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USOO868O136B2

(12) United States Patent (10) Patent No.: US 8,680,136 B2 Hirst et al. (45) Date of Patent: Mar. 25, 2014

(54) CYCLIC BORONIC ACID ESTER WO WOO3,O70714 8, 2003 DERVATIVES AND THERAPEUTCUSES WO WO 2004/039859 5, 2004 THEREOF WO WO 2005/033090 4/2005 WO WO 2007/095638 8, 2007 WO WO 2009/046098 A1 4, 2009 (75) Inventors: Gavin Hirst, San Diego, CA (US); Raja WO WO 2009/064413 A1 5, 2009 Reddy, San Diego, CA (US); Scott WO WO 2009/064414 A1 5.2009 Hecker, Del Mar, CA (US); Maxim WO WO 2009/140309 A2 11/2009 Totroy San Deigo, CA (US); David C. WO WO 2010/1307082010/075286 A1 11,T 2010 Griffith, San Marcos, CA (US): Olga WO WO 2011/O17125 A1 2/2011 Rodny, Mill Valley, CA (US); Michael N. Dudley, San Diego, CA (US); Serge OTHER PUBLICATIONS Boyer, San Diego, CA (US) Fanetal (2009): STN International HCAPLUS database, Columbus (73) Assignee: Rempex Pharmaceuticals, Inc., San (OH), accession No. 2009: 425839.* Diego, CA (US) Vasil'ev et al (1977): STN International HCAPLUS database, Columbus (OH), accession No. 1977: 72730.* (*) Notice: Subject to any disclaimer, the term of this Allen et al., “Ansel's Pharmaceutical Dosage Forms and Drug Deliv patent is extended or adjusted under 35 ery Systems', 8th Edition (2004). U.S.C. 154(b) by 9 days. Arya et al., “Advances in asymmetric enolate methodology'. Tetra hedron (2000) 56:917-947. (21) Appl. No.: 13/205,112 Biedrzycki et al., “Derivatives of tetrahedral boronic acids”. J. Organomet. Chem. (1992) 431:255-270. (22) Filed: Aug. 8, 2011 Bou et al., “Cloning, nucleotide sequencing, and analysis of the gene encoding an AmpC beta-lactamase in Acinetobacter baumannii'. (65) Prior Publication Data Antimicrob Agents Chemother (2000) 44(2):428-432. Bou et al., “OXA-24, a novel class D beta-lactamase with US 2012/OO40932 A1 Feb. 16, 2012 carbapenemase activity in an Acinetobacter baumannii clinical strain'. Antimicrob Agents Chemother (2000) 44(6): 1556-1561. Related U.S. Application Data Erratum (2006) 50(6) 2280. Brosz et al., “Resolution of alpha-aminoboronic esters by (60) Provisional application No. 61/372,296, filed on Aug. diastereoselective crystallization with pinanediols. Confirmation by 10, 2010, provisional application No. 61/488,655, x-ray analysis”, Tetrahedron: Asymmetry (1997) 8(9): 1435-1440. filed on May 20, 2011. Clinical and Laboratory Standards Institute (formerly NCCLS, National Committee for Clinical Laboratory Standards). “Methods (51) Int. Cl. for Dilution of Antimicrobial Susceptibility Tests for Bacteria That A6 IK3I/44 (2006.01) Grow Aerobically”, CLSI (Jan. 2006) M7-A726(2). A6 IK3I/38 (2006.01) Clinical and Laboratory Standards Institute (formerly NCCLS, National Committee for Clinical Laboratory Standards). “Methods CO7D 213/02 (2006.01) for Dilution of Antimicrobial Susceptibility Tests for Bacteria That CO7D 33.3/10 (2006.01) Grow Aerobically”, CLSI (Jan. 2009) M07-A829(2). (52) U.S. Cl. Farquhar et al., “Intensely potent doxorubicin analogues: structure USPC ...... 514/438: 549/29: 549/76; 548/146; activity relationship”. J. Med. Chem. (1998) 41(6):965-972. 548/194; 546/304:546/311: 514/352 Ghosh et al., “Enantioselective total synthesis of (+)-largazole, a (58) Field of Classification Search potent inhibitor of histone deacetylase'. Org Lett. (2008) USPC ...... 549/29, 74,76; 548/146, 190, 194; 10(17):3907-3909. 546/304,311 514/352, 365, 370, 438 Gossinger et al., “Towards EPC-syntheses of the structural class of See application file for complete search history. cochleamycins and macquarimicins. Part 3: EPC-syntheses of the beta-keto lactone subunits and first attempts towards the syntheses of the pentacyclic of this group”. Tetrahedron (2007) (56) References Cited 63:8336-83.50. U.S. PATENT DOCUMENTS (Continued) 4,822,786 A 4, 1989 Zama et al. 5,888,998 A 3, 1999 Maiti et al. 7,271,186 B1 9, 2007 Shoichet et al. Primary Examiner — Golam MM Shameem 7,582,621 B2 9, 2009 Baker et al. (74) Attorney, Agent, or Firm — Knobbe, Martens, Olson & 2004, OO19203 A1 1/2004 Micetich et al. Bear, LLP 2006, OO1911.6 A1 1/2006 Conley et al. 2006/0210883 A1 9, 2006 Chen et al. 2010, 0120715 A1 5, 2010 Burns et al. (57) ABSTRACT 2010, O256092 A1 10/2010 Xia et al. Disclosed herein are antimicrobial compounds compositions, FOREIGN PATENT DOCUMENTS pharmaceutical compositions, the use and preparation JP 2003-229277 8, 2003 thereof. Some embodiments relate to 1 cyclic boronic acid JP 2004-291253 10, 2004 ester derivatives and their use as therapeutic agents. WO WO 87/05297 9, 1987 WO WO 89.10961 11, 1989 WO WO O2/O83884 10, 2002 27 Claims, 4 Drawing Sheets US 8,680,136 B2 Page 2

(56) References Cited Montefour et al., “Acinetobacterbaumannii: an emerging multidrug resistant pathogen in critical care'. Crit Care Nurse (2008) 28(1): 15 OTHER PUBLICATIONS 25; quiz 26. Morandi et al., “Structure-based optimization of cephalothin-ana Ishii et al., “In vitro potentiation of with ME 1071, a logue boronic acids as beta-lactamase inhibitors'. Bioorg Med Novel metallo-B-lactamase inhibitor, against metallo-B-lactamase Chem. (2008) 16(3): 1195-205. Epub Nov. 7, 2007. producing clinical isolates.” Antimicrob. Panek et al., “Diastereoselectivity in the borane methyl sulfide pro Agents Chemother, doi:10.1128/AAC.01397-09 (2010) 54(9):3625 moted hydroboration of alpha-alkoxy-beta, gamma.-unsaturated 3629. esters. Documentation of an alkoxy-directed hydroboration reac Ito et al., “An efficient constructive method for a tricyclic system: an tion”. J. Org. Chem. (1992) 57(20):5288-5290. important intermediate for the synthesis of tricycloclavulone”, Tet Perez et al., “Why are we afraid of Acinetobacter baumannii?”. rahedron Lett. (2003) 44:1259-1261. Expert Rev Anti Infect Ther. (2008) 6(3):269-71. Jadhav et al., “Direct synthesis of alpha-(tert Reissig et al., "High diastereoselection in the alkylation of siloxy Butoxycarbonyl)aminoalkyI-boronates from Substituted methyl cyclopropanecarboxylates: consequence of a (alphaHaloalkyl)boronates”. Org Chem. (1996) 61(22):7951-7954. pyramidal ester enolate anion?”. J. Am. Chem. Soc. (1982) Kotha et al., “Recent applications of the Suzuki-miyaura cross-cou 104:1735-1737. pling reaction in organic synthesis'. Tetrahedron (2002) 58:9633 Sawyer et al., “Physical properties and synthetic utility of 9695. a-alkoxyorganolithium species as studied through ligand selectivity Kumar et al., “Synthesis of intermediates for the lactone moiety of in tin-lithium exchange'. J. Am. Chem. Soc. (1988) 110:842-853. mevinic acids via tellurium chemistry”. J. Org. Chem... (1994) Souto et al., “Synthesis and biological characterization of the histone 59(17):4760-4764. deacetylase inhibitor largazole and c7-modified analogues'. J. Med. Li et al., “Novel macrocyclic HCV NS3 protease inhibitors derived Chem. (2010) 53(12):4654-4667. from a-amino cyclic boronates'. Bioorganic Med Chem Lett. (2010) Spiegel et al., “CP-263,114 synthetic studies. Construction of an 20:5695-5700. isotwistane ring system via rhodium carbenoid C-H insertion'. Li et al., “Synthesis and evaluation of novel alpha-amino cyclic Tetrahedron (2002) 58:6545-6554. boronates as inhibitors of HCV NS3 protease'. Bioorg Med Chem Wang et al., “Recognition and resistance in TEM beta-lactamase'. Lett. (2010) 20:3550-3556. Biochemistry (2003) 42(28):8434-8444. Matteson, “Boronic esters in stereodirected synthesis”, Tetrahedron Wohlrab et al., “Total synthesis of plusbacin A: a depsipeptide (1989)45(7): 1859-1885. active against -resistant bacteria'. J. Am. Matteson et al., “A Stereospecific convergent coupling of Chem. Soc. (2007) 129:41.75-4177. nucleophilic and electrophilic chiral carbons'. J. Am. Chem. Soc. Yamamoto et al., “Iridium-catalyzed hydroboration of alkenes with (1989) 1 11:4399-4402. pinacolborane”, Tetrahedron (2004) 60:10695-10700. Matteson et al., “Synthesis of asymmetrically deuterated glycerol International Search Report and Written Opinion dated Sep. 14, 2011 and dibenzylglyceraldehyde via boronic esters”. J. Am. Chem. Soc. for International Patent Application No. PCT/US2011/046957, filed (1990) 112:3964-3969. Aug. 8, 2011. Meanwell. “Synopsis of Some recent tactical application of bioisosteres in drug design”. J. Med. Chem. (2011) 54:2529-2591. * cited by examiner U.S. Patent Mar. 25, 2014 Sheet 1 of 4 US 8,680,136 B2

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U.S. Patent Mar. 25, 2014 Sheet 4 of 4 US 8,680,136 B2

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FIG. 4 US 8,680,136 B2 1. 2 CYCLIC BORONCACDESTER tion of single amino acid substitutions in TEM-1 and SHV-1 DERVATIVES AND THERAPEUTIC USES resulted in the emergence of the extended-spectrum B-lacta THEREOF mase (ESBL) phenotype. New B-lactamases have recently evolved that hydrolyze RELATED APPLICATIONS the class of antimicrobials, including , , , , andertapenem, as well as This application claims the benefit of U.S. Provisional other B-lactam antibiotics. These carbapenemases belong to Application Nos. 61/372,296, filed Aug. 10, 2010, and molecular classes A, B, and D. Class A carbapenemases of the 61/488,655, filed May 20, 2011, both of which are incorpo KPC-type predominantly in Klebsiella pneumoniae but now rated herein by reference in their entirety. 10 also reported in other Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. The KPC carbap FIELD OF THE INVENTION enemase was first described in 1996 in North Carolina, but since then has disseminated widely in the US. It has been The present invention relates to antimicrobial compounds, particularly problematic in the New York City area, where compositions, their use and preparation as therapeutic agents. 15 several reports of spread within major hospitals and patient In particular, the present invention relates to cyclic boronic morbidity have been reported. These enzymes have also been acid ester compounds. recently reported in France, Greece, Sweden, United King dom, and an outbreak in Germany has recently been reported. BACKGROUND OF THE INVENTION Treatment of resistant strains with carbapenems can be asso ciated with poor outcomes. Antibiotics have been effective tools in the treatment of Another mechanism off-lactamase mediated resistance to infectious diseases during the last half-century. From the carbapenems involves combination of permeability or efflux development of antibiotic therapy to the late 1980s there was mechanisms combined with hyper production of beta-lacta almost complete control over bacterial infections in devel 25 mases. One example is the loss of a porin combined in hyper oped countries. However, in response to the pressure of anti production of ampC beta-lactamase results in resistance to biotic usage, multiple resistance mechanisms have become imipenem in Pseudomonas aeruginosa. Efflux pump over widespread and are threatening the clinical utility of anti expression combined with hyperproduction of the ampC bacterial therapy. The increase in antibiotic resistant strains B-lactamase can also result in resistance to a carbapenem Such has been particularly common in major hospitals and care 30 as meropenem. centers. The consequences of the increase in resistant strains Because there are three major molecular classes of serine include higher morbidity and mortality, longer patient hospi based f-lactamases, and each of these classes contains sig talization, and an increase in treatment costs nificant numbers off-lactamase variants, inhibition of one or Various bacteria have evolved -lactam deactivating a small number of B-lactamases is unlikely to be of therapeu enzymes, namely, B-lactamases, that counter the efficacy of 35 tic value. Legacy B-lactamase inhibitors are largely ineffec the various B-lactams. B-lactamases can be grouped into 4 tive against at least Class A carbapenemases, against the classes based on their amino acid sequences, namely, Ambler chromosomal and plasmid-mediated Class C cephalospori classes A, B, C, and D. Enzymes in classes A, C, and D nases and against many of the Class Doxacillinases. There include active-site serine B-lactamases, and class B enzymes, fore, there is a need for improved f-lactamase inhibitors. which are encountered less frequently, are Zn-dependent. 40 These enzymes catalyze the chemical degradation of B-lac SUMMARY OF THE INVENTION tamantibiotics, rendering them inactive. Some B-lactamases can be transferred within and between various bacterial The present invention relates to antimicrobial agents and strains and species. The rapid spread of bacterial resistance potentiators thereof. Some embodiments include com and the evolution of multi-resistant strains severely limits 45 pounds, compositions, pharmaceutical compositions, use and B-lactam treatment options available. preparation thereof. In particular, some embodiments, relate The increase of class D B-lactamase-expressing bacterium to cyclic boronic acid ester derivatives. strains such as Acinetobacter baumannii has become an Some embodiments include compounds having the struc emerging multidrug-resistant threat. A. baumannii strains ture of formula I: express A, C, and D class B-lactamases. The class D B-lacta 50 mases such as the OXA families are particularly effective at destroying carbapenem type B-lactam antibiotics, e.g., imi , the active carbapenems component of Merck's Pri maxin(R) (Montefour, K. et al. Crit. Care Nurse 2008, 28, 15: Perez, F. et al. Expert Rev. Anti Infect. Ther. 2008, 6, 269; 55 Bou, G.; Martinez-Beltran, J. Antimicrob. Agents Chemother. 2000, 40, 428. 2006, 50, 2280; Bou, G. et al., J. Antimicrob. Agents Chemother. 2000, 44, 1556). This has imposed a pressing threat to the effective use of drugs in that or a pharmaceutically acceptable salt thereof, wherein: category to treat and prevent bacterial infections. Indeed the 60 Y is a 1-4 atom alkylene or 2-4 atom alkenylene linker, number of catalogued serine-based B-lactamases has optionally substituted by one or more substituents selected exploded from less than ten in the 1970s to over 300 variants. from the group consisting of C1, F, CN, CFs, R, OR, These issues fostered the development of five "generations' - C(=O)NR'R'', and C(=O)CR, wherein said alky of . When initially released into clinical prac lene or alkenylene linker is optionally fused to an optionally tice, extended-spectrum cephalosporins resisted hydrolysis 65 substituted aryl, optionally substituted heteroaryl, optionally by the prevalent class A B-lactamases, TEM-1 and SHV-1. substituted carbocyclyl, or optionally substituted heterocy However, the development of resistant strains by the evolu clyl; US 8,680,136 B2 3 4 R" is selected from a group consisting of -Coalkyl, (ii) a geminal R and R together form —C-9 —Coalkenyl, -Coalkynyl, -NR'R''. —CoalkylR'', alkenylenylCOR, or —CoalkenylR'', —C-9alkynylR'', -carbocyclyl-R'', —CH(OH)CalkylR, —CH(OH)CalkenylR, —CH (iii) each R attached to a ring atom forming part of a (OH)CalkynylR, -CH(OH)carbocyclyl-R, C(=O) substituted or unsubstituted aryl is absent; R, —C(=O)CoalkylR, —C(=O)CoalkenylR. each R is independently selected from a group consisting —C(=O)CoalkynylR, —C(=O)Cocarbocyclyl-R, of H. —Coalkyl, Coalkenyl, —Coalkynyl, carbocyclyl, C(=O)NR'R'', N(R)C(=O)R, N(R)C(=O) —CoalkylR'', —CoalkenylR'', —CoalkynylR'', -car NR'R'', N(R)C(=O)CR, N(R)C(=O)C(-NR) bocyclyl-R', substituted or unsubstituted aryl, substituted or R, N(R)C(=O)C(—CRR)R, N(R)C(=O)Cl. 10 unsubstituted heteroaryl, substituted or unsubstituted car alkylN(R)C(=O)R, N(R)C(-NR)R, C(-NR) bocyclyl, and substituted or unsubstituted heterocyclyl: NR'R'', N=C(R)NR'R'', N(R)SOR, N(R) each R" is independently selected from a group consisting SONR'R'', N=CHR, substituted or unsubstituted aryl, of H, -Calkyl, -OR. -CH(=NH), —C(=O)CR, substituted or unsubstituted heteroaryl, substituted or unsub 15 substituted or unsubstituted aryl, substituted or unsubstituted stituted carbocyclyl, and substituted or unsubstituted hetero heteroaryl, substituted or unsubstituted carbocyclyl, and sub cyclyl; stituted or unsubstituted heterocyclyl: R is selected from a group consisting of H. —Coalkyl, each R'' is independently selected from a group consisting Coalkenyl, -Coalkynyl, carbocyclyl, -CoalkylR', of substituted or unsubstituted aryl, substituted or unsubsti —CoalkenylR'', —CoalkynylR'', carbocyclyl-R'', tuted heteroaryl, substituted or unsubstituted carbocyclyl, —C(=O)CR, CalkylCO.R. —CoalkenylCO.R. and substituted or unsubstituted heterocyclyl: —C-alkynylCO.R. and -carbocyclyl-CO.R. or alterna tively: X is selected from a group consisting of H. —CO.R', and (i) Randan Rare taken together with the atoms to which carboxylic acid isosteres; they are attached to form a substituted or unsubstituted 25 R'' is selected from a group consisting9. of H., Coalkyl,C., alkyl carbocyclyl or substituted or unsubstituted heterocyclyl, —(CH) R, C(R').OC(O)Coalkyl, -C(R').OC (ii) R' and a carbon atom in Y are taken together with (O)R'', C(R').OC(O)OC-alkyl and C(R').OC(O) intervening atoms to form a substituted or unsubstituted OR; carbocyclyl or substituted or unsubstitued heterocyclyl, each R" is independently selected from a group consisting O 30 (iii) R' is absent when the carbon to which it is attached is of H and Calkyl; and a ring atom in an aryl or heteroaryl ring: m is independently Zero or an integer from 1 to 2, wherein each R" is independently selected from a group consisting each Coalkyl, Coalkenyl, and Coalkynyl is independently of H. halo. —Coalkyl, —Coalkenyl, —Coalkynyl, optionally substituted. NRR 10, OR, —CalkylCO.R. C2-9 35 In some embodiments, the compound of formula I has the alkenylCO.R. —CoalkynylCO.R. and -carbocyclyl structure of formula II: CO.R. or independently: (i) Randan Rare taken together with the atoms to which

they are attached to form a substituted or unsubstituted II carbocyclyl or substituted or unsubstituted heterocyclyl, 40 (ii) Randan Rare taken together with the atoms to which they are attached to form a substituted or unsubstituted carbocyclyl or substituted or unsubstituted heterocyclyl, (iii) an R and a carbon atom in Y are taken together with intervening atoms to form a substituted or unsubstituted 45 carbocyclyl or substituted or unsubstitued heterocyclyl, (iv) each of the following conditions are met: (a) Y is a 3-4 atom alkylene or 3-4 atom alkenylene linker, or a pharmaceutically acceptable salt thereof, wherein: (b) R is absent, 50 the bond represented by a dashed and solid line represents (c) R' and a carbon atom in Y are taken together with a bond selected from the group consisting of a single bond and intervening atoms to form a Substituted or unsubsti a double bond with the proviso that the dashed and solid line tuted aryl or a substituted or unsubstituted heteroaryl, can only be a double bond when n is 1: and RandR are independently selected from a group consist (d) each Rattached to a ring atom forming part of the 55 ing of C1, F, CN, CF, R, OR, C(=O)NR'R'', and substituted or unsubstituted aryl or a substituted or —C(=O)CR'; or alternatively, R and Rare taken together unsubstituted heteroaryl formed by R and Y is with the atoms to which they are attached to form a substi absent; tuted or unsubstituted aryl, substituted or unsubstituted het each R is independently selected from a group consisting eroaryl, substituted or unsubstituted carbocyclyl or substi of H. halo. —Coalkyl, —Coalkenyl, —Coalkynyl, 60 NRR 10, OR, —CalkylCO.R. C2-9 tuted or unsubstituted heterocyclyl: alkenylCO.R. —CoalkynylCO.R. -carbocyclyl-CO.R. RandR are independently selected from a group consist or independently: ing of C1, F, CN, CF, R, OR, C(=O)NR'R'', and (i) an R and an R are taken together with the atoms to —C(=O)CR, with the proviso that if the bond represented which they are attached to form a substituted or unsub 65 by a dashed and solid line is a double bond then Rand Rare stituted carbocyclyl or substituted or unsubstituted het absent; and erocyclyl, n is independently Zero or an integer from 1 to 2. US 8,680,136 B2 5 6 In some embodiments, the compound of formula I the -continued structure of formula IIIa or IIIb: IVc

R4

IIIa

10

or a pharmaceutically acceptable salt thereof, wherein: IIIb. 15 the bond represented by a dashed and solid line represents a bond selected from the group consisting of a single bond and a double bond; each R and each R" are independently selected from a group consisting of C1, F, CN, CF, R, OR, —C(=O) NR'R'', and C(=O)CR: or alternatively, an Randan R' are taken together with the atoms to which they are attached to form a substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted car or a pharmaceutically acceptable salt thereof, wherein: bocyclyl or substituted or unsubstituted heterocyclyl: 25 each R and each R are independently selected from a the bond represented by a dashed and solid line represents group consisting of C1, F, CN, CF, R, OR, —C(=O) a bond selected from the group consisting of a single bond and NR'R'', and C(=O)CR, with the proviso that if the bond a double bond; represented by a dashed and solid line is a double bond then each R and Rare independently selected from a group the Rand Rattached to the carbonatoms bonded that bond consisting of C1, F, CN, CF, -R, OR, —C(=O) 30 are absent. NR'R'', and –C(=O)CR; or alternatively, an R and R' Some embodiments include a pharmaceutical composition are taken together with the atoms to which they are attached comprising a therapeutically effective amount of any one of to form a substituted or unsubstituted aryl, substituted or the foregoing compounds and a pharmaceutically acceptable unsubstituted heteroaryl, substituted or unsubstituted car excipient. bocyclyl or substituted or unsubstituted heterocyclyl: 35 Some embodiments include any one of the foregoing com pounds or compositions for use in the treatment or prevention each R and Rare independently selected from a group of a bacterial infection. consisting of C1, F, CN, CF, -R, OR, —C(=O) Some embodiments include methods for treating or pre NR'R'', and C(=O)CR, with the proviso that if the bond venting a bacterial infection comprising administering to a represented by a dashed and solid line is a double bond then 40 subject in need thereof, an effective amount of any one of the Rand Rare absent. foregoing compounds or compositions. In some embodiments, the compound of formula I has the Some embodiments include the use of any one of the fore structure of formula IVa, IVb, or IVc: going compounds or compositions in the preparation of a medicament for the treatment or prevention of a bacterial 45 infection. Some embodiments further comprise administering an IV a additional medicament, either is a separate composition or in the same composition. In Some embodiments, the additional medicament includes 50 an antibacterial agent, antifungal agent, an antiviral agent, an anti-inflammatory agent or an anti-allergic agent. In some embodiments, the additional medicament com prises an antibacterial agent such as a B-lactam. In some embodiments, the B-lactam includes , 55 (, , , , ), , (Car IVb indacillin), , , , , , (), , (G), Clometocillin, Benzathine benzylpeni 60 cillin, Procaine benzylpenicillin, , , (V), , Benzathine phe noxymethylpenicillin, , (Dicloxacil lin, ), , Meticillin, , Faro penem, Biapenem, Doripenem, , Imipenem, 65 Meropenem, , , , Cefazo lin, , , , , Cefalo nium, Cefaloridine, , , , US 8,680,136 B2 7 8 , , , , , hyicus Subsp. hyicus, Staphylococcus haemolyticus, Staphy , , Cefninox, , , lococcus hominis, or Staphylococcus saccharolyticus. , , , , Cefu Zonam, , , Cefnmetazole, , In some embodiments, the infection that is treated or pre , , , , Cefdal vented comprises a bacteria that includes Pseudomonas oxime, , , , Cefnmenoxime, aeruginosa, Pseudomonas fluorescens, Stenotrophomonas , , , Ce?pimizole, Cef maltophilia, Escherichia coli, Citrobacter freundii, Salmo piramide, , , , , nella typhimurium, Salmonella typhi, Salmonella paratyphi, , , . Latamoxe?, , Salmonella enteritidis, Shigella dysenteriae, Shigella flex , Ce?pirome, Cefauinome, , Ceftaro 10 neri, Shigella Sonnei, Enterobacter cloacae, Enterobacter line, CXA-101, RWJ-54428, MC-04,546, ME 1036, aerogenes, Klebsiella pneumoniae, Klebsiella Oxytoca, Ser BAL30072, SYN 2416, , Cefauinome, , , , , RWJ-442831, RWJ ratia marcescens, Acinetobacter calcoaceticus, Acineto 333441, or RWJ-333442. bacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, In some embodiments, the B-lactam includes Ceftazidime, 15 Yersinia pseudotuberculosis, Yersinia intermedia, Haemo Biapenem, Doripenem, Ertapenem, Imipenem, Meropenem, philus influenzae, Haemophilus parainfluenzae, Haemophi or Panipenem. lus haemolyticus, Haemophilus parahaemolyticus, Helico In some embodiments, the 3-lactam is selected from AZtre bacter pylori, Campylobacter fetus, Campylobacter jejuni, onam, Tigemonam, BAL30072, SYN 2416, or Carumonam. Campylobacter coli, Vibrio cholerae, Vibrio parahaemolyti In Some embodiments, the B-lactam Tigemonam, the com position is suitable for oral administration, X is COR'', cus, Legionella pneumophila, Listeria monocytogenes, Neis and R' is selected from a group consisting of Coalkyl, seria gonorrhoeae, Neisseria meningitidis, Moraxella, —(CH) R, C(R').OC(O)Coalkyl, -C(R').OC Bacteroides fragilis, Bacteroides vulgatus, Bacteroides (OR". —C(R').OC(O)CC-alkyl and C(R').OC(O) Ovalus, Bacteroides thetaiotaomicron, Bacteroides unifor OR''. 25 mis, Bacteroides eggerthii, or Bacteroides splanchnicus. In some embodiments, the infection that is treated or pre vented comprises a bacteria that includes Pseudomonas Some embodiments include a sterile container, comprising aeruginosa, Pseudomonas fluorescens, Pseudomonas aci any one of the foregoing compounds in Solid form and an dovorans, Pseudomonas alcaligenes, Pseudomonas putida, antibacterial agent in Solid form. In some embodiments, the Stenotrophomonas maltophilia, Burkholderia cepacia, Aero 30 antimicrobial agent is one of the additional medicaments monas hydrophilia, Escherichia coli, Citrobacter freundii, described above. Some embodiments include a method of Salmonella typhimurium, Salmonella typhi, Salmonella preparing a pharmaceutical composition for administration, paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shi comprising reconstituting the contents of the sterile container gella flexneri, Shigella sonnei, Enterobacter cloacae, Entero 35 using a pharmaceutically acceptable diluent. In some bacter aerogenes, Klebsiella pneumoniae, Klebsiella Oxy embodiments, the reconstituted Solution is administered foca, Serratia marcescens, Francisella tularensis, intravenously to a patient. Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia 40 BRIEF DESCRIPTION OF THE DRAWINGS Stuartii, Acinetobacter baumannii, Acinetobacter calcoace ticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia inter FIG. 1 is a graph depicting the plasma concentration profile of a cyclic boronic acid ester derivative as a function of time media, Bordetella pertussis, Bordetella parapertussis, Bor after administration to Sprague Dawley rats. detella bronchiseptica, Haemophilus influenzae, Haemophi 45 lus parainfluenzae, Haemophilus haemolyticus, FIG. 2 is a graph depicting the plasma concentration profile Haemophilus parahaemolyticus, Haemophilus ducreyi, Pas of a prodrug of the cyclic boronic acid ester derivative of FIG. teurella multocida, Pasteurella haemolytica, Branhamella 1 as a function of time after administration to Sprague Dawley catarrhalis, Helicobacter pylori, Campylobacter fetus, ratS. Campylobacter jejuni, Campylobacter coli, Borrelia burg 50 FIG.3 is an X-ray powder diffraction of a crystalline form dorferi, Vibrio cholerae, Vibrio parahaemolyticus, of a cyclic boronic acid ester derivative. Legionella pneumophila, Listeria monocytogenes, Neisseria FIG. 4 is a graph depicting an overlay of differential scan gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, ning calorimetry and thermogravimetric results for the crys Gardnerella vaginalis, Bacteroides fragilis, Bacteroides dis 55 talline form of FIG. 3. tasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides Ovalus, Bacteroides thetaiotaomicron, DETAILED DESCRIPTION Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile, Mycobacterium tubercu 60 The present invention relates to antimicrobial agents and losis, Mycobacterium avium, Mycobacterium intracellulare, potentiators thereof. Some embodiments include com Mycobacterium leprae, Corynebacterium diphtheriae, pounds, compositions, pharmaceutical compositions, uses Corynebacterium ulcerans, Streptococcus pneumoniae, thereof, including methods of preparation, and methods of Streptococcus agalactiae, Streptococcus pyogenes, Entero treatment. In particular, the present invention relates to cyclic coccus faecalis, Enterococcus faecium, Staphylococcus 65 boronic acid ester derivatives. In some embodiments, the aureus, Staphylococcus epidermidis, Staphylococcus cyclic boronic acid ester derivatives have the structure of Saprophyticus, Staphylococcus intermedius, Staphylococcus formula I, II, IIIa, IIIb, IVa, IVb, or IVc as described above. US 8,680,136 B2 9 10 Some embodiments of the compound of formula II have unsubstituted aryl, substituted or unsubstituted heteroaryl, the defined 3,6-cis-stereochemistry shown in formula IIa. Substituted or unsubstituted —(CH-)-carbocyclyl, and substituted or unsubstituted heterocyclyl: each R" is independently selected from a group consisting IIa 5 of H. Coalkyl, —OR, —CH(=NH), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl, and substituted or unsubstituted heterocyclyl; and X is selected from a group consisting of H. —COH and 10 carboxylic acid isosteres. In some embodiments of compounds of formulas II, IIa, or R7 R8 IIb, n is 1. In some embodiments of compounds of formulas II, IIa, or or a pharmaceutically acceptable salt thereof. 15 IIb, n is zero. In some embodiments of compounds of formulas II, IIa, or Some embodiments of the compound of formula II have IIb, n is 2. the defined 3,6-trans-stereochemistry shown in formula IIb: Some embodiments of the compounds of formula IIIa or IIIb have the 3.7-cis-stereochemistry shown in formula IIIc and IIId:

IIb

IIIc

25 or a pharmaceutically acceptable salt thereof. In one embodiment of the compound of formula II: 30 R" is selected from a group consisting of -Coalkyl, III - Coalkenyl, - Coalkynyl, NR'R'', CoalkylR'', —CoalkenylR'', —CoalkynylR'', —CH(OH)C-9 alkylR, —CH(OH)CoalkenylR, —CH(OH)Coalky nylR, C(=O)R. —C(=O)CalkylR, C(=O)Cl. 35 alkenylR, C(=O)C-alkynylR, C(=O)NR'R'', N(R)C(=O)R, N(R)C(=O)NR'R'', N(R)C (—O)OR, N(R)C(=O)C(—NR)R, N(R)C(=O) CalkylN(R)C(=O)R. N(R)C(—NR)R’, C(-NRNR'R'', N=C(R)NR'R'', N(R)SOR, 40 or a pharmaceutically acceptable salt thereof. -N(R)SONR'R'', substituted or unsubstituted aryl, sub Some embodiments of the compounds of formula IIIa or stituted or unsubstituted heteroaryl, substituted or unsubsti IIIb have the 3,7-trans-stereochemistry shown informula IIIe tuted carbocyclyl, and substituted or unsubstituted heterocy and IIIf: clyl; R is selected from a group consisting of H. —Coalkyl, 45

Coalkenyl, -Coalkynyl, -CoalkylR'', CoalkenylR', IIIe —C-alkynylR'', —C(=O)CR, and —CalkylCO.R. —CoalkenylCO.R. and —CoalkynylCO.R. or alterna tively Randan Rare taken together with the atoms to which they are attached to form a substituted or unsubstituted car 50 bocyclyl or substituted or unsubstituted heterocyclyl: each R" is independently selected from a group consisting of H, -NR'R''. -OR, and - CoalkylCO.R. C. alkenylCO.R. and CalkynylCOR, or independently, IIIf R and an R or independently an R and an R are taken 55 together with the atoms to which they are attached to form a substituted or unsubstituted carbocyclyl or substituted or unsubstituted heterocyclyl: each R is independently selected from a group consisting of H, -NR'R''. -OR, and - CoalkylCO.R. C. 60 alkenylCO.R. and CalkynylCOR, or independently, an Randan Rare taken together with the atoms to which they are attached to form a substituted or unsubstituted car bocyclyl or substituted or unsubstituted heterocyclyl: or a pharmaceutically acceptable salt thereof. each R is independently selected from a group consisting 65 Some embodiments of the compounds of formulas IVa, of H. —Coalkyl, Coalkenyl, -Coalkynyl. —C. IVb, or IVc have the 3,8-cis-stereochemistry shown in for 9alkylR'', CoalkenylR'', C-alkynylR'', substituted or mula IVd, IVe, and IVf: US 8,680,136 B2 11 12 -continued

IVd IV

10

or a pharmaceutically acceptable salt thereof.

IWe 15 In some embodiments of the compounds of formulas II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, each R. R. R. and Rare hydrogen. In some embodiments of the compounds of formulas II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, at least one R is substituted or unsubstituted aryl. In some embodiments of the compounds of formulas II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, at least one R is substituted or unsubstituted aryl. In some embodiments of the compounds of formulas II, 25 IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, at least one R and Rare taken together with the atoms to which they are attached to form a substituted or unsubsti tuted aryl. IVf In some embodiments of the compounds of formulas II, 30 IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, the bond represented by a dashed and solid line is a single bond. In other embodiments, the bond represented by a dashed and solid line is a double bond. In some embodiments of the compounds of formulas I, II, 35 IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, Rand each RandR is hydrogen. In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R' is NHC(=O)C-alkylR''. In some such embodi or a pharmaceutically acceptable salt thereof. 40 ments, R'' is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In some such embodiments, R' Some embodiments of the compounds of formulas IVa, is thien-2-yl. IVb, or IVc have the 3.8-trans-stereochemistry shown in for In some embodiments of the compounds of formulas I, II, mula IV.g., IVh, and IVi: IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and 45 IVi, R is NHC(=O)C(=NOR)R’, wherein R is selected from the group consisting of Coalkyl, Substituted or IVg unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl and substituted or unsubstituted heterocyclyl. 50 In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R is NHC(=O)CoalkylR''. In some such embodi ments, R'' is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted car 55 bocyclyl, or substituted or unsubstituted heterocyclyl. In some embodiments of the compounds of formulas I, II, IVh. IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R is NHC(=O)R’, wherein R is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, 60 substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R is NR'R''. 65 In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R is CalkylR''. US 8,680,136 B2 13 14 In some embodiments of the compounds of formulas I, II, Some embodiments include a compound selected from the IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and group consisting of: IVi, R is CH(OH)CalkylR. In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R is C(=O)Calkyl R. In some embodiments of the compounds of formulas I, II, O IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R is C(=O)NR'R''. In some embodiments of the compounds of formulas I, II, 10 HN IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and B-O OH, IVi, R is N(R)C(=O)NR'R''. / In some embodiments of the compounds of formulas I, II, HO O IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R is N(R)C(=O)C R. 15 In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and IVi, R is N(R)C(=O)CalkylN(R)C(=O)R’. In some embodiments of the compounds of formulas I, II, O IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and HN IVi, R is N(R)C(-NR)R. B-O OH, In some embodiments of the compounds of formulas I, II, W IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and HO O IVi, R is C(-NR)NR'R''. 25 O In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and HN IVi, R is N–C(R)NR'R''. In some embodiments of the compounds of formulas I, II, / IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and 30 HO O). IVi, R is C(=O)C(—NR)R. In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and O IVi, R is N(R)SO.R. In some embodiments of the compounds of formulas I, II, 35 D-( IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and HN IVi, R is N(R)SONR'R''. B - O OH, In some embodiments of the compounds of formulas I, II, / IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and HO O IVi, X is -COH. 40 e In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and / IVi, X is a carboxylic acid isostere. In some such embodi ments, the carboxylic acid isostere is selected from the group consisting of -P(O)(OR), -P(O)(R)(OR), -P(O) 45 O (OR''), P(O)(R)(OR), CON(R)OH, -SOH, HN —SON(R)OH, and B-O OH, / HO O 50 2 NN. // HN -N 55 wherein R' is selected from the group consisting of H. R. O —C(R').OC(O)Calkyl, -C(R').OC(O)R'', C(R'). OC(O)OC-alkyl and C(R').OC(O)OR''. HN B- O OH, In some embodiments of the compounds of formulas I, II, / IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and 60 HO O IVi, m is 1. C In some embodiments of the compounds of formulas I, II, IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and -- IVi, R. R7 and Rare H. HN B-O OH, In some embodiments of the compounds of formulas I, II, 65 / IIa, IIIa, IIIb, IIIc, IVa., IVb. IVc. IVd, IVe, IVf, IVg, IVh, and HO O IVi, R7 is H; R is - CoalkylCOR: and R is H.

US 8,680,136 B2 17 -continued -continued NH2 NH2 H N Ns O

O B 1 in HO O OH, HO1 No OH, 21 NH2

OH, 10 N N O

O B HO1 No OH,

15 N O H HO N 21 O HO1 B No OH,

NH HO O OH, H , N 1 O O B HO1 No OH, 25

O OH, 30 HO O OH, HO O

35 NH O B HO1 No OH, HN 2 N-1\s O 40 O B HO1 No OH,

45 rs O HO O OH, N 21 O HO1 B No OH, H OH,

50

55

60

1. HO1 No OH, 65 HO O OH, US 8,680,136 B2 19 20 -continued -continued H N 2 N O f N O B 5 S O 2 HO1 No OH, s H HN HN N / 21 O -o O Nan O B 10 HO O HO1 No OH, H F N NH2 O

O B Ns \( 15 HO1 No OH,

r O O O B 2O HO1 No OH, O N H nN.1N OH, B- O S. 25 / O HO

30 O N

r OH, 'litt B- O 35 / HO 4 o O 40 N / s OH, N N 't V H B- O O / HO 45

50 f

55 - /

60 N / N N

65

US 8,680,136 B2 25 26 -continued -continued

HN B-O OH, / O HO - - N-O OH, 10 HN B-O OH, W O HO

15

O

25 2 OH, 4 / HO O-N 30 OH, O a OH, 35

40

45 OH, HO

50 OH, HN B-O OH, O HO 55 OH

HN 60 B-O OH, W O HO N OH,

65 OH US 8,680,136 B2 27 28 -continued -continued "N-Nur O

O N H OH, B- O OH, / M 10 HO O HO O MeO-N O a S f O N HN 15 f \ B- O OH, HN S Ho O B-O OH, and HOOC / -)-./ HN HO O 21 HO N - 2 S f O (- Z NH O-N O 25 HO-N HN O B B- O OH, HO1 No OH, / --/ NH2 HO O OH 30 HO -" N N or a pharmaceutically acceptable salt thereof. Some embodiments include compounds selected from the Z group consisting of: O b-21 O 35 O B HO1 No OH, HOC 2 f O S O N O 40 HOC HN HO-N/ N w 'it > -o B- O OH, B-O / HO O HO 45 HOC \ / O a HOC 50 S O N O HN B- O OH, / HOOC / N v 't > -o HO O H B-O HOC O M 55 HO ... . B-O OH, / HO O 2 f 60 HOC O S O N O

HN HOHC / N w 't > -o H B- O B-O OH, / 65 / HO HO O US 8,680,136 B2

-continued -continued HOC

HO HOC HN B-O OH W O HN HO O Hi, m{ B-O 10 O HO

HN B-O OH, 15 / O HO O OH,

COH,

OH

25 OH,

30

HO O OH, 35

OH,

40 N -( M B- O OH, N O HO O

45

50 OH,

HO O OH,

55

60

65 O OH, US 8,680,136 B2 31 32 -continued not aromatic. In various embodiments, carbocyclyl groups can either be unsubstituted or substituted with one or more Substituents, e.g., halogen, alkoxy, acyloxy, amino, amido, cyano, nitro, hydroxyl, mercapto, carboxy, carbonyl, benzy loxy, aryl, heteroaryl, or other functionality that may be Suit O ably blocked with a protecting group. Typically, carbocyclyl groups will comprise 3 to 10 carbon atoms, preferably 3 to 6. o-( As used herein, “cycloalkyl means a fully saturated car HN bocyclyl ring system. Examples include cyclopropyl. B-O OH, 10 cyclobutyl, cyclopentyl, and cyclohexyl. M As used herein, "cycloalkenyl' means a carbocyclyl ring HO O system having at least one double bond. An example is cyclo hexenyl. or a pharmaceutically acceptable salt thereof. As used herein, “lower alkyl means a subset of alkyl, and Definitions 15 thus is a hydrocarbon substituent, which is linear, or Terms and Substituents are given their ordinary meaning branched. Preferred lower alkyls are of 1 to about 4 carbons, unless defined otherwise, and may be defined when intro and may be branched or linear. Examples of lower alkyl duced and retain their definitions throughout unless otherwise include butyl, propyl, isopropyl, ethyl, and methyl Likewise, specified, and retain their definitions whether alone or as part radicals using the terminology “lower” refer to radicals pref of another group unless otherwise specified. erably with 1 to about 4 carbons in the alkyl portion of the As used herein, “alkyl means a branched, or straight chain radical. saturated chemical group containing only carbon and hydro As used herein, “aryl' means an aromatic radical having a gen, Such as methyl, isopropyl, isobutyl, sec-butyl and pentyl. single-ring (e.g., phenyl) or multiple condensed rings (e.g., In various embodiments, alkyl groups can either be unsubsti naphthyl or anthryl) with only carbon atoms present in the tuted or Substituted with one or more Substituents, e.g., halo 25 ring backbone. In various embodiments, aryl groups can gen, hydroxyl, Substituted hydroxyl, acyloxy, amino, Substi either be unsubstituted or substituted with one or more sub tuted amino, amido, cyano, nitro, guanidino, amidino, stituents, e.g., amino, cyano, hydroxyl, lower alkyl, haloalkyl, mercapto, Substituted mercapto, carboxy, Sulfonyloxy, carbo alkoxy, nitro, halo, mercapto, carboxy, carbonyl, benzyloxy, nyl, benzyloxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, or aryl, heteroaryl, and other substituents. Some embodiments other functionality that may be suitably blocked with a pro 30 include Substitution with an alkoxy group, which may be tecting group. Typically, alkyl groups will comprise 1 to 20 further Substituted with one or more Substituents, e.g., amino, carbonatoms, 1 to 9 carbonatoms, preferably 1 to 6, and more cyano, hydroxyl, lower alkyl, haloalkyl, alkoxy, nitro, halo, preferably 1 to 5 carbon atoms. mercapto, and other Substituents. A preferred aryl is phenyl. As used herein, “alkenyl' means a straight or branched As used herein, the term "heteroaryl' means an aromatic chain chemical group containing only carbon and hydrogen 35 radical having one or more heteroatom(s) (e.g., N, O, or S) in and containing at least one carbon-carbon double bond. Such the ring backbone and may include a single ring (e.g., pyri as 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, dine) or multiple condensed rings (e.g., quinoline). In various 2-butenyl, and the like. In various embodiments, alkenyls can embodiments, heteroaryl groups can either be unsubstituted either be unsubstituted or substituted with one or more sub or Substituted with one or more Substituents, e.g., amino, stituents, e.g., halogen, hydroxyl. Substituted hydroxyl, acy 40 cyano, hydroxyl, lower alkyl, haloalkyl, alkoxy, nitro, halo, loxy, amino, Substituted amino, amido, cyano, nitro, guani mercapto, carboxy, carbonyl, benzyloxy, aryl, heteroaryl, and dino, amidino, mercapto, Substituted mercapto, carboxy, other substituents. Examples of heteroaryl include thienyl, Sulfonyloxy, carbonyl, benzyloxy, aryl, heteroaryl, carbocy pyrridyl, furyl, oxazolyl, oxadiazolyl, pyrollyl, imidazolyl, clyl, heterocyclyl, or other functionality that may be suitably triazolyl, thiodiazolyl pyrazolyl, isoxazolyl, thiadiazolyl, blocked with a protecting group. Typically, alkenyl groups 45 pyranyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thiaz will comprise 2 to 20 carbon atoms, 2 to 9 carbon atoms, olyl, quinolinyl, quinazolinyl and others. preferably 2 to 6, and more preferably 2 to 5 carbon atoms. In these definitions it is contemplated that substitution on As used herein, “alkynyl' means a straight or branched the aryl and heteroaryl rings is within the scope of certain chain chemical group containing only carbon and hydrogen embodiments. Where substitution occurs, the radical is called and containing at least one carbon-carbon triple bond, such as 50 substituted aryl or substituted heteroaryl. Preferably one to 1-propynyl, 1-butynyl, 2-butynyl, and the like. In various three and more preferably one or two substituents occur on embodiments, alkynyls can either be unsubstituted or substi the aryl ring. Though many Substituents will be useful, pre tuted with one or more substituents, e.g., halogen, hydroxyl, ferred substituents include those commonly found in aryl Substituted hydroxyl, acyloxy, amino, Substituted amino, compounds, such as alkyl, cycloalkyl, hydroxy, alkoxy, amido, cyano, nitro, guanidino, amidino, mercapto, Substi 55 cyano, halo, haloalkyl, mercapto and the like. tuted mercapto, carboxy, Sulfonyloxy, carbonyl, benzyloxy, As used herein, "amide' or "amido' includes both aryl, heteroaryl, carbocyclyl, heterocyclyl, or other function RNR'CO— (in the case of R-alkyl, alkaminocarbonyl-) and ality that may be suitably blocked with a protecting group. RCONR'— (in the case of R-alkyl, alkyl carbonylamino-). Typically, alkynyl groups will comprise 2 to 20 carbonatoms, “Amide' or “amido” includes a H CON , alkyl-CON 2 to 9 carbon atoms, preferably 2 to 6, and more preferably 2 60 carbocyclyl-CON , aryl-CON ... heteroaryl-CON.— or to 5 carbon atoms. heterocyclyl-CON.— group, wherein the alkyl, carbocyclyl, As used herein, 'carbocyclyl means a non-aromatic cyclic aryl or heterocyclyl group is as herein described. ring system containing only carbon atoms in the ring system As used herein, the term “ester' includes both ROCO - (in backbone, Such as cyclopropyl, cyclobutyl, cyclopentyl, the case of R-alkyl, alkoxycarbonyl-) and RCOO– (in the cyclohexyl, and cyclohexenyl. Carbocyclyls may include 65 case of R-alkyl, alkylcarbonyloxy-). multiple fused rings. Carbocyclyls may have any degree of As used herein, “acyl means an H CO. , alkyl-CO—, saturation provided that at least one ring in the ring system is carbocyclyl-CO—, aryl-CO—, heteroaryl-CO— or hetero US 8,680,136 B2 33 34 cyclyl-CO— group wherein the alkyl, carbocyclyl, aryl or wherein the alkyl, carbocyclyl, aryl, heteroaryl or heterocy heterocyclyl group is as herein described. Preferred acyls clyl group is as herein described. contain a lower alkyl. Exemplary alkyl acyl groups include As used herein, a substituted group is derived from the formyl, acetyl, propanoyl, 2-methylpropanoyl, t-butylacetyl, unsubstituted parent group in which there has been an butanoyl and palmitoyl. exchange of one or more hydrogenatoms for another atom or As used herein, "halo or halide' is a chloro, bromo, fluoro group. When Substituted, the Substituent group(s) is (are) or iodo atom radical. Chloro and fluoro are preferred halides. substituted with one or more substituent(s) individually and The term “halo' also contemplates terms sometimes referred independently selected from C-C alkyl, C-C alkenyl, to as “halogen', or “halide'. C-C alkynyl, C-C, carbocycle (optionally substituted with As used herein, "heterocyclyl means a non-aromatic 10 halo, alkyl, alkoxy, carboxyl, haloalkyl, CN, SO-alkyl, cyclic ring system comprising at least one heteroatom in the —CF, and —OCF), C-C heteroalkyl, 5-7 membered het ring system backbone. Heterocyclyls may include multiple erocyclyl (e.g., tetrahydrofuryl) (optionally substituted with fused rings. Heterocyclyls may have any degree of Saturation halo, alkyl, alkoxy, carboxyl, CN, -SO-alkyl, -CF, and provided that at least one ring in the ring system is not aro —OCF), aryl (optionally substituted with halo, alkyl, aryl matic. The heteroatom(s) may be present in either a non 15 optionally Substituted with C-C alkyl, arylalkyl, alkoxy, aromatic or aromatic ring in the ring system. In various carboxyl, CN, -SO-alkyl, -CF, and —OCF), arylalkyl embodiments, heterocyclyls may be substituted or unsubsti (optionally Substituted with halo, alkyl, alkoxy, aryl, car tuted with one or more Substituents, e.g., halogen, alkoxy, boxyl, CN, SO-alkyl, —CF, and —OCF), heteroaryl acyloxy, amino, amido, cyano, nitro, hydroxyl, mercapto, (optionally Substituted with halo, alkyl, alkoxy, aryl, aralkyl, carboxy, carbonyl, benzyloxy, aryl, heteroaryl, and other Sub carboxyl, CN, SO-alkyl, —CF, and—OCF), heteroary stituents, and are attached to other groups via any available lalkyl (optionally substituted with halo, alkyl, alkoxy, aryl, valence, preferably any available carbon or nitrogen. Pre carboxyl, CN, -SO-alkyl, -CF, and —OCF), halo (e.g., ferred heterocycles are of 5-7 members. In six membered chloro, bromo, iodo and fluoro), cyano, hydroxy, C-C, monocyclic heterocycles, the heteroatom(s) are selected from alkoxy, C-C alkoxyalkyl (i.e., ether), aryloxy, Sulfhydryl one up to three of O, Nor S, and when the heterocycle is five 25 (mercapto), halo(C-C)alkyl (e.g., —CF), C-C alkylthio. membered, preferably it has one or two heteroatoms selected arylthio, amino (-NH2), mono- and di-(C-C)alkyl amino, from O, N, or S. Examples of heterocyclyl include pyrrolidi quaternary ammonium salts, amino(C-C)alkoxy (e.g., nyl, piperidinyl, azepanyl, tetrahydrofuranyl, tetrahydropyra —O(CH)NH), amino(C-C)alkoxyalkyl (e.g., -CHO nyl, oxepanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl. (CH2)NH2), hydroxy(C-C)alkylamino, amino(C-C) thiepanyl, indolinyl and dihydrobenzofuranyl. 30 alkylthio (e.g. —S(CH2)NH), cyanoamino, nitro, car As used herein, “substituted amino” means an amino radi bamyl, oxo (=O), carboxy, glycolyl, glycyl, hydrazino, cal which is substituted by one or two alkyl, cycloalkyl, aryl, guanidinyl, sulfamyl, sulfonyl, sulfinyl, thiocarbonyl, thio heteroaryl or heterocyclyl groups, wherein the alkyl, aryl, carboxy, C-amide, N-amide, N-carbamate, O-carbamate, and heteroaryl, cycloalkyl or heterocyclyl are defined as above. urea. Wherever a group is described as “optionally substi As used herein, “substituted hydroxyl means RO group 35 tuted that group can be substituted with the above substitu wherein R is an alkyl, an aryl, heteroaryl, cycloalkyl or a entS. heterocyclyl group, wherein the alkyl, cycloalkyl, aryl, het In some embodiments, Substituted group(s) is (are) Substi eroaryl or heterocyclyl are defined as above. tuted with one or more substituent(s) individually and inde As used herein, “substituted thiol means RS group pendently selected from C-C alkyl, C-C, carbocycle, wherein R is an alkyl, an aryl, heteroaryl, cycloalkyl or a 40 amino (-NH), amino(C-C)alkoxy, carboxyl, oxo (=O). heterocyclyl group, wherein the alkyl, cycloalkyl, aryl, het C-C alkylthio, amino(C-C)alkylthio, guanidinyl, aryl, 5-7 eroaryl or heterocyclyl are defined as above. membered heterocyclyl, heteroarylalkyl, hydroxy, halo, As used herein, “sulfonyl' means an alkylSO, arylSO, amino(C-C)alkoxy, and amino(C-C)alkoxyalkyl. heteroarylSO, carbocyclylSO, or heterocyclyl-SO group In some embodiments, Substituted group(s) is (are) Substi wherein the alkyl, carbocyclyl, aryl, heteroaryl or heterocy 45 tuted with one or more substituent(s) individually and inde clyl are defined as above. pendently selected from C-C alkyl, amino (—NH), amino As used herein, “sulfamido” means an alkyl-N-S(O), (C-C)alkoxy, carboxyl, oxo (=O), C-C alkylthio, amino N—, aryl-NS(O)N heteroaryl-NS(O)N , carbocyclyl (C-C)alkylthio, guanidinyl, hydroxy, halo, amino(C-C) NS(O)N or heterocyclyl-NS(O)N group wherein the alkoxy, and amino(C-C)alkoxyalkyl. alkyl, carbocyclyl, aryl, heteroaryl or heterocyclyl group is as 50 In some embodiments, Substituted group(s) is (are) Substi herein described. tuted with one or more substituent(s) individually and inde As used herein, “sulfonamide' means an alkyl-S(O)N , pendently selected from C-C alkyl, amino ( NH), car aryl-S(O)N , heteroaryl-S(O)N , carbocyclyl-S(O) boxyl, oxo (=O), guanidinyl, hydroxy, and halo. N— or heterocyclyl-S(O)N group wherein the alkyl, car It is to be understood that certain radical naming conven bocyclyl, aryl, heteroaryl or heterocyclyl group is as herein 55 tions can include either a mono-radical or a di-radical, described. depending on the context. For example, where a Substituent As used herein, “ureido” means an alkyl-NCON , aryl requires two points of attachment to the rest of the molecule, NCON , heteroaryl-NCON , carbocyclyl-NCON , het it is understood that the substituent is a di-radical. For erocyclyl-NCON group or heterocyclyl-CON group example, a Substituent identified as alkyl that requires two wherein the heterocyclyl group is attached by a ring nitrogen, 60 points of attachment includes di-radicals such as —CH2—, and wherein the alkyl, carbocyclyl, aryl, heteroaryl or hetero —CHCH , —CHCH(CH)CH , and the like. Other cyclyl group is as herein described. radical naming conventions clearly indicate that the radical is As used herein, “guanidino” means an alkyl-NC(=NR) a di-radical. For example, as used herein, “alkylene' means a N-, aryl-NC(—NR)N , heteroaryl-NC(—NR)N , car branched, or straight chain Saturated di-radical chemical bocyclyl-NC(=NR)N- or heterocyclyl-NC(=NR)N- 65 group containing only carbon and hydrogen, such as methyl group wherein R' is an H, substituted or unsubstituted ene, isopropylene, isobutylene, sec-butylene, and pentylene, hydroxyl, CN, alkyl, aryl, heteroaryl or a heterocyclyl group, that is attached to the rest of the molecule via two points of US 8,680,136 B2 35 36 attachment. As used herein, “alkenylene' means a straight or when a carboxylic isostere is optionally substituted with one branched chain di-radical chemical group containing only or more moieties selected from Ras defined above, then the carbon and hydrogen and containing at least one carbon substitution and substitution position is selected such that it carbon double bond. Such as 1-propenylene, 2-propenylene, does not eliminate the carboxylic acid isosteric properties of 2-methyl-1-propenylene, 1-butenylene, and 2-butenylene, the compound. Similarly, it is also contemplated that the that is attached to the rest of the molecule via two points of placement of one or more R substituents upon a carbocyclic attachment. or heterocyclic carboxylic acid isostere is not a Substitution at As used herein, “isosteres' of a chemical group are other one or more atom(s) that maintain(s) or is/are integral to the chemical groups that exhibit the same or similar properties. carboxylic acid isosteric properties of the compound, if Such For example, tetrazole is an isostere of carboxylic acid 10 substituent(s) would destroy the carboxylic acid isosteric because it mimics the properties of carboxylic acid even properties of the compound. though they both have very different molecular formulae. Tetrazole is one of many possible isosteric replacements for Other carboxylic acid isosteres not specifically exempli carboxylic acid. Other carboxylic acid isosteres contem fied in this specification are also contemplated. plated include - SOH, -SOHNR, -PO.(R), -PO, 15 The skilled artisan will recognize that some structures (R), CONHNHSOR, COHNSOR, and described herein may be resonance forms or tautomers of —CONRCN, where R is as defined above. In addition, compounds that may be fairly represented by other chemical carboxylic acid isosteres can include 5-7 membered car structures, even when kinetically; the artisan recognizes that bocycles or heterocycles containing any combination of CH, Such structures are only a very Small portion of a sample of O, S, or N in any chemically stable oxidation state, where any of the atoms of said ring structure are optionally substituted in Such compound(s). Such compounds are considered within one or more positions. The following structures are non the scope of the structures depicted, though such resonance limiting examples of carbocyclic and heterocyclic isosteres forms or tautomers are not represented herein. contemplated. The atoms of said ring structure may be In some embodiments, due to the facile exchange of boron optionally substituted at one or more positions with R as 25 esters, the compounds described herein may convert to or defined above. exist in equilibrium with alternate forms. Accordingly, in Some embodiments, the compounds described herein may exist in combination with one or more of these forms. For example, Compound 5 may exist in combination with one or 30 more open-chain form (5a), dimeric form (5b), cyclic dimeric N N form (5c), trimeric form (5d), cyclic trimeric form (5e), and // the like. A HN-N \ , \- 1

H N N 35 A. N y- OH \ N{ \ SN{ 5 N-N S 21 HOC HS 40 O N N N N 2 2 /r, Y. HN F 45 B CO2H OH N HO1 No Aey N 6 No A W= N- S \ O{ OH 50 OH O O 21 O As N NH 55 NH - b 5a O O O O S 21

60 As O s OH O N-- CO2H It is also contemplated that when chemical Substituents are 65 B added to a carboxylic isostere, the compound retains the HO1NOH properties of a carboxylic isostere. It is contemplated that US 8,680,136 B2 37 -continued -continued 5e o O

S 2 O

O O1 B S HN O / 10 B o1 No O

B 15 O No O O NH

S N

Sc The compounds provided herein may encompass various 25 Stereochemical forms. The compounds also encompasses diastereomers as well as optical isomers, e.g. mixtures of S enantiomers including racemic mixtures, as well as indi vidual enantiomers and diastereomers, which arise as a con HN sequence of structural asymmetry in certain compounds. 30 Separation of the individual isomers or selective synthesis of B1 the individual isomers is accomplished by application of vari ous methods which are well known to practitioners in the art. O The term "agent” or “test agent' includes any substance, O molecule, element, compound, entity, or a combination 35 thereof. It includes, but is not limited to, e.g., protein, -B polypeptide, peptide or mimetic, Small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natu NH ral product, a synthetic compound, or a chemical compound, ora combination of two or more Substances. Unless otherwise 40 specified, the terms “agent”, “substance', and “compound are used interchangeably herein. The term “analog is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replac 45 ing a specific Substituent of the reference molecule with an alternate Substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screen ing of analogs, to identify variants of known compounds S 50 having improved characteristics (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry. The term “mammal’ is used in its usual biological sense. Thus, it specifically includes humans, cattle, horses, dogs, 55 cats, rats and mice but also includes many other species. The term “microbial infection” refers to the invasion of the host organism, whether the organism is a vertebrate, inverte brate, fish, plant, bird, or mammal, by pathogenic microbes. This includes the excessive growth of microbes that are nor 60 mally present in or on the body of a mammal or other organ ism. More generally, a microbial infection can be any situa tion in which the presence of a microbial population(s) is damaging to a host mammal. Thus, a mammal is “suffering from a microbial infection when excessive numbers of a 65 microbial population are present in or on a mammal’s body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of a mam US 8,680,136 B2 39 40 mal. Specifically, this description applies to a bacterial infec tion. “Curing means that the symptoms of active infection tion. Note that the compounds of preferred embodiments are are eliminated, including the elimination of excessive mem also useful in treating microbial growth or contamination of bers of viable microbe of those involved in the infection. cell cultures or other media, or inanimate Surfaces or objects, However, certain long-term or permanent effects of the infec and nothing herein should limit the preferred embodiments tion may exist even after a cure is obtained (such as extensive only to treatment of higher organisms, except when explicitly tissue damage). so specified in the claims. “Treat,” “treatment or “treating,” as used herein refers to The term “pharmaceutically acceptable carrier' or “phar administering a pharmaceutical composition for prophylactic maceutically acceptable excipient' includes any and all sol and/or therapeutic purposes. The term “prophylactic treat vents, dispersion media, coatings, antibacterial and antifun 10 ment” refers to treating a patient who is not yet infected, but gal agents, isotonic and absorption delaying agents and the who is susceptible to, or otherwise at risk of a particular like. The use of Such media and agents for pharmaceutically infection, whereby the treatment reduces the likelihood that active Substances is well known in the art. Except insofar as the patient will develop an infection. The term “therapeutic any conventional media or agent is incompatible with the treatment” refers to administering treatment to a patient active ingredient, its use in the therapeutic compositions is 15 already Suffering from an infection. contemplated. Supplementary active ingredients can also be Administration and Pharmaceutical Compositions incorporated into the compositions. In addition, various adju Some embodiments include pharmaceutical compositions vants such as are commonly used in the art may be included. comprising: (a) a safe and therapeutically effective amount of These and other such compounds are described in the litera the cyclic boronic acid ester derivative, or its corresponding ture, e.g., in the Merck Index, Merck & Company, Rahway, enantiomer, diastereoisomer or tautomer, or pharmaceuti N.J. Considerations for the inclusion of various components cally acceptable salt; and (b) a pharmaceutically acceptable in pharmaceutical compositions are described, e.g., in Gil carrier. man et al. (Eds.) (1990); Goodman and Gilmans: The Phar The cyclic boronic acid ester derivatives are administered macological Basis of Therapeutics, 8th Ed., Pergamon Press. at a therapeutically effective dosage, e.g., a dosage sufficient The term “pharmaceutically acceptable salt” refers to salts 25 to provide treatment for the disease states previously that retain the biological effectiveness and properties of the described. While human dosage levels have yet to be opti compounds of the preferred embodiments and, which are not mized for the compounds of the preferred embodiments, gen biologically or otherwise undesirable. In many cases, the erally, a daily dose for most of the cyclic boronic acid ester compounds of the preferred embodiments are capable of derivatives described herein is from about 0.25 mg/kg to forming acid and/or base salts by virtue of the presence of 30 about 120 mg/kg or more of body weight, from about 0.5 amino and/or carboxyl groups or groups similar thereto. Phar mg/kg or less to about 70 mg/kg, from about 1.0 mg/kg to maceutically acceptable acid addition salts can be formed about 50 mg/kg of body weight, or from about 1.5 mg/kg to with inorganic acids and organic acids. Inorganic acids from about 10 mg/kg of body weight. Thus, for administration to a which salts can be derived include, for example, hydrochloric 70 kg person, the dosage range would be from about 17 mg acid, hydrobromic acid, Sulfuric acid, nitric acid, phosphoric 35 per day to about 8000 mg per day, from about 35 mg per day acid, and the like. Organic acids from which salts can be or less to about 7000 mg per day or more, from about 70 mg derived include, for example, acetic acid, propionic acid, per day to about 6000 mg per day, from about 100 mg per day glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic to about 5000 mg per day, or from about 200 mg to about 3000 acid, Succinic acid, fumaric acid, tartaric acid, citric acid, mg per day. The amount of active compound administered benzoic acid, cinnamic acid, mandelic acid, methanesulfonic 40 will, of course, be dependent on the Subject and disease state acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic being treated, the severity of the affliction, the manner and acid, and the like. Pharmaceutically acceptable base addition schedule of administration and the judgment of the prescrib salts can be formed with inorganic and organic bases. Inor ing physician. ganic bases from which salts can be derived include, for Administration of the compounds disclosed herein or the example, sodium, potassium, lithium, ammonium, calcium, 45 pharmaceutically acceptable salts thereof can be via any of magnesium, iron, Zinc, copper, manganese, aluminum, and the accepted modes of administration for agents that serve the like; particularly preferred are the ammonium, potassium, similar utilities including, but not limited to, orally, Subcuta Sodium, calcium and magnesium salts. Organic bases from neously, intravenously, intranasally, topically, transdermally, which salts can be derived include, for example, primary, intraperitoneally, intramuscularly, intrapulmonarily, vagi secondary, and tertiary amines, Substituted amines including 50 nally, rectally, or intraocularly. Oral and parenteral adminis naturally occurring Substituted amines, cyclic amines, basic trations are customary in treating the indications that are the ion exchange resins, and the like, specifically Such as isopro subject of the preferred embodiments. pylamine, trimethylamine, diethylamine, triethylamine, The compounds useful as described above can be formu tripropylamine, and ethanolamine. Many such salts are lated into pharmaceutical compositions for use in treatment known in the art, as described in WO 87/05297, Johnston et 55 of these conditions. Standard pharmaceutical formulation al., published Sep. 11, 1987 (incorporated by reference herein techniques are used, such as those disclosed in Remington's in its entirety). The Science and Practice of Pharmacy, 21st Ed., Lippincott “Solvate” refers to the compound formed by the interaction Williams & Wilkins (2005), incorporated by reference in its of a solvent and an EPI, a metabolite, or salt thereof. Suitable entirety. Solvates are pharmaceutically acceptable solvates including 60 In addition to the selected compound useful as described hydrates. above, come embodiments include compositions containing “Subject' as used herein, means a human or a non-human a pharmaceutically-acceptable carrier. The term “pharmaceu mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a tically-acceptable carrier, as used herein, means one or more pig, a goat, a non-human primate or a bird, e.g., a chicken, as compatible Solid or liquid filler diluents or encapsulating well as any other vertebrate or invertebrate. 65 Substances, which are suitable for administration to a mam Atherapeutic effect relieves, to some extent, one or more of mal. The term “compatible’, as used herein, means that the the symptoms of the infection, and includes curing an infec components of the composition are capable of being com US 8,680,136 B2 41 42 mingled with the Subject compound, and with each other, in a Dosage Forms: Tablets (1989); and Ansel, Introduction to manner Such that there is no interaction, which would Sub Pharmaceutical Dosage Forms 8th Edition (2004). stantially reduce the pharmaceutical efficacy of the composi Various oral dosage forms can be used, including Such tion under ordinary use situations. Pharmaceutically-accept Solid forms as tablets, capsules, granules and bulk powders. able carriers must, of course, be of sufficiently high purity and These oral forms comprise a safe and effective amount, usu sufficiently low toxicity to render them suitable for adminis ally at least about 5%, with a maximum of about 90%, of the tration preferably to an animal, preferably mammal being compound. Tablets can be compressed, tablet triturates, treated. enteric-coated, Sugar-coated, film-coated, or multiple-com Some examples of Substances, which can serve as pharma pressed, containing Suitable binders, lubricants, diluents, dis ceutically-acceptable carriers or components thereof, are 10 integrating agents, coloring agents, flavoring agents, flow Sugars, such as lactose, glucose and Sucrose; starches, such as inducing agents, and melting agents. Liquid oral dosage corn starch and potato starch; cellulose and its derivatives, forms include aqueous solutions, emulsions, Suspensions, Such as sodium carboxymethyl cellulose, ethyl cellulose, and Solutions and/or Suspensions reconstituted from non-effer methyl cellulose; powdered tragacanth; malt, gelatin; talc; Vescent granules, and effervescent preparations reconstituted Solid lubricants, such as Stearic acid and magnesium Stearate; 15 from effervescent granules, containing suitable solvents, pre calcium Sulfate; vegetable oils, such as peanut oil, cottonseed servatives, emulsifying agents, Suspending agents, diluents, oil, sesame oil, olive oil, corn oil and oil of theobroma; poly Sweeteners, melting agents, coloring agents and flavoring ols such as propylene glycol, glycerine, Sorbitol, mannitol, agents. and polyethylene glycol; alginic acid; emulsifiers, such as the The pharmaceutically-acceptable carrier suitable for the TWEENS: wetting agents, such sodium lauryl sulfate; color preparation of unit dosage forms for peroral administration is ing agents; flavoring agents; tableting agents, stabilizers; well-known in the art. Tablets typically comprise conven antioxidants; preservatives; pyrogen-free water, isotonic tional pharmaceutically-compatible adjuvants as inert dilu saline; and phosphate buffer Solutions. ents, such as calcium carbonate, sodium carbonate, mannitol, The choice of a pharmaceutically-acceptable carrier to be lactose and cellulose; binders such as starch, gelatin and used in conjunction with the Subject compound is basically 25 Sucrose; disintegrants such as starch, alginic acid and cros determined by the way the compound is to be administered. carmelose; lubricants such as magnesium Stearate, Stearic The compositions described herein are preferably provided acid and talc. Glidants such as silicon dioxide can be used to in unit dosage form. As used herein, a “unit dosage form' is a improve flow characteristics of the powder mixture. Coloring composition containing an amount of a compound that is agents, such as the FD&C dyes, can be added for appearance. Suitable for administration to an animal, preferably mammal 30 Sweeteners and flavoring agents, such as aspartame, saccha Subject, in a single dose, according to good medical practice. rin, menthol, peppermint, and fruit flavors, are useful adju The preparation of a single or unit dosage form however, does vants for chewable tablets. Capsules typically comprise one not imply that the dosage form is administered once per day or or more solid diluents disclosed above. The selection of car once per course of therapy. Such dosage forms are contem rier components depends on secondary considerations like plated to be administered once, twice, thrice or more per day 35 taste, cost, and shelf stability, which are not critical, and can and may be administered as infusion over a period of time be readily made by a person skilled in the art. (e.g., from about 30 minutes to about 2-6 hours), or admin Peroral compositions also include liquid Solutions, emul istered as a continuous infusion, and may be given more than sions, Suspensions, and the like. The pharmaceutically-ac once during a course of therapy, though a single administra ceptable carriers suitable for preparation of Such composi tion is not specifically excluded. The skilled artisan will rec 40 tions are well known in the art. Typical components of ognize that the formulation does not specifically contemplate carriers for syrups, elixirs, emulsions and Suspensions the entire course of therapy and such decisions are left for include ethanol, glycerol, propylene glycol, polyethylene those skilled in the art of treatment rather than formulation. glycol, liquid Sucrose, Sorbitol and water. For a Suspension, The compositions useful as described above may be in any typical Suspending agents include methyl cellulose, sodium of a variety of suitable forms for a variety of routes for 45 carboxymethyl cellulose, AVICEL RC-591, tragacanth and administration, for example, for oral, nasal, rectal, topical Sodium alginate; typical wetting agents include lecithin and (including transdermal), ocular, intracerebral, intracranial, polysorbate 80; and typical preservatives include methyl intrathecal, intra-arterial, intravenous, intramuscular, or other paraben and sodium benzoate. Peroral liquid compositions parental routes of administration. The skilled artisan will may also contain one or more components such as Sweeten appreciate that oral and nasal compositions comprise compo 50 ers, flavoring agents and colorants disclosed above. sitions that are administered by inhalation, and made using Such compositions may also be coated by conventional available methodologies. Depending upon the particular methods, typically with pH or time-dependent coatings. Such route of administration desired, a variety of pharmaceuti that the Subject compound is released in the gastrointestinal cally-acceptable carriers well-known in the art may be used. tract in the vicinity of the desired topical application, or at Pharmaceutically-acceptable carriers include, for example, 55 various times to extend the desired action. Such dosage forms Solid or liquid fillers, diluents, hydrotropies, Surface-active typically include, but are not limited to, one or more of cel agents, and encapsulating Substances. Optional pharmaceu lulose acetate phthalate, polyvinylacetate phthalate, hydrox tically-active materials may be included, which do not sub ypropyl methyl cellulose phthalate, ethyl cellulose, Eudragit stantially interfere with the inhibitory activity of the com coatings, waxes and shellac. pound. The amount of carrier employed in conjunction with 60 Compositions described herein may optionally include the compound is Sufficient to provide a practical quantity of other drug actives. material for administration per unit dose of the compound. Other compositions useful for attaining systemic delivery Techniques and compositions for making dosage forms use of the Subject compounds include Sublingual, buccal and ful in the methods described herein are described in the fol nasal dosage forms. Such compositions typically comprise lowing references, all incorporated by reference herein: Mod 65 one or more of Soluble filler Substances such as Sucrose, ern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Sorbitol and mannitol; and binders such as acacia, microcrys Rhodes, editors, 2002); Lieberman et al., Pharmaceutical talline cellulose, carboxymethyl cellulose and hydroxypropyl US 8,680,136 B2 43 44 methyl cellulose. Glidants, lubricants, Sweeteners, colorants, drates such as dextrose, mannitol, and dextran. Further antioxidants and flavoring agents disclosed above may also acceptable excipients are described in Powell, et al., Com be included. pendium of Excipients for Parenteral Formulations, PDA J A liquid composition, which is formulated for topical oph Pharm Sci and Tech 1998, 52 238-311 and Nema et al., thalmic use, is formulated Such that it can be administered Excipients and Their Role in Approved Injectable Products: topically to the eye. The comfort should be maximized as Current Usage and Future Directions, PDAJ Pharm Sci and much as possible, although sometimes formulation consider Tech 2011,65287-332, both of which are incorporated herein ations (e.g. drug stability) may necessitate less than optimal by reference in their entirety. Antimicrobial agents may also comfort. In the case that comfort cannot be maximized, the be included to achieve a bacteriostatic or fungistatic solution, liquid should beformulated such that the liquid is tolerable to 10 the patient for topical ophthalmic use. Additionally, an oph including but not limited to phenylmercuric nitrate, thimero thalmically acceptable liquid should either be packaged for sal, benzethonium chloride, benzalkonium chloride, phenol, single use, or contain a preservative to prevent contamination cresol, and chlorobutanol. over multiple uses. The resulting composition may be infused into the patient For ophthalmic application, Solutions or medicaments are 15 over a period of time. In various embodiments, the infusion often prepared using a physiological Saline solution as a time ranges from 5 minutes to continuous infusion, from 10 major vehicle. Ophthalmic solutions should preferably be minutes to 8 hours, from 30 minutes to 4 hours, and from 1 maintained at a comfortable pH with an appropriate buffer hour to 3 hours. In one embodiment, the drug is infused over system. The formulations may also contain conventional, a 3 hour period. The infusion may be repeated at the desired pharmaceutically acceptable preservatives, stabilizers and dose interval, which may include, for example, 6 hours, 8 Surfactants. hours, 12 hours, or 24 hours. Preservatives that may be used in the pharmaceutical com The compositions for intravenous administration may be positions disclosed herein include, but are not limited to, provided to caregivers in the form of one more solids that are benzalkonium chloride, PHMB, chlorobutanol, thimerosal, reconstituted with a suitable diluent such as sterile water, phenylmercuric, acetate and phenylmercuric nitrate. A useful 25 saline or dextrose in water shortly prior to administration. surfactantis, for example, Tween 80. Likewise, various useful Reconstituted concentrated solutions may be further diluted vehicles may be used in the ophthalmic preparations dis into a parenteral Solutions having a Volume of from about 25 closed herein. These vehicles include, but are not limited to, to about 1000 ml, from about 30 ml to about 500 ml, or from polyvinyl alcohol, povidone, hydroxypropyl methyl cellu about 50 ml to about 100 ml. In other embodiments, the lose, poloxamers, carboxymethyl cellulose, hydroxyethyl 30 compositions are provided in solution ready to administer cellulose and purified water. parenterally. In still other embodiments, the compositions are Tonicity adjustors may be added as needed or convenient. provided in a solution that is further diluted prior to admin They include, but are not limited to, salts, particularly sodium istration. In embodiments that include administering a com chloride, potassium chloride, mannitol and glycerin, or any bination of a compound described herein and another agent, other Suitable ophthalmically acceptable tonicity adjustor. 35 the combination may be provided to caregivers as a mixture, Various buffers and means for adjusting pH may be used so or the caregivers may mix the two agents prior to administra long as the resulting preparation is ophthalmically accept tion, or the two agents may be administered separately. able. For many compositions, the pH will be between 4 and 9. The actual dose of the active compounds described herein Accordingly, buffers include acetate buffers, citrate buffers, depends on the specific compound, and on the condition to be phosphate buffers and borate buffers. Acids or bases may be 40 treated; the selection of the appropriate dose is well within the used to adjust the pH of these formulations as needed. knowledge of the skilled artisan. In a similar vein, an ophthalmically acceptable antioxidant Kits for Intravenous Administration includes, but is not limited to, sodium metabisulfite, sodium Some embodiments include a kit comprising a compound thiosulfate, acetylcysteine, butylated hydroxyanisole and described herein and an additional agent, such as an antimi butylated hydroxytoluene. 45 crobial agent. In one embodiment, both components are pro Other excipient components, which may be included in the vided in a single sterile container. In the case of Solids for ophthalmic preparations, are chelating agents. A useful reconstitution, the agents may be pre-blended and added to chelating agent is edetate disodium, although other chelating the container simultaneously or may be dry-powder filled into agents may also be used in place or in conjunction with it. the container in two separate steps. In some embodiments, the For topical use, creams, ointments, gels, Solutions or Sus 50 solids are sterile crystalline products. In other embodiment, pensions, etc., containing the compound disclosed herein are the solids are lyophiles. In one embodiment, both compo employed. Topical formulations may generally be comprised nents are lyophilized together. Non-limiting examples of of a pharmaceutical carrier, co-solvent, emulsifier, penetra agents to aid in lyophilization include Sodium or potassium tion enhancer, preservative system, and emollient. phosphates, citric acid, tartaric acid, gelatin, and carbohy For intravenous administration, the compounds and com 55 drates Such as dextrose, mannitol, and dextran. One embodi positions described herein may be dissolved or dispersed in a ment includes non-sterile solids that are irradiated either pharmaceutically acceptable diluent, Such as a saline or dex before or after introduction into the container. trose solution. Suitable excipients may be included to achieve In the case of a liquid, the agents may be dissolved or the desired pH, including but not limited to NaOH, sodium dispersed in a diluent ready for administration. In another carbonate, sodium acetate, HCl, and citric acid. In various 60 embodiment, the solution or dispersion may be further diluted embodiments, the pH of the final composition ranges from 2 prior to administration. Some embodiments include provid to 8, or preferably from 4 to 7. Antioxidant excipients may ing the liquid in an IV bag. The liquid may be frozen to include sodium bisulfite, acetone sodium bisulfite, sodium improve stability. formaldehyde, sulfoxylate, thiourea, and EDTA. Other non In one embodiment, the container includes other ingredi limiting examples of Suitable excipients found in the final 65 ents such as a pHadjuster, a solubilizing agent, or a dispersing intravenous composition may include sodium or potassium agent. Non-limiting examples of pH adjusters include NaOH, phosphates, citric acid, tartaric acid, gelatin, and carbohy Sodium carbonate, Sodium acetate, HCl, and citric acid. US 8,680,136 B2 45 46 The molar ratio of compound described herein to addi Phenoxymethylpenicillin (V), Propicillin, Benzathine phe tional agent (e.g., antibacterial agent) may be from about 10:1 noxymethylpenicillin, Pheneticillin, Cloxacillin (e.g., to 1:10, 8:1 to 1:8, 5:1 to 1:5, 3:1 to 1:3, 2:1 to 1:2, or about , Flucloxacillin), Oxacillin, , Nafcil 1:1. In various embodiments the amount of compound lin, , Biapenem, Doripenem, Ertapenem, Imi described herein may be from 100 mg to 5 g, 500 mg to 2 g, 5 penem, Meropenem, Panipenem, Tomopenem, Razupenem, or about 1 g. Similarly, in various embodiments the amount of , Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin, additional agent may be from 100 mg to 5g, 500 mg to 2 g, or , Cefaloridine, Cefalotin, Cefapirin, Cefatrizine, about 1 g. Cefazedone, Cefazaflur, Cefradine, Cefroxadine, Ceftezole, In an alternative embodiment, the two components may be Cefaclor, Cefamandole, Cefninox, Cefonicid, Ceforanide, provided in separate containers. Each container may include 10 Cefotiam, Cefprozil, Cefbuperazone, Cefuroxime, Cefu a solid, Solution, or dispersion. In such embodiments, the two Zonam, Cefoxitin, Cefotetan, Cefnmetazole, Loracarbef, containers may be provided in a single package or may be Cefixime, Ceftazidime, Ceftriaxone, Cefcapene, Cefdal provided separately. In one embodiment, the compound oxime, Cefdinir, Cefditoren, Cefetamet, Cefnmenoxime, described herein is provided as a solution while the additional Cefodizime, CefoperaZone, Cefotaxime, Ce?pimizole, Cef agent (e.g., antibacterial agent) is provided as a solid ready for 15 piramide, Cefpodoxime, Cefsulodin, Cefteram, Ceftibuten, reconstitution. In one such embodiment, the solution of the Ceftiolene, Ce?tizoxime, Flomoxef. Latamoxe?, Cefepime, compound described herein is used as the diluent to reconsti Cefozopran, Ce?pirome, Cefauinome, Ceftobiprole, Ceftaro tute the other agent. line, CXA-101, RWJ-54428, MC-04,546, ME 1036, Methods of Treatment BAL30072, SYN 2416, Ceftiofur, Cefauinome, Cefovecin, Some embodiments of the present invention include meth Aztreonam, Tigemonam, Carumonam, RWJ-442831, RWJ ods of treating bacterial infections with the compounds and 333441, and RWJ-333442. compositions comprising cyclic boronic acid ester deriva Preferred embodiments include B-lactams such as Ceftazi tives described herein. Some methods include administering dime, Biapenem, Doripenem, Ertapenem, Imipenem, Mero a compound, composition, pharmaceutical composition penem, ME 1036, Tomopenem, Razupenem, and Panipenem. described hereinto a subject in need thereof. In some embodi 25 Some embodiments include co-administration of the com ments, a Subject can be an animal, e.g., a mammal, a human. pounds, compositions and/or pharmaceutical compositions In some embodiments, the bacterial infection comprises a bacteria described herein. As will be appreciated from the described herein with an additional agent, wherein the addi foregoing, methods of treating a bacterial infection include tional agent comprises a . Examples of mono methods for preventing bacterial infection in a subject at risk 30 bactams include aztreonam, tigemonam, BAL 30072, SYN thereof. 2416 (BAL 19764), and carumonam. Some embodiments include co-administration of the com Further embodiments include administering a combination pounds, compositions and/or pharmaceutical compositions of compounds to a subject in need thereof. A combination can described herein with an additional agent, wherein the addi include a compound, composition, pharmaceutical composi tional agent comprises a Class A, B, C, or D beta-lactamase tion described herein with an additional medicament. 35 inhibitor. An example of a class B beta lactamase inhibitor Some embodiments include co-administering a com includes ME 1071 (Yoshikazu Ishii et al., “In Vitro Potentia pound, composition, and/or pharmaceutical composition tion of Carbapenems with ME 1071, a Novel Metallo-B-Lac described herein, with an additional medicament. By “co tamase Inhibitor, against Metallo-B-lactamase Producing administration, it is meant that the two or more agents may Pseudomonas aeruginosa Clinical Isolates. Antimicrob. be found in the patient’s bloodstream at the same time, 40 regardless of when or how they are actually administered. In Agents Chemother, doi:10.1128/AAC.01397-09 (July one embodiment, the agents are administered simultaneously. 2010)). Other examples of beta-lactamase inhibitors admin In one such embodiment, administration in combination is istered as an additional agent include , taZo accomplished by combining the agents in a single dosage bactam, , (NXL-104), MK-765, and form. When combining the agents in a single dosage form, 45 BAL29880. MK-7655 has the following structure: they may be physically mixed (e.g., by co-dissolution or dry mixing) or may form an adduct or be covalently linked Such that they split into the two or more active ingredients upon MK-7655 administration to the patient. In another embodiment, the agents are administered sequentially. In one embodiment the 50 agents are administered through the same route, such as orally. In another embodiment, the agents are administered N through different routes, such as one being administered orally and another being administered i.v. Examples of additional medicaments include an antibac 55 terial agent, antifungal agent, an antiviral agent, an anti-in flammatory agent and an anti-allergic agent. Some embodiments include co-administration of a com Indications pound, composition or pharmaceutical composition The compounds and compositions comprising cyclic described herein with an antibacterial agent Such as a B-lac 60 boronic acid ester derivatives described herein can be used to tam. Examples of such B-lactams include Amoxicillin, Ampi treat bacterial infections. Bacterial infections that can be cillin (e.g., Pivampicillin, Hetacillin, Bacampicillin, treated with the compounds, compositions and methods Metampicillin, Talampicillin), Epicillin, Carbenicillin (Car described herein can comprise a wide spectrum of bacteria. indacillin), Ticarcillin, Temocillin, AZlocillin, Piperacillin, Example organisms include gram-positive bacteria, gram Mezlocillin, Mecillinam (Pivmecillinam), Sulbenicillin, 65 negative bacteria, aerobic and anaerobic bacteria, Such as Benzylpenicillin (G), Clometocillin, Benzathine benzylpeni Staphylococcus, Lactobacillus, Streptococcus, Sarcina, cillin, Procaine benzylpenicillin, Azidocillin, Penamecillin, Escherichia, Enterobacter; Klebsiella, Pseudomonas, Acine US 8,680,136 B2 47 48 tobacter, Mycobacterium, Proteus, Campylobacter, Citro skilled artisanto carry out these manipulations. These include bacter, Nisseria, Baccillus, Bacteroides, Peptococcus, reduction of carbonyl compounds to their corresponding Clostridium, Salmonella, Shigella, Serratia, Haemophilus, alcohols, oxidations, acylations, aromatic Substitutions, both Brucella and other organisms. electrophilic and nucleophilic, etherifications, esterification More examples of bacterial infections include Pseudomo and Saponification and the like. These manipulations are dis nas aeruginosa, Pseudomonas fluorescens, Pseudomonas cussed in Standard texts such as March Advanced Organic acidovorans, Pseudomonas alcaligenes, Pseudomonas Chemistry (Wiley), Carey and Sundberg, Advanced Organic putida, Stenotrophomonas maltophilia, Burkholderia cepa Chemistry (incorporated herein by reference in its entirety) cia, Aeromonas hydrophilia, Escherichia coli, Citrobacter and the like. freundii, Salmonella typhimurium, Salmonella typhi, Salmo 10 The skilled artisan will readily appreciate that certain reac nella paratyphi, Salmonella enteritidis, Shigella dysenteriae, tions are best carried out when other functionality is masked Shigella flexneri, Shigella Sonnei, Enterobacter cloacae, or protected in the molecule, thus avoiding any undesirable Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella side reactions and/or increasing the yield of the reaction. Oxytoca, Serratia marcescens, Francisella tularensis, Mor Often the skilled artisan utilizes protecting groups to accom ganella morganii, Proteus mirabilis, Proteus vulgaris, Provi 15 plish Such increased yields or to avoid the undesired reac dencia alcalifaciens, Providencia rettgeri, Providencia stu tions. These reactions are found in the literature and are also artii, Acinetobacter baumannii, Acinetobacter calcoaceticus, well within the scope of the skilled artisan. Examples of many Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia of these manipulations can be found for example in T. Greene pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bor and P. Wuts Protecting Groups in Organic Synthesis, 4th Ed., detella pertussis, Bordetella parapertussis, Bordetella bron John Wiley & Sons (2007), incorporated herein by reference chiseptica, Haemophilus influenzae, Haemophilus parainflu in its entirety. enZae, Haemophilus haemolyticus, Haemophilus The following example schemes are provided for the guid parahaemolyticus, Haemophilus ducreyi, Pasteurella multo ance of the reader, and represent preferred methods for mak cida, Pasteurella haemolytica, Branhamella Catarrhalis, ing the compounds exemplified herein. These methods are Helicobacter pylori, Campylobacter fetus, Campylobacter 25 not limiting, and it will be apparent that other routes may be jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio employed to prepare these compounds. Such methods spe cholerae, Vibrio parahaemolyticus, Legionella pneumophila, cifically include Solid phase based chemistries, including Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria combinatorial chemistry. The skilled artisan is thoroughly meningitidis, Kingella, Moraxella, Gardnerella vaginalis, equipped to prepare these compounds by those methods given Bacteroides fragilis, Bacteroides distasonis, Bacteroides 30 the literature and this disclosure. The compound numberings 3452A homology group, Bacteroides vulgatus, Bacteroides used in the synthetic schemes depicted below are meant for Ovalus, Bacteroides thetaiotaOmicron, Bacteroides unifor those specific schemes only, and should not be construed as or mis, Bacteroides eggerthii, Bacteroides splanchnicus, confused with same numberings in othersections of the appli Clostridium difficile, Mycobacterium tuberculosis, Mycobac cation. terium avium, Mycobacterium intracellulare, Mycobacte 35 Trademarks used herein are examples only and reflect rium leprae, Corynebacterium diphtheriae, Corynebacte illustrative materials used at the time of the invention. The rium ulcerans, Streptococcus pneumoniae, Streptococcus skilled artisan will recognize that variations in lot, manufac agalactiae, Streptococcus pyogenes, Enterococcus faecalis, turing processes, and the like, are expected. Hence the Enterococcus faecium, Staphylococcus aureus, Staphylococ examples, and the trademarks used in them are non-limiting, cus epidermidis, Staphylococcus saprophyticus, Staphyllo 40 and they are not intended to be limiting, but are merely an coccus intermedius, Staphylococcus hyicus Subsp. hyicus, illustration of how a skilled artisan may choose to perform Staphylococcus haemolyticus, Staphylococcus hominis, or one or more of the embodiments of the invention. Staphylococcus saccharolyticus. ("H) nuclear magnetic resonance spectra (NMR) were The following examples will further describe the present measured in the indicated solvents on either a Bruker NMR invention, and are used for the purposes of illustration only, 45 spectrometer (Avance TM DRX500, 500 MHz for 1H) or and should not be considered as limiting. Varian NMR spectrometer (Mercury 400BB, 400 MHz for 1H). Peak positions are expressed in parts per million (ppm) EXAMPLES downfield from tetramethylsilane. The peak multiplicities are denoted as follows, S, singlet; d, doublet, t, triplet, q, quartet; General Procedures 50 quin, quintet; sex, sextet; Sep, Septet, non, nonet; dd, doublet of doublets; td, triplet of doublets; m, multiplet. Materials used in preparing the cyclic boronic acid ester The following abbreviations have the indicated meanings: derivatives described herein may be made by known methods n-BuLi-n-butyllithium or are commercially available. It will be apparent to the t-Bu-tert-butyl skilled artisan that methods for preparing precursors and 55 DCM-dichloromethane functionality related to the compounds claimed herein are DMF=N,N-dimethylformamide generally described in the literature including, for example, DIPEA-diisopropylethylamine procedures described in U.S. Pat. No. 7,271,186 and EDCI=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide WO2009064414, each of which is incorporated by reference ESBL-extended-spectrum B-lactamase in its entirety. In these reactions, it is also possible to make use 60 ESIMS-electron spray mass spectrometry of variants which are themselves known to those of ordinary EtOAc-ethyl acetate skill in this art, but are not mentioned in greater detail. The EtOH=ethanol skilled artisan given the literature and this disclosure is well HATU-2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetram equipped to prepare any of the compounds. ethyluronium hexafluorophosphate It is recognized that the skilled artisan in the art of organic 65 HCl-hydrochloric acid chemistry can readily carry out manipulations without further HOBt-hydroxybenzotriazole direction, that is, it is well within the scope and practice of the Im-imidazole US 8,680,136 B2 49 50 LiHMDS-lithium bis(trimethylsilyl)amide TMS-trimethylsilyl MeCN=acetonitrile TPPB-tris(pentafluorophenyl)borane monohydrate NaHCO=sodium bicarbonate The following example schemes are provided for the guid NaSO sodium sulfate ance of the reader, and collectively represent an example NMM-N-methylmorpholine method for making the compounds provided herein. Further more, other methods for preparing compounds described NMR nuclear magnetic resonance herein will be readily apparent to the person of ordinary skill Pd/C-palladium on carbon in the art in light of the following reaction schemes and TBDMSC1=tert-butyldimethylsilyl chloride examples. Unless otherwise indicated, all variables are as TBS-tert-butyldimethylsilyl defined above. TFA=trifluoroacetic acid 10 Compounds of formula I where R' is an acylamino group THF-tetrahydrofuran and X is a carboxylic acid can be prepared as depicted in TLC-thin layer chromatography Scheme 1.

Scheme 1

TBSC

CHCl2, 5h. -e-

VI -e- Ir(COD)Cl] TPPB CHCl2, 16 h VII J. Org. Chem. (1994), Tet. (2004), 60(47), 59(17),4760-4764 10695-10700

OH THF 50° C. 16h OH

VIII

O B R R' O 5 H R 3 R R6 XI

O RCOC RCOH -78°C., 1.5 h HATU, DIPEA, rt, 1.5 h CHCl2, 16 h US 8,680,136 B2

-continued O O OM NCOR 2 R4 R8 Dioxane3NHCI B R 110° C., 1.5 h / R7 -- O R5 H R2 TBSO R6 XII

The addition of enolates to substituted C.B-unsaturated acid chloride to result directly in analogs of structure XII. ketones or aldehydes to form B-hydroxy esters is a well Such analogs of XII can also be made via coupling of the known reaction (Scheme 1). Substituents R and R of for bis-TMS amine with commercially available carboxylic mula I may be controlled by use of the appropriate C-mono or 15 acids under typical amide coupling conditions (e.g., carbodi di-substituted ester III. Similarly, substituents R. R., and R' imide or HATU coupling). Compounds of Formula I where may be controlled by use of the appropriate substituted C.B- R" is substituted with - N(R)C(=O)C(=NOR)R’ may be unsaturated ketones or aldehydes analog II. Precursors of synthesized from corresponding carboxylic acids via cou structure IV, where R and R' or Rare combined together, pling of XI to XII as in scheme 1. Such carboxylic acids can may be made following the known procedures J. Am. Chem. be made by following the procedures described U.S. Pat. No. Soc. (1982), 104, 1735-7, Tetrahedron Lett. (2003), 44, 1259 5,888.998, U.S. Application Publication No. 2004/0019203, 62. The B-hydroxy ester of structure IV is protected with an and U.S. Pat. No. 4,822.786, all of which are incorporated acid-sensitive protecting group, affording V; this selection herein by reference in their entirety. Simultaneous deprotec allows simultaneous deprotection of the boronate ester and tion of the pinane ester, the tert-butyldimethylsilyloxy group hydroxyl protecting group in the final step, resulting in a 25 and the tert-butyl estergroup and concomitant cyclization are cyclized product. The pinacol boronate VII is formed from achieved by heating with dilute HCl, affording the desired substituted Vusing iridium catalysis Tetrahedron (2004), 60. oxaborinane derivatives of structure XIII. This transforma 10695-700. Trans-esterification was readily achieved with tion may also be achieved by treatment with BC1 or BBr. optically active pinane diol VIII to result in IX Tetrahedron. 30 Alternatively, the deprotection may be attained via trans Asymmetry, (1997), 8, 1435-40. Transesterification may also esterification with isobutylboronic acid in presence of dilute be achieved from the catechol ester analog of VII. Such cat HC1 (WO09064413).

Scheme 2

R4 R6 V. N. J. Am. Chem. Soc. O R O -e-1990, 112,3964-969 N WO2009,46098 B R OTBS M O R3 H DX J. Am. Chem. Soc. 1989, 111,4399-402 J. Am. Chem. Soc. 1988, 110,842-53

Re= - CoalkylR -NR'R10 echolesters can be made by reaction of V with commercially Compounds of structure XVI where R' of Formula I is an available catecholborane Tetrahedron (1989), 45, 1859-85. 60 alkyl, aralkyl or aminoaryl group may be made from bromo Homologation of IX to give chloromethylene addition prod intermediate XIV as shown in Scheme 2 J. Organomet. uctX with good stereocontrol may be achieved via Matteson Chem. (1992), 431, 255-70. Such bromo derivatives may be reaction conditions (WO0946098). The chloro derivative X made as analogously to the chloro compounds of Scheme 1, can be utilized to introduce a substituted amine group at the utilizing dibromomethane J. Am. Chem. Soc. (1990), 112, C3-position of the oxaborinane-2-ol. Stereospecific substitu 65 3964-969. Displacement of the bromo group in XIV can be tion with hexamethyldisilaZane gives the corresponding bis achieved by O-alkoxy Substituted alkyllithium agents J. Am. (trimethylsilyl)amide XI which may be reacted in situ with an Chem. Soc. (1989), 111,4399-402, J. Am. Chem. Soc. (1988), US 8,680,136 B2 53 54 110,842-53 or organomagnesium reagents (WO0946098) or Such single isomers result in enantiomerically pure cis-com by the sodium salt of alkyl or aryl carbamate derivatives J. pounds XIII or XVI when used in the sequences depicted in Org. Chem. (1996), 61, 7951-54), resulting in XV. Cycliza Schemes 1 and 2. tion of XV to afford XVI may be achieved under the condi tions described in Scheme 1. 5 Scheme 4

R7 R8 O -e- 10 Her pi He O 2 HO R6 XIX OH 15

XVII

25

30

35

40 XVIII The sequence shown in Scheme 1 also allows for varied ring sizes in formula I Such as 7- and 8-membered rings. For example, a seven-membered analog XX where n=1 can be Compounds of formula XIII and XVI are mixtures of 3.6- achieved by using the corresponding allyl intermediate (XIX) cis- and 3.6-trans-isomers. These analogs can be made in as a starting material (Scheme 4). Such allyl derivatives as enantiomerically pure form aS single isomers by starting (as 45 XIX can be made utilizing one of several well known f3-hy in Scheme 1) with a single enantiomer (XVII), as shown in droxy ester preparations Tetrahedron (2007), 63, 8336-50. Scheme 3. A variety of methods to prepare such enantiomeri- Intermediate XIX where n=2 can be prepared as described in cally pure B-hydroxy esters are known in literature, for Scheme 1 to give corresponding 8-membered compound of example via resolution Org. Lett., (2008), 10, 3907-09) or structure XX starting from pent-4-ene-1-al J. Med. Chem. stereoselective synthesis Tetrahedron, (2000), 56,917-47. (1998), 41(6), 965-972).

Scheme 5

R2 Rb Grubb's R g Catalyst -- b Ro? BNani,OR XXI US 8,680,136 B2 56 -continued

1. (+)-Pinanediol -e- COR" 2. Hydroxy protection

XXIV

1. DCM Matteson n-BuLi, THF Homologation- 2. LHMDS Amide formation 3. R9COCI or RCOH, EDCI HOBt

XXVI

Compounds of formula XXVI and XXVII can be made -continued following the sequence depicted in Scheme 5. Ring-Closing Metathesis reaction with boronated olefins (XXI) and olefin 40 substituted f-hydroxy esters (XXII) result in cyclic boronates of formula XXIII. Such cyclic boronates (XXIII) undergo ready esterification with (+)-pinane diol to give required Mat teson reaction precursors upon protection of the resulting alcohol with groups such as t-butyldimethylsilyl- or benzylor 45 trity1. Matteson homologation followed by amide formation result in compounds of formula XXV with high stereoselec tivity, as described above. Acid mediated hydrolysis of com pounds of XXV upon deprotection give cyclic boronate (XXVI). Double bond substitution of XXVI can be further 50 modified to other analogs such as Saturated cyclic boronate (XXVII) by catalytic hydrogenation. The above sequence can be utilized to make 7- or 8-membered rings with double bond at a desired position by varying p and q of XXI and XXII. 55 Compounds of formula I where R and R taken together Scheme 6 form an aryl ring can be made from commercially available Y 60 substituted aryl precursors as XXVIII. Substitution of the Z bromine atom by a boronate ester may be done under palla /x -- -e- o -e- dium catalyzed conditions Tetrahedron (2002), 58, 9633 95. The steps of hydroxy ester formation, C.-amidoboronate Br CHO 65 XXVIII preparation and cyclization can be attained by Synthetic steps analogous to those in Scheme 1 to give compounds XXIX. US 8,680,136 B2 57 58

Scheme 7

Compounds of formula I where RandR are substituted as Compounds of Formula I where X is a carboxylic acid maleate (XXXV) or succinate (XXXVI) may be made fol isostere can be prepared following the protocols described in lowing the sequence shown in Scheme 7. Maleate intermedi the literature (see J. Med. Chem. 2011, 54, 2529-2591, which ates such as XXXII can be transformed to analogs XXXV 45 analogously to the steps in Scheme 1. Analogs of XXXV can is incorporated herein by reference in its entirety). be further transformed to the corresponding Succinic acids of structure XXXVI by catalytic hydrogenation. Maleate inter mediate XXXII may be assembled from intermediate XXXI by successive deprotection of the TBS group, oxidation to the Illustrative Compound Examples aldehyde, addition of vinyl Grignard and reprotection as a 50 TBS ether. Intermediate XXXI may be formed from a pro Synthesis of 2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)ac tected propargylic alcohol XXX following methods known in etamido)-1,2-oxaborinan-6-yl)acetic acid. An example syn the literature Tetrahedron, (2002), 58,6545-54). thesis of 1 is depicted in Scheme 8 and Example 1.

US 8,680,136 B2 61 62 Example 1 The resulting was cooled to -78° C. and 5-thiopheneacetyl chloride was added and the solution stirred at -78°C. for 1.5 Step 1 h. Then, the cooling bath was removed and the solution stirred at ambient temperature for 1.5 h. The reaction was quenched A round-bottom flask charged with Ir(cod)Cl]- (350 mg. with water and extracted twice with EtOAc. The organic 0.52 mmol) and 1,4-bis(diphenylphosphanyl)butane (446 layers were combined, washed with water, brine, dried mg, 1.04 mmol) was flushed with argon. DCM (60 mL), (NaSO) and concentrated in vacuo to afford a pale yellow pinacolborane (3 mL, 21 mmol) and tert-butyl-3-(tert-bu Solid as crude product. The residue was chromatographed on tyldimethylsilyloxy)pent-4-enoate XXXVII J. Org. Chem. a silica column (100% DCM->40% EtOAc/DCM) to afford (1994), 59(17), 4760-4764 (5 g, 17.48 mmol) in 5 mL of 10 570 mg of 6-(tert-butoxy)-4-(tert-butyldimethylsilyl)oxy DCM were added successively at room temperature. The 6-oxo-1-(thiophen-2-ylacetamido)-1-(2S,6R)-2,9.9-trim mixture was then stirred at room temperature for 16 h. The ethyl-3,5-dioxa-4-boratricyclo6.1.1.02.6 decan-4-ylhexy reaction was quenched with MeOH (3 mL) and water (10 lidyne XLII as a white solid (570 mg. 0.92 mmol, 39.5% mL), the product was extracted with ether, and dried. Chro yield). matography on silica gel (100% DCM->50% EtOAc/DCM 15 gave tert-butyl 3-(tert-butyldimethylsilyloxy)-5-(4.4.5.5-tet ramethyl-1,3,2-dioxaborolan-2-yl)pentanoate XXXVIII (5.5 Step 6 g, 13.2 mmol. 75.5% yield). Method D: To a solution of amide XLII (250 mg, 0.40 Step 2 mmol) in 1,4-dioxane (10 mL) was added 10 mL of 3 NHC1. To a solution of tert-butyl 3-(tert-butyldimethylsilyloxy)- The mixture was heated to 110° C. for 90 min. The solution 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pentanoate was cooled and diluted with 10 mL of water and extracted XXXVIII (5.4 g, 13 mmol) in THF (25 mL) was added twice with 10 mL of diethyl ether. The aqueous layer was (1S,2S,3R,5S)-2,6,6-trimethylbicyclo[3.1.1 heptane-2,3- 25 concentrated to afford a sticky residue as crude product. The diol (2.4 g. 14.3 mol) at room temperature. The reaction residue was rinsed with 5 mL of water, dissolved in 10% mixture was stirred for 16 hand then was concentrated under MeCN-water and lyophilized to afford 2-((3R)-2-hydroxy-3- vacuum. The residue was purified by column chromatogra (2-(thiophen-2-yl)acetamido)-1,2-oxaborinan-6-yl)acetic phy (100% hexane->40% EtOAc/hexane) on silica gel to give acid 1 as white powder (100 mg, 0.337 mmol. 84.1% yield). 1-(tert-butoxy)-3-(tert-butyldimethylsilyl)oxy-1-oxo-6- 30 H NMR (CDOD) 8 ppm 0.94-1.35 (m. 1H), 1.35-1.54 (m, (2S,6R)-2,9.9-trimethyl-3,5-dioxa-4-boratricyclo6.1.1.02, 1H), 1.54-1.68 (m. 1H), 1.68-2.00 (m. 1H), 2.20-2.67 (m, 6 decan-4-ylhexan-3-yl XXXIX (5.5 g, 11 mmol, 84.6% 3H), 3.93 (s, 1H), 3.98 (s, 1H), 402-4.23 (m, 2H), 6.98-7.05 yield). (m. 2H), 7.32-7.36 (m, 1H); ESIMS found for CHBNOs m/z 280 (100%) (M-HO)+. Step 3 35 Alternative procedures for Steps 5 and 6 are shown in To a solution of DCM (1.5 mL, 23.6 mmol) in THF (30 mL) Scheme 9. at -100°C. was added 2.5M n-butyl lithium in hexane (5.19 mL, 12.98 mmol) slowly under nitrogen and down the inside wall of the flask whilst maintaining the temperature below 40 Scheme 9 -90° C. The resulting white precipitate was stirred for 30 Method B minutes before the addition of 1-(tert-butoxy)-3-(tert-bu O O RCOH, EDCI, tyldimethylsilyl)oxy-1-oxo-6-(2S,6R)-2,9.9-trimethyl-3, O N(TMS) HOBT, NMM 5-dioxa-4-boratricyclo6.1.1.02.6 decan-4-ylhexan-3-yl Ye CHCl2 XXXIX (5.5g, 11 mmol) in THF (10 mL) at -90° C. Zinc 45 o --Method C chloride (23.6 mL, 0.5M in diethyl ether, 11.86 mmol) was then added to the reaction mixture at -90° C. and then the H TBSO RCOH, HATU III DIPEA reaction was allowed to warm to room temperature where it CHCl2 was stirred for 16 h. The reaction was quenched with a satu rated Solution of ammonium chloride and the phases were 50 separated. The aqueous phase was then extracted with diethyl ether (3x50 mL) and the combined organic extracts were R - O O Method E dried over NaSO filtered and concentrated under reduced o NH 90% aq. TFA pressure. The concentrated material was then chromato Hess 55 Yp Anisole graphed (100% hexane->50% EtOAc/hexane) to obtain M 6-(tert-butoxy)-4-(tert-butyldimethylsilyl)oxy-1-chloro-6- O oxo-1-(2S,6R)-2,9.9-trimethyl-3,5-dioxa-4-boratricyclo H TBSO 6.1.1.02.6 decan-4-ylhexyl XL (5.6 g. 10.5 mmol. 95.4% XXXV yield). O 60 Step 4-5 R-( HN Chloro intermediate XL (1.2g, 2.33 mmol) in THF (10 B- O OH mL) was cooled to -78° C. under nitrogen. A solution of M LiHMDS (2.33 mL, 1.0 M in THF, 2.33 mmol) was added 65 HO slowly and the reaction flask was then allowed to warm to XXXVI room temperature where it was stirred for 16 h. Method A: US 8,680,136 B2 64 -continued Method B RCOH, EDC1, O. O HOBT, NMM

O N(TMS): k HCHCl2 C B Method C O RCOH, HATU H TBSO DIPEA XLI CHCl2 10 R O -Q O O Method E O NH k 90% aq.TFA M B Anisole M O 15 2-((3R)-2-hydroxy-3-(2-phenylacetamido)-1,2-oxabori H TBSO nan-6-yl)acetic acid 2. 'H NMR (CDOD) 8 ppm 0.82-1.33 XLIII (m. 1H), 1.33-1.51 (m, 1H), 1.51-1.68 (m, 1H), 1.69-2.00 (m, O 1H), 2.14-2.34 (m. 1H), 2.34-2.69 (m, 2H), 3.74-3.76 (m, 2H), 3.98-4.20 (m, 1H), 7.22-7.41 (m, 5H); ESIMS found for R-( C14H18BNO5 m/z 274 (100%) (M-HO)". HN B- O OH / HO O XLIV 25

Step 5, Method B To a solution of the acid (0.36 mmol) in DCM (10 mL) at 0° 30 C. under nitrogen was added EDCI (86 mg, 0.45 mmol) and HOBT (48 mg, 0.36 mmol). After stirring at 0°C. for 30 minutes, a solution of the bis-silyl amide intermediate XLI (0.3 mmol) in DCM (2 mL) followed by N-methyl-morpho line (65 uL, 0.6 mmol) were sequentially added at 0°C. The 35 reaction flask was then allowed to warm to room temperature. After stirring at room temperature overnight, the reaction mixture was washed with water, then brine, dried (NaSO), filtered and concentrated under vacuum. The residue was purified by column chromatography to produce intermediate 40 XLIII. 2-((3R)-3-acetamido-2-hydroxy-1,2-oxaborinan-6-yl) acetic acid 3. 'H NMR (CDOD) 8 ppm 1.07-1.36 (m. 1H), Step 5, Method C 1.36-1.59 (m, 1H), 1.59-1.73 (m. 1H), 1.73-2.09 (m. 1H), A solution of bis-silyl amide XLI (0.5 mmol) and acid in 2.15-2.16 (d. 3H), 2.35-2.69 (m, 3H), 4.01-4.23 (m. 1H): dry DCM (10 mL) were cooled to 0°C. Then DIPEA (1.5 45 ESIMS found for CHBNOs m/z 198 (100%) (M-HO)". mmol) was added drop wise followed HATU (0.75 mmol). The mixture was then allowed to warm to room temperature.

After TLC has indicated complete conversion (-3 h) of the starting materials, the reaction was diluted with additional DCM (20 mL). The reaction mixture was washed with water 50 (3x5 mL), brine (10 mL), and dried over NaSO. After removal of the solvent, the residue was subjected to flash column chromatography to produce intermediate XLIII. Step 6, Method E 55 To a solution of amide (XLIII) (0.1 mmol) in dichloroet hane (2 mL) at 0°C. was treated with pre-cooled 90% aq.TFA 2-((3R)-3-(cyclopropanecarboxamido)-2-hydroxy-1,2- (4 mL) and stirred at room temperature for 3 hrs. The reaction oxaborinan-6-yl)acetic acid 4. 'H NMR (CDOD) 8 ppm mixture was evaporated in vacuo, azeotroped with MeCN 0.98-1.32 (m, 5H), 1.32-1.67 (m, 2H), 1.67-2.06 (m, 2H), (3x5 mL) and the residue was triturated with ether (5 mL). 60 2.27-2.66 (m, 3H), 3.98-4.16 (m, 1H); ESIMS found for The product separated was filtered, dissolved in dioxane CHBNOs m/z 224 (100%) (M-HO)". water mixture and freeze dried to give the final productXLIV as a fluffy solid. The following compounds are prepared starting from enan The following compounds are prepared in accordance with 65 tiomerically pure (R)-tert-butyl 3-hydroxypent-4-enoate (J. the procedure described in the above Example 1 using meth Am. Chem. Soc. 2007, 129,4175-4177) in accordance with ods A and D. the procedure described in the above Example 1.

US 8,680,136 B2 75 76 Synthesis of 2-((3R)-3-amino-2-hydroxy-1,2-oxaborinan Example 2 6-yl)acetic acid hydrochloride 7. An example synthesis of 7 is depicted in Scheme 10 and Example 2. Step 1

5 6-(tert-butoxy)-4-(tert-butyldimethylsilyl)oxy-1-chloro Scheme 10 6-oxo-1-(2S,6R)-2,9.9-trimethyl-3,5-dioxa-4-boratricyclo

O LHMDS 6.1.1.02.6 decan-4-ylhexane XLI (515 mg, 0.97 mmol) in THF THF (5 mL) was cooled to -78°C. under nitrogen. A solution -78° C. - rt of LiHMDS (1 mL 1.0 Min THF, 1 mmol. 1.0 eq) was added 16h 10 slowly and the reaction flask was then allowed to warm to -e- room temperature where it was stirred for 16 h. The yellow H TBSO Solution was concentrated under reduced pressure to give an XLI oil. After hexane (10 mL) was added to the oil, a precipitate 3NHC Dioxane 15 formed. This was then filtered through Celite and the filtrate O O 110° C., concentrated under reduced pressure to give 1-bis(trimeth O N(TMS) 1.5 ylsilyl)amino-6-(tert-butoxy)-4-(tert-butyldimethylsilyl) B oxy-6-oxo-1-(2S,6R)-2,9.9-trimethyl-3,5-dioxa-4-boratri M cyclo6.1.1.02.6 decan-4-ylhexyl XLII. O H TBSO Step 2 XLII C The procedure is identical to that found in Example 1 2. method D. Compound 7 was isolated as a white powder (120 HN 25 mg, 0.573 mmol. 59.1% yield). H NMR (CDOD) 8 ppm B-O OH 1.43-1.66 (m, 1H), 1.66-1.79 (m. 1H), 1.79-1.97 (m. 1H), / 1.97-2.30 (m, 1H), 2.40-2.71 (m, 3H), 4.34-4.54 (m, 1H): HO O ESIMS found for CHBNO, m/z. 174 (63%) (M+H). 7 Synthesis of 2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)ac etamido)-1,2-oxaborepan-7-yl)acetic acid 63. An example synthesis of 63 is depicted in Scheme 11 and Example 3.

US 8,680,136 B2 79 80 Example 3 reaction was allowed to warm to room temperature where it was stirred for 16 h. The reaction was quenched with a satu Step 1 rated Solution of ammonium chloride and the phases were separated. The aqueous phase was then extracted with diethyl To a solution of tert-butyl 3-hydroxypent-4-enoate, XLVI ether (2x10 mL) and the combined organic extracts were (674 mg, 3.92 mmol) in DCM (15 mL) was added diisopro dried over NaSO filtered and concentrated under reduced pylallylboronate XLV (2 g, 11.76 mmol) via syringe. To the pressure. The concentrated material was then chromato mixture was then added Grubbs' first generation catalyst (260 graphed (100% hexane->20% EtOAc-hexane) to obtain tert mg, 0.31 mmol. 7.5 mol%) and the vessel was purged with butyl (7S)-3-(tert-butyldimethylsilyl)oxy-7-chloro-7-(1R, argon. The reaction was heated at 65° C. under nitrogen for 18 10 2R,6S,8R)-6.9.9-trimethyl-3,5-dioxa-4-boratricyclo h. The mixture was concentrated under vacuum and the resi 6.1.1.0° decan-4-ylheptanoate LI (740 mg, 1.40 mmol. due was purified by flash column chromatography (100% 67.2% yield). hexane->30% EtOAc/hexane) to afford tert-butyl 2-(2-hy droxy-3,6-dihydro-2H-1,2-oxaborinin-6-yl)acetate XLVII Step 6 (770 mg, 3.63 mmol, 92.7% yield). 15 Chloro intermediate LI (727 mg, 1.37 mmol) in THF (7 Step 2 mL) was cooled to -78°C. under nitrogen. A solution of 1M To a solution of tert-butyl 2-(2-hydroxy-3,6-dihydro-2H LiHMDS solution in THF (1.37 mL, 1.37 mmol) was added 1.2-oxaborinin-6-yl)acetate XLVII (670 mg, 3.16 mmol) in slowly at -78°C. Upon completion of the addition, the reac EtOAc (45 mL) was added 10% Pd/C (135 mg). The vessel tion flask was allowed to warm to room temperature. After was evacuated by applying vacuum and flushed with hydro stiffing at room temperature for 16 h, the reaction mixture was gen gas. The reaction was stirred under hydrogen for 2h. The concentrated under vacuum and hexane (20 mL) was added. mixture was filtered through a Celite pad and which was The precipitated lithium salts were filtered off through a washed with additional EtOAc (15 mL). Concentration of the 25 Celite pad, rinsed with additional hexane and the combined filtrate gave pure tert-butyl 2-(2-hydroxy-1,2-oxaborinan-6- filtrates were concentrated under vacuum to give crude tert yl)acetate XLVIII (641 mg, 3.00 mmol, 94.8% yield). butyl (7S)-7-bis(trimethylsilyl)amino-3-(tert-butyldim ethylsilyl)oxy-7-(1R,2R,6S,8R)-6.9.9-trimethyl-3,5-di Step 3 oxa-4-boratricyclo[6.1.1.0° decan-4-ylheptanoate LII. 30 To a solution of tert-butyl 2-(2-hydroxy-1,2-oxaborinan-6- Step 7 yl)acetate XLVIII (641 mg, 3.00 mmol) in THF (20 mL) was added (1S,2S,3R,5S)-2,6,6-trimethylbicyclo[3.1.1 heptane To a stirred solution of 2-thiophenacetic acid (232 mg, 1.64 2,3-diol (509 mg, 3 mol) at room temperature. The reaction mmol) in DCM (45 mL) at 0°C. under nitrogen was added mixture was stirred for 16 hand concentrated under vacuum. 35 EDCI (391 mg, 2.05 mmol) and HOBT (221 mg, 1.64 mmol). The residue was purified by column chromatography (100% After stirring at 0°C. for 30 minutes, a solution of the bis-silyl hexane->40% EtOAc/hexane) on silica gel to give tert-butyl amide LII intermediate (1.37 mmol) in DCM (10 mL) fol 3-hydroxy-6-(1R,2R,6S,8R)-6.9.9-trimethyl-3,5-dioxa-4- lowed by N-methyl-morpholine (0.3 mL, 2.74 mmol) were boratricyclo[6.1.1.0 decan-4-ylhexanoate XLIX (790 sequentially added at 0°C. Upon completion of the addition, mg, 2.16 mmol. 71.9% yield). 40 the reaction flask was allowed to warm to room temperature. After stirring at room temperature overnight, the reaction Step 4 mixture was washed with water, dried and concentrated under vacuum. The residue was purified by column chromatogra To a solution of alcohol XLIX (790 mg, 2.16 mmol) in phy (100% DCM->50% EtOAc/DCM) to afford tert-butyl DMF (7.5 mL) was added imidazole (548 mg, 8.06 mmol) 45 (7S)-3-(tert-butyldimethylsilyl)oxy-7-2-(thiophen-2-yl) followed by TBDMSC1 (580 mg, 3.87 mol). The reaction acetamido-7-(1R,2R,6S,8R)-6.9.9-trimethyl-3,5-dioxa-4- mixture was stirred at room temperature for 16 hand concen boratricyclo[6.1.1.0 decan-4-ylheptanoate LIII (340 mg. trated under vacuum. The white slurry was dissolved in 100 0.54 mmol. 39.4% yield for 2 steps). mL of EtOAc and washed with saturated NaHCO solution (20 mL), water (2x10 mL) and dried (NaSO). The organic 50 Step 8 extract was concentrated under vacuum and the residue was purified by column chromatography (100% hexane->30% To a solution of amide LIII (300 mg, 0.47 mmol) in 1,4- EtOAc/hexane) on silica gel to give tert-butyl 3-(tert-bu dioxane (9 mL) was added 9 mL of 3 NHC1. The reaction tyldimethylsilyl)oxy-6-(1R,2R,6S,8R)-6.9.9-trimethyl-3, mixture was heated at reflux for 90 minutes. The cooled 5-dioxa-4-boratricyclo[6.1.1.0° decan-4-ylhexanoate L (1 55 reaction mixture was then diluted with water (10 mL) and g, 2.08 mmol, 96.3% yield). extracted with diethyl ether (2x10 mL). The aqueous layer was concentrated to afford a sticky Solid which was azeo Step 5 troped with MeCN (3x10 mL). The residue was dissolved in 40% dioxane-water and lyophilized to afford 2-((3R)-2-hy To a solution of DCM (0.26 mL, 4.16 mmol) in THF (5 mL) 60 droxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborepan-7- at -100°C. was added 2.5Mn-butyl lithium in hexane (1 mL, yl)acetic acid 63 as an off-white solid (100 mg. 32.1 mmol. 2.5 mmol) slowly under nitrogen and down the inside wall of 68.4% yield). H NMR (CDOD) 8 ppm 1.21-1.38 (m, 2H), the flask whilst maintaining the temperature below -90° C. 1.42-1.60 (m, 2H), 1.60- 1.72 (m. 1H), 1.80-1.94 (m. 1H), The resulting white precipitate was stirred for 30 minutes 2.32-2.47 (m, 2H), 2.54-2.58 (dd, J=15 Hz, J-6 Hz, 1H), before the addition of L (1 g, 2.08 mmol) in THF (3 mL) at 65 3.97-3.98 (d. J=8 Hz, 1H), 4.05 (s. 2H), 6.97-7.01 (m. 1H), -90° C. Zinc chloride (5 mL, 0.5M in THF, 2.5 mmol) was 7.02-7.10 (m, 1H), 7.33-7.37 (m, 1H); ESIMS found for then added to the reaction mixture at -90° C. and then the CHsBNOS m/z 294.0 (M-HO)". US 8,680,136 B2 81 82 Synthesis of 2-((3R)-2-hydroxy-3-(2-(thiophen-2-yl)ac The organic extract was concentrated under vacuum and the etamido)-2,3,4,7-tetrahydro-1,2-oxaborepin-7-yl)acetic acid residue was purified by column chromatography (100% hex 64. An example synthesis of 64 is depicted in Scheme 12 and ane->20% EtOAc/hexane) on silica gel to give tert-butyl Example 4. (4Z)-3-(tert-butyldimethylsilyl)oxy-6-(1R,2R,6S,8R)-6,

Scheme 12 O O N O B (+)-Pinanediol l HO1 NO oB - - ... wO B /-, -on TBSCIIs e wO B /-, -on l THF C to HO DMF C O TBSO XLVII LIV LV

DCMon-BuLI THF, -100° C. S O O A NH /= O O'Bu (MeSi)Nspy o O'Bu Cl o O'Bu O Thiophenacetic O-B TBSO TBSO sg-R TBSO aacid s b S. s. EDCI, HOBT S 3) DCM

LVII LVI LVIII

3NHC Dioxane

O N OH

B- O M HO 64

Example 4 9.9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0 decan-4- ylhex-4-enoate LV (800 mg, 1.67 mmol. 93.9% yield). Step 1 45 To a stirred solution of tert-butyl 2-(2-hydroxy-3,6-dihy Step 3 dro-2H-1,2-oxaborinin-6-yl)acetate XLVII (770 mg. 4.58 mmol) in THF (25 mL) was added (1S,2S,3R,55)-2,6,6-tri 50 To a solution of DCM (0.3 mL, 4.68 mmol) in THF (8 mL) methylbicyclo[3.1.1 heptane-2,3-diol (980 mg, 4.58 mmol) at -100°C. was added 2.5M n-butyl lithium in hexane (1.12 at room temperature. The reaction mixture was stirred for 16 mL, 2.8 mmol) slowly under nitrogen and down the inside h and concentrated under vacuum. The residue was purified wall of the flask whilst maintaining the temperature below by column chromatography (100% hexane->30% EtOAc/ -90° C. The resulting white precipitate was stirred for 30 hexane) on silica gel to give tert-butyl (4Z)-3-hydroxy-6- 55 minutes before the addition of LV (1.12g, 2.34 mmol) in THF (1R,2R,6S,8R)-6,9.9-trimethyl-3,5-dioxa-4-boratricyclo (3 mL) at -90° C. and the reaction was allowed to warm to 6.1.1.0 decan-4-ylhex-4-enoate LIV (1 g, 2.75 mmol, room temperature where it was stirred for 16 h. The reaction 59.9% yield). was quenched with a saturated Solution of ammonium chlo ride and the phases were separated. The aqueous phase was Step 2 60 then extracted with diethyl ether (2x10 mL) and the combined organic extracts were dried over NaSO, filtered and con To a solution of alcohol LIV (650 mg, 1.78 mmol) in DMF centrated under reduced pressure. The concentrated material (10 mL) was added imidazole (484 mg, 7.12 mmol) followed was then chromatographed (100% hexane->20% EtOAc/ by TBDMSC1 (534 mg., 3.56 mol). The reaction mixture was hexane) to obtaintert-butyl (4Z.7S)-3-(tert-butyldimethylsi stirred at room temperature for 16 hand concentrated under 65 lyl)oxy-7-chloro-7-(1R,2R,6S,8R)-6.9.9-trimethyl-3,5-di vacuum. The white slurry was dissolved in 100 mL of EtOAc oxa-4-boratricyclo[6.1.1.0° decan-4-ylhept-4-enoate LVI and washed with water (2x10 mL), brine and dried (Na2SO). (820 mg, 1.56 mmol. 66.5% yield). US 8,680,136 B2 83 Step 4 Scheme 13 Chloro intermediate LVI (790 mg, 1.49 mmol) in THF (10 mL) was cooled to -78°C. under nitrogen. A solution of 1M LiHMDS solution in THF (1.5 mL, 1.5 mmol) was added S f O slowly at -78°C. Upon completion of the addition, the reac 1M HCl in EtOAC -3- tion flask was allowed to warm to room temperature. After HN ETOH, rt, 16 h stirring at room temperature for 16 h, the reaction mixture B- O OH was concentrated under vacuum and hexane (20 mL) was 10 / added. The precipitated lithium salts were filtered off through HO O a Celite pad, rinsed with additional hexane and the combined 5 filtrates were concentrated under vacuum to give crude tert butyl (4Z,7S)-7-bis(trimethylsilyl)amino-3-(tert-bu 15 S f O tyldimethylsilyl)oxy-7-(1R,2R,6S,8R)-6.9.9-trimethyl-3, 5-dioxa-4-boratricyclo[6.1.1.0 decan-4-ylhept-4-enoate LVII. HN - B-O O / Step 5 -)-.HO O 65 To a stirred solution of 2-thiophenacetic acid (252 mg, 1.78 mmol) in DCM (35 mL) at 0°C. under nitrogen was added 25 EDCI (426 mg, 2.23 mmol) and HOBT (240 mg, 1.78 mmol). After stirring at 0°C. for 30 minutes, a solution of the crude Example 5 bis-silyl amide LVII intermediate in DCM (10 mL) followed by N-methyl-morpholine (0.32 mL, 3 mmol) were sequen Step 1 tially added at 0°C. Upon completion of the addition, the 30 reaction flask was allowed to warm to room temperature. To a solution of 5 (400 mg, 1.35 mmol) in 4 mL of absolute After stirring at room temperature overnight, the reaction ethanol was added anhydrous 1M HCl in EtOAc (4 mL, 4 mixture was washed with water, dried and concentrated under mmol). The reaction was stirred at room temperature for 16 h. vacuum. The residue was purified by column chromatogra 35 The mixture was then concentrated and azeotroped with phy (100% DCM->25% EtOAc/DCM) to afford tert-butyl acetonitrile (3x10 mL) to give a sticky solid. Ether (10 mL) (4Z,7S)-3-(tent-butyldimethylsilyl)oxy-7-2-(thiophen-2- was added to the azeotroped Sticky Solid and the resulting yl)acetamido-7-(1R,2R,6S,8R)-6.9.9-trimethyl-3,5-dioxa precipitate was filtered. The filtered solid was rinsed with 4-boratricyclo6.1.1.0 decan-4-ylhept-4-enoate LVIII additional ether (5 mL) and dried to give ethyl 2-((3R,6S)-2- 40 hydroxy-3-(2-(thiophen-2-yl)acetamido)-1,2-oxaborinan-6- (600 mg, 0.95 mmol, 63.7% yield for 2 steps). yl)acetate 65 (300 mg, 0.92 mmol. 68.5% yield). H NMR (CDOD) 8 ppm 0.98-1.09 (q, J=14 Hz, 1H), 1.23-1.26 (t, J=7 Hz, 3H), 1.49-1.54 (dd, J=14 Hz, J=3 Hz, 1H), 1.57-1.64 (dt, Step 6 J=11 Hz, J-2 Hz, 1H), 1.72-1.78 (brd, J=14 Hz, 1H), 2.24 45 2.28 (dd, J=15 Hz, J-6 Hz, 1H), 2.34-2.39 (dd, J=15 Hz, J=8 HZ, 1H), 2.63 (brs, 1H), 3.99 (s. 2H), 4.07-4.13 (q, J–4 Hz, A solution of amide LVIII (100 mg, 0.15 mmol) in anisole 3H), 6.99-7.01 (t, J=4 Hz, 1H), 7.05-7.06 (d. J=3 Hz, 1H), (5 mL) at 0°C. was treated with pre-cooled 90% aq trifluo 7.35-7.36 (dd, J=5 Hz, J=1.3 Hz, 1H); ESIMS found for roacetic acid (10 mL). The reaction mixture was warmed to CHBNOS m/z,308.1 (M-HO)". room temperature and stirred for 16 h. The mixture was 50 Synthesis of 2-((3R,7R)-2-hydroxy-3-(2-(thiophen-2-yl) evaporated in vacuo, azeotroped with MecN (3x5 mL). The acetamido)-2,3,4,7-tetrahydro-1,2-oxaborepin-7-yl)acetic residue was sonicated in water (10 mL) and ether (10 mL). acid 67. An example synthesis of 67 is depicted in Scheme 14 The aqueous phase was separated, washed with ether (2x5 and Example 6. mL) and freeze dried to give fluffy solid 2-((3R)-2-hydroxy 3-(2-(thiophen-2-yl)acetamido)-2,3,4,7-tetrahydro-1,2-ox 55 aborepin-7-yl)acetic acid 64 (15 mg, 0.05 mmol. 32.3% Scheme 14 yield). H NMR (CDOD) 8 ppm 2.23-2.35 (m, 2H), 2.40 2.61 (m, 2H), 2.76-2.83 (m. 1H), 3.96-4.03 (m, 1H), 4.10 (s, 2H), 5.34-5.40 (m. 1H), 5.53-5.74 (m, 1H), 6.97-7.08 (m, 60 Grubb's 2H), 7.32-7.39 (m. 1H); ESIMS found for CHBNOS m/z Catalyst 1. B -- -e- 292 (M-HO)". O HODJ O'Bu DCM, reflux Synthesis of ethyl 2-((3R,6S)-2-hydroxy-3-(2-(thiophen -N N LIX 2-yl)acetamido)-1,2-oxaborinan-6-yl)acetate 65. An 65 example synthesis of 65 is depicted in Scheme 13 and XLV Example 5. US 8,680,136 B2 85 86 -continued -continued

a

S f 8.N y

10

OH

15 Example 7

Step 1 Prepared starting from enantiomerically pure (S)-tert-butyl Example 6 3-hydroxypent-4-enoate J. Med. Chem. (2010), 53, 4654 4667 in accordance with the procedure described in the Step 1 above Step 1 of Example 3 25 Prepared starting from enantiomerically pure (R)-tert-bu Steps 2-7 tyl 3-hydroxypent-4-enoate J. Am. Chem. Soc. (2007), 129, 4175-4177 in accordance with the procedure described in the Prepared in accordance with the procedure described in the above Step 1 of Example 3 30 above Steps 1-6 of Example 4. White fluffy solid (45 mg, 0.146 mmol. 39% yield). "H Steps 2-7 NMR (CDOD) 8 ppm 2.15-2.18 (m. 1H), 2.29-2.38 (m, 2H), 2.66-2.72 (m, 2H), 3.88-3.91 (m, 1H)4.00 (s. 2H), 5.24-5.27 Prepared in accordance with the procedure described in the 35 (m. 1H), 5.57-5.63 (m, 1H), 6.87-6.96 (m, 2H), 7.24-728 (m, above Steps 1-6 of Example 4. 1H); ESIMS found for CHBNOS m/z 292 (M-HO)". White fluffy solid (23 mg, 0.074 mmol, 47% yield). "H Synthesis of 2-((3R,6S)-3-(benzyloxycarbonylamino)-2- NMR (CDOD) 8 ppm 2.29-2.31 (m. 1H), 2.40-2.68 (m, 4H), hydroxy-1,2-oxaborinan-6-yl)acetic acid 69. An example 4.10 (m, 2H), 4.74-4.82 (m, 1H), 5.35-5.38 (m, 1H), 5.53 synthesis of 69 is depicted in Scheme 16 and Example 8. 5.58 (m, 1H), 6.98-7.05 (m, 2H), 7.32-7.36 (m, 1H); ESIMS 40 found for CHBNOS m/z 292 (M-HO)". Synthesis of 2-((3R,7S)-2-hydroxy-3-(2-(thiophen-2-yl) Scheme 16 acetamido)-2,3,4,7-tetrahydro-1,2-oxaborepin-7-yl)acetic O O acid 68. An example synthesis of 68 is depicted in Scheme 15 45 O N(TMS) BOCOC N CHCl2 and Example 7. B M O H TBSO Scheme 15 XLI 50

N O Grubb's Catalyst 1. B -- '', -e- lsO s HO w O'Bu DCM, reflux 55 XLV O 60 o- O O 90% ad TFA O NH NY N O M Anisole B -- M B -e- O Ho1 No1 "", O'Bu Step 2-7 H TBSO 65 XLIII US 8,680,136 B2 87 88 -continued Synthesis of 2-((3R,6S)-2-hydroxy-3-(phenylsulfona mido)-1,2-oxaborinan-6-yl)acetic acid 71. An example syn thesis of 71 is depicted in Scheme 17 and Example 9.

Scheme 17 O O O N(TMS) PhS(O)Cl N CH.C. OH 10 B M O 69 H TBSO XLI 15 Example 8

Step 1 e O o2S3 O O 90% aq. TFA O NH He A solution of bis-silyl amide XLI (0.2 mmol) in DCM (5 v Anisole B mL) was cooled to 0° C. and benzyl chloroformate (0.056 M mL, 0.4 mmol) was added. Then, the cooling bath was O removed and the solution stirred at ambient temperature for H TBSO LXIV 16 h. The reaction was quenched with water and extracted 25 twice with EtOAc. The organic layers were combined, washed with water, brine, dried (NaSO) and concentrated in vacuo to afford a pale yellow oil as crude product. The residue O was chromatographed on a silica column (100% DCM->40% M OES EtOAc/DCM) to afford carbamate LXIII (90 mg, 0.143 30 V mmol, 71.5% yield). HN B- O OH Step 2 M HO O A solution of carbamate LXIII (70 mg, 0.11 mmol) in 35 71 anisole (5 mL) at 0°C. was treated with pre-cooled 90% aq trifluoroacetic acid (10 mL). The reaction mixture was warmed to room temperature and stirred for 16h. The mixture was evaporated in vacuo, azeotroped with MeCN (3x5 mL). Example 9 The residue was sonicated in water (10 mL) and ether (10 40 mL). The aqueous phase was separated, washed with ether (2x5 mL) and freeze dried to give 2-((3R,6S)-3-(benzyloxy Step 1-2 carbonylamino)-2-hydroxy-1,2-oxaborinan-6-yl)acetic acid 69 as a fluffy solid (10 mg 0.033 mmol. 29.6% yield). ESIMS Prepared in accordance with the procedure described in the found for C.H.BNOS m/z 289.9 (M-HO)". 45 above Steps 1-2 of Example 8. The following compound is prepared in accordance with Off-white solid (30 mg, 0.096 mmol, 43% yield). "H NMR the procedure described in the above Example 8. (CDOD) 8 ppm 1.57-1.83 (series of m, 4H), 2.49-2.71 (se ries of m, 3H), 4.35-4.89 (m, 1H), 7.51-7.59 (m, 3H), 7.85 50 7.89 (m, 2H); ESIMS found for CHBNOS m/z 296.1 70 (M-HO)". Synthesis of 2-((3R,6S)-2-hydroxy-3-(3-phenylureido)-1, 2-oxaborinan-6-yl)acetic acid 72. An example synthesis of 72 is depicted in Scheme 18 and Example 10. 55

Scheme 18

O O 1. TFA 60 O N(TMS) CHCl2 2-((3R,6S)-2-hydroxy-3-(isobutoxycarbonylamino)-1,2- B -e-2. PhNECEO oxaborinan-6-yl)acetic acid 70 as a off-white solid (20 mg. O DIEA 0.073 mmol, 27% yield). "H NMR (CDOD) 8 ppm 0.95 (d. H TBSO CHCl2 XLI J=7 Hz, 6H), 1.62-1.67 (m, 1H), 1.70-1.75 (m, 2H), 1.87-1.90 65 (m. 2H), 2.42-2.60 (m,3H), 3.77-3.86 (m, 2H), 4.35-4.38 (m, 1H); ESIMS found for CHBNOS m/z 256 (M-HO)". US 8,680,136 B2 90 -continued TABLE 1 Example Structure

NH 1 a S o- O O 90% ad TFA o O O NH NY M Anisole B M HN O 10 H TBSO B-O OH / LXV HO

2 15 N-(O HN O HN B-O OH 72 / HO

25 3 O Example 10 HN B-O OH Step 1 / 30 HO

To a solution of bis-silyl amide XLI (0.2 mmol) in DCM (5 4. mL) at 0°C. was added a solution of TFA in hexane (0.6 mmol). The reaction was stirred at 0°C. for 20 min before adding phenyl isocayanate (0.04 mL, 0.4 mmol) followed by 35 N,N-diisopropylethylamine (0.18 mL, 1 mmol). The cooling bath was then removed and the solution was stirred at ambient temperature for 16 h. The reaction was quenched with water O and extracted twice with EtOAc. The organic layers were combined, washed with water, brine, dried (NaSO) and 40 concentrated in vacuo to afford a pale yellow oil as crude product. The residue was chromatographed on a silica col umn (100% DCM->25% EtOAc/DCM) to afford the pure urea (50 mg, 0.081 mmol. 40.7% yield). 45 Step 2 Deprotection was performed following the procedure described above in step 2 of example 8 to give 2-((3R,6S)-2- 50 hydroxy-3-(3-phenylureido)-1,2-oxaborinan-6-yl)acetic acid 72 as a white solid (20 mg, 0.068 mmol, 86% yield). "H NMR (CDOD) 8 ppm 1.24-1.31 (m, 1H), 1.56-1.64 (m, 2H) 1.78-1.81 (m, 1H), 2.36-2.40 (dd, J=15 Hz, J-6 Hz, 1H), 2.46-2.58 (dd, J=13 Hz, J-7 Hz, 1H), 2.68-2.71 (m, 1H), 55 4.07-4.12 (m. 1H), 7.15-7.18 (m, 1H), 7.34-7.37 (m, 4H); ESIMS found for CHBNOs m/z 275.1 (M-HO)". Illustrative compounds of Formula (I) are shown in Table 1. Some structures are shown with defined configurations at 60 selected stereocenters but the shown stereochemistries are not meant to be limiting and all possible Stereoisomers of the shown structures are to be considered encompassed herein. Compounds of any absolute and relative configurations at the Stereocenters as well as mixtures of enantiomers and diaste 65 reoisomers of any given structure are also encompassed herein. US 8,680,136 B2 91 92 TABLE 1-continued TABLE 1-continued

Example Structure Example Structure

8 O 5 16 O HN O HN p- O OH HN 10 HO O B- O OH / 9 HN O HO O

17 HN 15 B- O OH NH / HO O

HN 10 O 2O p- O OH HN HO O O B - O OH /-/ HO O 18

11 HOC O HN

30 B- O OH W \ -)-.HO/ O S 19 O 12 O \, 29 35 V H HN HN B-O OH y -O OH Ho? O HO O 40 2O 13 O R / NS HN 45 HN HN \ / /B-O OH B-O OH HO O Ho? O

14 pMe 50 21 W O

N \ HN y -O OH 55 HN S HO O

15 22 O 60 HOC

O NH HN HOC - B- O OH HN O HG O B-O OH HN 65 HO O US 8,680,136 B2 93 TABLE 1-continued TABLE 1-continued

Example Structure Example Structure 30 23 N= 5 HOC \ A. O HOC HN HN B-O OH B- O OH 10 M HO O W \ O HO O

24 HOC O S ... . 15 31 a B-O OH S 4 O M N HO O W OH 2O N N 'it 25 HOC O V H B- O O / HO HOC HN B-O OH / 25 32 HO O

26 HOC B- O OH W 30 O HO O HOC HN W \ B-O OH M HO 33 NH 35 H 27 HO N O

O B OH H OH

40 34

28 OH

B-O 45 / HN O H

35 H HO N 50

O OH

29 36 HN 55

60 N 21

OH

65 HO US 8,680,136 B2 95 96 TABLE 1-continued TABLE 1-continued

Example Structure Structure 37 NH H w N

O B HO1 No OH 38 46 NH

O OH OH

39 47 OH

25 48

40

30 49

35 50

O OH

41 40 51

O OH

42 45 52

50 O OH 53 43

55 1 in OH O OH

44 S4

60

N 65 OH US 8,680,136 B2 97 98 TABLE 1-continued TABLE 1-continued

Example Structure Example Structure 55 HN 5

63 N -( WB-O OH N O HO O

10 O

56 OUB-O ANN N H 15 W NaN N O HO

O OH 64

57 N O 2O O N O OH OH

58 B- O 25 M HO

65 2

30 OH

59

35

66

40

O OH

60 45 67

O OH

61 50

O OH

62 NH2 55

68

60 O O OH r > -o

O 65 US 8,680,136 B2 99 100 TABLE 1-continued TABLE 1-continued

Example Structure Example Structure

69 75

O B-O 10 W HO

B-O OH

HO O 15

70 76

O

B-O / HO B- O OH M HO O 25

71

77 30 N

B-O W 35 HO

72

40

78

B-O OH / HO O 45

73

O N 50

w OH 't N B-O 79 HO 55

O 74 N OH HN 'tt N B- O 60 W HO OH

65 US 8,680,136 B2 101 102 TABLE 1-continued TABLE 1-continued Example Structure Example Structure 8O ar ? O O

HO O-N N it X-/ B-O NH2 O HO 10 O N 81 4 O y OH N 'it S O O H B-O N 15 W HO / • OH N N "it V H B- O O / 88 HO 2O OH

82 a / N s O H B-O A w y-on 25 Hof HO O-N N t V / H B-O HO 89 83 HN \o M 30

S s O

O N it > -on 35 H B-O A HO

84

40 90 O N

v. OH 'tti B-O 45 / HO

85

50

O y) O 91 't H 55 O B- O Ho HN -o OH 86 HN O YN- 60 HO O s O

O N "ft lyson H B-O 65 US 8,680,136 B2 103 104 TABLE 1-continued TABLE 1-continued

Example Structure Example Structure

92 98 O N B-O OH / HO "it > -on 10 BHO / HO

99

15 B-O OH 93 / O HO O B-O - OH / HO 1OO HN B-O OH / 25 O HO O / 94 Nt \O 30 B-O?. " ul OH 101 / HO ( , 35 / N. CH2OH B-O OH / HO O 95 40 102 O O

OH N 't O y 45 H B-O / HO B-O OH / HO O 96 50 103

55 OH B-O OH 97 HO O

104 60

B-O OH HO O

65 US 8,680,136 B2 105 106 TABLE 1-continued TABLE 1-continued

Example Structure Example Structure

105

OH O 10 B-O OH

112 15 "N-N- l 106 O

O O N H A HO O

11 MeO-N O 3 \ 25 N HN

107 HN S Ho O

( , 30 114 HN W N HN HO W) -s V N 2 O B-O / HO 35 O H O 2 N OH O O B NN HO1 OuNo O 108 40 a 115 NH2 $ 4 O OH is HO-MN N KN M Z O-N HN H OH 45 O O- a N X-/ A B-O N O HO O B HO1 NO OH 109 116 a ( , 50

S f O O N / OH HO-N N 'tt H B- O 55 W HO 117 2 f 110 a 60 S O O S N / v OH HO O HOOC N 'til \ / H B-O OH M 65 HO US 8,680,136 B2 107 108 TABLE 1-continued The checkerboard (CB) assay is performed in microtiter plates. Ceftazidime orbiapenem are diluted in the X axis, each Example Structure column containing a single concentration of antibiotic. BLIS 118 2 are diluted in the y axis, each row containing an equal con 5 centration of BLI. The result of these manipulations is that 4 o O each well of the microtiter plate contains a unique combina N tion of concentrations of the two agents. The assay is per formed in MHB with a final bacterial inoculum of 5x105 HOHC / N v 'tti > -o CFU/mL (from an early-log phase culture). Microtiter plates H B-O 10 are incubated during 20 h at 35° C. and are read using a M microtiter plate reader (Molecular Devices) at 650 nm as well HO as visual observation using a microtiter plate reading minor. 119 a The MIC is defined as the lowest concentration of antibiotics, within the combination, at which the visible growth of the S f O 15 organism is completely inhibited. Activity of BLIs is reported at MPC8, or the minimal potentiation concentration to reduce the MIC of antibiotic 8-fold. HOHC / HN Ceftazidime potentiation is studied in strains of various B- O OH bacteria that are resistant to ceftazidime due to expression of / B-lactamase hydrolyzing enzymes. The panel of strains used HO O in checkerboard experiments contains B-lactamases that belong to all the known classes of these enzymes: A, B, C and 120 a D. Activity of Compound I is tested at the maximum concen S f O tration of 40 g/mL. At this concentration it shows no inhi 25 bition of growth of any bacteria tested, however at concen tration as low as 0.6 ug/mL it reduced MICs to ceftazidime 8-fold in some bacteria (Table 2). Based on CB results, 1 has HOOC / HN a broad-spectrum B-lactam potentiation activity against the B-O OH strains expressing B-lactamases. Compound I was the most / HO O 30 potent against the strains expressing KPCs and other class A enzymes (CTX-M-3), and some class C (MIR-1, CMY-2), 121 r and class D (OXA-2) enzymes. S O TABLE 2 / Strain Organism Description PCR Class MPC8 HO-N HN KP1005 Klebsielia ESBL CTX-M- A Z. B- O OH pneumoniae 14 KP1009 Kiebsielia ESBL CTX-M- A Y / pneumoniae 15 HO O 40 EC1008 Escherichia Coi ESBL CTX-M-3 A X KP1004 Kiebsielia Serine KPC-2 A. X pneumoniae carbapenemase KP1008 Kiebsielia Serine KPC-2 A. X Example 11 pneumoniae carbapenemase EC1007 Escherichia Coi Serine KPC-3 A. X The potency and spectrum of B-lactamase inhibitors was 45 carbapenemase KP1010 Kiebsielia ESBL SHV 12 A Y determined by assessing their antibiotic potentiation activity. pneumoniae The potentiation effect is observed by the reduction of the KP1012 Klebsielia ESBL SHV-18. A Y minimum inhibitory concentration off-lactam antibiotics in pneumoniae the presence of B-lactamase inhibitors (BLIs). The activity of ec306 Escherichia coii First ESBL SHV-2 A. Y 50 described BLIs in combination with ceftazidime or biapenem is ec307 Escherichia coi Common SHV SHV-4 A. Y assessed by the checkerboard assay (Antimicrobial Combi ESBL nations. In Antibiotics in Laboratory Medicine, Ed. Victor ec3O8 Escherichia coi Common SHV SHV-5 A. Y ESBL Lorian, M.D., Fourth edition, 1996, pp. 333-338) using broth EC1009 Escherichia Coi ESBL TEM-10 A Z. microdilution method performed as recommended by the 55 ec3O2 Escherichia coi Common ESBL TEM-10 A Z. NCCLS (National Committee for Clinical Laboratory Stan in US EC1012 Escherichia Coi ESBL TEM-12 A Y dards (NCCLS). 1997. Methods for Dilution of Antimicrobial ec303 Escherichia coi Common ESBL TEM-12 A Y Susceptibility Tests for Bacteria That Grow Aerobically— in US Fourth Edition; Approved Standard. NCCLS. Document EC1011 Escherichia Coi ESBL TEM-26 A Z. ec304 Escherichia coi Common ESBL TEM-26 A Z. M7-A4, Vol 17 No. 2). In this assay, multiple dilutions of two 60 in US drugs, namely BLI and B-lactam (ceftazidime or biapenem), ec3OO Escherichia coi Common ESBL TEM-3 A. Y are being tested, alone and in combination, at concentrations in France equal to, above and below their respective minimal inhibitory ec301 Escherichia Coi ESBL TEM-6 A. Z. CF1OOO Citrobacter Hyper AmpC C Y concentrations (MICs). BLIs are solubilized in 10% DMSO freundii expression at 10 mg/mL. Stock solutions are further diluted, according to 65 ECL1003 Enterobacier Hyper AmpC C Z. the needs of a particular assay, in Mueller Hinton Broth cloacae expression (MHB). Stock solution can be stored at -80° C. US 8,680,136 B2 109 TABLE 2-continued TABLE 2-continued Strain Organism Description PCR Class MPC8 Strain Organism Description PCR Class MPC8 KX1000 Kiebsielia ESBL OXA-2 D X ec310 Escherichia coi E. cloacae-like ACT-1 C X 5 Oxytoca Amp-C X = MPC8 of 2.5 lug/mL or less. EC1004 Escherichia coli pampC CMY-2 C X Y= MPC8 of greater than 2.5 g/mL to 10 ugmL. Z = MPC8 of greater than 10 ugmL. EC1010 Escherichia coli pampC CMY-6 C Y EC1014 Escherichia coli pampC DHA-1 C Z. 10 Next, ceftazidime potentiation activity of several cyclic EC1 OO6 Escherichia coli pampC FOX-5 C Y boronic acid ester derivatives was tested using a larger panel EC1016 Escherichia coli pampC FOX-5 C Z. of strains expressing B-lactamase hydrolyzing enzymes. Ceftazidime MICs were determined alone and in the presence ec309 Escherichia coi E. cloacae-like MIR-1 C X of fixed concentrations of various cyclic boronic acid ester Amp-C derivatives. Most compounds were tested at 10 ug/mL. Cyclic KP1007 Kiebsiella ESBL OXA-10, D Y boronic acid ester derivatives were capable of reducing pneumoniae qnrB4 ceftazidime MICs 4 to >64-fold depending on B-lactamase (Table 3). TABLE 3 Ceftazidime MIC (ig/mL) with or without cyclic boronic acid ester derivative

3 at 10 4 at 10 Sat 10 6 at 1.O 7 at 10 Strain Organism Description PCR Class Alone Ig/mL g/mL Lig/mL g/mL g/mL, KP1005 Klebsielia ESBL CTX-M-14 A. Z. Z. Z. Z. Z. Z. pneumoniae KP1009 Kiebsielia ESBL CTX-M-15 A. Z. Z. Z. Z. Z. Z. pneumoniae KP1006 Kiebsielia ESBL CTX-M2 A. Y X X X X ND pneumoniae EC1008 Escherichia ESBL CTX-M3 A. Z. Y Y Y Y Z. coi ba. 1063 Pseudomonas ESBL GES-1 A. Z Z Z Z Z Z aeruginosa KP1004 Kiebsielia Serine KPC-2 A. Z. Y Y Y Y Z. pneumoniae carbapenemase KP1008 Kiebsielia Serine KPC-2 A. Y X X X X Z. pneumoniae carbapenemase EC1007 Escherichia Serine KPC-3 A. Z. X X X X Z. coi carbapenemase KP1010 Kiebsielia ESBL SHV-12 A. Z. Z. Z. Y Y Z. pneumoniae KP1012 Klebsielia ESBL SHV-18 A. Z. Z. Z. Y Y Z. pneumoniae ec306 Escherichia First ESBL SHV-2 A. Z. Z. Z. Z. Z. Z. coi described ec307 Escherichia Common SHW SHV-4 A. Z. Z. Z. Y Z. Z. coi ESBL ec3O8 Escherichia Common SHW SHV-5 A. Z. Z. Z. Z. Z. Z. coi ESBL KP1011 Kiebsielia ESBL SHV-5 A. Y X X X X ND pneumoniae EC1009 Escherichia ESBL TEM-10 A. Z. Z. Z. Z. Z. Z. coi ec3O2 Escherichia Common ESBL TEM-10 A. Z. Z. Z. Z. Z. Z. coi in US EC1012 Escherichia ESBL TEM-12 A. Z. Z. Z. Y Y Z. coi ec3O3 Escherichia Common ESBL TEM-12 A. Z. Z. Z. Y Y Z. coi in US EC1011 Escherichia ESBL TEM-26 A. Z. Z. Z. Z. Z. Z. coi ec300 Escherichia Common ESBL TEM-3 A. Z. Z. Z. Y Z. Z. coi in France ec3O1 Escherichia ESBL TEM-6 A. Z. Z. Z. Z. Z. Z. coi KP1014 Klebsielia Metallo B Vim-1 B Z. Z. Z. Z. Z. ND pneumoniae 8088Se. CF1OOO Citrobacter Hyper AmpC C Z. Z. Z. Y Y Z. freundi expression CF1001 Citrobacter Hyper AmpC C Z. Z. Z. Y Y ND freundi expression ECL1002 Enterobacier Hyper AmpC C Z. Z. Y Y Y Z. cloacae expression US 8,680,136 B2

TABLE 3-continued Ceftazidime MIC (ig/mL) with or without cyclic boronic acid ester derivative

3 at 10 4 at 10 S at 10 6 at 10 7 at 10 Strain Organism Description PCR Class Alone Lig/mL Lig/mL g/mL Lig/mL g/mL ECL1003 Enterobacier Hyper AmpC C Z Z. Z. Z. Z. ND cloacae expression ec310 Escherichia E. cloacae-like ACT1 C Z Y Y X X Z. coi Amp-C EC1004 Escherichia pAmpC CMY-2 C Z Y Y Y Y Z. coi SA1000 Saimoneiia pAmpC CMY-2 C Z Z. Y Y X ND KP1013 Klebsielia pAmpC CMY-2 C Z Z. Y Y Y ND pneumoniae EC1010 Escherichia pAmpC CMY-6 C Z Z. Y Y Y Z. coi EC1014 Escherichia pAmpC DHA-1 C Z Y Y X X Z. coi EC1006 Escherichia pAmpC FOX-5 C Z Y Z. Y Y Z. coi EC1016 Escherichia pAmpC FOX-5 C Z Z. Z. Z. Z. ND coi ec309 Escherichia E. cloacae-like MIR-1 C Z Y Y X X Z. coi Amp-C PAM2005 Pseudomonas ampC C Z Z. Z. Z. Z. Z. aeruginosa PAM2035 Pseudomonas ampC mex A: tet C Z Z. Z. Y Y Z. aeruginosa KP1007 Kiebsielia ESBL OXA-10 D Z Z. Z. Y Y Z. pneumoniae KX1000 Kiebsielia ESBL OXA-2 D Z Z. Y Y Y Y Oxytoca AB1054 Acinetobacter OXA-carbapenemase OXA-23 D Z Z. Z. Z. Z. Z. battmannii AB1052 Acinetobacter OXA-carbapenemase OXA-24 D Z Z. Z. Z. Z. ND battmannii AB1057 Acinetobacter OXA-carbapenemase OXA-58 D Z Z. Z. Z. Z. Z. battmannii X = MIC of 1 ugmL or less. Y= MIC of greater than 1 lug/mL to 8 LugmL. Z = MIC of greater than 8 LugmL. NTD = NotDetermined.

Biapenem is a carbapenem B-lactam; only selected B-lac 40 expressing class A carbapenemases: MPC8 (minimal poten tamases confer resistance to this class of antibiotics. Among tiation concentration of cyclic boronic acid ester derivative them are serine carbapemenases that belong to class A and class D. Biapenem potentiation is studied in Strains express (ug/mL) to reduce the MIC of Biapenem 8-fold) varied from ing various carbapenemases from these classes using CB 0.02 ug/mL to 0.16 ug/mL (Table 4). Cyclic boronic acid ester assays. Various cyclic boronic acid ester derivatives showed derivatives were capable of reducing biapenem MICs up to significant potentiation of biapenem against the strains 1000-fold (Table 4). TABLE 4 Strain Organism Description PCR Class Compound MPC8 ECL1004 Enterobacter cloacae Serine carbapenemase NMC-A A. 1 Y EC1007 Escherichia coi Serine carbapenemase KPC-3 A. 1 X KP1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A. 1 Y SM1000 Serraia marcescens Serine carbapenemase SME-2 A. 1 Y ECL1004 Enterobacter cloacae Serine carbapenemase NMC-A A. 2 Y EC1007 Escherichia coi Serine carbapenemase KPC-3 A. 2 X KP1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A. 2 X SM1000 Serraia marcescens Serine carbapenemase SME-2 A. 2 Y ECL1004 Enterobacter cloacae Serine carbapenemase NMC-A A. 3 X EC1007 Escherichia coi Serine carbapenemase KPC-3 A. 3 X KP1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A. 3 X KP1008 Klebsiella pneumoniae Serine carbapenemase KPC-2 A. 3 X SM1000 Serraia marcescens Serine carbapenemase SME-2 A. 3 Y AB1052 Acinetobacter baumannii OXA-carbapenemase OXA-24 D 3 Z. AB1054 Acinetobacter baumannii OXA-carbapenemase OXA-23 D 3 Z. AB1057 Acinetobacter baumannii OXA-carbapenemase OXA-58 D 3 Z. ECL1004 Enterobacter cloacae Serine carbapenemase NMC-A A. 4 X EC1007 Escherichia coi Serine carbapenemase KPC-3 A. 4 X KP1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A. 4 X KP1008 Klebsiella pneumoniae Serine carbapenemase KPC-2 A. 4 X US 8,680,136 B2 113 114 TABLE 4-continued

Strain Organism Description PCR Class Compound MPC8 S M OOO Serraia marcescens Serine carbapenemase SME-2 A. 4 X B1052 Acinetobacter battmannii OXA-carbapenemase OXA-24 D 4 Z. B1054 Acinetobacter battmannii OXA-carbapenemase OXA-23 D 4 Z. B1057 Acinetobacter battmannii OXA-carbapenemase OXA-58 D 4 Z. CL1004 Enterobacter cloacae Serine carbapenemase NMC-A A. 5 Y C1007 Escherichia coi Serine carbapenemase KPC-3 A. 5 X P1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A. 5 X OOO Serraia marcescens Serine carbapenemase SME-2 A. 5 Y B1052 Acinetobacter battmannii OXA-carbapenemase OXA-24 D 5 Z. B1054 Acinetobacter battmannii OXA-carbapenemase OXA-23 D 5 Z. B1057 Acinetobacter battmannii OXA-carbapenemase OXA-58 D 5 Z. CL1004 Enterobacter cloacae Serine carbapenemase NMC-A A. 6 Y C1007 Escherichia coi Serine carbapenemase KPC-3 A. 6 X P1004 Klebsiella pneumoniae Serine carbapenemase KPC-2 A. 6 X OOO Serraia marcescens Serine carbapenemase SME-2 A. 6 Y B1052 Acinetobacter battmannii OXA-carbapenemase OXA-24 D 6 Z. B1054 Acinetobacter battmannii OXA-carbapenemase OXA-23 D 6 X B1057 Acinetobacter battmannii OXA-carbapenemase OXA-58 D 6 Z. X = MPC8 of less than 0.16 ugmL. Y= MPC8 of 0.16 lug/mL to 1 lug/mL. Z = MPC8 of greater than 1 ugmL.

Example 12 ceftazidime and biapenem was measured at 250 nm and 296 nm, respectively. This experiment was used to determine the The ability of B-lactamase inhibitors to inhibit hydrolysis 25 optimal lysate dilution which produced a linear curve of of ceftazidime and biapenem was studied. Lysates were pre- relative UV signal that decreased to approximately OD=0.3- pared from bacteria expressing various B-lactamases as a 0.5 over 1 hour. Source of enzymes. Bacterial lysates were prepared as fol- Finally, the potency of cyclic boronic acid ester derivative lows. A single colony from the fresh over-night plate was to inhibit the cleavage of ceftazidime and biapenem cleavage transferred to 5 mL of LB broth and grown to ODoo-0.6-0.8. by bacterial lysates was determined. 100 ul of buffer A (50 Next, this culture was transferred to 500 mL of LB and grown mMSodium Phosphate pH-7: 0.5% glucose, 1 mM MgCl) to ODoo-0.7–0.9. Cells were pelleted by centrifugation at was added to each well of 96-well UV-transparent plate. 50 ul 5000 RPM (JA-14 rotor) for 15 minutes at room temperature. of 6x cyclic boronic acid ester derivative solution in buffer A The pellet was resuspended in 10 mL of PBS. Five freeze- was titrated vertically in 96-well plate column to generate thaw cycles by putting cells at -20° C. and thawing them at 3-fold dilutions. 50 ul of diluted lysate in buffer A (optimal the room temperature were next applied. After the last thaw dilution is determined in experiment above) was added, and step cells were spun down at 18K for 30 minutes and the the plate was incubated in the plate reader at 37° C. for 15 supernatant was collected. This lysate was stored at -20°C. minutes. 50 ul of 50 ug/mL solutions of ceftazidime or biap Next, the activity of bacterial lysates was optimized for enem in buffer A (pre-incubated at 37° C. for 15 minutes) ceftazidime and biapenem cleavage as follows. 50 ul of buffer were next added to each well and hydrolysis of ceftazidime or A (50 mM Sodium Phosphate pH=7: 0.5% glucose, 1 mM biapenem was recorded at 250 nm and 296 nm, respectively. MgCl) was added to each well of 96-well UV-transparent ECs of inhibition was determined by plotting the rate of plate. 50 ul of lysate was titrated vertically in 96-well plate ceftazidime or biapenem cleavage vs. cyclic boronic acid column to generate 2-fold lysate dilutions. 100 ul of buffer A as ester derivative concentration. was added to each well, placed in plate reader at 37° C. and The results of these experiments are presented in Table 5 incubated for 15 minutes. 50 ul of 50 g/mL solutions of and Table 6. These experiments demonstrate that the ceftazidime or biapenem in buffer A (pre-incubated at 37°C. described compounds are inhibitors with a broad-spectrum for 15 minutes) were added to each well. Hydrolysis of activity towards various B-lactamases. TABLE 5 ICso (g/mL) of inhibition - of slavicine hydrolysis Strain Organism Description PCR Class 3 4 5 6 7

KP1005 Klebsielia ESBL CTX-M-14 A. X Z Z, X Y Z. pneumoniae KP1009 Kiebsielia ESBL CTX-M-15 A. X Z Z, X Y Y pneumoniae pa. 1063 Pseudomonas ESBL GES-1 A. Y Z. Y X Y Y aeruginosa KP1004 Kiebsielia Serine KPC-2 A. Z. X X X X Z. pneumoniae carbapenemase KP1008 Kiebsielia Serine KPC-2 A. Z. Y X X X Z. pneumoniae carbapenemase EC1007 Escherichia Serine KPC-3 A. Z. Y X X X Z. coi carbapenemase US 8,680,136 B2 115 116 TABLE 5-continued ICso (ugmL) of inhibition of Ceftazidime hydrolysis

Strain Organism Description PCR Class Tazobactam 3 4 5 6 7

KP1010 Kiebsielia ESBL SHV-12 A. X Z Z. Y Z Z. pneumoniae KP1012 Klebsielia ESBL SHV-18 A. X Z Z. Y Y Z. pneumoniae ec306 Escherichia First ESBL SHV-2 A. Y Z Z. Y Y Z. coi described ec3O8 Escherichia Common SHW SHV-5 A. X Z, Z, Z, Z, Z. coi ESBL ec3O2 Escherichia Common ESBL in TEM-10 A. X Y Z, X Y Y coi US ec303 Escherichia Common ESBL in TEM-12 A. X Z Z. Y. Z. Y coi US ec304 Escherichia Common ESBL in TEM-26 A. X Z, Z. Y Y Y coi US ec3OO Escherichia Common ESBL in TEM-3 A. X Z Z. Y Z Z. coi France ec301 Escherichia ESBL TEM-6 A. X Z, Z. Y Y Y coi ECL1003 Enterobacier Hyper AmpC C ND Z Z. Y Z Z. cloacae expression EC1014 Escherichia pAmpC DHA-1 C ND Z Z. Y Z Z. coi KP1007 Kiebsielia ESBL OXA-10 D Y Z Z. Y Z Z. pneumoniae KX1000 Kiebsielia ESBL OXA-2 D X Z Z, X Y Z. Oxytoca

X = IC50 of less than 0.1 ugmL, Y = IC50 of 0.1 ugmL to 1 LugimTL, Z = IC50 of greater than 1 LugmL, NTD = NotDetermined.

TABLE 6 ICso (ugmL) of inhibition of biapenen hydrolysis Strain Organism Description PCR Class Tazobactam 3 4 5 6 7

EC1007 Escherichia Serine KPC-3 A. Z. Y Y X X Z. coi carbapenemase KP1004 Kiebsielia Serine KPC-2 A. Z. Z. Y X Y ND pneumoniae carbapenemase KP1008 Kiebsielia Serine KPC-2 A. Z. Z Z. Y Y ND pneumoniae carbapenemase SM1OOO Serraia Serine SME-2 A. Y Z. Y X Y Z. marcescens carbapenemase X = IC50 of less than 0.1 ugmL, Y = IC50 of 0.1 ugmL to 1 LugimTL, Z = IC50 of greater than 1 ugmL. NTD = NotDetermined.

The potency and spectrum off-lactamase inhibitors is also 55 est concentration of 40 ug/mL. In this assay potency of com determined by assessing their aztreonam potentiation activity pounds is determined as a concentration of BLIs to inhibit in a dose titration potentiation assay using strains of various growth of bacteria in the presence of 4 ug/mL of aztreonam (MPC). Tables 7,8 and 9 summarize BLI potency of aztre bacteria that are resistant to aztreonam due to expression of onam potentiation (MPC) for various strains overexpress various ?-lactamases. Aztreonam is a monobactamantibiotic 60 ing class A (ESBLs), class A (KPCs), and class C and class D and, similar to ceftazidime, is hydrolyzed by the majority of beta-lactamases, respectively. Aztreonam MIC for each strain beta-lactamases that belong to class A, C or D (but not class is also shown. Table 7 summarizes activity of BLIs to poten B). The potentiation effect is observed as the ability of BLI tiate aztreonam against Strains expressing class A ESBLs. compounds to inhibit growth in the presence of sub-inhibi Table 8 summarizes activity of BLIs to potentiate aztreonam tory concentration of aztreonam. MIC of test strains vary 65 against Strains expressing class A KPCs. Table 9 summarizes from 32 g/mL to > 128 g/mL. Aztreonam is present in the activity of BLIS to potentiate aztreonam against Strains test medium at 4 Lig/mL. Compounds were tested at the high expressing class C and D enzymes.

US 8,680,136 B2 119 120 TABLE 8-continued TABLE 9-continued

Aztreonam MIC 62 >128 64 >128 63 AZT AZT AZT 64 MPC MPC MPC 65 KPC-2 KPC-2 KPC-3 66 KP1004 KP1008 EC1007 53 Y X X X = MPC24 of less than 5ug/mL. S4 Z. X Y 10 55 Y X X Y= MPC24 of 5ug/mL to 20 Lig/mL. 56 Y X X Z = MPC24 of greater than 20 Lig/mL. 57 X X X NTD = NotDetermined. 58 Z. Z. Z. 59 Z. Z. Z. The potency and spectrum off-lactamase inhibitors is also 60 Z. Y Y 15 61 X X X determined by assessing their biapenem potentiation activity 62 Y X Y in a dose titration potentiation assay using strains expressing 63 Z. Y Y serine carbapemenases (such as KPC). The potentiation 64 Z. X Y 65 Z. Z. Z. effect is observed as the ability of BLI compounds to inhibit 66 Y X X growth in the presence of sub-inhibitory concentration of biapenem. MIC of test strains vary from 4 ug/mL to >1 X = MPC24 of less than 5ug/mL. ug/mL. Biapenem is present in the test medium at 1 lug/mL. Y= MPC24 of 5ug/mL to 20 ugmL. Compounds tested at the highest concentration of 40 ug/mL. Z = MPC24 of greater than 20 ugmL. NTD = NotDetermined. In this assay potency of compounds is determined as a con 25 centration of BLIs to inhibit growth of bacteria in the pres ence of 1 ug/mL of biapenem (MPCa). Table 10 summarizes TABLE 9 BLI potency of biapenem potentiation (MPC). Biapenem Class MIC for each strain is also shown.

C C C D D 30 TABLE 10 Aztreonam 64 >128 32 128 128 Biapenen MIC MIC >8 8 4 8 AZT BPM BPM BPM BPM AZT MPC4 AZT 35 MPC MPC, MPC, MPC AZT MPC24 AZT OXA-10 MPC24 KP1004 KP1008 EC1007 ECL1004 MPC24 CMY-6 MPC24 qnrB4 OXA-2, KPC-2 KPC-2 KPC-3. NMC-A ECL1002 EC1010 PAM2O3S KP1007 KPX1001 TaZobactam Clavulanic >40 40 >40 O.O8 5 3 Acid 4 40 Tazobactam >40 2O 2O 5 >40 5 3 Z. Z. Z. Z. Z. 6 4 Y Y Z. Z. Z. 33 5 Y Y X X Y 34 6 Y Z. Y Y Z. 35 33 Z. Z. Z. Z. Z. 36 34 Z. Z. Z. Z. Z. 45 37 35 Z. Z. Z. Z. Z. 38 36 Z. Z. Z. Y Z. 39 37 Z. Z. Z. Z. X 40 38 Z. Z. Z. Z. Z. 41 39 Z. Z. Z. Z. Z. 42 40 Z. Z. Z. Z. Z. 50 43 41 Z. Z. Z. Z. Z. 45 42 Z. Z. Z. Z. Z. 46 43 Y Y Y Z. Y 47 45 Z. Z. Z. Z. Z. 48 46 Z. Z. Z. Z. Z. 49 47 Z. Z. Z. Z. Z. 55 50 48 Z. Z. Z. Z. Z. 51 49 Z. Z. Y Z. Y 52 50 Z. Z. Z. Z. Y 53 51 Z. Z. Z. Z. Y S4 52 Z. Z. Z. Z. Z. 55 53 Z. Z. Z. Z. Y 56 S4 Z. Z. Z. Z. Z. 60 57 55 Z. Z. Z. Z. Z. 58 56 Z. Z. Z. Z. Y 59 57 Z. Z. Z. Z. Z. 60 58 Z. Z. Z. Z. Z. 61 59 Z. Z. Z. Z. Z. 62 60 Z. Z. Z. Z. Z. 65 63 61 Y Y Y Y Y 64 US 8,680,136 B2 121 122 TABLE 10-continued Example 13 Biapenen MIC Selected B-lactamase inhibitors were also tested for their >8 8 4 8 ability to potentiate the monobactam tigemonam. The poten BPM BPM BPM BPM tiation effect is observed as the ability of BLI compounds to MPC MPC, MPC, MPC inhibit growth in the presence of sub-inhibitory concentration KP1004 KP1008 EC1007 ECL1004 oftigemonam. MIC of test strains vary from 8 ug/mL to >128 KPC-2 KPC-2 KPC-3 NMC-A ug/mL. Tigemonam is present in the test medium at 4 Lig/mL. 65 Y X Y Z. Compounds tested at the highest concentration of 40 ug/mL. 66 X X X X 1 O In this assay potency of compounds is determined as a con X FCol of less than 1 Lig/mL. centration of BLIs to inhibit growth of bacteria in the pres Y FCol of 1 ugmL to 5ug/mL. Z. FCol of greater than 5ug/mL. ence of 4 Lig/mL of aztreonam (MPCa). Tables 12 and 13 NTD = NotDetermined. Summarize BLI potency of tigemonam potentiation Some bacterial lysates were also optimized for the cleav 1 5 (MPC) for various strains overexpressing class A (ESBLs), age of aztreonam and nitrocefin. ECso of inhibition was deter class C and class D beta-lactamases, respectively. Tige mined by plotting the rate of aztreonam or nitrocefin cleavage monam MIC for each strain is also shown. Table 12 Summa vs. BLI concentration. The results of these experiments are rizes activity of BLIS to potentiate tigemonam against Strains presented in Table 11. These experiments confirmed that the expressing class A ESBLs. Table 13 Summarizes activity of described compounds are inhibitors with a broad-spectrum 2 O BLIS to potentiate aztreonam against strains expressing class activity towards various B-lactamases. C and D enzymes. TABLE 11 AZT ICso AZT ICso AZT ICso AZTICso AZTICso AZTICso AZT ICso AZTICso AZT ICso NCF ICso KP1OOS KP1009 ec3O2 ec304 KP1004 KP1008 EC1007 KP1007 KPX1001 EC1010 CTX-M-14 CTX-M-15 TEM-10 TEM-26 KPC-2 KPC-2 KPC-3 OXA-10 OXA-2 pAmpC (CMY-6)

Clavulanic O.OS48 O.247 O.O27 O.O27 O.74 2.22 148 148 O.08 ND Acid TaZobactam <0.0274 O.O27 0.055 O.O27 O 4 2 O 4.44 O.O274 ND 3 4 5 6 33 34 35 36 37 38 39 40 41 42 43 45 46 47 48 49 50 51 52 53 S4 55 56 57 58 59 60 61 62 63 65 66

X = IC50 of less than 0.5 ugmL, Y = IC50 of 0.5 ugmL to 2 ugmL, Z = IC50 of greater than 2 LugmL, NTD = NotDetermined. US 8,680,136 B2

TABLE 12

Tigemonam MIC mL

512 2S6 >512 2S6 64 256 >512 512 MPC MPC MPC MPC, MPC, MPC MPC MPC CTX-M-14 CTX-M-15 SHVS, SHV-12 SHV-18 TEM-10 TEM-10 TEM-26 KP1OOS KP1009 ec3O8 KP1010 KP1012 EC1009 ec3O2 ec3O4

Tazobactam 10 10 5 1.25 1.25 2.5 5 1.25 Clavulanic 2.5 1.25 <=0.6 <=0.6 <=0.6 <=0.6 2.5 <=0.6 Acid 5 Z. Z. Z. Z. Z. Z. Z. Z. 9 Z. Z. Z. Z. Z. Z. Z. Z. 18 X X Y X X X Y Y 37 Z. Z. Z. Z. Z. Z. Z. Z. 48 Z. Z. Z. Z. Z. Z. Z. Z. 63 Z. Y Z. Y Y Z. Z. Z. 64 Z. Y Y X Y Y Z. Z. 67 Z. Y Z. Y Y Z. Z. Z. 68 Y Y Y X X Y Y Y X = MPC24 of less than 2 Lig/mL. Y= MPC24 of 2 Lig/mL to 10 ugmL. Z = MPC24 of greater than 10 ugmL. NTD = NotDetermined.

TABLE 13 TABLE 13-continued Class 25 64 X X Y X Y 67 Y X X Z. X C C C D S 68 Y X Y X X Tigemonam 32 16 8 256 8 X = MPC24 of less than 2 Lig/mL. MIC Y= MPC24 of 2 Lig/mL to 10 ug/mL. (Lig/mL) 30 Z = MPC of greater than 10 ug/mL. NTD = NotDetermined. MPC MPC MPC MPC CMY-6, MPC OXA-10, OXA-2, ECL1002 EC1010 PAM2O3S KP1007 KPX1001 Example 14

TaZobactam 10 2.5 5 5 40 - Clavulanic >40 40 >40 <=206 1.25 Checkerboard assays were used to evaluate the ability of Acid Compound 5 to potentiate various carbapenems (biapenem, 5 Y X X Z. X doripenem, ertapenem, imipenem, and meropenem) against 9 18 y X X % & the strains expressing KPC alone or in combination with 37 X X X Z. X additional beta-lactamases. The highest concentration of 48 Y X Y Z. X 40 Compound 5 was 10 mg/L. The results are present in the Table 63 Y X Y Y X 14. Compound 5 was capable to significantly potentiate mul tiple carbapenems. TABLE 1.4 Concentration of Compound 5 (mg/L) to Potentiate Carbapenen (mg/L Organism Strain Enzymes Antibiotic O O.16 0.31 O.62S 1.25 2.5 S 10 Kiebsielia KP1004 KPC-2 Biapenem Z. X X X X X X X pneumoniae Kiebsielia KP1004 KPC-2 Doripenem Y Y X X X X X X pneumoniae Kiebsielia KP1004 KPC-2 Ertapenem Z. Z. Y Y X X X X pneumoniae Kiebsielia KP1004 KPC-2 mipenem Z. X X X X X X X pneumoniae Kiebsielia KP1004 KPC-2 Meropenem Z. Y Y X X X X X pneumoniae Kiebsielia KP1008 KPC-2 Biapenem Z. X X X X X NG NG pneumoniae Kiebsielia KP1008 KPC-2 Doripenem Y X X X X X NG NG pneumoniae Kiebsielia KP1008 KPC-2 Ertapenem Z. X X X X X NG NG pneumoniae Kiebsielia KP1008 KPC-2 mipenem Y X X X X X NG NG pneumoniae Kiebsielia KP1008 KPC-2 Meropenem Y X X X X X NG NG pneumoniae Kiebsielia KP1082 KPC-2, Biapenem Y X X X X X X X pneumoniae SHV-1

US 8,680,136 B2 127 128 TABLE 14-continued Concentration of Compound 5 (mg/L) to Potentiate Carbapenen (mg/L Organism Strain Enzymes Antibiotic O 0.16 O.31 O.62S 1.25 2.5 S 10 Escherichia EC1007 KPC-3 Meropenem Y X X X X X X X coi Enterobacter ECL1058 KPC-3, Biapenem Z. Y Y Y X X X X cloacae SHV-11, TEM Enterobacter ECL1058 KPC-3, Doripenem Z. Y Y Y Y X X X cloacae SHV-11, TEM Enterobacter ECL1058 KPC-3, Ertapenem Z. Z. Z. Z. Y Y X X cloacae SHV-11, TEM Enterobacter ECL1058 KPC-3, Imipenem Z. Y Y Y X X X X cloacae SHV-11, TEM Enterobacter ECL1058 KPC-3, Meropenem Z. Y Y Y Y X X X cloacae SHV-11, TEM Enterobacter ECL1059 KPC-3, Biapenem Y X X X X X X X cloacae SHV-12, TEM Enterobacter ECL1059 KPC-3, Doripenem Y X X X X X X X cloacae SHV-12, TEM Enterobacter ECL1059 KPC-3, Ertapenem Y X X X X X X X cloacae SHV-12, TEM Enterobacter ECL1059 KPC-3, Imipenem Y X X X X X X X cloacae SHV-12, TEM Enterobacter ECL1059 KPC-3, Meropenem Y X X X X X X X cloacae SHV-12, TEM Kiebsiella KP1083 KPC-3, Biapenem Z. Y X X X X X X pneumoniae SHV-1, TEM Kiebsiella KP1083 KPC-3, Doripenem Z. Y X X X X X X pneumoniae SHV-1, TEM Kiebsiella KP1083 KPC-3, Ertapenem Z. Y X X X X X X pneumoniae SHV-1, TEM Kiebsiella KP1083 KPC-3, Imipenem Z. Y X X X X X X pneumoniae SHV-1, TEM Kiebsiella KP1083 KPC-3, Meropenem Z. Y X X X X X X pneumoniae SHV-1, TEM Kiebsiella KP1084 KPC-3, Biapenem Z. Z. Z. Z. Z. Y X X pneumoniae SHV-11, TEM Kiebsiella KP1084 KPC-3, Doripenem Z. Z. Z. Z. Y Y Y X pneumoniae SHV-11, TEM Kiebsiella KP1084 KPC-3, Ertapenem Z. Z. Z. Z. Z. Z. Y Y pneumoniae SHV-11, TEM Kiebsiella KP1084 KPC-3, Imipenem Z. Z. Z. Y Y Y X X pneumoniae SHV-11, TEM Kiebsiella KP1084 KPC-3, Meropenem Z. Z. Z. Z. Z. Y Y X pneumoniae SHV-11, TEM Kiebsiella KP1088 KPC-3, Biapenem Z. Y X X X X X X pneumoniae SHV-11, TEM Kiebsiella KP1088 KPC-3, Doripenem Y Y Y X X X X X pneumoniae SHV-11, TEM Kiebsiella KP1088 KPC-3, Ertapenem Z. Z. Y X X X X X pneumoniae SHV-11, TEM Kiebsiella KP1088 KPC-3, Imipenem Z. Y X X X X X X pneumoniae SHV-11, TEM US 8,680,136 B2 129 TABLE 14-continued Concentration of Compound 5 (mg/L) to Potentiate Carbapenen (mg/L

Organism Strain Enzymes Antibiotic O 0.16 O.31 O.625 1.25 2.5 S 10 Kiebsiella KP1088 KPC-3, Meropenem Z. Y Y X X X X X pneumoniae SHV-11, TEM-1 X = MIC of less than 0.5 mg/L. Y= MIC of 0.5 mg/L to 4 mg/L. Z = MIC of greater than 4 mg/L. NG = No Growth.

Example 15 TABLE 1.5 15 An in vivo model can be used to evaluate the single dose Route of Dosef T1/2 Cmax CLF AUC pharmacokinetic properties and absolute oral bioavailability Adm (mg/kg) (hir) (mg/L) (L/h/kg) (mg * h L) IV 2O 1.56 1982 1.65 12.15 of a test compound. As described more specifically below, a IV 50 4.53 45.93 1.77 28.19 test compound is administered to Sprague-Dawley (SD) rats 2O PO 50 1.55 O.29 6038 O.81 either intravenously or orally in a crossover study design and the resulting pharmacokinetic properties and oral bioavail In this experiment, three male Sprague dawley rats were ability are measured. given Compound 5 by intravenous or Compound 62 orally For intravenous administration, male rats were given a 30 (pro-drug for Compound 5). Plasma samples were collected minutes intravenous infusion dose of 20 or 50 mg/kg of 25 at designated time points and analyzed for the presence of Compound 5 via femoral vein cannula. Plasma samples (0.3 Compound 5. This study was designed to determine the oral ml) were collected from jugular vein cannula at 0.17, 0.33, bioavailability of Compound 62a pro-drug of Compound 5. 0.47, 0.58, 0.68, 0.75, 1,2,3,4, and 6hrs after the dosing. For Male rats (non-fasted) were orally administered 50 mg/kg of the prodrug Compound 62. As shown in FIG. 2, the pro-drug oral administration, male rats were given 50 mg/kg of Com 30 of Compound 5 has oral bioavailability of greater than 80%. pound 5 (in saline) or Compound 62 (in 100% ethanol) orally Polymorphs can be detected, identified, classified and using an oral gavage tip. Plasma samples were collected from characterized using well-known techniques such as, but not each rat at 0.08, 0.17, 0.25, 0.33, 0.50, 0.75, 1, 2, 3, 4, and 6 limited to, differential scanning calorimetry (DSC), thermo hrs after the dosing. 35 gravimetry (TGA) and powder X-ray diffractometry Plasma concentrations of the compounds were tested using (PXRD). LC/MS/MS method with a lower limit of quantification of 10 ng/mL for Compound 5 and 100 ng/mL for Compound 62. Example 16 Extraction: 50LL Volumes of plasma from samples and stan 40 The crystal structure of Compound 5 was analyzed using dards were extracted using 200 uL of methanol with 100 mM X-ray powder diffraction (“PXRD). The X-ray diffraction ammonium acetate, 2 ug/mL gatifloxacin (internal standard data were collected at room temperature using a PANalytical for Compound 62) and 2 ug/mL Compound 38 (internal stan XPert Pro diffractometer (Cu KO. radiation) fitted with an dard for Compound 5). The samples were mixed and centri automatic sample changer, a theta-theta goniometer, auto fuged for 30 min at 3000xg. 150 uL of supernatant was 45 matic beam divergence slits, a secondary monochromator and removed and added to 450 uL of water. a scintillation counter. Samples were prepared for analysis by HPLC mass spectrometry: An Agilent 1100HPLC pump, packing the powder into a 12 mm diameter, 0.25 mm deep HTC PAL autosampler and a Sciex 3200O mass spectrometer cavity that had been cut into a Si Zero-background wafer specimen mount. The sample was rotated while being irradi were used for separation and quantification. Compound 62 ated with copper K-alpha 1x-rays (wavelength=1.5406 Ang and its internal standard were detected using +ESI. Com stroms) with the X-ray tube operated at 45 kV/40 mA. The pound 5 and its internal standard were detected using ESI. analyses were performed with the goniometer running in LC/MS/MS: 1) Column: Chromolith FastGradient RP-18e. continuous mode set for a 5 second count per 0.02 step over 50x2 mm; 2) Mobile phase A: Aqueous Water with 0.1% a two theta range of 2° to 55°. The illustrative PXRD pattern TFA, Orgainic phase B: Acetonitrile with 0.1% TFA; Flow 55 for Compound 5 is shown in FIG. 3. Rate: 600 u/min: Injection volume: 10 uL: HPLC gradient: As will be appreciated by the skilled crystallographer, the relative intensities of the various peaks reported in FIG.3 may 5% B-e60% B, 0.01-e 1.5 min: 60% B, 1.5-e 1.6 min: 60% vary due to a number of factors such as orientation effects of B-5% B, 1..6-> 1.7 min; 5% B, 1.7->2.7 min. crystals in the X-ray beam or the purity of the material being Plasma concentrations were modeled using WinNonlinP) 60 analyzed or the degree of crystallinity of the sample. The peak (Pharsight Corp, Mountain View, Calif.). positions may also shift for variations in Sample height but the In this experiment, three male Sprague Dawley rats were peak positions will remain substantially as defined in FIG. 3. given Compound 5 by intravenous or oral route. At desig The skilled crystallographer also will appreciate that mea nated time points, bloods were collected and analyzed. As surements using a different wavelength will result in different shown in the above Table 15 and FIG. 1, Compound 5 has a 65 shifts according to the Bragg equation—nw2d sin 0. Such linear PK in rats. However, Compound 5 it is not orally further PXRD patterns generated by use of alternative wave absorbed. lengths are considered to be alternative representations of the US 8,680,136 B2 131 132 PXRD patterns of the crystalline materials of the present by monitoring the weight change as the sample is heated. If invention and as such are within the scope of the present infrared spectroscopy is conducted on the Volatile compo invention. nents outgassed during TGA analysis of a pseudopolymorph Table 16 lists peak positions and relative intensities for the (TGA-IR), then the molecular composition of the PXRD pattern of FIG. 3. Accordingly, some embodiments pseudopolymorph can be determined. These techniques are include a crystalline form of Compound 5 having three or thus useful for characterizing Solid state forms existing as more, four or more, five or more, six or more, seven or more, Solvates and/or hydrates. eight or more, nine or more, or ten or more characteristic Compound 5 was analyzed using differential scanning PXRD (wavelength=1.5406 A) peaks selected from 9.0°, calorimetry (DSC). ATA Instruments Q100 differential scan 15.7°, 17.30, 17.6°, 18.10, 21.30, 22.4°, 23.5°, 24.99, 27.20, 10 ning calorimeter equipped with an autosampler and a refrig 27.4, 28.1°, 29.1°, 31.2°, and 35.7° 20. Some embodiments erated cooling system under 40 mL/min N purge was used to include a crystalline form of Compound 5 having three or perform the analysis. Each sample was heated from 25 to more, four or more, five or more, or six characteristic PXRD 300° C. at 15° C. per minute in an aluminium pan with the lid (wavelength=1.5406 A) peaks selected from 9.0°, 17.3°, laid on top, with a nitrogen purge gas. The data from DSC 17.6°, 18.1°, 22.4, and 27.2°20. Some embodiments include 15 analyses are dependent on several factors, including the rate a crystalline form of Compound 5 having characteristic of heating, the purity of the sample, crystal size, and sample PXRD (wavelength=1.5406 A) peaks at 9.1°, 17.3°, 17.6°, size. The DSC thermogram obtained for the sample of Com and 18.1° 20. pound 5 is shown in FIG. 4 overlayed with the TGA thermo gram. These data reveal a single endothermic transition at TABLE 16 1550 C. o 26 Areacts 20 d-spacing A) Thermogravimetric-infrared (TG-IR) Analysis was pre formed on a TA Instruments Q5000 thermogravimetric ana 9.0O88 870.8 9.8O831 12.O132 29.19 7.361.18 lyzer interfaced to a Nicolet 6700 FT-IR spectrometer 13.2369 1912 6.68332 25 (Thermo Electron) equipped with an external TGA-IR mod 15.4527 55.73 S.72961 ule with a gas flow cell and DTGS detector. The FT-IR wave 16.6911 41.97 5.30719 length verification was performed using polystyrene, and the 17.3464 285.76 S.10815 TG calibration standards were nickel and AlumelTM. The 17.59 171.25 S.O3794 18.1212 475.59 4.891.45 sample was placed in a platinum or aluminium sample pan, 19958S 23.95 4.4451 30 and the pan was inserted into the TG furnace. The TG instru 201214 18.12 4.40949 ment was started first, immediately followed by the FT-IR 21.3328 84.5 4.16175 22403S 147.38 3.96S21 instrument. The TG instrument was operated under a flow of 22.9212 39.45 3.87681 helium at 90 and 10 cc/min for the purge and balance, respec 23.48 60.99 3.78579 tively. The furnace was heated under nitrogen at a rate of 15° 24.8881 77.52 3.5747 35 C./minute to a final temperature of 23.0°C. IR spectra were 26.1352 20.92 3.40689 26.3458 20.23 3.38O13 collected approximately every 32 seconds for approximately 27.2278 162.9 3.27261 13 minutes. Each IR spectrum used 32 co-added scans col 27.357 SO.29 3.25744 lected at a spectral resolution of 4 cm. The TGA thermo 28.0871 54.62 3.17441 gram obtained for the sample of Compound 5 is shown in 29.0644 51.29 3.06985 40 29.63 30.23 3.01253 FIG. 4 overlayed with the DSC thermogram. These TGA data 3O.1989 19.34 2.957O6 with IR analysis of the evolved gas indicate that the input 31.2457 65.66 2.86033 material is non-Solvated but loses one mole-equivalent of 32.1641 32.04 2.78073 water between 135 and 181° C. 33.7983 19.84 264992 35.1614 21.23 2.55O25 All references cited herein, including but not limited to 35.6871 57.8 2.51388 45 published and unpublished applications, patents, and litera 36.5979 22 2.45338 ture references, are incorporated herein by reference in their 37.7599 33.73 2.3805 entirety and are hereby made a part of this specification. To 39.8439 31.99 226066 the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained As is well understood in the art, because of the experimen 50 in the specification, the specification is intended to Supersede tal variability when X-ray diffraction patterns are measured and/or take precedence over any such contradictory material. on different instruments, the peak positions are assumed to be The term “comprising as used herein is synonymous with equal if the two theta (20) values agree to within 0.2 (i.e., “including.” “containing, or “characterized by, and is inclu +0.2). For example, the United States Pharmacopeia states sive or open-ended and does not exclude additional, unrecited that if the angular setting of the 10 strongest diffraction peaks 55 elements or method steps. agree to within +0.2° with that of a reference material, and the All numbers expressing quantities of ingredients, reaction relative intensities of the peaks do not vary by more than 20%, conditions, and so forth used in the specification are to be the identity is confirmed. Accordingly, peak positions within understood as being modified in all instances by the term 0.2 of the positions recited herein are assumed to be identi "about. Accordingly, unless indicated to the contrary, the cal. 60 numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be Example 17 obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any DSC measure thermal transition temperatures at which a claims in any application claiming priority to the present crystalline form absorbs or releases heat when its crystal 65 application, each numerical parameter should be construed in structure changes or it melts. TGA is used to measure thermal light of the number of significant digits and ordinary rounding stability and the fraction of volatile components of a sample approaches. US 8,680,136 B2 133 134 The above description discloses several methods and mate Randan Rare taken together with the atoms to which rials that are Susceptible to modifications, as well as alter they are attached to form a substituted or unsubsti ations in the fabrication methods and equipment. Such modi tuted carbocyclyl or substituted or unsubstituted het fications will become apparent to those skilled in the art from erocyclyl, a consideration of this disclosure or practice of the methods (ii) R7 and an R are taken together with the atoms to disclosed herein. Consequently, it is not intended that this which they are attached to form a substituted or invention be limited to the specific embodiments disclosed unsubstituted carbocyclyl or substituted or unsubsti herein, but that it cover all modifications and alternatives tuted heterocyclyl, coming within the true scope and spirit of the invention. (iii) an Randa carbonatom in Y are taken together with 10 intervening atoms to form a substituted or unsubsti What is claimed is: tuted carbocyclyl or substituted or unsubstitued het 1. A compound having the structure of formula I: erocyclyl, (iv) each of the following conditions are met: 15 (a) Y is a 3-4 atom alkylene or 3-4 atom alkenylene R linker, Y R8 (b) R is absent, (c) R' and a carbon atom in Y are taken together with 1 'NOB iii. HO NR6 R7 intervening atoms to form a substituted or unsub stituted aryl or a substituted or unsubstituted het eroaryl, and or a pharmaceutically acceptable salt thereof, wherein: (d) each Rattached to a ring atom forming part of the Y is a 1-4 atom alkylene, optionally substituted by one or substituted or unsubstituted aryl or a substituted or more Substituents selected from the group consisting of unsubstituted heteroaryl formed by R and Y is C1, F, CN, CF, R, OR, C(=O)NR'R'', and 25 absent; —C(=O)CR, wherein said alkylene or alkenylene each R is independently selected from a group consisting linker is optionally fused to an optionally substituted of H, halo. —Coalkenyl, -Coalkynyl, - NR'R''. aryl, optionally Substituted heteroaryl, optionally Sub —OR. - CoalkylCO.R. —CoalkenylCO.R. stituted carbocyclyl, or optionally substituted heterocy —CoalkynylCO.R. -carbocyclyl-CO.R., or indepen clyl; 30 dently: R" is selected from a group consisting of NR'R''. an R and an R are taken together with the atoms to - Co alkylR'', CoalkenylR'', CoalkynylR', which they are attached to form a substituted or -carbocyclyl-R'', -CH(OH)CalkylR, —CH(OH) unsubstituted carbocyclyl or substituted or unsubsti CoalkenylR, —CH(OH)C-alkynylR, —CH(OH) tuted heterocyclyl, carbocyclyl-R, C(=O)R. —C(=O)C alkylR. 35 (ii) a geminal R7 and R together form —Co. —C(=O)CoalkenylR. —C(=O)CoalkynylR, alkenylenylCO.R. or —C(=O)C-9-carbocyclyl-R, C(=O)NR'R'', (iii) each Rattached to a ring atom forming part of a N(R)C(=O)R, N(R)C(=O)NR'R'', N(R) substituted or unsubstituted aryl is absent; C(=O)CR, N(R)C(=O)C(-NR)R, N(R)C each R is independently selected from a group consisting (=O)C(=CRR)R, N(R)C(=O)CalkylN(R) 40 of H. —C-alkyl, Calkenyl, —C-alkynyl, carbocy C(=O)R, N(R)C(—NR)R, C(-NR) clyl, -CoalkylR'', —CoalkenylR'', —Coalky NRR, N C(R)NR'R'', N(R)SOR, nylR'', -carbocyclyl-R', substituted or unsubstituted N(R)SONR'R'', N=CHR, substituted or aryl, substituted or unsubstituted heteroaryl, substituted unsubstituted aryl, substituted or unsubstituted het or unsubstituted carbocyclyl, and substituted or unsub eroaryl, substituted or unsubstituted carbocyclyl, and 45 stituted heterocyclyl: substituted or unsubstituted heterocyclyl: each R" is independently selected from a group consisting R is selected from a group consisting of H. —Coalkyl, of H, Calkyl, -OR. -CH(—NH), —C(=O) Coalkenyl, -Coalkynyl, carbocyclyl, -Co OR, substituted or unsubstituted aryl, substituted or alkylR'', —CoalkenylR'', —CoalkynylR'', car unsubstituted heteroaryl, substituted or unsubstituted bocyclyl-R'', —C(=O)CR, CalkylCOR, 50 carbocyclyl, and substituted or unsubstituted heterocy —CoalkenylCO.R. —CoalkynylCO.R. and -car clyl; bocyclyl-COR, or alternatively: each R'' is independently selected from a group consisting (i) R' and an R are taken together with the atoms to of substituted or unsubstituted aryl, substituted or which they are attached to form a substituted or unsubstituted heteroaryl, substituted or unsubstituted unsubstituted carbocyclyl or substituted or unsubsti 55 carbocyclyl, and substituted or unsubstituted heterocy tuted heterocyclyl, clyl; (ii) R' and a carbon atom in Y are taken together with X is selected from a group consisting of -COR', and intervening atoms to form a Substituted or unsubsti carboxylic acid isosteres; tuted carbocyclyl or substituted or unsubstitued het R" is selected from a group consisting of H. Coalkyl, erocyclyl, or 60 -(CH2)os R. —C(R').OC(O)Coalkyl, (iii) R is absent when the carbon to which it is attached —C(R'),OC(O)R'', C(R'),OC(O)CC-alkyland is a ring atom in an aryl or heteroaryl ring; C(R'),OC(O)OR'': each R" is independently selected from a group consisting each R" is independently selected from a group consisting of H, halo. —Coalkyl, —Coalkenyl, —Coalkynyl, of H and C-alkyl, and NR'R''. -OR. - CoalkylCO.R. C. 65 m is independently an integer from 1 to 2. alkenylCO.R.—CoalkynylCOR, and-carbocyclyl wherein each Coalkyl, Coalkenyl, and Coalkynyl is CO.R. or independently: independently optionally substituted. US 8,680,136 B2 135 136 2. The compound of claim 1, having the structure of for SO.NR'R'', substituted or unsubstituted aryl, substi mula II: tuted or unsubstituted heteroaryl, substituted or unsub stituted carbocyclyl, and substituted or unsubstituted heterocyclyl: II R is selected from a group consisting of H. —Coalkyl, Coalkenyl, -Coalkynyl, -CoalkylR'', —Coalk enylR'', —CoalkynylR'', —C(=O)CR, and Co alkylCO.R. —CoalkenylCO.R. and —C-9 alkynylCO.R. or alternatively Randan Rare taken 10 together with the atoms to which they are attached to form a substituted or unsubstituted carbocyclyl or sub stituted or unsubstituted heterocyclyl: each R is independently selected from a group consisting or a pharmaceutically acceptable salt thereof, wherein: of H, -NR'R'', OR, and –CalkylCO.R. 15 —C-9 alkenylCO.R. and —C-9alkynylCO.R. or the bond represented by a dashed and solid line represents: independently, Randan R or independently an R and R and Rare independently selected from a group consist an Rare taken together with the atoms to which they are ing of C1, F, CN, CF, R, OR, C(=O)NR'R'', attached to form a substituted or unsubstituted carbocy and –C(=O)CR: or alternatively, RandR are taken clyl or substituted or unsubstituted heterocyclyl: together with the atoms to which they are attached to each R is independently selected from a group consisting form a substituted or unsubstituted aryl, substituted or of H, -NR'R'', OR, and –CalkylCO.R. unsubstituted heteroaryl, substituted or unsubstituted —Cs alkenylCO.R. and —CoalkynylCO.R. or carbocyclyl or substituted or unsubstituted heterocyclyl; independently, an Randan Rare taken together with RandR are independently selected from a group consist the atoms to which they are attached to form a substi ing of C1, F, CN, CF, R, OR, C(=O)NR'R'', 25 tuted or unsubstituted carbocyclyl or substituted or and –C(=O)CR; and unsubstituted heterocyclyl: n is independently Zero or an integer from 1 to 2. each R is independently selected from a group consisting 3. The compound of claim 2 having the defined 3.6-cis of H. Coalkenyl, -Coalkynyl, -CoalkylR'', stereochemistry shown in formula IIa: —CoalkenylR'', —CoalkynylR', Substituted or 30 unsubstituted aryl, substituted or unsubstituted het eroaryl. Substituted or unsubstituted —(CH-)-car IIa bocyclyl, and substituted or unsubstituted heterocyclyl; each R" is independently selected from a group consisting of H. —Coalkyl, OR, —CH(=NH), substituted or 35 unsubstituted aryl, substituted or unsubstituted het eroaryl, substituted or unsubstituted carbocyclyl, and substituted or unsubstituted heterocyclyl; and X is selected from a group consisting of —COH and carboxylic acid isosteres. 40 6. The compound of claim 2, wherein n is 1. 7. The compound of claim 2, wherein R,R,R, and Rare or a pharmaceutically acceptable salt thereof. hydrogen. 4. The compound of claim 2 having the defined 3,6-trans 8. The compound of claim 1, having the structure of for stereochemistry shown in formula IIb: mula IIIa: 45

IIb

IIIa

50

55 or a pharmaceutically acceptable salt thereof. 5. The compound of claim 2, wherein: or a pharmaceutically acceptable salt thereof, wherein: the bond represented by a dashed and solid line represents a single bond; 60 each R and Rare independently selected from a group consisting of C1, F, CN, CF, -R, OR, —C(=O) NR'R'', and –C(=O)CR; or alternatively, an R and R" are taken together with the atoms to which they are attached to form a substituted or unsubstituted aryl, sub 65 stituted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl or substituted or unsubsti tuted heterocyclyl: US 8,680,136 B2 137 138 each R and Rare independently selected from a group 12. The compound of claim 11, having the 3,8-cis-stere consisting of C1, F, CN, CF, -R, OR, —C(=O) ochemistry shown in formula IVd: NR'R'', and C(=O)CR. 9. The compound of claim 8, having the 3.7-cis-stere ochemistry shown in formula IIIe: 5 IVd

IIIc 10

15 or a pharmaceutically acceptable salt thereof. 13. The compound of claim 11, having the 3.8-trans-stere or a pharmaceutically acceptable salt thereof. ochemistry shown in formula IVg: 10. The compound of claim 1, having the 3,7-trans-stere ochemistry shown in formula IIIe:

IVg

IIIe 25

30

or a pharmaceutically acceptable salt thereof. or a pharmaceutically acceptable salt thereof. 35 14. The compoundclaim 1, wherein Rand each RandR is hydrogen. 11. The compound of claim 1, having the structure of formula IVa: 15. The compound of claim 14, wherein m is 1. 16. The compound of claim 15, wherein R' is NHC (=O)CalkylR''. 40 IV a 17. The compound of claim 16, wherein R'' is substituted or unsubstituted aryl or substituted or unsubstituted het eroaryl. 18. The compound of claim 17, wherein R'' is thien-2-yl. 19. The compound of claim 1, wherein R is NHC(=O) 45 C(=NOR)R’, wherein R is selected from the group con sisting of Coalkyl, Substituted or unsubstituted aryl, Substi tuted or unsubstituted heteroaryl, substituted or unsubstituted carbocyclyl and substituted or unsubstituted heterocyclyl. 50 20. The compound of claim 1, wherein X is CO.H. 21. The compound of claim 1, wherein X is a carboxylic or a pharmaceutically acceptable salt thereof, wherein: acid isostere. 22. The compound of claim 21, wherein the carboxylic acid the bond represented by a dashed and solid line represents isostere is selected from the group consisting of —P(O) a single bond; 55 (OR), P(O)(R)(OR), -P(O)(OR), P(O)(R) each R and each R" are independently selected from a (OR''), CONCR)OH, -SOH, -SON(R)OH, and group consisting of C1, F, CN, CF, R. —OR, —C(=O)NR'R'', and –C(=O)CR: or alternatively, an R and an R are taken together with the atoms to which they are attached to form a substituted or unsub 60 a NN. stituted aryl, substituted or unsubstituted heteroaryl, // substituted or unsubstituted carbocyclyl or substituted HN-N or unsubstituted heterocyclyl: each R and each R are independently selected from a 65 wherein R' is selected from the group consisting of H. R'', group consisting of C1, F, CN, CF, R. —OR, - C(R'),OC(O)Calkyl, C(R'),OC(O)R'', C(R') C(=O)NR'R'', and C(=O)CR. OC(O)OC-alkyl and C(R').OC(O)CR''. US 8,680,136 B2 139 140 23. The compound of claim 1, wherein m is 1. -continued 24. The compound of claim 1, having a structure selected O from the group consisting of

2 S

o O 10

HN B-O OH, / HO O 15 O

2O O

O HN /-/ HN -o OH, HOC O HO O 25 O HN ( / \ B- O OH, HN S / 30 HO O B- O OH, / O HO O H O HN D ( 35 -o OH, HO O

H e 40 O J/ N HN

HN \ / -o OH, O 45 HO O HN B-O OH, / OMe HO O 50 W O

N HN f \ -o OH, O 55 HN S HO O

HN

A B-O OH, ( ) O

-o OH, /B- O OH, as O ) HO O HO O HN US 8,680,136 B2 141 142 -continued -continued HN H O HO N O 5 HN HO B-O OH, OH, / HO O

10

H N

65 HO1 No OH, US 8,680,136 B2 143 144 -continued -continued

O B 21 1. n HO O OH,

O

10 O O B HO1 O OH,

HO O OH, H

15 N O

HO O O H,

O

OH, NH O B B- O HO1 No 25 / OH, HO HN 2 N-1\s O

O B HO1 No OH, 30

O rs O N O B 35 a 'litt lso 2 HO1 No OH, B- O / H HO O

O B HNY COUHO1 No OH, 40 e NH2 H S / O N O

O B 45 HN HO1 No OH, B-O O H M O s O

NH O B 50 HO1 No OH, H O N O O OH, NH O B HO1 No OH, 55

O r O N O B 60 2 HO1 No OH, OH,

HN 2 21 O

65 NS O - BS OH,

US 8,680,136 B2 147 148 -continued -continued 2

S O

HO-N HN S B- O OH, W HO O N H B-O OH, / or a pharmaceutically acceptable salt thereof. HO O 25. The compound of claim 1, having a structure selected "N-N- l 15 from the group consisting of O O

O N H HN B - O OH, M B- O OH, HO O / MeO -N O HO O 25

O / -HO -,O . 30 HN B-O OH, / - HO O HO Na 35 (- Z NH

O B O-N HO1 OJNo OH, S 2 NH2 40 OH / HO Z N s ( N N O HN 45 8 N21 O B-O OH, and / O B HO O HO1 No OH, 50

O

HN 55 B-O OH, / O HO O HN B-O OH, 60 / O HO O

HN and or a pharmaceutically acceptable salt thereof. B-O OH, and / 65 26. A pharmaceutical composition comprising a therapeu HO O tically effective amount of a compound of claim 1 and a pharmaceutically acceptable excipient. US 8,680,136 B2 149 150 27. The compound of claim 1, having the structure:

B- O OH or a pharmaceutically acceptable salt thereof. 15

k k k k k