HK1190150B - Pyrimidine gyrase and topoisomerase iv inhibitors - Google Patents

Description

Gyrase and topoisomerase IV inhibitors

CROSS-REFERENCE TO RELATED APPLICATIONS

United states provisional patent application serial No. 61/432,965, filed 2011, 1, month 14, as required by the present application under 35u.s.c. § 119; U.S. provisional patent application serial No. 61/515,174 filed on 8/4/2011; U.S. provisional patent application serial No. 61/515,249 filed on 8/4/2011; and the benefit of U.S. provisional patent application serial No. 61/499,134 filed on 20/6/2011; the entire contents of each application are incorporated herein by reference.

Background

Bacterial resistance to antibiotics has long been recognized and is today considered a serious world-wide hygiene problem. Some bacterial infections are either difficult to treat with antibiotics or even untreatable due to resistance. This problem has become particularly serious with the recent development of multidrug resistance in specific bacterial strains, such as Streptococcus Pneumoniae (SP), mycobacterium tuberculosis (mycobacterium tuberculosis), and Enterococcus (Enterococcus). The emergence of vancomycin resistant enterococci is of particular concern because vancomycin has previously been the only effective antibiotic for treating this infection, and has been considered the "last resort" drug for many infections. Although many other drug-resistant bacteria, such as enterococci, do not cause life-threatening diseases, there is concern that the genes that induce resistance may spread to more lethal organisms, such as Staphylococcus aureus (Staphyloccocusaurus) where methicillin resistance has become prevalent (Decerq, et al, Current OptioninionIn-InfectionIn-InnovationIndustrials, 1999, 1, 1; Levy, "the Challenge of antimicrobial resistance Resistence", scientific American, March, 1998).

Another concern is how rapidly antibiotic resistance can spread. For example, before the sixties of the twentieth century, SPs were universally sensitive to penicillin, and in 1987, only 0.02% of SP strains in the united states were resistant. However, by 1995, SP resistance to penicillin was reported to be about seven percent and as high as 30% in some parts of the United states (Lewis, FDAConsumermasazine (9 months 1995); Gershmann the medical reporter, 1997).

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

Due to the need for fighting drug-resistant bacteria and the increasing failure of available drugs, there has been renewed interest in developing new antibiotics. An attractive strategy for the development of new antibiotics is the inhibition of DNA gyrase and/or topoisomerase IV, bacterial enzymes required for DNA replication and thus bacterial cell growth and division. Gyrase and/or topoisomerase IV are also associated with events in DNA transcription, repair and recombination.

Gyrases are one of the topoisomerases, a group of enzymes that catalyze the interconversion of topoisomers of DNA (see generally, Kornberg and Baker, DNAsreptilization, 2 nd edition, Chapter 12, 1992, W.H.Freeman and Co.; Drica, molecular microbiology, 1992, 6, 425; Drica and ZHao, Microbiolandmolecular biology reviews, 1997, 61, 377-392). Gyrase itself controls DNA supercoiling and relieves the topological stress that occurs when the DNA strands of the parent duplex unwind during the replication process. Gyrase also catalyzes the conversion of relaxed, closed circular duplex DNA into a negative supercoiled form that is more favorable for recombination. The mechanism of the supercoiling reaction involves the packaging of gyrase around a region of DNA, double-strand breaking in that region, passing through a second region of DNA by breaking, and rejoining the broken strands. Such cleavage mechanisms are specific for type II topoisomerases. The supercoiling reaction is driven by the binding of ATP to gyrase. ATP is subsequently hydrolyzed during the reaction. This ATP binding and subsequent hydrolysis causes a conformational change in the DNA-bound gyrase, which is essential for its activity. It has also been found that the level of DNA supercoiling (or relaxation) is dependent on the ATP/ADP ratio. In the absence of ATP, gyrase is only able to relax supercoiled DNA.

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

Topoisomerase IV mainly resolves chromosomal dimers that are linked at the end of DNA replication.

The widely used quinolone antibiotics inhibit bacterial DNA gyrase (GyrA) and/or topoisomerase iv (parc). Examples of quinolones include early compounds such as nalidixic acid and oxolinic acid, and later, more potent fluoroquinolones such as norfloxacin, ciprofloxacin and trovafloxacin. These compounds bind to GyrA and/or ParC and stabilize the cleaved complex, thereby inhibiting overall gyrase function, resulting in cell death. Fluoroquinolones inhibit the catalytic subunit of gyrase (gyrA) and/or topoisomerase IV (ParC) (see Drlica and Zhao, microbiology and molecular biology reviews, 1997, 61, 377-392). However, resistance has also been recognized as a problem with such compounds (WHORreport, "UseofQuinolonesenfoodImidalsandPotenTemplainn Impactor Humanhealth", 1998). For quinolones, as with other classes of antibiotics, bacteria exposed to earlier compounds often rapidly develop cross-resistance against more potent compounds in the same class.

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

There are fewer known inhibitors that bind to GyrB. Examples include coumarin, novobiocin and coumaromycin A1, cyclothialidine, cinodin and clooxetine. Coumarin has been shown to bind very tightly to GyrB. For example, novobiocin makes hydrogen bonds to the network of proteins and several hydrophobic contacts. While novobiocin and ATP do not appear to bind within the ATP binding site, there is minimal overlap in the direction of binding of the two compounds. The overlapping parts are the sugar unit of novobiocin and ATP adenine (Maxwell, trends microbiology, 1997, 5, 102).

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

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

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

Summary of The Invention

The present invention relates 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 a salt thereof:

wherein R is hydrogen or fluoro; x is hydrogen, -PO (OH)₂、－PO（OH）O^-M⁺、－PO（O^-）₂·2M⁺or-PO (O)^-）₂·D^{2 +}；M⁺Is a pharmaceutically acceptable monovalent cation; and D²⁺Is a pharmaceutically acceptable divalent cation. The compounds of formula (I) have a wide range of antibacterial activity and advantageous toxicological properties or are prodrugs of compounds having said activity.

The invention also relates to compounds of formula (IA) or a pharmaceutically acceptable salt thereof, useful as gyrase and/or topoisomerase IV inhibitors. The compounds of formula (IA) are encompassed by formula (I). The compound of formula (IA) may be represented by:

wherein R is hydrogen or fluorine. The compounds of formula (IA) have a wide range of antibacterial activity and advantageous toxicological properties.

The invention also relates to compounds of formula (IB) or a pharmaceutically acceptable salt thereof, useful as gyrase and/or topoisomerase IV inhibitors. The compounds of formula (IB) are encompassed by formula (I). The compound of formula (IB) may be represented by:

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 compound of formula (IB) is the compound (R) -1-ethyl-3- (6-fluoro-5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d]Phosphoester prodrugs of imidazol-2-yl) urea having a wide range of antibacterial activity and favorable toxicological properties. In addition to the compounds provided herein, the present invention further provides pharmaceutical compositions comprising a compound of formula (I), including 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 invention relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and an additional therapeutic agent selected from the group consisting of: antibiotics, anti-inflammatory agents, matrix metalloproteinase inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressive agents, anticancer agents, antiviral agents, cytokines, growth factors, immunomodulators, prostaglandins or anti-vascular hyperproliferative compounds.

In another embodiment, 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 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 antiviral agent, a cytokine, a growth factor, an immunomodulator, a prostaglandin, or an anti-vascular hyperproliferation compound, as part of a multiple dosage form together with the compound or as a separate dosage form.

Brief Description of Drawings

Figure 1 is a thermal ellipsoid plot of two symmetrically independent molecules of compound 12.

Figure 2 is a thermal ellipsoid plot of two symmetrically independent molecules of compound 23.

Detailed description of the invention

As used herein, the term "halogen" means F, Cl, Br or I.

Unless otherwise indicated, the structures described herein are also intended to include all stereochemical forms of the structures; i.e., the R and S configurations for each asymmetric center. Thus, individual stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the compounds presented are within the scope of the invention.

Isotopically labeled forms of the 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 isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen and fluorine, for example²H、³H、¹³C、¹⁴C、¹⁵N、¹⁸O and¹⁷and O. Such radiolabeled and stable isotopically labeled compounds are useful, for example, as research or diagnostic tools or gyrase and/or topoisomerase IV inhibitors with improved therapeutic profiles. When appropriate, the structure also comprises a zwitterionic form of the compound or salt.

In one embodiment, the compound of formula (I) comprises a compound of formula (IC)

Wherein R is as defined above.

In another embodiment, the compounds of formula (I) include compounds of formulae (ID) and (IE) as shown below:

(R) -1-ethyl-3- (5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea or a pharmaceutically acceptable salt thereof; and

(R) -1-ethyl-3- (6-fluoro-5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea or a pharmaceutically acceptable salt thereof. Unless otherwise specified, the phrase "compound of formula (I)" is intended to include other formulae shown herein encompassed by formula (I), including formulae (IA), (IB), (IC), (ID), and (IE).

The compounds of formula (IB) are prodrugs of the parent compound 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea. Thus, the activity exhibited following prodrug administration is primarily due to the presence of the parent compound resulting from prodrug cleavage.

The term "prodrug" refers to a compound that is a prodrug that releases a drug in vivo via some metabolic process after administration and absorption. In general, a prodrug has 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 by reducing toxicity and undesirable effects of the drug by controlling its absorption, blood levels, metabolic profile and cellular uptake.

The term "parent compound" or "parent drug" refers to a biologically active entity that is released upon prodrug administration either enzymatically via metabolic or catalytic processes or via chemical processes. The parent compound may also be the starting material for the preparation of its corresponding prodrug.

By M⁺Monovalent cations as defined include ammonium, alkali metal ions such as sodium, lithium and potassium ions, dicyclohexylamine ions and N-methyl-D-glucamine ions. From D²⁺Divalent cations as defined include alkaline earth metal ions such as aluminum, calcium and magnesium ions. Also included are ions of amino acid cations such as arginine, lysine, ornithine, and the like. If M is⁺Is a monovalent cation, it is recognized thatFruit Presence definition 2M⁺Then M is⁺Each may be the same or different. Furthermore, it is similarly recognized that 2M, if any, is defined⁺Then, conversely, divalent cations D may be present²⁺. In addition, basic nitrogen-containing groups may be quaternized with such agents as: lower alkyl halides such as methyl, ethyl, propyl and butyl chloride, bromide and iodide; dialkyl sulfates such as dimethyl, diethyl, dibutyl; diamyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromine and iodine; aralkyl halides such as benzyl bromide and others.

Various embodiments of the present invention include compounds or salts of formula (IB) as shown below:

(1) a compound wherein X is

（a）－PO（OH）O^-M⁺；

（b）－PO（O^-）₂·2M⁺(ii) a Or

（c）－PO（O^-）₂·D²⁺；

(2) A compound in which M⁺Is that

（a）Li⁺、Na⁺、K⁺N-methyl-D-glucamine, or N (R)⁹）₄ ⁺(ii) a Or

（b）Na⁺；

(c) Each R⁹Independently is hydrogen or C₁-C₄An alkyl group;

(3) a compound of formula (I) wherein D²⁺Is that

（a）Mg²⁺、Ca²⁺And Ba²⁺(ii) a Or

（b）Ca²⁺；

(4) The compound (R) -2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-ylphosphate; and

(5) the compound (R) -disodium 2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate.

It will be appreciated that a number of alternative embodiments of the compound or salt of formula (IB) may be selected by requiring one or more of the alternative embodiments listed above in (1) through (3). For example, a further embodiment of the invention may be achieved by combining (1) (a) and (2) (a); (1) (a) and (2) (b); (1) (c) and (3) (a); (1) (c) and (3) (b); (1) (b) and (2) (a); (1) (b) and (2) (b); and the like.

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

One embodiment of the present invention is directed to a method of treating a bacterial infection in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a compound having formula (I) or a pharmaceutically acceptable salt thereof.

According to another embodiment, the present invention provides a method of reducing or inhibiting the number of bacteria in a biological sample. Such methods comprise contacting the biological sample with a compound of formula (I) or a pharmaceutically acceptable salt thereof.

As used herein, the term "biological sample" includes cell cultures or extracts thereof; biopsy material or extract thereof from a mammal; and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof. The term "biological sample" also includes living organisms, in which case "contacting a compound of the invention with a biological sample" is synonymous with the term "administering said compound or a composition comprising said compound) to a mammal".

One embodiment comprises contacting the biological sample with a compound selected from the group consisting of: (R) -1-ethyl-3- (5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea or a pharmaceutically acceptable salt thereof; and (R) -1-ethyl-3- (6-fluoro-5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea or a pharmaceutically acceptable salt thereof. Pharmaceutical compositions useful for such methods are described below.

One embodiment comprises contacting the biological sample with a phosphate prodrug of (R) -1-ethyl-3- (6-fluoro-5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea as defined by formula (IB). Pharmaceutical compositions useful for such methods are described below.

The antimicrobial activity of the compounds of formula (I) can be demonstrated in an antimicrobial sensitivity assay. The details of the conditions used for the microbial susceptibility assay are set forth in the examples below.

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

Agents that increase the susceptibility of bacterial organisms to antibiotics are known. For example, U.S. patent No. 5,523,288, U.S. patent No. 5,783,561, and U.S. patent No. 6,140,306 describe methods of using bactericidal/permeability increasing proteins (BPI) for increasing antibiotic susceptibility of gram-positive and gram-negative bacteria. Agents which increase the outer membrane permeability of bacterial organisms have been described by Vaara, M. in microbiological reviews (1992) p 395-411, and the sensitisation of gram-negative bacteria has been described by Tsubery, H. et al in J.Med.chem. (2000) p 3085-3092.

Another embodiment of the present invention relates to a method of preventing, controlling, treating, or reducing the progression, severity, or effect of a bacterial infection in a mammal in need thereof as described above, but further comprising the step of administering to the mammal an agent that increases the sensitivity of the bacterial organism to an antibiotic.

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

The pharmaceutical compositions and methods of the present invention will generally be used to control bacterial infections in vivo. Examples of bacterial organisms that can be controlled by the compositions and methods of the invention include, but are not limited to, the following: streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus pyogenes (Streptococcus pyogenes), enterococcus faecalis (Enterococcus faecis), enterococcus faecium (Enterococcus faecium), Klebsiella pneumoniae (Klebsiella pneumoniae), Enterobacter species (Enterobacterpp), Proteus sp., Pseudomonas aeruginosa (Pseudomonas aeruginosa), Escherichia coli (E.coli), Serratia marcescens (Serratia seriforme), Staphylococcus aureus (Staphylococcus aureus), Staphylococcus negavirus (Coag. Neg. Staphyloccci), Haemophilus influenzae (Haemophilus flavidus), Bacillus anthracis (Bacillus pneumoniae), Streptococcus pneumoniae (Streptococcus pneumoniae), Staphylococcus aureus (Streptococcus pneumoniae), Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus aureus (Clostridium difficile), Staphylococcus aureus (Clostridium pneumoniae), Staphylococcus epidermidis), Staphylococcus aureus (Clostridium difficile), Staphylococcus epidermidis), Staphylococcus aureus (Clostridium pneumoniae), Staphylococcus epidermidis (Clostridium), Staphylococcus epidermidis), Staphylococcus aureus (Clostridium), Staphylococcus epidermidis), Staphylococcus (Clostridium), Streptococcus) Neisseria meningitidis (Neisseria meningitidis), Mycobacterium avium complex (Mycobacterium avium complex), Mycobacterium abscessus (Mycobacterium abscesses), Mycobacterium kansasii (Mycobacterium kansasii) and Mycobacterium ulcerosa (Mycobacterium ulcerans).

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

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

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

In another embodiment, the compositions and methods will therefore be used to control, treat or reduce the progression, severity or effects of: upper respiratory tract infections, lower respiratory tract infections, ear infections, pleuropneumoniae and bronchial infections, complicated urinary tract infections, non-complicated urinary tract infections, intra-abdominal infections, cardiovascular infections, bloodstream infections, sepsis, bacteremia, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital infections, eye infections or granuloma infections, non-complicated skin and skin structure infections (uSSSI), complicated skin and skin structure infections (cSSSI), catheter infections, pharyngitis, sinusitis, otitis externa, otitis media, bronchitis, empyema, pneumonia, community-acquired bacterial pneumonia (CABP), hospital-acquired pneumonia (HAP), hospital-acquired bacterial pneumonia, respirator-associated pneumonia (VAP), diabetic foot infections, vancomycin-resistant enterococcus infections, cystitis and pyelonephritis, kidney stones, kidney-derived pneumonia (cabs), and liver inflammation, Prostatitis, peritonitis, complex intraperitoneal infections (cIAI) and other intraperitoneal infections, dialysis-related peritonitis, visceral abscesses, endocarditis, myocarditis, pericarditis, infusion-related sepsis, meningitis, encephalitis, brain abscesses, osteomyelitis, arthritis, genital ulcers, urethritis, vaginitis, cervicitis, gingivitis, conjunctivitis, keratitis, endophthalmitis, infections in cystic fibrosis patients, or infections in febrile neutropenia patients.

In another embodiment, the bacterial infection is characterized by the presence of one or more of the following: streptococcus pneumoniae, Streptococcus pyogenes, enterococcus faecalis, enterococcus faecium, Staphylococcus aureus, coagulase-negative staphylococci, Bacillus anthracis, Staphylococcus epidermidis, Staphylococcus saprophyticus or Mycobacterium tuberculosis.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

According to another embodiment, the beta-lactam resistant moraxella is beta-lactam resistant moraxella catarrhalis.

According to another embodiment, the bacterial infection is characterized by the presence of one or more of the following: methicillin-resistant Staphylococcus aureus, fluoroquinolone-resistant Staphylococcus aureus, vancomycin intermediate-resistant Staphylococcus aureus, linezolid-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, macrolide-resistant Streptococcus pneumoniae, fluoroquinolone-resistant Streptococcus pneumoniae, vancomycin-resistant enterococcus faecalis, linezolid-resistant enterococcus faecalis, fluoroquinolone-resistant enterococcus faecalis, vancomycin-resistant enterococcus faecium, linezolid-resistant enterococcus faecium, fluoroquinolone-resistant enterococcus faecium, ampicillin-resistant enterococcus faecium, macrolide-resistant Haemophilus influenzae, beta-lactam-resistant Haemophilus influenzae, fluoroquinolone-resistant Haemophilus influenzae, beta-lactam-resistant Moraxella catarrhalis, methicillin-resistant Staphylococcus epidermidis, vancomycin-resistant Staphylococcus epidermidis, fluoroquinolone-resistant staphylococcus epidermidis, macrolide-resistant mycoplasma pneumoniae, isoniazid-resistant mycobacterium tuberculosis, rifampin-resistant mycobacterium tuberculosis, fluoroquinolone-resistant neisseria gonorrhoeae or cephalosporin-resistant neisseria gonorrhoeae.

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

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

In addition to the compounds of the present invention, pharmaceutically acceptable derivatives or prodrugs of the compounds of the present invention may also be used in compositions for the treatment or prevention of the disorders identified above.

By "pharmaceutically acceptable derivative or prodrug" is meant any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of the invention which, upon administration to a recipient, is capable of providing, directly or indirectly, a compound of the invention or an inhibitory active metabolite or residue thereof. Particularly advantageous derivatives or prodrugs are those that increase the bioavailability of the compounds of the 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 enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.

Pharmaceutically acceptable prodrugs of the compounds of the present invention include, but are not limited to, esters, amino acid esters, phosphate esters, metal salts, and sulfonate esters.

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

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

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

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

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

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

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

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

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

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

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

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

The compounds of formula (I) may also be co-administered with other antibiotics to increase the effect of treatment or prevention against a variety of bacterial infections. When the compounds of the invention are administered in combination therapy with other agents, they may be administered to the patient sequentially or simultaneously. Alternatively, the pharmaceutical or prophylactic composition according to the invention comprises a combination of a compound of formula (I) and another therapeutic or prophylactic agent.

In some embodiments, the additional one or more therapeutic agents is an antibiotic selected from the group consisting of: natural penicillins, penicillinase-resistant penicillins, pseudomonad-resistant penicillins, aminopenicillins, first-generation cephalosporins, second-generation cephalosporins, third-generation cephalosporins, fourth-generation cephalosporins, carbapenems, cephamycins, quinolones, fluoroquinolones, aminoglycosides, macrolides, ketolides, polymyxins, tetracyclines, glycopeptides, streptogramins, oxazolidinones, rifamycins, or sulfonamides.

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

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

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

In some embodiments, a solid form of a compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered for the treatment of gram-positive infections. In some embodiments, the composition is a solid, liquid (e.g., suspension), or iv (e.g., a form of a compound of formula (I) or a pharmaceutically acceptable salt thereof is dissolved into a liquid and administered iv) composition. In some embodiments, a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an additional antibiotic agent, such as a natural penicillin, a penicillinase-resistant penicillin, an anti-pseudomonas penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a glycopeptide, a streptogramin, an oxazolidinone, a rifamycin, or a sulfonamide. In some embodiments, a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in solid form is administered orally, and iv. an additional antibiotic agent such as a natural penicillin, a penicillinase resistant penicillin, an anti-pseudomonas penicillin, an aminopenicillin, a first generation cephalosporin, a second generation cephalosporin, a third generation cephalosporin, a fourth generation cephalosporin, a carbapenem, a cephamycin, a quinolone, a fluoroquinolone, an aminoglycoside, a macrolide, a ketolide, a polymyxin, a tetracycline, a glycopeptide, a streptogramin, an oxazolidinone, a rifamycin, or a sulfonamide is administered.

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

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

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

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

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

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

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

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

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

The delivery of therapeutic agents from implantable or indwelling devices is known in the art. See, e.g., "RecentreprenementationCoatedSteps" published by Hofma et al in Current International Cardiology reports2001, 3:28-36, the entire contents of which, including the references cited therein, are incorporated herein by reference. Other descriptions of implantable devices can be found in U.S. patent nos. 6,569,195 and 6,322,847; and U.S. patent application nos. 2004/0044405, 2004/0018228, 2003/0229390, 2003/0225450, 2003/0216699 and 2003/0204168, each of which is incorporated by reference herein in its entirety.

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

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

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

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

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

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

When the compositions of the present invention comprise a combination of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic or prophylactic agents, the compound and the additional agent should be present at dosage levels of from about 10% to 80% of the dosage normally administered in a monotherapy regimen.

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

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

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

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

The compounds of the present invention can be prepared according to conventional methods known to those skilled in the art for analogous compounds, such as those described in U.S. Pat. nos. RE 40245E; U.S. patent nos. 7,495,014B 2; U.S. patent nos. 7,569,591B 2; U.S. patent nos. 7,582,641B 2; U.S. patent nos. 7,618,974B 2; and U.S. patent No. 7,727,992B 2. All 6 of these patents are incorporated herein by reference in their entirety. The detailed conditions for preparing the compounds of the present invention are further illustrated in the examples.

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

The following definitions describe the terms and abbreviations described herein:

example 1

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

1-bromo-2-nitrobenzene (1) (600 g, 99%, 2.941mol, AlfaAesca 11686), 1, 3-bis (diphenylphosphino) propane (62.50 g, 97%, 147.0mmol, AlfaAesca A12931), 1, 4-dioxane (2.970L, Sigma-Aldrich 360481), potassium carbonate (812.9 g, 5.882mol, JT-Baker 301201), and 2, 3-dihydrofuran (2) (1.041 kg, 99%, 1.124L, 14.70mol, Aldrich 018) were combined. The nitrogen stream was bubbled through the stirred mixture for 4 hours, then palladium (II) acetate (16.51 g, 73.52mmol, Strem 461780) was added and deoxygenation continued for another 10 minutes. The reaction mixture was stirred under reflux under nitrogen overnight (work-up) aliquotsSample NMR showed complete consumption of aryl bromide). It was allowed to cool, diluted with hexane (1L) and passed through FlorisilWas filtered (500 g, -200 mesh) and eluted with EtOAc. The filtrate was concentrated under reduced pressure (2- (2-nitrophenyl) -2, 3-dihydrofuran is volatile under high vacuum and may be slightly unstable at room temperature) to give a mixture of (3 a) and (3 b) as a dark brown oil (654.0 g). The crude material was stored in a refrigerator and advanced without further purification.

Example 2

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

5% Palladium on carbon (16.3 g, 50% wet, 3.83mmol, Aldrich 330116) was placed in a Parr bottle under nitrogen followed by MeOH (100 mL, JT-Baker 909333). 2- (2-Nitrophenyl) -2, 5-dihydrofuran and 2- (2-Nitrophenyl) -2, 3-dihydrofuran (3 a) dissolved in MeOH (389 mL) were added&3b) (163 g) of the crude mixture, followed by NEt₃(237.6 mL, 1.705mol, Sigma-Aldrich 471283). Place the container on a Parr shaker and use H₂And (4) saturation. Adding 30psiH₂And shaken until consumption was complete (LCMS and NMR showed complete reaction). The reaction mixture was purged with nitrogen and passed through Celite^TMFiltered and washed with EtOAc. The filtrate was concentrated on a rotary evaporator to give a brown oil. The reaction was repeated three more times on the same scale and the batches were combined for purification. The crude product was vacuum distilled (about 15 torr) and the distillate was collected at 108 ℃ and 129 ℃ to give (4) (427.9 g, average yield 84%; 98% GCMS purity) as a clear pale yellow oil. LCMS (over 5 min)10-90%CH₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 163.95 (1.46 min).¹HNMR（300MHz，CDCl₃）：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。

Example 2a

Preparation of (R) -2- (tetrahydrofuran-2-yl) aniline (4 a).

33g of compound (4) was dissolved in MeOH (265 ml), which resulted in a concentration of about 125 mg/ml. The mixture was filtered through a 0.2 micron membrane filter, followed by chromatography on a ChiralPak at 100 bar using a Bergermultigram supercritical fluid chromatography systemChromatography was performed on an IC column (30 mmX150mm, column temperature 35 ℃, Chiraltechnologies). The mobile phase was eluted with 350 ml/min (90: 10) CO₂:CH₃OH with UV monitoring at 220 nm. 15.64g of the desired product (4 a) were obtained as a green oil. Analysis of SFC ([ 90: 10)]CO₂:CH₃OH, at 5 ml/min on a chiralpak ic column (4.6X 100 mm) maintained at 35 ℃ and operated at 100 bar pressure with UV monitoring at 220 nm) showed 95.5% ee with 95% overall purity.

Example 3

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

To a stirred solution of 2-tetrahydrofuran-2-yl-aniline (4) (53.45 g, 327.5 mmol) in methyl tert-butyl ether (MTBE, 641.4 mL) and acetonitrile (213.8 mL) cooled to 2 ℃ was added 4 parts of N-bromosuccinimide (58.88 g, 99%, 327.5mmol, aldrich b 81255), maintaining the internal temperature below about 8 ℃. The reaction mixture was stirred while cooling with an ice water bath for 30 minutes (NMR of working aliquot showed complete consumption of starting material). Adding aqueous 1NNa₂S₂O₃(330 mL), the cold water bath was removed and stirred for 20 minutes. The mixture was diluted with EtOAc and the layers were separated. The organic phase is washed with saturated aqueous NaHCO₃(2X), water, brine wash, over MgSO₄Dry above, filter through a short plug of silica, elute with EtOAc, and concentrate under reduced pressure to give (5) (82.25 g, 77-94% HPLC purity) as a very dark amber oil. Proceed without further purification. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 242.10 (2.89 minutes).¹HNMR（300MHz，CDCl₃）7.22（d，J=2.3Hz，1H），7.16（dd，J=8.4，2.3Hz，1H），6.54（d，J=8.4Hz，1H），4.79–4.73（m，1H），4.15（s，2H），4.10–4.01（m，1H），3.93–3.85（m，1H），2.26–2.13（m，1H），2.12–1.97（m，3H）ppm。

Example 4

Preparation of N- (4-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl) -2,2, 2-trifluoroacetamide (6).

Trifluoroacetic anhydride (455.3 mL, 3.2) stirred at 2 ℃ was added over 15 minutes (reaction temperature increased to 14 ℃ C.) via an addition funnel75mol, Sigma-Aldrich 106232) was slowly added 4-bromo-2-tetrahydrofuran-2-yl-aniline (5) (79.29 g, 327.5 mmol) as a viscous oil. The remaining oil was washed into the reaction mixture with anhydrous 2-methyltetrahydrofuran (39.6 mL, Sigma-Aldrich 414247). The cold bath was removed and ammonium nitrate (34.08 g, 425.8mmol, Aldrich 467758) was added. The reaction temperature was raised to 40 ℃ over about 30 minutes, at which time a cold water bath was used to control the exothermicity and bring the reaction to room temperature. The cold bath was then removed and stirring continued for an additional 40 minutes (HPLC showed very little non-nitrated material remaining). The reaction mixture was slowly poured into a stirred mixture of crushed ice (800 g). The solid precipitate was collected by filtration, washed with water, saturated aqueous NaHCO₃(to pH 8), water and hexane washes again. The wet solid was first dried in a convection oven at 50 ℃ for several hours and then under reduced pressure in an oven at 40 ℃ overnight to give (6) as a pale brown solid (77.86 g, 62% yield; 98% HPLC purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 383.19 (3.27 min).¹HNMR（300MHz，CDCl₃）9.81（s，1H），8.08（d，J=2.2Hz，1H），7.73（d，J=2.2Hz，1H），4.88（dd，J=9.0，6.5Hz，1H），4.17–4.08（m，1H），4.03–3.95（m，1H），2.45–2.34（m，1H），2.17–2.06（m，2H），1.96–1.83（m，1H）ppm。

Example 5

Preparation of 4-bromo-2-nitro-6-tetrahydrofuran-2-yl-aniline (6 a).

N- (4-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl) -2,2, 2-trifluoroacetamide (6) (54.00 g, 140.9 mmol) was dissolved in 1, 4-dioxane (162 mL) and aqueous 6m naoh (70.45 mL, 42 mL) was added2.7mmol, JT-Baker 567202). The reaction mixture was stirred at reflux for 2 days (HPLC showed complete consumption), allowed to cool, diluted with MTBE (800 mL), diluted with water (2 × 200 mL), saturated aqueous NH₄Cl, water and brine. The mixture was stirred over MgSO₄Dried, filtered and concentrated under reduced pressure to give (6 a) (40.96 g, 93% yield; overall 92% HPLC plus NMR purity) as dark amber oil. LCMS (C18 column eluted with 10-90% MeOH/water gradient from 3-5 min using formic acid modifier) M + 1: 287.28 (3.44 min).¹HNMR（300MHz，CDCl₃）8.24（d，J=2.4Hz，1H），7.41（d，J=2.3Hz，1H），6.91（s，2H），4.80（t，J=7.2Hz，1H），4.14–4.05（m，1H），3.98–3.90（m，1H），2.36–2.19（m，1H），2.15–2.01（m，3H）ppm。

Example 6

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

Mixing 4-bromo-2-nitro-6-tetrahydrofuran-2-yl-aniline (6 a) (40.40 g, 92%, 129.5 mmol), 1, 4-dioxane (260 mL, Sigma-Aldrich 360481), 2- [5- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrimidin-2-yl]Propyl-2 alcohol (7) (41.05 g, 155.4 mmol) and aqueous 2.7MNa₂CO₃(143.9 mL, 388.5 mmol). A nitrogen stream was bubbled through the stirred mixture for 1 hour, followed by the addition of tetrakis (triphenylphosphine) palladium (0) (7.48 g, 6.47mmol, Strem 462150). The reaction mixture was stirred at reflux for 2 hours (HPLC showed complete reaction), allowed to cool, diluted with EtOAc, water, saturated aqueous NH₄Cl, brine, over MgSO₄Dried and passed through FlorisiShort plug filtration, eluting with EtOAc. The filtrate was concentrated under reduced pressure to give a dark brown oil. Dissolving oil in CH₂Cl₂In, and CH for short plug through silica gel₂Cl₂Followed by EtOAc elution. The desired fractions were concentrated on a rotary evaporator until a precipitate formed, giving a thick brown paste which was triturated with MTBE. The solid was collected by filtration, washed with MTBE, and dried under high vacuum to give (8) (35.14 g, 99+% HPLC purity) as a yellow solid. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 345.00 (2.69 min).¹HNMR（300MHz，CDCl₃) 8.88 (s, 2H), 8.36 (d, J =2.2Hz, 1H), 7.56 (d, J =2.1Hz, 1H), 7.09 (s, 2H), 4.92 (t, J =7.2Hz, 1H), 4.62 (s, 1H), 4.20-4.11 (m, 1H), 4.03-3.94 (m, 1H), 2.39-2.26 (m, 1H), 2.23-2.08 (m, 3H), 1.64 (s, 6H) ppm. The filtrate was further concentrated and purified by ISCO silica gel chromatography eluting with 0-80% EtOAc/hexanes to give the product (8) as a second crop of amber solid (4.46 g, 88% overall yield; 88% HPLC purity.

Example 7

Alternative preparation of 2- [5- (4-amino-3-nitro-5-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl ] propyl-2-ol (8).

Mixing N- (4-bromo-2-nitro-6-tetrahydrofuran-2-yl-phenyl) -2,2, 2-trifluoroacetamide (6) (19.00 g, 49.59 mmol), 2- [5- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrimidin-2-yl]Propyl-2 alcohol (7) (14.41 g, 54.55 mmol), aqueous 2.7M sodium carbonate (73.48 mL, 198.4 mmol) and 1, 4-dioxane (190 mL, Sigma-Aldrich360481). A nitrogen stream was bubbled through the stirred mixture for 40 minutes, followed by addition of 1, 1' -bis (diphenylphosphino) ferrocene palladium dichloride dichloromethane adduct (2.025 g, 2.480mmol, Strem 460450). The reaction mixture was refluxed under N₂Stirring was continued for 7 hours, an additional 50mL of saturated aqueous sodium carbonate was added, and heating at reflux for an additional 16 hours. The mixture was allowed to cool, then diluted with EtOAc (500 mL) and water (200 mL). The layers were separated and the aqueous phase was extracted with EtOAc (200 mL). The combined organic phases were washed with water (500 mL), brine (500 mL) and washed with Na₂SO₄Drying by FlorisilThe plug was filtered and concentrated on a rotary evaporator to give the crude product (8) as an orange oil. The crude product was purified by ISCO silica gel chromatography eluting with 20-90% EtOAc/hexanes to give (8) as an orange solid (15.00 g, 87% yield; 81-88% purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 345.35 (2.68 min).¹HNMR（300MHz，CDCl₃）8.88（s，2H），8.36（d，J=2.2Hz，1H），7.56（d，J=2.1Hz，1H），7.09（s，2H），4.92（t，J=7.2Hz，1H），4.62（s，1H），4.20–4.11（m，1H），4.03–3.94（m，1H），2.39–2.26（m，1H），2.23–2.08（m，3H），1.64（s，6H）ppm。

Example 8

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

To 2- [5- (4-amino-3-nitro-5-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl under nitrogen]Suspension of propyl-2 alcohol (8) (30.10 g, 87.41 mmol) and THF (90 mL) in a Parr bottle was added a slurry of 5% palladium on charcoal (3.01 g, 50% wet, 0.707mmol, Aldrich 330116) in MeOH (90 mL, JT-Baker 909333) followed by NEt₃(24.37 mL, 174.8mmol, Sigma-Aldrich 471283). Place the container on a Parr shaker and use H₂And (4) saturation. Adding 45psiH₂And shaken until consumption was complete (HPLC showed complete conversion). The reaction mixture was purged with nitrogen and passed through Celite^TMFiltered and washed with EtOAc. The filtrate was refiltered through a 0.5 micron glass fiber filter paper sandwiched between two sheets of P5 paper and concentrated under reduced pressure to give (9) as a light brown foam (28.96 g, 98% yield; 93% NMR purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 315.32 (1.54 min).¹HNMR（300MHz，CDCl₃）8.83（s，2H），6.92（d，J=1.8Hz，1H），6.88（d，J=1.8Hz，1H），4.90（dd，J=7.9，6.2Hz，1H），4.72（s，1H），4.18（s，2H），4.17–4.08（m，1H），3.99–3.89（m，1H），3.46（s，2H），2.34–2.19（m，1H），2.17–2.05（m，3H），1.63（s，6H）ppm。

Example 9

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

To 2- [5- (3, 4-diamino-5-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl]To a stirred solution of propyl-2 alcohol (9) (32.10 g, 102.1 mmol) in 1, 4-dioxane (160.5 mL, Sigma-Aldrich 360481) was added ph3.5 buffer (240.8 mL) prepared by dissolving NaOAc trihydrate (34.5 g) in 1N aqueous H₂SO₄Prepared (240 mL). 1-Ethyl-3- (N- (ethylcarbamoyl) -C-methylthio-carboimino) urea (10) (28.46 g, 122.5mmol, CBResearch and development) was added and stirred under reflux overnight (HPLC showed 99% consumption of starting diamine). The reaction mixture was cooled to room temperature and poured (bubbled) with aqueous saturated NaHCO in portions₃(480 mL) and water (120 mL) to give a pH of 8-9. This was stirred for 30 minutes, the solid was collected by filtration, washed well with water to neutral pH, and then washed more sparingly with EtOH. The solid was dried under reduced pressure to give (11) (34.48 g, 82% yield; 99.4% HPLC purity) as an off-white solid. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 411.41 (1.73 minutes).¹HNMR（300MHz，MeOD）9.02（s，2H），7.62（s，1H），7.37（s，1H），5.31（s，1H），4.23（dd，J=14.5，7.3Hz，1H），4.01（dd，J=15.0，7.1Hz，1H），3.38–3.28（m，2H），2.58–2.46（m，1H），2.16–2.05（m，2H），2.02–1.88（m，1H），1.63（s，6H），1.22（t，J=7.2Hz，3H）ppm。

Example 10

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

CHIRALPAK eluted with DCM/MeOH/TEA (60/40/0.1) at 35 deg.CICResolution of 1-Ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl on a column (by Chiraltechnologies)]-7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl]A racemic sample of urea (11) (24.60 g) gave the desired enantiomer (12) (11.35 g, 45% yield; 99+% HPLC purity, 99+% ee) as a white solid. Analytical chiral HPLC retention time was 6.2 min (CHIRALPAK)IC4.6X250mm column, 1 mL/min flow rate, 30 ℃).

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

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

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

Example 11

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

Cooling of 1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] with an ice water bath]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]A stirred suspension of urea (12) (9.32 g, 22.71 mmol) in absolute ethanol (93.2 mL). Methanesulfonic acid (1.548 mL, 23.85mmol, Sigma-Aldrich 471356) was added, the cold bath removed and stirred at room temperature for 20 minutes. It is rotary steamed at 35 deg.CConcentrate on the vessel to a thick slurry, dilute with EtOAc, collect the solid by filtration, wash with EtOAc, and dry under reduced pressure to give the initial harvest (13) (8.10 g) as a white solid. The filtrate was concentrated on a rotary evaporator (rotavap) to give a yellowish glassy foam, which was dissolved in EtOH, concentrated to a solid slurry and concentrated with EtOAc/Et₂Triturate and collect by filtration. The solid was washed with EtOAc/Et₂O washes, combined with the first harvest, and dried under reduced pressure gave (13) as a white solid (9.89 g, 86% yield; 99+% HPLC purity, 99+% ee). Analytical chiral HPLC is shown in CHIRALPAKICOne enantiomer, having a retention time of 6.3 minutes, was eluted on a 4.6X250mm column with DCM/MeOH/TEA (60/40/0.1) using a flow rate of 1 mL/min at 30 ℃. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 411.53 (1.74 min).¹HNMR（300MHz，MeOD）9.07（s，2H），7.79（s，1H），7.62（s，1H），5.30（t，J=7.3Hz，1H），4.24（dd，J=14.6，7.3Hz，1H），4.04（dd，J=15.0，7.6Hz，1H），3.40–3.30（m，2H），2.72（s，3H），2.65–2.54（m，1H），2.20–2.07（m，2H），2.04–1.90（m，1H），1.64（s，6H），1.23（t，J=7.2Hz，3H）ppm。

Example 12

Preparation of 2- (2-fluoro-6-nitro-phenyl) -2, 3-dihydrofuran (15A) and 2- (2-fluoro-6-nitro-phenyl) -2, 5-dihydrofuran (15B).

2-bromo-1-fluoro-3-nitro-benzene (14) (200.3 g, 98%, 892.3mmol, BoscheF 6657), 1, 4-dioxane (981.5 mL, Sigma-Aldrich 360481), and 2, 3-dihydrofuran (2) (341.1 mL, 99%, 4.462mol, Aldrich 200018) were charged to a reaction flask followed by N, N-diisopropylethylamine (155.4 mL, 892.3mmol, Sigma-Aldrich 550043) and bromine (tri-tert-butylphosphine) palladium (I) dimer (6.936 g, 8.923mmol, johnson matthey c 4099). The mixture was stirred at reflux for 2 hours (HPLC showed 98% consumption of the starting aryl bromide). It was allowed to cool, the precipitate was removed by filtration, rinsed with EtOAc, and the filtrate was concentrated in vacuo to a dark reddish brown semi-solid oil. This was dissolved in CH₂Cl₂By having CH₂Cl₂Eluted off the silica plug and concentrated in vacuo to give a mixture of 15A and 15B as dark amber oil (291.3 g). The crude product was advanced without further purification. The major product was 2- (2-fluoro-6-nitro-phenyl) -2, 3-dihydrofuran (15A) (96%): LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 210.23 (3.13 minutes);¹HNMR（300MHz，CDCl₃) 7.54 (dt, J =8.0, 1.2Hz, 1H), 7.43 (td, J =8.2, 5.2Hz, 1H), 7.32 (ddd, J =9.7, 8.3, 1.3Hz, 1H), 6.33 (dd, J =4.9, 2.4Hz, 1H), 5.80 (t, J =10.9Hz, 1H), 5.06 (q, J =2.4Hz, 1H), 3.18-3.07 (m, 1H), 2.94-2.82 (m, 1H) ppm. The minor product was 2- (2-fluoro-6-nitro-phenyl) -2, 5-dihydrofuran (15B) (4%): GCMS (agilent hp-5MS30mx250 μmx0.25 μ M column heated at 60 ℃ for 2 minutes to 300 ℃ for more than 15 minutes using a flow rate of 1 mL/min) M + 1: 210 (11.95 min).¹HNMR（300MHz，CDCl₃）7.47（d，J=8.0Hz，1H），7.43–7.34（m，1H），7.30–7.23（m，1H），6.21–6.15（m，1H），6.11–6.06（m，1H），5.97–5.91（m，1H），4.89–4.73（m，2H）ppm。

Example 13

Preparation of 3-fluoro-2-tetrahydrofuran-2-yl-aniline (16).

5% Palladium on carbon (37.3 g, 50% wet, 8.76mmol, Aldrich 330116) was placed in a Parr bottle under nitrogen followed by MeOH (70 mL, JT-Baker 909333). 2- (2-fluoro-6-nitro-phenyl) -2, 3-dihydrofuran and 2- (2-fluoro-6-nitro-phenyl) -2, 5-dihydrofuran (15A) dissolved in MeOH (117 mL) were added&15B) (186.6 g, 892.1 mmol) of crude mixture, followed by NEt₃(124.3 mL, 892.1mmol, Sigma-Aldrich 471283). Place the container on a Parr shaker and use H₂And (4) saturation. After adding 45psiH₂After this time, the reaction mixture was shaken until the starting material consumption was complete (HPLC and LCMS showed complete reaction). The reaction mixture was purged with nitrogen and passed through Celite^TMFiltered and washed with EtOAc. The filtrate was concentrated on a rotary evaporator to give a brown oil, which was dissolved in Et₂O and washed with water (2 ×). The ether phase was extracted with aqueous 1N HCl (5X 250 mL) and the ether phase was treated with Et₂O (3X) washed and then basified with aqueous 6NNaOH to pH 12-14. By CH₂Cl₂(4X) extracting the alkaline aqueous phase and diluting with saturated aqueous NH₄The combined organic extracts were washed with Cl over MgSO₄Dried and filtered through a pad of silica, using CH₂Cl₂Elute to 25% EtOAc/hexanes. The desired filtrate was concentrated under reduced pressure to give 16 as a light brown oil (121.8 g, 84% GCMS plus NMR purity). GCMS (agilent hp-5MS30mx250 μmx0.25 μ M column heated at 60 ℃ for 2 minutes to 300 ℃ for more than 15 minutes using a flow rate of 1 mL/min) M + 1: 182.0 (11.44 min). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 182.10 (2.61 min).¹HNMR（300MHz，CDCl₃）6.97（td，J=8.1，6.3Hz，1H），6.43–6.35（m，2H），5.21–5.13（m，1H），4.54（s2H), 4.16-4.07 (m, 1H), 3.90-3.81 (m, 1H), 2.23-2.00 (m, 4H) ppm. Additional harvests were obtained as follows: the combined ether phases were treated with saturated aqueous NaHCO₃Washing with brine in Na₂SO₄Dry, decant and concentrate under reduced pressure. The oil was vacuum distilled (about 15 torr) and the distillate was collected at 101-108 ℃. To a stirred solution of the distillate oil in EtOH (1 volume) was slowly added 5MHCl (1 equivalent) in iPrOH at 2 ℃. The resulting suspension was brought to room temperature, diluted with EtOAc (3 vol, vol/vol) and stirred for 2 hours. The white solid was collected by filtration, washed with EtOAc, and dried under reduced pressure to give the product as a second harvest of HCl salt. The mother liquor was concentrated to a slurry, diluted with EtOAc and the solid was collected by filtration, washed with EtOAc, and dried in vacuo to give the product as a third harvest of HCl salt. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 182.10 (2.58 min).¹HNMR（300MHz，CDCl₃) 10.73 (br.s, 3H), 7.66 (d, J =8.1Hz, 1H), 7.33 (td, J =8.2, 5.9Hz, 1H), 7.13-7.05 (m, 1H), 5.26 (dd, J =9.0, 6.5Hz, 1H), 4.38-4.28 (m, 1H), 4.00-3.91 (m, 1H), 2.59-2.46 (m, 1H), 2.30-1.95 (m, 3H) ppm. The overall yield from three harvests was 76%.

Example 14

Preparation of 4-bromo-3-fluoro-2-tetrahydrofuran-2-yl-aniline (17).

To a stirred solution of 3-fluoro-2-tetrahydrofuran-2-yl-aniline (16) (131.9 g, 92%, 669.7 mmol) in methyl tert-butyl ether (1.456L) and acetonitrile (485 mL) cooled to-20 deg.C was added 3 parts of N-bromosuccinimide (120.4 g, 99%, 669.7mmol,aldrich b 81255) maintaining the reaction temperature below about-15 ℃. After complete addition, stirring was continued for 30 minutes at-15 to-10 ℃. Working aliquots¹HNMR showed 96% consumption of the starting aniline, so an additional 4.82g nbs was added and stirred at-10 ℃ for an additional 30 minutes. Mixing aqueous 1NNa₂S₂O₃(670 mL) was added to the reaction mixture. The cold water bath was removed and the mixture was stirred for 20 minutes and subsequently diluted with EtOAc. The layers were separated and washed with saturated aqueous NaHCO₃(2 x), washing the organic phase with water and brine in Na₂SO₄Dry, decant and concentrate under reduced pressure to give a dark amber oil. The residue was diluted with hexane and eluted through a short plug of silica, 25% EtOAc in hexane to 50% EtOAc in hexane. The desired filtrate was concentrated in vacuo to give 17 as a dark brown oil (182.9 g, 90% yield; 86% NMR purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 260.12 (3.20 min).¹HNMR（300MHz，CDCl₃）7.15（dd，J=8.6，7.6Hz，1H），6.30（dd，J=8.7，1.3Hz，1H），5.19–5.12（m，1H），4.58（s，2H），4.16–4.07（m，1H），3.90–3.81（m，1H），2.23–1.99（m，4H）ppm。

Example 15

Preparation of N- (4-bromo-3-fluoro-6-nitro-2-tetrahydrofuran-2-yl-phenyl) -2,2, 2-trifluoro-acetamide (18).

Neat 4-bromo-3-fluoro-2-tetrahydrofuran-2-yl-aniline (17) (123.0 g, 86%, 406.7 mmol) as a thick oil was slowly added via addition funnel to trifluoroacetic anhydride (565.3 mL, 4.067mol, Sigma-Aldrich 106232) stirred at 2 ℃ over about 20 minutes (reaction temperature increased to 13 ℃). The remaining oil was washed with anhydrous THF (35 mL) until the reaction mixtureIn the material. The cold bath was removed and the reaction was heated to 35 ℃ followed by the addition of NH portion by portion over 2.5 hours₄NO₃(4.88 gx20 parts, 1.22mol, Sigma-Aldrich A7455), the reaction temperature was maintained at 30-41 ℃ and an ice-water bath was used only when needed to control the exothermicity. After complete addition, the reaction mixture was stirred for another 10 minutes (HPLC showed 99% completion of the reaction). It was slowly poured into crushed ice (1.23 kg) and stirred for 1 hour to allow a filterable solid precipitate to form, which was collected and washed with water, with saturated aqueous NaHCO₃Washed sparingly and again with water (to pH 7). The product was dried in a convection oven at 40 ℃ overnight and then in an oven at 50 ℃ overnight under reduced pressure to give 18 as a beige solid (152.5 g, 90% yield; 96% HPLC purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 401.30 (3.41 minutes).¹HNMR（300MHz，CDCl₃）10.56（s，1H），8.19（d，J=6.6Hz，1H），5.22（dd，J=10.3，6.4Hz，1H），4.22（dd，J=15.8，7.2Hz，1H），3.99（dd，J=16.1，7.5Hz，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-fluoro-6-nitro-2-tetrahydrofuran-2-yl-aniline (19).

The reaction flask was charged with N- (4-bromo-3-fluoro-6-nitro-2-tetrahydrofuran-2-yl-phenyl) -2,2, 2-trifluoro-acetamide (18) (242.3 g, 604.1 mmol), 1, 4-dioxane (1.212L), aqueous 2M 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 and washed with saturated aqueous NaHCO₃Neutralizing and separatingThe layers were separated and the aqueous phase was re-extracted with EtOAc (2 ×). The combined organic phases were washed with brine (2 ×), over MgSO₄Dried, filtered and concentrated in vacuo to afford 19 as a brown-green solid (181.7 g, 94% yield; 95% HPLC purity). The product was carried forward to the next step without further purification. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 305.20 (3.63 min).¹HNMR（300MHz，CDCl₃）8.35（d，J=7.3Hz，1H），7.45（s，2H），5.23–5.16（m，1H），4.23–4.14（m，1H），3.93–3.84（m，1H），2.31–1.96（m，4H）ppm。

Example 17

Preparation of 2- [5- (4-amino-2-fluoro-5-nitro-3-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl ] propyl-2-ol (20).

To a stirred solution of 4-bromo-3-fluoro-6-nitro-2-tetrahydrofuran-2-yl-aniline (19) (525.0 g, 1.721mol, bridgeorganic co.) in 1, 4-dioxane (4.20L, Sigma-Aldrich 360481) was added 1.2m nahco₃Aqueous solution (4.302L, 5.163 mol). Bubbling a stream of nitrogen through the stirred mixture for 2 hours, followed by addition of 2- [5- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrimidin-2-yl]Propyl-2-ol (7) (545.4 g, 2.065mol, bridgeorganics co.) and 1, 1' -bis (diphenylphosphino) ferrocene palladium dichloride dichloromethane adduct (42.16 g, 51.63mmol, Strem 460450). The reaction mixture was stirred at reflux, allowed to cool, diluted with EtOAc (8.4L), and the layers were separated. With saturated aqueous NH₄Cl and subsequent washing of the organic phase with brine. The aqueous phase was re-extracted with EtOAc (4L) and the organic extract was washed with brine. The combined organic phases were washed with water and brine₄Drying by FlorisilShort plug filtration, eluting with EtOAc, and concentration of the filtrate on a rotary evaporator gave a dark brown moist solid. This was dissolved in CH₂Cl₂Loaded onto a pad of silica gel, eluting with hexanes, then 25% EtOAc/hexanes, and then 50% EtOAc/hexanes. The desired filtrate was concentrated on a rotary evaporator to a thick suspension, and the solid was collected by filtration, triturated with MTBE, and dried in vacuo to give 20 as a bright yellow solid (55.8% yield, 90-97% HPLC purity). The filtrate was concentrated and the above purification repeated to give a second harvest 20 as a bright yellow solid (19.7% yield). The filtrate was concentrated again to give a dark brown oil and this was loaded with toluene and minimal CH₂Cl₂On the silica column of (2). It was eluted with EtOAc/hexanes (0% to 50%). The desired fraction was concentrated to a syrup and diluted with MTBE/hexanes. The solid was collected by filtration and washed with minimal MTBE to give a third harvest 20 as a bright yellow solid (4.9% yield), with an overall yield from the three harvests of 80%. LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 363.48 (2.95 min).¹HNMR（300MHz，CDCl₃）8.84（d，J=1.6Hz，2H），8.27（d，J=8.0Hz，1H），7.62（s，2H），5.31–5.24（m，1H），4.63（s，1H），4.27–4.18（m，1H），3.97–3.87（m，1H），2.33–2.05（m，4H），1.64（s，6H）ppm。

Example 18

Preparation of 2- [5- (4, 5-diamino-2-fluoro-3-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl ] propyl-2-ol (21).

5% Palladium on carbon (14.21 g, 50% wet, 3.339mmol, Aldrich 330116) was placed in a Parr bottle under nitrogen, followed by MeOH (242 mL, JT-Baker 909333) and NEt₃(46.54 mL, 333.9mmol, Sigma-Aldrich 471283). Reacting 2- [5- (4-amino-2-fluoro-5-nitro-3-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl]Propyl-2-ol (20) (121.0 g, 333.9 mmol) was dissolved in hot THF (360 mL), allowed to cool, added to the reaction mixture, and rinsed with another portion of THF (124 mL). Place the container on a Parr shaker and use H₂And (4) saturation. Adding 45psiH₂And shaken until consumption was complete (HPLC and LCMS showed complete reaction). The reaction mixture was purged with nitrogen and passed through Celite^TMFiltered and washed with EtOAc. It was refiltered through paper (glass microfiber) and the filtrate was concentrated in vacuo. The reaction was repeated three more times on the same scale and the fractions were combined to give 21 as a brown solid (447 g, 99% yield; 93% HPLC purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 333.46 (1.79 min).¹HNMR（300MHz，CDCl₃）8.81（d，J=1.4Hz，2H），6.69（d，J=7.3Hz，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（m，1H），2.18–1.99（m，3H），1.63（s，6H）ppm。

Example 19

Preparation of 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl ] urea (22).

To 2- [5- (4, 5-diamino-2-fluoro-3-tetrahydrofuran-2-yl-phenyl) pyrimidin-2-yl]Propyl-2-ol (21) (111.3)g, 334.9 mmol) and 1, 4-dioxane (556.5 mL, Sigma-Aldrich 360481) was added 1-ethyl-3- (N- (ethylcarbamoyl) -C-methylthio-carboimino) urea (10) (93.36 g, 401.9mmol, CBResearch process development) followed by dissolution of NaOAc trihydrate (158.1 g) in 1N aqueous H₂SO₄pH3.5 buffer (1.113L) prepared in (1.100L). The reaction mixture was stirred at reflux overnight (HPLC showed complete conversion), cooled to room temperature, and poured (bubbled) portionwise into aqueous saturated NaHCO₃(2.23L) to the starting solution, giving a pH of 8-9. This was stirred for 30 minutes, the solid was collected by filtration, washed well with water to neutral pH, and then washed more sparingly with EtOH. The solid was dried under reduced pressure to give 22 as an off-white yellowish solid (135.2 g, 94% yield; 99% HPLC purity). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 429.58 (2.03 min).¹HNMR（300MHz，MeOD）8.95（d，J=1.6Hz，2H），7.45（d，J=6.5Hz，1H），5.38（br.s，1H），4.27（dd，J=14.9，7.1Hz，1H），4.01（dd，J=15.1，7.0Hz，1H），3.37–3.29（m，2H），2.55（br.s，1H），2.19–2.07（m，2H），2.02–1.82（br.s，1H），1.63（s，6H），1.21（t，J=7.2Hz，3H）ppm。

Example 20

Chiral chromatographic separation of 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea (23).

CHIRALPAK eluted with DCM/MeOH/TEA (60/40/0.1) at 25 deg.CICResolution of 1-Ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl on a column (by Chiraltechnologies)]-7-tetrahydrofuran-2-yl-1H-benzimidazol-2-yl]A racemic sample of urea (22) (133.60 g) gave 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 minutes (CHIRALPAK)IC4.6X250mm column, 1 mL/min flow rate, 30 ℃). The solid was suspended in 2:1EtOH/Et₂O (5 vol), stirred for 10 min, collected by filtration, washed with 2:1EtOH/Et₂O washed and dried under reduced pressure to give a white solid (60.6 g).

The structure and absolute stereochemistry of 23 was confirmed by single crystal x-ray diffraction analysis, after preparation of a sealed tube source of CuK- α (CuK α radiation,and single crystal diffraction data were obtained on a bruker apexiii diffractometer with an apexiccd detector. Crystals with dimensions of 0.15 × 0.10mm were selected, cleaned using mineral oil, fixed on MicroMount and concentrated on a bruker apex iii system. Three batches of 40 frames separated in reciprocal space were obtained to provide the orientation matrix and initial cell parameters. The final cell parameters were obtained and refined based on the full dataset after data collection was complete. P2 for structural and centerless based system extinction and intensity statistics₁Refining in space group.

Diffraction data sets of reciprocal space were obtained using 0.5 ° steps for 30 seconds exposure per frame toThe resolution of (2). Data was collected at 100 (2) K. Integration of intensity and refinement of lattice parameters were done using APEXII software. Observation of the crystals after data collection showed no signs of decomposition. As shown in fig. 2, there are two symmetrically independent molecules in the structure and both symmetrically independent molecules are R isomers.

Data were collected, refined and simplified using apex ii software. Using SHELXS97 (Sheldrick, 1990); the program solved the structure and refined it using the SHELXL97 (Sheldrick, 1997) program. The crystal exhibits a P2₁A space group of monoclinic cells. The lattice parameter isβ=102.826（1）°。

Example 21

Preparation of the mesylate salt of 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea (23A).

To 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]To a stirred suspension of urea (23) (15.05 g, 35.13 mmol) in dichloromethane (60 mL, J.T. Baker 931533) and absolute ethanol (15 mL, Pharmco-AAPER 111000200) was added methanesulfonic acid (2.392 mL, 36.89mmol, Sigma-Aldrich 471356). Stir at room temperature until a clear solution is observed. Heptane (300 mL) was added slowly over about 1 hour, and the solid precipitate was collected by filtration (Whatman qualitative #3 paper over whatmann gf/F glass microfiber paper was used). Drying under reduced pressure in a vacuum oven (dehydration with calcium sulfate and potassium hydroxide) at 40 ℃ overnight gave 23A (13.46 g, 99+% HPLC purity, 99+% ee) as a white solid. Analytical chiral HPLC is shown in CHIRALPAKIC4.6X250mm column on CH₂Cl₂One enantiomer, having a retention time of 8.6 minutes, was eluted with MeOH/TEA (60/40/0.1) using a flow rate of 1 mL/min at 30 ℃. A second harvest of the white solid product 23A was obtained from the filtrate (4.36 g, 98% HPLC purity, 99+% ee). LCMS (10-90% CH over 5 min)₃CN/water gradient elution C18 column, using formic acid modifier) M + 1: 429.58 (2.03 min).¹HNMR（300MHz，MeOD）9.00（d，J=1.6Hz，2H），7.67（d，J=6.1Hz，1H），5.39（t，J=7.7Hz，1H），4.30（dd，J=14.9，6.9Hz，1H），4.03（dd，J=14.8，7.7Hz，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=7.2Hz，3H）ppm。

(R) -1-ethyl-3- (6-fluoro-5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea 23 can then be converted to a phosphate or phosphate prodrug according to the scheme described below.

Scheme 1

Reagents and conditions: (a) dibenzyl N, N-diisopropyl phosphoramidite, tetrazole, 23 ℃ and DMF; (b) mCPBA, 0-23 ℃ and DMF; (c) h₂、Pd/C、M⁺OH^-Or D²⁺（OH^-）₂、EtOH、H₂O; (d) containing water H⁺(ii) a (e) Containing water M⁺OH^-。

Compounds of formula (IB) can be prepared from compound 23 as shown in scheme 1. In step 1, compound 23, followed by m-chloroperoxybenzoic acid (mCPBA), is treated with dibenzyl N, N-diisopropylphosphoramidite and tetrazole to provide dibenzyl phosphate 24. In step 2, at M⁺OH^-Or D²⁺（OH^-）₂Hydrogenolysis of 24 in the presence of (b) provides 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) Can be obtained by treating the dianionic form with an aqueous acid. The monoanionic form (X = PO (OH) O) of the compound of formula (IB)^-M⁺) Can be prepared by using an equivalent of M⁺OH^-Obtained by treating the free acid form.

Scheme 2

Reagents and conditions: (a) boc₂O, DMF, 23 ℃; (b) dibenzyl N, N-diisopropyl phosphoramidite, tetrazole, 23 ℃ and DMF;（c）mCPBA、0-23℃、DMF；（d）TFA、H₂O、MeOH、DCM、23℃；（e）H₂、Pd/C、M⁺OH^-or D²⁺（OH^-）₂、EtOH、H₂O; (f) containing water H⁺；（_g) Containing water M⁺OH^-。

Alternatively, compounds of formula (IB) may be prepared from compound 23 as shown in scheme 2. In step 1, di-tert-butyl dicarbonate (Boc) is used₂O) treating compound 23 to provide a protected benzimidazole compound 25. In step 2, compound 25 is treated with dibenzyl N, N-diisopropylphosphoramidite and tetrazole, followed by mCPBA, to provide protected dibenzyl phosphate 26. In step 3, compound 26 is treated with trifluoroacetic acid (TFA) to remove the protecting group and provide dibenzyl phosphate 24. In step 4, at M⁺OH^-Or D²⁺（OH^-）₂Hydrogenolysis of 24 in the presence of (b) provides 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) Can be obtained by treating the dianionic form with an aqueous acid. The monoanionic form (X = PO (OH) O) of the compound of formula (I)^-M⁺) Can be prepared by using an equivalent of M⁺OH^-Obtained by treating the free acid form.

Scheme 3

Reagents and conditions: (a) boc₂O, DMAP, DMF, 23 ℃; (b) dibenzyl N, N-diisopropyl phosphoramidite, tetrazole, 23 ℃ and DMF; (c) mCPBA, 0-23 ℃ and DMF; (d) TFA, DCM; (e) containing water M⁺OH^-Or D²⁺（OH^-）₂(ii) a (f) Containing water H⁺(ii) a (g) Containing water M⁺OH^-。

Compounds of formula (IB) can also be prepared from compound 23 as shown in scheme 3. In step 1, two equivalents of Boc are used in the presence of N, N-Dimethylaminopyridine (DMAP)₂Compound 23 is treated to provide a double protected benzimidazole compound 28. In step 2, compound 28 is treated with dibenzyl N, N-diisopropylphosphoramidite and tetrazole, followed by mCPBA, to provide the double protected dibenzyl phosphate 29. In step 3, compound 29 is treated with TFA to remove the protecting group. With aqueous M⁺OH^-Or D²⁺（OH^-）₂Treating the resulting intermediate provides 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) Can be obtained by treating the dianionic form with an aqueous acid. The monoanionic form (X = PO (OH) O) of the compound of formula (I)^-M⁺) Can be prepared by using an equivalent of M⁺OH^-Obtained by treating the free acid form.

Example 22

Preparation of (R) -dibenzyl 2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (24).

To a 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl in a 1-L round bottom flask]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]Urea (23) (10.24 g, 23.66 mmol) in N₂DMF (200 mL) was added at 23 ℃ followed by a tetrazole solution (105.2 mL of 0.45M in MeCN, 47.32mmol), followed by N-dibenzyloxyphosphonyl-N-isopropyl-propyl-2-amine (12.26 g, 11.93mL, 35.49 mmol). After 4.5 hours, more N-dibenzyloxyphosphonyl-N-isopropyl-propyl-2-amine (4 mL) was added. After stirring for a further 16 hours, the reaction was cooled to 0 ℃ (ice water bath) and subsequently treated with mCPBA (15.17 g, 61.52 mmol). The mixture was stirred at 0 ℃ for 30 min, then at 23 ℃ for 30 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 (500 mL), and brine (500 mL), then dried (magnesium sulfate), filtered, and concentrated. The residue was purified by MPLC using an ISCOCOMBIFLASH brand flash chromatography purification system (330 g column) eluting with 0-10% EtOH in a linear gradient of DCM over a 16.5 column volume at a flow rate of 200 mL/min. After concentration in vacuo, (R) -dibenzyl 2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] was obtained as a white solid]Imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (24) (13.89 g, 20.17mmol, 85.27%). ESMS (M + 1) = 689.5;¹HNMR（300MHz，CD₃OD) 8.88 (d, J =1.6Hz, 2H), 7.37 (d, J =6Hz, 1H), 7.30 (m, 10H), 5.38-5.33 (m, 1H), 5.12-5.01 (m, 4H), 4.24 (dd, J =6.8, 14.9Hz, 1H), 3.98 (dd, J =6.9, 15.1Hz, 1H), 3.35-3.27 (m, 3H), 2.52 (q, J =5.9Hz, 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) -2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (W).

The 1LPArr vessel was filled with water (200 mL), Pd/C (4 g, 10 wt% dry weight, wet, Degussa type), (R) -dibenzyl 2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ], (R) -dibenzyl]Imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (24) (13.89 g, 20.17 mmol), EtOH (400 mL), and aqueous 1M NaOH (40.34 mL, 40.34 mmol). The resulting mixture was placed in 50psiH₂The hydrogenation was carried out for 40 minutes on a Parr shaker. The reaction mixture was filtered through a 0.22 μm Polyphenylene Ether Sulfone (PES) membrane to give a dark filtrate. Water washing results in darker material across the filter membrane. The resulting filtrate was passed through a pad of celite and the dark material was not eluted until the pad was washed with water. The resulting dark solution (ca 700 mL) was diluted with three volumes of EtOH (2100 mL), filtered through 0.22 μmPES membrane (using 4 disposable Corning polystyrene filtration 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 μmPES membrane to give a clear yellow solution, then passed through a plug of celite (26 mm diameter x60mm height, pre-wetted with EtOH), rinsed with EtOH (50 mL), and then the filtrate was concentrated. The resulting residue was dissolved in water (250 mL) in a 1L round bottom flask and 1M aqueous HCl (40 mL) was added slowly over 15 minutes with stirring to give a slurry of a white solid. Twenty minutes after the HCl addition was complete, the solid was collected via filtration through a 0.22 μmPES membrane. The collected solid was washed with water (100 mL) before transferring (still wet) to a 1L round bottom flask and slurrying in MeOH (150 mL) for 30 min. The resulting fine white precipitate was collected via filtration and subsequently dried in vacuo overnight. The resulting free acid (9.17 g, 18.0 mmol) was treated with water (80 mL) followed by 1.0N aqueous NaOH (36.0 mL, 2 equiv.). The resulting solution was frozen and lyophilized to give [1- [5- [2- (ethylcarbamoylamino) -6-fluoro-7- [ (2R) -tetrahydrofuran-2-yl ] as a pale cream solid]-1H-benzimidazol-5-yl]Pyrimidin-2-yl]-1-methyl-ethyl]Disodium phosphate (W) (10.206 g, 18.08mmol, 89.66%) with consistent analytical data. ESMS (M + 1) =509.4；¹HNMR（300MHz，D₂O) 8.58 (s, 2H), 6.92 (d, J =6.3Hz, 1H), 5.13 (t, J =7.5Hz, 1H), 3.98-3.81 (m, 2H), 3.04 (q, J =7.2Hz, 2H), 2.26 (t, J =5.7Hz, 1H), 1.97-1.92 (m, 2H), 1.67 (s, 6H) and 1.01 (t, J =7.2Hz, 3H) ppm.

Example 24

Preparation of Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea (25).

To 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl at 23 ℃]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]Boc was added to a solution/suspension of urea (23) (10.72 g, 25.02 mmol) in DMF (250 mL)₂O (6.11 g, 28.00 mmol). After 2 hours, 2M ammonia in MeOH (2 mL) was added to quench any excess Boc₂And O. The quenched reaction mixture was partitioned between EtOAc and water (400 mL each), the organic layer was separated, washed with water (2 × 400 mL) and brine (400 mL), then over MgSO₄Dried, filtered and concentrated to give Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl used without further purification]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]Urea (25) (12.69 g, 23.58mmol, 94.26%). ESMS (M + 1) = 529.3;¹HNMR（300.0MHz，CDCl₃）9.50（s，1H），9.02（t，J=5.3Hz，1H），8.91（d，J=1.6Hz，2H），7.74（d，J=6.5Hz，1H），5.58（t，J=7.8Hz，1H），4.64（s，1H），4.22（q，J=7.4Hz，1H），4.05（td，J=7.8，4.3Hz，1H），3.47（td，J=7.2，4.3Hz，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 =7.3Hz, 3H) ppm.

Example 25

Preparation of Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea dibenzyl phosphate (26).

In N₂To Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl at 23 deg.C]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]To urea (25) (12.69 g, 23.58 mmol) and tetrazole (3.304 g, 47.16 mmol) was added DCM (240 mL) followed by N-dibenzyloxyphosphono-N-isopropyl-propyl-2-amine (9.775 g, 9.509mL, 28.30 mmol). After 3 hours at 23 ℃, the reaction was cooled to 0 ℃, followed by the addition of mCPBA (6.977 g, 28.30 mmol). The reaction solution was stirred at 0 ℃ for 45 minutes, followed by stirring at 23 ℃ for 20 minutes. The reaction 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 bisulfite (63 g in 350mL 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 ISCOMBIFLASH brand flash chromatography purification system (330 g silica column), eluting with 0-100% EtOAc in a linear gradient of hexane over 16 column volumes at 200 mL/min. The product-containing fractions were evaporated in vacuo to afford Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]Urea dibenzyl phosphate (26) (11.92 g, 15.11mmol, 64.09%). ESMS (M + 1) = 789.2;¹HNMR（300.0MHz，CDCl₃）9.51（s，1H），9.03（t，J=5.4Hz，1H），8.91（d，J=1.6Hz，2H），7.73（d，J=6.5Hz，1H）7.37-7.28 (m, 10H), 5.58 (t, J =7.8Hz, 1H), 5.17-5.05 (m, 4H), 4.23 (t, J =7.5Hz, 1H), 4.05 (td, J =7.8, 4.3Hz, 1H), 3.53-3.44 (m, 2H), 2.39 (dd, J =7.9, 14.5Hz, 2H), 2.28-2.15 (m, 2H), 1.98 (s, 6H), 1.72 (m, 9H) and 1.31 (t, J =7.2Hz, 3H) ppm.

Example 26

Preparation of (R) -dibenzyl 2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (24).

Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl in DCM (300 mL) at 23 deg.C]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]A solution of urea dibenzyl phosphate (26) (11.9 g, 15.09 mmol) was added water (2.325 mL, 129.1 mmol) followed by TFA (3.441 g, 2.325mL, 30.18 mmol). After 1 hour, only partial conversion was observed by thin layer chromatography (tlc), so more TFA (3.441 g, 2.325mL, 30.18 mmol) was added. After a further 2.5 hours MeOH (2 mL) was added and the mixture was stirred for a further 18 hours. The reaction mixture was washed with 1:1 brine, saturated aqueous sodium bicarbonate (200 mL). The aqueous layer was re-extracted with DCM (150 mL), the organic layers were combined, then dried (over magnesium sulfate), filtered and concentrated in vacuo. The resulting residue was redissolved in EtOAc (200 mL), washed with water (150 mL) and brine (100 mL), then dried (over magnesium sulfate), filtered, and concentrated to give (R) -dibenzyl 2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] as a white solid]Imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (24) (10.21 g, 14.83mmol, 98.27%). ESMS (M +1）=689.4；¹HNMR（300MHz，CD₃OD) 8.88 (d, J =1.6Hz, 2H), 7.37 (d, J =6Hz, 1H), 7.30 (m, 10H), 5.38-5.33 (m, 1H), 5.12-5.01 (m, 4H), 4.24 (dd, J =6.8, 14.9Hz, 1H), 3.98 (dd, J =6.9, 15.1Hz, 1H), 3.35-3.27 (m, 3H), 2.52 (q, J =5.9Hz, 1H), 2.14-2.05 (m, 2H), 1.91 (s, 6H) and 1.22-1.14 (m, 3H) ppm.

Example 27

Preparation of disodium (R) -2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (W).

A1L round bottom flask was charged with (R) -dibenzyl 2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ]]Imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (24) (9.37 g, 13.61 mmol), EtOH (300 mL), water (150 mL), Pd/C (10 wt% dry weight, wet, Degussa type, 3 g), and 1M aqueous NaOH (27.22 mL, 27.22 mmol). The suspension was evacuated for 3 minutes (needle to pump) and then placed under an atmosphere of hydrogen (balloon). After stirring at 23 ℃ for 2.5 h, the reaction was filtered through 0.22 μm PES membrane (disposable Corning filtration System, 1L, polystyrene, # 431098) to remove the catalyst and washed with EtOH (50 mL). The resulting solution was concentrated and the residue was dissolved in water (80 mL), treated with MeCN (80 mL), then frozen and lyophilized to give (R) -2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] as a white solid]Imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl disodium phosphate (W) (7.10 g, 12.81mmol, 94.12%). ESMS (M + 1) = 509.3;¹HNMR（300MHz，D₂O）8.58（s，2H），6.92（d，J=6.3Hz，1H），5.13 (t, J =7.5Hz, 1H), 3.98-3.81 (m, 2H), 3.04 (q, J =7.2Hz, 2H), 2.26 (t, J =5.7Hz, 1H), 1.97-1.92 (m, 2H), 1.67 (s, 6H) and 1.01 (t, J =7.2Hz, 3H) ppm.

Example 28

Preparation of di-Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea (28).

To 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl in DMF (30 mL)]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]DMAP (38.01 mg, 0.3111 mmol) followed by Boc (23) (1.333 g, 3.111 mmol) was added to the solution/suspension of urea (23) (1.333 g, 3.111 mmol)₂O (1.426 g, 1.501mL, 6.533 mmol). After 30 min, the reaction mixture was diluted with water and EtOAc (300 mL each), the organic layer was 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 ISCOCOMBIFLASH brand flash chromatography purification system (80 g silica column) eluting with 0-60% EtOAc in a linear gradient of hexane over a 20 column volume at a flow rate of 60 mL/min. The desired product fractions were combined and evaporated to give the di-Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl as a clear foam]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]Urea (28) (1.43 g, 2.275mmol, 73.11%). ESMS (M + 1) = 629.3;¹HNMR（300.0MHz，CDCl₃）8.95（d，J=1.6Hz，2H），8.31-8.27（m，1H），8.05（d，J=6.5Hz，1H），5.80-5.68（m，1H），4.70（s，1H），4.21-4.09（m，1H），3.98（d，J=6.4Hz，1H），3.42-3.37（m，2H），2.45-2.00（m，4H），1.65（s，6H），1.62（s，9H），1.37（s，9H) and 1.28-1.21 (m, 3H) ppm.

Example 29

Preparation of di-Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea dibenzyl phosphate (29).

In N₂di-Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl at 23 deg.C]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]To urea (28) (1.13 g, 1.797 mmol) and tetrazole (251.8 mg, 3.594 mmol) was added DCM (30 mL) followed by N-dibenzyloxyphosphonoyl-N-isopropyl-propyl-2-amine (744.7 mg, 724.4. mu.L, 2.156 mmol). After stirring for 18 h, the reaction was cooled to 0 ℃ and subsequently treated with mCPBA (531.5 mg, 2.156 mmol). The reaction was stirred at 0 ℃ for 15 minutes, followed by 23 ℃ for 30 minutes. The resulting solution was then partitioned between EtOAc and saturated aqueous sodium bicarbonate (300 mL each), the organic layer was 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 ISCOCOMBIFLASH brand flash chromatography purification system (80 g silica column) eluting with 0-80% EtOAc in a linear gradient of hexane over a 20 column volume at a flow rate of 60 mL/min. The desired product fractions were combined and evaporated to give the di-Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl as a clear, glassy oil]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]Urea dibenzyl phosphate (29) (1.03 g, 1.159mmol, 64.50%). ESMS (M + 1) = 889.2;¹HNMR（300.0MHz，CDCl₃）8.93（d，J=1.5Hz，2H），8.31（s，1H），8.04（d，J=6.4Hz，1H），7.36-7.26（m，10H），5.83-5.70 (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 (m, 9H), 1.40 (s, 9H) and 1.26 (t, J =7.2Hz, 3H) ppm.

Example 30

Preparation of sodium (R) -2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (W).

di-Boc-1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl in DCM (10 mL) at 23 deg.C]-7- [ (2R) -tetrahydrofuran-2-yl]-1H-benzimidazol-2-yl]To a solution of urea dibenzyl phosphate (29) (121 mg, 0.1361 mmol) was added TFA (5 mL). After 2 hours, the reaction mixture was concentrated in vacuo. The residue was dissolved in MeOH (6 mL) and taken up in approximately 0.5mL2MNH in MeOH₃Treatment (to completely dissolve the material.) reaction solution was purified on preparative HPLC, reverse phase, Sunfireprep C18OBD5 μ M column 19 × 100mm in 6 injections; linear gradient with 10-90% aqueous MeCNw/0.1% TFA buffer for 15 min elution at 20 mL/min flow rate. fractions containing product were combined and lyophilized. the resulting material was suspended in MeOH (3 mL), stirred at 23 ℃ for 30 min, then the precipitate was collected via filtration through a plastic frit, the resulting white solid was re-slurried with MeOH (3 mL), then collected via filtration to give 68mg white solid after drying. the white solid was treated with 0.10M aqueous NaOH (2.68 mL, 2 equivalents NaOH) to give a solution that was then passed through an AcrocCR 13mm syringe filter with 0.45 μ TFE membrane, washed with water (2 mL), the resulting solution was treated with MeCN (3 mL) and the resulting solution was lyophilized as a white powder (R5-Freeze-2- (R5) lyophilized powder(3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d]Imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate sodium salt (W). ESMS (M + 1) = 509.2;¹HNMR（300MHz，D₂o) 8.58 (s, 2H), 6.92 (d, J =6.3Hz, 1H), 5.13 (t, J =7.5Hz, 1H), 3.98-3.81 (m, 2H), 3.04 (q, J =7.2Hz, 2H), 2.26 (t, J =5.7Hz, 1H), 1.97-1.92 (m, 2H), 1.67 (s, 6H) and 1.01 (t, J =7.2Hz, 3H) ppm.

Example 31

Sensitivity testing in liquid media

The compounds of the invention were tested for antimicrobial activity by sensitivity testing in liquid media. Such assays are performed within the guidance of the latest CLSI documents that manage such practices: "M07-A8 methods for DiluetionImitrificationLiscalabilityTestsformasteriatathGrowAerobacter; approval criteria-eighth edition (2009) ". Other publications such as "antibiotic laboratory medicine" (edited by v. lowian, publishers Williams and Wilkins, 1996) provide the basic practice in laboratory antibiotic testing. The specific protocol used was as follows:

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

Materials:

round bottom 96-well microtiter plate (Costar 3788)

Mueller HintonII agar plates (MHII; BBL premix)

Mueller Hinton II liquid broth (MHII; BBL premix)

BBLPromptInoculationSystem（FisherB26306）

Test reading mirror (Fisher)

Agar plates of bacteria streaked to single colonies, freshly prepared

Sterile DMSO

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

Cracking horse blood (Quadfive 270-100)

Resazurin 0.01%

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

Pooled mouse serum (Valley Biomedical AS 3054)

Strain (medium, broth and agar):

1. staphylococcus aureus ATCC #29213

a.MHII

MHII +50% human serum

MHII +50% rat serum

MHII +50% mouse serum

2. Staphylococcus aureus ATCC #29213GyrBT173I (MHII)

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

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

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

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

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

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

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

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

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

12. Bacillus cereus (Bacillus cereus) ATCC10987 (MHII)

13. Bacillus cereus ATCC14579 (MHII)

14. Bacillus subtilis ATCC6638 (MHII)

15. Bacillus subtilis (168) ATCC6051 (MHII)

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

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

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

3. For each plate of compound tested, 0.15mL of cells were transferred to 15mL (. about.10)⁶cells/mL) sterile broth (or see below), the suspension 1/100 was diluted and then vortex mixed. If test compound(s) (ii)>8 compounds) over 1 plate, the volume can be increased accordingly.

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

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

Drug dilution, inoculation, MIC determination:

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

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

3. 25 μ L of LDMSO was added to columns 2-12 of all rows of microtiter plates containing 200x stock of compound and 25 μ L was serially diluted from column 1 through column 11, changing the tip after each column. I.e., 25 μ L compound +25 μ LDMSO =2x dilution. A "no compound" DMSO well was left at the end of the series for control.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Materials:

round bottom 96-well microtiter plate (Costar 3788)

Mueller HintonII agar plates (MHII; BBL premix)

Mueller Hinton II liquid broth (MHII; BBL premix)

BBLPromptInoculationSystem（Fisherb26306）

Test reading mirror (Fisher)

Agar plates of bacteria streaked to single colonies, freshly prepared

Sterile DMSO

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

1. escherichia coli ATCC #25922

2. Coli, JMI Collection Strain, see Table 5

3. Escherichia coli AG100WT

4. Escherichia coli AG100tolC

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

6. Acinetobacter baumannii ATCC #19606

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

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

9. Klebsiella pneumoniae ATCC #700603

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

11. Moraxella catarrhalis ATCC #25238

12. Moraxella catarrhalis ATCC #49143

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

14. Haemophilus influenzae ATCC49247

15. Haemophilus influenzae (Rd 1KW 20) ATCC51907

16. Haemophilus influenzae Rd0894 (AcrA-)

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

18. Pseudomonas aeruginosa PAO1

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

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

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

22. Stenotrophomonas maltophilia (Stenotrophormonastra) ATCCBA-84

23. Stenotrophomonas maltophilia ATCC13637

Preparation of inoculum:

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

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

3. For each plate of compound tested, 0.15mL of cells were transferred to 15mL (. about.10)⁶cells/mL) sterile broth (see below), the suspension 1/100 was diluted and vortex mixed. If test compound(s) (ii)>8 compounds) over 1 plate, the volume is increased accordingly.

4.50 μ l cells (. about.5X 10) were used⁴Cells) to inoculate a broth containing 50. mu.l of dilution in brothFor each microtiter well of drug (see below).

Drug dilution, inoculation, MIC determination:

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

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

3. 25 μ L of LDMSO was added to columns 2-12 of all rows of microtiter plates containing 200x stock of compound and 25 μ L was serially diluted from column 1 through column 11, changing tips after each column. I.e., 25 μ L compound +25 μ LDMSO =2x dilution. At the end of the series, "no compound" DMSO wells were left for control.

4. For each strain tested, two microtiter plates were prepared with 50 μm shii broth using Matrix pipettes.

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

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

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

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

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

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

Materials:

petri dish 60x15mm (Thermoscientific catalog # 12567100)

Centrifuge tube, 15mL (Costar)

BBLPromptInoculationSystem（FisherB26306）

Agar plates with streaked bacteria to single colonies, freshly prepared

Sterile DMSO

GasPak^TMIncubation container (BD catalog # 260672)

GasPak^TMEzanarobe container system pouch (BD catalog # 260678)

GasPak^TMEZC02 Container system Small bag (BD catalogue # 260679)

GasPak^TMEZCampy Container System pouch (BD Cat # 260680)

The strain is as follows:

1. clostridium difficile ATCCBA-1382;

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

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

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

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

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

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

8. Neisseria gonorrhoeae ATCC35541

9. Neisseria gonorrhoeae ATCC49226

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

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

Media preparation and growth conditions:

according to CLSI publication' M11-A7methods for the inhibition of microbiological Susctionalitinityingof Anaerobic Bacteria; approval criteria-seventh edition (2007)' preparation of growth media recommended for each microbial species, in addition to neisseria gonorrhoeae and neisseria meningitidis, the media therefor was according to "M07-A8 methods for dilution of microbiological viability test for bacterial growth aerobically; the approval standard, eighth edition (2009) ″, was prepared.

Pouring a flat plate:

1. a 100x stock of drug was prepared for each test compound as described in table 1. Using a 15mL centrifuge tube, 100uL of each drug stock was added to 10mL of agar melt (cooled to 55 ℃ in a water bath). Mix by inverting the tube 2-3X and then pour into individually labeled 60X15mm petri dishes.

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

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

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

5. Corresponding marker plates were placed for drug concentration and strain.

Growth of cells requiring maintenance of an anaerobic environment:

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

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

Plating (performed in biosafety cabinet):

1. each strain was streaked onto individual agar plates as described above. The time required for incubation and the environmental conditions (i.e. anaerobic, oxygen demand in trace amounts, etc.).

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

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

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

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

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

6. The inoculum was allowed to dry (as short as needed) and then placed upside down in GasPak in an appropriate number of sachets and incubated.

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

MIC determination:

the test plates were examined after the correct incubation time and the MIC end-point was read at a concentration where a significant reduction in the appearance of growth on the test plates occurred, compared to that grown on the positive control plates.

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

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

^**Compounds were dissolved and diluted in 100% DMSO

Scheme 4. MIC determination procedure for Mycobacterium species

Material

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

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

Middlebrook7H10 broth with 0.2% glycerol

Middlebrook7H10 agar with 0.2% glycerol

MiddlebrookOADCEnrichment

Inoculum preparation for mycobacterium tuberculosis:

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

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

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

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

1. using the frozen stock culture prepared or in 10Klett or 5X10⁷Fresh cultures grown at a concentration of 7H10 broth/ml.

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

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

Plate preparation:

1. the plate is labeled.

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

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

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

5. 50 μ l of 4x working solution was added to the first well of the indicated row. Continue for all compounds tested. Using a multi-channel electrokinetic pipette, mix 4X and serially dilute the compound through the 11 th well. The remaining 50. mu.l were discarded. The 12 th well was used as a positive control.

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

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

Scheme 5 protocol for M.tuberculosis seroconversion MIC assay

Materials and reagents:

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

Middlebrook7H9 broth with 0.2% glycerol (BD 271310)

MiddlebrookOADCEnrichment

Fetal bovine serum

Catalase (SigmaC 1345)

Dextrose

NaCl₂

BBLPromptInoculationSystem（Fisherb26306）

Agar plates with streaking of bacteria to single colonies (Middlebrook 7H11 with 0.2% glycerol and OADC enrichment)

Sterile DMSO

Preparing a culture medium:

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

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

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

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

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

Preparation of inoculum:

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

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

3. Cultures in each of the three media formulations were prepared by diluting the bbl prot suspension (e.g.: 0.2ml cells were transferred to 40ml medium) at 1/200,to obtain-10⁶Initial cell density of cells/ml.

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

Drug dilution, inoculation, MIC determination:

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

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

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

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

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

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

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

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

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

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

The percent inhibition by a given compound was calculated as follows: percent inhibition =100- ([ pore fluorescence-mean background fluorescence ]/[ DMSO control-mean background fluorescence ] x 100). For all three medium conditions, the MICs score was the lowest compound concentration at which inhibition of Resazurin reduction ('% -inhibition) signal ≧ 70% at the given medium condition.

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

In table 2 and subsequent tables and examples, "compound 12" corresponds to 1-ethyl-3- [5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea, and "compound 13" relates to the mesylate salt of compound 12. Similarly, "compound 23" corresponds to 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea, and "compound 23A" relates to the mesylate salt of compound 23. These are the same numbers used in the examples above to identify the compounds and salts.

TABLE 2 MIC values of selected Compounds

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

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

Table 3A also shows the MIC assay results for selected compounds of the invention. In table 3A, "compound 23A" corresponds to the mesylate salt of 1-ethyl-3- [ 6-fluoro-5- [2- (1-hydroxy-1-methyl-ethyl) pyrimidin-5-yl ] -7- [ (2R) -tetrahydrofuran-2-yl ] -1H-benzimidazol-2-yl ] urea (23A). Similarly, the "compound W" corresponds to disodium (R) -2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate (W). These are the same numbers used to identify the compounds in the synthetic examples above.

TABLE 3A MIC values for selected Compounds

¹American type culture center

²Built by vortexing

³ColiGeneticStockCenter

⁴All tolC constructs are tolC:: Tn10 derived from CAG12184 (ColiGenetic Stockcenter)

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

TABLE 4 panel of anaerobic organisms used to generate MIC90 data

CMI#	Biological body
		A2380	Bacteroides fragilis
A2381	Bacteroides fragilis
		A2382	Bacteroides fragilis
A2486	Bacteroides fragilis
		A2487	Bacteroides fragilis
A2489	Bacteroides fragilis
		A2527	Bacteroides fragilis
A2529	Bacteroides fragilis
		A2562	Bacteroides fragilis
A2627	Bacteroides fragilis
		A2802	Bacteroides fragilis
A2803	Bacteroides fragilis
		A2804	Bacteroides fragilis
A2805	Bacteroides fragilis
		A2806	Bacteroides fragilis
A2807	Bacteroides fragilis
		A2808	Bacteroides fragilis
A2809	Bacteroides fragilis
		A2810	Bacteroides fragilis
A2811	Bacteroides fragilis
		A2812	Bacteroides fragilis
A2813	Bacteroides fragilis

CMI#	Biological body
		A2814	Bacteroides fragilis
A2460	Bacteroides thetaiotaomicron (B. Thetaioomicron)
		A2462	Bacteroides thetaiotaomicron
A2463	Bacteroides thetaiotaomicron
		A2464	Bacteroides thetaiotaomicron
A2536	Bacteroides thetaiotaomicron
		A2591	Bacteroides simplex (B.uniformis)
A2604	Bacteroides vulgare (B.vulgatus)
		A2606	Bacteroides vulgaris
A2613	Oval bacterium (B.ovatus)
		A2616	Leptobacterium ovatus
A2815	Bacteroides camouflagus (Bacteroides tectom)
		A2816	Ureolyticus (B. ureolyticus)
A2817	Flavobacterium sp. (Bacteroides capitosus)
		A2818	Urea decompositionBacteroides
A2824	Parabacter distasonis
		A2825	Leptobacterium ovatus
A2826	Bacteroides simplex
		A2827	Bacteroides simplex
A2828	Bacteroides vulgaris
		A2829	Bacteroides vulgaris
A2830	Leptobacterium ovatus
		A2831	Bacteroides thetaiotaomicron
A2832	Parabacter distasonis
		A2833	Bacteroides thetaiotaomicron
A2767	Clostridium difficile
		A2768	Clostridium difficile
A2769	Clostridium difficile
		A2770	Clostridium difficile
A2771	Clostridium difficile
		A2772	Clostridium difficile
A2773	Clostridium difficile
		A2774	Clostridium difficile
A2775	Clostridium difficile
		A2776	Clostridium difficile
A2777	Clostridium difficile
		A2778	Clostridium difficile

CMI#	Biological body
		A2779	Clostridium difficile
A2780	Clostridium difficile
		A2140	Clostridium perfringens
A2203	Clostridium perfringens
		A2204	Clostridium perfringens
A2227	Clostridium perfringens
		A2228	Clostridium perfringens
A2229	Clostridium perfringens
		A2315	Clostridium perfringens
A2332	Clostridium perfringens
		A2333	Clostridium perfringens
A2334	Clostridium perfringens
		A2389	Clostridium perfringens
A2390	Clostridium perfringens
		A864	Fusobacterium necrophorum (F. necrophorum)
A871	Fusobacterium nucleatum (F.nucleolus)
		A1667	Fusobacterium necrophorum
A1666	Fusobacterium necrophorum
		A2249	Fusobacterium nucleatum
A2716	Fusobacterium species
		A2717	Fusobacterium species
A2719	Fusobacterium species
		A2721	Fusobacterium species
A2722	Fusobacterium species
		A2710	Fusobacterium species
A2711	Fusobacterium species
		A2712	Fusobacterium species
A2713	Fusobacterium species
		A2714	Fusobacterium species
A2715	Fusobacterium species
		A1594	Anaerobic digestion of Streptococcus (Peptostreptococcus anaerobius)
A2158	Streptococcus magnus (Peptostreptococcus magnus)
		A2168	Anaerobic digestion of streptococci
A2170	Streptococcus macrodigestive system
		A2171	Streptococcus macrodigestive system
A2575	Digestion of Streptococcus species
		A2579	Streptococcus undenatured (Peptostreptococcus asaccharolyticus)

CMI#	Biological body
		A2580	Streptococcus undenatured
A2614	Streptococcus undenatured
		A2620	Streptococcus undenatured
A2629	Digestion of Streptococcus species
		A2739	Prevotella denticola (Prevotella denticola)
A2752	Two-way Prevotella (Prevotella bivia)
		A2753	Prevotella intermedia (Prevotella intermedia)
A2754	Prevotella intermedia
		A2756	Prevotella bifidus
A2759	Prevotella bifidus
		A2760	Prevotella denticola
A2761	Prevotella intermedia
		A2762	Prevotella nigricans (Prevotella melaninogenica)
A2765	Prevotella nigricans producing bacterium
		A2766	Prevotella nigricans producing bacterium
A2821	Prevotella bifidus
		A2822	Prevotella bifidus
QCBF	Bacteroides fragilis
		QCBT	Bacteroides thetaiotaomicron
QCCD	Clostridium difficile
		QCBF	Bacteroides fragilis
QCBT	Bacteroides thetaiotaomicron
		QCCD	Clostridium difficile

In Table 5 below, the term "JMI" represents Jones microbiology institute at Northliberty, Iowa.

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

JMI isolate #	JMI biological coding	Biological body
			394	ACB	Acinetobacter baumannii
2166	ACB	Acinetobacter baumannii
			3060	ACB	Acinetobacter baumannii
3170	ACB	Acinetobacter baumannii
			9328	ACB	Acinetobacter baumannii
9922	ACB	Acinetobacter baumannii
			13618	ACB	Acinetobacter baumannii
14308	ACB	Acinetobacter baumannii

JMI isolate #	JMI biological coding	Biological organismsBody 64->
			17086	ACB	Acinetobacter baumannii
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 cloacae
12735	ECL	Enterobacter cloacae
			13057	ECL	Enterobacter cloacae
18048	ECL	Enterobacter cloacae
			25173	ECL	Enterobacter cloacae
29443	ECL	Enterobacter cloacae
			44	EF	Enterococcus faecalis
355	EF	Enterococcus faecalis
			886	EF	Enterococcus faecalis
955	EF	Enterococcus faecalis
			1000	EF	Enterococcus faecalis
1053	EF	Enterococcus faecalis
			1142	EF	Enterococcus faecalis
1325	EF	Enterococcus faecalis
			1446	EF	Enterococcus faecalis
2014	EF	Enterococcus faecalis
			2103	EF	Enterococcus faecalis
2255	EF	Enterococcus faecalis
			2978	EF	Enterococcus faecalis
2986	EF	Enterococcus faecalis
			5027	EF	Enterococcus faecalis
5270	EF	Enterococcus faecalis
			5874	EF	Enterococcus faecalis
7430	EF	Enterococcus faecalis
			7904	EF	Enterococcus faecalis
8092	EF	Enterococcus faecalis
			8691	EF	Enterococcus faecalis

JMI isolate #	JMI biological coding	Organism 65->
			9090	EF	Enterococcus faecalis
10795	EF	Enterococcus faecalis
			14104	EF	Enterococcus faecalis
16481	EF	Enterococcus faecalis
			18217	EF	Enterococcus faecalis
22442	EF	Enterococcus faecalis
			25726	EF	Enterococcus faecalis
26143	EF	Enterococcus faecalis
			28131	EF	Enterococcus faecalis
29765	EF	Enterococcus faecalis
			30279	EF	Enterococcus faecalis
31234	EF	Enterococcus faecalis
			31673	EF	Enterococcus faecalis
115	EFM	Enterococcus faecium
			227	EFM	Enterococcus faecium
414	EFM	Enterococcus faecium
			712	EFM	Enterococcus faecium
870	EFM	Enterococcus faecium
			911	EFM	Enterococcus faecium
2356	EFM	Enterococcus faecium
			2364	EFM	Enterococcus faecium
2762	EFM	Enterococcus faecium
			3062	EFM	Enterococcus faecium
4464	EFM	Enterococcus faecium
			4473	EFM	Enterococcus faecium
4653	EFM	Enterococcus faecium
			4679	EFM	Enterococcus faecium
6803	EFM	Enterococcus faecium
			6836	EFM	Enterococcus faecium
8280	EFM	Enterococcus faecium
			8702	EFM	Enterococcus faecium
9855	EFM	Enterococcus faecium
			10766	EFM	Enterococcus faecium
12799	EFM	Enterococcus faecium
			13556	EFM	Enterococcus faecium
13783	EFM	Enterococcus faecium
			14687	EFM	Enterococcus faecium

JMI isolate #	JMI biological coding	Organism 66->
			15268	EFM	Enterococcus faecium
15525	EFM	Enterococcus faecium
			15538	EFM	Enterococcus faecium
18102	EFM	Enterococcus faecium
			18306	EFM	Enterococcus faecium
19967	EFM	Enterococcus faecium
			22428	EFM	Enterococcus faecium
23482	EFM	Enterococcus faecium
			29658	EFM	Enterococcus faecium
597	EC	Escherichia coli
			847	EC	Escherichia coli
1451	EC	Escherichia coli
			8682	EC	Escherichia coli
11199	EC	Escherichia coli
			12583	EC	Escherichia coli
12792	EC	Escherichia coli
			13265	EC	Escherichia coli
14594	EC	Escherichia coli
			22148	EC	Escherichia coli
29743	EC	Escherichia coli
			30426	EC	Escherichia coli
470	BSA	Group A streptococcus
			2965	BSA	Group A streptococcus
3112	BSA	Group A streptococcus
			3637	BSA	Group A streptococcus
4393	BSA	Group A streptococcus
			4546	BSA	Group A streptococcus
4615	BSA	Group A streptococcus
			5848	BSA	Group A streptococcus
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

JMI isolate #	JMI biological coding	67->
			12	BSB	Group B streptococcus
10366	BSB	Group B streptococcus
			10611	BSB	Group B streptococcus
16786	BSB	Group B streptococcus
			18833	BSB	Group B streptococcus
30225	BSB	Group B streptococcus
			10422	BSC	Group C streptococcus
14209	BSC	Group C streptococcus
			29732	BSC	Group C streptococcus
8544	BSG	Group G streptococci
			18086	BSG	Group G streptococci
29815	BSG	Group G streptococci
			147	HI	Haemophilus influenzae
180	HI	Haemophilus influenzae
			934	HI	Haemophilus influenzae
970	HI	Haemophilus influenzae
			1298	HI	Haemophilus influenzae
1819	HI	Haemophilus influenzae
			1915	HI	Haemophilus influenzae
2000	HI	Haemophilus influenzae
			2562	HI	Haemophilus influenzae
2821	HI	Haemophilus influenzae
			3133	HI	Haemophilus influenzae
3140	HI	Haemophilus influenzae
			3497	HI	Haemophilus influenzae
3508	HI	Haemophilus influenzae
			3535	HI	Haemophilus influenzae
4082	HI	Haemophilus influenzae
			4108	HI	Haemophilus influenzae
4422	HI	Haemophilus influenzae
			4868	HI	Haemophilus influenzae
4872	HI	Haemophilus influenzae
			5858	HI	Haemophilus influenzae
6258	HI	Haemophilus influenzae
			6875	HI	Haemophilus influenzae
7063	HI	Haemophilus influenzae
			7600	HI	Haemophilus influenzae

JMI isolate #	JMI biological coding	68->
			8465	HI	Haemophilus influenzae
10280	HI	Haemophilus influenzae
			10732	HI	Haemophilus influenzae
10850	HI	Haemophilus influenzae
			11366	HI	Haemophilus influenzae
11716	HI	Haemophilus influenzae
			11724	HI	Haemophilus influenzae
11908	HI	Haemophilus influenzae
			12093	HI	Haemophilus influenzae
12107	HI	Haemophilus influenzae
			13424	HI	Haemophilus influenzae
13439	HI	Haemophilus influenzae
			13672	HI	Haemophilus influenzae
13687	HI	Haemophilus influenzae
			13792	HI	Haemophilus influenzae
13793	HI	Haemophilus influenzae
			14440	HI	Haemophilus influenzae
15351	HI	Haemophilus influenzae
			15356	HI	Haemophilus influenzae
15678	HI	Haemophilus influenzae
			15800	HI	Haemophilus influenzae
17841	HI	Haemophilus influenzae
			18614	HI	Haemophilus influenzae
25195	HI	Haemophilus influenzae
			27021	HI	Haemophilus influenzae
28326	HI	Haemophilus influenzae
			28332	HI	Haemophilus influenzae
29918	HI	Haemophilus influenzae
			29923	HI	Haemophilus influenzae
31911	HI	Haemophilus influenzae
			428	KPN	Klebsiella pneumoniae
791	KPN	Klebsiella pneumoniae
			836	KPN	Klebsiella pneumoniae
1422	KPN	Klebsiella pneumoniae
			1674	KPN	Klebsiella pneumoniae
1883	KPN	Klebsiella pneumoniae
			6486	KPN	Klebsiella pneumoniae

JMI isolate #	JMI biological coding	Organism 69->
			8789	KPN	Klebsiella pneumoniae
10705	KPN	Klebsiella pneumoniae
			11123	KPN	Klebsiella pneumoniae
28148	KPN	Klebsiella pneumoniae
			29432	KPN	Klebsiella pneumoniae
937	MCAT	Moraxella catarrhalis
			1290	MCAT	Moraxella catarrhalis
1830	MCAT	Moraxella catarrhalis
			1903	MCAT	Moraxella catarrhalis
4346	MCAT	Moraxella catarrhalis
			4880	MCAT	Moraxella catarrhalis
6241	MCAT	Moraxella catarrhalis
			6551	MCAT	Moraxella catarrhalis
7074	MCAT	Moraxella catarrhalis
			7259	MCAT	Moraxella catarrhalis
7544	MCAT	Moraxella catarrhalis
			8142	MCAT	Moraxella catarrhalis
8451	MCAT	Moraxella catarrhalis
			9246	MCAT	Moraxella catarrhalis
9996	MCAT	Moraxella catarrhalis
			12158	MCAT	Moraxella catarrhalis
13443	MCAT	Moraxella catarrhalis
			13692	MCAT	Moraxella catarrhalis
13817	MCAT	Moraxella catarrhalis
			14431	MCAT	Moraxella catarrhalis
14762	MCAT	Moraxella catarrhalis
			14842	MCAT	Moraxella catarrhalis
15361	MCAT	Moraxella catarrhalis
			15741	MCAT	Moraxella catarrhalis
17843	MCAT	Moraxella catarrhalis
			18639	MCAT	Moraxella catarrhalis
241	GC	Neisseria gonorrhoeae
			291	GC	Neisseria gonorrhoeae
293	GC	Neisseria gonorrhoeae
			344	GC	Neisseria gonorrhoeae
451	GC	Neisseria gonorrhoeae
			474	GC	Neisseria gonorrhoeae

JMI isolate #	JMI biological coding	Organism 70->
			491	GC	Neisseria gonorrhoeae
493	GC	Neisseria gonorrhoeae
			503	GC	Neisseria gonorrhoeae
521	GC	Neisseria gonorrhoeae
			552	GC	Neisseria gonorrhoeae
573	GC	Neisseria gonorrhoeae
			592	GC	Neisseria gonorrhoeae
25	NM	Neisseria meningitidis
			813	NM	Neisseria meningitidis
1725	NM	Neisseria meningitidis
			2747	NM	Neisseria meningitidis
3201	NM	Neisseria meningitidis
			3335	NM	Neisseria meningitidis
7053	NM	Neisseria meningitidis
			9407	NM	Neisseria meningitidis
10447	NM	Neisseria meningitidis
			12685	NM	Neisseria meningitidis
12841	NM	Neisseria meningitidis
			14038	NM	Neisseria meningitidis
1127	PM	Proteus mirabilis
			3049	PM	Proteus mirabilis
4471	PM	Proteus mirabilis
			8793	PM	Proteus mirabilis
10702	PM	Proteus mirabilis
			11218	PM	Proteus mirabilis
14662	PM	Proteus mirabilis
			17072	PM	Proteus mirabilis
19059	PM	Proteus mirabilis
			23367	PM	Proteus mirabilis
29819	PM	Proteus mirabilis
			31419	PM	Proteus mirabilis
1881	PSA	Pseudomonas aeruginosa
			5061	PSA	Pseudomonas aeruginosa
7909	PSA	Pseudomonas aeruginosa
			8713	PSA	Pseudomonas aeruginosa
14318	PSA	Pseudomonas aeruginosa
			14772	PSA	Pseudomonas aeruginosa

JMI isolate #	JMI biological coding	71->
			15512	PSA	Pseudomonas aeruginosa
17093	PSA	Pseudomonas aeruginosa
			17802	PSA	Pseudomonas aeruginosa
19661	PSA	Pseudomonas aeruginosa
			29967	PSA	Pseudomonas aeruginosa
31539	PSA	Pseudomonas aeruginosa
			82	SA	Staphylococcus aureus
99	SA	Staphylococcus aureus
			138	SA	Staphylococcus aureus
139	SA	Staphylococcus aureus
			140	SA	Staphylococcus aureus
141	SA	Staphylococcus aureus
			142	SA	Staphylococcus aureus
272	SA	Staphylococcus aureus
			287	SA	Staphylococcus aureus
354	SA	Staphylococcus aureus
			382	SA	Staphylococcus aureus
1112	SA	Staphylococcus aureus
			1687	SA	Staphylococcus aureus
1848	SA	Staphylococcus aureus
			2031	SA	Staphylococcus aureus
2159	SA	Staphylococcus aureus
			2645	SA	Staphylococcus aureus
3256	SA	Staphylococcus aureus
			3276	SA	Staphylococcus aureus
4044	SA	Staphylococcus aureus
			4214	SA	Staphylococcus aureus
4217	SA	Staphylococcus aureus
			4220	SA	Staphylococcus aureus
4231	SA	Staphylococcus aureus
			4240	SA	Staphylococcus aureus
4262	SA	Staphylococcus aureus
			4370	SA	Staphylococcus aureus
4665	SA	Staphylococcus aureus
			4666	SA	Staphylococcus aureus
4667	SA	Staphylococcus aureus
			5026	SA	Staphylococcus aureus

JMI isolate #	JMI biological coding	72->
			5666	SA	Staphylococcus aureus
6792	SA	Staphylococcus aureus
			7023	SA	Staphylococcus aureus
7461	SA	Staphylococcus aureus
			7899	SA	Staphylococcus aureus
7901	SA	Staphylococcus aureus
			8714	SA	Staphylococcus aureus
9374	SA	Staphylococcus aureus
			9437	SA	Staphylococcus aureus
10056	SA	Staphylococcus aureus
			10110	SA	Staphylococcus aureus
11379	SA	Staphylococcus aureus
			11629	SA	Staphylococcus aureus
11659	SA	Staphylococcus aureus
			12788	SA	Staphylococcus aureus
12789	SA	Staphylococcus aureus
			13043	SA	Staphylococcus aureus
13086	SA	Staphylococcus aureus
			13721	SA	Staphylococcus aureus
13742	SA	Staphylococcus aureus
			13932	SA	Staphylococcus aureus
14210	SA	Staphylococcus aureus
			14384	SA	Staphylococcus aureus
15428	SA	Staphylococcus aureus
			15430	SA	Staphylococcus aureus
17721	SA	Staphylococcus aureus
			18688	SA	Staphylococcus aureus
19095	SA	Staphylococcus aureus
			20195	SA	Staphylococcus aureus
22141	SA	Staphylococcus aureus
			22689	SA	Staphylococcus aureus
27398	SA	Staphylococcus aureus
			29048	SA	Staphylococcus aureus
29051	SA	Staphylococcus aureus
			30491	SA	Staphylococcus aureus
30538	SA	Staphylococcus aureus
			25	SEPI	Staphylococcus epidermidis

JMI isolate #	JMI biological coding	Organism 73->
			53	SEPI	Staphylococcus epidermidis
385	SEPI	Staphylococcus epidermidis
			398	SEPI	Staphylococcus epidermidis
701	SEPI	Staphylococcus epidermidis
			713	SEPI	Staphylococcus epidermidis
1381	SEPI	Staphylococcus epidermidis
			2174	SEPI	Staphylococcus epidermidis
2286	SEPI	Staphylococcus epidermidis
			2969	SEPI	Staphylococcus epidermidis
3417	SEPI	Staphylococcus epidermidis
			3447	SEPI	Staphylococcus epidermidis
4753	SEPI	Staphylococcus epidermidis
			7241	SEPI	Staphylococcus epidermidis
9366	SEPI	Staphylococcus epidermidis
			10665	SEPI	Staphylococcus epidermidis
11792	SEPI	Staphylococcus epidermidis
			12311	SEPI	Staphylococcus epidermidis
13036	SEPI	Staphylococcus epidermidis
			13227	SEPI	Staphylococcus epidermidis
13243	SEPI	Staphylococcus epidermidis
			13621	SEPI	Staphylococcus epidermidis
13638	SEPI	Staphylococcus epidermidis
			13800	SEPI	Staphylococcus epidermidis
14078	SEPI	Staphylococcus epidermidis
			14392	SEPI	Staphylococcus epidermidis
15007	SEPI	Staphylococcus epidermidis
			16733	SEPI	Staphylococcus epidermidis
18871	SEPI	Staphylococcus epidermidis
			23285	SEPI	Staphylococcus epidermidis
27805	SEPI	Staphylococcus epidermidis
			29679	SEPI	Staphylococcus epidermidis
29985	SEPI	Staphylococcus epidermidis
			30259	SEPI	Staphylococcus epidermidis
31444	SEPI	Staphylococcus epidermidis
			268	SPN	Streptococcus pneumoniae
1264	SPN	Streptococcus pneumoniae
			2482	SPN	Streptococcus pneumoniae

JMI isolate #	JMI biological coding	74->
			2653	SPN	Pneumonia chain ballBacteria
2994	SPN	Streptococcus pneumoniae
			3123	SPN	Streptococcus pneumoniae
3124	SPN	Streptococcus pneumoniae
			4336	SPN	Streptococcus pneumoniae
4858	SPN	Streptococcus pneumoniae
			5606	SPN	Streptococcus pneumoniae
5881	SPN	Streptococcus pneumoniae
			5897	SPN	Streptococcus pneumoniae
5900	SPN	Streptococcus pneumoniae
			6051	SPN	Streptococcus pneumoniae
6216	SPN	Streptococcus pneumoniae
			6556	SPN	Streptococcus pneumoniae
7270	SPN	Streptococcus pneumoniae
			7584	SPN	Streptococcus pneumoniae
8479	SPN	Streptococcus pneumoniae
			8501	SPN	Streptococcus pneumoniae
9256	SPN	Streptococcus pneumoniae
			9257	SPN	Streptococcus pneumoniae
10246	SPN	Streptococcus pneumoniae
			10467	SPN	Streptococcus pneumoniae
10886	SPN	Streptococcus pneumoniae
			11217	SPN	Streptococcus pneumoniae
11228	SPN	Streptococcus pneumoniae
			11238	SPN	Streptococcus pneumoniae
11757	SPN	Streptococcus pneumoniae
			11768	SPN	Streptococcus pneumoniae
12121	SPN	Streptococcus pneumoniae
			12124	SPN	Streptococcus pneumoniae
12149	SPN	Streptococcus pneumoniae
			12767	SPN	Streptococcus pneumoniae
12988	SPN	Streptococcus pneumoniae
			13321	SPN	Streptococcus pneumoniae
13393	SPN	Streptococcus pneumoniae
			13521	SPN	Streptococcus pneumoniae
13544	SPN	Streptococcus pneumoniae
			13700	SPN	Streptococcus pneumoniae

JMI isolate #	JMI biological coding	75->
			13704	SPN	Streptococcus pneumoniae
13822	SPN	Streptococcus pneumoniae
			13838	SPN	Streptococcus pneumoniae
14131	SPN	Streptococcus pneumoniae
			14413	SPN	Streptococcus pneumoniae
14744	SPN	Streptococcus pneumoniae
			14808	SPN	Streptococcus pneumoniae
14827	SPN	Streptococcus pneumoniae
			14835	SPN	Streptococcus pneumoniae
14836	SPN	Streptococcus pneumoniae
			15832	SPN	Streptococcus pneumoniae
17336	SPN	Streptococcus pneumoniae
			17343	SPN	Streptococcus pneumoniae
17349	SPN	Streptococcus pneumoniae
			17735	SPN	Streptococcus pneumoniae
18060	SPN	Streptococcus pneumoniae
			18567	SPN	Streptococcus pneumoniae
18595	SPN	Streptococcus pneumoniae
			19082	SPN	Streptococcus pneumoniae
19826	SPN	Streptococcus pneumoniae
			22174	SPN	Streptococcus pneumoniae
22175	SPN	Streptococcus pneumoniae
			27003	SPN	Streptococcus pneumoniae
28310	SPN	Streptococcus pneumoniae
			28312	SPN	Streptococcus pneumoniae
29890	SPN	Streptococcus pneumoniae
			29910	SPN	Streptococcus pneumoniae

Example 32

Mouse staphylococcus aureus kidney infection model

Animals: female CD-1 mice (8-10 weeks old; 6/group) were obtained from Charles river laboratories and were maintained and maintained according to the guidelines for care and use of laboratory animals (GuidetotheCareardrendsUseof Experimental animals).

Bacterial strains and stocks

Methicillin-susceptible staphylococcus aureus (MSSA) strain ATCC29213 was obtained from the american type culture center. To prepare stocks for animal studies, staphylococcus aureus was plated on mueller hinton agar plates and incubated overnight at 37 ℃. 3-4 colonies from the plate were used to inoculate 5mM MulGluere HintonBroth (MHB) with incubation at 37 ℃ for 8 hours with shaking at 300 RPM. 5mL of 8h culture was 20-fold diluted into 100mL and incubated overnight (12-14 h). The bacteria were pelleted at 3000x for 20 minutes and washed twice with Phosphate Buffered Saline (PBS) containing 0.5% Bovine Serum Albumin (BSA). Will contain 1x10 in PBS/20% glycerol¹⁰Aliquots of cfu (1 mL) were frozen and stored at-80 ℃ until the day of use. Stock titers were confirmed by serial dilution and plating on mueller hinton agar plates.

Mouse staphylococcus aureus kidney infection model

Prior to inoculation, bacterial stocks were thawed and washed once with PBS/BSA. The stock was then diluted to 1X10 with PBS/BSA⁹Final concentration of cfu/mL to give about 1-2X10 in 100uL volume⁸cfu/inoculum of mice. This inoculum was found to be optimal in preliminary experiments, saidPreliminary experiment using 10⁶–10⁹Challenge dose of S.aureus organisms/mouse to establish-10 hours after infection⁶cfu/kidney load without mortality (data not shown). In these preliminary studies, the amount of water used was less than 10⁷challenge with Staphylococcus aureus in cfu/mouse induced incompatible infection or no chronic infection, 10⁹The dose of cfu/mouse resulted in rapid disease and mortality in a high percentage of mice. Injections were given intravenously via the tail vein using a sterile 30 gauge needle. After completion of infection of the mice, bacterial stocks used to challenge the mice were plated and counted to verify the inoculum concentration.

To evaluate bacterial load at the indicated time post-infection (most commonly 2-26 hours), mice were euthanized and kidneys were aseptically harvested and placed in sterile PBS/BSA (5 mL/pair of kidneys). The kidneys were homogenized aseptically using a hand-held homogenizer (Powergen 125; Fisher scientific). During collection and homogenization, all samples were kept on ice, and the homogenizer was thoroughly washed and sterilized between each sample. The homogenates were serially diluted in sterile PBS/BSA and plated on MH agar to determine bacterial counts/pair kidneys.

CD-1 mice (6/group) were treated with Staphylococcus aureus (ATCC 29213) at 2X10⁸cfu/mouse IV challenge. Two hours after challenge, mice were treated with 10ml/kg vehicle (10% VitETPGS) or with 10, 30, 100mg/kg of compound 23A via oral gavage. The 30 min and 6 hour treatment groups were treated once, while the 24 hour group received a second treatment 10 hours after the first dose. After an increased amount of time post-treatment (30 minutes, 6 hours or 24 hours), mice were euthanized and the kidneys were harvested, homogenized and plated to quantify staphylococcus aureus load. The load of the kidney pairs from each mouse and the median for each group of mice were calculated.

As a result: compound 23A administered orally showed in vivo efficacy against experimentally induced infection of renal MSSA (SA 29213). There was no difference in renal load between compound and vehicle treated mice at 30 minutes after the initial treatment. The 30 min vehicle-treated group served as an early control for comparing the effect of the compounds at later time points. All compound treatments reduced bacterial load in the kidneys by 0.4-0.6 log compared to time matched vehicle controls 6 hours after the first dose. In addition, compound 23A administered at 30 and 100mg/kg provided a 0.3-0.4 log reduction compared to the 30 minute early control.

After a 24-hour treatment period (which included the second treatment dose administered at 10 hours), a decrease in bacterial density compared to the time-matched vehicle control was observed for all treatment groups. Compound 23A administered at 30 and 100mg/kg bid provided a 2.8-2.9 log reduction, while compound 23ABID at 10mg/kg was more variable and provided a 1.8 log reduction compared to the 24 hour vehicle treated control. In addition, compound 23A administered at 30 and 100mg/kg showed a 1.5 log reduction compared to the 30 min early control, indicating bacterial activity.

Total sum as shown in table 6 above, at 6 hours and 24 hours evaluation time, BID administration of 30 and 100mg/kg compound 23A reduced bacterial growth of methicillin-sensitive staphylococcus aureus (MSSA) strain ATCC29213 compared to the 30 minute control group, whereas treatment with 10mg/kg compound 23A restricted bacterial growth to 6 hours, but was less effective than the other treatment groups at 24 hours.

CD-1 mice (6/group) were treated with Staphylococcus aureus (ATCC 29213) at 2X10⁸cfu/mouse IV challenge. After 2 hours, a single group of mice (early control (EC)) was euthanized and the kidneys were harvested, homogenized and plated out to quantifyStaphylococcus aureus load. Additional groups of infected mice were treated with 10, 30, 60, 100mg/kg of compound 23A via oral gavage with 10ml/kg of vehicle (10% VitETPGS; late stage control, LC). After 24 hours, treatment groups of mice were euthanized and kidneys harvested, homogenized and plated to quantify staphylococcus aureus load. The load of the kidney pairs from each mouse and the median for each group of mice were summarized.

As a result: total sum as shown in table 7 above, a single oral dose of compound 23A showed in vivo efficacy against experimentally induced infection of renal MSSA (SA 29213). After 24 hours, all treatments showed a decrease in bacterial density compared to the time-matched vehicle control. Compound 23A demonstrated a dose-dependent reduction of 1.9, 2.3, 3.1, and 3.3 log reduction compared to vehicle controls when administered at 10, 30, 60, or 100 mg/kg. In addition, doses of 60 and 100mg/kg of compound 23A reduced the bacterial load by 1.2-1.4 log compared to the early control, suggesting that compound 23A has bacterial activity.

CD-1 mice (8/group) were treated with Staphylococcus aureus (ATCC 29213) at 2X10⁸cfu/mouse IV challenge. After 2 hours, a single group of mice (early control (EC)) was euthanized and the kidneys were harvested, homogenized and plated to quantify the staphylococcus aureus load. Additional groups of infected mice were treated with 10ml/kg of vehicle (water; late control, LC) via oral gavage, compound W was administered at a nominal dose level of 16, 49, 99 or 166mg/kg, which is expected to deliver 10, 30, 60, 100mg/kg of compound 23A, at an active partial dose equivalent of 10, 30, 60, 100mg/kg after complete conversion. After 24 hours, treatment groups of mice were euthanized and kidneys harvested, homogenized and plated to quantify staphylococcus aureus load. The load of the kidney pairs from each mouse and the median for each group of mice were summarized.

As a result: total sum as shown in table 8 above, a single oral dose of compound W showed in vivo efficacy against experimentally induced infection of renal MSSA (SA 29213). After 24 hours, all treatments showed a decrease in bacterial density compared to the time-matched vehicle control. When administered at 16, 49, 99, and 166mg/kg (which provide equivalent exposure of 10, 30, 60, or 100mg/kg compound 24), compound W demonstrated 1.9, 2.3, 2.7, 2.6, and 3.0 log reduction in dose-dependent reduction compared to vehicle control. In addition, doses of 16, 49, 99, and 166mg/kg compound W reduced the bacterial load by 0.7-1.5 log compared to the early control, suggesting that compound 23A had bacterial activity.

Example 33

Neutropenia rat thigh infection model

Animals: specific pyrogen-free, male sprague dawley rats between 76-100 grams were obtained from charles river laboratories, inc. (Wilmington, MA) and used in this experiment. Animals were allowed a minimum of seven (7) days of acclimation before study initiation.

Bacteria: methicillin-susceptible staphylococcus aureus (MSSA) ATCC strain 29213 was used for in vivo experiments. Test isolates were subcultured twice onto standard microbial agar medium (trypticase soy agar with 5% sheep blood). The second transfer was performed less than 24 hours prior to use in the preparation of the thigh infection model inoculum.

Neutropenia rat thigh infection model: to induce neutropenia, three days prior to infection, rats were treated with the immunosuppressive cyclophosphamide at 150mg/kg administered in 1ml by intraperitoneal Injection (IP). 10 in physiological saline⁷A suspension of cfu/methicillin-sensitive Staphylococcus aureus 29213 was injected Intramuscularly (IM) 0.2ml into two post-thigh infected rats. At an increased amount of timeAfterwards (2-26 hours), two hind thighs of each animal were harvested, rinsed with sterile saline, weighed, then placed in 50ml sterile normal saline and placed on wet ice until homogenization. Approximately half of the total homogenized sample volume was passed through a macroporous filter (to remove cartilage and large clumped pieces of tissue), diluted in saline and cultured on agar medium plates (trypticase soy agar with 5% sheep blood). All plates were incubated at about 37 ℃ for 18-24 hours. Colony forming units (in cfu/ml homogenates) were counted, and the median for each treatment and control group was counted. Typically, each group has n = 6; each thigh is considered a discrete number. The median cfu/ml/group was compared to that of the vehicle control group (late control; LC) matched at the initial bacterial density of 2 hours (early control) or time of simultaneous harvest.

TABLE 8 Compound 23A demonstrates dose-related and time-dependent reduction in thigh burden of Staphylococcus aureus in neutropenic rats

Chinese character shao (a Chinese character of 'shao')

Neutropenia rats (3/group) were treated with staphylococcus aureus (ATCC 29213) at 2x10⁶cfu/thigh infections were performed by Intramuscular (IM) challenge. After 2 hours, a single group of rats (early control (EC) was euthanized and thighs harvested, homogenized and plated to quantify staphylococcus aureus load. additional infected rats were treated via oral gavage with 10ml/kg of vehicle (20% Cavitron/1% HPMCAS-MG; late control, LC) or with 10, 30, 60MG/kg of compound 23A. the 8 hour treatment group received a single treatment (QD) and euthanized, and thighs harvested 8 hours after treatment (QD) for cfu assay, while the 24 hour treatment group received 2 doses 12 hours apart (q 12 h) and euthanized, and thighs harvested 24 hours after treatmentLoad per individual thigh and summarize cfu/ml and median values from the 3 rat group.

As a result: as shown in table 8 above, compound 23A administered orally showed in vivo efficacy against MSSA (SA 29213). All groups had a reduction in load compared to time matched controls at 8 hours after the first dose, a 1.3 log reduction for compound 23A at 10mg/kg, and a2 log reduction for compound 23A at 30 and 60 mg/kg. Bacterial growth of SA29213 was maintained to at least the point of stagnation with 10mg/kg of compound 23A, while bacterial loads were slightly reduced by-0.5-0.6 log with 60 and 100mg/kg of compound 23A, compared to the early control.

After the second treatment dose, administered at 24 hours and at 12 hours, a 2.4-2.8 log reduction in bacterial density compared to the late control was observed for all treatment groups. About a 0.8 log reduction was observed for compound 23A at 10mg/kg compared to the early control, while compound 23A at 30 and 60mg/kg provided-1.1-1.2 log reductions.

Neutropenia rats (3/group) were treated with staphylococcus aureus (ATCC 29213) at 2x10⁶cfu/thigh infections were performed by Intramuscular (IM) challenge. After 2 hours, a single group of rats (early control (EC)) was euthanized and the thighs were harvested, homogenized and plated to quantify the staphylococcus aureus load. Additional infected rats were treated with 10ml/kg vehicle (10% vitamin E/TPGS; late stage control, LC) or with 30, 60, 100mg/kg of Compound 13 via oral gavage. The 8 hour treatment group received a single treatment (QD) and was euthanized, and the thighs were harvested 8 hours after treatment (QD) for cfu determination, while the 24 hour treatment group received 2 doses 12 hours apart (q 12 h) and was euthanized, and the thighs were harvested 24 hours after treatment. Measuring the load from each individual thighAnd cfu/individual thigh and median values from the 3 rat group were summarized for each group.

As a result: as shown in table 9 above, compound 13 administered orally showed in vivo efficacy against MSSA (SA 29213). Differences in the degree of antimicrobial activity were seen between the three treatment groups. All groups had a reduction in load compared to the time matched controls at 8 hours after the first dose, a 1.5 log reduction for compound 13 at 10mg/kg, and 1.7 and 1.8 log reductions for compound 13 at 60 and 100 mg/kg. Bacterial growth of SA29213 was maintained to at least the stagnation point 8 hours after the first administration with 10mg/kg of compound 13, while compound 13 at 60 and 100mg/kg slightly reduced the bacterial load by-0.4 and-0.5 log, respectively, compared to the earlier control. After the second treatment dose, administered at 24 hours and at 12 hours, an approximately 1 log reduction in bacterial density compared to the early control was shown by compound 13 at 60 and 100 mg/kg. In contrast, compound 13 administered at 30mg/kg did not appear to be effective, with variable cfu levels averaging 0.3 log greater than the early control. However, all dose levels reduced bacterial density compared to the late control. About-1.1 log reduction was observed for the 10mg/kg treatment group, while 2.85 and-3 log reductions were observed for the 60 and 100mg/kg dose levels.

In summary, at the 8 hour and 24 hour evaluation time, the q12h doses of 60 and 100mg/kg compound 13 reduced bacterial growth of SA29213 compared to the initial control group, while treatment with 30mg/kg limited bacterial growth at 8 hours, but was less effective than the other treatment groups at 24 hours.

Example 34

Seven day oral (gavage) toxicity and toxicology in rats

The purpose of this study was: 1) evaluation of potential toxicity of compound 13 and compound 23A when administered orally to male rats by gavage for 7 consecutive days, and 2) evaluation of the toxicology kinetics of compound 13 and compound 23A after the first and seventh doses.

Animal(s) production

Species, origin, history and demonstration

CD (SD) rats from Charles river laboratory SofStoneRidge, NY. Animals were laboratory bred and experimentally first used for experimentation. Rats were chosen because they are the species commonly used for non-clinical toxicity assessment.

Number, sex, age and weight ranges

Forty rats were ordered (20 uncased males and 20 males with jugular vein cannulation). From these animals, 15 non-cannulated males and 15 cannulated males were used). The animals were as consistent as possible in age. Rats were pre-pubertal to young adults, about 9 weeks of age at the beginning of dosing. Their supplier calculated birth dates were kept in the study records. The weight of the animals at the time of distribution to the groups ranged from 218.5-306.3 g.

Design of research

Rats were designated as shown in table 10 below. Animals received the test article or vehicle by oral gavage for 7 consecutive days and terminated the day after dosing was complete. The first day of dosing was designated study day 1. Animals were assessed for changes in clinical signs, body weight, and other parameters as described below.

TABLE 10 group assignment and dosage levels

Route/dose

For groups a and B-D, the vehicle and test article were administered once daily for 7 consecutive days by oral gavage at a dose volume of 10mL/kg body weight, respectively. For groups E and F, the test article and vehicle were administered twice daily by oral gavage at a dose volume of 10mL/kg body weight, approximately 8 hours apart, for 7 consecutive days, respectively. The actual volume administered to each animal was calculated and adjusted based on the most recent weight of each animal.

Life (In-Life) observation and measurement

Observation of

Throughout the study, animals were observed for viability at least once in the morning and once in the middle of the day, separated by at least 4 hours. During the treatment period, daily cage-side observations were made and recorded before and after dosing (only after the first dose). Based on C for two compounds from a previous study_max/T_maxPost-dose observations were made during the treatments that occurred at the following times:

1 hour after dosing for groups A-F.

One cage-side observation was made at necropsy on the day.

Unscheduled observations

Recording any findings observed at times other than the scheduled observation time, either on an unscheduled observation or in the proventis; however, no abnormalities were observed throughout the study. Proventis is an electronic data collection, management and reporting system commonly used in the art.

Body weight

Body weight was measured for randomization on day 1 before dosing began. During the treatment, body weights were measured on days 1 and 7. In addition, fasting body weight was measured prior to necropsy for calculation of organ/body weight ratio.

Food consumption

Throughout the study, food consumption was measured daily starting 3 days before dosing began.

Clinical pathology assessment

At necropsyAnterior suborbital plexus (in CO)₂/O₂Blood samples were collected from all animals under anesthesia, for the main study animals) or jugular vein cannulation (for the toxicological kinetic animals) for assessment of hematological, coagulation and serum chemistry parameters. Coagulation samples from these rats cannot be analyzed due to residual heparin used to keep the cannula open to toxicological kinetic animals. Animals were fasted overnight prior to blood collection. On day of blood collection for clinical pathology analysis, animals were not necropsied until after blood collection and samples judged acceptable by the clinical pathology group.

Hematology

Appropriate amounts of blood were collected into EDTA-containing tubes. The whole blood samples were analyzed for the parameters shown in table 11 below.

TABLE 11 Whole blood parameters

Coagulation

Appropriate amounts of blood were collected into tubes containing sodium citrate and subsequently centrifuged to obtain plasma for determination of Prothrombin Time (PT) and partially activated prothrombin time (APTT).

Serum chemistry

Appropriate amounts of blood were collected into tubes without anticoagulant. The sample was allowed to coagulate and then centrifuged to obtain serum. The sera were analyzed for the parameters shown in table 12 below.

TABLE 12 serum parameters

Toxicological kinetics

Blood samples (approximately 0.5 mL/sample) were collected from jugular vein cannulas to contain K on days 1 and 7 of administration for all toxicological kinetic animals at the time points listed below₃In an EDTA tube. Toxicological kinetic animals from the control group (group F) had only a single blood collection sample from each collection day at the 1 hour time point (after the first dose administration on the day). Before each collection, a small blood sample (with heparin lock solution) was removed from the cannula and discarded. A new syringe was placed on the cannula and the protocol required samples were obtained. The syringe with the blood sample was removed and a new syringe with saline was attached to the cannula. The blood volume was replaced with an equal volume of saline and then the blocking solution was placed in the cannula. Each animal was returned to its cage until the next collection time point.

All samples collected during this study were placed in labeled containers. Each tag contains the following information: 1) study number, 2) animal number, 3) collection interval, 4) group and gender, and 5) collection date.

Blood samples were mixed immediately by inversion, then placed on wet ice and cold centrifuged (-1500 g, -10 min, -5 ℃) to obtain plasma. Plasma was aliquoted as 2 aliquots into 96-well 1.4-mL polypropylene tubes with a penetrable TPEcapcluster-tested RNase, no DNase cap and stored frozen (. ltoreq. -70 ℃ C.).

TABLE-13 sample Collection time points

Point in time	Window (Refreshment window)1
		Before administration	After administration
1 hour	4 minutes
		2 hours2	5 minutes
4 hours	5 minutes
		8 hours3	5 minutes
12 hours	10 minutes
		24 hours	Plus or minus 20 minutes
48 hours4	Plus or minus 40 minutes

¹All samples were collected in the collection window.

²Only after the administration on day 1.

³Groups E and F were obtained before PM dose administration.

⁴Only after the 7 th day dosing.

Terminate

No animals were considered moribund during the study. All study animals were euthanized and necropsied after the treatment days specified in the protocol. All animals were bled (in depth CO)₂/O₂Anesthesia undercuttingSevered aorta) is terminated.

Autopsy

All animals terminated during the study were necropsied and histologically collected. Necropsy included the following examinations:

cadaver and muscle/bone systems; all external surfaces and orifices;

cranial cavity and outer surface of the brain;

a neck with associated organs and tissues; and

thoracic, abdominal and pelvic cavities with their associated organs and tissues.

All exceptions are described and recorded.

Organ weight

Kidneys, liver and prostate were weighed for all animals euthanized at scheduled necropsy. After weighing, approximately 1 gram of liver and kidney samples were weighed, transferred to a Precellys7mLCK28 tissue homogenization tube (catalog No. 0904-01), snap frozen and analyzed.

Organ/body ratios were calculated using the terminal fasting body weights obtained prior to necropsy.

Tissue preservation and bone marrow harvesting

Tissues and organs shown in table 14 below were collected from all animals and preserved in 10% neutral-buffered formalin, except testis, epididymis, and eye. Testis, epididymis and eyes with optic nerve attachments were fixed in modified Davidson's solution for-24-48 hours, rinsed with water, and then transferred to 10% neutral buffered formalin for storage.

TABLE 14 tissue Collection

^aThe thyroid gland was weighed together with the attached parathyroid gland.

Histopathology

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

Statistical analysis

Where appropriate, numerous animal data were statistically evaluated.

For comparative statistics, group a (control group) was compared to groups B and C (treatment group, QD dosing) and group F (control group, BID dosing) was compared to group E (treatment group, BID dosing). The data were evaluated for homogeneity of variance using the Leven test and for normality distribution using the Shapiro-Wilks test, with significance at p ≦ 0.05. Data determined to be uniform and normally distributed were evaluated by analysis of variance (ANOVA). If ANOVA verifies significance at p.ltoreq.0.05, a pair-wise comparison of each treatment group with the respective control group is made using the parametric test (Dunnett test) to identify statistical differences (p.ltoreq.0.05). Data determined to be non-uniform or non-normal distributions were evaluated for population factor significance using the Kruskal-Wallis test. If there is significance between the groups (p.ltoreq.0.05), a nonparametric test (Wilcoxon with Bonferroni-Holm) is used to compare the treated groups with the control group. Food consumption data from animals that underwent spillover were excluded from the applicable time period. The comparative statistics of food consumption data are limited to Dunnett's test (parameters). Statistics were not performed on pre-test food consumption (day 4 to day 1).

Results

Exposure to different dose levels for compound 23A and compound 13 was dose-related. No adverse observation or effect on average body weight was observed in animals treated with compound 13 or compound 23A. For animals treated once daily with compound 13 (100 or 200 mg/kg) and twice daily with compound 23A (300 mg/kg), average food consumption was reduced during the different intervals of the study. However, these effects were not considered adverse or biologically significant because the reduced food consumption was not associated with body weight changes in the compound 13 and compound 23A groups. The mean calcium ion Concentration (CA) was statistically lower when compared to the twice daily treated control, while the mean ALT and AST were statistically higher for the group of rats administered 300mg/kg compound 23A twice daily. For animals receiving compound 13 or compound 23A at any dosage regimen, histopathological findings associated with the test article were not noted.

Within the scope of this study and based on the absence of changes in body weight, clinical pathology and histopathology, the NOEL (no observed level of effect) for compound 13 administered orally via gavage to male rats once daily for 7 days was 200mg/kg (844 μ g hr/ml day7 AUC) while the NOEL for compound 23A administered once daily was 100mg/kg (82 μ g hr/ml AUC). NOAEL (no observed level of effect) for compound 23A administered orally to male rats twice daily via gavage for 7 days was 300mg/kg (291 μ g hr/ml auc).

Thus, compounds 13 and 23A did not demonstrate adverse toxicity in the study range at dose levels up to 200 mg/kg/day and 600 mg/kg/day, respectively.

Example 35

Oral range finding toxicity and toxicological kinetics studies in male cynomolgus monkeys

The purpose of this study was: 1) evaluation of potential toxicity of compound 23 when administered orally to male cynomolgus monkeys by gavage for 7 consecutive days, and 2) evaluation of the toxicology kinetics of compound 23 after the first and seventh dosing.

Animal(s) production

Species, origin, history and demonstration

Cynomolgus monkey (macaca fascicularis) was obtained from primus bio-resources inc. of pincourt, Quebec, canada. Cynomolgus monkeys were chosen because they are a non-rodent species commonly used for non-clinical toxicity assessment.

Number, sex, age and weight ranges

Eight (2 naive and 6 non-naive) males were used in this study. The animals were young adults and weighed between 2-4 kg before dosing began.

Design of research

Animals were designated as shown in table 15 below. Animals received compound 23 or vehicle once daily for 7 consecutive days by oral gavage and terminated the day after dosing was complete. The first day of dosing was designated study day 1. The actual volume administered to each animal was calculated and adjusted based on the most recent weight of each animal.

TABLE 15 group assignment and dosage levels

^*Control animals received control/vehicle alone (20% captisol/1% HPMCAS/1% PVP in 0.01MKCl/HCL buffer)

Life observation and measurement

Observation of

Clinical signs (unhealthy, behavioral changes, etc.) were recorded at the cage at least once daily during the study.

Body weight

Body weights were recorded for all animals before group assignment and at days 1 (before dosing), 3 and 7 and finally before necropsy (fasting).

Electrocardiography (ECG)

Electrocardiograms (bipolar limb leads I, II and III, and enhanced unipolar leads aVR, aVL, and aVF) were obtained again for all monkeys once during the pretreatment period and at day7 (post-dose).

Tracing is evaluated for macroscopic changes indicative of cardiac electrical dysfunction. The potential presence of abnormalities involving heart rate (lead II), sinus and atrioventricular rhythm or conduction is determined. Heart rate, PR interval, QRS duration, QT and QTc interval values were measured.

Toxicological kinetics

A series of 7 blood samples (about 0.5mL each) were collected from each monkey on days 1 and 7 at the following time points: pre-dose, 30 minutes and 2,3, 6, 12 and 24 hours post-dose. For this purpose, each monkey bleeds by venipuncture and the sample is collected into a tube containing the anticoagulant K2 EDTA. The tubes were placed on wet ice until ready for processing.

Clinical pathology

Laboratory examinations (hematology, coagulation, clinical chemistry and urinalysis) were performed on all animals before treatment began and before termination at day 8.

Blood samples were collected by venipuncture after an overnight period of food deprivation by at least 12 hours but not more than 20 hours. Urine was collected from animals deprived of food and water overnight (at least 12 hours but not 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, cell morphology, white blood cell count, platelet count, white blood cell classification (absolute), reticulocyte (absolute and percent), and mean corpuscular hemoglobin concentration (calculated).

Coagulation

The partially activated prothrombin time and prothrombin time are measured on a blood sample collected into a citrate anticoagulant.

Clinical chemistry

The following parameters were measured on a blood sample collected in a tube containing clotting activator: a/g ratio (calculated), creatinine, alanine aminotransferase, globulin (calculated), albumin, glucose, alkaline phosphatase, phosphorus (inorganic), aspartate aminotransferase, potassium, bilirubin (total), sodium, calcium, total protein, chloride, triglycerides, cholesterol (total), urea, gamma glutamyltransferase and sorbitol dehydrogenase.

Urine analysis

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

Terminate

All animals were euthanized after completion of the treatment period at day 8 after the overnight period without food. Monkeys were pre-anesthetized with ketamine and then euthanized by intravenous overdose of sodium pentobarbital, followed by exsanguination by transection of the major vessels.

Autopsy

All animals terminated during the study were necropsied and histologically collected. Necropsy included the following examinations:

cadaver and muscle/bone systems;

all external surfaces and orifices;

cranial cavity and outer surface of the brain;

a neck with associated organs and tissues; and

thoracic, abdominal and pelvic cavities with their associated organs and tissues.

All exceptions are described and recorded.

Tissue preservation

After visual inspection and weighing of the selected organs was complete, the tissues and organs were retained as shown in table 16 below. Neutral buffered 10% formalin was used for fixation and preservation unless otherwise indicated.

TABLE 16 tissue and organ Retention

Histopathology

For all animals, all tissues indicated above were embedded in paraffin, sectioned and stained with hematoxylin and eosin for examination by light microscopy.

Results

Exposure to different dose levels for compound 23 was dose-related.

There were no clinical signs, or changes in body weight, electrocardiographic parameters, clinical pathology parameters, or organ weight, which could be attributed to administration of compound 23 at doses up to 200 mg/kg/day. Similarly, there was no macroscopic or microscopic findings that could be unambiguously ascribed to compound 23 administration at doses up to 200 mg/kg/day. The level of No Observed Effect (NOEL) for compound 23 in male cynomolgus monkeys was determined to be 200 mg/kg/day.

Example 36

Pharmacokinetic Studies

Pharmacokinetic parameters of selected compounds of the invention were determined in the experiments described below. General analytical procedures and specific experimental protocols were used as follows:

general analysis program

The following general analytical procedure was used in the pharmacokinetic experiments described below:

and (6) analyzing the sample. The concentration of compound 23 and compound W in plasma was determined using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS) method. Prior to extraction, plasma samples were diluted 2, 4,5 or 10 fold with blank plasma as needed depending on dose level or formulation. Compound 23 and compound W together with Internal Standard (IS), each 100 μ L, were extracted from (diluted) plasma by direct protein precipitation with acetonitrile (1: 4 ratio plasma/acetonitrile). After centrifugation, the supernatant extract (10 μ L) was injected into the LC/MS/MS system. The HPLC system included a waters xterramsc18 column, 5 microns, 2.1mm diameter x50mm length, eluted with a gradient mobile phase consisting of 0.1% formic acid in water or acetonitrile.

Analytes were detected by MS/MS in a Multiple Reaction Monitoring (MRM) mode with Atmospheric Pressure Chemical Ionization (APCI). The lower limit of quantitation (LLOQ) is 1, 2, 4,5, 10 or 20ng/mL depending on the sample dilution factor. The linear range of the assay is 1-5000 ng/mL. The day-to-day and day-to-day measurement accuracy was within 2% of the rated value. The intra-and inter-day assay variability was < 10%.

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

Pharmacokinetic data analysis. Use ofProfessional version software, version 5.1.1 (pharsight corporation, mountain view, CA), analyzed the plasma concentration time profiles of compound 23 and compound W by a non-compartmental pharmacokinetic method.

Determination of key pharmacokinetic parameters, including AUC_all、AUC_extrap、C_max、t_maxCl _ obs, Vss _ obs and t_1/2。

And (5) analyzing the statistical data. Descriptive statistics including mean, Standard Deviation (SD) and coefficient of variation (% CV) were calculated for plasma concentrations and estimates of pharmacokinetic parameters using WinNonlin software, version 5.1.1 or microsoft excel 2000.

Oral study of monkeys

Male cynomolgus monkeys (n = 3/dose group) were given 3, 30 and 300mg/kg of compound W at single nominal PO doses by gavage. Compound W was formulated in 0.5% MC (microcrystalline cellulose). Animals were free to eat and enter water before and after dosing.

Blood samples (approximately 0.25mL each) were collected via carotid cannulation before dosing and at 0 (pre-dosing), 0.25, 0.5, 1, 2,3, 4, 6, 8, 12, 24, 48 hours post-dosing. Each blood sample was collected into a tube, which was kept on wet ice and contained potassium EDTA as an anticoagulant. Plasma was separated and stored at-70 ℃ until analysis.

Plasma samples were analyzed using liquid chromatography/tandem mass spectrometry (LC/MS) methods to determine the concentration of compound 23 and compound W, depending on the sample dilution factor, with a lower limit of quantitation (LLOQ) of 1-20 ng/mL. Non-compartmental Pharmacokinetic (PK) analysis was performed on plasma concentrations versus time data. The results of this analysis are provided in table 17.

TABLE 17 pharmacokinetic data from monkey oral study

Monkey IV study

Male cynomolgus monkeys (n = 3/dose group) were administered a single nominal IV bolus dose of 1mg/kg of compound W via jugular vein cannulation. Compound W was formulated in D5W (5% dextrose in water). Animals were free to eat and enter water before and after dosing.

Blood samples (approximately 0.25mL each) were collected via carotid cannulae before dosing and at 0 (pre-dose), 5 minutes, 10 minutes, 0.25, 0.5, 1, 2,3, 4, 6, 8, 12, 24, 48 hours post-dose. Each blood sample was collected into a tube, which was kept on wet ice and contained potassium EDTA as an anticoagulant. Plasma was separated and stored at-70 ℃ until analysis.

Plasma samples were analyzed using liquid chromatography/tandem mass spectrometry (LC/MS) methods to determine the concentration of compound 23 and compound W, depending on the sample dilution factor, with a lower limit of quantitation (LLOQ) of 1-20 ng/mL. Non-compartmental Pharmacokinetic (PK) analysis was performed on plasma concentrations versus time data. The results of this analysis are provided in table 18.

TABLE 18 pharmacokinetic data from monkey IV study

Oral Studies in rats

A group of male sprague dawley rats (n =3 per dose group) was administered a single nominal oral dose of 3, 10, 30, 300mg/kg of compound W by gavage. Compound W was formulated in 0.5% MC (microcrystalline cellulose) or 20% Captisol, 1% HPMC-AS (hydroxypropylmethylcellulose acetate succinate), 1% PVP (polyvinylpyrrolidone). Animals were free to eat and enter water before and after dosing. Blood samples (approximately 0.25mL each) were collected via carotid cannulae before dosing and at 0 (pre-dosing), 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 hours after dosing. Each blood sample was collected into a tube, which was kept on wet ice and contained potassium EDTA as an anticoagulant. Plasma was separated and stored at-70 ℃ until analysis.

Plasma samples were analyzed using liquid chromatography/tandem mass spectrometry (LC/MS) methods to determine the concentration of compound 23 and compound W, depending on the sample dilution factor, with a lower limit of quantitation (LLOQ) of 1-20 ng/mL. Non-compartmental Pharmacokinetic (PK) analysis was performed on plasma concentrations versus time data. The results of this analysis are provided in table 19.

TABLE 19 pharmacokinetic data from rat oral studies

Rat IV study

A group of male sprague dawley rats (n =3 per dose group) were administered a single nominal IV bolus dose of 1 and 5mg/kg of compound W via jugular vein intubation. Compound W was formulated in D5W. Animals were free to eat and enter water before and after dosing. Blood samples (approximately 0.25mL each) were collected via carotid cannulae before dosing and at 0 (pre-dose), 5 minutes, 10 minutes, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 hours post-dose. Each blood sample was collected into a tube, which was kept on wet ice and contained potassium EDTA as an anticoagulant. Plasma was separated and stored at-70 ℃ until analysis.

Plasma samples were analyzed using liquid chromatography/tandem mass spectrometry (LC/MS) methods to determine the concentration of compound 23 and compound W, depending on the sample dilution factor, with a lower limit of quantitation (LLOQ) of 1-20 ng/mL. Non-compartmental Pharmacokinetic (PK) analysis was performed on plasma concentrations versus time data. The results of this analysis are provided in table 20. TABLE 20 pharmacokinetic data from rat IV study

Oral study in mice

A group of female CD-1 mice (n = 3/dose group) was administered a single nominal oral dose of 10, 30, 100mg/kg of compound W by gavage. Compound W was formulated in 0.5% MC. Animals were free to eat and enter water before and after dosing. Blood samples (about 0.025mL each) were collected from the mandibular veins prior to dosing and at 0 (pre-dosing), 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 hours post-dosing. Each blood sample was collected into a tube, which was kept on wet ice and contained potassium EDTA as an anticoagulant. Plasma was separated and stored at-70 ℃ until analysis.

Plasma samples were analyzed using a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method, with a lower limit of quantitation (LLOQ) of 1-20 ng/mL, depending on the sample dilution factor. Non-compartmental Pharmacokinetic (PK) analysis was performed on plasma concentrations versus time data. The results of this analysis are provided in table 21.

TABLE 21 pharmacokinetic data from mouse oral studies

The studies described above demonstrate that compound W is converted in vivo to compound 23 in at least rat, dog and monkey.

Example 37

Enzymology study

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

DNA gyrase ATPase assay

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

ATPase assay at 30 ℃ with 100mM Tris pH7.6, 1.5mM MgCl₂150mM KCl in a buffer solution. The conjugate system contained final concentrations of 2.5mM phosphoenolpyruvate, 200. mu.M Nicotinamide Adenine Dinucleotide (NADH), 1mMDTT, 30ug/ml pyruvate kinase, and 10ug/ml lactate dehydrogenase. Enzyme (90 nM final concentration) and DMSO solution of the selected compound (3% final concentration) were added. The reaction mixture was allowed to incubate at 30 ℃ for 10 minutes. The reaction was initiated by adding ATP to a final concentration of 0.9mM and the rate of NADH disappearance was monitored over the course of 10 minutes at 340 nm. Determination of K from the comparison of the Rate with the concentration Profile_iAnd IC₅₀The value is obtained.

Selected compounds of the invention were found to inhibit staphylococcus aureus DNA gyrase. Table 22 shows the inhibitory activity of these compounds in a staphylococcus aureus DNA gyrase inhibition assay.

TABLE 22 inhibition of Staphylococcus aureus DNA gyrase

Selected compounds	Ki（nM）	IC50（nM）
			Compound 23	9
Compound W	< 9	54

DNOTOPOIVO ATPase assay

The ATP to ADP conversion by the staphylococcus aureus TopoIV enzyme is coupled with the NADH to NAD + conversion, and the progress of the reaction is measured by the change in absorbance at 340 nm. TopoIV (64 nM) was incubated with the selected compound (final 3% DMSO) in buffer for 10 min at 30 ℃. The buffer consisted of 100mM Tris7.5, 1.5mM MgCl2, 200mM MK glutamate, 2.5mM phosphoenolpyruvate, 0.2mM ADH, 1mM DTT, 5. mu.g/mL linearized DNA, 50. mu.g/mL LBSA, 30. mu.g/mL pyruvate kinase, and 10. mu.g/mL Lactate Dehydrogenase (LDH). The reaction was initiated with ATP and the rate was monitored continuously on a molecular devices SpectraMAX plate reader at 30 ℃ for 20 minutes. Determination of inhibition constants, Ki and IC from a plot of rates fitted to the Morrison equation for tight binding inhibitors compared to the concentration of selected compounds₅₀。

Selected compounds of the invention were found to inhibit staphylococcus aureus DNATopoIV. Table 23 shows the inhibitory activity of these compounds in a staphylococcus aureus DNA gyrase inhibition assay.

TABLE 23 inhibition of Staphylococcus aureus DNOToIV

Selected compounds	Ki（nM）	IC50（nM）
			Compound 23	12
Compound W	30	150

Example 38

Water solubility study

The water solubility of compound 23 and compound W was determined according to the following procedure.

And (4) preparing a sample. Aqueous samples of each compound were prepared as follows. Compound (20-30 mg compound) was weighed into a 4mL clean vial before adding water (0.5 mL) and stirring 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 filter, Duraporo PVDF0.22 μm, Cat # UFC30 GVNB). The filtrate was collected and the pH was measured with a pH meter. The filtrate containing compound W was diluted 10-fold to provide a suitable concentration for HPLC analysis. The filtrate containing compound 23 did not require dilution.

Preparation of standard solution. Standard solutions of each compound were prepared according to the following procedure. 1-2mg of each compound was weighed accurately into a 10mL volumetric flask and water (for compound W) or 1:1 methanol: 0.1NHCl (for compound 23) was added to dissolve the compound completely. Sonication was performed on compound 23 to aid dissolution in 1:1 methanol: 0.1n hcl. When all solids were dissolved, additional solvent was added to adjust the volume of each solution to 10 ml. The resulting solution was mixed well to give a standard solution of each compound. Each standard solution was then diluted 2-fold, 10-fold, and 100-fold with solvent.

And (4) analyzing the solubility. Analysis by HPLC (Agilent 1100, injection volume 10. mu.L, wavelength 271nm, column XTerra)Phenyl5 μm, 4.6 × 50mm, part No. 186001144, mobile phase a: 0.1% TFA in water, 0.1% TFA in AcN) analyzed aliquots of each sample and each standard solution. Each standard solution was injected three times and each sample was injected twice. A standard curve was obtained by plotting the peak area average from HPLC compared to the concentration of a standard solution (the weight of the standard was suitably corrected based on the total water content of the solid, as determined by elemental analysis). The concentration of each sample was calculated from the peak area of the aqueous sample from the HPLC results and the slope and intercept of the standard curve. The solubility values listed in table 24 below are derived from the product of the sample concentration and the sample dilution factor.

TABLE 24 Water solubility of Compounds 23 and W

Compound (I)	Solid forms	pH	Solubility (mg/mL)
				Compound 23	Crystalline form	>3.0	<0.001
Compound W	Crystalline form	4.39	0.25

Example 39

In vivo metabolism studies in liver and liver S9 cells

Conversion of compound W to compound 23 was studied in liver and intestinal S9 fractions from rat, dog, monkey and human. Compound W was incubated at 0.1, 0.3, 1,3, 10, 20, 40, 100, 200, 300 μ M in the liver S9 fraction and at 1,3, 10, 20, 100, 300, 500, 1000 μ M in the intestine S9 fraction. Incubations were completed for 0, 5, 10, 15, 30, 45 or 60 minutes. Formation of compound 23 was quantified by LC/MS-MS and the data was fitted to the MichaelisMenten equation. The data in table 25 below indicates that compound W is rapidly converted to compound 23 in these liver and intestine S9 fractions.

TABLE 25 formation Rate of Compound 23 from Compound W in liver and intestine S9 (V)_MAX）

^*ND: parameters were not determined and the formation rate was not saturated

Claims (40)

1. A compound of the formula,

wherein R is hydrogen or fluoro; 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 pharmaceutically acceptableA divalent cation; or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, having the formula,

wherein R is hydrogen or fluoro; or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1, having the formula,

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; or a pharmaceutically acceptable salt thereof.

4. A compound according to claim 1, having the formula,

wherein R is hydrogen or fluoro; or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 4, wherein the compound is (R) -1-ethyl-3- (5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea or a pharmaceutically acceptable salt thereof.

6. The compound according to claim 4, wherein the compound is (R) -1-ethyl-3- (6-fluoro-5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea or a pharmaceutically acceptable salt thereof.

7. The salt according to claim 4, wherein the salt is the mesylate salt of (R) -1-ethyl-3- (5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea.

8. The salt according to claim 4, wherein the salt is the mesylate salt of (R) -1-ethyl-3- (6-fluoro-5- (2- (2-hydroxypropyl-2-yl) pyrimidin-5-yl) -7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-2-yl) urea.

9. A compound according to claim 3, wherein X is-PO (OH) O^-M⁺、–PO(O^-)₂·2M⁺or-PO (O)^-)₂·D²⁺；M⁺Selected from Li⁺、Na⁺、K⁺N-methyl-D-glucamine and N (R)⁹)₄ ⁺Wherein each R is⁹Independently is hydrogen or C₁-C₄An alkyl group; d²⁺Selected from Mg^{2 +}、Ca²⁺And Ba²⁺。

10. The compound according to claim 9, wherein X is-PO (OH) O^-M⁺or-PO (O)^-)₂·2M⁺；M⁺Selected from Li⁺、Na⁺、K⁺N-methyl-D-glucamine and N (R)⁹)₄ ⁺Wherein each R is⁹Independently is hydrogen or C₁-C₄An alkyl group.

11. The compound according to claim 9, wherein X is-PO (O)^-)₂·2M⁺；M⁺Selected from Li⁺、Na⁺、K⁺N-methyl-D-glucamine and N (R)⁹)₄ ⁺Wherein each R is⁹Independently is hydrogen or C₁-C₄An alkyl group.

12. A compound according to claim 9, wherein M⁺Is Na⁺。

13. A compound according to claim 3, wherein said compound is disodium (R) -2- (5- (2- (3-ethylureido) -6-fluoro-7- (tetrahydrofuran-2-yl) -1H-benzo [ d ] imidazol-5-yl) pyrimidin-2-yl) propyl-2-yl phosphate.

14. A pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

15. A pharmaceutical composition comprising a compound according to claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

16. A pharmaceutical composition comprising a compound according to claim 3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

17. A method of reducing or inhibiting the number of streptococcus pneumoniae, staphylococcus epidermidis, enterococcus faecalis, staphylococcus aureus, clostridium difficile, moraxella catarrhalis, neisseria gonorrhoeae, neisseria meningitidis, mycobacterium avium complex, mycobacterium abscessus, mycobacterium kansasii, mycobacterium ulcerosa, chlamydophila pneumoniae, chlamydia trachomatis, haemophilus influenzae, streptococcus pyogenes, or β -hemolyticus bacteria in a biological sample, comprising contacting the biological sample with a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the biological sample is a cell culture or an extract thereof, a biopsy material from a mammal or an extract thereof, or a bodily fluid or an extract thereof.

18. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for controlling, treating or reducing the progression, severity or effect of a nosocomial or non-nosocomial bacterial infection in a patient.

19. Use according to claim 18, wherein the bacterial infection is characterised by the presence of one or more of: mycobacterium tuberculosis, Streptococcus pneumoniae, Staphylococcus epidermidis, enterococcus faecalis, Staphylococcus aureus, Clostridium difficile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerosa, Chlamydia pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Streptococcus pyogenes or beta-hemolytic streptococcus.

20. Use according to claim 19, wherein the bacterial infection is selected from one or more of the following: upper respiratory tract infections, lower respiratory tract infections, ear infections, pleuropneumoniae infections, concurrent urinary tract infections, non-concurrent urinary tract infections, intra-abdominal infections, cardiovascular infections, bloodstream infections, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital infections, eye infections or granuloma infections, catheter infections, sinusitis, diabetic foot infections, vancomycin-resistant enterococcal infections, visceral abscesses, gingivitis, infections in cystic fibrosis patients or infections in febrile neutropenia patients.

21. Use according to claim 20, wherein the bacterial infection is selected from one or more of the following: community-acquired bacterial pneumonia, hospital-acquired bacterial pneumonia, bacteremia, diabetic foot infection, catheter infection, non-concurrent skin and skin structure infection, or vancomycin-resistant enterococcus infection.

22. A pharmaceutical composition comprising a compound according to claim 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle.

23. Use of a compound according to claim 6, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for controlling, treating or reducing the progression, severity or effect of a nosocomial or non-nosocomial bacterial infection in a patient.

24. Use according to claim 23, wherein the bacterial infection is characterised by the presence of one or more of: mycobacterium tuberculosis, Streptococcus pneumoniae, Staphylococcus epidermidis, enterococcus faecalis, Staphylococcus aureus, Clostridium difficile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerosa, Chlamydia pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Streptococcus pyogenes or beta-hemolytic streptococcus.

25. Use according to claim 24, wherein the bacterial infection is selected from one or more of the following: upper respiratory tract infections, lower respiratory tract infections, ear infections, pleuropneumoniae infections, concurrent urinary tract infections, non-concurrent urinary tract infections, intra-abdominal infections, cardiovascular infections, bloodstream infections, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital infections, eye infections or granuloma infections, catheter infections, sinusitis, diabetic foot infections, vancomycin-resistant enterococcal infections, visceral abscesses, gingivitis, infections in cystic fibrosis patients or infections in febrile neutropenia patients.

26. Use according to claim 25, wherein the bacterial infection is selected from one or more of the following: community-acquired bacterial pneumonia, hospital-acquired bacterial pneumonia, bacteremia, diabetic foot infection, catheter infection, non-concurrent skin and skin structure infection, or vancomycin-resistant enterococcus infection.

27. A pharmaceutical composition comprising a compound according to claim 13, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

28. Use of a compound according to claim 13, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for controlling, treating or reducing the progression, severity or effect of a nosocomial or non-nosocomial bacterial infection in a patient.

29. The use according to claim 28, wherein the bacterial infection is characterized by the presence of one or more of: mycobacterium tuberculosis, Streptococcus pneumoniae, Staphylococcus epidermidis, enterococcus faecalis, Staphylococcus aureus, Clostridium difficile, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Mycobacterium avium complex, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium ulcerosa, Chlamydia pneumoniae, Chlamydia trachomatis, Haemophilus influenzae, Streptococcus pyogenes or beta-hemolytic streptococcus.

30. Use according to claim 29, wherein the bacterial infection is selected from one or more of the following: upper respiratory tract infections, lower respiratory tract infections, ear infections, pleuropneumoniae infections, concurrent urinary tract infections, non-concurrent urinary tract infections, intra-abdominal infections, cardiovascular infections, bloodstream infections, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital infections, eye infections or granuloma infections, catheter infections, sinusitis, diabetic foot infections, vancomycin-resistant enterococcal infections, visceral abscesses, gingivitis, infections in cystic fibrosis patients or infections in febrile neutropenia patients.

31. Use according to claim 30, wherein the bacterial infection is selected from one or more of the following: community-acquired bacterial pneumonia, hospital-acquired bacterial pneumonia, bacteremia, diabetic foot infection, catheter infection, non-concurrent skin and skin structure infection, or vancomycin-resistant enterococcus infection.

32. Use according to claim 19, wherein the bacterial infection is selected from one or more of the following: otitis externa, otitis media, bronchitis, nosocomial pneumonia, ventilator-associated pneumonia, complicated intra-abdominal infections and other intraperitoneal infections, osteomyelitis, arthritis, genital ulcers, urethritis, vaginitis, or cervicitis.

33. Use according to claim 24, wherein the bacterial infection is selected from one or more of the following: otitis externa, otitis media, bronchitis, nosocomial pneumonia, ventilator-associated pneumonia, complicated intra-abdominal infections and other intraperitoneal infections, osteomyelitis, arthritis, genital ulcers, urethritis, vaginitis, or cervicitis.

34. Use according to claim 29, wherein the bacterial infection is selected from one or more of the following: otitis externa, otitis media, bronchitis, nosocomial pneumonia, ventilator-associated pneumonia, complicated intra-abdominal infections and other intraperitoneal infections, osteomyelitis, arthritis, genital ulcers, urethritis, vaginitis, or cervicitis.

35. Use according to claim 19, wherein the bacterial infection is selected from one or more of the following: pharyngitis, bronchial infections, pneumonia, empyema, septicemia, bacteremia, endocarditis, myocarditis, pericarditis, meningitis, encephalitis, brain abscesses, non-concurrent skin and skin structure infections, prostatitis, cystitis and pyelonephritis, nephrolithiasis, peritonitis, conjunctivitis, keratitis or endophthalmitis.

36. Use according to claim 19, wherein the bacterial infection is selected from one or more of the following: community-acquired bacterial pneumonia, hospital-acquired bacterial pneumonia, infusion-related sepsis, or dialysis-related peritonitis.

37. Use according to claim 24, wherein the bacterial infection is selected from one or more of the following: pharyngitis, bronchial infections, pneumonia, empyema, septicemia, bacteremia, endocarditis, myocarditis, pericarditis, meningitis, encephalitis, brain abscesses, non-concurrent skin and skin structure infections, prostatitis, cystitis and pyelonephritis, nephrolithiasis, peritonitis, conjunctivitis, keratitis or endophthalmitis.

38. Use according to claim 24, wherein the bacterial infection is selected from one or more of the following: community-acquired bacterial pneumonia, hospital-acquired bacterial pneumonia, infusion-related sepsis, or dialysis-related peritonitis.

39. Use according to claim 29, wherein the bacterial infection is selected from one or more of the following: pharyngitis, bronchial infections, pneumonia, empyema, septicemia, bacteremia, endocarditis, myocarditis, pericarditis, meningitis, encephalitis, brain abscesses, non-concurrent skin and skin structure infections, prostatitis, cystitis and pyelonephritis, nephrolithiasis, peritonitis, conjunctivitis, keratitis or endophthalmitis.

40. Use according to claim 29, wherein the bacterial infection is selected from one or more of the following: community-acquired bacterial pneumonia, hospital-acquired bacterial pneumonia, infusion-related sepsis, or dialysis-related peritonitis.