymatic action of a metabolic or a catabolic process, or via a al process following administration of the prodrug. The parent compound may also be the starting material for the preparation of its corresponding prodrug.

The monovalent cations defined by M+ include ammonium, alkali metal ions such as sodium, lithium and potassium ions, ohexylamine ion, and yl-D-glucamine ion. The nt cations defined by D2+ e, alkaline earth metal ions such as aluminum, calcium and magnesium ions. Also included are amino acid cations such as ions of arginine, WO 97269 lysine, ne, and so forth. If M+ is a monovalent cation, it is recognized that if the definition 2M+ is present, each of M+ may be the same or different. In addition, it is similarly recognized that if the definition 2M+ is present, a divalent cation D2+ may instead be t.

Also, the basic nitrogen-containing groups may be quatemized with such agents as: lower alkyl halides, such as methyl, ethyl, propyl, and butyl de, bromides and iodides, dialkyl es like yl, diethyl, dibutyl, diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl bromide and others.

Various ments of the invention, include compounds or salts of formula (IB) as set forth below: (1) compounds wherein X is (a) —PO(OH)O'M+, (b) —P0(0')2-2M+, or (c) —PO(O')2-D2+, (2) compounds wherein M+ is (a) Lil, Nal, Kt, N—methyl-D-glucamine, or +, or (b) Nat; (c) each R9 is independently hydrogen or a C1-C4 alkyl group, (3) nds wherein D2+ is (a) Mgzi Ca2+, and Bazl, or (b) Ca2+, (4) the compound (R)(5-(2-(3-ethylureido)fluoro(tetrahydrofuran—2- -benzo[d]imidazolyl)pyrimidin—2-yl)propanyl phosphate, and (5) the compound disodium (R)(5-(2-(3-ethylureido)fluoro (tetrahydrofuran—2-yl)- l H-benzo [d]imidazol-5 -yl)pyrimidin—2-yl)propanyl phosphate.

It is understood that various alternative embodiments of the compounds or salts of formula (IB) can be selected by requiring one or more of the alternate embodiments listed in (1) through (3) above. For example, further embodiments of the invention can be ed by combining (l)(a) and (2)(a); (l)(a) and (2)(b); (l)(C) and (3)(a); (l)(C) and (3)(b); (l)(b) and (2)(a), (l)(b) and (2)(b), and the like.

The prodrugs of the present invention are characterized by unexpectedly high aqueous solubility. This solubility facilitates administration of higher doses of the prodrug, resulting in a greater drug load per unit dosage.

One embodiment of this invention relates to a method of treating a bacterial infection in a mammal in need thereof, comprising administering to said mammal a therapeutically effective amount of a compound having the formula (I) or a pharmaceutically acceptable salt thereof According to r embodiment, the ion provides a method of decreasing or inhibiting bacterial quantity in a biological sample. This method comprises contacting said biological sample with a compound of formula (I) or a pharmaceutically acceptable salt thereof The term "biological sample", as used herein, includes cell cultures or extracts thereof, biopsied material obtained from a mammal or extracts thereof, and blood, saliva, urine, feces, semen, tears, or other body fluids or ts thereof The term "biological sample" also includes living sms, in which case "contacting a compound of this invention with a biological sample" is synonymous with the term "administering said compound or composition comprising said compound) to a mamma ".

One embodiment comprises contacting said biological sample with a compound ed from the group consisting of (R)-l-ethyl(5-(2-(2-hydroxypropan—2-yl)pyrimidin- -yl)(tetrahydrofuranyl)-lH-benzo[d]imidazolyl)urea, or a pharmaceutically acceptable salt thereof, and (R)-l-ethyl(6-fluoro(2-(2-hydroxypropanyl)pyrimidin yl)(tetrahydrofuranyl)-lH-benzo[d]imidazolyl)urea, or a pharmaceutically acceptable salt thereof ceutical compositions useful for such methods are bed below.

One embodiment comprises contacting said biological sample with a ate ester prodrug of (R)-l -ethyl(6-fluoro-5 -(2-(2-hydroxypropanyl)pyrimidinyl) (tetrahydrofuran—2-yl)-lH-benzo[d]imidazolyl)urea, as defined by formula (IB).

Pharmaceutical itions useful for such methods are described below.

The antimicrobial activity of the nds of formula (I) may be demonstrated in an antimicrobial susceptibility assay. The details of the conditions used for the antimicrobial tibility assays are set forth in the Examples below.

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

Agents which increase the susceptibility of ial organisms to antibiotics are known. For example, US. Pat. No. 5,523,288, US. Pat. No. 561 and US. Pat. No. 6,140,306 describe methods of using bactericidal/perrneability—increasing protein (BPI) for increasing antibiotic susceptibility of gram-positive and gram-negative bacteria. Agents that increase the permeability of the outer membrane of bacterial organisms have been described by Vaara, M. in Microbiological Reviews (1992) pp. 395-411, and the ization of gram- negative bacteria has been described by Tsubery, H., et al, in J. Med. Chem. (2000) pp. 3085- 3092.

Another embodiment of this invention relates to a method, as described above, of preventing, controlling, treating or ng the ement, severity or effects of a bacterial infection in a mammal in need thereof, but further comprising the step of administering to said mammal an agent which increases the susceptibility of bacterial organisms to antibiotics.

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

The ceutical compositions and s of this invention will be useful generally for controlling bacterial infections in vivo. Examples of ial organisms that may be lled by the compositions and methods of this invention include, but are not limited to the following organisms: Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcusfaecalis, coccusfaecium, Klebsiella pneumoniae, Enterobacter spp.

Proteus spp. Pseudomonas aeruginosa, E. coli, Serratia marcescens, Staphylococcus aureus, Coag. Neg. Staphylococci, Haemophilus influenzae, Bacillus anthracis, Mycoplasma pneumoniae, Moraxella catarrhalis, Chlamydophila pneumoniae, Chlamydia trachomatis, Legionella pneumophila, Mycobacterium tuberculosis, Helicobacter pylori, Staphylococcus saprophyticus, Staphylococcus epidermidis, Francisella tularehsis, Yersihia pestis, Closz‘ridl'um dlfificile, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacteriumabscessus, Mycobacterium kansasil' andMycobacterium ulcerans.

The compositions and methods will therefore be useful for controlling, treating or reducing the advancement, severity or effects of mial or non-nosocomial infections. es of nosocomial and non-nosocomial infections include but are not limited to upper respiratory infections, lower atory infections, ear infections, pleuropulmonary and bronchial infections, complicated urinary tract infections, uncomplicated urinary tract infections, intra-abdominal infections, cardiovascular infections, a blood stream infection, sepsis, bacteremia, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital infections, eye infections, or granulomatous infections. Examples of specific bacterial infections include but are not limited to uncomplicated skin and skin structure infections ), complicated skin and skin structure infections ), er infections, pharyngitis, sinusitis, otitis extema, otitis media, bronchitis, a, pneumonia, community-acquired ial pneumoniae (CABP), hospital-acquired pneumonia (HAP), hospital-acquired ial pneumonia, ventilator-associated pneumonia (VAP), diabetic foot infections, vancomycin resistant enterococci infections, cystitis and pyelonephritis, renal calculi, prostatitis, peritonitis, complicated intra-abdominal infections (cIAI) and other inter-abdominal infections, dialysis-associated peritonitis, visceral abscesses, endocarditis, myocarditis, pericarditis, transfusion-associated sepsis, meningitis, encephalitis, brain abscess, osteomyelitis, tis, l , urethritis, vaginitis, cervicitis, itis, conjunctivitis, tis, endophthalmitisa, an infection in cystic fibrosis patients or an infection of febrile neutropenic patients.

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

In one embodiment, the compositions and s will therefore be useful for controlling, treating or reducing the advancement, severity or effects of community-acquired bacterial pneumoniae (CABP), hospital-acquired nia (HAP), hospital-acquired bacterial pneumonia, ventilator-associated pneumonia (VAP), bacteremia, diabetic foot infections, catheter infections, uncomplicated skin and skin structure infections (uSSSI), complicated skin and skin ure infections (cSSSI), vancomycin resistant cocci infections or osteomyelitis.

In another embodiment, the compositions and methods will therefore be useful for controlling, treating or reducing the advancement, severity or effects of upper respiratory infections, lower respiratory infections, ear ions, pleuropulmonary and ial infections, complicated urinary tract infections, uncomplicated urinary tract infections, intra- abdominal infections, cardiovascular infections, a blood stream infection, sepsis, bacteremia, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital ions, eye infections, or granulomatous infections, uncomplicated skin and skin structure infections (uSSSI), complicated skin and skin structure infections (cSSSI), catheter infections, pharyngitis, sinusitis, otitis extema, otitis media, itis, empyema, nia, community-acquired bacterial pneumoniae (CABP), al-acquired pneumonia (HAP), hospital—acquired bacterial pneumonia, ventilator-associated pneumonia (VAP), diabetic foot infections, vancomycin resistant cocci infections, cystitis and ephritis, renal calculi, prostatitis, peritonitis, complicated intra-abdominal infections (cIAI) and other inter-abdominal infections, dialysis-associated peritonitis, visceral ses, endocarditis, ditis, pericarditis, transfusion-associated sepsis, meningitis, alitis, brain abscess, osteomyelitis, arthritis, genital ulcers, itis, vaginitis, cervicitis, gingivitis, conjunctivitis, keratitis, endophthalmitisa, an infection in cystic fibrosis patients or an infection of febrile neutropenic patients.

In another embodiment, the bacterial infection is characterized by the presence of one or more of Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcusfaecalis, Enterococcusfaecium, Staphylococcus aureus, Coag. Neg. Staphlococci, Bacillus anthracis, Staphylococcus epidermiclis, Staphylococcus saprophyticus, or Mycohacterium tuberculosis.

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

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

In r embodiment, the bacterial infection is characterized by the presence of one or more of Clostriclium clifiicile, ria gonorrhoeae, Neisseria meningiticlis, Mycohacterium avium complex, Mycohacterium sus, Mycohacterium kansasii, Mycohacterium ulcerans, Chlamydophila pneumoniae and Chlamydia tracomatis.

In another embodiment, the bacterial infection is characterized by the presence of one or more of Streptococcus pneumoniae, Staphylococcus epidermiclis, Enterococcus faecalis, Staphylococcus aureus, Clostriclium ile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningiticlis, cterium avium x, Mycohacterium abscessus, Mycohacterium kansasii, Mycohacterium ulcerans, Chlamydophila pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Streptococcus pyogenes or B-haemolytic streptococci.

In some embodiments, the bacterial infection is characterized by the presence ofone or more of Methicillin resistant Staphylococcus , Fluoroquinolone resistant Staphylococcus aureus, Vancomycin intermediate resistant Staphylococcus aureus, Linezolid resistant Staphylococcus , Penicillin resistant Streptococcus pneumoniae, Macrolide resistant Streptococcus pneumoniae, Fluoroquinolone ant Streptococcus pneumoniae, Vancomycin resistant coccusfaecalis, Linezolid resistant Enterococcusfaecalis, Fluoroquinolone resistant Enterococcus faecalis, Vancomycin ant Enterococcus faecium, Linezolid ant Enterococcusfaecium, Fluoroquinolone resistant Enterococcus faecium, Ampicillin resistant Enterococcusfaecium, Macrolide resistant Haemophilus influenzae, B-lactam resistant Haemophilus influenzae, Fluoroquinolone resistant Haemophilus influenzae, B-lactam resistant Moraxella catarrhalis, Methicillin resistant Staphylococcus epidermiclis, Methicillin resistant lococcus epidermiclis, Vancomycin resistant lococcus epidermidis, Fluoroquinolone resistant Staphylococcus epidermiclis, Macrolide ant Mycoplasma pneumoniae, lsoniazid resistant Mycobacterium tuberculosis, Rifampin resistant Mycobacterium tuberculosis, Methicillin resistant Coagulase ve staphylococcus, Fluoroquinolone resistant ase negative lococcus, Glycopeptide intermediate ant Staphylococcus aureus, Vancomycin resistant Staphylococcus , Hetero vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin resistant Staphylococcus aureus, Macrolide-Lincosamide-Streptogramin resistant lococcus, B-lactam resistant coccusfaecalis, B-lactam resistant Enterococcusfaecium, Ketolide resistant Streptococcus pneumoniae, Ketolide resistant Streptococcus pyogenes, Macrolide resistant Streptococcus pyogenes, Vancomycin resistant staphylococcus epidermiclis, Fluoroquinolone resistant Neisseria gonorrhoeae, Multidrug Resistant Pseudomonas aeruginosa or Cephalosporin resistant Neisseria gonorrhoeae.

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

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

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

According to r embodiment, the quinolone resistant Staphylococci are selected from Fluoroquinolone resistant Staphylococcus aureus, Fluoroquinolone resistant Staphylococcus epidermiclis, or Fluoroquinolone resistant Coagulase negative staphylococcus.

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

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

According to another embodiment, the Linezolid resistant Enterococciare selected from Linezolid resistant Enterococcusfaecalis, or Linezolid resistant Enterococcusfaecium.

According to another embodiment, the Glycopeptide resistant Enterococci are ed from Vancomycin resistant Enterococcusfaecium or Vancomycin resistant Enterococcusfaecalis.

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

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

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

According to another embodiment, the Ketolide resistant Streptococci are selected from Macrolide resistant Streptococcus pneumoniae and de resistant Streptococcus According to another ment, the Fluoroquinolone resistant ococci is Fluoroquinolone resistant Streptococcus pneumoniae. ing to r embodiment, the B-lactam resistant Haemophilus is B-lactam resistant Haemophilus influenzae. -l6- According to another embodiment, the Fluoroquinolone resistant Haemophilus is Fluoroquinolone resistant hilus influenzae.

According to another ment, the Macrolide resistant Haemophilus is Macrolide resistant Haemophilus influenzae.

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

According to another embodiment, the Isoniazid resistant Mycobacterium is zid resistant Mycobacterium ulosis.

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

According to another embodiment, the B-lactam resistant lla is B-lactam resistant Moraxella catarrhalis.

According to another embodiment, the bacterial infection is characterized by the ce of one or more of the following: Methicillin resistant Staphylococcus , Fluoroquinolone resistant Staphylococcus aureus, Vancomycin intermediate resistant Staphylococcus , Linezolid resistant Staphylococcus aureus, Penicillin ant ococcus pneumoniae, Macrolide resistant Streptococcus pneumoniae, Fluoroquinolone resistant Streptococcus niae, Vancomycin resistant Enterococcusfaecalis, Linezolid resistant Enterococcus faecalis, Fluoroquinolone resistant Enterococcus faecalis, ycin resistant Enterococcusfaecium, Linezolid resistant Enterococcusfaecium, Fluoroquinolone ant Enterococcus faecium, Ampicillin resistant Enterococcus faecium, Macrolide resistant Haemophilus influenzae, B-lactam resistant Haemophilus zae, Fluoroquinolone resistant Haemophilus influenzae, B-lactam resistant Moraxella catarrhalis, Methicillin ant Staphylococcus epidermiclis, Methicillin resistant Staphylococcus epidermiclis, Vancomycin resistant Staphylococcus epidermiclis, Fluoroquinolone resistant Staphylococcus epidermiclis, Macrolide resistant Mycoplasma pneumoniae, Isoniazid resistant Mycobacterium tuberculosis, Rifampin resistant Mycobacterium ulosis, 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 ant Staphylococcus epidermiclis, Methicillin resistant Coagulase negative staphylococcus, Fluoroquinolone resistant Staphylococcus aureus, quinolone resistant Staphylococcus epidermtdts, Fluoroquinolone resistant Coagulase negative staphylococcus, Vancomycin resistant Staphylococcus aureus, Glycopeptide intermediate resistant Staphylococcus aureus, Vancomycin resistant Staphylococcus aureus, Vancomycin intermediate resistant lococcus aureus, Hetero vancomycin intermediate resistant Staphylococcus aureus, Hetero vancomycin resistant lococcus aureus, Vancomycin resistant Enterococcusfaect'um, Vancomycin resistant coccusfaecalt's, Penicillin ant Streptococcus pneumohtae, Macrolide resistant ococcus pneumohtae, Fluoroquinolone resistant Streptococcus pneumohtae, Macrolide resistant Streptococcus pyogenes, or B-lactam resistant Haemopht'lus influenzae.

According to another ment, the bacterial infection is characterized by the presence of one or more of the following: Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcusfaect'um, Vancomycin resistant Enterococcusfaecalt's, ycin ant Staphylococcus aureus, Vancomycin intermediate ant lococcus aureus, Hetero vancomycin intermediate resistant Staphylococcus , Hetero vancomycin resistant Staphylococcus aureus, Multidrug Resistant Pseudomohas aerugthosa, Isoniazid resistant Mycobactertum tuberculosis, and Rifampin resistant Mycobactert'um tuberculosis.

In on to the compounds of this invention, pharmaceutically acceptable derivatives or prodrugs of the compounds of this ion may also be employed in compositions to treat or prevent the above-identified disorders.

A "pharmaceutically acceptable derivative or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof. Particularly favored tives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e. g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. ceutically acceptable prodrugs of the nds of this ion include, without limitation, esters, amino acid esters, phosphate esters, metal salts and sulfonate esters.

Pharmaceutically acceptable salts of the compounds of this ion include those derived from pharmaceutically acceptable inorganic and organic acids and bases. es of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, rsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, oate, hexanoate, hloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, e, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as , while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the ion and their pharmaceutically acceptable acid addition salts.

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

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

Such compositions may ally comprise an additional therapeutic agent. Such agents include, but are not limited to, an antibiotic, an anti-inflammatory agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an suppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a grth , an immunomodulator, a prostaglandin or an anti-vascular roliferation compound.

The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof Pharmaceutically acceptable carriers that may be used in the pharmaceutical itions of this ion include, but are not limited to, ion exchangers, alumina, aluminum stearate, in, serum proteins, such as human serum albumin, buffer substances such as ates, glycine, sorbic acid, potassium sorbate, l glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, 2012/021270 disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene , sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, wool fat and self-emulsifying drug delivery s (SEDDS) such as alpha-tocopherol, polyethyleneglycol 1000 ate, or other similar polymeric delivery matrices.

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

Depending upon the particular condition, or disease state, to be treated or prevented, additional therapeutic agents, which are normally administered to treat or prevent that condition, may be administered er with the inhibitors of this invention. Such therapeutic agents include, but are not limited to, an otic, an anti-inflammatory agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a grth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound.

The compounds of this invention may be ed in a conventional manner for controlling bacterial infections levels in vivo and for treating diseases or reducing the advancement or severity of effects which are mediated by bacteria. Such methods of treatment, their dosage levels and requirements may be ed by those of ry skill in the art from available methods and techniques.

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

Alternatively, the compounds of this invention may be used in compositions and methods for treating or protecting individuals against bacterial infections or diseases over extended periods of time. In one embodiment, the compounds of this invention may be used in compositions and methods for ng or protecting individuals against bacterial infections or diseases over a 1-2 week period. In another embodiment, the compounds of this ion may be used in compositions and methods for treating or protecting individuals against bacterial infections or diseases over a 4-8 week period (for e, in the treatment of patients with or at risk for developing endocarditis or osteomyelitis). In another embodiment, the compounds of this invention may be used in compositions and methods for treating or protecting individuals against bacterial infections or diseases over an 8-12 week period. The compounds may be employed in such compositions either alone or together with other compounds of this invention in a manner consistent with the conventional ation of enzyme inhibitors in pharmaceutical compositions. For example, a compound of this ion may be combined with pharmaceutically able adjuvants conventionally employed in vaccines and administered in prophylactically ive s to protect duals over an ed period of time against bacterial ions or diseases.

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

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

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

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

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

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

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

In other embodiments, the additional eutic agents are selected from a natural penicillin including Benzathine llin G, llin G and Penicillin V, from a penicillinase-resistant penicillin including illin, Dicloxacillin, Nafcillin and Oxacillin, from a antipseudomonal penicillin including Carbenicillin, Mezlocillin, Pipercillin, Pipercillin/tazobactam, Ticaricillin and Ticaricillin/Clavulanate, from an aminopenicillin including Amoxicillin, llin and Ampicillin/Sulbactam, from a first generation cephalosporin including Cefazolin, oxil, CephaleXin and rine, from a second generation cephalosporin including Cefaclor, or-CD, Cefamandole, cid, Cefprozil, Loracarbef and xime, from a third generation cephalosporin including Cefdinir, Cefixime, Cefoperazone, Cefotaxime, Cefpodoxime, idime, Ceftibuten, Ceftizoxme and Ceftriaxone, from a fourth generation cephalosporin including Cefepime, Ceftaroline and Ceftobiprole, from a Cephamycin including Cefotetan and Cefoxitin, from a carbapenem including Doripenem, Imipenem and nem, from a monobactam including nam, from a quinolone including Cinoxacin, NalidiXic acid, Oxolininc acid and Pipemidic acid, from a fluoroquinolone including Besifloxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, xacin, Norfloxacin, Ofloxacin and Sparfloxacin, from an aminoglycoside including Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Spectinomycin, Streptomycin and Tobramycin, from a macrolide including Azithromycin, Clarithromycin and Erythromycin, from a ketolide including Telithromycin, from a ycline including Chlortetracycline, ocycline, Doxycycline, Minocycline and Tetracycline, from a glycopeptide including Oritavancin, Dalbavancin, Telavancin, Teicoplanin and Vancomycin, from a streptogramin including Dalfopristin/quinupristin, from an oxazolidone including Linezolid, from a cin including Rifabutin and Rifampin and from other antibiotics including bactitracin, colistin, l, Daptomycin, chloramphenicol, clindamycin, isoniazid, metronidazole, mupirocin, polymyXin B, pyrazinamide, trimethoprim/sulfamethoxazole and xazole.

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

WO 97269 In some embodiments, a solid form of a nd of formula (I), can be administered for the treatment of a gram positive infection. In some embodiments, the composition is a solid, liquid (e.g., a suspension), or an iv (e.g., a form of the formula (I) compound is dissolved into a liquid and stered iv) ition. In some embodiments, the composition ing a formula (I) compound, is administered in ation with an additional antibiotic agent, for example, a natural penicillin, a penicillinase-resistant penicillin, an antipseudomonal penicillin, an enicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a quinolone, a fluoroquinolone, an lycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a glycopeptide, a streptogramin, an oxazolidinone, a rifamycin, or a sulfonamide. In some embodiments, the composition including a solid form of a formula (I) compound is administered orally, and the additional otic agent, for example, a natural penicillin, a penicillinase-resistant penicillin, an antipseudomonal llin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third tion cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a quinolone, a uinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a eptide, a streptogramin, an oxazolidinone, a rifamycin, or a sulfonamide is administered iv.

In some embodiments, a solid form of a formula (I) compound, can be stered for the treatment of a gram negative infection. In some embodiments, the composition is a solid, liquid (e.g., a suspension), or an iv (e.g., a form of a formula (I) compound is dissolved into a liquid and administered iv) composition. In some ments the composition including a formula (I) compound is administered in combination with an additional antibiotic agent, selected from a: natural penicillin, a penicillinase-resistant penicillin, an antipseudomonal penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a monobactam, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, tetracycline or a amide. In some embodiments, the composition including a solid form of a formula (I) compound is administered orally, and the onal antibiotic agent, for example, a natural penicillin, a penicillinase-resistant penicillin, an antipseudomonal penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a monobactam, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, tetracycline or a sulfonamide is administered orally. In some embodiments, the additional therapeutic agent is administered iv.

] The onal therapeutic agents described above may be administered separately, as part of a multiple dosage regimen, from the inhibitor-containing composition.

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

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes aneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.

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

Among the able vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium de on. In addition, sterile, fixed oils are conventionally employed as a solvent or ding 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 hain alcohol t or dispersant, such as those described in Pharmacopeia Helvetica, or a similar alcohol.

WO 97269 The pharmaceutical itions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, rs which are commonly used e lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn . When aqueous suspensions and solutions and propylene glycol are administered orally, the active ingredient is combined with fying and ding agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

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

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

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

According to another embodiment, compounds of formula (I) may also be delivered by implantation (e.g., surgically), such as with an implantable or indwelling device.

An implantable or indwelling device may be designed to reside either permanently or temporarily in a subject. Examples of implantable and indwelling devices include, but are not d to, contact lenses, central venous catheters and needleless connectors, endotracheal tubes, intrauterine devices, ical heart valves, kers, peritoneal dialysis catheters, prosthetic joints, such as hip and knee replacements, tympanostomy tubes, y ers, voice prostheses, stents, delivery pumps, vascular filters and implantable control release compositions. ms can be detrimental to the health of patients with an implantable or ling medical device because they introduce an artificial substratum into the body and can cause persistent infections. Thus, providing compounds of formula (I) in or on the table or ling device can prevent or reduce the production of a biofilm. In addition, implantable or indwelling devices may be used as a depot or reservoir of compounds of a (I). Any implantable or indwelling device can be used to deliver compounds of formula (I) provided that a) the device, compounds of a (I) and any pharmaceutical composition including compounds of formula (I) are biocompatible, and b) that the device can deliver or release an effective amount of compounds of formula (I) to confer a therapeutic effect on the treated patient.

Delivery of therapeutic agents via implantable or indwelling devices is known in the art. See for example, “Recent Developments in Coated Stents” by Hofma et al. hed in Current Interventional Cardiology Reports 2001, 3:28-36, the entire contents of which, including references cited therein, incorporated herein by reference. Other descriptions of implantable devices can be found in US. Patent Nos. 195 and 6,322,847, and US.

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

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

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

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

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

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

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

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

When the compositions of this invention comprise a combination of a compound of a (I) and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 10% to 80% of the dosage normally administered in a monotherapy regime.

Upon improvement of a patient's condition, a maintenance dose of a nd, composition or combination of this ion may be administered, if necessary. uently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is ed when the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence or disease As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any ular t will depend upon a variety of factors, including the activity of the specific compound ed, the age, body weight, general health status, seX, diet, time of administration, rate of ion, drug combination, the severity and course of the disease, and the patient's disposition to the disease and the judgment of the treating physician.

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

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

The compounds of this invention may be ed in accordance with general methods known to those skilled in the art for analogous compounds, as taught by US. Pat.

No. RE40245 E, US. Pat. No. 7,495,014 B2, US. Pat. No. 7,569,591 B2, US. Pat. No. 641 B2, US. Pat. No. 974 B2, and US. Pat. No. 7,727,992 B2. All six of said patents are incorporated by reference as if fully set forth herein. The details of the conditions used for preparing the compounds of the t invention are further set forth in the Examples.

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

The following tions describe terms and abbreviations used herein: Ac acetyl Bu butyl Et ethyl Ph phenyl Me methyl THF tetrahydrofuran DCM dichloromethane CH2C12 dichloromethane EtOAc ethyl acetate CH3CN acetonitrile EtOH 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 ide Eth triethylamine DIPEA diisopropylethylamine DIEA diisopropylethylamine K2C03 potassium carbonate Na2C03 sodium carbonate Na2S203 sodium thiosulfate CS2C03 cesium carbonate NaHC03 sodium bicarbonate NaOH sodium hydroxide Na2SO4 sodium sulfate MgSO4, magnesium sulfate K3PO4 potassium phosphate NH4Cl ammonium chloride LC/MS liquid chromatography/mass spectra GCMS gas chromatography mass spectra HPLC high performance liquid chromatography GC gas chromatography LC liquid chromatography IC ion chromatography IM intramuscular u colony forming units MIC minimum tory concentration Hr or h hours atm atmospheres rt or RT room temperature TLC thin layer chromatography HCl hydrochloric acid H2O water EtNCO ethyl nate Pd/C palladium on carbon NaOAc sodium acetate H2SO4 ic acid N2 nitrogen gas H2 hydrogen gas n-BuLi n-butyl lithium DI de-ionized Pd(OAc)2 palladium(II)acetate PPh3 triphenylphosphine i-PrOH isopropyl alcohol NBS N—bromosuccinimide WO 97269 Pd[(Ph3)P]4 tetrakis(triphenylphosphine)palladium(0) PTFE polytetrafluoroethylene rpm tions per minute SM starting material Equiv. equivalents lH-NMR proton nuclear magnetic resonance HPMCAS ypropylmethylcellulose acetate PVP polyvinylpyrrolidone EDTA ethylenediaminetetraacetic acid KZEDTA dibasic potassium ethylenediaminetetraacetate mCPBA meta-chloroperoxyb enzoic acid aq aqueous BoczO di-tert-butyl dicarbonate DMAP methylaminopyridine mL milliliters L liters mol moles g grams LCMS liquid chromatography-mass spectrometry MHZ megahertz CDC13 deuterochloroform NEE triethylamine mmol millimoles psi pounds per square inch l'PrOH isopropylalcohol ppm parts per million NH4N03 ammonium nitrate Hz hertz Pd(dppDC12 [1,1 ’-Bis(diphenylphosphino)ferrocene] dichloropalladium(II) L liters MeOD deutero-methanol CD30D deutero-methanol ee enantiomeric excess min minutes Bn benzyl RBF round-bottom flask MeCN acetonitrile PES hersulfone mm millimeters um micrometers M molar N normal Boc tert—butoxycarbonyl ESMS electrospray mass spectrometry CV column volume D20 deuterium oxide NH3 ammonia OBD optimum bed density mg milligrams CLSI Clinical and Laboratory Standards Institute ATCC American Type Culture Collection MHII Mueller Hinton II uL microliters WT wild type CGSC Coli Genetic Stock Center MS mass ometry IS internal standard APCI heric pressure chemical ionization MRM multiple reaction monitoring m/z mass-to-charge ratio LLOQ lower limit of tation ng nanograms UV ultraviolet SD standard deviation % CV coefficient of variation PO perioral MC microcrystalline cellulose EDTA nediaminetetraacetic acid or nediaminetetraacetate PK pharmacokinetic IV intravenous D5W 5% dextrose in water solution HPMC-AS hydroxypropyl methylcellulose acetyl succinate PVP polyvinylprrolidone CAPT captisol ATP adenosine triphosphate ADP adenosine diphosphate NADH nicotinamide adenine dinucleotide (reduced form) NAD+ nicotinamide adenine dinucleotide (oxidized form) TRIS tris(hydroxymethyl)aminomethane mM millimolar MgClz magnesium chloride KCl potassium chloride uM micromolar DTT threitol nM nanomolar Ki dissociation constant ICso half maximal inhibitory concentration ug micrograms BSA bovine serum albumin LDH lactate dehydrogenase PVDF polyvinylidene fluoride AcN acetonitrile VMAx the maximum initial velocity or rate of a on Example 1 ation of 2-(2-nitrophenyl)-2,5-dihydrofuran and 2-(2-nitrophenyl)-2,3- dihydrofuran (3a&3b).

WO 97269 Pd(OAC)2, dppp + m + Br 0 1,4-dioxane, \ 0 / N02 K2C03, reflux N02 No2 o 1 2 3a 3b Mixed 1-bromonitro-benzene (1) (600 g, 99%, 2.941 mol, Alfa Aesar A11686), 1,3-bis(diphenylphosphino)propane (62.50 g, 97%, 147.0 mmol, Alfa Aesar A12931), 1,4- dioxane (2.970 L, Sigma-Aldrich 360481), potassium carbonate (812.9 g, 5.882 mol, JT- Baker 301201), and 2,3-dihydrofuran (2) (1.041 kg, 99%, 1.124 L, 14.70 mol, h 200018). A stream of nitrogen was bubbled through the ng mixture for 4 hrs, followed by addition of palladium (II) acetate (16.51 g, 73.52 mmol, Strem 461780) and uation of deoxygenation for another 10 minutes. The reaction mixture was stirred at reflux under nitrogen overnight (NMR of a -up aliquot showed complete consumption of arylbromide). It was d to cool, diluted with hexane (1 L), filtered through a short plug of Florisil® (500 g, -200 mesh), and eluted with EtOAc. The filtrate was concentrated under reduced re (2-(2-nitropheny1)-2,3-dihydrofuran is volatile under high vacuum and may be somewhat unstable at room temperature) giving a mixture of (3a) and (3b) as a dark brown oil (654.0 g). The crude material was stored in the refrigerator and carried forward without further purification.

Example 2 Preparation of 2-tetrahydrofuranyl-aniline (4). psi H2, Pd/C NEt3, MeOH rt N02 02 O NH2 0 Placed 5% palladium on carbon (16.3 g, 50% wet, 3.83 mmol, Aldrich 330116) in a Parr bottle under nitrogen, followed by MeOH (100 mL, JT-Baker 909333). Added the crude mixture of 2-(2-nitrophenyl)-2,5-dihydrofuran and 2-(2-nitrophenyl)-2,3-dihydrofuran (3a&3b)) (163 g) dissolved in MeOH (389 mL), followed by NEt3 (237.6 mL, 1.705 mol, Sigma-Aldrich 471283). Placed the vessel on a Parr shaker and ted with H2. Added 30 psi H2 and shook until consumption complete (LCMS and NMR showed complete reaction).

The reaction e was purged with nitrogen, filtered through CeliteTM and rinsed with EtOAc. The filtrate was concentrated on a rotary evaporator giving a brown oil. Repeated the reaction three more times on the same scale and the batches were combined for purification. The crude product was vacuum distilled (ca. 15 torr) ting the distillate at 108 - 129°C to give (4) as a clear faint yellow oil (427.9 g, average yield was 84%; 98% GCMS purity). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 163.95 (1.46 min). 1H NMR (300 MHz, CDClg): 5 7.15 — 7.04 (m, 2H), 6.77 — 6.62 (m, 2H), 4.85 — 4.77 (m, 1H), 4.18 (s, 2H), 4.12 — 4.02 (m, 1H), 3.94 — 3.85 (m, 1H), 2.25 — 1.95 (m, 4H) ppm. e 2a Preparation of (R)(tetrahydrofuranyl)aniline (4a).

SFC chiral tion —> NH2 0 NH2 0 (4a) 95.5% ee 37% yield ved 33g of compound (4) into MeOH (265 ml) which resulted in a concentration of imately 125mg/ml. The mixture was filtered through a 0.2 micron membrane filter then chromatographed on a ChiralPak® IC column (30mm X 150mm, column temp 350C, Chiral Technologies) at 100 bar using a Berger multigram supercritical fluid chromatographic system. Mobile phase was (90:10) COzzCH30H eluting at 350ml/min with UV monitoring at 220 nanometers. ed 15.64g of desired product (421) as a green oil. Analytical SFC ([90:10] COzzCH30H, at 5ml/min on a ChiralPak IC column (4.6 X 100mm) held at 35°C and run at 100bar pressure with UV monitoring at 220nm) showed 95.5% ee with 95% overall purity.

Example 3 Preparation of 4-bromotetrahydrofuranyl-aniline (5).

NH2 0 MTBE, CH3CN NH? O NBS, 2°C 4 5 To a stirring solution of ahydrofuranyl-aniline (4) (53.45 g, 327.5 mmol) in methyl tert-butyl ether(MTBE, 641.4 mL) and acetonitrile (213.8 mL) cooled to 2°C was added N—bromosuccinimide (58.88 g, 99%, 327.5 mmol, Aldrich B81255) in 4 portions maintaining internal temperature below about 8°C. The reaction mixture was stirred while cooling with an ice-water bath for 30 minutes (NMR of a worked-up aliquot showed complete consumption of starting material). Added aqueous 1 N Na28203 (330 mL), removed the cold bath and stirred for 20 s. The mixture was diluted with EtOAc and the layers were separated. The organic phase was washed with ted aqueous NaHC03 (2x), water, brine, dried over MgSO4, filtered through a short plug of silica, eluted with EtOAc, and concentrated under reduced pressure to give (5) as a very dark amber oil (82.25 g, 77-94% HPLC purity). Carried forward t further purification. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 242.10 (2.89 min). 1H NMR (300 MHz, CDC13) 5 7.22 (d, J = 2.3 Hz, 1H), 7.16 (dd, J = 8.4, 2.3 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H), 4.79 — 4.73 (m, 1H), 4.15 (s, 2H), 4.10 — 4.01 (m, 1H), 3.93 — 3.85 (n1, 1H), 2.26 — 2.13 (m, 1H), 2.12 — 1.97 (m, 3H) ppm.

Example 4 Preparation of N—(4-bromonitrotetrahydrofuranyl-phenyl)-2,2,2-trifluoro- ide (6).

TFAA, Me-THF 2°C to rt , rt to 40°C 0 NH 0 NHZO Y CF3 To trifluoroacetic anhydride (455.3 mL, 3.275 mol, Sigma-Aldrich ) stirring at 2°C was slowly added 4-bromotetrahydrofuranyl-aniline (5) (79.29 g, 327 .5 mmol) as a thick oil via addition funnel over 15 minutes (reaction temperature rose to 14°C).

The remaining oil was rinsed into the reaction mixture with anhydrous 2- tetrahydrofuran (39.6 mL, Sigma-Aldrich 414247). The cold bath was removed and ammonium nitrate (34.08 g, 425.8 mmol, Aldrich ) was added. The reaction temperature rose to 40°C over about 30 minutes at which time a cold water bath was used to control the exotherm and bring the reaction to room temperature. The cold bath was then 2012/021270 removed and stirring continued for another 40 s (HPLC showed very little remaining un-nitrated material). The reaction mixture was slowly poured into a stirring mixture of d ice (800 g). The solid precipitate was collected by filtration, washed with water, saturated aqueous NaHC03 (to pH 8), water again, and hexane. The wet solid was dried first in a convection oven at 50°C for several hours and then under reduced pressure in an oven at 40°C overnight giving (6) as a light brown solid (77.86 g, 62% yield, 98% HPLC purity).

LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 383.19 (3.27 min). 1H NMR (300 MHz, CDClg) 5 9.81 (s, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H), 4.88 (dd, J = 9.0, 6.5 Hz, 1H), 4.17 — 4.08 (m, 1H), 4.03 — 3.95 (m, 1H), 2.45 — 2.34 (m, 1H),2.17 — 2.06 (m,2H), 1.96 — 1.83 (n1, 1H) Example 5 Preparation of 4-bromonitrotetrahydrofuranyl-aniline (6a). aq NaOH OZN 1,4-dioxane reflux OZN O NH O Y NH2 O Dissolved N—(4-bromonitrotetrahydrofuranyl-phenyl)-2,2,2-trifluoroacetamide (6) (54.00 g, 140.9 mmol) in 1,4-dioxane (162 mL) and added aqueous 6 M NaOH (70.45 mL, 422.7 mmol, JT-Baker 567202). The reaction mixture was stirred at reflux for 2 days (HPLC showed complete conversion), allowed to cool, diluted with MTBE (800 mL), washed with water (2 x 200 mL), ted aqueous NH4Cl, water and brine. The mixture was dried over MgSO4, filtered, and concentrated under reduced pressure to give (621) as a dark amber oil (40.96 g, 93% yield, overall 92% HPLC plus NMR ). LCMS (C18 column eluting with 10-90% MeOH / water gradient from 3-5 minutes with formic acid modifier) M+1: 287.28 (3.44 min). 1H NMR (300 MHz, CDClg) 5 8.24 (d, J = 2.4 Hz, 1H), 7.41 (d, J = 2.3 Hz, 1H), 6.91 (s, 2H), 4.80 (t, J = 7.2 Hz, 1H), 4.14 — 4.05 (m, 1H), 3.98 — 3.90 (m, 1H), 2.36 — 2.19 (m, 1H), 2.15 — 2.01 (n1, 3H) ppm.

Example 6 Preparation of 2-[5-(4-aminonitrotetrahydrofuran—2-yl-phenyl)pyrimidin pan—2-ol (8).

Br iOH N \N Pd(PPh3)4 NI | aq Na C02 3 / 1,4-dioxane OZN O/B\O reflux NH2 0 H NH2 0 7 8 Mixed 4-bromonitrotetrahydrofuran—2-yl-aniline (621) (40.40 g, 92%, 129.5 mmol), l,4-dioxane (260 mL, Sigma-Aldrich 360481), 2-[5 -(4,4,5,5-tetramethyl-l,3,2- dioxaborolan—2-yl)pyrimidinyl]propanol (7) (41.05 g, 155.4 mmol), and aqueous 2.7 M Na2C03 (143.9 mL, 388.5 mmol). A stream of nitrogen was bubbled through the stirring mixture for 1 hr, followed by addition of tetrakis(triphenylphosphine)palladium (0) (7.48 g, 6.47 mmol, Strem 462150). The reaction mixture was stirred at reflux for 2 hrs (HPLC showed complete reaction), allowed to cool, diluted with EtOAc, washed with water, saturated aqueous NH4Cl, brine, dried over MgSO4, and filtered through a short plug of Florisil® eluting with EtOAc. The filtrate was concentrated under d re giving adark brown oil. Dissolved in CH2C12 and eluted through a short plug of silica gel with CH2C12 and then EtOAc. The desired fraction was concentrated on a rotary evaporator until a precipitate formed giving a thick brown , which was triturated with MTBE. The solid was collected by ion, washed with MTBE, and dried under high vacuum giving (8) as a yellow solid (35.14 g, 99+% HPLC purity,). LCMS (C18 column eluting with 10-90% CH3CN / water nt over 5 minutes with formic acid modifier) M+1: 345.00 (2.69 min). 1H NMR (300 MHz, CDC13)5 8.88 (s, 2H), 8.36 (d, J = 2.2 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.09 (s, 2H), 4.92 (t, J = 7.2 Hz, 1H), 4.62 (s, 1H), 4.20 — 4.11 (n1, 1H), 4.03 — 3.94 (m, 1H), 2.39 — 2.26 (n1, 1H), 2.23 — 2.08 (m, 3H), 1.64 (s, 6H) ppm.The filtrate was further trated and purified by ISCO silica gel chromatography eluting with 0 to 80% EtOAc/ hexane giving a second crop of product (8) as an amber solid (4.46 g, 88% overall yield, 88 % HPLC purity.

Example 7 Alternate preparation of 2-[5 -(4-aminonitro-5 -tetrahydrofuran—2-yl- phenyl)pyrimidinyl]propanol (8).

N \ N \N N Pd(dppf)C|2 | I / / aq Na2CO3 OZN + B 1,4-dioxane OYNH O O/ \o reflux CF3 M 6 NH2 0 Mixed N—(4-bromonitro tetrahydrofuran—2-yl-phenyl)-2,2,2-tn'fiuoro-acetamide (6) (19.00 g, 49.59 mmol), 2-[5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan—2-yl)pyrimidinyl]propan—2-ol (7) (14.41 g, 54.55 mmol), aqueous 2.7 M sodium carbonate (73.48 mL, 198.4 mmol), and 1,4-dioxane (190 mL, Aldrich 360481). A stream of nitrogen was bubbled through the stirring mixture for 40 minutes, followed by on of 1,1’-bis(diphenylphosphino)ferrocene dichloropalladium dichloromethane adduct (2.025 g, 2.480 mmol, Strem 460450). The reaction mixture was stirred at reflux under N2 for 7 hrs, added another 50 mL of saturated s sodium carbonate, and refluxed for another 16 hrs. The mixture was allowed to cool, then diluted with EtOAc (500 mL) and water (200 mL). The layers were separated and the aqueous phase extracted with EtOAc (200 mL). The ed organic phase was washed with water (500 mL), brine (500 mL), dried over Na2804, filtered through a Florisil® plug, and concentrated on a rotary ator to give crude (8) as an orange oil. Crude product was purified by ISCO silica gel chromatography g with 20-90% EtOAc / hexane to give (8) as an orange solid (15.00 g, 87% yield; 81-88% purity). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 345.35 (2.68 min). 1H NMR (300 MHz, 5 8.88 (s, 2H), 8.36 (d, J = 2.2 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.09 (s, 2H), 4.92 (t, J = 7.2 Hz, 1H), 4.62 (s, 1H), 4.20 — 4.11 (m, 1H), 4.03 — 3.94 (m, 1H), 2.39 — 2.26 (m, 1H), 2.23 — 2.08 (m, 3H), 1.64 (s, 6H) ppm.

Example 8 Preparation of 2-[5-(3,4-diamino-5 -tetrahydrofuranyl-phenyl)pyrimidin yl]propan—2-ol (9). 2%ZOI OI Z Z I 45 psi H2 Pd/C I \ \ NEt3 MeOH THF To a suspension of 4-aminonitro-5 -tetrahydrofiiranyl- phenyl)pyrimidinyl]propanol (8) (30.10 g, 87.41 mmol) and THF (90 mL) in a Parr bottle under nitrogen was added a slurry of 5% ium on carbon (3.01 g, 50% wet, 0.707 mmol, Aldrich 330116) in MeOH (90 mL, JT-Baker 909333), followed by NEt3 (24.37 mL, 174.8 mmol, Sigma-Aldrich 471283). Placed the vessel on a Parr shaker and saturated with H2. Added 45 psi H2 and shook until consumption was complete (HPLC showed complete sion). The reaction mixture was purged with nitrogen, filtered through CeliteTM and rinsed with EtOAc. The filtrate was re-filtered through a 0.5 micron glass fiber filter paper ched between two P5 papers, and concentrated under reduced pressure giving (9) as a light brown foam (28.96 g, 98% yield, 93% NMR purity). LCMS (C18 column eluting with -90% CH3CN / water nt over 5 minutes with formic acid modifier) M+1: 315.32 (1.54 min). 1H NMR (300 MHz, CDC13)5 8.83 (s, 2H), 6.92 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 1.8 Hz, 1H), 4.90 (dd, J = 7.9, 6.2 Hz, 1H), 4.72 (s, 1H), 4.18 (s, 2H), 4.17 — 4.08 (m, 1H), 3.99 — 3.89 (n1, 1H), 3.46 (s, 2H), 2.34 — 2.19 (n1, 1H), 2.17 — 2.05 (m, 3H), 1.63 (s, 6H) ppm.

Example 9 Preparation of 1-ethyl-3 -[5-[2-(1-hydroxymethyl-ethyl)pyrimidin-5 -yl] tetrahydrofuran—2-yl-1H-benzimidazolyl]urea (1 1). :0H :0H EtHNO s/ o JQN JLNHEt pH 3.5 buffer dioxane reflux HN:NHHN>:11 To a stirring solution of 2-[5-(3,4-diaminotetrahydrofuranyl- phenyl)pyrimidinyl]propanol (9) (32.10 g, 102.1 mmol) in 1,4-dioxane (160.5 mL, Sigma-Aldrich 360481) was added pH 3.5 buffer (240.8 mL), prepared by dissolving NaOAc trihydrate (34.5 g) in IN aqueous H2804 (240 mL). Added 1-ethyl(N-(ethylcarbamoyl)-C- methylsulfanyl-carbonimidoyl)urea (10) (28.46 g, 122.5 mmol, CB Research and pment) and stirred at reflux overnight (HPLC showed 99% consumption of starting e). The reaction mixture was cooled to room temperature and poured portion-wise (frothing) into a stirring solution of aqueous saturated NaHC03 (480 mL) and water (120 mL) giving pH 8-9. This was stirred for 30 minutes, the solid was collected by filtration, washed copiously with water to l pH, and then more sparingly with EtOH. The solid was dried under reduced pressure giving (11) as an off-white solid (34.48 g, 82% yield; 99.4% HPLC purity). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid r) M+1: 411.41 (1.73 min). 1H NMR (300 MHz, MeOD) 5 9.02 (s, 2H), 7.62 (s, 1H), 7.37 (s, 1H), 5.31 (s, 1H), 4.23 (dd, J = 14.5, 7.3 Hz, 1H), 4.01 (dd, J = .0, 7.1 Hz, 1H), 3.38 — 3.28 (m, 2H),2.58 — 2.46 (m, 1H),2.16 — 2.05 (m, 2H), 2.02 — 1.88 (m, 1H), 1.63 (s, 6H), 1.22 (t, J = 7.2 Hz, 3H) ppm.

Example 10 Chiral chromatographic isolation of l-3 - [5 -[2-(1-hydroxymethyl- ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea (12). iOH fm N ‘\N N ‘\N I I / / chiral chrom N N\ >LNH o >’NH o HN HN >20 /EO HN 11 HN ) ) 12 A racemic sample of 1-ethyl[5-[2-(1-hydroxymethyl-ethyl)pyrimidinyl]- 7-tetrahydrofuranyl-1H—benzimidazolyl]urea (11) (24.60 g) was resolved on a CHIRALPAK® IC® column (by Chiral Technologies) g with DCM / MeOH / TBA (60 / 40 / 0.1) at 35°C giving the desired enantiomer (12) as a white solid (11.35 g, 45% yield; 99+% HPLC purity, 99+% ee). Analytical chiral HPLC retention time was 6.2 min (CHIRALPAK® IC® 4.6 X 250 mm column, 1 mL/min flow rate, 30°C).

] The structure and te stereochemistry of 12 were confirmed by single-crystal x-ray diffraction analysis. Single crystal diffraction data were acquired on a Bruker Apex II diffractometer equipped with sealed tube Cu K-alpha source (Cu Kd radiation, y = 1.54178 A) and an Apex II CCD detector. A crystal with dimensions of 1/2x 0.05 x 0.05 mm was selected, cleaned using mineral oil, mounted on a MicroMount and centered on a Bruker APEXTI system. Three batches of 40 frames separated in reciprocal space were obtained to provide an orientation matrix and initial cell ters. Final cell parameters were obtained and refined after data collection was completed based on the full data set. Based on systematic absences and intensities statistics the structure was solved and refined in acentric P21 space group.

A diffraction data set of reciprocal space was obtained to a resolution of 0.9 A using 05° steps using 60 s exposure for each frame. Data were collected at 100 (2) K.

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

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

The data were collected, refined and d using the Apex 11 software. The structure was solved using the SHELXS97 (Sheldrick, 1990), program(s) and the structure refined using the SHELXL97 (Sheldrick, 1997) m. The crystal shows inic cell with P21 space group. The lattice parameters are a = 9.8423(4) A, b = 10.8426(3) A, c = 19.4441 (7) A, [5: 6(3)°. Volume = 9(12) A3.

Example 11 Preparation of the esulfonic acid salt of l-ethyl[5-[2-(l-hydroxy-l - methyl-ethyl)pyrimidinyl][(2R)-tetrahydrofuran—2-yl]-lH-benzimidazolyl]urea (13).

N \N MeSO3H >’NH DCM,EtOH 2°C—rt ) 12 A stirring sion of 1-ethyl[5-[2-(1-hydroxymethyl-ethyl)pyrimidin yl][(2R)-tetrahydrofuran—2-yl]-1H-benzimidazolyl]urea (12) (9.32 g, 22.71 mmol) in absolute ethanol (93.2 mL) was cooled with an ice-water bath. Added methanesulfonic acid (1.548 mL, 23.85 mmol, Sigma-Aldrich 471356), removed cold bath and stirred at room temperature for 20 minutes. It was concentrated on a rotary evaporator at 35°C to a thick slurry, d with EtOAc, ted the solid by filtration, washed with EtOAc, and dried under reduced pressure giving an initial crop of (13) as a white solid (8.10 g). The filtrate was concentrated on a rotavap giving a yellowish glassy foam, which was dissolved in EtOH, concentrated to a solid slurry, triturated with EtOAc / Et20, and collected by filtration. The solid was washed with EtOAc / Et20, combined with the first crop, and dried under reduced re giving (13) as a white solid (9.89 g, 86% yield; 99+% HPLC purity, 99+% ee).

Analytical chiral HPLC shows one omer with retention time of 6.3 min eluting with DCM / MeOH / TEA (60 / 40 / 0.1) on a CHIRALPAK® IC® 4.6 X 250 mm column with 1 mL/min flow rate at 30°C. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 411.53 (1.74 min). 1H NMR (300 MHz, MeOD) 5 9.07 (s, 2H), 7.79 (s, 1H), 7.62 (s, 1H), 5.30 (t, J = 7.3 Hz, 1H), 4.24 (dd, J = 14.6, 7.3 Hz, 1H), 4.04 (dd, J = 15.0, 7.6 Hz, 1H), 3.40 — 3.30 (m, 2H),2.72 (s, 3H), 2.65 — 2.54 (m, 1H),2.20 — 2.07 (m, 2H), 2.04 — 1.90 (m, 1H), 1.64 (s, 6H), 1.23 (t, J = 7.2 Hz, 3H) Example 12 Preparation of 2-(2-fluoronitro-phenyl)-2,3-dihydrofuran (15A) and 2-(2- fluoronitro-phenyl)-2,5 -dihydrofuran (15B). szBr2(tBu3P)2 + m + Br 0 EtN(iPr)2, dioxane, reflux \ N02 N02 0 N02 0 15A 15B 2-Bromofluoronitro-benzene (14) (200.3 g, 98%, 892.3 mmol, Bosche F6657), 1,4-dioxane (981.5 mL, Sigma-Aldrich 360481), and 2,3-dihydrofuran (2) (341.1 mL, 99%, 4.462 mol, Aldrich 200018) were d in a reaction flask, followed by N,N— diisopropylethylamine (155.4 mL, 892.3 mmol, Sigma-Aldrich 550043) and bromo(tri-tertbutylphosphine )pa11adium(I) dimer (6.936 g, 8.923 mmol, n Matthey C4099). The mixture was stirred at reflux for 2 hrs (HPLC showed 98% consumption of starting arylbromide). It was allowed to cool, the precipitate was removed by filtration, rinsed with EtOAc, and the filtrate concentrated in vacuo to a dark h brown semi-solid oil. This was dissolved in CH2C12, eluted through a plug of silica with CH2C12, and concentrated in vacuo giving a mixture of 15A and 15B as a dark amber oil (291.3 g). The crude product was d forward without further purification. The major product was 2-(2-fluoronitro- pheny1)-2,3-dihydrofuran (15A) (96%): LCMS (C18 column eluting with 10-90% CH3CN/ water gradient over 5 minutes with formic acid modifier) M+1: 210.23 (3.13 min), 1H NMR (300 MHZ, CDC13) 5 7.54 (dt, J = 8.0, 1.2 Hz, 1H), 7.43 (td, J = 8.2, 5.2 Hz, 1H), 7.32 (ddd, J = 9.7, 8.3, 1.3 Hz, 1H), 6.33 (dd, J = 4.9, 2.4 Hz, 1H), 5.80 (t, J = 10.9 Hz, 1H), 5.06 (q, J = 2.4 Hz, 1H), 3.18 — 3.07 (m, 1H), 2.94 — 2.82 (m, 1H) ppm. The minor product was 2-(2- fluoronitro-pheny1)-2,5-dihydrofuran (15B) (4%): GCMS (Agilent HP-5MS 30 m x 250 um x 0.25 pm column g at 60°C for 2 min to 300°C over 15 min with a 1 mL/min flow rate) M+1: 210 (11.95 min). 1H NMR (300 MHz, CDC13)6 7.47 (d, J = 8.0 Hz, 1H), 7.43 — 7.34 (m, 1H), 7.30 — 7.23 (m, 1H), 6.21 — 6.15 (n1, 1H), 6.11 — 6.06 (m, 1H), 5.97 — 5.91 (m, 1H), 4.89 — 4.73 (m, 2H) ppm.

Example 13 ation of 3-fluorotetrahydrofurany1—aniline (16). psi H2, Pd/C No2 NEt3 MeOH rt NH2 0 Placed 5% palladium on carbon (37.3 g, 50% wet, 8.76 mmol, Aldrich 330116) in a Parr bottle under nitrogen, followed by MeOH (70 mL, JT-Baker 909333). Added the crude mixture of 2-(2-fluoronitro-phenyl)-2,3-dihydrofuran and 2-(2-fluoronitro- )-2,5-dihydrofuran (15A&15B) (186.6 g, 892.1 mmol) dissolved in MeOH (117 mL), followed by NEt3 (124.3 mL, 892.1 mmol, Sigma-Aldrich 471283). Placed the vessel on a Parr shaker and saturated with H2. After adding 45 psi H2, the reaction mixture was shaken until consumption of the ng material was complete (HPLC and LCMS showed te reaction). The reaction mixture was purged with nitrogen, filtered through CeliteTM and rinsed with EtOAc. The filtrate was concentrated on a rotary evaporator giving a brown oil, which was dissolved in EtzO and washed with water (2x). The ether phase was extracted mmmmmm1N1EM5xfiOmDJmMnmmwmwwwflmoGammmmbwmw with aqueous 6 N NaOH to pH 12-14. The basic aqueous phase was extracted with CH2C12(4x), and the combined c extract washed with saturated aqueous NH4Cl, dried over MgSO4, and filtered h a pad of silica eluting with CH2C12 to 25% EtOAc / hexane.

The desired te was trated under reduced pressure giving 16 as a light brown oil (121.8 g, 84% GCMS plus NMR purity). GCMS (Agilent HP-5MS 30 m x 250 um x 0.25 pm column heating at 60°C for 2 min to 300°C over 15 min with a 1 mL/min flow rate) M+1: 182.0 (11.44 min). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 182.10 (2.61 min). 1H NMR (300 MHz, CDClg) 5 6.97 (td, J = 8.1, 6.3 Hz, 1H), 6.43 — 6.35 (n1, 2H), 5.21 — 5.13 (m, 1H), 4.54 (s, 2H), 4.16 — 4.07 (m, 1H), 3.90 — 3.81 (m, 1H), 2.23 — 2.00 (m, 4H) ppm. Additional crops were obtained as follows: the combined ether phase was washed with saturated aqueous NaHC03, brine, dried over NaZSO4, decanted, and concentrated under reduced pressure. The oil was vacuum distilled (ca. 15 torr) collecting the distillate at 101 — 108°C. To a stirring solution of the distilled oil in EtOH (1 volume) at 2°C was slowly added 5 M HCl (1 eq) in iPrOH. The resulting suspension was brought to room ature, diluted with EtOAc (3 volumes, vol/vol), and stirred for 2 hrs. The white solid was collected by filtration, washed with EtOAc, and dried under d pressure giving a second crop of product as the HCl salt.

The mother liquor was concentrated to a slurry, diluted with EtOAc and the solid ted by filtration, washed with EtOAc, and dried in vacuo giving the HCl salt as a third crop of the product. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 182.10 (2.58 min). 1H NMR (300 MHz, CDCl3) 5 10.73 (br.s, 3H), 7.66 (d, J = 8.1 Hz, 1H), 7.33 (td, J = 8.2, 5.9 Hz, 1H), 7.13 — 7.05 (m, 1H), 5.26 (dd, J = 9.0, 6.5 Hz, 1H), 4.38 — 4.28 (m, 1H), 4.00 — 3.91 (n1, 1H), 2.59 — 2.46 (m, 1H), 2.30 — 1.95 (m, 3H) ppm. The overall yield from the three crops was 76%.

Example 14 Preparation of 4-bromo-3 -fluorotetrahydrofuran—2-yl-aniline (17).

F F MTBE, CH3CN NBS, -15°C NH2 0 NH2 0 16 17 To a ng solution of 3-fluorotetrahydrofuran—2-yl-aniline (16) (131.9 g, 92%, 669.7 mmol) in methyl tert—butyl ether (1.456 L) and acetonitrile (485 mL) cooled to - °C was added N—bromosuccinimide (120.4 g, 99%, 669.7 mmol, Aldrich B81255) in 3 portions maintaining a reaction temperature below about -15°C. After complete addition ng was ued at -15 to -10°C for 30 minutes. 1H NMR of a worked-up aliquot showed 96% consumption of starting aniline so added another 4.82 g NBS and stirred at - °C for another 30 minutes. Aqueous 1 N Na28203 (670 mL)was added to the reaction mixture. The cold bath was removed, the mixture stirred for 20 minutes, then diluted with EtOAc. The layers were separated and the organic phase was washed with saturated aqueous NaHC03 (2x), water, brine, dried over NaZSO4, decanted, and concentrated under reduced re giving a dark amber oil. The residue was diluted with hexane and eluted through a short plug of silica eluting with 25% EtOAc / hexane to 50% EtOAc / hexane. The d filtrate was concentrated in vacuo giving 17 as a dark amber oil (182.9 g, 90% yield, 86% NMR purity). LCMS (C18 column eluting with 10-90% CH3CN/ water gradient over 5 minutes with formic acid modifier) M+1: 260.12 (3.20 min). 1H NMR (300 MHz, CDClg) 5 7.15 (dd, J = 8.6, 7.6 Hz,1H), 6.30 (dd, J = 87,13 Hz,1H), 5.19 — 5.12 (m, 1H), 4.58 (s, 2H), 4.16 — 4.07 (n1, 1H), 3.90 — 3.81 (m, 1H), 2.23 — 1.99 (m, 4H) ppm. e 15 Preparation of N—(4-bromo-3 -fluoronitrotetrahydrofuranyl-phenyl)-2,2,2- trifluoro-acetamide (18).

WO 97269 Br Br F i) TFAA, THF 2°C to rt ii) NH4NO3, 30 to 41°C 02N NH2 0 OYNH o 17 CF3 To trifluoroacetic anhydride (565.3 mL, 4.067 mol, Sigma-Aldrich 106232) stirring at 2°C was slowly added neat 4-bromo-3 -fluorotetrahydrofuran—2-yl-aniline (17) (123.0 g, 86%, 406.7 mmol) as a thick oil via addition funnel over about 20 minutes ion temperature rose to 13°C). The remaining oil was rinsed into the reaction mixture with ous THF (35 mL). The cold bath was removed and the on was heated to 35°C, followed by portion-wise addition ofNH4N03 (4.88 g x 20 portions, 1.22 mol, Sigma- Aldrich A7455) over 2.5 hrs maintaining the reaction temperature between 30 and 41°C using an ice-water bath only as needed to control the exotherm. After te addition the reaction mixture was stirred for another 10 minutes (HPLC showed reaction 99% complete).

It was slowly poured into crushed ice (1.23 kg) and d for 1 hr to allow formation of a filterable solid precipitate, which was collected and washed with water, sparingly with saturated aqueous NaHC03, and water again (to pH 7). The product was dried in a convection oven overnight at 40°C and then under reduced pressure in an oven at 50°C overnight giving 18 as a beige solid (152.5 g, 90% yield, 96% HPLC purity). LCMS (C18 column g with 10-90% CH3CN/ water gradient over 5 minutes with formic acid modifier) M+l: 401.30 (3.41 min).1H NMR (300 MHz, CDC13)5 10.56 (s, 1H), 8.19 (d, J = 6.6 Hz, 1H), 5.22 (dd, J = 10.3, 6.4 Hz, 1H), 4.22 (dd, J = 15.8, 7.2 Hz, 1H), 3.99 (dd, J = 16.1, 7.5 Hz, 1H), 2.50 — 2.38 (m, 1H), 2.22 — 2.11 (m,2H), 1.86 — 1.71 (m, 1H) ppm.

Example 16 Preparation of 4-bromo-3 -fluoronitrotetrahydrofuran—2-yl-aniline (19).

Br Br F F aq st04 1,4-dioxane OQN —>OQN reflux, 5 days OYNH O NH2 0 A reaction flask was charged with N—(4-bromo-3 -fluoronitrotetrahydrofuran- 2-yl-phenyl)-2,2,2-trifluoro-acetamide (18) (242.3 g, 604.1 mmol), 1,4-dioxane (1.212 L), aqueous 2 M sulfuric acid (362.4 mL, 724.9 mmol), and stirred at reflux for 5 days (HPLC showed 98% conversion). Allowed to cool, diluted with EtOAc, neutralized with saturated aqueous NaHC03, separated the layers, and re-extracted the aqueous phase with EtOAc (2x).

The combined organic phase was washed with brine (2x), dried over MgSO4, filtered and concentrated in vacuo giving 19 as a greenish brown solid (181.7 g, 94% yield, 95% HPLC purity). The product was carried to the next step without further cation. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 305.20 (3.63 min). 1H NMR (300 MHz, CDClg) 5 8.35 (d, J = 7.3 Hz, 1H), 7.45 (s, 2H), 5.23 — 5.16 (m, 1H), 4.23 — 4.14 (n1, 1H), 3.93 — 3.84 (m, 1H), 2.31 — 1.96 (m, 4H) ppm.

Example 17 Preparation of 4-aminofluoronitrotetrahydrofuranylphenyl idinyl]propanol (20).

E OH Br E:OH N \N Pd(dppf)C|2 N \ N F '/ fl, ' /B\ oxane, reflux F O O NH2 0 4+ 19 7 ngz To a stirring solution of 4-bromofluoronitrotetrahydrofuranyl-aniline (19) (525.0 g, 1.721 mol, Bridge Organics Co.) in 1,4-dioxane (4.20 L, Sigma-Aldrich 360481) was added a 1.2 M aqueous solution ofNaHC03 (4.302 L, 5.163 mol). A stream of nitrogen was bubbled through the stirring mixture for 2 hrs, followed by addition of 2-[5- ,5-tetramethyl-1,3,2-dioxaborolan—2-yl)pyrimidinyl]propan—2-ol (7) (545.4 g, 2.065 mol, Bridge Organics Co.) and 1,1’-bis(diphenylphosphino)ferrocene dichloropalladium dichloromethane adduct (42.16 g, 51.63 mmol, Strem 460450). The reaction mixture was stirred at reflux overnight, allowed to cool, diluted with EtOAc (8.4 L), and the layers were separated. The c phase was washed with saturated s NH4Cl and then brine. The aqueous phase was re-extracted with EtOAc (4 L) and washed this organic extract with brine.

The combined organic phase was dried over MgSO4, d through a short plug of Florisil®, eluted with EtOAc, and the filtrate concentrated on a rotary evaporator giving a dark brown wet solid. This was dissolved in CHzClz, loaded on a pad of silica gel, eluted with hexane, then 25% EtOAc / hexane, and then 50% EtOAc / hexane. The desired e was concentrated on a rotary evaporator to a thick suspension, and the solid was collected by filtration, triturated with MTBE, and dried in vacuo giving 20 as a bright yellow solid (55.8% yield, 90-97% HPLC purity). The filtrate was concentrated and the above purification was repeated giving a second crop of 20 as a bright yellow solid (19.7% yield). The filtrate was again trated giving a dark brown oil and this was loaded on a silica column with toluene and minimal . It was eluted with EtOAc / hexane (0% to 50%). The desired fractions were concentrated to a slurry and diluted with MTBE / hexane. The solid was collected by filtration and washed with minimal MTBE giving a third crop of 20 as a bright yellow solid (4.9% yield) with an overall yield of 80% from the three crops. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 363.48 (2.95 min). 1H NMR (300 MHz, CDClg) 5 8.84 (d, J = 1.6 Hz, 2H), 8.27 (d, J = 8.0 Hz, 1H), 7.62 (s, 2H), 5.31 — 5.24 (m, 1H), 4.63 (s, 1H), 4.27 — 4.18 (m, 1H), 3.97 — 3.87 (n1, 1H), 2.33 — 2.05 (m, 4H), 1.64 (s, 6H) ppm.

Example 18 Preparation of 2-[5-(4,5-diaminofluorotetrahydrofuran—2-yl- phenyl)pyrimidinyl]propanol (21). fi:OH 5:0H 45 psi H2, Pd/C, NEt3 MeOH, THF Placed 5% palladium on carbon (14.21 g, 50% wet, 3.339 mmol, Aldrich 330116) in a Parr bottle under nitrogen, followed by MeOH (242 mL, er 909333) and NEt3 (46.54 mL, 333.9 mmol, Sigma-Aldrich 471283). Dissolved 2-[5-(4-aminofluoronitro- 3-tetrahydrofuranyl-phenyl)pyrimidinyl]propanol (20) (121.0 g, 333.9 mmol) in hot THF (360 mL), allowed to cool, added to the reaction mixture, and rinsed with another portion of THF (124 mL). Placed the vessel on a Parr shaker and saturated with H2. Added 45 psi H2 and shook until consumption was complete (HPLC and LCMS showed complete reaction). The reaction e was purged with nitrogen, filtered through CeliteTM and rinsed with EtOAc. It was re-filtered through paper (glass microfibre) and the filtrate trated in vacuo. Repeated the reaction three more times on the same scale and the batches were combined giving 21 as a brown solid (447 g, 99% yield, 93% HPLC purity).

LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid r) M+1: 333.46 (1.79 min). 1H NMR (300 MHz, CDClg) 5 8.81 (d, J = 1.4 Hz, 2H), 6.69 (d, J = 7.3 Hz, 1H), 5.27 — 5.20 (m, 1H), 4.73 (s, 1H), 4.70 (s, 2H), 4.23 — 4.14 (m, 1H), 3.94 — 3.86 (m, 1H), 3.22 (s, 2H), 2.32 — 2.22 (n1, 1H), 2.18 — 1.99 (m, 3H), 1.63 (s, 6H) ppm. e 19 ation of l-3 - [6-fluoro-5 -[2-(1-hydroxymethyl-ethyl)pyrimidin-5 - yl] tetrahydrofuranyl- 1 H-benzimidazolyl] urea (22). 0 ’0 10 JL i JL / EtHN M ‘N NHEt pH 3.5 buffer dioxane, reflux NH2 0 To a stirring suspension of 2-[5 -(4,5-diaminofluoro-3 -tetrahydrofuran—2-yl- phenyl)pyrimidinyl]propanol (21) (111.3 g, 334.9 mmol) and 1,4-dioxane (556.5 mL, Sigma-Aldrich 360481) was added 1-ethyl(N—(ethylcarbamoyl)-C-methylsulfanyl- carbonimidoyl)urea (10) (93.36 g, 401.9 mmol, CB Research and Development) followed by a pH 3.5 buffer (1.113 L), prepared by dissolving NaOAc rate (158.1 g) in 1N aqueous H2804 (1.100 L). The reaction mixture was stirred at reflux overnight (HPLC showed complete conversion), cooled to room temperature, and poured portion-wise (frothing) into a stirring solution of aqueous saturated NaHC03 (2.23 L) giving pH 8-9. This was stirred for minutes, the solid was collected by filtration, washed sly with water to neutral pH, and then more sparingly with EtOH. The solid was dried under reduced pressure giving 22 as an off-white yellowish solid (135.2 g, 94% yield, 99% HPLC purity). LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 429.58 (2.03 min). 1H NMR (300 MHz, MeOD) 5 8.95 (d, J = 1.6 Hz, 2H), 7.45 (d, J = 6.5 Hz, 1H), 5.38 (br.s, 1H), 4.27 (dd, J =14.9, 7.1 Hz, 1H), 4.01 (dd, J = 15.1, 7.0 Hz, 1H), 3.37 — 3.29 (n1, 2H), 2.55 (br.s, 1H), 2.19 — 2.07 (m, 2H), 2.02 — 1.82 (br.s, 1H), 1.63 (s, 6H), 1.21 (t, J = 7.2 Hz, 3H) ppm.

Example 20 Chiral chromatographic isolation of 1-ethyl-3 - [6-fluoro-5 -[2-(1-hydroxy -ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea (23).

\N N\N chiral chrom A racemic sample of 1-ethyl[6-fluoro[2-(1-hydroxymethyl- ethyl)pyrimidinyl]tetrahydrofuranyl-1H-benzimidazolyl]urea (22) (133.60 g) was resolved on a CHIRALPAK® IC® column (by Chiral Technologies) eluting with DCM / MeOH / TEA (60 / 40 / 0.1) at 25°C giving the desired enantiomer 23 as an off-white solid (66.8 g, 45% yield, 99.8% HPLC purity, 99+% ee). Analytical chiral HPLC retention time was 7.7 min LPAK® IC® 4.6 X 250 mm column, 1 mL/min flow rate, 30°C). The solid was suspended in 2:1 EtOH / EtzO (5 volumes), stirred for 10 minutes, collected by filtration, washed with 2:1 EtOH / EtzO, and dried under d pressure giving a white solid (60.6 g).

The structure and absolute stereochemistry of 23 were confirmed by single-crystal X-ray diffraction analysis. Single crystal diffraction data were acquired on a Bruker Apex II _ 51 _ diffractometer equipped with sealed tube Cu K-alpha source (Cu K0t radiation, y = 1.54178 A) and an Apex II CCD detector. A crystal with dimensions of 0.15 x 0.15 x 0.10 mm was ed, cleaned using mineral oil, mounted on a MicroMount and ed on a Bruker APEXTI system. Three s of 40 frames separated in reciprocal space were obtained to provide an orientation matrix and l cell parameters. Final cell parameters were obtained and refined after data tion was completed based on the full data set. Based on systematic absences and intensities statistics the structure was solved and refined in acentric P21 space group.

A diffraction data set of reciprocal space was obtained to a tion of 0.85 A using 050 steps using 30 s exposures for each frame. Data were collected at 100 (2) K.

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

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

The data were collected, refined and d using the Apex 11 software. The structure was solved using the SHELXS97 (Sheldrick, 1990), program(s) and the structure refined using the SHELXL97 (Sheldrick, 1997) program. The crystal shows monoclinic cell with P21 space group. The lattice parameters are a = 9.9016(2) A, b = 10.9184(2) A, c = 19.2975(4) A, [5: 102.826(1)°. Volume = 2034.190) A3.

Example 21 Preparation of the methanesulfonic acid salt of 1-ethyl[6-fluoro[2-(1-hydroxy methyl-ethyl)pyrimidinyl][(2R)-tetrahydrofuran—2-yl]-1H-benzimidazolyl]urea (23A).

M3803H N\ >’NH -M so He 3 DCM, EtOH 2°C—rt >:O ) 23A To a stirring suspension of l[6-fluoro[2-(1-hydroxymethyl- ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea (23) (15.05 g, 35.13 mmol) in romethane (60 mL, J.T. Baker 931533) and absolute ethanol (15 mL, Pharmco-AAPER 111000200) was added methanesulfonic acid (2.392 mL, 36.89 mmol, Sigma-Aldrich 471356). d at room ature until a clear solution was observed.

Added heptane (300 mL) slowly over about 1 hr and collected the solid precipitate by filtration (using a Whatman qualitative # 3 paper on top of a Whatman GF/F glass microfibre paper). Dried under reduced pressure in a vacuum oven (desiccated with calcium sulfate and ium hydroxide) overnight at 40°C giving 23A as a white solid (13.46 g, 99+% HPLC purity, 99+% ee). Analytical chiral HPLC shows one enantiomer with retention time of 8.6 min eluting with CHzClz / MeOH / TBA (60 / 40 / 0.1) on a CHIRALPAK® IC® 4.6 X 250 mm column with 1 mL/min flow rate at 30°C. A second crop of white solid product 23A (4.36 g, 98% HPLC purity, 99+% ee) was obtained from the filtrate. LCMS (C18 column eluting with 10-90% CH3CN / water gradient over 5 minutes with formic acid modifier) M+1: 429.58 (2.03 min). 1H NMR (300 MHz, MeOD) 5 9.00 (d, J = 1.6 Hz, 2H), 7.67 (d, J = 6.1 Hz, 1H), 5.39 (t, J = 7.7 Hz, 1H), 4.30 (dd, J = 14.9, 6.9 Hz, 1H), 4.03 (dd, J =14.8,7.7 Hz, 1H), 3.40 — 3.31 (m, 2H), 2.72 (s, 3H), 2.70 — 2.60 (m, 1H), 2.21 — 2.08 (m, 2H), 1.98 — 1.84 (m, 1H), 1.65 (s, 6H), 1.22 (t, J = 7.2 Hz, 3H) ppm.

The (R)ethyl(6-fluoro(2-(2-hydroxypropanyl)pyrimidinyl) (tetrahydrofuran—2-yl)-1H-benzo[d]imidazolyl)urea 23 may then be ted to the phosphate or phosphate salt prodrugs according to the schemes set forth below.

Scheme 1 OH o—'Fl'>—OBn ox | I I / / / F F F (a), then (b) (C) N Step 1 N Step 2 N \ \ \ NH 0 NH 0 NH 0 HN HN HN >:o >:o )zo HN) 23 HN) 24 HN) (”3) X = F>0(o-)2 2M+ or X = F>0(o-)2 02+ — x = F>0(0H)2 + x = PO(OH)O'M+ Reagents and conditions: (a) dibenzyl N,N-diisopropylphosphoramidite, tetrazole, 23 0C, DMF; (b) mCPBA, 0-23 0C, DMF; (c) H2, Pd/C, M+0H' or ')2, EtOH, H20; (d) aq H+; (e) aq M+OH'.

Compounds of formula (IB) may be prepared from compound 23 as shown in Scheme 1. In Step 1, compound 23 is treated with dibenzyl N,N-diisopropylphosphoramidite and tetrazole, followed by meta-chloroperoxybenzoic acid (mCPBA), to afford dibenzyl phosphate 24. In Step 2, hydrogenolysis of 24 in the presence of M+OH' or D2+(OH')2 s the dianionic form of the compound of formula (IB) (X = —PO(O')2-2M+ or —PO(O' )2-D2+). The free acid form of the compound of formula (IB) (X = PO(OH)2) may be obtained by treating the dianionic form with aqueous acid. The ionic form of the compound of formula (IB) (X = PO(OH)O'M+) may be ed by treating the free acid form with one equivalent of M+OH'.

Scheme 2 2012/021270 N N (b), then (c) N Step 2 NH 0 /FN HN Boc N \N F F N Step4 N >LNH o >LNH o HN HN >=o >=o HN HN ) "B’ ) PO(O')2 2M+ or X F>0(o-)2 02+ — x = PO(OH)2 + x = PO(OH)O'M+ Reagents and conditions:(a) BOC2O, DMF, 23 °C,(b)dibenzyl N,N- diisopropylphosphoramidite, tetrazole, 23 °C, DMF, (c) mCPBA, 0-23 °C, DMF, (d) TFA, H20, MeOH, DCM, 23 0C, (e) H2, Pd/C, MIOH' or D2+(0H')2, EtOH, H20, (f) aq HI, (g) aq MIOH'.

Alternatively, the compounds of formula (IB) may be prepared from compound 23 as shown in Scheme 2. In Step 1, compound 23 is treated with t-butyl onate (B0020) to afford protected benzimidazole compound 25. In Step 2, compound 25 is treated with dibenzyl N,N-diisopropylphosphoramidite and tetrazole, followed by mCPBA, to afford protected dibenzyl phosphate 26. In Step 3, compound 26 is treated with trifluoroacetic acid _ 55 _ (TFA) to remove the ting group and afford dibenzyl ate 24. In Step 4, hydrogenolysis of 24 in the presence of M+OH' or D2+(OH')2 affords the dianionic form of the compound of formula (IB) (X = —PO(O')2°2M+ or —PO(O')2-D2+). The free acid form of the compound of formula (IB) (X = PO(OH)2) may be obtained by treating the dianionic form with aqueous acid. The monoanionic form of the compound of formula (I) (X = PO(OH)O'M+) may be obtained by treating the free acid form with one equivalent of M+OH'.

Scheme 3 HN 23 Step 2 (b), then (c) (d), then (e) N Step 3 (| B) x = PO(O')2 2M+ or x = PO(O')2 D2+ — x = PO(OH)2 —> X = PO(OH)O'M+ Reagents and ions:(a)B002O, DMAP, DMF, 23 oC; (b)dibenzyl N,N— diisopropylphosphoramidite, tetrazole, 23 0C, DMF; (c) mCPBA, 0-23 0C, DMF; (d) TFA, DCM; (e) aq M+OH' or D2+(OH')2; (f) aq H+; (g) aq M+OH'.

The compounds of formula (IB) may also be prepared from compound 23 as shown in Scheme 3. In Step 1, nd 23 is treated with two equivalents of B0020 in the presence of N,N—dimethylaminopyridine (DMAP) to afford bis-protected benzimidazole compound 28. In Step 2, compound 28 is d with dibenzyl N,N— diisopropylphosphoramidite and tetrazole, followed by mCPBA, to afford otected dibenzyl phosphate 29. In Step 3, compound 29 is treated with TFA to remove the protecting groups. Treatment of the resulting intermediate with aqueous M+OH' or ')2 affords the dianionic form of the nd of formula (IB) (X = )2-2M+ or —PO(O')2-D2+).

The free acid form of the compound of formula (IB) (X = PO(OH)2) may be obtained by treating the dianionic form with aqueous acid. The monoanionic form of the compound of formula (I) (X = PO(OH)O'M+) may be obtained by treating the free acid form with one equivalent of M+OH.

Example 22 Preparation of (R)-dibenzyl 2-(5-(2-(3-ethylureido)fluoro(tetrahydrofuran yl)- lH-benzo [d]imidazolyl)pyrimidin—2-yl)propanyl phosphate (24). fif’” o—fE—OBn \ OBn NI N N i N / I i) dibenzyl N,N-diisopropyl- / F phosphoramidite, tetrazole, 23 °C, DMF N -- o >\’NH II) mCPBA, 0 23 C, DMF 0 N\ NH O )=O HM):O HN) HN) 23 24 To l-3 - [6-fluoro-5 -[2-(l -hydroxy- l -methyl-ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl]-lH-benzimidazolyl]urea (23) (10.24 g, 23.66 mmol) in a l L round bottom flask under N2 at 23 °C was added DMF (200 mL) followed by a solution of tetrazole (105.2 mL of 0.45 M in MeCN, 47.32 mmol) followed by N- dibenzyloxyphosphanyl-N-isopropyl-propanamine (12.26 g, 11.93 mL, 35.49 mmol).

After 4.5 h more N—dibenzyloxyphosphanyl-N-isopropyl-propanamine (4 mL) was added.

After stirring a r 16 h the reaction was cooled to 0 °C (ice-water bath) then treated with mCPBA (15.17 g, 61.52 mmol). The mixture was stirred at 0 0C for 30 min then at 23 0C for min after which the reaction mixture was partitioned between water (400 mL) and EtOAc (700 mL). The organic layer was separated, washed with saturated aqueous sodium bicarbonate (500 mL), 10% aqueous sodium bisulfite (500 mL), saturated aqueous sodium bicarbonate (5 00 mL), and brine (5 00 mL) then dried (magnesium e), filtered and concentrated. The residue was purified by MPLC using an ISCO COMBIFLASH brand flash chromatography purification system (330 g column) eluting with a 0-10% EtOH in DCM linear gradient over 16.5 column volumes at a 200 mL/min flow rate. After concentration in vacuo, (R)-dibenzyl 2-(5-(2-(3-ethylureido)fluoro(tetrahydrofuranyl)-1H- d]imidazolyl)pyrimidinyl)propanyl phosphate(24) (13.89 g, 20.17 mmol, 85.27%) was obtained as a white solid. ESMS (M + 1) = 689.5,1H NMR (300 MHz, CD30D) 8.88 (d, J = 1.6 Hz, 2H), 7.37 (d, J = 6 Hz, 1H), 7.30 (m, 10H), 5.38 - 5.33 (n1, 1H), 5.12 - .01 (m, 4H), 4.24 (dd, J = .9 Hz, 1H), 3.98 (dd, J = 6.9, 15.1 Hz, 1H), 3.35 - 3.27 (m, 3H), 2.52 (q, J = 5.9 Hz, 1H), 2.14 - 2.05 (m,2H), 1.91 (s, 6H) and 1.22 - 1.14 (m, 3H) ppm.

] Example 23 Preparation of disodium (R)(5-(2-(3-ethylureido)fluoro(tetrahydrofuran— 2-yl)-1H—benzo[cflimidazolyl)pyrimidinyl)propanyl phosphate (W). ‘3 ‘P.

O-Fl’-OBn O-E-ONa OBn ONa N \N N \N I I / / H2,Pd/C F F NaOH N EtOH,H20 N \ \ NH 0 NH 0 HN HN >=O >=O HN) HN) 24 w A 1 L Parr vessel was charged with water (200 mL), Pd/C (4 g, 10 wt% dry basis, wet, a type), (R)-dibenzyl 2-(5 -(2-(3-ethylureido)fluoro(tetrahydrofuran—2-yl)- 1H-benzo[cflimidazolyl)pyrimidinyl)propanyl ate(24) (13.89 g, 20.17 mmol), EtOH (400 mL) and aqueous 1 M NaOH (40.34 mL, 40.34 mmol). The resulting mixture was hydrogenated under 50 psi H2 on a Parr shaker apparatus for 40 min. The reaction mixture was filtered through a 0.22 pm polyethersulfone (PES) membrane giving a dark colored filtrate. A water rinse resulted in more dark material ng the filter membrane.

The resulting filtrate was passed through a pad of Celite and the dark material did not elute 2012/021270 until the pad was rinsed with water. The resulting dark solution (approx. 700 mL) was diluted with three volumes of EtOH (2100 mL), filtered through a 0.22 mm PES membrane (using 4 disposable Corning polystyrene filter systems, #431098) and the filtrate concentrated in vacuo. The resulting residue was dissolved in water (100 mL) and EtOH (300 mL), filtered through a 0.22 mm PES membrane to give a clear yellow solution, then passed through a Celite plug (26 mm diameter X 60 mm height, pre-wet with EtOH) rinsing with EtOH (50 mL) and the filtrate then trated. The resulting residue was ved in water (250 mL) in a 1 L round bottom flask, then 1 M aqueous HCl (40 mL) was slowly added over 15 min with stirring to give a slurry of white solid. Twenty minutes following tion of the HCl addition, the solid was collected via filtration through a 0.22 mm PES membrane. The collected solid was washed with water (100 mL), then transferred (still wet) to a 1 L round bottom flask and slurried in MeOH (150 mL) for 30 min. The resulting fine white precipitate was collected via ion then dried in vacuo overnight. The resulting free acid (9.17 g, 18.0 mmol) was treated with water (80 mL) then 1.0 N aq NaOH (36.0 mL, 2 equiv). The resulting solution was frozen and lyophilized to give disodium [1-[5-[2- (ethylcarbamoylamino)fluoro[(2R)-tetrahydrofuranyl]-1H-benzimidazol yl]pyrimidinyl]methyl-ethyl] phosphate (W) (10.206 g, 18.08 mmol, 89.66%) as a pale, cream-colored solid with consistent analytical data. ESMS (M + 1) = 5094, 1H NMR (300 MHz, D20) 5 8.58 (s, 2H), 6.92 (d, J = 6.3 Hz, 1H), 5.13 (t, J = 7.5 Hz, 1H), 3.98 - 3.81 (m, 2H), 3.04 (q, J = 7.2 Hz, 2H), 2.26 (t, J = 5.7 Hz, 1H), 1.97 - 1.92 (m, 2H), 1.67 (s, 6H) and 1.01 (t, J = 7.2 Hz, 3H) ppm.

Example 24 ] Preparation of Boc-l -ethyl-3 - [6-fluoro-5 -hydroxymethylethyl )pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea (25). 2012/021270 N \N Boc20 N\ DMF,23°C NH 0 HN Boc To a solution/suspension of 1-ethyl[6-fluoro[2-(1-hydroxymethyl- ethyl)pyrimidin—5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea (23) (10.72 g, 25.02 mmol) in DMF (250 mL) at 23 0C was added Boc20 (6.11 g, 28.00 mmol). After 2 hours, 2 M ammonia in MeOH (2 mL) was added to quench any excess BoczO. The quenched reaction mixture was partitioned between EtOAc and water (400 mL each), the organic layer separated, washed with water (2 X 400 mL) and brine (400 mL), then dried over MgSO4, filtered and concentrated to give Bocethyl[6-fluoro[2-(1-hydroxymethyl- ethyl)pyrimidin—5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea (25) (12.69 g, 23.58 mmol, 94.26%)which was used without fithher purification. ESMS (M + 1) = 529.3, 1H NMR (300.0 MHz, CDClg) 5 9.50 (s, 1H), 9.02 (t, J = 5.3 Hz, 1H), 8.91 (d, J = 1.6 Hz, 2H), 7.74 (d, J = 6.5 Hz, 1H), 5.58 (t, J = 7.8 Hz, 1H), 4.64 (s, 1H), 4.22 (q, J = 7.4 Hz, 1H), 4.05 (td, J = 7.8, 4.3 Hz, 1H), 3.47 (td, J = 7.2, 4.3 Hz, 2H), 2.42 - 2.35 (m, 2H), 2.28 - 2.16 (m, 2H), 1.75 (s, 9H), 1.68 (s, 6H) and 1.31 (t, J = 7.3 Hz, 3H) ppm.

Example 25 ] Preparation of Bocethyl-3 - [6-fluoro-5 -[2-(1-hydroxymethylethyl idin—5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea dibenzyl phosphate (26). a5)” E ,O—Ililg—OBn N \N / NI \N i) dibenzyl N,N-diisopropyl- / F phosphoramidite, tetrazole, DMF, 23 °C /N)\_’_N ii) mCPBA, DMF, 0—23 °c N Boc HN Boc4;N O 26 To Boc-l-ethyl[6-fluoro[2-(1-hydroxymethyl-ethyl)pyrimidinyl] [(2R)-tetrahydrofuranyl]-1H-benzimidazolyl]urea (25) (12.69 g, 23.58 mmol) and tetrazole (3.304 g, 47.16 mmol) under N2 at 23 °C was added DCM (240 mL) followed by N- dibenzyloxyphosphanyl-N-isopropyl-propanamine (9.775 g, 9.509 mL, 28.30 mmol).

After 3 hours at 23 0C, the reaction was cooled to 0 0C then mCPBA (6.977 g, 28.30 mmol) was added. The resulting on was stirred for 45 min at 0 0C then for 20 min at 23 OC.

The on mixture was then partitioned between DCM (50 mL) and saturated aqueous sodium bicarbonate (400 mL). The organic layer was separated, then washed successively with aqueous sodium te (63 g in 350 mL water) and saturated aqueous sodium bicarbonate (400 mL), then dried over magnesium sulfate, filtered and concentrated in vacuo.

The residue was purified by MPLC using an ISCO COMBIFLASH brand flash chromatography purification system (330 g silica column) eluting with a 0-100% EtOAc in hexanes linear gradient over 16 column volumes at 200 mL/min. Product containing fractions were evaporated in vacuo to give Bocethyl[6-fluoro[2-(1-hydroxymethyl- pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea dibenzyl ate (26) (11.92 g, 15.11 mmol, 64.09%). ESMS (M + 1) = 7892; 1H NMR (300.0 MHz,CDC13)5 9.51 (s, 1H), 9.03 (t, J = 5.4 Hz, 1H), 8.91 (d, J = 1.6 Hz, 2H), 7.73 (d, J = 6.5 Hz, 1H), 7.37 - 7.28 (m, 10H), 5.58 (t, J = 7.8 Hz, 1H), 5.17 - 5.05 (m, 4H), 4.23 (t, J = 7.5 Hz, 1H), 4.05 (td, J = 7.8, 4.3 Hz, 1H), 3.53 = 79,145 Hz, - 3.44 (m, 2H), 2.39 (dd, J 2H), 2.28 - 2.15 (m, 2H), 1.98 (s, 6H), 1.72 (m, 9H) and 1.31 (t, J = 7.2 Hz, 3H) ppm.

Example 26 WO 97269 Preparation of (R)-dibenzyl 2-(5-(2-(3-ethylureido)fluoro (tetrahydrofuran—2-yl)- 1H-benzo [d]imidazolyl)pyrimidinyl)propanyl phosphate (24). ‘3 ‘3 O-Fl’-OBn O-Fl’-OBn OBn OBn N \ N N \ N I I / / TFA, H20 F F MeOH N DCM, 23 °C N flN ‘ o NH 0 B06 HN HN )=o )=o HN) HN) To a solution of ethyl[6-fluoro[2-(1-hydroxy-1 -methyl- ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea dibenzyl phosphate (26) (11.9 g, 15.09 mmol) in DCM (300 mL) at 23 0C was added water (2.325 mL, 129.1 mmol) then TFA (3.441 g, 2.325 mL, 30.18 mmol). After 1 h, only partial conversion was observed by tlc, so more TFA (3.441 g, 2.325 mL, 30.18 mmol) was added. After a further 2.5 h, MeOH (2 mL) was added and the e stirred a further 18 hours. The reaction mixture was washed with 1:1 brinezsaturated s sodium bicarbonate (200 mL).

The aqueous layer was re-extracted with DCM (150 mL), the organic layers combined, then dried (over ium sulfate), edand concentrated in vacuo. The resulting residue was re-dissolved in EtOAc (200 mL)washed with water (150 mL) and brine (100 mL), then dried (magnesium sulfate) filtered and concentrated to give (R)-dibenzyl 2-(5-(2-(3-ethylureido) fluoro(tetrahydrofuranyl)-1H-benzo[d]imidazolyl)pyrimidinyl)propanyl phosphate (24) (10.21 g, 14.83 mmol, 98.27%) as a white solid. ESMS (M + 1) = 6894; 1H NMR (300 MHz, CDgOD) 5 8.88 (d, J = 1.6 Hz, 2H), 7.37 (d, J = 6 Hz, 1H), 7.30 (m, 10H), .38 = 68,149 Hz, 1H), 3.98 (dd, J = 6.9, - 5.33 (m, 1H), 5.12 - 5.01 (m, 4H), 4.24 (dd, J .1 Hz, 1H), 3.35 = 5.9 Hz, 1H), 2.14 - 3.27 (m, 3H), 2.52 (q, J - 2.05 (m,2H), 1.91 (s, 6H) and 1.22 - 1.14 (m, 3H) ppm.

Example 27 Preparation of disodium (R)(5-(2-(3-ethylureido)fluoro (tetrahydrofuran—2-yl)-1H-benzo [d]imidazolyl)pyrimidinyl)propanyl phosphate (W). ‘3 9 O-Ii’-OBn 0Na OBn ONa N \ N N \ N I I / / H2, Pd/C F F aq NaOH N EtOH, 23 °C N \ \ NH 0 NH 0 HN HN >=o >=o HN) HN) 24 W A 1 L round bottom flask was charged with (R)-dibenzyl 2-(3- ethylureido)fluoro(tetrahydrofuran—2-yl)-lH-benzo[d]imidazolyl)pyrimidin—2- yl)propan—2-yl phosphate (24) (9.37 g, 13.61 mmol), EtOH (300 mL), water (150 mL), Pd/C (10 wt% dry basis, wet, Degussa type, 3 g) and 1 M aqueous NaOH (27.22 mL, 27.22 mmol).

The suspension was evacuated for 3 min (needle to pump) then placed under an atmosphere of hydrogen gas (balloon). After stirring 2.5 h at 23 0C, the reaction was filtered through a 0.22 um PES membrane (disposable Corning filter system, 1 L, polystyrene, #431098) to remove catalyst and washed with EtOH (50 mL). The resulting solution was concentrated, the e ved in water (80 mL), treated with MeCN (80 mL), then frozen and lyophilized to give disodium (R)(5-(2-(3-ethylureido)fluoro(tetrahydrofuran—2-yl)- 1H-benzo[d]imidazolyl)pyrimidinyl)propanyl phosphate (W) (7.10 g, 12.81 mmol, 94.12%) as a white solid. ESMS (M + 1) = 5093, 1H NMR (300 MHz, D20) 5 8.58 (s, 2H), 6.92 (d, J = 6.3 Hz, 1H), 5.13 (t, J = 7.5 Hz, 1H), 3.98 = 7.2 Hz, - 3.81 (m, 2H), 3.04 (q, J 2H), 2.26 (t, J = 5.7 Hz, 1H), 1.97 - 1.92 (n1, 2H), 1.67 (s, 6H) and 1.01 (t, J = 7.2 Hz, 3H) ppm.

Example 28 Preparation of l -ethyl-3 - [6-fluoro-5 -[2-(1-hydroxymethylethyl )pyrimidin-5 -yl] -7 -[(2R)-tetrahydrofuranyl]-1H-benzimidazol-2 -yl]urea (28).

N \ N N \ N F Boc20 DMAP \ DMF 23 °C NH 0 ’ To a solution/suspension of 1-ethyl[6-fluoro[2-(1-hydroxymethyl- ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea (23) (1 .3 33 g, 3.111 mmol) in DMF (30 mL) was added DMAP (38.01 mg, 0.3111 mmol) ed by Boc20 (1.426 g, 1.501 mL, 6.533 mmol). After 30 min, the reaction mixture was diluted with water and EtOAc (300 mL each), the organic layer separated, washed with water and brine (300 mL each), then dried over magnesium sulfate, filtered and concentrated. The residue was purified by MPLC using an ISCO COMBIFLASH brand flash chromatography purification system (80 g silica column) eluting with a 0-60% EtOAc in hexanes linear gradient over 20 column volumes at 60 mL/min flow rate. Desired product ons were combined and evaporated to give diBocethyl[6-fluoro[2-(1-hydroxymethyl- ethyl)pyrimidinyl][(2R)-tetrahydrofuranyl]-1H-benzimidazolyl]urea (28) (1.43 g, 2.275 mmol, ) as a clear foam. ESMS (M + 1) = 629.3, 1H NMR (300.0 MHz, CDC13) 5 8.95 (d, J = 1.6 Hz, 2H), 8.31 = 6.5 Hz, 1H), 5.80 - 8.27 (m, 1H), 8.05 (d, J - 5.68 (m, 1H), 4.70 (s, 1H), 4.21 = 6.4 Hz, 1H), 3.42 - 4.09 (m, 1H), 3.98 (d, J - 3.37 (n1, 2H), 2.45 - 2.00 (m, 4H), 1.65 (s, 6H), 1.62 (s, 9H), 1.37 (s, 9H) and 1.28 - 1.21 (n1, 3H) ppm.

Example 29 Preparation of diBocethyl-3 - [6-fluoro-5 -[2-(1-hydroxymethyl- ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea dibenzyl phosphate (29). fi:O—Fl’—0BnOBn N \N i)dibenzy| N,N-diisopropyl- / oramidite, tetrazole, DMF, 23 °C ii) mCPBA, DMF, 0—23 °C N ”N o (Boc)2 N 28 29 To diBoc-l -ethyl-3 - [6-fluoro-5 -[2-(1-hydroxymethyl-ethyl)pyrimidin-5 - yl][(2R)-tetrahydrofuranyl]-1H-benzimidazolyl]urea (28) (1.13 g, 1.797 mmol) and ole (251.8 mg, 3.594 mol) at 23 °C under N2 was added DCM (30 mL) followed by N—dibenzyloxyphosphanyl-N-isopropyl-propan—2-amine (744.7 mg, 724.4 uL, 2.156 mmol).

After stirring for 18 h, the reaction was cooled to 0 °C then treated with mCPBA (531.5 mg, 2.156 mmol). The reaction was stirred for 15 min at 0 0C, then for 30 min at 23 °C. The resulting solution was then ioned between EtOAc and saturated s sodium bicarbonate (3 00 mL each), the organic layer separated, then washed with 10% aqueous sodium bisulfite, saturated aqueous sodium bicarbonate and brine (300 mL each), then dried over magnesium sulfate filtered and concentrated. The residue was purified by MPLC using an ISCO COMBIFLASH brand flash chromatography purification system (80 g silica column) eluting with a 0-80% EtOAc in s linear gradient over 20 column volumes at 60 mL/min flow rate. Desired product fractions were combined and evaporated to give diBoc-l -3 - [6-fluoro-5 -[2-(1-hydroxymethyl-ethyl)pyrimidin-5 -yl] -7 - [(2R)- tetrahydrofuran—2-yl]—1H-benzimidazolyl]urea dibenzyl phosphate (29) (1.03 g, 1.159 mmol, 64.50%) as a clear, glassy oil. ESMS (M + 1) = 8895, 1H NMR (300.0 MHz, CDClg) 8.93 (d, J = 1.5 Hz, 2H), 8.31 (s, 1H), 8.04 (d, J = 6.4 Hz, 1H), 7.36 - 7.26 (m, 10H), 5.83 - .70 (m, 1H), 5.16 - 5.05 (m, 4H), 4.24 - 4.18 (m, 1H), 4.03 - 3.97 (m, 1H), 3.42 - 3.36 (m, 2H), 2.43 - 2.05 (m, 4H), 1.98 (s, 6H), 1.64 (s, 9H), 1.40 (s, 9H) and 1.26 (t, J = 7.2 Hz, 3H) ppm.

Example 30 WO 97269 Preparation of sodium (R)(5-(2-(3-ethylureido)fluoro hydrofuran—2-yl)-lH-benzo [d]imidazolyl)pyrimidin—2-yl)propanyl phosphate (W). ‘3 ‘3 O-Fl’-OBn O-Fl’-ONa OBn ONa N \ N N \ N 1) TFA, DCM F 2) NaOH aq N NH 0 29 W To a solution of diBoc-l-ethyl[6-fluoro[2-(1-hydroxymethyl- ethyl)pyrimidin-5 -yl] -7 - [(2R)-tetrahydrofuranyl] - 1 H-benzimidazolyl]urea dibenzyl phosphate (29) (121 mg, 0.1361 mmol) in DCM (10 mL) at 23 0C was added TFA (5 mL).

After 2 h, the reaction mixture was concentrated in vacuo. The residue was dissolved in MeOH (6 mL) and treated with approx 0.5 mL 2 M NH3 in MeOH (to fully dissolve the al). The resulting solution was purified in 6 injections on preparative HPLC, reverse phase, Sunfire prep C18 OBD 5 uM column 19 X 100 mm, g with a10-90% aq MeCN w/ 0.1% TFA buffer, linear gradient over 15 min at 20 mL/min flow rate. Fractions containing product were pooled and lyophilized. The resulting material was suspended in MeOH (3 mL), stirred at 23 0C for 30 min, then the precipitate was collected via ion through a plastic frit. The resulting white solid was re-subjected to a MeOH slurry (3 mL), then collected via filtration to give 68 mg of white solid after drying. The white solid was treated with 0.10 M aq NaOH (2.68 mL, 2 equiv NaOH) to give a solution that was then passed through an sc CR 13 mm syringe filter with 0.45 pm PTFE membrane, flushing with water (2 mL). The resulting solution was treated with MeCN (3 mL), frozen and lyophilized to give sodium (R)(5-(2-(3-ethylureido)fluoro(tetrahydrofuranyl)-1H- benzo[d]imidazolyl)pyrimidin—2-yl)propan—2-yl phosphate (W) as a white powder. ESMS (M + 1) = 5092, 1H NMR (300 MHz, D20) 5 8.58 (s, 2H), 6.92 (d, J = 6.3 Hz, 1H), 5.13 (t, J = 7.5 Hz, 1H), 3.98 = 7.2 Hz, 2H), 2.26 - 3.81 (m, 2H), 3.04 (q, J (t, J = 5.7 Hz, 1H), 1.97 - 1.92 (m, 2H), 1.67 (s, 6H) and 1.01 (t, J = 7.2 Hz, 3H) ppm.

] Example 31 Susceptibility Testing in Liquid Media nds of this invention were tested for antimicrobial ty by susceptibility testing in liquid media. Such assays were performed within the guidelines of the latest CLSI document governing such practices: "M07-A8 Methods for Dilution crobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard-- Eighth Edition (2009)". Other publications such as "Antibiotics in Laboratory Medicine" (Edited by V. Lorian, Publishers Williams and Wilkins, 1996) provide essential practical techniques in laboratory antibiotic testing. The specific protocols used were as follows: ol #1: Gyrase MIC determination of compounds using microdilution broth method Materials: ] Round bottom 96-well iter plates (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 with bacteria streaked to single colonies, freshly prepared Sterile DMSO Human serum (US. Biologicals S1010-51) Laked horse blood (Quad Five 270-100) Resazurin 0.01% Sprague Dawley Rat serum (U.S. Biologicals 1011-90B or Valley ical AS3061 SD) Pooled Mouse serum (Valley BioMedical AS3054) Strains (media, broth and agar): 1. lococcus aureus ATCC #29213 a. MHII b. MHII -- 50% human serum c. MHII -- 50% rat serum d. MHII -- 50% mouse serum 2. Staphylococcus aureus ATCC #29213 GyrB T1731 (MHII) 3. Staphylococcus aureus, JMI collection s; see table 5(MHII) 9.09:“ Staphylococcus epidermidis, JMI collection strains; see table 5 (MHII) Enterococcusfaecalis ATCC #29212 (MHII -- 3% laked horse blood) Enterococcusfaecium ATCC #49624 (MHII -- 3% laked horse blood) Enterococusfaecalis, JMI collection strains; see table 5 (MHII -- 3% laked horse blood) cocusfaecium, JMI collection strains; see table 5 (MHII -- 3% laked horse blood) Streptococcus pneumoniae ATCC #10015 (MHII + 3% laked horse blood) . Streptococcus pneumoniae,JMI collection strains; see table 5 (MHII + 3% laked horse blood) 11. fl-haemolytic streptococci, Groups A, B, C, G) JMI tion strains; see table 5 (MHII + 3% laked horse blood) 12. Bacillus cereus ATCC 10987 (MHII) 13. Bacillus cereus ATCC 14579(MHII) 14. Bacillus subtilis ATCC 6638 (MHII) . Bacillus subtilis (168) ATCC 6051 (MHII) [00264 um prep (for all strains other than S. aureus + 50% sera): 1. Usingthe BBL Prompt kit; picked 5 big or 10 small; well separated colonies from culture grown on the appropriate agar medium as indicated above and inoculated 1 mL of sterile saline provided in the kit. 2. Vortexed the wells for ~ 30 s to provide a suspension of ~108 cells/mL. Actual density could be confirmed by plating out ons of this suspension. 3. Diluted the suspension 1/ 100 by transferring 0.15 mL of cells into 15 mL (~106 cells/mL) sterile broth (or see below) for each plate of compounds tested; then swirled to mix. If more than 1 plate of nds (> 8 compounds) were tested; s were increased accordingly. a. For E. faecalis; E. faecium and S. niae: 14.1 mL MHII + 0.9 mL laked horse blood was used. 4. Used 50 ul cells (~5 X 104 cells) to inoculate each microtiter well containing 50 ul of the drug diluted in broth (see below).

Drug dilutions, inoculation, MIC ination: 1. All drug/compound stocks were prepared at 12.8 mg/mL concentration; usually in 100% DMSO.

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

If ng concentration of MICs was 8 ug/mL final concentration, then required 6.25 uL of stock + 43.75 uL DMSO. Each 200x stock was placed in a te row of column 1 of a new 96 well microtiter plate.

Added 25 uL ofDMSO to columns 2 -12 of all rows of the microtiter plate containing 200x compound stocks and serially diluted 25 uL from column 1 through column 11, changed tips after each column. i.e. 25 uL compound + 25 uL DMSO = 2x dilution.

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

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

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

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

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

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

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

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

Used the same BBL Prompt inoculum of S. aureus ATCC #29213 as described above, diluted 1/200 by transferring 0.15 mL of cells into 30 mL (~5x105 cells/mL) of the 50% human serum media prepared above and swirled to mix. 2012/021270 Filled all test wells of the desired number of microtiter plates with 100 uL cells in 50% serum media.

Transferred 0.5 uL of each compound dilution (w/Matrix auto-pipettor) to 100 uL of cells/media. The usual starting concentration of compound was 8 ug/mL after the 1/200 dilution into medium + cells — compound concentrations decreased in 2x steps across the rows of a microtiter plate. All MICs were done in duplicate.

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

Plates were incubated at 37°C for at least 18 hours. After incubation, added 25 [LL of 0.01% Resazurin to each well and continued to incubate at 37°C for at least 1 additional hour or until the Resazurin color changes.

Plates were Viewed with a test g mirror and the MIC was recorded. When using Resazurin, the color of the dye changed from a dark blue to a bright pink in wells with no growth. The lowest tration of drug that turned the dye pink was the MIC. ol 2: Gyrase MIC determination of compounds against Gram negatives using microdilution broth method Materials: Round bottom 96-well iter plates (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 with bacteria ed to single colonies, freshly prepared Sterile DMSO Strains (MHII media for all; broth and agar): l. Escherichia coli ATCC # 25922 2. Escherichia coli, JMI tion strains, see table 5 3. Escherichia coliAG100 WT 4. Escherichia coli AG100 tolC . Acineiohacier baumannii ATCC # BAA-1710 6. Acineiohacier baumannii ATCC # 19606 7. obacter baumannii, JMI collection strains, see table 5 8. Klebsiella pneumoniae ATCC # BAA-1705 9. Klebsiella pneumoniae ATCC # 700603 . Klebsiella pneumoniae, JMI collection s, see table 5 11. Moraxella catarrhalis ATCC# 25238 12. Moraxella catarrhalis ATCC# 49143 13. Moraxella catarrhalz‘s, JMI collection strains, see table 5 14. Haemophilus zae ATCC 49247 . Haemophilus influenzae (Rdl KW20) ATCC 51907 16. Haemophilus influenzae Rd0894 ) 17. Haemophilus influenzae, JMI collection strains, see table 5 18. Pseudomonas aeruginosa PAOl 19. Pseudomonas aeruginosa, HVII collection strains, see table 5 . Proteus mirabilis, HVII collection strains, see table 5 21. Enterobacter e, JMI collection strains, see table 5 22. Stenoz‘rophomonas maltophl'll'a ATCC BAA-84 23. Stenoz‘rophomonas maltophl'll'a ATCC13637 Inoculum prep: 1. Using the BBL Prompt kit, picked 5 big or 10 small, well separated colonies from cultures grown on agar medium and inoculated 1 mL sterile saline that came with the kit. 2. ed the wells for ~ 30 s to give a suspension of ~108 cells/mL. Actual density could be confirmed by plating out dilutions of this suspension. 3. Diluted the suspension 1/ 100 by erring 0.15 mL of cells into 15 mL (~106 cells/mL) sterile broth (see below) for each plate of compounds tested, swirled to mix.

If more than 1 plate of compounds (> 8 compounds) was to be tested, increased volumes accordingly. 4. Used 50 ul cells (~5 X 104 cells) to inoculate each iter well containing 50 ul of the drug d in broth (see below).

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

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

Left “no nd” DMSO well at the end of the series for control. 4. For each strain tested, prepared two microtiter plates with 50 [LL of MHII broth using a Matrix or.

. Transferred 0.5 uL of each dilution riX auto-pipettor) to 50 [LL of medium/microtiter well prior to the addition of 50 pl of cells. The usual ng concentration of compound was 8 ug/mL after the 1/200 dilution into medium + cells — compound trations decreased in 2X steps across the rows of a microtiter plate.

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

Protocol #3: Gyrase MIC determination of compounds using Agar dilution method Materials: Petri plates 60 X 15 mm (Thermo Scientific Cat. # 12567100) Centrifuge tubes, 15 mL (Costar) BBL Prompt Inoculation System (Fisher b26306) Agar plates with bacteria streaked to single colonies, freshly prepared Sterile DMSO GasPakTM incubation containers (BD Cat. #260672) GasPak TM EZ Anaerobe container system sachets (BD Cat. #260678) GasPak TM EZ CO2 container system sachets (BD Cat. #260679) GasPak TM EZ Campy container system sachets (BD Cat. #260680) 1. Closz‘ridl'um dlfificile ATCC BAA-1382, 2 Closz‘ridz'um dzfificz'le, CMI collection s, see table 4 3 Closz‘ridz'um gens, CMI collection strains, see table 4 4 . Bacteroidesfragz‘lz‘s and Bacteroz'des spp., CMI collection strains, see table 4 . Fusobacterium spp., CMI collection s, see table 4 6. Peptostreptococcus, spp., CMI collections s, see table 4 7. Prevotella spp., CMI collection strains, see table4 8. N gonorrhoeae ATCC 35541 9. N gonorrhoeae ATCC 49226 . ria gonorrhoeae, JMI collection strains, see table 4 11. Neisseria meningitidis, HVII collection strains, see table 4 Media preparation and growth conditions: Growth medium ended for each microbial species was prepared according to the CLSI publication ‘Ml 1-A7 Methods for crobial tibility Testing of Anaerobic Bacteria, Approved Standard - Seventh Edition (2007)’ with the exception of N gonorrhoeae and N meningitidis for which media was prepared according to"M07-A8 Methods for Dilution crobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved Standard--Eighth Edition (2009)".

Plate pouring: 1. Prepared 100x drug stocks of each test compound as described in Table 1. Used a 15 mL centrifuge tube, added 100 uL of each drug stock to 10 mL of molten agar (cooled to ~ 55°C in water bath). Mixed by ing tube 2 -3X then pour into individually labeled 60X15 mm Petri dish. 2. Routine test concentrations were: 0.002 ug/mL — 16 ug/mL (14 plates). 3. Poured 4 drug free plates: 2 as positive control, 2 as aerobic control. 4. Allowed plates to dry. Used same day or stored overnight at RT or stored up to 3 days at 4°C.

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

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

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

Plate inoculation (performed in biosafety cabinet): I. Streaked each strain onto individual agar plates as described above. Incubated for required time and environmental condition (i.e. anaerobic, erophilic, etc).

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

Adjusted suspension to O.D.600 0.05 (5x10e7 cfu/mL). Vortexed to mix.

Transferred ~02 mL of adjusted, mixed cultures to a 96 well plate. When S 5 s were tested, all strains were lined together in a single row. When testing > 5 s, erred strains into plate with no more that 5 strains in a single row. This was ary to fit on the small plates.

Used multi-channel pipettor, spotted 0.002 mL of each strain from prepared 96 well plates onto each MIC test plate. This resulted in ~ 1x10e5 cfu/spot. When testing C. dzfificile, strains d when grown, however distance between channel or spots was far enough such that swarming cells did not impair assay results. a. Inoculated2 drug free plates first, while the other 2 drug free plates were inoculated last after the MIC test plates. The former and latter served as growth and inoculation controls. Incubated one plate from each set of drug- free controls under required atmospheric ions with MIC plates and one set aerobically to test for contamination with aerobic bacteria. Aerobic culture was negative for growth when working with strict anaerobe or microaerophilic strain. Some growth was visible with N gonorrhoeae. 6. Allowed inoculum to dry (for as short a time as necessary), then placed upside down in GasPak with appropriate number of sachets and incubate. 7. Neisserz'a spp. were incubated at 37°C in a5% C02 environment for 24h.

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

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

Final Volume Volume Diluent, Intermediate Conc. (uL) added Stock from stock DMSO Conc. At 1:100 to 10 mL Step (ug/ml) Source (uL) (uL)** (ug/mL) (ug/mL) agar -—-7—7_—_—1,600 Stock 1,600 Stock 1,600 Stock *1,600 ug/ml = 64 ul (10mg/ml stock) + 336 ul DMSO, 400 ul total volume to start **compound dissolved and diluted in 100% DMSO Protocol 4. MIC Determination Procedure for Mycobacterium species Materials Round bottom 96-well microtiter plates r 3788) or similar Film plate seals (PerkinElmer, TopSeal-A #6005250 or similar) Middlebrook 7H10 broth with 0.2% ol Middlebrook 7H10 agar with 0.2% glycerol brook OADC Enrichment Inoculum Preparation for M. tuberculosis: Used prepared frozenM ulosis stock stored at -700C. M tuberculosis was grown in 7H10 broth + 10% OADC, then frozen at a concentration of 100 Klett or 5 X 107cfiJ/ml, Prepared a 1:20 dilution by removal of 1 ml of the frozen stock and added it to 19 ml of 7H10 broth + 10% OADC (final concentration 2.5 X 106cfu/ml).

From this dilution prepared a second 1:20 dilution, removed 1 ml and added it to 19 ml of fresh broth. This was the final inoculum to add to the l plates.

Inoculum Preparation for M. kansasii, M. avium, M. abscessus and Nocardia spc.: 1. Used prepared frozen stock of culture or a fresh culture grown in 7H10 broth at a tration of 10 Klett or 5 X 107/ml.

Prepared a 1:20 dilution by removing 1.0 ml of the culture stock and added it to 19 ml of 7H10 broth (final concentration 2.5 X 106cfu/ml).

From this dilution prepared a 1:20 dilution, removed 1 ml and added it to 19 ml of fresh broth (final suspension).

Plate Preparation: 1. Labeled plates. 2. Added 50 ul of 7H10 broth + 10% OADC to all wells being utilized for MIC determination using a multichannel electronic pipettor.

Prepared stock solutions of drugs (e. g. 1 mg/ml concentration) to be tested.

Thawed and diluted frozen stock solutions using 7H10 broth + 10% OADC to obtain a g solution 4X the maXimum concentration tested (e.g. final concentration 32 ug/ml, highest tration tested was 8 ug/ml). Dilutions were made from the stock solution. To start at a concentration of l ug/ml, the drugs were prepared at 4 ug/ml, so the starting tration was 1 ug/ml. Removed 25 ul of the 1mg/ml stock and added to 6.2 ml of broth. All dilutions of drugs were done in broth.

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

Continued for all compounds to be tested. Using a multichannel electronic pipettor, mixed 4X and serial diluted compounds through the 11th well. Discarded remaining 50 pl. Used the 12th well as the ve control.

Incubated plates at 37° C M tuberculosis for ~18 days; M avium andM kansasil‘ for ~7 days; Nocardl'a and M abcessus for ~4 days; with film seals. 7. Read visually and recorded the results. The MIC was recorded as the lowest concentration of drug where no growth was observed (optical clarity in the well). ol 5. Protocol for Mycobacterium tuberculosisSerum Shift MIC Assay Materials and reagents: Costar #3904 Black-sided, flat-bottom 96-well microtiter plates Middlebrook 7H9 broth (BD27l310) with 0.2% glycerol brook OADC Enrichment Fetal Bovine Serum Catalase (Sigma Cl 345) Dextrose NaClz BBL Prompt Inoculation System (Fisher b26306) Agar plates (Middlebrook 7Hll with 0.2% ol and OADC enrichment) with bacteria streaked to single colonies Sterile DMSO Media prep: 1. For serum shifted MICs, three different media were required which all had a base of 7H9 + 0.2% ol. It was important that all media and supplements were sterilized prior to MICs. 2. ed all media below and inoculated as described in next section. Tested all compounds against Mtb using each media. a. 7H9 -- 0.2% glycerol -- 10% OADC dard” MIC . b. 7H9 -- 0.2% glycerol -- 2 g/L dextrose + 0.85 g/L NaCl + 0.003 g/L catalase (0% FBS). c. 2x 7H9 + 0.2% glycerol + 2 g/L dextrose + 0.85 g/L NaCl + 0.003 g/L catalase combined with equal volume Fetal Bovine Serum (50% FBS).

Inoculum prep: 1. Using BBL Prompt, picked 5-10 eparated colonies and inoculated 1 ml sterile saline that came in the kit. Typically plates were two to three weeks of age when used for this assay due to the slow growth of this organism in culture. 2. ed well, then sonicated in water bath for 30 sec providing a suspension of ~108 cells/n11. Actual density could be confirmed by plating out dilutions of this sion.

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

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

Drug dilutions, inoculation, MIC determination: 1. Control drug stocks Isoniazid and Novobiocin were prepared at 10 mM in 100% DMSO while Ciprofloxacin and in were prepared at 1 mM in 50% DMSO and 100% DMSO, respectively. Prepared dilutions- dispensed 100 pL of the stock solution into the first column of a 96-well plate. Prepared 11-step, 2-fold serial dilutions across the row for each compound by transferring 50 pl from column 1 into 50 pl of DMSO in column 2. Continued to transfer 50 pL from column 2 through column 11 while mixing and changing tips at each column. Left column 12 with DMSO only as a control.

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

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

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

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

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

As bed above for control compounds, generated 2-fold, 11-point dilutions of each compound using DMSO as diluent. In all cases, left column 12 as DMSO only for a control. Once all dilutions were complete, again transferred 1 pl of each on into an empty iter well prior to the addition of 100 pl of cells as done for the l compounds. 0 All wells were inoculated with 100 pl of diluted cell suspension (see above).

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

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

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

After 24 hours the fluorescence of each well was measured at Excitation 492 nm, on 595 nm. t inhibition by a given compound was ated as follows: t inhibition=100-([well fluorescence-average background fluorescence]/[DMSO control - e background fluorescence] x100). MICs were scored for all three medium conditions as the lowest compound concentration that inhibited resazurin reduction (‘%—inhibition’) signal 270% at a given medium condition.

Table 2 shows the results of the MIC assay for selected compounds of this invention.

In Table 2 and in subsequent Tables and Examples, “Compound 12” corresponds to 1-ethyl[5-[2-(1-hydroxymethyl-ethyl)pyrimidinyl][(2R)- ydrofuran—2-yl]-1H-benzimidazolyl]urea and “Compound 13” relates to the mesylate salt of Compound 12. Similarly, “Compound 23” corresponds to 1-ethyl[6-fluoro[2-(1- hydroxy-1 -methyl-ethyl)pyrimidin-5 -yl][(2R)-tetrahydrofuranyl]-1H-benzimidazol a and “Compound 23A” relates to the mesylate salt of Compound 23. These are the same numbers used to identify said compounds and salts as used in the Examples above.

Table 2 — MIC Values of Selected Compounds MIC (”g/ml) Strain/Special Condition Protocol Compound Compound 13 23A Staphylococcus aureus 1 0.13 0.021 ATCC 29213 Staphylococcus aureusATCC 1 0.31 0.15 29213 with Human Serum Staphylococcus aureus 1 0.53 0.18 ATCC 29213 with Rat Serum Staphylococcus aureus 1 2 0.5 ATCC 29213 with Mouse Serum Staphylococcus aureus 1 1.29 0.3 ATCC 29213 GyrB T173I Enterococcusfaecalis ATCC 1 0.081 0.028 MIC (”g/ml) Strain/Special Condition Protocol Compound Compound 13 23A 29212, with Laked Horse Blood Enterococcusfaecium ATCC 0.39 0.11 49624 with Laked Horse Blood Enterococcusfaecium ATCC 0.25 0.11 49624 Streptococcus pneumoniae 0.022 0.01 ATCC 10015, with Laked Horse Blood Bacillus cereus ATCC 10987 0.031 Bacillus cereus ATCC 14579 HHHH 0.031 Bacillus subtilis ATCC 6638 Bacillus subtilis (168) ATCC 6051 Closiriclium le ATCC 0.38 BAA-1382 hilus influenzae 0.5 ATCC 49247 hilus influenzae (Rdl 2.5 1.3 KW20) ATCC 51907 Haemophilus zae 0.14 0.041 Rd0894 (AcrA-) Moraxella caiarrhalis ATCC 0.071 $0.016 25238 Moraxella caiarrhalis ATCC 0.04 30.016 49143 Neisseria gonorrhoeae 1.3 0.42 ATCC 35541 Neisseria hoeae 2.3 ATCC 49226 Escherichia coli AG100 WT N >16 Escherichia coli AG100 iolC 0.11 0.063 Escherichia coli ATCC NN 16 12 25922 Escherichia coli CHE30 Escherichia coli CHE30 iolC NNNN 0.125 Escherichia coli MC4100 >16 Escherichia coli MC4100 0.25 iolC Klebsiella pneumoniae N 16 ATCC 700603 Klebsiella pneumoniae 12 ATCC BAA-1705 Aciheiobacier baumahhii ATCC 19606 MIC (”g/ml) Strain/Special Condition ol Compound Compound 13 23A obacter baumannii >16 6 ATCC BAA-1710 Pseudomonas aeruginosa >16 >16 PAOl Pseudomonas aeruginosa 0.33 0.25 PAO750 Stenotrophomonas Not done >8 maltoplzilia ATCC BAA-84 Stenotrophomonas N Not done >8 maltoplzilia ATCC13637 Mycobacterium avium 103 0.47 0.18 M avium Far 0.94 0.23 M avium 3404.4 0.94 0.23 Nocardia caviae 2497 2 0.125 N asteroids 2039 ##4>4>4>4>4>4>4>4> 8 1 N nova 10 8 1 M. kansasii 303 Not Done 0.03 M kansasii 316 Not Done 0.06 M ii 379 Not Done <0.015 M tuberculosis H37RV 0.37 0.015 ATCC 25618 M tuberculosis Erdman 4; 0.25 0.06 ATCC 35801 M tuberculosis Erdman 0.045 0.03 ATCC 35801 M tuberculosis Erdman 2 0.5 ATCC 35801 with Mouse Serum M sus BB2 \ot Done 1 M abscessus MC 6005 \0t Done 1 M abscessus MC 5931 \0t Done 0.5 M abscessus MC 5605 \ot Done 1.5 M abscessus MC 6025 \ot Done 0.75 M abscessus MC 5908 \ot Done 1.5 M sus BB3 \ot Done 0.5 M abscessus BB4 \ot Done 2 M abscessus BB5 \0t Done 0.5 M abscessus MC 5922 \ot Done 0.25 M abscessus MC 5960 \ot Done 0.5 M abscessus BB1 \ot Done 2 M abscessus MC 5812 \0t Done 1 M abscessus MC 5901 \0t Done 1 M abscessus BB6 \0t Done 0.5 M abscessus BB8 \ot Done 0.5 M abscessus MC 5908 ##4>4>4>4>4>4>4>4>4>4>4>4>4>4>4>4> \ot Done 1 M abscessus LT 949 \0t Done 1 MIC (ug/ml) Strain/Special Condition Protocol Compound Compound 13 23A M abscessus BB10 Not Done 0.015 M abscessus MC 6142 Not Done 0.5 M sus MC 6136 ##4>4>4> Not Done 0.5 M abscessus MC 6111 Not Done 0.5 M abscessus MC 6153 Not Done 1 Table 3 shows the results of the MIC90 assay for selected nds of this invention.

Table 3 — MIC90 Values of Selected Compounds with Panels of Gram Positive, Gram Negative and Anaerobic Pathogens nd 13 Compound 23A Organism Number Protocol Range MIC90 Range MIC90 of (ug/ml) (ug/ml) (rig/ml) (”g/ml) Isolates Tested Aerobic Gram- positive Staphylococcus 67 0.03- 0.25 0.008- 0.03 aureus 0.5 0.06 ococcus 35 0.03- 0.12 0.008- 0.03 epidermidis 0.25 0.03 Enterococcusfaecalis 34 0.03- 0.25 0.015- 0.06 0.25 0.12 Enterococcusfaecium 33 0.12- 0.5 0.003- 0.12 0.5 0.25 ococcus 67 0.015- 0.06 0.008- 0.015 pneumoniae 0.06 0.03 olytic 28 0.06- 0.25 0.015- 0.12 streptococci (Groups 0.5 0.12 A, B, C and G) Aerobic Gram- negative Haemophilus 55 0.25- 8 0.06- 2 1 influenzae Moraxella catarrhalis 26 0.015- £0.004- 0.03 0.12 0.03 Acinetobacter 12 >8- >8 4 - >8 >8 baumannii Pseudomonas 12 >8- >8 >8- >8 >8 aeruginosa Escherichia coli 12 - >8 >8 >8- >8 2 12 NN Klebsiella >8- >8 2 - >8 >8 pneumoniae Compound 13 Compound 23A Organism Number Protocol Range MIC90 Range MIC90 of (”g/ml) (ug/ml) (”g/ml) (”g/ml) Isolates Tested s mirahl'll's 12 N >8- >8 >8 4 - >8 >8 Enterohacter cloacae 12 >8- >8 >8 >8- >8 >8 Neisseria 13 UJN 0.5- 1 1 0.12- 0.25 hoeae 0.25 Neisserl'a meningitidts 12 0.015- 0.12 0.008- 0.03 0.25 0.06 Anaerobes Bacteroicles and 26 2- >16 >16 0.12- 16 16 Parahacter spp.

Bacterol'desfragtll's 25 8- >16 >16 1- 16 16 Clostrl'cll'um dzfltctle 16 05- 16 1 0.06- 4 0.25 l'cll'um 12 0.12- 0.5 0.12- 0.5 0.5 perfrtngens 0.5 Fusohacterl'um spp. 16 1-4 2 0.015- >16 Peptostreptococcus ll 0.06- >16 0.03- >16 spp. >16 >16 ella spp. 13 3 05- >16 >16 0.06- 16 16 Table 3A also shows the results of the MIC assays for selected compounds of this invention. In Table 3A, “Compound 23A” corresponds to the methanesulfonic acid salt of 1-ethyl-3 - [6-fluoro-5 -[2-(1-hydroxymethyl-ethyl)pyrimidin-5 -yl] [(2R)- tetrahydrofuran—2-yl]—1H-benzimidazolyl]urea (23A). Similarly, “Compound W” corresponds to disodium (R)(5-(2-(3-ethylureido)—6-fluoro(tetrahydrofuran—2-y1)-1H- benzo[d]imidazoly1)pyrimidin—2-y1)propan—2-y1 phosphate (W). These are the same numbers used to identify said compounds in the synthetic Examples above.

Table 3A. MIC Values of Selected Compounds MIC (ug/ml) Strain Compound Compound Strain/Special Condition Source ol 23A W lococcus aureus ATCC ATCC1 1 0.021 8 29213 Staphylococcus aureus ATCC ATCC 1 0.15 8 29213 with Human Serum Staphylococcus aureus ATCC ATCC 1 0.18 8 29213 with Rat Serum Staphylococcus aureus ATCC ATCC 1 0.5 8 29213 with Mouse Serum MIC (ug/ml) Strain Compound nd Strain/Special Condition Source Protocol 23A W Staphylococcus aureus ATCC 2 1 0.3 >8 29213 GyrB T1731 Enterococcusfaecalts ATCC ATCC 1 0.028 1 29212, with Laked Horse Blood Enterococcusfaectum ATCC ATCC 1 0.11 2 49624 with Laked Horse Blood Streptococcus pneumoniae ATCC 1 0.01 0.25 ATCC 10015, with Laked Horse Blood Haemophtlus influenzae (Rd1 ATCC 2 1.3 8 KW20) ATCC 51907 Haemophtlus influenzae Hiroshi 2 0.041 0.25 Rd0894 (AcrA-) Nikaido Escherichia coli AG100 WT CGsc3 2 4 >16 Escherichia colt AG100 tolC4 Vertex 2 0.063 8 1American Type Culture Collection 2Constructed by Vertex 3Coli Genetic Stock Center 4all tolC constructs are toZszTn10 derived from CAG12184 (Coli Genetic Stock Center) In Table 4 below, the term “CMI” stands for The Clinical Microbiology ute located in Wilsonville, Oregon.

] Table 4 Panels of Anaerobic Organism Used to GenerateMIC90 Data CMI# ORGANISM A2380 B. fragilis A2381 B. is A23 82 B. fragilis A2486 B. fragilis A2487 B. fragilis A2489 B. is A2527 B. fragilis A2529 B. fragilis A2562 B. fragilis A2627 B. fragilis A2802 B. fragilis A2803 B. fragilis A2804 B. fragilis A2805 B. fragilis A2806 B. fragilis A2807 B. fragilis A2808 B. fragilis A2809 B. fragilis CMI# SM A2810 B. fragilis A2811 B. fragilis A2812 B. fragilis A2813 B. fragilis A2814 B. fragilis A2460 B. thetaioz‘aomicron A2462 B. thetaioz‘aomicron A2463 B. thetaioz‘aomicron A2464 B. thetaioz‘aomicron A2536 B. thetaioz‘aomicron A2591 B. mis A2604 B. vulgatus A2606 B. vulgatus A2613 B. ovatus A2616 B. ovatus A2815 Bacteroides rectum A2816 B. ureolyticus A2817 Bacteroides capillosus CMI# ORGANISM A2818 B. ureolyticus A2824 Parabacter distasom's A2825 B. ovatus A2826 B. uniformis A2827 B. mis A2828 B. vulgatus A2829 B. vulgatus A2830 B. ovatus A2831 B. thetaioz‘aomicron A2832 Parabacter distasom's A2833 B. thetaioz‘aomicron A2767 C. dzflzcz‘le A2768 C. dzflzcz‘le A2769 C. dzflzcz‘le A2770 C. dzflzcz‘le A2771 C. dzflzcz‘le A2772 C. ‘le A2773 C. dzflzcz‘le CMI# ORGANISM A2774 C. dzflzcz‘le A2775 C. dzflzcz‘le A2776 C. dzflzcz‘le A2777 C. ‘le A2778 C. dzflzcz‘le A2779 C. dzflzcz‘le A2780 C. dzflzcz‘le A2140 C. ngens A2203 C. perfringens A2204 C. perfringens A2227 C. perfringens A2228 C. perfringens A2229 C. perfringens A2315 C. perfringens A2332 C. perfringens A2333 C. perfringens A2334 C. perfringens A2389 C. perfringens CMI# ORGANISM A2390 C. perfringens A864 F. necrophorum A871 F. nucleaium A1667 F. necrophorum A1666 F horum A2249 F nucleaium A2716 Fusobacierium species A2717 Fusobacierium species A2719 Fusobacierium species A2721 Fusobacierium species A2722 Fusobacierium species A2710 Fusobacierium s A2711 Fusobacierium species A2712 Fusobacierium species A2713 Fusobacierium s A2714 Fusobacierium species A2715 Fusobacierium species A1594 Pepiosirepiococcus anaerobius CMI# ORGANISM A2158 Peptostreptococcus magnus A2168 Peptostreptococcus anaerobius A2170 Peptostreptococcus magnus A2171 Peptostreptococcus magnus A2575 treptococcus spp.

A2579 Peptostreptococcus asaccharolyticus A2580 Peptostreptococcus asaccharolyticus A2614 treptococcus asaccharolyticus A2620 Peptostreptococcus asaccharolyticus A2629 Peptostreptococcus spp.

A2739 Prevotella denticola A2752 Prevotella bivz‘a A2753 ella intermedia A2754 Prevotella intermedia A2756 Prevotella bivz‘a A2759 Prevotella bivz‘a A2760 Prevotella denticola A2761 Prevotella intermedia CMI# ORGANISM A2762 Prevotella melaninogenica A2765 Prevotella melaninogenica A2766 Prevotella melaninogenica A2821 Prevotella bivz‘a A2822 Prevotella bivz‘a QCBF B. is QCBT B. thetaioz‘aomicron QCCD C. dlfificz'le QCBF B. is QCBT B. thetaioz‘aomicron QCCD C. dlfificz'le In Table 5 below, the term “JMI” stands for The Jones Microbiology Institute located in North Liberty, Iowa.

Table 5: Panels of Gram ve and Gram Negative Organism Used to GenerateMIC90 Data JMI Organism JMI Isolate # Code Organism 394 ACB Acinetobacter baumannii 2166 ACE Acinetobacter baumannii 3060 ACE Acinetobacter baumannii 3170 ACE Acinetobacter baumannii 9328 ACE Acinetobacter baumannii JMI Organism JMI e # Code Organism 9922 ACE obacter baumannii 13618 ACB Acinetobacter baumannii 14308 ACB Acinetobacter baumannii 17086 ACB Acinetobacter nii 17176 ACB Acinetobacter baumannii 30554 ACB Acinetobacter baumannii 32007 ACB Acinetobacter baumannii 1192 ECL Enterobacter cloacae 3096 ECL Enterobacter cloacae 5534 ECL Enterobacter cloacae 6487 ECL Enterobacter cloacae 9592 ECL Enterobacter cloacae 11680 ECL Enterobacter cloacae 12573 ECL Enterobacter e 12735 ECL Enterobacter cloacae 13057 ECL Enterobacter cloacae 18048 ECL Enterobacter cloacae 25173 ECL Enterobacter e 29443 ECL Enterobacter cloacae 44 EF Zhuerococcusjhecahs 355 EF Zhuerococcusjhecahs 886 EF Zhuerococcusjhecahs 955 EF Zhuerococcusjhecahs 1000 EF Zhuerococcusjhecahs 1053 EF Zhuerococcusjhecahs 1142 EF Zhuerococcusjhecahs 1325 EF Zhuerococcusjhecahs 1446 EF Zhuerococcusjhecahs 2014 EF Zhuerococcusjhecahs 2103 EF Zhuerococcusjhecahs JMI Organism JMI Isolate # Code Organism 2255 EF Zhuerococcusjhecahs 2978 EF Zhuerococcusjhecahs 2986 EF Zhuerococcusjhecahs 5027 EF Zhuerococcusjhecahs 5270 EF Zhuerococcusjhecahs 5874 EF Zhuerococcusjhecahs 7430 EF Zhuerococcusjhecahs 7904 EF coccusjhecahs 8092 EF Zhuerococcusjhecahs 8691 EF Zhuerococcusjhecahs 9090 EF Zhuerococcusjhecahs 10795 EF Zhuerococcusjhecahs 14104 EF Zhuerococcusjhecahs 16481 EF Zhuerococcusjhecahs 18217 EF Zhuerococcusjhecahs 22442 EF coccusjhecahs 25726 EF Zhuerococcusjhecahs 26143 EF coccusjhecahs 28131 EF Zhuerococcusjhecahs 29765 EF Zhuerococcusjhecahs 30279 EF Zhuerococcusjhecahs 31234 EF Zhuerococcusjhecahs 31673 EF Zhuerococcusjhecahs 115 EF‘A Zhuerococcusjhecnun 227 EF‘A Zhuerococcusjhecnun 414 EF‘A Zhuerococcusjhecnun 712 EF‘A Zhuerococcusjhecnun 870 EF‘A Zhuerococcusjhecnun 911 EF‘A Zhuerococcusjhecnun 2356 EF‘A Zhuerococcusjhecnun JMI sm JMI Isolate # Code Organism 2364 EF‘A coccusjaecnhn 2762 EF‘A lhaerococcusjaecnhn 3062 EF‘A lhaerococcusjaecnhn 4464 EF‘A lhaerococcusjaecnhn 4473 EF‘A lhaerococcusjaecnhn 4653 EF‘A lhaerococcusjaecnhn 4679 EF‘A lhaerococcusjaecnhn 6803 EF‘A lhaerococcusjaecnhn 6836 EF‘A lhaerococcusjaecnhn 8280 EF‘A lhaerococcusjaecnhn 8702 EF‘A lhaerococcusjaecnhn 9855 EF‘A lhaerococcusjaecnhn 10766 EF‘A lhaerococcusjaecnhn 12799 EF‘A lhaerococcusjaecnhn 13556 EF‘A lhaerococcusjaecnhn 13783 EF‘A lhaerococcusjaecnhn 14687 EF‘A lhaerococcusjaecnhn 15268 EF‘A coccusjaecnhn 15525 EF‘A lhaerococcusjaecnhn 15538 EF‘A lhaerococcusjaecnhn 18102 EF‘A lhaerococcusjaecnhn 18306 EF‘A lhaerococcusjaecnhn 19967 EF‘A lhaerococcusjaecnhn 22428 EF‘A lhaerococcusjaecnhn 23482 EF‘A lhaerococcusjaecnhn 29658 EF‘A lhaerococcusjaecnhn 597 EC Escherichia coli 847 EC Escherichia coli 1451 EC Escherichia coli 8682 EC: Exchenchuzcoh 2012/021270 JMI Organism JMI Isolate # Code Organism 11199 EC Escherichia coli 12583 EC Escherichia coli 12792 EC Escherichia coli 13265 EC Escherichia coli 14594 EC ichia coli 22148 EC ichia 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 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 2012/021270 JMI sm JMI Isolate # Code Organism 10422 BSC Group C ococcus 14209 BSC Group C Streptococcus 29732 BSC Group C Streptococcus 8544 BSG Group G Streptococcus 18086 BSG Group G Streptococcus 29815 BSG Group G Streptococcus 147 HI Haemophl'lus tnfluenzae 180 HI Haemophl'lus tnfluenzae 934 HI Haemophl'lus tnfluenzae 970 HI Haemophl'lus tnfluenzae 1298 HI Haemophl'lus tnfluenzae 1819 HI Haemophl'lus tnfluenzae 1915 111 I7aenuymhflustufluenzae 2000 HI Haemophl'lus tnfluenzae 2562 HI Haemophl'lus tnfluenzae 2821 HI Haemophl'lus tnfluenzae 3133 HI Haemophl'lus tnfluenzae 3140 HI Haemophl'lus tnfluenzae 3497 HI Haemophl'lus tnfluenzae 3508 HI Haemophl'lus tnfluenzae 3535 HI Haemophl'lus tnfluenzae 4082 HI Haemophl'lus tnfluenzae 4108 HI Haemophl'lus tnfluenzae 4422 HI Haemophl'lus tnfluenzae 4868 HI Haemophl'lus tnfluenzae 4872 HI Haemophl'lus zae 85 8 HI Haemophl'lus tnfluenzae 625 8 HI Haemophl'lus tnfluenzae 6875 111 I7aenuymhflustufluenzae 7063 HI Haemophl'lus tnfluenzae JMI Organism JMI Isolate # Code Organism 7600 HI hilus influenzae 8465 HI Haemophilus influenzae 10280 HI Haemophilus influenzae 10732 111 ymhflusinfluenzae 10850 HI Haemophilus influenzae 1 1366 HI Haemophilus influenzae 11716 HI Haemophilus influenzae 1 1724 HI Haemophilus influenzae 1 1908 HI hilus influenzae 12093 HI Haemophilus influenzae 12107 HI Haemophilus influenzae 13424 HI Haemophilus zae 13439 HI Haemophilus influenzae 13672 HI Haemophilus influenzae 13687 HI Haemophilus influenzae 13792 111 I1aenuymhflusinfluenzae 13793 HI Haemophilus influenzae 14440 HI Haemophilus influenzae 153 51 HI Haemophilus influenzae 15356 HI Haemophilus influenzae 15678 HI Haemophilus influenzae 15800 111 I1aenuymhflusinfluenzae 17841 HI Haemophilus influenzae 18614 111 I1aenuymhflusinfluenzae 25195 HI Haemophilus influenzae 27021 HI Haemophilus influenzae 28326 111 ymhflusinfluenzae 28332 HI Haemophilus influenzae 29918 HI Haemophilus influenzae 29923 HI hilus influenzae JMI Organism JMI Isolate # Code Organism 31911 HI Haemophilus influenzae 428 KP\ Klebsiella pneumoniae 791 KP\ ella pneumoniae 836 KP\ Klebsiella pneumoniae 1422 KP\ Klebsiella pneumoniae 1674 KP\ Klebsiella niae 1883 KP\ Klebsiella pneumoniae 6486 KP\ Klebsiella pneumoniae 8789 KP\ Klebsiella pneumoniae 10705 KP\ Klebsiella pneumoniae 11123 KP\ Klebsiella pneumoniae 28148 KP\ Klebsiella pneumoniae 29432 KP\ Klebsiella pneumoniae 937 VICAT Moraxella catarrhalis 1290 VICAT Moraxella catarrhalis 1830 VICAT Moraxella catarrhalis 1903 VICAT Moraxella catarrhalis 4346 VICAT Moraxella catarrhalis 4880 VICAT Moraxella catarrhalis 6241 VICAT Moraxella catarrhalis 6551 VICAT Moraxella catarrhalis 7074 VICAT Moraxella catarrhalis 7259 VICAT Moraxella halis 7544 VICAT Moraxella halis 8142 VICAT Moraxella catarrhalis 8451 VICAT Moraxella catarrhalis 9246 VICAT Moraxella halis 9996 VICAT lla catarrhalis 12158 VICAT Moraxella catarrhalis 13443 VICAT Moraxella catarrhalis JMI Organism JMI Isolate # Code Organism 13692 VICAT Moraxella catarrhalis 13817 VICAT Moraxella halis 14431 VICAT Moraxella catarrhalis 14762 VICAT Moraxella catarrhalis 14842 VICAT Moraxella catarrhalis 15361 VICAT Moraxella catarrhalis 15741 VICAT Moraxella catarrhalis 17843 VICAT Moraxella catarrhalis 18639 VICAT Moraxella catarrhalis 241 GC Neisseria gonorrhoeae 291 GC Neisseria hoeae 293 GC Neisseria gonorrhoeae 344 GC Neisseria gonorrhoeae 451 GC Neisseria gonorrhoeae 474 GC Neisseria hoeae 491 GC Neisseria gonorrhoeae 493 GC Neisseria gonorrhoeae 503 GC Neisseria gonorrhoeae 521 GC Neisseria gonorrhoeae 552 GC Neisseria gonorrhoeae 573 GC Neisseria hoeae 592 GC Neisseria gonorrhoeae \VI Neisseria meningitidis 813 \VI Neisseria meningitidis 1725 \VI ria meningitidis 2747 \VI Neisseria meningitidis 3201 \VI Neisseria meningitidis 3335 \VI Neisseria itidis 7053 \VI Neisseria meningitidis 9407 \VI Neisseria meningitidis 2012/021270 JMI Organism JMI Isolate # Code Organism 10447 NM Net'sserl'a mentngtttdts 12685 NM Net'sserl'a mentngtttdts 12841 NM Net'sserl'a mentngtttdts 14038 NM Net'sserl'a mentngtttdts 1127 PVI Proteus mtrabl'll's 3049 PVI Proteus mtrabl'll's 4471 PVI Proteus mtrabl'll's 8793 PVI Proteus mtrabl'll's 10702 PVI Proteus mtrabl'll's 11218 PVI Proteus 'll's 14662 PVI Proteus mtrabl'll's 17072 PVI Proteus 'll's 19059 PVI Proteus mtrabl'll's 23367 PVI Proteus mtrabl'll's 29819 PVI Proteus mtrabl'll's 31419 PVI Proteus mtrabl'll's 1881 PSA monas aerugl'nosa 5061 PSA monas aerugl'nosa 7909 PSA Pseudomonas aerugl'nosa 8713 PSA Pseudomonas aerugl'nosa 14318 PSA Pseudomonas aerugl'nosa 14772 PSA Pseudomonas aerugl'nosa 15512 PSA Pseudomonas aerugl'nosa 17093 PSA Pseudomonas aerugl'nosa 17802 PSA Pseudomonas aerugl'nosa 19661 PSA Pseudomonas aerugl'nosa 29967 PSA Pseudomonas aerugl'nosa 31539 PSA Pseudomonas 'nosa 82 SA Staphylococcus aureus 99 SA Staphylococcus aureus WO 97269 JMI sm JMI Isolate # Code Organism 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 3 82 SA Staphylococcus aureus 1 112 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 4231 SA lococcus 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 JMI Organism JMI Isolate # Code Organism 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 lococcus aureus 10056 SA Staphylococcus aureus 10110 SA Staphylococcus aureus 11379 SA Staphylococcus aureus 1 1629 SA Staphylococcus aureus 11659 SA Staphylococcus aureus 12788 SA lococcus 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 143 84 SA Staphylococcus aureus 15428 SA Staphylococcus aureus 15430 SA Staphylococcus aureus 17721 SA lococcus aureus 18688 SA Staphylococcus aureus 19095 SA Staphylococcus aureus 20195 SA Staphylococcus aureus 22141 SA Staphylococcus aureus 22689 SA Staphylococcus aureus JMI Organism JMI Isolate # Code Organism 27398 SA Staphylococcus aureus 29048 SA Staphylococcus aureus 29051 SA Staphylococcus aureus 30491 SA Staphylococcus aureus 30538 SA Staphylococcus aureus SEPI Staphylococcus epidermtdts 53 SEPI lococcus epidermtdts 3 85 SEPI Staphylococcus epidermtdts 398 SEPI Staphylococcus epidermtdts 701 SEPI Staphylococcus epidermtdts 713 SEPI Staphylococcus epidermtdts 1381 SEPI Staphylococcus epidermtdts 2174 SEPI Staphylococcus epidermtdts 2286 SEPI Staphylococcus epidermtdts 2969 SEPI Staphylococcus epidermtdts 3417 SEPI Staphylococcus mtdts 3447 SEPI Staphylococcus epidermtdts 4753 SEPI Staphylococcus epidermtdts 7241 SEPI Staphylococcus epidermtdts 9366 SEPI Staphylococcus epidermtdts 10665 SEPI lococcus epidermtdts 1 1792 SEPI lococcus epidermtdts 12311 SEPI Staphylococcus epidermtdts 13036 SEPI Staphylococcus epidermtdts 13227 SEPI Staphylococcus epidermtdts 13243 SEPI Staphylococcus epidermtdts 13621 SEPI Staphylococcus epidermtdts 13638 SEPI Staphylococcus epidermtdts 13800 SEPI Staphylococcus epidermtdts 14078 SEPI Staphylococcus epidermtdts —104— JMI Organism JMI Isolate # Code sm 14392 SEPI Staphylococcus epidermidis 15007 SEPI Staphylococcus epidermidis 16733 SEPI Staphylococcus ml'cll's 18871 SEPI Staphylococcus epiderml'cll's 23285 SEPI Staphylococcus epiderml'cll's 27805 SEPI Staphylococcus epiderml'cll's 29679 SEPI lococcus epiderml'cll's 29985 SEPI Staphylococcus epiderml'cll's 30259 SEPI Staphylococcus epiderml'cll's 31444 SEPI Staphylococcus epiderml'cll's 268 SP\ Streptococcus pneumoniae 1264 SP\ Streptococcus pneumoniae 2482 SP\ Streptococcus pneumoniae 2653 SP\ Streptococcus pneumoniae 2994 SP\ Streptococcus pneumoniae 3123 SP\ Streptococcus pneumoniae 3124 SP\ Streptococcus pneumoniae 4336 SP\ Streptococcus pneumoniae 4858 SP\ Streptococcus pneumoniae 5606 SP\ Streptococcus pneumoniae 5881 SP\ Streptococcus pneumoniae 5897 SP\ Streptococcus pneumoniae 5900 SP\ Streptococcus niae 6051 SP\ Streptococcus pneumoniae 6216 SP\ ococcus pneumoniae 6556 SP\ Streptococcus pneumoniae 7270 SP\ Streptococcus pneumoniae 7584 SP\ Streptococcus pneumoniae 8479 SP\ Streptococcus pneumoniae 8501 SP\ Streptococcus pneumoniae WO 97269 JMI sm JMI e # Code Organism 9256 SP\ Streptococcus pneumoniae 9257 SP\ Streptococcus pneumoniae 10246 SP\ Streptococcus pneumoniae 10467 SP\ Streptococcus pneumoniae 10886 SP\ Streptococcus pneumoniae 1 1217 SP\ Streptococcus pneumoniae 11228 SP\ Streptococcus pneumoniae 11238 SP\ Streptococcus pneumoniae 1 1757 SP\ Streptococcus pneumoniae 1 1768 SP\ Streptococcus pneumoniae 12121 SP\ Streptococcus pneumoniae 12124 SP\ ococcus pneumoniae 12149 SP\ Streptococcus pneumoniae 12767 SP\ ococcus pneumoniae 12988 SP\ Streptococcus pneumoniae 13321 SP\ Streptococcus pneumoniae 13393 SP\ Streptococcus pneumoniae 13521 SP\ Streptococcus pneumoniae 13544 SP\ Streptococcus pneumoniae 13700 SP\ Streptococcus pneumoniae 13704 SP\ Streptococcus niae 13822 SP\ Streptococcus pneumoniae 13838 SP\ Streptococcus pneumoniae 14131 SP\ Streptococcus pneumoniae 14413 SP\ Streptococcus pneumoniae 14744 SP\ Streptococcus pneumoniae 14808 SP\ Streptococcus pneumoniae 14827 SP\ Streptococcus pneumoniae 14835 SP\ Streptococcus pneumoniae 14836 SP\ Streptococcus pneumoniae WO 97269 JMI sm JMI Isolate # Code Organism 15832 SP\ Streptococcus pneumoniae 17336 SP\ Streptococcus pneumoniae 17343 SP\ Streptococcus pneumoniae 17349 SP\ Streptococcus pneumoniae 17735 SP\ ococcus pneumoniae 18060 SP\ Streptococcus niae 18567 SP\ Streptococcus pneumoniae 18595 SP\ Streptococcus pneumoniae 19082 SP\ Streptococcus pneumoniae 19826 SP\ Streptococcus pneumoniae 22174 SP\ Streptococcus pneumoniae 22175 SP\ Streptococcus pneumoniae 27003 SP\ Streptococcus pneumoniae 28310 SP\ Streptococcus pneumoniae 28312 SP\ Streptococcus pneumoniae 29890 SP\ Streptococcus pneumoniae 29910 SP\ Streptococcus pneumoniae Example 32 Mouse S. aureus Kidney Infection Model s: female CD-1 mice (8—10 weeks of age; 6/group), were obtained from Charles River Laboratories and were housed and ined in accordance with the Guide to the Care and Use ofExperimental Animals.

] Bacterial strain and stocks Methicillin—sensitive S. aureus (MS SA) strain ATCC 29213 was obtained from the American Type Culture Collection. To prepare stocks for animal studies S. aureus was plated on Mueller Hinton Agar plates and incubated at 37°C overnight. 3-4 colonies from the plate were used to inoculate 5 mL Mueller Hinton Broth (MHB) that was incubated for 8 hours at 37°C with shaking at 300 RPM. The 5 mL 8-hour culture was diluted 20-fold into 100 mL and incubated ght (12-14 hours). The bacteria were pelleted for 20 minutes at 3000 X and washed twice with Phosphate-Buffered Saline (PBS) containing 0.5% Bovine Serum Albumin (BSA). Aliquots (lmL) containing ~l fu in PBS/20%Glycerol were frozen and stored at -800C until the day of use. Stock titers were confirmed by serial dilution and plating on Mueller Hinton agar plates.

Mouse S. aureus kidney ion model Prior to inoculation, bacterial stocks were thawed and washed once with PBS/BSA. The stocks were then diluted with PBS/BSA to a final tration of 1X 109cfu/mL to give an inoculum of approximately 1-2 X 108cfu per mouse in a volume of 100 uL. This um was found to be optimal in preliminary experiments that ed challenge doses of 106—109S. aureus organisms/mouse to establish a ~106cfu/kidneys burden at 26 hours post infection without mortality (data not shown). In these preliminary studies challenge with S. aureus at less than 107cfu/mouse induced istent or no chronic ions, whereas a dose of 109cfu/mouse resulted in rapid illness and ity in a high percentage of the mice. Injections were given intravenously via the tail vein using a sterile gauge needle. Upon completion of the mouse infections, the bacterial stocks used to challenge the mice were plated and counted to verify the concentration of the inocula.

] To assess the bacterial burden at the indicated times post infection (most commonly 2 to 26 hours), mice were euthanized and the s were aseptically harvested, and placed into sterile PBS/BSA (5 mL/pair kidneys). Kidneys were homogenized under e conditions, using a hand held homogenizer (Powergen 125, Fisher ific). During collection and homogenization all of the samples were kept on ice and the homogenizer was washed thoroughly and sterilized between each sample. Homogenates were serially diluted in sterile PBS/BSA and plated on MH agar to determine bacterial counts per pair of kidneys.

Table 6. Compound 23A Reduces S. aureus Burdens in the Mouse S. aureus Kidney Infection Model Time of Treatment Minutes 6 Hours 24 Hours Median Log Median Log Median Log Kidney Difference Kidney Difference Kidney Difference Burden (Log vs. Vehicle Burden (Log vs. Vehicle Burden (Log vs. cfu/ kidneys) Control cfu/ kidneys) Control cfu/ kidneys) Vehicle Control Vehicle 4.97 5.21 6.35 mg/kg Compound 4.84 -0.14 4.84 -0.37 4.48 -l.87 -lO8- Table 6. Compound 23A Reduces S. aureus Burdens in the Mouse S. aureus Kidney ion Model Time of Treatment Minutes 6 Hours 24 Hours Median Log Median Log Median Log Kidney Difference Kidney Difference Kidney Difference Burden (Log vs. Vehicle Burden (Log vs. Vehicle Burden (Log vs. cfu/ kidneys) l cfu/ kidneys) Control cfu/ kidneys) Vehicle Control mg/kg Compound 4.97 0.00 4.62 -0.59 3.44 -2.90 100 mg/kg Compound 4.89 -0.08 4.55 -0.66 3.49 -2.86 CD-1 mice (6/group) were challenged IV with S. aureus (ATCC 29213) at 2X108cfu/mouse. Two hours post challenge mice were treated via oral gavage with Vehicle (10% VitE TPGS) at 10 ml/kg or Compound 23A at 10, 30, 100 mg/kg. The 30-minute and 6-hour treatment groups were treated once, while the 24-hour group received a second treatment 10 hours after the first dose. After increasing amounts of time post treatment (30 s, 6 hours or 24 hours), the mice were euthanized and the kidneys harvested, homogenized and plated to quantitate S. aureus burdens. Burdens from pairs of kidneys for each mouse and the median for each group of mice werecalculated.

Results: Orally administered Compound 23A exhibited in viva efficacy t mentally induced kidney MSSA (SA 29213) infection. At 30 minutes after the initial treatment there was no difference in kidney burden between compound- and vehicle-treated mice. The 30 minute vehicle-treated group served as early control for comparison of nd effects at later time points.At 6hours after the first dose, all compound ents reduced bacterial burdens in the kidneys by 0.4-0.6 logs versus time-matched vehicle control.

Additionally, Compound 23A administered at 30 and 100 mg/kg ed 0.3-0.4 10g reduction versus the 30 minute early control.

After a treatment period of 24hours (that included a second dose of treatment administered at 10hours), a decrease in ial density compared to time-matched vehicle control was observed for all treatment groups. Compound 23A administered at 30 and 100 mg/kg BID provided 2.8-2.9 log reduction, s 10 mg/kg Compound 23A BID was more variable and provided a 1.8 log reduction versus the 24-hour vehicle-treated control. In addition, Compound 23A administered at 30 and 100 mg/kg showed approximately1.5 log reductions versus the 30-minute vehicle-treated control, indicative of bactericidal activity.

] In summary and as shown above in Table 6, BID dosing of 30 and 100 mg/kg Compound 23A diminished bacterial grth of methicillin-sensitive S. aureus (MS SA) strain ATCC 29213 as compared to the 30 minute control group at both the 6hours and 24hour assessment times while ent with 10 mg/kg Compound 23A limited bacterial growth at 6hours but was less effective than other treatment groups at 24hours.

Table 7. A Single Dose of Compound 23A Reduces S. aureus Burdens in the Mouse S. aureus Kidney Infection Model Median Kidney Log Reduction vs. Log ion vs. ent Group Burden (Log . Early Control Late Control cfu/k1dneys) Early Control 4.73 Late Control 6.63 1.90 mg/kg Compound 23A 4.73 0.00 -1.90 mg/kg Compound 23A 4.32 -0.40 -2.31 60 mg/kg Compound 23A 3.52 -1.21 -3.11 100 mg/kg Compound 3.31 -1.42 -3.32 ] CD-1 mice (6/group) were challenged IV with S. aureus (ATCC 29213) at 2x108 cfu/mouse. After 2 hours a single group of mice (Early Control (EC)) was euthanized and the kidneys harvested, homogenized and plated to quantitate S. aureus burdens. The additional groups of infected mice were treated via oral gavage with Vehicle at 10 ml/kg (10% VitE TPGS, Late Control, LC), Compound 23A at 10, 30, 60, 100 mg/kg. After 24 hours the groups of d mice were euthanized and the s harvested, homogenized and plated to quantitate S. aureus burdens. Burdens from pairs of kidneys for each mouse and the median for each group of mice were summarized.

Results: In summary andas shown above in Table 7,a single oral dose of Compound 23A exhibited in viva efficacy against an experimentally induced kidney MSSA (SA 29213) infection. After 24 hours all treatments showed decreases in bacterial density compared to time-matched vehicle control. Compound 23A demonstrated dose-dependent reductions of 1.9, 2.3, 3.1 and 3.3 log reductions versus vehicle control when administered at , 30, 60 or 100 mg/kg. In addition, doses of 60 and 100 mg/kg Compound 23A reduced bacterial burdens versus the early control by 1.2-1.4 logs suggesting Compound 23A has bactericidal activity.

Table 7A A Single Dose of Compound W Reduces Bacterial Burdens in the Mouse MSSA Kidney Infection Model Treatment Group Median Kidney Loglo Difference .

. Loglo Difference (Compound W Single Dose Burden (Loglo vs. Early vs Late Control Eguivalent) CFU/kidneys) Control ° Early Control 4.40 Late Control 5.94 1.54 16 (10) mg/kg Compound W 3.69 -0.71 -2.25 49 (30) mg/kg Compound W 3.22 -1.18 -2.72 99 (60) mg/kg Compound W 3.32 -1.08 -2.62 166 (100) mg/kg nd W 2.94 -1.46 -3.00 CD-1 mice (8/group) were challenged IV with S. aureus (ATCC 29213) at 2x108 cfu/mouse. After 2 hours a single group of mice (Early Control (EC)) was ized and the s harvested, homogenized and plated to quantitate S. aureus burdens. The onal groups of infected mice were treated via oral gavage with Vehicle at 10 ml/kg (Water, Late Control, LC), Compound W was administered at 16, 49, 99 or 166 mg/kg nominal dose levels that were expected to deliver 10, 30, 60 or 100 mg/kg of Compound 23A, the active moiety dose equivalents upon complete conversion at 10, 30, 60, 100 mg/kg. After 24 hours the groups of treated mice were euthanized and the kidneys harvested, homogenized and plated to quantitate S. aureus burdens. Burdens from pairs of s for each mouse and the median for each group of mice were summarized. s: In summary andas shown above in Table 8,a single oral dose of Compound W ted in viva efficacy against an experimentally induced kidney MSSA (SA 29213) infection. After 24 hours all treatments showed decreases in bacterial density compared to atched vehicle control. nd W demonstrated dose-dependent reductions of 1.9, 2.3, 2.7, 2.6 and 3.0 log reductions versus vehicle control when administered at 16, 49, 99 and 166 mg/kg that provided lent exposures of 10, 30, 60 or 100 mg/kg compound 24. In addition, doses of 16, 49, 99 and 166 mg/kg Compound W reduced bacterial burdens versus the early l by 0.7-1.5 logs suggesting Compound 23A has bactericidal activity.

] Example 33 NeutropenicRat Thigh Infection Model Animals: Specific-pathogen-free, male Sprague Dawley rats weighing between 76 to 100 grams were ed from Charles River Laboratories, Inc. (Wilmington, MA) and utilized in this experiment. s were allowed to acclimate for a minimum of seven (7) days before study commencement.

] Bacteria: The methicillin-sensitive Staphylococcus aureus(MS SA) ATCC strain 29213 was utilized for in vivo experimentation. The test isolate was subcultured twice onto standard microbiological agar media (trypticase soy agar with 5% sheep blood). The second transfer was made less than 24hours before use in the preparation of the thigh infection model Neutropenic Rat Thigh Infection Model: To induce neutropenia, rats were treated with the immunosuppressant cyclophosphamide at 150 mg/kg, administered in 1 ml by intraperitoneal (IP) injection, three day prior to infection. Rats were ed by an uscular (IM) 0.2 ml injection into both rear thighs with a 107 cfu/mlmethicillin- sensitiveStaphylococcus aureus 29213 suspension in normal saline. After increasing amounts of time (2-26 hours) the two rear thighs of each animal were harvested, rinsed with sterile saline, weighed, then placed in 50 ml sterile normal saline and placed on wet ice until homogenization. Approximately one-half of the total homogenized sample volume was passed through a large pore filter (to remove cartilage and large clumped pieces of tissue), diluted in saline and cultured onto agar media plates (trypticase soy agar with 5% sheep blood). All culture plates were incubated at approximately 37°C for 18-24 hours. Colonyforrning units were enumerated (in cfu/ml of homogenate) and the median for each treatment and control group was calculated. Typically each group had an n = 6, each thigh was considered a discrete . The median cfu/mlper group was compared to the l bacterial density at 2 hours (Early l) or that of the time-matched vehicle control group (Late Control, LC) group harvested simultaneously.

Table 8. Compound 23A Demonstrates Dose-Related and Time-Dependent Reductions in S. aureusThigh Burdens in Neutropenic Rats ent Time 8 Hours 24 Hours Median Log Median Log Treatment Thigh Log Reduction Thigh Log Difference Group Burden Difference vs. Late Burden Difference vs. Late (Log cfu/ vs. Early Control (Log cfu/ vs. Early Control mL) Control (8-hour) mL) Control (24-hour) Early .37 5.37 Control Late l 6.77 1.41 6.99 1.62 mg/kg Compound 5.42 0.05 -l.36 4.59 -0.78 -2.40 mg/kg Compound 4.75 -0.62 -2.02 4.31 -l.06 -2.68 60 mg/kg Compound 4.80 -0.57 -l.97 4.19 -l.l8 -2.80 Neutropenic rats (3/group) were infected by intramuscular (IM) challenge with S. aureus (ATCC 29213) at ~2X106 igh. After 2 hours a single group of rats (Early l (EC) was euthanized and the thighs harvested, homogenized and plated to quantitate S. aureus burdens. The additional infected rats were treated by oral gavage with Vehicle at 10 ml/kg (20% Cavitron/1% HPMCAS-MG, Late Control, LC) or Compound 23A at 10, 30, 60 mg/kg. The 8-hour treatment groups received a single treatment (QD) and were euthanized and thighs collected for cfu determination 8 hours post treatment (QD), while the 24-hour treatment groups received 2 doses 12 hours apart (q12h) and were euthanized and thighs collected 24 hours post treatment. Burdens from each individual thigh were determined and the cfu/ml and the median from the group of 3 rats was summarized.

Results: As shown above in Table ly administered nd 23A exhibited in viva efficacy against the MSSA (SA 29213). At 8 hours after the first dose all of the groups had ions in burdens compared to time-matched control, ~13 log reduction for Compound 23A at 10 mg/kg and ~2 log reduction for Compound 23A at 30 and 60 mg/kg. Compared to early control, Compound 23A at 10 mg/kg held the bacterial grth of SA 29213 to at least the point of stasis, whereas nd 23A at 60 and 100 mg/kg slightly decreased the ial burden by .6 logs.

After 24 hours and a second dose of treatment administered at 12hours, a decrease in bacterial y ed to late control of .8 logs was observed for all treatment groups. Compared to early control an approximately 0.8 log reduction was observed for Compound 23A at 10 mg/kg, while Compound 23A at 30 and 60 mg/kg provided ~ 1.1-1.2 log reductions.

Table 9. nd 13 Demonstrates Dose-Related and Time-Dependent Reductions in S. aureusThigh Burdens in Neutropenic Rats Treatment Time 8 Hours 24 Hours Median Log Median Log Thigh Log Difference Thigh Log Difference Treatment Burden Difference vs. Late Burden Difference vs. Late Group (Log cfu/ vs. Early Control (Log cfu/ vs. Early Control mL) Control (8-hour) mL) Control (24-hour) Early Control 5.30 5.30 Late Control 6.64 1.34 7.16 1.86 mg/kg .15 -0.15 -1.49 6.02 0.72 -1.14 Compound 13 60 mg/kg 4.95 -0.35 -1.69 4.31 -0.99 -2.85 Compound 13 100 mg/kg 4.82 -0.48 -1.82 4.17 -1. 13 -2.99 Compound 13 Neutropenic rats (3/group) were infected by intramuscular (1M) challenge with S. aureus (ATCC 29213) at ~2X106 cfu/thigh. After 2 hours a single group of rats (Early Control (EC)) was euthanized and the thighs harvested, nized and plated to quantitate S. aureus burdens. The additional infected rats were treated by oral gavage with Vehicle at 10 ml/kg (10% Vitamin E/TPGS, Late Control, LC) or Compound 13 at 30, 60, 100 mg/kg.

The 8-hour treatment groups received a single treatment (QD) and were euthanized and thighs ted for cfu determination 8 hours post treatment (QD), while the 24 hour treatment groups received 2 doses 12 hours apart (q12h) and were euthanized and thighs collected 24 hours post treatment. Burdens from each individual thigh were determined and the cfu/individual thigh and the median from the group of 3 rats was summarized for each group.

Results: As shown above in Table 9,orally stered Compound 13 exhibited in vivo efficacy against the MS SA (SA 29213). Differences in the extent of antibacterial activity were seen between the three treatment groups. At 8 hours after the first dose all of the groups had reductions in burdens compared to time-matched control, ~15 log ion for nd 13 at 10 mg/kg and ~1.7 and 1.8 log reduction for Compound 13 at 60 and 100 mg/kg.At 8 hours after the first dose, Compound 13 at 10mg/kg held the ial growth of SA 29213 to at least the point of stasis, whereas nd 13 at 60 and 100 mg/kg slightly decreased the bacterial burden versus early control by ~0.4 and ~05 logs respectively. After 24 hours and the second dose of treatment administered at 12 hours, a decrease in bacterial y compared to early control of approximately 1 log was exhibited by Compound 13 at 60 and 100 mg/kg. In contrast, Compound 13 administered at 30 mg/kg did not appear effective with variable cfu levels averaging 0.3 logs greater than the early control. However, all dose levels decreased bacterial density compared to late control. A reduction of ~1.1 log was observed for the 10 mg/kg ent group, whereas a reduction of 2.85 and ~3 logs was observed for the 60 and 100 mg/kg dose .

In summary, the q12h doses of 60 and 100 mg/kg Compound 13 diminished bacterial grth of SA 29213 as compared to the initial control group at both the 8 hours and 24 hours assessment times while treatment with 30 mg/kg limited ial grth at 8hours but was less ive than other ent groups at 24 hours.

Example 34 Seven-Day Oral (Gavage) Toxicity and Toxicokinetics Study in Rats The objectives of this study were: 1) to evaluate the potential toxicity of Compound 13 and Compound 23A when administered orally by gavage to male rats for 7 consecutive days and 2) to assess the toxicokinetics of Compound 13, and Compound 23A after the first and seventh doses.

Animals Species Source History and Justification Crl:CD(SD) rats were ed from Charles River Laboratories of Stone Ridge, NY. The animals were laboratory bred and experimentally na’1’ve. Rats were chosen because they are a species that is commonly used for nonclinical toxicity evaluations.

Number Sex Age and BodyWeightRange WO 97269 Forty rats (20 nulated males and 20 males with jugular vein cannulas) were ordered. From these animals, 15 noncannulated males and 15 cannulated males were used.

Animals were as uniform in age as possible. The rats were prepubertal to young adult, approximately 9 weeks of age at initiation of dosing. Their er-calculated birth date was retained in the study records. The weight range for the animals at the time of allocation to groups was 2185-3063 g.

Study Design ] The rats were assigned as shown in the Table 10 below. Animals received the test article or vehicle by oral gavage for 7 consecutive days and were terminated the day following completion of dosing. The first day of dosing was ated as Day 1 of the study.The animals were evaluated for changes in clinical signs, body weight, and other parameters as described below.

Table 10 - Group Assignment and Dose Levels N0. Ammals Dose N0. . Dose Dose Animals for Dose als (M) Test Doses (M) Level Concentration. Volume Necropsy G“mp Toxicokinetics Article Per Day Main Study (mg/kg/day) (mg/mL) (mL/kg) (Day 8) A 3 0 Vehicle 0 l 0 10 3 B 3 3 comlgound 100 1 10 10 6 C 3 3 comlgound 200 1 20 10 6 Compound D 3 3 100 l 10 10 6 Compound E 3 3 300 2 30 10 6 F 0 3 Vehicle 0 2 0 10 3 ] Route/Dose The vehicle and test article were administered by oral gavage once daily for 7 consecutive days at a dose volume of 10 mL/kg body weight for Group A and Groups B-D, tively. The test article and vehicle were administered by oral gavage twice daily, approximately 8 hours apart, for 7 consecutive days at a dose volume of 10 mL/kg body weight for Group E and Group F, respectively. The actual volume administered to each animal was calculated and adjusted based on the most recent body weight of each animal.

] In-Life Observations and Measurements Observations ] Animals were observed for viability at least once in the morning and once in the afternoon, at least 4 hours apart, throughout the study. During the treatment period, daily -ll6- cageside ations were made and ed predose and postdose (following the first dose only). The postdosing ations made during treatment ed at the following times based on CmaX/Tmax for the two compounds from previous studies: 1 hour postdose for Groups A-F.

One cageside observation was made on the day of necropsy.

Unscheduled Observations Any findings observed at times other than scheduled observation times were to be recorded on an unscheduled observation or in Provantis, however, no abnormalities were observed throughout the study. Provantis is an electronic data collection, ment and reporting system that is commonly used in the art.

Body Weights Prior to start of dosing, body weights were measured for randomization on Day 1.

During the treatment, body weights were measured on Day 1 and Day 7. In addition, fasted body s were measured prior to necropsy for calculation of organ/body weight ratios.

Food Consumption Throughout the study, food consumption was measured daily starting 3 days prior to start of dosing.

Clinical Pathology Evaluation Blood s for evaluation of hematology, coagulation, and serum chemistry parameters were collected from all animals from the retro-orbital plexus (under COz/Oz anesthesia, for the main study s) or jugular vein cannula (for the toxicokinetic animals) prior to necropsy. Due to residual heparin used to keep the cannulas patentfor the toxicokinetic animals, coagulation samples from these rats, were not able to be analyzed.

The animals were fasted overnight prior to blood collection. On the day of blood collection for clinical pathology analyses, the animals were not necropsied until after the blood was collected and the samples judged to be acceptable by the clinical ogy group.

Hematology An appropriate amount of blood was collected in ontaining tubes. The whole blood samples were ed for the parameters indicated below in Table 11.

Table 11— Whole Blood ters Red blood cells (RBC) Mean corpuscular volume (MCV) (count and morphology) -ll7- White blood cells (WBC) Mean corpuscular hemoglobin (MCH) (total and differential) Hemoglobin concentration Mean corpuscular hemoglobin (HGB) concentration (MCHC) Hematocrit (HCT) Platelet count (PLAT) Reticulocyte count Mean platelet volume (MPV) (ABSRET) Coagulation An appropriate amount of blood was collected in tubes containing sodium citrate and then centrifuged to obtain plasma for the determination of prothrombin time (PT) and activated partial thromboplastin time (APTT).

Serum Chemistry An appropriate amount of blood was ted in tubes without anticoagulant.

The sample was allowed to clot and then was centrifuged to obtain serum. The serum was analyzed for the parameters ted below in Table 12.

Table 12 — Serum Parameters Sodium (NA) Calcium (CA) Potassium (K) Inorganic phosphorus (PHOS) de (CL) Glucose (GLU) Total bilirubin (TBILI) Urea nitrogen (BUN) Alkaline phosphatase Total n (TPRO) (ALKP) Lactate dehydrogenase Albumin (ALB) (LDH) Aspartate aminotransferase Globulin (GLOB) (AST) Alanine aminotransferase Albumin/globulin ratio (ALT) (A/G) Gamma-glutamyltransferase Cholesterol (CHOL) (GGT) Creatine phosphokinase (CK) Triglycerides (TRIG) Creatinine (CREA) Toxicokinetics On the lSt and 7th day of dosing, blood samples (approximately 0.5 mL/sample) were collected from the jugular vein cannula for all toxicokinetic animals at the timepoints listed below into K3EDTA-containing tubes. kinetic s from the control group (Group F) only had a single blood tion ng from each collection day at the l-hour timepoint (following the first dose administration of the day). Prior to each collection, a small sample of blood (with heparin blocking on) was removed from the cannula and discarded. A new syringe was placed on the cannula, and the protocol-required sample was taken. The syringe with the blood sample was removed, and a new syringe with saline attached to the cannula. Blood volume was replaced with an equal volume of saline and then blocking solution placed in the cannula. Each animal was returned to its cage until the next collection int.

All samples collected during this study were placed in labeled containers. Each label contained the following ation: 1) Study number, 2) Animal number, 3) collection interval, 4) Group and Sex, and 5) Date of collection.

] The blood samples were mixed immediately by inverting, then placed on wet ice and centrifuged cold g, ~10 s, ~ 5 °C) to obtain . The plasma was split into 96-well 1.4-mL polypropylene tubes with pierceable TPE capcluster certified RNase, DNase free caps as2 aliquots and stored frozen (3-70 °C).

Table — 13 - Sample Collection Timepoints Timepoint Window1 Predose Predose l h : 4 min 2 h2 :: 5 min 4 h : 5 min 8 h3 :: 5 min 12 h :: 10 min 24 h :: 20 min 48 h4 40 min IAll samples were collected within the collection window. 2Following Day 1 dosing only.

SObtainedfrom Groups E and Fprior to PM dose administration. 4Following Day 7 dosing only.

Termination No animal was deemed moribund during the study.All study animals were euthanized and ted to a necropsy following the protocol-prescribed number of days of treatment. All animals were terminated by exsanguination (severing the abdominal aorta while under deep COz/Oz anesthesia).

Necropsy -ll9- A necropsy with tissue collection was conducted on all animals terminated during the study. The necropsy included examination of: carcass and muscular/skeletal system; all al surfaces and orifices; cranial cavity and external surface of the brain; neck with associated organs and tissues; and thoracic; abdominal; and pelvic cavities with their associated organs and tissues.

All abnormalities were bed and recorded.

Organ Weights For all animals euthanized at scheduled necropsies; the kidneys; liver; and prostate gland were weighed. ing weighing; an approximate 1 gram sample of liver and kidney was weighed; erred to Precellys 7 mL CK28 Tissue Homogenizing tubes (Cat. No. 1); snap-frozen; and analyzed.

Organ/body ratios were calculated using the terminal fasted body weight obtained prior to necropsy.

Tissue Preservation and Bone Marrow Collection The tissues and organs indicated below in Table 14 were collected from all animals and were ved in 10% neutral-buffered in with the exception of the testes; ymides; and eyes. Testes; epididymides; and eyes with optic nerve attached were fixed in Modified Davidson’s Solution for ~24-48 hours; rinsed with water; and then transferred to % neutral-buffered formalin for storage.

Table 14 — Tissue Collection Tissue Submitted at Organ Weight Histopathology Necropsy Animal ID X Adrenal gland (2) X Aorta X , mesenteric X Bone ) X Bone marrow (sternum) X Brain X Epididymides (2) X Esophagus X Eyes (2) X Gross lesions X Heart X Intestine, cecum X Intestine, colon X Intestine, duodenum X Intestine, jejunum X Tissue Submitted at Organ Weight Histopathology Necropsy Intestine, ileum X Intestine, rectum X s (2) X X X Liver X X X Lungs X Lymph node, mandibular X Lymph node, mesenteric X Mammary gland X Nerve, optic X Nerve, sciatic X Parathyroid gland (2)a X Pancreas X Pituitary X Prostate X X X Seminal vesicles X al muscle (biceps X femorl‘s) Skin (abdominal) X Spinal cord, cervical X Spinal cord, thoracic X Tissue Submitted at Organ Weight Histopathology Necropsy Spinal cord, lumbar X Spleen X Stomach X Testes (2) X Thymus X d gland (2)21 X Tongue X Trachea X Urinary bladder X “Thyroid weighed with parathyroids attached.

Histopathology For all animals scheduled for the terminal necropsy, the kidneys, liver, and prostate were embedded in paraffin, sectioned and d with hematoxylin and eosin for further examination by light microscopy. For Groups A, D, E, and F only, the remaining tissues listed above were embedded in paraffin, ned and stained with xylin and eosin for fiirther ation by light microscopy.

Statistical Analysis Where appropriate, numeric animal data were evaluated statistically.

For comparative statistics, Group A (control group) was compared to Groups B and C (treated groups, dosed QD) and Group F (control group, dosed BID) was compared to Group E (treated group, dosed BID). Data were evaluated using the Levene Test for neity of variances and the Shapiro-Wilks Test for normality of distributions, with significance at p S 0.05. Data determined to be homogeneous and of normal distribution were ted by analysis of variance ). If the ANOVA verified significance at p: 0.05, pairwise comparisons of each treated group with the tive control group were made using a tric test (Dunnett Test) to identify statistical differences (p S 0.05). Data determined to be nonhomogeneous or of nonnormal distribution were evaluated using a Kruskal-Wallis Test for group factor significance. If icance (p S 0.05) existed between groups, a nonparametric test (Wilcoxonwith Bonferroni-Holm), was used to compare treatment groups to the control group. Food consumption data from animals where spillage occurred was excluded from the applicable time period. Comparative statistics of food consumption data were limited to the Dunnett Test (parametric). Statistics were not performed on pretest food consumption (Day 4 to Day 1).

Results The exposures for different dosage levels of Compound 23A and nd 13 were dose related. No adverse observations or effects on mean body weight were observed in animals treated with either Compound 13 or nd 23A. Mean food consumption was reduced during different intervals of the study for animals treated once daily with Compound 13 (100 or 200 mg/kg) and twice daily with Compound 23A (300 . However, as the decreased food consumption was not correlated with body weight changes in the Compound 13 and Compound 23A groups, these effects were not considered to be adverse or biologically significant. The mean calcium ion tration (CA) was statistically lower, while the mean ALT and the AST for the group of rats administered 300 mg/kg Compound 23A twice a day were statistically higher when ed to the controls treated twice a day.

No test article-related histopathological findings were noted for animals receiving either Compound 13 or Compound 23A at any dose regimen.

Within the scope of this study and based on the absence of changes in body weight, clinical pathology, and histopathology, the NOEL (No-Observable—Effect-Level) for nd 13 administered to male rats once a day for 7 days orally via gavage was 200 mg/kg (844 ug*hr/ml Day 7 AUC), while the NOEL for Compound 23A administered once a day was 100 mg/kg (82 ug*hr/ml AUC). The NOAEL (No-Observable-Adverse-Effect- Level) for Compound 23A administered to male rats twice a day for 7 days orally via gavage was 300 mg/kg (291 ug*hr/ml AUC).

Therefore, Compounds 13 and 23A did not demonstrate adverse toxicity within the scope of the study at dose levels up to 200 mg/kg/day and 600 mg/kg/day, respectively. —124— Example 35 An Oral Range g Toxicity and Toxicokinetic Study in Male Cynomolgus s The objectives of this study were 1) to evaluate the potential toxicity of Compound 23 when administered orally by gavage to male Cynomolgus s for 7 consecutive days; and 2) to assess the toxicokinetics of Compound 23 after the first and seventh doses.

Animals Species Source History and Justification Cynomolgus monkeys (Macaca Fascicularis) were obtained from Primus Bio- Resources Inc. of PinCourt, Quebec, Canada. Cynomolgus monkeys were chosen because they are a non-rodent species that is ly used for nonclinical toxicity evaluations.

Number Sex Age and Body Weight Range Eight (2 naive and 6 non-naive) males were used in the study. The animals were young adults and weighed between 2 to 4 kg at the onset of .

Study Design The animals were assigned as shown in Table 15 below. Animals received Compound 23 or vehicle by oral gavage once per day for 7 consecutive days and were terminated the day following completion of dosing. The first day of dosing was designated as Day 1 of the study. The actual volume administered to each animal was ated and adjusted based on the most recent body weight of each animal.

Table 15 — Group Assignment and Dose Levels Dose Level Concgfiffation Dose Volume Number of ““313 (mu/k (m mL) mUk) l * 0 5 2 50 10 5 2 100 20 5 2 200 40 5 2 * The Control animals received the l/vehicle (20% captisol/1% HPMCAS/1%PVP in 0.01M KCl/HCL buffer) alone In-Life Observations and Measurements Observations Cage-side clinical signs (ill health, behavioral s etc.) were recorded at least once daily during the study.

Body s Body weights were recorded for all s prior to group assignment and on Days 1 (prior to dosing), 3 and 7 as well as terminally prior to necropsy (fasted).

Electrocardiography (ECG) Electrocardiograms ar limb leads I, II and III, and augmented unipolar leads aVR, aVL and aVF) were obtained for all monkeys once during the pre—treatment period and again on Day 7 (post-dosing).

] The tracings were assessed for gross changes indicative of cardiac ical dysfunction. The potential presence of abnormalities involving heart rate (lead 11), sinus and atrioventricular rhythm or tivity were determined. Heart rate, PR interval, QRS duration, QT and QTc intervals values were measured.

Toxicokinetics A series of 7 blood samples (approximately 0.5 mL each) were ted from each monkey on Days 1 and 7 at the following time : predose, 30 minutes and 2, 3, 6, 12 and 24 hours post-dose. For this purpose, each monkey was bled by venipuncture and the samples were collected into tubes containing the anticoagulant, K2EDTA. Tubes were placed on wet ice until ready for processing.

Clinical Pathology Laboratory investigations (hematology, coagulation, clinical try and urinalysis) were performed on all s prior to start of treatment and prior to termination on Day 8.

Blood samples were collected by venipuncture following an overnight period of food deprivation consisting of at least 12 hours but no more than 20 hours. Urine was collected from animals deprived of food and water, overnight (at least 12 hours but no more than 20 hours).

Hematology ] The following parameters were measured on blood samples collected into EDTA anticoagulant: red blood cell count, mean corpuscular hemoglobin (calculated), hematocrit (calculated), mean corpuscular volume, hemoglobin, morphology of cells, white blood cell count, platelet count, white blood cell differential (absolute), reticulocyte (absolute and tage) and mean corpuscular hemoglobin concentration (calculated).

Coagulation WO 97269 ted partial thromboplastin time and prothrombin time were measured on blood s collected into citrate anticoagulant. al Chemistry The following parameters were measured on blood samples collected into tubes containing clotting activator: a/g ratio (calculated), creatinine, e aminotransferase, globulin lated), albumin, e, alkaline phosphatase, phosphorus (inorganic), aspartate aminotransferase, potassium, bilirubin (total), sodium, calcium, total protein, chloride, cerides, cholesterol (total), urea, gamma glutamyltransferase and sorbitol dehydrogenase.

Urinalysis The following parameters were measured on urine samples: bilirubin, protein, blood, sediment microscopy, color and appearance, specific gravity, glucose, urobilinogen, ketones, volume and pH.

Termination All animals were euthanized upon completion of the treatment period on Day 8 following an overnight period without food.The monkeys were pre-anesthetized with Ketamine and then euthanized by an intravenous overdose of sodium pentobarbital followed by exsanguination by transsection of major blood s.

Necropsy A necropsy with tissue tion was conducted on all s terminated during the study. The necropsy included examination of: carcass and muscular/skeletal system, all external surfaces and orifices, cranial cavity and al surface of the brain, neck with associated organs and tissues, and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

All abnormalities were described and recorded.

Tissue Preservation On completion of the gross examination and selected organ weighing, the tissues and organs were retained as noted below in Table 16. Neutral buffered 10% in was used for fixation and preservation unless otherwise indicated.

Table 16. Tissue and Organ Retention VYYSSUES “[2]?" mg)“ N3"? rungs mE3 Adrenals Pituitary Animal identification Prostate Aorta (thoracic) -. € € Rectum mmm’afi —Blood Salivary Gland, mandibular —Bone marrow smears (3) Sciatic nerve —Brain Seminal vesicles Cecum al muscle Colon Skin & subcutis cic) —Epididymides Duodenum —Esophagus um —Eyes Ileum Femur & marrow Spinal Cord, cervical Gallbladder Spleen 4 —Heart Stemum & marrow —Kidneys Stomach —Liver (2 lobes) .4estes 4 mmmmmmmmmmmmm — 05-... Lungs (2 lobes) Thymus 4 OD —Lymph Node, mandibular Thyroid gland/parathyroids 4 —Lymph Node, mesenteric .4O500c(D o Mammary gland cic a 6 Optic nerves Urinary bladder mmmm Pancreas ‘ bnormal findings —Davidson’s fluid used for fixation and preservation b Lungs were infused with 10% neutral buffered formalin used for fixation and preservation C Lungs were weighed with trachea -—Bouin’s fluid used for fixation and preservation Examined microscopically Histopathology For all animals, all tissues indicated above were embedded in n, sectioned and stained with hematoxylin and eosin and examined by light microscopy.

Results The exposures for different dosage levels of Compound 23 were dose related.

There were no clinical signs, or changes in body weights, electrocardiography parameters, clinical pathology ters, or organ weights that could be attributed to the administration of Compound 23 at doses up to 200 mg/kg/day. Similarly, there were no copic or microscopic findings that could clearly be attributed to the administration of Compound 23 at doses up to 200 mg/kg/day. The no observed effect level (NOEL) for Compound 23 in male lgus monkeys was determined to be 200 mg/kg/day.

Example 36 PHARMACOKINETIC STUDIES -l28- The pharmacokinetic parameters of selected compounds of this invention were determined in the experiments described below. General analytic procedures and specific experimental protocols were employed as follows: l Analytic Procedures The following general analytic ures were employed in the pharmacokinetic experiments described below: Sample Analysis. Concentrations of Compound 23and Compound W in plasma were determined using a high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method. Before extraction, plasma samples were diluted using blank plasma 2-, 4-, 5-, or 10-fold, as necessary, ing on the dose level or formulation.

Compound 23 and Compound W along with the internal standard (IS) were extracted from (diluted) plasma, 100 uL each, by direct protein itation with acetonitrile (1 :4 ratio of /acetonitrile). After centrifugation, the supernatant extract (10 uL) was injected onto the LC/MS/MS system. The HPLC system included a Waters Xterra MS C18 column, 5 , 2.1 mm er x 50 mm long eluted with a gradient mobile phase consisting of 0.1% formic acid in water or in acetonitrile.

The es were detected by MS/MS with Atmospheric Pressure Chemical Ionization (APCI) in the mode of multiple reaction ring (MRM). The lower limit of quantitation (LLOQ) was 1, 2, 4, 5, 10, or 20 ng/mL, depending on the sample dilution factor.

The linear range of the assay was from 1 to 5000 ng/mL. The intra-day and inter-day assay cy was within 2% of the nominal values. The intra- and inter-day assay variability was <10%.

Samples of the dose suspension formulation of Compound W were assayed with an HPLC/UV method after 10-fold to 500- or 1000-fold of dilution with DMSO:acetonitrile:water (33:33:33) depending on the dose level or formulation. Samples of the dose solution formulation of Compound W were d with an HPLC/UV method after -, 50-, 100 or 500-fold of dilution with DMSO:water (50:50) depending on the dose level or formulation.

Pharmacokinetic Data Analysis. Plasma concentration-time profiles of Compound 23 and nd W were analyzed by noncompartmental pharmacokinetic methods using WinNonlin® Professional Edition software, Version 5.1.1 (Pharsight Corporation, Mountain View, CA).

Key pharmacokinetic ters including AUCau, AUCextrap, Cmax, tmax, Cl_obs, Vss_obs and tl/z were ined.

Statistical Data Analysis. Descriptive statistical data of plasma concentrations and pharmacokinetic parameter estimates were calculated, including the mean, standard deviation (SD), and coefficient of variation (%CV) using WinNonlin re, Version 5.1.1 or Microsoft Excel 2000.

Monkey Oral Study Malecynomolgus s (n=3 per dose group)were administered single nominal PO doses of 3, 30 and 300 mg/kg of Compound W by . Compound W was formulated in 0.5% MC (microcrystalline cellulose). Animals had free access to food and water before and after dosing.

Blood s (approximately 0.25 mL each) were collected via a carotid artery catheter prior to dosing and at 0 (predose), 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48 hours post dose. Each blood sample was collected into a tube that was kept on wet ice and contained potassium EDTA as the agulant. Plasma was separated and stored at approximately -70°C until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine the trations of nd 23 and Compound W with a lower limit of quantitation (LLOQ) of 1 to 20 ng/mL, depending on the sample dilution factor. Plasma concentration vs. time data was subjected to noncompartmental pharmacokinetic (PK) analysis. The results of this analysis are provided in Table 17.

Table 17. Pharmacokinetic Data from Monkey Oral Study Dose Cmax AUC AUCextrap Tmax t1/2 (mg/kg) Route Formulation Analyte (ug/ml) (ug * hr/ml) (ug* hr/ml) (hr) (hr) Compound P0 0.5% MC 23 14.4 24.7 24.8 1.7 13.9 Compound 100 P0 0.5% MC 23 20.9 76.7 76.9 2.3 8.3 Compound 300 P0 0.5% MC 23 23.8 155.1 155 1.2 5.6 Compound P0 0.5% MC W 0.0264 0.0453 0.206 0.83 — Compound 100 P0 0.5% MC W 0.322 0.432 0.437 0.67 5.31 300 P0 0.5% MC Compound 4 3.69 3.76 0.58 13.15 -l30- Monkey IV Study Male cynomolgus monkeys (n=3 per dose group) were administered a single nominal IV bolus dose of 1 mg/kg of Compound W via a jugular vein cannula. Compound W was formulated in D5W (5% dextrose in water solution). Animals had free access to food and water before and after dosing.

] Blood samples (approximately 0.25 mL each) were collected via a carotid artery catheter prior to dosing and at 0 (predose), 5min, 10min, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48 hours postdose. Each blood sample was collected into a tube that was kept on wet ice and contained potassium EDTA as the anticoagulant. Plasma was separated and stored at approximately -70°C until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine the concentrations of Compound 23 and Compound W, with a lower limit of quantitation (LLOQ) of 1 to 20 ng/mL, depending on the sample dilution factor. Plasma tration vs. time data were subjected to partmental pharmacokinetic (PK) analysis. The results of this analysis are provided in Table 18.

Table 18. Pharmacokinetic Data from Monkey IV Study C0 AUC Cl Dose (ug/ (ug*hr/ rap n/ t1/2 Vss (mg/kg) Route Formulation Analyte ml) ml) (ug*hr/ml) kg) (hr) (L/kg) Compound IV D5W 23 10.9 3.78 3.81 23.4 6.17 2.09 Compound IV D5W W 62.4 5.79 5.83 18.2 5.35 1.88 -l3l- Rat Oral Study Groups of maleSprague Dawley rats (n=3 per dose group) were administered single nominal oral doses of 3, 10, 30, 300 mg/kg of Compound W by gavage. Compound W was formulated in either0.5% MC (microcrystalline cellulose) or 20% Captisol, 1% S (hydroxypropyl methylcellulose acetyl succinate), 1% PVP inylpyrrolidone). Animals had free access to food and water before and after dosing.

Blood samples (approximately 0.25 mL each) were collected Via a carotid artery catheter prior to dosing and at 0 (predose), 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 hours post dose. Each blood sample was collected into a tube that was kept on wet ice and contained potassium EDTA as the anticoagulant. Plasma was separated and stored at approximately -70°C until Plasma samples were analyzed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine the concentrations of Compound 23 and Compound W with a lower limit of quantitation (LLOQ) of 1 to 20 ng/mL, depending on the sample dilution factor. Plasma concentration vs. time data was subjected to partmental pharmacokinetic (PK) is. The results of this analysis are provided in Table 19.

Table 19. Pharmacokinetic Data from Rat Oral Study Dose CmaX/C0 AUC AUCextrap TmaX t1/2 (mg/kg) Formulation Analyte (ug/ml) (ug* hr/ml) (ug* hr/ ml) (hr) (hr) Compound 3 0.5% VIC 23 0.117 0.311 0.314 0.58 4.06 Compound 0.5% VIC 23 2.9 22.5 22.6 1.7 2.6 Compound 100 0.5% VIC 23 6.6 77.1 77.4 2.5 2.7 300 0.5% VIC 23 11.7 222.8 307.6 — 17.9 % CAPT, 1% HPVIC- Compound 300 AS, 1% PVP 23 16.2 294.6 — 5 — Compound 3 0.5% VIC W — — — — — Compound 0.5% VIC W 0.022 0.178 0.058 3.3 3.1 Compound 100 0.5% VIC W 0.021 0.061 0.066 0.8 7.2 Compound 300 0.5% VIC W 2.33 0.324 0.464 1.2 11.3 % CAPT, 1% HPVIC- Compound 300 AS, 1% PVP W 0.6 2.37 4.27 1.8 — Rat IV Study ] Groups of male Sprague Dawley rats (n=3 per dose group) were administered single nominal IV bolus doses of 1 and 5 mg/kg of Compound W via a jugular vein a.

Compound W was formulated in D5W. Animals had free access to food and water before and after dosing. Blood samples (approximately 0.25 mL each) were collected via a carotid artery catheter prior to dosing and at 0 (predose), 5min, 10min, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24hours post dose. Each blood sample was collected into a tube that was kept on wet ice and contained potassium EDTA as the agulant. Plasma was separated and stored at approximately -70°C until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to determine the concentrations of Compound 23 and Compound W with a lower limit of tation (LLOQ) of 1 to 20 ng/mL, depending on the sample dilution factor. Plasma tration vs. time data were subjected to noncompartmental pharmacokinetic (PK) analysis. The results of this analysis are provided in Table 20.

Table 20. Pharmacokinetic Data from Rat IV Study Dose AUC (mg/ Cmax/C0 (ug*hr/ rap t1/2 Cl_obs Vss_obs kg) Formulation Analyte (ug/ml) ml) (ug*hr/ ml) (hr) (ml/min/kg) (L/kg) Compound 1 DSW 23 0.247 0.306 0.31 1.8 54.9 3.8 Compound DSW 23 1.2 3.04 3.06 3.6 27.3 4.08 1 DSW W 4.8 0.416 0.419 0.9 46.7 0.38 Compound DSW W 9.03 1.11 1.12 7.2 84.6 5.8 Mouse Oral Study Groups of femaleCD-1 mice (n=3 per dose group) were administered single nominal oral doses of 10, 30, 100 mg/kg of Compound W by gavage. Compound W was formulated in 0.5% MC. Animals had free access to food and water before and after dosing.

Blood samples (approximately 0.025 mL each) were collected from the sub-mandibular vein prior to dosing and at 0 (predose), 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 hours postdose. Each blood sample was collected into a tube that was kept on wet ice and ned potassium EDTA as the anticoagulant. Plasma was separated and stored at approximately -70°C until analysis. -l33- Plasma samples were analyzed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with a lower limit of quantitation (LLOQ) of 1 to 20 ng/mL, depending on the sample dilution factor. Plasma concentration vs. time data was subjected to noncompartmental cokinetic (PK) analysis. The results of this analysis are provided in Table 21.

Table 21. Pharmacokinetic Data from Vlouse Oral Study Dose AUC (0-t) Cmax (mg/ kg) Formulation (ug*hr/mL) (ug*hr/ ml) Tmax (hr) 0.5% MC 1.7 1.2 0.3 0.5% MC 4.1 2.1 0.3 100 0.5% MC 26.6 9.1 0.4 The studies described above, demonstrate that Compound W is converted in viva into Compound 23 in at least rats, dogs and s.

Example 37 ENZYMOLOGY STUDIES The enzyme tion activities of selected nds of this invention were determined in the experiments described below: DNA Gyrase ATPase Assay The ATP hydrolysis activity of S. aureus DNA gyrase was measured by coupling the production of ADP through pyruvate kinase/lactate dehydrogenase to the oxidation ofNADH. This method has been described previously (Tamura and t, 1990, J. Biol. Chem, 265, 21342).

ATPase assays were carried out at 30°C in buffered solutions containing 100 mM TRIS pH 7.6, 1.5 mM MgC12, 150 mM KCl. The coupling system contains final concentrations of 2.5 mM phosphoenol pyruvate, 200 uM nicotinamide adenine eotide (NADH), 1 mM DTT, 30 ug/ml pyruvate kinase, and 10 ug/ml lactate dehydrogenase. The enzyme (90 nM final concentration) and a DMSO solution (3 % final concentration) of the selected compound were added. The reaction mixture was d to incubate for 10 minutes at 30°C. The reaction was initiated by the addition of ATP to a final concentration of 0.9 mM, and the rate ofNADH disappearance was monitored at 340 ters over the course of 10 minutes. The K and IC50 values were determined from rate versus concentration profiles. —134— Selected compounds of the present invention were found to inhibit S. aureus DNA gyrase. Table 22 shows the inhibitory activity of these compounds in the S. aureus DNA gyrase inhibition assay.

Table 22. Inhibition of S. aureus DNA Gyrase Selected nd Ki (nM) ICSO (nM) Compound 23 9 Compound W < 9 54 DNA Topo IV ATPase Assay The sion of ATP to ADP by S. aureus TopoIV enzyme was coupled to the conversion ofNADH to NAD+, and the progress of the reaction was measured by the change in ance at 340 nm. TopoIV (64 nM) was incubated with the selected compound (3% DMSO final) in buffer for 10 s at 30 °C. The buffer consisted of 100 mM Tris 7.5, 1.5 mM MgCl2, 200 mM K-Glutamate, 2.5 mM phosphoenol pyruvate, 0.2 mM NADH, 1 mM DTT, 5 ug/mL linearized DNA, 50 ug/mL BSA, 30 ug/mL pyruvate kinase, and 10 ug/mL lactate dehyrodgenase (LDH). The reaction was ted with ATP, and rates were monitored continuously for 20 minutes at 30°C on a Molecular Devices SpectraMAX plate reader. The inhibition constant, Ki, and the IC50 were determined from plots of rate vs. concentration of selected compound fit to the Morrison Equation for tight binding inhibitors. ed compounds of the present invention were found to inhibit S. aureus DNA Topo IV. Table 23 shows the inhibitory activity of these nds in the S. aureus DNA gyrase inhibition assay.

Table 23. Inhibition of S. aureus DNA Topo IV Selected nd Ki (nM) IC50 (nM) Compound 23 12 Compound W 30 150 Example 38 AQUEOUS SOLUBILITY STUDY -l35- The aqueous solubilities of compound 23 and compound W were determined according to the following procedure.

] Preparation of Samples. Aqueous samples of each nd were prepared as follows. Compounds were weighed (20 — 30mg nd) in 4 ml clear vials prior to adding water (0.5mL) and ng by magnetic stirrer. 1.0N HCl was added to the suspension to adjust the pH to the desired range. After stirring for 96 hours at room temperature, the suspension was filtered through a 0.22 micron filter (Millipore, Ultrafree centrifugal filters, Durapore PVDF 0.22pm, Cat# UFC30GVNB). The filtrate was ted and the pH measured with a pH meter. The filtrate containing compound W was diluted 10-fold to provide an appropriate concentration for HPLC analysis. The filtrate containing compound 23 did not require dilution. ation of Standard Solutions. Standard solutions of each compound were prepared according to the ing procedure. 1 to 2 mg of each compound was accurately weighed into a 10mL volumetric flask and either water (for compound W) or 1:1 methanol:0.1N HCl (for compound 23) was added to completely ve the nds.

Sonication was performed for compound 23 to assist with the dissolution in 1:1 methanol:0.1N HCl. When all solids dissolved, additional solvent was added to adjust the volume of each solution to 10ml. The resulting solutions were thoroughly mixed to give the standard ons of each compound. Each standard solution was then diluted with solvent by 2-fold, 10-fold, and 100-fold.

Solubility Analysis. Aliquots of each sample and each standard solution were analyzed by HPLC analysis (Agilent 1100, injection volume 10 uL, wavelength 271nm, column XTerra® Phenyl 5pm, 4.6X50mm, Part No. 186001144, mobile phase: A: 0.1% TFA in water 0.1% TFA in AcN). Each standard solution was injected three times, and each of the samples was ed twice. Standard curves were obtained by plotting the average of the peak area from the HPLC versus the concentrations of the standard solutions (with appropriate corrections of the weights of the standards based on total water content of the solid as determined by elemental is). The concentration of each sample was ated from the peak area of the aqueous sample from the HPLC results and the slope and intercept of the standard curves. The solubility values listed in Table 24 below were derived from the product of the concentration of the sample and the dilution factor of the sample.

Table 24. s lity of Compounds 23 and W Solubility Com n ound Solid form n m_lmL Compound 23 < 0.001 Compound W lline 4.39 0.25 Example 39 IN VIVO METABOLISM STUDY IN HEPATIC AND LIVER S9 CELLS The conversion of Compound W to Compound 23 was studied in liver and intestinal S9 ons from rats, dogs, monkeys and humans. Compound W was ted at 0.1, 0.3, 1, 3, 10, 20, 40, 100, 200, 300 pM in liver S9 fractions and at 1, 3, 10, 20, 100, 300, 500, 1000 “M in intestinal S9 fractions. The incubations were done for 0, 5, 10, 15, 30, 45 or 60 minutes. The formation of Compound 23 was quantified by MS and data were fitted to the Michaelis Menten equation. The data in Table 25 below indicates that Compound W rapidly converts to Compound 23 in these hepatic and intestinal S9 fractions.

Table 25. ty of formation (VMAX) of Compound 23 from Compound W in Liver and Intestinal S9 VMAX (liver) VMAX (intestine) (pmoles/min/mg) (pmoles/min/mg) Dog 19.3 1 162 Monkey 25 .2 1974 Rat 45.5 958 Human 45.8 ND * *ND: Parameters not determined, rate of formation did not saturate

Claims (24)

WHAT IS CLAIMED IS:

1. A compound of formula n R is hydrogen or fluorine; X is hydrogen, –PO(OH)2, –PO(OH)O-M+, –PO(O-)2•2M+, or –PO(O-)2•D2+; M+ is a pharmaceutically acceptable lent cation; and D2+ is a pharmaceutically acceptable divalent ; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 having the formula (IA) wherein R is hydrogen or fluorine; or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 having the formula (IB) wherein X is –PO(OH)2, –PO(OH)O-M+, –PO(O-)2•2M+, or –PO(O-)2•D2+; M+ is a pharmaceutically acceptable monovalent cation; and D 2+ is a pharmaceutically acceptable divalent cation; or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1 having the formula (IC) wherein R is en or fluorine; or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 4, wherein the compound is ethyl (5-(2-(2-hydroxypropanyl)pyrimidinyl)(tetrahydrofuranyl)-1H-benzo[d]imidazol- 2-yl)urea, or a pharmaceutically able salt thereof.

6. The compound ing to claim 4, wherein the compound is (R)ethyl (6-fluoro(2-(2-hydroxypropanyl)pyrimidinyl)(tetrahydrofuranyl)-1H- benzo[d]imidazolyl)urea, or a pharmaceutically acceptable salt thereof.

7. The salt according to claim 4, wherein the salt is a esulfonic acid salt of (R)ethyl(5-(2-(2-hydroxypropanyl)pyrimidinyl)(tetrahydrofuranyl)-1H- benzo[d]imidazolyl)urea.

8. The salt according to claim 4, wherein the salt is a methanesulfonic acid salt of (R)ethyl(6-fluoro(2-(2-hydroxypropanyl)pyrimidinyl)(tetrahydrofuran yl)-1H-benzo[d]imidazolyl)urea.

9. The compound according to claim 3, wherein X is –PO(OH)O -M+, –PO(O -) +, or –PO(O-) 2+ 2•2M 2•D ; M+ is selected from a group consisting of Li+, Na+, K+, N- methyl-D-glucamine, and N(R9)4+, wherein each R9 is independently hydrogen or a C1-C4 alkyl group; D2+ is selected from a group consisting of Mg2+ , Ca2+ , and Ba2+ .

10. The compound according to claim 9, wherein X is –PO(OH)O-M+ or –PO(O -) +; M+ is selected from a group ting of Li+, Na+, K+, N-methyl-D- 2•2M glucamine, and N(R9)4+, wherein each R9 is independently hydrogen or a C1-C4 alkyl group.

11. The compound according to claim 9, wherein X is –PO(O-)2•2M +; M+ is selected from a group consisting of Li+, Na+, K+, N-methyl-D-glucamine, and +, wherein each R9 is independently en or a C1-C4 alkyl group.

12. The compound according to any one of claims 9 to 11, n M+ is Na+.

13. The compound according to claim 3, wherein the nd is disodium (R)- 2-(5-(2-(3-ethylureido)fluoro(tetrahydrofuranyl)-1H-benzo[d]imidazol yl)pyrimidinyl)propanyl phosphate.

14. The compound according to claim 3, wherein the compound is dihydrogen (R)(5-(2-(3-ethylureido)fluoro(tetrahydrafuranyl)-1H-benzo[d]imidazol yl)pyrimidinyl)propanyl phosphate.

15. A pharmaceutical composition comprising a compound ing to any one of claims 1 to 14 or a pharmaceutically able salt thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

16. A method of decreasing or inhibiting Streptococcus pneumoniae, Mycobacterium tuberculosis, Staphylococcus epidermidis, Enterococcus is, Staphylococcus aureus, Clostridium difficile, Moraxella catarrhalis, ria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerans,Chlamydophila pneumoniae, Chlamydia trachomatis , Haemophilus influenzae, Streptococcus pyogenes or b-haemolytic streptococci bacterial quantity in a biological sample in vitro comprising contacting said biological sample with a compound according to any one of claims 1 to 14.

17. The use of a compound according to any one of claims 1 to 14 in the manufacture of a medicament for controlling, treating or reducing the advancement, severity or effects of a nosocomial or a non-nosocomial ial infection.

18. The use according to claim 17, wherein the bacterial infection is characterized by the presence of one or more of Streptococcus pneumoniae, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus , Clostridium difficile, Moraxella catarrhalis, ria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium x, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerans, cterium tuberculosis, Chlamydophila pneumoniae, Chlamydia matis, Haemophilus influenzae, ococcus pyogenes or b-haemolytic streptococci.

19. The use according to claim 18, wherein the bacterial infection is ed from one or more of the following: upper respiratory infections, lower respiratory infections, ear infections, pleuropulmonary and ial infections, complicated y tract infections, uncomplicated urinary tract infections, intra-abdominal infections, cardiovascular infections, a blood stream infection, sepsis, bacteremia, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital ions, eye infections, or granulomatous infections, uncomplicated skin and skin structure infections (uSSSI), complicated skin and skin structure infections (cSSSI), er infections, gitis, sinusitis, otitis externa, otitis media, bronchitis, empyema, pneumonia, community-acquired bacterial pneumoniae (CABP), hospital-acquired pneumonia (HAP), hospital-acquired bacterial nia, ventilator-associated pneumonia (VAP), diabetic foot infections, ycin resistant enterococci infections, cystitis and pyelonephritis, renal calculi, prostatitis, peritonitis, complicated intra-abdominal infections (cIAI) and other inter-abdominal infections, dialysisassociated peritonitis, visceral abscesses, endocarditis, myocarditis, pericarditis, transfusionassociated sepsis, meningitis, encephalitis, brain abscess, osteomyelitis, arthritis, genital ulcers, urethritis, vaginitis, cervicitis, gingivitis, ctivitis, keratitis, endophthalmitisa, an infection in cystic fibrosis patients or an infection of febrile neutropenic patients.

20. The use according to claim 19, wherein the bacterial ion is selected from one or more of the following: community-acquired bacterial pneumoniae (CABP), hospitalacquired nia (HAP), hospital-acquired bacterial pneumonia, ventilator-associated pneumonia (VAP), emia, diabetic foot infections, catheter infections, uncomplicated skin and skin structure ions (uSSSI), complicated skin and skin structure infections (cSSSI), vancomycin ant enterococci infections or osteomyelitis.

21. A compound according to claim 1, substantially as herein bed with reference to any one of the es and/or figures.

22. A pharmaceutical composition according to claim 15, substantially as herein described with reference to any one of the examples and/or figures.

23. A method according to claim 16, substantially as herein described with reference to any one of the es and/or figures.

24. A use according to claim 17, substantially as herein described with reference to any one of the examples and/or figures.