OF BACTERIAL INFECTIONS

Background

Antibiotic tolerance and resistance has become a grave threat to the successful treatment of many common baciersa! infections. Indeed, according to the infectious Disease Society of America, methiciSlin resistant Staphylococcus aureus ( RSA) kills more

Americans every year than emphysema, HiV/AlDS, Parkinson's disease and homicide combined. Not only is multi-drug resistance in common infectious Gram-positive and -negative pathogens such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acineiobacter bau annii, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Entembacter species on the rise, but evidence of resistance is being seen in Salmonella and Clostridium difficile, and increasingly Neisseria gonorrhoeae (Gerard D. Wright, "Antibiotics: A New Hope," 19 (20 2), 3-10). Due to this increase in resistance, the development of new antibacterial medicines is an important medical need.

Summary

There remains a need for new therapies for treating bacterial infections. There is provided the compound (2R,4S,4aS)-11 -f!uoro-2,4-dtmethyi-8-[{4S)-4-methyl-2-oxo-1 ,3- oxa20lidin-3-y!3-1 ,2 ₎ 4,4a-tetrahydro-2'H _i 8H-spiro[1 _i 4-oxazino[4 _> 3-aI1 ,2]oxazoio[4,5- gJquinoline-S.S'- yrimidinej^'^'.G'il' S'HHnone, or a pharmaceutically acceptable salt thereof, for potential use for treating bacterial infections.

In one aspect, there is provided a method for treating a bacteria! infection caused by Bacillus anthracis, Bacillus cereus, Burkholderia spp., Brucella spp,, Franci sella spp., Ye ina spp. , Mycoplasma spp., Ureaplasma spp., Chlamydia trachomatis or Chlamydia pneumoniae in a subject in need thereof comprising administering an effective amount of (2R,4S,4aS)-11-ftuoro-2,4-dimethyl-8-[{4S}-4-methyl-2-oxo-1 ,3-oxazolidin-3-yfji-1 ,2,4,4a- tetrahydro-2'H,6H-spiro[1 ,4-oxazino[4,3-a3[1 ,2]oxazolo 4,5-g]quinoline-5,5'-pyr!rnidine]- 2 4 Q'{VH,d'H}-tnom, or a pharmaceutically acceptable salt thereof, to the subject.

In one aspect, there is provided the use of (2 4$,4aS)-11-fiuoro~2,4-dimethyl~8~ (4S}-4-methyi-2-oxo-1 ,3-oxazoiidin-3-yi]-1 ,2,4,4a-tetrahydro-2'H,6H-spiro[1 ,4-oxazino[4,3- a][1 ^JoxazoloH.S-gJqufnoline-S^'-pyrimidinej- ^'^'i 'H,3'H}-trione, or a pharmaceutically acceptable salt thereof, for treating a bacterial infection caused by one or more bacterium selected from Bacillus anthracis. Bacillus cereus, Burkholderia spp., Brucella spp. ,

Francisella spp., Yersina spp., Mycoplasma spp., Ureaplasma spp., Chlamydia trachomatis or Chlamydia pneumoniae.

in one aspect ther is provided the use of {2R,4S,4aS)-11 -fluoro-2,4-dimethyl-8- [(4$)-4-methyl-2-oxo-1 ,3-oxa20lidin~3~ylj[~1 a][1 ^joxazo^^-gjqutnoltne-S.S -pyrimidinej-Z^'.e'il Ή,3Ή)-ίποπθ, or a pharmaceuticaiiy acceptable salt thereof, in the manufacture of a medicament for treating a bacterial infection caused by one or more bacterium selected from Badiius anthracis. Bacillus cereus,

Burkholderia spp.. Brucella spp. , Frandsella spp,, Yersina spp , Mycoplasma spp. ,

Ureaplasma spp.. Chlamydia trachomatis or Chlamydia pneumonias.

in one aspect, there is provided a pharmaceutical composition comprising

(2 4S,4aS)-1 1-fiuorc~2,4-dimethyi~8-[{4SH-methyi-2-oxo-1 ,3-oxazoiidin~3~yS]-1 ,2,4,4a- ieirahydro-2'W,8H~spira[1 ^-oxazino^^-ajJI ^Joxazoio^^gjquinofine-S.S'-pyrimidine]- 2 ^, ,4',8 ^, {1 ^, H _! 3'H)-ti1one _i or a pharmaceuticaiiy acceptabie salt thereof, for treating a bacterial infection caused by one or more bacterium selected from Bacillus anthracis, Bacillus cereus, Burkholderia spp.. Brucella spp., Frandsella spp. , Yersina spp. , Mycoplasma spp...

Ureaplasma spp., Chlamydia trachomatis or Chlamydia pneumoniae.

Detailed Description

There are provided methods of treating bacteria! infections caused by one or more bacterium selected from Bacillus anthracis, Bacillus cereus, Burkholderia spp., Brucella spp., Frandsella spp., Yersina spp. , Mycoplasma spp,, Ureaplasma spp. , Chlamydia trachomatis or Chlamydia pneumoniae by administering to a subject in need thereof an effective amount of (2 ,4S,4aS)-11-fiuoro-2,4-dimethyt-8- (4S)-4-methyi-2-oxo-1 ,3-oxazQlidin-3-yl3-1 ,2,4,4a- tetrahydrQ~2'H,6H-spirGl1 ,4~oxazino[4,3-a3^

2 ^! ,4\δ'(1 ^! Η ₍ 3 ^! ΗΜποηβ, or a pharmaceuticaiiy acceptabie salt thereof.

The compound (2R,4S,4aS)~11 -fiuoro-2,4-dimethyi-8-[{4S}-4-methyi-2~oxo-1 ,3- oxazoJidin- -ylKI ,2,4,48-ί©δ¾Γ ν ΐΓθ-2Ή6Η-5ρίΓθ[1 ,4-oxazino[4,3-aH1 ,2]oxazoio|4,5- g]quinoiine-5 _! 5'-pyrimidinei~2\4\6 ^as the following structure:

The aforementioned compound, and its method of synthesis, is disclosed in international Appiication No. PCT/GB201 /050164, which is expressly incorporated herein in its entirety.

The language "bacterial infection" includes infections caused by one or more species of Gram-negative, Gram-positive, or atypical bacteria.

in some embodiments, the bacteria! infection is caused by Bacillus anthracis or Bacillus cereus.

in some embodiments, the bacterial infection is caused by Burkholderia spp,, for example, Burkholderia mallei, Burkholderia pseudomelia! and Burkhofderia cepacia.

in some embodiments, the bacteria! infection is caused by Brucella spp., for example, Brucella meliiensis, Brucella abortus, Brucella cams, Brucella suis and Brucella ovis.

in some embodiments, the bacterial infection is caused by Francisella spp. , for example, Francisella iularensis, Francisella novicida and Francisella philomiragia,

in some embodiments, the bacterial infection is caused by Yersina spp., for example, Yersinia pestis and Yersinia enteroco!ltica.

in some embodiments, the bacterial infection is caused by Mycoplasma spp., for example Mycoplasma gallisepticum, Mycoplasma genitalium, Mycoplasma aemofe!ls. Mycoplasma hominls. Mycoplasma hyopneumonlae, Mycoplasma ovipneumoniae and Mycoplasma pneumoniae.

in some embodiments, the bacterial infection is caused by Ureaplasma spp., for example, Ureaplasma parvum and Ureaplasma urealytscum

in some embodiments, the bacterial infection is caused by Chlamydia trachomatis or Chlamydia pneumoniae.

in some embodiments, the bacteria are resistant to one or more antibacteria!s other than (2/? ₅ 4S,4aS)-11 -f!uoro-2,4-dimethyl-8-[{4S)-4-methyl-2-oxo-1 ,3-0xa2oiidin-3-yi]- 1 ,2 A4a ^« tetrahydro2'W,6W-spiro[1 ,4-oxazino[4,3-a][1 ^Joxazoto^S-oJquinoline-S,^- pyrimidinei~2\4\6 1¾3'HHrione. The language "resistance" and "antibacterial resistance" refers to bacteria that are able to survive exposure to one or more antibacterial agents. In one embodiment, the bacteria is resistant to one or more of an aminoglycoside antibiotic (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, spectinomycin), an ansamycin antibiotic {e.g., rifaximin, streptomycin), a carbapenem antibiotic {e.g., ertapenem, doripenem, imipenem/cilastatin, meropenem), a cephalosoprin antibiotic (e.g., cefadroxil, cefaxoiin, cefatoiin, cefalexin, cefaclor, cefamandoie, cefoxitin, cefprozil, cefuroxime, ceftsime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, certibuten, ceftizoxime, ceftriaxone, cefepime, ceftaro!in fosamil, ceftobiprole), a giycopeptide antibiotic (e.g., teicoplanin, vancomycin, telavancin), a lincosamide anitbiotic (e.g., clindamycin, ((neomycin), dapfomycin, a macroiide antibiotic {e.g., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin), aztreonam, furazolidone, nitrofuantoin, an oxazolidinone antibiotic (e.g., linezolid, posizolid, radezolid, torezo!id), a peniciiiin antibiotic (e.g. , amoxaciliin, ampicillin, azlociilin, carbenicillin, cioxaciliin, dicloxaciS!in, fiucloxacillin, mezlocillin, methiciliin, nafcillin, oxacillin, penicillin, piperaciflin, temociliin, ticarciiiin), amoxici!iin/ciavu!ante,

ampiciSin/sulbactam, piperacilSin/tazobactam, ticarcillin/ciavuianate, a quino!one antibacterial (e.g., ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levoffoxacin, lomefioxacin, oxifSoxacin, nalidixic acid, norfloxacin, ofloxacin, trovafioxin, grepafloxacin, sparfioxacin, temafloxacin), a suf!onamide antibiotic (e.g. , mafenide. sulfacetamide, sulfadiazine, silver sulfadiazine, suifadimethoxine, sulfamethizole, sulfamethoxazole, sulfanamide,

sulfasalazine, sulisoxazo!e, trimethoprim/sulfamethoxazole - TMP-S X) and a tetracycline antibiotic (e.g., demec!ocyc!ine, doxycycline, minocycline, oxytetracycSine, tetracycline, tigecSycSine). In some embodiments, the bacteria is resistant to doxycycline. in some embodiments, the bacteria is resistant to levofloxacin and/or ciprofloxacin. In some embodiments, the bacteria is resistant to azithromycin. In some embodiments, the bacteria is resistant to tetracycline.

In some embodiments, there is provided a method of treating a subject suffering from a sexually transmitted bacteria! infection comprising administering to the subject an effective amount of a (2f?,4S,4a$)-1 1-fluoro-2 _! 4-dimethyl-8-[(4S)-4-methyi-2-oxo-1 ,3- oxazolidin-3-y!]-1 ,4-oxazino[4,3-a][1 ,2]oxa2oSo[4,5- g]quinoline-5 ₍ 5-pynmidine]-2 ^, _l 4 ^, ,6 ^, (1 ^* H _l 3 ^, H)-trione, or a pharmaceutically acceptable salt thereof.

In some embodiments, there is provided (2R,4S,4aS)~11 luoro-2,4-dimeihyi-8-- [(4SM-meth l-2-oxo-1 ,3-oxazolidin-3-yi]-1 ,2 A4a4etrahydro-2 6H-spiro[1 _t 4-oxazino[4,3~ a][1 ^joxazo^^-gjqutnoltne-S.S -pyrimidinej-Z^'.e'il Ή,3ΉΗηοηβ, or a pharmaceutically acceptable salt thereof, for use in treating a bacterial infection caused by one or more bacterium selected from Bacillus anthracis, Bacillus cereus, Burkhoidena spp., Brucella spp. , Francisella spp., Y&rsina spp.. Mycoplasma spp., Ureaplasma spp., Chlamydia trachomatis or Chlamydia pneumoniae.

in one aspect, there is provided a method for treating an anthrax infection, glanders, melioidosis, a pulmonary infection in a subject suffering from cystic fibrosis, brucellosis, tularemia, plague, sepsis, yersiniosis, pelvic inflammatory disease, atypical pneumonia, nonspecific urethritis, pneumonia, bronchopulmonary dysplasia or meningitis in a subject in need thereof comprising administering an effective amount of (2 4S,4aS}-11-fiuoi -2,4- dimethyl-8-[(4S)-4-methyl-2-oxo-1 ,3-oxazoiidsn-3-yli-1 ,2,4 _> 4a-ietrahydro-2'H _> eH-sp!ro[1 _> 4- o azί o[4,3- ][1 ,23oxazolo 4,5-g] ui oiine-5,5'- yrimidsπe]~2 4^6 Ή,3 ^, H) rione _f or a pharmaceutically acceptable salt thereof, to the subject.

The language "treat," "treating" and "treatment" includes the reduction or inhibition of enzyme or protein activity related to a bacterial infection in a subject, amelioration of one or more symptoms of a bacterial infection in a subject, or the slowing or delaying of progression of a bacterial infection in a subject. The language "treat ," "treating" and

"treatment" also includes the reduction or inhibition of the bacterial growth, replication or a reduction or inhibition of the bacterial load of bacteria in a subject.

The term "subject" includes, for example, primates, cows, horses, pigs, sheep, dogs, cats, rabbits, rats, birds (including wild and domestic birds, such as turkeys, geese, chickens, ducks and the like) and mice, in some embodiments, the subject is a primate, for example, a human, in some embodiments, the subject is suffering from a Gram-positive bacterial infection, in some embodiments, the subject is suffering from a Gram-negaitve bacteriai infection, in some embodiments, the subject is suffering from an atypical bacteriai infection, in some embodiments, the subject is in need of treatment (e.g., the subject would benefit biologically or medicai!y from treatment). In some embodiments, the subject is suffering from a significant underlying disease state that complicates the response to treatment of a bacteriai infection, for example cystic fibrosis, in some embodiments, the subject is suffering from one or more bacteriai infections (e.g., co-infecied by two or more bacterial infections). In some embodiments, the subject is suffering from an infection caused by Neisseria gonorrhoeae, in some embodiments, the subject is co-infected with Chlamydia trachomatis and Neisseria gonorrhoeae. In some embodiments, the subject is at risk of contracting a sexually transmitted bacterial infection (e.g., a Chlamydia trachomatis or Neisseria gonorrhoeae infection).

The language "effective amount" includes an amount of (2 ,4S,4aS)-11-fluoro-2,4- di methy!-8-[{4$H-methyl-2^^

Qxazi o^S-aHl^jaxazoSo^S-Q qui oli e-S.S'-p ^ or a pharmaceutically acceptable salt thereof, that wiii eiicit a biological or medical response of a subject, fo example, the reduction or inhibition of enzyme or protein activity related to a bacterial DNA gyrase or a bacteriai infection, amelioration of symptoms of a bacteria! infection, or the slowing or delaying of progression of a bacteriai infection. In some embodiments, the language "effective amount" includes the amount of (2 ?,4S,4aS)-11- fiuorQ~2 ₍ 4-d!methyi~8-[(4 SH^

spiro[1 ,4-oxazino[4,3~3][1 ^joxazoio^.S-gjquinoiine-S.S -pyrimidinej- ^'^'i 1 Ή,3Ή)-ίήοηβ, or a pharmaceutically acceptable salt thereof, that when administered to a subject, is effective to at least partially alleviate, inhibit, and/or ameisoraie a bacteriai infection or inhibit bacteriai DNA gyrase, and/or reduce or inhibit the bacterial growth, replication or bacteria! load of a bacteria in a subject.

Exemplification o azq!i^

Q quinoline-S.S'-pyrimidSnel^'^'.e'd'tf.S'ffl-trione {Compound 1)

Compound 1 was synthesized as described below:

Intermediate 1

3-Chloro-6-[(2 ?,6S)-2,8-dimethyimorpholin-4~yl)-7-fiuoro-1.2^

To an ice cooled solution of 3-ch!oro-6J-difluoro-1 ,2-benz0X0z {prepared according to the procedure described in International Application Publication No. WO 2010/043893, 5.0 g, 23.0 mmoi) in anhydrous acetonitrile (50 mi) was added

diisopropy!ethylamine (5.9 g, 45.9 mmo!) followed by cis-2,6-dimethyimorpholine {2.8 g, 23.0 mmol) and the mixture was heated at 85 °C for 12 hours in a sealed tube. The soiution was cooled to room temperature and the volatile® were removed under vacuum. The the residue qwas dissolved in Ethyl acetate, washed with water followed by brine and then dried over anhydrous Na ₂ S0 ₄ . Removal of solvent under vacuum afforded the crude product, which was purified over silica gel column using a gradient of ethyl acetate in pet. ether to give title compound as solid. Yield: 6.0 g (84%). ¹ H MR (400 MHz, DMSO-cfe) 5: 1.0 (d, 6H), 2.9 (i, 2H), 3.1 (d, 2H), 3.8 (m, 2H), 7.7 (s, 1 H), 10.2 (s, 1 H). MS (ES) H ^' : 313 for

intermediate 2

3~Chioro~6-fi 2R ,6S)~2.6-dimeth ylmorphoiin-4-yll-5-( 1.3-dioxoian-2-yl -7-f 1 uoro- 1.2-

A soiution of intermediate 1 (16.3 g, 52.2 mmol), ethylene glycol (8.1 g, 130.8 mmol) and pyridinium p-toluenesulfonate (1 ,31 g, 5,2 mmoi) in toluene (300 ml) was heated at reflux in a Dean-Stark apparatus for 16 hours. The solvents were removed under vacuum and the residue was dissolved in diethyl ether (75 mL), washed with water (3 x 25 mL) and aqueous brine (25 ml). The organic layers were dried over anhydrous Na ₂ S0 and filtered. Removal of solvents unde vacuum afforded the title compound, which was further purified by trituration with hot hexane. Yield: 18,0 g (80 %}. H NMR (400 MHz, DMSO-cfe) S: 1.1 (d, 6H). 2.8 (t, 2H), 3.0 (d, 2H), 3.3 (m, 4H), 3.8 (m, 2H), 5.7 (s, 1 H), 7.6 (s, 1 H).

Intermediate 3

i4ffl-3-{6 f2 6^2.6-Dimethylm^^ en2oxaml-3-yl}-4-methyi-1 ,3^

To a stirred solution of NaH (0,24 g, 9,9 mmol) in dimethyiformamide (10 mL), a solution of (4R)-4-methy!-1 ,3-oxazo!idin-2-one (synthesized according to the procedure described in Nishiyama, T.; Matsui, Shtgekt; Yamada, F. J. Het, Chem, (1986), 23(5), 1427-9) (10 g, 9,9 mmol) in dimethyiformamide (10 mL) was added slowly at 0 'C over a period of 10 minutes. The mixture was stirred at the room temperature for 30 minutes and a solution of

Intermediate 2 (1.1 g. 3.1 mmol) in dimethyiformamide (5 ml) was added at the same temperature. This mixture was heated at 80X for 12 hours and poured into ice-cooied water and extracted with ethyl acetate (2 x 20 ml). The organic layers were dried over anhydrous Na ₂ S0 ₄ and the solvents were removed under vacuum. The crude product was purified by silica ge! column chromatography using a gradient of ethyl acetate in pet, ether. Yield: 0.15 g (12%). MS (ES) Mhf: 422,4 for C2oH ₂ FN ₃ Q ₈ . Intermediate 4

(4S)-3 6 (2R6S)~2,e~Dime^

benzoxamtr-3-yl -methy

intermediate 4 was prepared from Intermediate 2 using (4S)-4-methyl-1 ,3-oxazoitdin-2- one (synthesized according to the procedure described in Nishiyama, T.; Matsui, Shigeki; Yamada, F. J. Het. Chem, (1986), 23(5), 1427-9) in a method similar to the one described for the synthesis of intermediate 3. MS (ES) MH ^* : 422.4 for C20H24FN3O6.

Compound 1

i2 4$.4aS)-1 1-F!uoro-2.4-dimethyi~8^

-2 6^spirof1 ,4 >xazino^

A stirred mixture of Intermediate 4 (0.36 mmoS) and barbituric acid {0.3 mmo!) in acetic acid (1 mi) was heated at 85 °C for 16 hours. The solvents were evaporated, the residue was dissolved in methanol (2 mi) and water (5 ml) was added. The precipitated solids were filtered and purified by reverse phase HPLC (10 mM ammonium acetate in water, CHsCN}, eluting two components. The second eiuting component was isolated as a solid and identified as the title compound. The title compound was isolated by reverse phase HPLC (10 m ammonium acetate in water, CH3CN) as the first eluting of two components. ^r H R (400 MHz, DMSO-efe) 6: 0.9 (d, 3H), 1.15 (d, 3H), 1 .4 (d, 3H), 2,9 (d. 1 H), 3.1 (t, 1 H), 3.5-3.6 (m, 2H), 3.8 (m, 1 H), 3.9 (d, 1 H), 4.0 (d, 1 H), 4.2 (q, 1 H), 4.6-4.7 (m, 2H), 7.6 (s, 1 H), 11 .5 (s, 1 H), 1 1 .8 {s, 1 H). MS (ES) MH*: 488.4 for C ₂ 2H ₂₂ F s0 _7; [ ] _Q ^2Q ~ -92 (c ~ 1 ; MeOH).

Also isolated from the synthesis of Compound 1 as the second eiuting component from HPLC purification was (2S _! 4R ₍ 4aR)- 1 -fiu »o-2 ₍ -djnnre^ W {4SM^ettiyl-2- ⁾ o-1 ,3- oxazotidin-3-yt]~ ,2,4,4a-teirahydro-2'H,6H-spiro[1 ,4-oxazino[4,3-a][1 ,2]oxazoio[4 ,5- g]quinoline-5 _> 5 ^, -pyrimidine]~2\4\ 'H _i 3'H)-trione

¹ H NMR (400 MHz, DMSO-cf ₆ ) 6 ^" . 0.9 (d, 3H), 1.15 (d, 3H), 1.4 (d, 3H), 2.8 (d, 1 H), 3.1 (t 1H), 3.6-3.7 (m, 2H), 3.8-4.0 (m, 1 H), 3.9 (d, 1 H), 4.1 (d, 1 H), 4,2 (q. 1 H), 4.8-4,7 (m, 2H), 7.6 (s, 1 H), 1 1 .5 (s, 1 H), 1 1 .8 {S, 1 H). MS (ES) MH ^!" : 488.4 +224 (c = 1 ; MeOH).

Example 2, In Vitm AntibacAerjai

Compound 1 is an investigational inhibitor of the supercoi!ing and decatenation activity of the DNA gyrase and topoisomerase iV with activities against several different types of bacteria. Preliminary data suggest this agent maintains activity against organisms that are resistant to other agents such as fluoroquinolones and tetracyclines, including agents of sexually transmitted infections such as Neisseria gonorrhoeae. The present study was undertaken to increase knowledge of the in vitro activities of Compound 1 against additional human pathogens by testing a small number of clinical isolates and reference strains representing five species of moi!icutes that are important human pathogens.

Organisms tested included Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureapiasma ureaiyticum and Ureapiasma parvum. While M, pneumoniae is primarily a pathogen of the respiratory tract causing illnesses such as pharyngitis, tracheobronchitis, and pneumonia, the remaining species are important pathogens of the urogenital tracts in adult men and women and can aiso cause systemic disease in neonates when transmitted vertically during pregnancy or at defivery. Susceptibility testing was performed in accordance with guidelines of the Clinical Laboratory Standards Institute (CLSi) (CLSI 2011 ). Strains tested included organisms that contained the tet gene, which mediates tetracycline resistance, mutations in 23S ribosomal RNA that confers macrolide resistance, and others that contained mutations in DMA gyrase and/or topoisomerase IV that confer resistance to fluoroquinolones.

METHODS

Antsbacterials. Drugs included in the investigation are summarized in Table 1. An appropriate amount of each powdered drug was weighed to prepare 10 ml of a stock solution, allowing for the percentage purity of each. Antimicrobial agents were dissolved according to each manufacturer's instructions.

Table 1. Test Compound and Control/Reference Compounds

Bacterial strains. Pure cultures of clinical isolates of known titer derived from various body sites of adults and children that have been stored at minus 70 °C in the culture collections of the UAB Diagnostic Mycoplasma Laboratory were used in this investigation. Original sources of the isolates and year of isolation, when available, as well as specific resistance profiles, where relevant, are summarized in Tabie 2.

Table 2. Bacteria? Strains Tested

Mycoplasma genttatium (n - 5)

Mycoplasma hominis (n - 12)

Umap!asma species (n - 15) Notes for Table _: 2

Abbreviations: Uu = Ureapiasma urealyticum. Up = Ureapiasma parvum, BAl = bronchoa!veoiar Savage fluid, CSF = cerebrospinal fluid, ETA - endotracheal aspirate

Ureapiasma species were identified by real-time PGR as previously described (Xiao et al. Detection and characterization of human Ureapiasma species and serovars by real-time PCR. J. Clin. Microbiol. 2010,48, 2715-2723). Three clinical isolates were shown to be a mixture of both species, which sometimes occurs (Xiao et at Extensive horizontal gene transfer in ureap!asmas from humans questions the utility of serotyping for diagnostic purposes. J. Clin, Microbiol. 2011 , 49, 2818-2826).

Presence of tetM in M. omsnis and Ureapiasma species was determined by PCR in the UAB Diagnostic Mycoplasma Laboratory.

In vitro susceptibility test methods:

The assa employed for this investigation was the broth micro-dilution minima! inhibitory concentration (MIC) assay that was published in "Methods for Antimicrobial Susceptibility Testing of Human Mycoplasmas. Approved Guideline, CLSI Document M43-A" (CIS! 20 ), This assay employs 96 well microtiter plates into which a defined inoculum of the organism to be tested is added to doubling dilutions of antimicrobial agents in smail volumes. P!ates were incubated until the growth control changed color. The MIC endpoint was then determined by lack of color change in broth containing a pH indicator. Specific aspects of the procedures that were used follow.

Medi , SP4 broth and SP4 agar were used for testing M. pneumoniae and geniialium. Modified Hayflick's Mycoplasma broth and agar were used for testing . ho inis. Shepard's 1QB Broth and A8 agar were used for testing Ureapiasma species. These media and their formulations are described in the CLSI document (CLSI 2011 ).

Preparation of Inoculum. Organisms were thawed to room temperature and diluted in appropriate prewarmed media in 50 mL conical tubes to yield a final inoculum of approximately 10 ⁴ CFU/mL. At least 5 mis of inoculum was prepared for each drug, based on testing 8 dilutions in duplicate and appropriate controls. If more dilutions were needed to achieve endpoint ICs, an additional volume of inoculum was prepared, inoculated broths were incubated aerobicalSy at 37 ⁰ C for 2 hours prior to use to allow mycoplasmas to become metabolicaiiy active prior to inoculating microtiter plates. Due to their more rapid growth rates, ureaplasmas were incubated for only one hour prior to inoculating the plates.

Performance of Broth MtcrodHut ion Assay. A single microtiter p!ate was used for 4 drugs. Each drug was tested in duplicate (Drug 1-rows A, B; Drug 2-rows C, D; Drug 3- rows E, F. Wells 9, 10, 1 1 and 12 wer used for solvent, media, drug and growth controls, respectively. 0.025 mL of appropriate broth medium was added to rows 2-8 and 10 and 12 of ihe microliter plate, 0.025 mL of the highest concentration of drug to be tested was added to we!!s 1 , 2 and 11 in rows A, B. Weii 11 served as the drug control. The other drugs to be tested were added the same way in their respective rows. The highest drug concentration was prepared by performing an appropriate dilution on the stock solution. Antimicrobia! agents were serially diluted using a 0.025 mL multichannel pipette, beginning at the 2nd well, and continuing through well 8, discarding the final 0.025 ml. A solvent control was prepared in well 9 by incorporating 0,025 mL of the highest concentration (1 :10 dilution in sterile deionized water) of solvent used to dissolve the antimicrobial agent being tested if any substance other than water was used as a solvent. 0. 75 mL of the desired dilution of inoculated media that has been prewarmed for 2 hours was added to each well in rows 1 -9 and 12. Well 12 served as the growth control. Inocula were added starting with well 12 and working backwards to well 1 to prevent drug carryover. 0.175 mL of appropriate uninocuiated media was added to wells 10 and 11 {total of 0.2 mL) for media and drug controls. A final determination of the CFU/mL of the working dilution used to inoculate each microliter piate was made by preparing 6 serial dilutions of the inoculum (0.1 mL inoculum in 0.9 mL of the appropriate broth) and pipetting 20 μ! of each dilution onto the appropriate agar plate to check that a proper dilution was made and that the inoculum contained 10 -T0 ^S CFU/mL. Agar plates were incubated at 37 °C in air plus 5% CG ₂ untii colonies were visible and could be counted. Time required until growth becomes visible varies according to species, ranging from 24-72 hours for Ureaplasma species and M. hominis up to several days for

pneumoniae and M. genitalium. Sv scrodi!ution trays were incubated aerobicaliy at 37 °C and examined after 18-24 hours and then daily for color change in the growth control wells.

Determination of MiC Endpoints. Quality Control, and Assay Validation. M!Cs were recorded as the lowest concentration of antimicrobial agent inhibiting color change in broth medium at the time when the organism control well first showed color change. A positive reaction for growth of Ureaplasma spp. in 10B broth was evidenced by a color change from yellow to pink in the organism control well (i.e. well 12). A positive reaction for M. hominis in Mycoplasma broth was evidenced by a color change from pink to deeper red in the organism growth control well {i.e. well 2). A positive reaction for M. pneumoniae and M. genitalium in SP4 broth was evidenced by a color change from pink to yeiiow in the growth control well Results were considered valid if the control agar plate for organism's concentration indicated that there were between 10 ⁴ and 10 ^s CFU/mL Control wells and expected results were: well 9 (solvent control) ~ no color change; well 10 (media control) - no color change; well 1 1 (drug control) - no color change; well 12 (growth control) - growth and color change according to which organism is being tested, without turbidity. By performtng CFU quantification on the inoculum of each isolate tested, purity of the organisms was verified. SP4 agar detects contaminants or mixed cultures with Mycoplasma species when inocu Sated with M. pneumoniae and M. genitalium, M hominis grows on either SP4 or mycopiasma agar; M hominis and commensal respiratory Mycoplasma species produce fried egg colonies whereas; M. pneumoniae and M. genit&lium produce small spherical colonies. AS agar plates yield brown granular colonies for U aptasma species and would also detect contaminating Mycopiasma species or bacteria. Any turbidity in the growth control well indicates bacterial contamination and invalidates the results.

Broth Microdiiution MIC Quality Controi Limits, For quality control (QC) purposes, American Type Culture Collection (ATCC) strains designated by the CLSI (CLSi 2011 ) for each organism being tested were included with each assay every day of performance. MIC reference ranges for several antimicrobial agents have been established for these strains (CLSI 2011 ). QC strains that were used were; M. pneumoniae ATCC 29342, M. hominis ATCC 23114, and U. urealyttcum ATCC 33175. There is no genitalium type strain recommended by the CLSI since susceptibility testing has not been standardized fo this organism. Therefore, we chose the type strain ATCC 33530 for this organism. This strain has been used in our laboratory for other investigations and has predictable MiCs for several antimicrobial agents. Acceptable MIC QC limits for a single test {singSe-drug/singSe organism combination) are listed in Table 3 as derived from the CLSI document (CLSI 2011 ). QC strains performed as expected for all MIC assays for which data are presented. Table 3. MIC Limits (pg/mL) for Qua! Sty Control Strains for Mycoplasma hominis,

Mycoplasma pneumoniae and Ureapiasma urealyttcum Tested by Broth fVHcrodilution

Note for Table 3

Data in Table 3 were derived from the -43-A CLSI Document (CIS! 2011 ).

RESULTS

genitalium ... Compound 1 showed in vitro activity comparable to that of levofloxacin and doxycycline. The overall MIC range for these three drugs was within 4 2~ fold dilutions = 0.25-2 g/mL. Compound 1 MIC range 0.5-1 pg/mL) was less potent than azithromycin (MiC range 0.001 pg/mL}. M, pn m n e. The MICgo for Compound 1 (1 pg/mL) was equivalent to that of ievofioxacin and 4-foid higher than doxycyc!ine (0.25 pg/mL). Most M. pneumoniae isolates had azithromycin MiCs < 0.001 pg/mL, but two strains were chosen for testing because they had azithromycin MICS of 16 and 32 pg/mL and contained mutations in 23S ribosomai RNA. Compound 1 maintained in vitm potency against these two macroiide-resistant isolates comparable to that for those isolates that were fully macro! ide-susceptibie.

M, hominis. Compound 1 had the lowest overall activity against M. hominis with the MICgo of 4 pg/mL and a maximum MIC value of 8 pg/mL Doxycycline MiCs for M. hominis isolates without tetM ranged from 0.018-0.083 pg/mL, while M!Cs for those three with tetM were 4 pg/mL. Corresponding tetracycline MiCs were 32 pg/mL for those isolates.

Compound 1 MiCs for doxycycline not affected by the presence of tetM. MiC for

Compound 1 (4 pg/mL) was 8~fold greater than that of levofloxacin (0.25 pg/mL) and was equivaient to that of azithromycin, a drug that is not usually very active against this species. Without having information on achievable drug concentrations for Compound 1 , it is not possible to indicate whether these M!Cs would be considered susceptible or resistant.

Umaptasma species. The ICso for Compound 1 was 1 pg/mL, making it comparable to levofloxacin in potency. There was no difference in Compound 1 MiCs against levofloxacin-resistant ureap!asmas and levofloxacin-susceptible isolates. Similarly, among three Ureaplasma isolates containing tetM, M!Cs for Compound 1 were not affected with its MiCs ranging from 0.5-2 pg/mL versus 4-8 pg/mL for doxycycline, but M!C _¾ > for doxycycline-susceptible organisms (0.125 pg/mL) was 8-fold more active than Compound 1 (1 pg/mL). The Compound 1 MIC for the single macroiide-resistant isolate of U. u a!yticum (azithromycin MIC = 32 pg/mL) was 2 pg/mL, which was 2-fold dilution higher than the MICso for this drug, but overall. Compound 1 was 4-foid more potent than azithromycin (MICso of 1 vs 4 pg/mL).

Table 4. MiC Dataset for Compound 1 and Three Comparators Tested Against Human Mycoplasmas

Mycoplasma genitaitum (n - 5) MICs {pg/ml)

Mycoplasma pneumoniae (n - 12) ICs ( g/mL)

18 Mycoplasma hominis (n - 12) MiCs (Mg L)

Ureapiasma species (n - 15} MiCs (Mg/mL) Table 5. Data Summary for Compound 1 and Three Comparators Tested Agamst Human Myco lasmas

Notes for Tables 4 and 5

Abbreviations; AZi - azithromycin, DOX - doxycycline, LEV = levof!oxacin, Uu - Ureaplasma uraa!yticurn, Up = Ureaplasma parvum.

The 3 M, hommis isolates containing the tetM gene were aiso tested against tetracycline at the same time as doxycycline. All 3 isolates had MICs of 32 pg/mL for tetracycline. Discussion

Mycoplasma and Ureapiasma species thai infect humans can cause significant disease in the respiratory tracts as well as the urogenital tracts, in addition to N.

gono hoeae and Chlamydia trachomatis, both M. genitalium and Umapiasma u alyticum can cause male urethritis and M. genitalium also causes female cervicitis and pelvic inflammatory disease (Wastes KB, Taylor-Robinson D. Mycoplasma and Ureapiasma.

Manual of Clinical Microbiology, 10th Ed. Washington, D.C., ASM Press: 970-985, 2011 ). Invasive infections of the bloodstream, CSF, and lungs sometimes occur due to M. hominis and Ureaplasma species in neonates (Wastes and Taylor-Robinso 2011 ). invasive disease may also occur in adults in the setting of immunodeficiency (Wastes and Taylor-Robinson 2011 ).

Treatment options for mycoplasmal and ureapiasma! infections are no longer clear- cut since macro! ide resistance is becoming very common in M. pneumoniae in Asia and is spreading gradually to Europe and North America; ietracyiine resistance rates may approach 50% in M. hominis and Ureaplasma species i some areas; and resistance to macroiides and fluoroquinolones has been well documented among the genital mycoplasmas (Wastes KB, Lysynyansky I, Bebear CM. (2014). Emerging antimicrobial resistance in

mycoplasmas of humans and animals. Molscutes Molecular Biology and Pathogenesis. G. Browning and C. Citti. Norfolk, UK, Caister Academic Press; 289-322). Patients who are immune-suppressed and those who have received numerous courses of antibiotics over time are at greater risk for having infections with drug-resistant organisms (Wastes 2014). For these reasons, new agents that are not affected by cross-resistance to other drug classes such as macroiides, tetracyclines, and fluoroquinolones are needed.

This small preliminary study has demonstrated that Compound 1 has in vitro activity against M. genita!ium, M. pneumoniae, U. ureaiyticum and U, parvu that is comparable to ievofioxacin, another agent targeting DMA replication, and its potency was unaffected by presence of mutations conferring fluoroquinolone resistance. Furthermore, resistance to macroiides and tetracyclines in Mycoplasma and Ureapiasma species appeared not to have any significant measurable effect on MiCs of Compound 1 , though more isolates should be tested to confirm this observation. Azithromycin was the most potent agent tested against M. genitaiium and M. pneumoniae in the absence of mutations that affect macroiide binding to the nbosomes. The MiC ₈₀ for Compound 1 was 4-fold less than azithromycin against Ureaplasma species, making it the most active drug among the four agents tested. Conclusions

Compound 1 activity in vitro against . pneumoniae, M. genitalium and Ureapiasma species was similar overall to ievofioxacin with ail iCs < 2 pg/mL, while its potency against M. hominis was somewhat less in terms of MIC _S o {4 pg/mL).

* The SCso (1 pg/ml) of Compound 1 was 4-foid iower than that of azithromycin against Ureapiasma species, making it the most potent of the four agents tested against these organisms.

The activity of Compound 1 in vitro against M. pneumoniae, M. hominis and Ureapiasma species was not affected by mutations conferring macro! ide or fluoroquinolone resistance, or by the presence of te† in the small number of isolates tested.

Compound 1 may be a potentially useful agent for further development as a possible treatment for infections caused by human mycoplasmas and ureaplasmas in the urogenital tract or respiratory tract.

Example 3. In Vitr Antibacterial Activity of Compound 1 against Potential Agents of Bioterrorism

The potential of Category A and B Seiect Agents for use as agents of bioterrorism is we!! documented. To this end, we established antimicrobial susceptibility profiles for compounds from multiple drug classes and for Compound 1 against multiple isolates each of Bacillus anthracis {B. anthracis), Burkhoideria mallei (S, mallei), Burkhoideria pseudomaliei (S. pseudomailei), Brucella abortus {B. abortus), Brucella meiitensis (B. melitensis), Brucella $uis (B. suis), Franciseila tularensis (F. tuiarensis) and Yersina pestis ( Y, pestis). Testing was conducted in a broth microdiiution: assay format following Clinical and Laboratory Standards Institute (CLSi) guidelines. Results were reported as the lowest concentration (pg/mL) of antimicrobiai agent that completely inhibited growth of the organism in the microdiiution wells visually.

MATERIALS AND METHODS

Antibactertats

Three (3) comparator compounds (doxycycline, Ievofioxacin and chloramphenicol) and Compound 1 were screened for antibacterial activity against multiple isolates each of Bacillus anthracis (B. anthracis), Burkhoideria mallei (B. mallei), Burkhoideria pseudomall&i (S. pseudomaliei), Brucella abortus (B. abortus), Brucella melitensis (B. melitensis), Brucella suis (B. suis), Franciseila tuiarensis {F. tularensis) and Yersina pestis (Y. pestis).

Compounds were prepared according to instructions provided by the Sponsor and in accordance with CLSi guidelines. A total of 12 concentrations each for ail test and comparator compounds were tested i triplicate. The concentration range was a two-foid diiution scheme with a starting concentration of 64 pg/rnL and an ending concentration of 0.031 MQ/mL. Bacteria! strains

Ten isolates each of B. anthracis, Y. pestis, B, mallei, B, pseudomallei, B, suls, 8. melitensis, B. abortus and 3 isoiaies of F. tuiar&nsis were utilized for drug screening (Tabie 6). In addition, the following quality control strains were included: E. coli 25922, S. aureus 29213, P. aeruginosa 27853, S, pneumoniae 49619 and £ coil 35218,

Tabie 6. Bacterial isolates Screened

ffi vitro susceptibHity test methods (as appropriate)

Testing was conducted utilizing the broth microdslution methodology outlined b CLSI guidelines. Briefly, testing was conducted using 96-wefl, U-bottom microplates with an assay volume of 0.2 ml/weli. Plates containing appropriate broth and two-fold dilutions of the test compounds were inoculated wit a targeted concentration of 5.0 x 105 CFU/ ^' mL (5.0 x 104 CFU/welS) of bacterial agent and subsequently incubated for 24 -72 hours depending on the agent. Following incubation, the plates were read visually and individual welis scored for turbidity, partial clearing or complete clearing. The MIC was reported as the lowest concentration ^g/mL) of drug that visually inhibited growth of the organism. Growth medium, inoculum preparation and incubation conditions are provided below in Table 7. Table 7. Growth Medium, inoculum Preparation and incubation Conditions

The results of the screen described above are shown in Table 8 and 9. Table 8. Antimicrobial Susceptibility of Compound 1 and Three Comparators Against Select Bacteria

≤ 0.031

< 0.031

0.5 0.031 < 0.125

< 0.031

0.063 4 0.25

0,25 0,5

0.25 0.25

0.125 0.25

4 0.125 0.25

4 0.25 0.25

Brucella abortus

0.063 0.063 0.5 0.25

1 0,25 0.25

0.5 0.063 0.25

0.063 0.25

16 0.5 0.5

0.5 0.25

0.5 0.25

0.5 < 0.25

>64 0.25

0.25

Yersinia pestis

< 0.25

0.5 0.25

0.5 0.25

0.5 < 0.25

16 < 0.125

FranciseHa

≤ 0.125 tularensis

16 0.125

0.5 & 0.031 < 0.25

0.125 < 0.031 < 0.25

0.125 < 0.031 4 ≤ 0.25

0.25 ≤ 0.031 < 0.25

Bacillus 0.5 0.031 4 < 0.25 stnt racis 0.125 < 0.031 ≤ 0.25

1 0.031 0.25

0.5 0.031 < 0.25

0,05 < 0.031 ≤ 0.25

0.125 < 0.031 < 0.25

8 0.25 0.5

0.125 0.5

Bru lla

16 0.25

meiitensis

0.25 0.25 0.125 0.5 8 0,125 4 0.5

8 0.125 4 0.5

8 0.125 2 0.25

8 0.125 4 0.5

16 0.125 4 0,5

2 0.063 2 0.5

1 0.063 1 0.25

1 0.063 1 0,5

1 0.125 2 0.5

1 0.063 2 0,5

Brucella suis

2 0.125 2 0.25

1 0.063 4 0.5

1 0.063 2 0,5

1 0.063 1 0.25

1 0.063 2 0.5

Table 9. hi vitro activity of Compound 1 (Mg/mi)