(12) United States Patent (10) Patent No.: US 8,933,232 B2 Gu Et Al

USOO8933232B2

(12) United States Patent (10) Patent No.: US 8,933,232 B2 Gu et al. (45) Date of Patent: *Jan. 13, 2015

(54) 1,3,4-OXADIAZOLE AND 1,3,4-THIADIAZOLE (58) Field of Classification Search BETA-LACTAMASE INHIBITORS USPC ...... 546/183: 514/359 See application file for complete search history. (71) Applicant: Cubist Pharmaceuticals, Inc., Lexington, MA (US) (56) References Cited (72) Inventors: Yu Gui Gu, Acton, MA (US); Yong He, Bedford, MA (US); Ning Yin, U.S. PATENT DOCUMENTS Lexington, MA (US); Dylan C. T112,592 B2 9, 2006 L ilas etal de SatyM.S.," 7,612,087J. K. 4 B2 11/2009 ASZodiampilas et etal. al. Metcalf, III, Needham, MA (US) (Continued) (73) Assignee: Cubist Pharmaceuticals, Inc., FOREIGN PATENT DOCUMENTS Lexington, MA (US) EP 2135959 A1 12/2012 (*) Notice: Subject to any disclaimer, the term of this FR 2835 186 A 8, 2003 patent is extended or adjusted under 35 (Continued) U.S.C. 154(b) by 0 days. This patent is Subject to a terminal dis- OTHER PUBLICATIONS claimer. Crompton, et al: Beta-Lactamase inhibitors, the inhibition of serine (21) Appl. No.: 13/853,439 beta-lactamases by specific boronic acids; Biochem J., 1988, vol. ppl. No.: 9 251, pp. 453-459. (22) Filed: Mar. 29, 2013 (Continued) (65) Prior Publication Data US 2013/0296291 A1 Nov. 7, 2013 Primary Examiner-John Mabry Related U.S. Application Dat Assistant Examiner — Daniel Carcanague elated U.S. Application Uata (74) Attorney, Agent, or Firm — Lathrop & Gage LLP: Brian (60) Provisional application No. 61/618,131, filed on Mar. C. Trinque 30, 2012, provisional application No. 61/790,579, filed on Mar. 15, 2013. (57) ABSTRACT (51) 557,A08 (2006.01) B-Lactamase inhibitor compounds (BLIS) are disclosed, A6 IK3I/546 (200 6. 01) including compounds that have activity against class A, class C or class D B-lactamases. Methods of manufacturing the (Continued) BLIS, and uses of the compounds in the preparation of phar (52) U.S. Cl. maceutical compositions and antibacterial applications are CPC ...... A61 K3I/546 (2013.01); A61K31/439 also disclosed. (2013.01); A61 K3I/436 (2013.01); (Continued) 5 Claims, 31 Drawing Sheets

Table Compounds of Formula A-II Cupd. R R No. 70 H -Osor 702 -1 -Osos 703 1N-g - co NH 704 -1N1. - QSOH

705 l --a OSH 708 1, –0so 707 -NH2 -oso, 708 *O- -oso NH 709 HMls,(>- -oson 710 ")-- –0so US 8,933,232 B2 Page 2

(51) Int. Cl. WO WO 201 is A1 $38: WO WO2011 101710 A1 8, 2011 A6 IK3I/439 (2006.01) WO WO2012,086241 A1 6, 2012 A6 IK3I/545 (2006.01) WO WO2012/172368 A1 12/2012 A6 IK3I/407 (2006.01) WO WO 2013/O14496 A1 1/2013 A6 IK 45/06 (2006.01) WO WO 2013/O14497 A1 1, 2013 A6 IK3I/436 (2006.01) W8 W858RSA: 583 A6 IK3I/427 (2006.01) WO WO2013,180.197 A1 12/2013 (52) U.S. Cl. CPC ...... C07D471/08 (2013.01); A61K31/407 OTHER PUBLICATIONS (2013.01); A61K 45/06 (2013.01); A61 K 3 1/427 (2013.01); A61 K3I/545 (2013.01) its such Report, PCT/US2013/034562, dated Jul. 30, USPC ...... 546/183:54.6/121 : 514/359:514/203: Written Opinion, PCT/US2013/034562, dated Jul 30, 2013, 5 pages. 514/202: 514/300 Patani, et al: Bioisosterism: A Rational Approach in Drug Design; Chem Rev. 1996, vol. 96, pp. 3147-3176. (56) References Cited International Search Report, PCT/US2013/034589, dated Jul. 29, 2013, 4 pages. U.S. PATENT DOCUMENTS Written Opinion, PCT/US2013/034589, dated Jul. 29, 2013, 5 pages. Mangion, et al: A Concise of a beta-Lactamase Inhibitor; Organic 7,732,610 B2 6/2010 Lampilas et al. Letters, Oct. 21, 2011, 13(2), pp. 5480-5483. 8, 178,554 B2 5/2012 Lampilas et al. Blizzard et al. Side chain SAR of bicyclic beta-lactamase inhibitors 8,288,553 B2 10/2012 Priour et al. (BLIs). 1. discovery of a class CBLI for combination with imipinem; 8,471,025 B2 6/2013 Dedhiya et al. Bioorganic & Medicinal Chemistry Letters: 2010, vol. 20 918 8.487,093 B2 7/2013 Blizzard et al. gorg ry ; ZULU, Vol. ZU, pp. 2010/O197928 A1 8, 2010 Priour et al. 2011, 00461 O2 A1 2/2011 Ledoussal et al. Coleman: Diazabicyclooctanes (DBOs): a potent new class of non 2012fOO16553 A1 2/2012 Abe et al. beta-lactam beta-lactamase inhibitors; Current Opinion in 2012/0053350 A1 3/2012 Mangion et al. Microbiology; 2011, vol. 14, pp. 1-6. 2012/0323.01.0 A1 12/2012 Ronsheim et al. Milleretal:Practical and Cost-Effective Manufacturing Route for the 2013, OO12712 A1 1/2013 Priour et al. Synthesis of a beta-Lactamase Inhibitor; Organic Letters, 2014, vol. 2013,0059.774 A1 3/2013 Patel et al. 16, No. 1, pp. 174-177. 2013/0225.554 A1 8/2013 Maiti, et al. Yoshizawa, H. et al.; “New broad-spectrum parenteral 2013,0267480 A1 ck 10/2013 Dedhiya et al. cephalosporins exhibiting potent activity against both methicillin

2013/0289012 A1* 10, 2013 Gu et al...... 514,203 resistant Staphylococcus aureus (MRSA) and Pseudomonas 2013,0296290 A1* 11/2013 Gu et al. .. 514,202 2013,0296292 A1* 11, 2013 Gu et al. .. 514,202 aeruginosa. Part 2: Synthesis and structure-activity relationships 2013,0296,293 A1* 11, 2013 Gu et al. .. 514,202 the S-3578 series': Bioorganic and Medicinal Chemistry 2004, vol. 2013,0296555 A1* 11, 2013 Gu et al. .. 544/127 12, pp. 4211-4219. 2013/0303504 A1* 11, 2013 Gu et al. .. 514,202 Yoshizawa, H. et al.; “New broad-spectrum parenteral 2013/0345190 A1* 12/2013 Gu et al...... 514,207 cephalosporins exhibiting potent activity against both methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas FOREIGN PATENT DOCUMENTS aeruginosa. Part 3: 7b-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2- ethoxyiminoacetamidocephalosporins bearing 4-3-(aminoalkyl)- FR 2812 635 A 2, 2008 ureido)-1-pyridinium at C-3”; Bioorganic and Medicinal Chemistry FR 2.930 553 A1 10/2009 2004, vol. 12, pp. 4221-4231. FR 2951 171 A 4/2011 Yoshizawa, H. et al.: "S-3578, A New Broad Spectrum Parenteral KR 2010 130176 A 12/2010 Cephalosporin Exhibiting Potent Activity Against both Methicillin WO WO O2, 10172 A1 T 2002 resistant Staphylococcus aureus (MRSA) and Pseudomonas WO WO 03/063864 A2 T 2003 aeruginosa Synthesis and Structure-activity Relationships': The WO WO2005,108391 A1 11, 2005 WO WO2006/125974 A1 11, 2006 Journal of Antibiotics 2002, vol. 55, No. 11, pp.975-992. WO WO2007/1291.76 A2 11, 2007 Ida, T. et al. “CP6679, a new injectable cephalosporin with broad WO WO 2009/09.1856 A2 T 2009 spectrum and potent activities against methicillin-resistant WO WO2009/133442 A1 11, 2009 Staphylococcus aureus and Pseudomonas aeruginosa; Journal of WO WO 2010-118361 A1 1, 2010 Infection and Chemotheraphy 2002, vol. 8, pp. 138-144. WO WO 2010-056827 A1 5, 2010 WO WO2010, 126820 A1 11, 2010 * cited by examiner U.S. Patent Jan. 13, 2015 Sheet 1 of 31 US 8,933,232 B2

FIGURE 1A

Table Compounds of Formula A-II Cmpd p X R R No. 701 O H –0so 702 O HN 1 –0so 703 O H2N 1N3, –0so

NH 704 O us –0so HN 1.n NH 705 O ls –0so H H2N -Nce 3 O 706 O HN ulRn –0so 707 O -NH2 –0so 708 O "KO- –0so

709 O HN ls N O- – –0so 710 O "O- –0so

U.S. Patent Jan. 13, 2015 Sheet 3 of 31 US 8,933,232 B2

FIGURE 1C

Table Compounds of Formula A-II Cmpd p X R R No. NH / 7232 O | ul- r’s –0so 3 H

HN 724 O vy- s –0so

725 O f –0so HN

726 O Nu. –0so HN 727 O t-O-- –0so

728 O -O- n –0so HN 729 || O O- –0so

OH 730 O "' -- –0so 731 O ---, –0so

732 O N-y –0so U.S. Patent Jan. 13, 2015 Sheet 4 of 31 US 8,933,232 B2

FIGURE 1D

Table Compounds of Formula A-II Cmnd. p X R R N0.

OH 733 O n-is- –0so 734 O 1-1š. –0so 735 O 1\-1Nig –0so 736 O 1--N-8. –0so 737 O D-- –0so

NH 738 O ls –0so H2N N--

739 O ul- NH –0so

740 O -O-- -: 74 o u Cr' | - 742 O re –0so

743 O HNCr- o

O Y 744 O C? Nu, HN

745 O Dr Nu-1 D U.S. Patent Jan. 13, 2015 Sheet 5 of 31 US 8,933,232 B2

FIGURE 1E

Table Compounds of Formula A-II

R R

HN Y

HN Afr’sN -o on (>- –0so NH

748 O HN ls HNili II { X - –0so

NH 749 O ls –0so HN -O-- 750 O "{OXO – –0so

751 O st-(Os a. –0so

NH 752 O --O –oso wY-3- 753 O 17 –0so H2N

754 O –oso HN 1 R 755 O N- –0so H2N

W R Y 756 O Of N- –0so w U.S. Patent Jan. 13, 2015 Sheet 6 of 31 US 8,933,232 B2

FIGURE 1 F

Table Compounds of Formula A-II Cmpd p X R R No.

H 757 O 17 w"N-3-6.N Y –oso

HN2 2. 758 O O): –0so 759 O C-- –0so 760 O x- –oso N 761 O C) –0so N

765 O O-re –0so

766 O C-1, Y D

767 O 5, D U.S. Patent Jan. 13, 2015 Sheet 7 of 31 US 8,933,232 B2

FIGURE 1G

Table Compounds of Formula A-II Cmpd X R R No. 768 O HoNo. () - --OSOH

769 O HN S. --OSOH

770 O HN N-1 --OSOH

H 771 O HN N-1aul's, --OSOH

775 O in ()--

776 O HM -1- --OSOH \ /

777 O HN N-g --OSOH N v n

778 O ()-- D 779 O ( ); 780 O u-O- U.S. Patent Jan. 13, 2015 Sheet 8 of 31 US 8,933,232 B2

FIGURE 1H

Table Compounds of Formula A-II Cmnd p X R R N). 781 O --O): –0so 782 O / \ – –0son 783 S to-O- –0so

784 S HN us –0so

785 S H2N us -g XII & IIII --Osor 786 S -( ): –0so 787 O to-Ox>- –0so 788 O "OXO- –0so O N 1 789 O –0so HN 790 O "O I "Sr. –0so 791 O "O –0so

792 O HN\ / 1-1) –0so U.S. Patent Jan. 13, 2015 Sheet 9 of 31 US 8,933,232 B2

FIGURE I

Table Compounds of Formula A-II Cmpd. R No. 793 -->

794

795 to-O--

796

797

798 U.S. Patent Jan. 13, 2015 Sheet 10 of 31 US 8,933,232 B2

09.0IJN

U.S. Patent Jan. 13, 2015 Sheet 14 of 31 US 8,933,232 B2

090IJN U.S. Patent US 8,933,232 B2

U.S. Patent Jan. 13, 2015 Sheet 16 of 31 US 8,933,232 B2

:punoduuoouogeleduuoos?ODO O |-

a ----as a -a ------w

3 U.S. Patent US 8,933,232 B2

() U.S. Patent US 8,933,232 B2

U.S. Patent Jan. 13, 2015 Sheet 19 Of 31 US 8,933,232 B2

SOINÍN()IWOO

?I-VXO

U.S. Patent Jan. 13, 2015 Sheet 21 of 31 US 8,933,232 B2

HIIIIIIIiini NN

punodtuo3Ioneleduuoos?OOO

U.S. Patent US 8,933,232 B2

U.S. Patent Jan. 13, 2015 US 8,933,232 B2

U.S. Patent Jan. 13, 2015 Sheet 28 of 31 US 8,933,232 B2

4. 4. --

N |- U.S. Patent Jan. 13, 2015 Sheet 29 Of 31 US 8,933,232 B2

N HIIIIIm?????N ?? © U.S. Patent Jan. 13, 2015 Sheet 30 of 31 US 8,933,232 B2

U.S. Patent Jan. 13, 2015 Sheet 31 of 31 US 8,933,232 B2

Çpunoduuoouoleueduuoos?OOO 6ETH(10.IH US 8,933,232 B2 1. 2 1,3,4-OXADIAZOLE AND 1,3,4-THADIAZOLE wherein X is selected from O and S; BETA-LACTAMASE INHIBITORS R is selected from

RELATED APPLICATIONS OSOH, SOH, OPOH, This application claims priority to U.S. Provisional Appli cation No. 61/618,131, filed Mar. 30, 2012, and U.S. Provi sional Application No. 61/790,579, filed Mar. 15, 2013. The entire contents of these applications are incorporated herein 10 CHCO2H, and CF2CO2H by reference in their entireties. and, TECHNICAL FIELD R" is selected from: 15 a. hydrogen; This disclosure is directed to B-lactamase inhibitors (BLIS) which are effective as inhibitors of B-lactamases and, when used in combination with B-lactam antibiotics are useful in b. the treatment of bacterial infections. The compounds when combined with a B-lactam antibiotic are effective in treating infections caused by bacteria that are resistant to B-lactam pi R2, antibiotics due to the presence of B-lactamases. Pharmaceu tical compositions comprising Such compounds, methods of using Such compounds, and processes for preparing Such wherein R is selected from compounds are also disclosed. 25

BACKGROUND NR

Bacterial resistance to B-lactam antibiotics, especially in 30 Gram-negative bacteria, is most commonly mediated by --e. and \s. B-lactamases. B-lactamases are enzymes that catalyze the hydrolysis of the B-lactam ring, which inactivates the anti wherein each of R, R and R is independently selected bacterial activity of the B-lactam antibiotic and allows the from hydrogen, (C-C)-alkyl, aminoalkyl, aminocy bacteria to become resistant Inhibition of the B-lactamase 35 with a BLI slows or prevents degradation of the B-lactam cloalkyl, and hydroxyalkyl, and n is selected from 1, 2 antibiotic and restores -lactam antibiotic susceptibility to and 3; B-lactamase producing bacteria. Many of these B-lactamases are not effectively inhibited by BLIs currently on the market rendering the B-lactam antibiotics ineffective in treating bac 40 teria that produce these B-lactamases. There is an urgent need for novel BLIs that inhibit B-lactamases that are not effec tively inhibited by the current clinical BLIs (e.g. KPC, class C and class D B-lactamases) and that could be used in combi nation with B-lactam antibiotics to treat infections caused by 45 B-lactam resistant bacteria. wherein R is H., (C-C)alkylamino, and SUMMARY OF INVENTION NH 50 The present invention provides, in one aspect, compounds of chemical formula (I), orpharmaceutically-acceptable salts NH2: thereof, which are BLIs and are useful in combination with B-lactam antibiotics for the treatment of bacterial infections. 55 d. amino; (I) NN R-( C. X 60

N H

JY. N O R 65 wherein R is selected from H., (C-C)-unsubstituted alkyl, amino-(C-C)-alkyl, aminocycloalkyl, hydroxy alkyl, US 8,933,232 B2

and

NH ls HN iii. HN N iii. and H s NH2, each of R*, R*, R* and R* is independently selected and each of p and q is independently selected from 1 and 2: 10 from hydrogen or (C-C)-alkyl, aminoalkyl, aminocy and cloalkyl, and hydroxyalkyl, provided that at least one of f. CH(R)CH-NH, R*, R*, R* and R7* hydrogen, wherein R is selected from amino and hydroxyl. n is selected from 1, 2, 3 and 4, and In another aspect, the invention provides compounds of 15 m is selected from 1, 2 and 3; chemical Formula (A-I) or a pharmaceutically acceptable salt thereof, which are BLIs and are useful in combination with B-lactam antibiotics for the treatment of bacterial infections.

(A-I) NS N

R"-(X 25 N H wherein R* is selected from NH, !' N. O R NR6" 30 wherein X* is selected from O and S: or X O ... X R* is selected from wherein each of R*, R*, R* and R* is as described 35 previously and each of R. R', and R'' is independently Selected from hydrogen or (C-C)-alkyl, provided that OSOH, SO3H, OPOH, at least one of R. R', and R'' is hydrogen;

40 d. amino CHCO2H and CF2COH:

and 45 R18 R* is selected from: y q* a. hydrogen; Z (T)-(CH2), s pe

b. 50 wherein Z is selected from CR'R' or NR'', each of R'' and R' is independently selected from H, NH, and 55 wherein R* is selected from

NHR3, and R'R''N l, X wherein each of R*, R*, R* and R* is as described previously, 65 alternatively, R'' and R' together with the carbon to which R* is selected from hydrogen, (C-C)-alkyl, aminoalkyl, they are attached, form a cycloalkyl or heterocyclyl ring aminocycloalkyl, hydroxyalkyl, containing 4-6 ring members, US 8,933,232 B2 5 R" is selected from Hand i. NR17 rt-fix2 wherein R is selected from NH, -NH(C-C)-alkyland wherein each of R', R'' and R7 is independently 10 Selected from hydrogen, (C-C)-alkyl, aminoalkyl, aminocycloalkyl, and hydroxyalkyl, provided that at least one of R', Rand R7 is hydrogen,

R" is selected from NH and 15 wherein each of R*, R*, R* and R* is as described previously, X s is selected from 0 and 1, and V is selected from 0, 1, 2, and 3; wherein each of R*, R*, R* and R* is as described previously, j. each of p and q is independently selected from 0, 1, 2 and 25 3 s T is selected from NH and O ICD - t is selected from 0, 1, 2, 3, and 4, and k each of r and y is independently selected from 0 and 1: 30 N-(CH2) s

35 wherein M is selected from NR’, CR'R'' and O, wherein R’ is Hor

wherein R' is selected from NH and 40

X 45 wherein each of R', RandR'7 is as described previ ously, wherein each of R*, R*, R* and R7* is as described each of RandR is independently selected from H, NH, previously, and R" is selected from amino and hydroxyl, and 50 w is selected from 0 and 1:

9. 55 wherein each of R*, R*, R* and R* is as described previously, and h. u is selected from 0, 1 and 2: 60 X l. 65 wherein each of R*, R*, R* and R7* is as described K2 previously; US 8,933,232 B2 7 8 -continued FIG. 8 shows Table VIII, Standard BLI Potentiation MIC Assay of Compounds Partnered with Aztreonam Against a Panel of Isogenic and Clinical Strains Expressing B-Lacta ; and aSS FIG. 9 shows Table IX, Standard BLI Potentiation MIC Assay of Compounds Partnered with Meropenem Against a Panel of Isogenic and Clinical Strains Expressing B-Lacta aSS

10 DETAILED DESCRIPTION

Definitions In one embodiment, the invention provides use of a com pound of Formula I for inhibiting B-lactamases. Molecular terms, when used in this application, have their In one embodiment, the invention provides use of a com 15 common meaning unless otherwise specified. pound of Formula A-I for inhibiting B-lactamases. The term “alkyl is defined as a linear or branched, satu In one embodiment, the invention provides compounds of rated radical having one to about twenty carbon atoms unless Formula I with high binding affinity for B-lactamase otherwise specified. Preferred alkyl radicals are “lower alkyl enzymes. In one embodiment, the invention provides compounds of radicals having one to about five carbon atoms. Examples of Formula A-I with high binding affinity for B-lactamase alkyl groups include, without limitation, methyl, ethyl, tert enzymes. butyl, isopropyl, and hexyl. A subset of the term alkyl is In one embodiment, the present invention also provides “(C-C)-unsubstituted alkyl” which is defined as an alkyl antibacterial compositions comprising compounds of For group that bears no substituent groups. Examples of (C-C)- mula I and at least one B-lactam antibiotic. 25 unsubstituted alkyl groups include methyl, ethyl, propyl and In one embodiment, the present invention also provides isopropyl. It is understood that ifa (C-C)-alkyl is “substi antibacterial compositions comprising compounds of For tuted that one or more hydrogen atoms is replaced by a mula A-I and at least one B-lactam antibiotic. substitutent. In one embodiment, the present invention provides phar The term amino denotes a NH2 radical. maceutical compositions comprising compounds of Formula 30 The term “aminoalkyl denotes an alkyl in which one or I and at least one B-lactam antibiotic and methods of use more of the alkyl hydrogen atoms has been replaced by an thereof. amino group. In one embodiment, the present invention provides phar The term "aminocycloalkyl denotes a cycloalkyl in which maceutical compositions comprising compounds of Formula one of the cycloalkyl hydrogenatoms has been replaced by an A-I and at least one 3-lactam antibiotic and methods of use 35 amino group. thereof. The term “cycloalkyl or “cycloalkyl ring is defined as a In one embodiment, the invention provides methods of use saturated or partially unsaturated carbocyclic ring in a single of the compounds of Formula I to treat bacterial infections in or fused carbocyclic ring system having from three to twelve a subject. ring members. In a preferred embodiment, a cycloalkyl is a In one embodiment, the invention provides methods of use 40 ring system having three to seven ring members. Examples of of the compounds of Formula A-I to treat bacterial infections a cycloalkyl group include, without limitation, cyclopropyl. in a Subject. cyclobutyl, cyclohexyl, and cycloheptyl. The term “hydroxyalkyl denotes an alkyl radical in which BRIEF DESCRIPTION OF THE DRAWINGS one or more of the alkyl hydrogenatoms has been replaced by 45 a hydroxyl group. FIGS. 1A-1I show Table I, Representative Compounds of It will be understood by one of skill in the art that a Formula A-II FIGS. 2A-2G show Table II, Standard BLI potentiation MIC assay against a panel of isogenic and clinical strains expressing B-lactamases. 50 or — FIGS. 3A-3F show Table III, the synergy MIC of represen tative compounds of Formula II-A against a panel of isogenic denote the point of attachment of a Substituent group where and clinical Strains expressing 3-lactamases. indicated. For example FIGS. 4A-4C show Table IV, an assay to determine inhi bition kinetics of representative compounds of Formula II-A 55 for the KPC-2 B-lactamase. FIGS.5A-5B show Table V. Synergy MIC of Comparator Compounds Against a Panel of Isogenic and Clinical Strains Expressing B-lactamases FIG. 6 shows Table VI, Standard BLI Potentiation MIC 60 Assay of Compounds Partnered with CXA-101 Against a Panel of Isogenic and Clinical Strains Expressing B-Lacta represent that the point of attachment of the amide moiety is aSS at the carbonyl carbon. FIG. 7 shows Table VII, Standard BLI Potentiation MIC The functional classification of B-lactamases and terms Assay of Compounds Partnered with Ceftazidime Against a 65 “Class A. “Class C, and “Class D B-lactamases are under Panel of Isogenic and Clinical Strains Expressing B-Lacta stood by one of skill in the art and are described in “Updated aSS Functional Classification of B-Lactamases’, Bush, K.: US 8,933,232 B2 10 Jacoby, G. A.; Antimicrob. Agents Chemother: 2010, 54,969 or mass number different from the atomic mass or mass 976, herein incorporated by reference. number usually found in nature. Examples of isotopes Suit The salts of the compounds of the invention include acid able for inclusion in the compounds described herein include addition salts and base addition salts. In a one embodiment, and are not limited to H, H, C, C, C, C1, F, 'I, the salt is a pharmaceutically acceptable salt of the compound PI, N, N, O, 7O, O, P, and S. In one embodi of Formula I. The term “pharmaceutically acceptable salts' ment, isotopically-labeled compounds are useful in drug and/ embraces salts commonly used to form alkali metal salts and or substrate tissue distribution studies. In another embodi to form addition salts of free acids or free bases. The nature of ment, Substitution with heavier isotopes such as deuterium the salt is not critical, provided that it is pharmaceutically affords greater metabolic stability (for example, increased in acceptable. Suitable pharmaceutically acceptable acid addi 10 tion salts of the compounds of the invention may be prepared Vivo half-life or reduced dosage requirements). In yet another from an inorganic acid or an organic acid. Examples of Such embodiment, Substitution with positron emitting isotopes, inorganic acids include, without limitation, hydrochloric, such as 'C, F, 'O and 'N, is useful in Positron Emission hydrobromic, hydroiodic, nitric, carbonic, Sulfuric and phos Topography (PET) studies for examining substrate receptor phoric acid. Examples of appropriate organic acids may be 15 occupancy. Isotopically-labeled compounds are prepared by selected from aliphatic, cycloaliphatic, aromatic, aryla any suitable method or by processes using an appropriate liphatic, heterocyclic, carboxylic and Sulfonic classes of isotopically-labeled reagent in place of the non-labeled organic acids, examples of which include, without limitation, reagent otherwise employed. formic, acetic, propionic, succinic, glycolic, gluconic, The invention also embraces isolated compounds. An iso maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, lated compound refers to a compound which represents at 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, least 10%, such as at least 20%, such as at least 50% and toluenesulfonic, Sulfanilic, mesylic, cyclohexylaminosul further such as at least 80% of the compound present in the fonic, Stearic, algenic, B-hydroxybutyric, malonic, galactic, mixture. In one embodiment, the compound, a pharmaceuti and galacturonic acid. Suitable pharmaceutically-acceptable cally acceptable salt thereof, or a pharmaceutical composi base addition salts of compounds of the invention include, but 25 tion comprising the compound exhibits detectable (i.e. statis are not limited to, metallic salts made from aluminum, cal tically significant) activity when tested in conventional cium, lithium, magnesium, potassium, Sodium and Zinc or biological assays Such as those described herein. organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, B-Lactamase Inhibitors (BLIs) N-methylglucamine, lysine and procaine. All of these salts 30 In one aspect, the invention provides compounds of For may be prepared by conventional means from the correspond mula I or pharmaceutically-acceptable salts thereof: ing compound of the invention by treating, for example, the compound of the invention with the appropriate acid or base. (I) The compounds of the invention can possess one or more NN asymmetric carbon atoms and are thus capable of existing in 35 N the form of optical isomers as well as in the form of racemic or non-racemic mixtures thereof. The compounds of the X invention can be utilized in the present invention as a single isomer or as a mixture of stereochemical isomeric forms. N H Diastereoisomers, i.e., nonsuperimposable stereochemical 40 isomers, can be separated by conventional means such as ! N chromatography, distillation, crystallization or Sublimation. O R The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for The substituent X of Formula I is selected from O and S. In example by formation of diastereoisomeric salts by treatment 45 one aspect of the invention X is S. In another aspect of the with an optically active acid or base. Examples of appropriate invention X is O. acids include, without limitation, tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic Substituent R of Formula I is selected from acid. The mixture of diastereomers can be separated by crys tallization followed by liberation of the optically active bases 50 from the optically active salts. An alternative process for separation of optical isomers includes the use of a chiral OSOH, SOH, OPOH, chromatography column optimally chosen to maximize the separation of the enantiomers. Still another available method involves synthesis of covalent diastereoisomeric molecules 55 CHCO2H, and CF2CO2H. by treating compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conven tional means such as chromatography, distillation, crystalli In a preferred embodiment, R is zation or sublimation, and then hydrolyzed to obtain the 60 enantiomerically pure compound. The optically active com pounds of the invention can likewise be obtained by utilizing optically active starting materials. These isomers may be in OSOH. the form of a free acid, a free base, an ester or a salt. Compounds described herein also include isotopically-la 65 beled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass US 8,933,232 B2 11 12 The group R' of Formula I is selected from: In one aspect of the invention R' is selected from H. —CH-NH, CHCH-NH – CONH, -NH, -CH —CHCH-NHCHCH-NH.

10 ~y HN Sry s NH wherein R is selected from H2 !--->H NH NR 15 --se and x.

wherein each of R. R. and R is independently selected from hydrogen, (C-C)-alkyl, aminoalkyl, aminocy o'C - r) – cloalkyl, and hydroxyalkyl, and n is selected from 1, 2 and 3; 25 HN s

C. O 30

wherein R is H, (C-C)alkylamino, and 35

NH In one embodiment of the invention R' is selected from H and x's 40 d. amino; HN ~y C. 45 g In one embodiment of the invention, the compounds of the NR7, invention are of the stereochemistry disclosed in Formula II. p

wherein R is selected from H. (C-C)-unsubstituted 50 (II) alkyl, amino-(C-C)-alkyl, aminocycloalkyl, hydroxy alkyl, and

55 NH

NH2 60 In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R' is H. and each of p and q is independently selected from 1 and 2: In another embodiment of the invention, the compound is and of Formula II and X is O, R is OSOH and R' is f. CH(R)CH-NH, - CH-NH. wherein R is selected from amino and hydroxyl. 65 In another embodiment of the invention, the compound is In one aspect of the invention n is 1. In another aspect of the of Formula II and X is O, R is OSOH and R' is invention n is 2. In another aspect of the invention n is 3. - CHCH-NH. US 8,933,232 B2 13 14 In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R is NH

NH 5 --O ls NH2.

cks 1.2 In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R' is 10 In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R' is -- NH 15 \ / NH In another embodiment of the invention, the compound is x- 1s.2 of Formula II and X is O, Ris-OSOH and R' is

In another embodiment of the invention, the compound is NH of Formula II and X is O, Ris-OSOH and R' is CONH. In another embodiment of the invention, the compound is >n-r -- 2 of Formula II and X is O, Ris-OSOH and R' is -NH. 25 In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R is In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R' is -CH(OH) CH-NH. 30 In another embodiment of the invention, the compound is of Formula II and X is O, R is OSOH and R' is NH. CHCH-NHCHCH-NH. In another embodiment of the invention, the compound is of Formula II and X is O, R is OSOH and R' is —CH 35 (NH2)CH-NH. In another embodiment of the invention, the compound is In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R' is of Formula II and X is S, R is —OSOH and R' is - CHCH-NH. In another embodiment of the invention, the compound is NH 40 of Formula II and X is S, R is OSOH and R' is N ls NH2.

45 NH. In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R' is In another embodiment of the invention, the compound is 50 of Formula II and X is O, Ris-OSOH and R' is NH. HN ~y 55 In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R' is In another embodiment of the invention, the compound is of Formula and II X is S, R is OSOH and R' is 60 NH NH. 1s. 65 cars 2 In another embodiment of the invention, the compound is of Formula II and X is O, Ris-OSOH and R' is US 8,933,232 B2 15 16 Preferred compounds of Formula I are the compounds: In a preferred embodiment, R* is

OSOH.

and The group R'* of Formula I is selected from: 10

a hydrogen; b.

15 R21 'n

R* is selected from

25 NHR', and - X.

It will be understood by one of skill in the art that depend R* is selected from hydrogen, (C-C)-alkyl, aminoalkyl, ing on the nature of R' and R, compounds of Formula I may 30 aminocycloalkyl, hydroxyalkyl, exist in a salt or Zwitterionic form. In one aspect, the invention provides compounds of For mula A-I or pharmaceutically-acceptable salts thereof: NH 35 HN 'm es iii. and H s (A-I) NN N R"-( each of R*, R*, R* and R* is independently selected X" 40 from hydrogen or (C-C)-alkyl, aminoalkyl, aminocy cloalkyl, and hydroxyalkyl, provided that at least one of N H R*, R*, R* and R7* is hydrogen, Z NN. n is selected from 1, 2, 3 and 4, and O R 45 m is selected from 1, 2 and 3;

The substituent X* of Formula A-I is selected from O and S. In one aspect of the invention X* is S. In another aspect of the invention X* is O. 50 Substituent R* of Formula A-I is selected from

--ol --ol --ol 55 wherein R* is selected from NH,

wherein each of R*, R*, R* and leis as described pre --not and -- to 65 viously and each of R. R', and R'' is independently Selected from hydrogen or (C-C)-alkyl, provided that at least one of R. R', and R'' is hydrogen; US 8,933,232 B2 17 18 wherein R' is selected from NH and d. amino;

C. y

pe-in-la-- wherein each of 10 Z is selected from CR'R' or NR'', R*, R*, R* and R7* is as described previously, each of R'' and R' is independently selected from H, NH, R’ is selected from amino and hydroxyl, and and w is selected from 0 and 1: 15

wherein each of R*, R*, R* and R* is as described h. previously, alternatively, R'' and R' together with the carbon to which they are attached, form a cycloalkyl or heterocyclyl ring 25 containing 4-6 ring members, R" is selected from Hand wherein each of R*, R* and R* is as described pre 30 viously; NR17

35 R21 each of R', RandR'7 is independently selected from hydrogen, (C-C)-alkyl, aminoalkyl, aminocycloalkyl, and hydroxyalkyl, provided that at least one of R', R' and R7 is hydrogen, 40 wherein R is selected from NH, -NH(C-C)-alkyland R" is selected from NH and

wherein each of R*, R*, R* and R* is as described previously, wherein each of R*, R*, R* and R* is as described 50 previously, s is selected from 0 and 1, and each of p and q is independently selected from 0, 1, 2 and V is selected from 0, 1, 2, and 3; 3 s T is selected from NH and O 55 j. t is selected from 0, 1, 2, 3, and 4, and each of r and y is independently selected from 0 and 1:

60 k.

M N-(CH2) s

65 US 8,933,232 B2 19 20 wherein M is selected from NR, CR'R'' and O, -continued wherein R’ is Hand NH

NR17 5 HN s 2s -

HN N s s s 10 \ / H wherein each of R', Rand R7 is as described previ ously, each of RandR is independently selected from H, NH, HN and HN1)-1 15 s s

NR6" s s l HN HN R4R5°N NR7 wherein each of R*, R*, R* and R* is as described HN ) s ~y previously, and u is selected from 0, 1 and 2: NH

* HN -l N l. H , -N s H HN NH HN

( NH s 30 HN ls N s Yx l H N HN ; and 35 s s HN HN

l HN 40 HN-(O- re-O-4- - X “) II I I III.iii, 45 OH In one aspect of the invention R* is selected from re--X

HN ~% 50 H OH H, H.N s HN N NH NH r

HN N HN N 55 OH

HN , -NH2, HN s 60 OH l HN -N-X s o NH s

HN N HN s 65 s HN US 8,933,232 B2 21 22 -continued -continued ~~yH N HN l

10 -y-.NH O>-- 15

25 30 R - P - 35 HN HN

N HN -- -H - is KO-- 40 NH 45 --H

65 US 8,933,232 B2 23 24 -continued -continued NH is-O s HN !--->N

HN N 1. s \ /

HN \ / N 15 HN\ / --

In one embodiment of the invention R'* is selected from HN , HN III III,

lNH -NH ~y s HN -{O- , 30 NH NH ls HN2 l HN NH s s HN N-O la III III, 35 N W s s -N" \ s HN HN

40 HN , HN s HNKO in , and HN s 45 X O -X * Y . . . . . III . HN O? 50 In one embodiment of the invention R'* is selected from HN ... I I II

"C-4 H, ~y s H--X s HNO N 60 HN , and Nuck H s N --X 65 In one embodiment of the invention,HN the compounds of the invention are of the stereochemistry disclosed in Formula A-II. US 8,933,232 B2 25 26 -continued N-N (A-II) ? R-4 y, 5 o-1, X N I I IIH HN N IIH 2 N O N YOSO,H, O R 10 NS N In another embodiment of the invention, X*, R* and R'* NO- -( J'', are chosen from the substituents listed in Table I (See FIG. 1). Preferred compounds of Formula A-I are 15 N I IIH O 1 YOSOH, N- N N- N W HN 7 A. 2O O '. O i

N I I IIH N . I I IIH

O NYOS,H, 25 Ou1 YOSO,H, HN N NN NS S-( J W O % O '. 30 \\ N IIH N IIH H2 NS u N O YOSOH, N n HN2 N o? OSOH, 35 O-( \N N O JA. .. H / NN N III I IIH o-1, 40 N

N II I I IIH O YOSO,H,

N O YOSO,H, It will be understood by one of skill in the art that depend NH ing on the nature of R'* and R*, compounds of Formula I may HN ( exist in a salt or Zwitterionic form. 2 HN Enzyme Inhibition- and Binding- Affinity /N NN The compounds of the invention (e.g. compounds of For J 50 mula I, compounds of Formula A-I, compounds of Formula O II, compounds of Formula A-II) are effective in inhibiting- B-lactamase. In one aspect of the invention the compounds of N . IIH Table I are effective f3-lactamase inhibitors.

O usOSOH, ss. In one aspect the compound

HN N N-N 2 ( s 4 y. O %. 60 O N H N ... I I IIH N us O OSOH O OSOH, 65 is effective in inhibiting B-lactamase. US 8,933,232 B2 27 28 In one aspect the compound from class A, class C or class D3-lactamases. In one aspect of the invention the compounds of Formula II inhibit B-lacta mases selected from class A, class C or class D B-lactamases. HN N-N In one aspect of the invention the compounds of Formula A-II inhibit B-lactamases selected from class A, class C or class D Y-\ul O >. B-lactamases. In one aspect of the invention the compounds N ... H of Table I inhibit B-lactamases selected from class A, class C or class D B-lactamases. In one aspect of the invention the compound of the Formula a N 10 O OSOH,

N-N is effective in inhibiting B-lactamase. In one aspect the compound 4 y, O ... 15 N I I IIH N N-N NN-KH y%, O 1. Yoso,H, O N II I I IIH inhibits 3-lactamases selected from class A, class C or class D / N B-lactamases. In one aspect of the invention the compound of O OSOH the Formula 25 is effective in inhibiting B-lactamase. In one aspect the compound HN N-N

Y-\ul O >. 30 N . . H A N NO- NH N . I IIH O OSOH, 35 O ! NOSOH inhibits 3-lactamases selected from class A, class C or class D B-lactamases. In one aspect of the invention the compound of the Formula is effective in inhibiting B-lactamase. In one aspect the compound 40 N N-N NN-KH y'', O

O >. 45 N II I I IIH HN N H 2 N O OSOH,

O/ NOSOH 50 inhibits 3-lactamases selected from class A, class C or class D is effective in inhibiting B-lactamase. B-lactamases. In one aspect of the invention the compound of When used in combination with B-lactam antibiotics, the the Formula compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, com 55 pounds of Formula A-II) potentiate the activity of the B-lac tam antibiotic against microorganisms that are normally resistant to B-lactam antibiotics due to the presence of a B-lactamase or multiple B-lactamases. INO- N H In one aspect of the invention the compounds of the inven 60 tion (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) O !- N OSOH, inhibit B-lactamases selected from class A, class C or class D B-lactamases. In one aspect of the invention the compounds of Formula I, inhibit B-lactamases selected from class A, class 65 inhibits 3-lactamases selected from class A, class C or class D C or class D B-lactamases. In one aspect of the invention the B-lactamases. In one aspect of the invention the compound of compounds of Formula A-I inhibit B-lactamases selected the Formula US 8,933,232 B2 29 30 Synergy MIC assay or a BLI potentiation assay (e.g. as N-N described herein), both of which are run in the presence of a B-lactam. The lower the sMIC or MIC value the more active the BLI, regardless of the mechanism of action of the BLI HN O compound (e.g., including inhibition of B-lactamases by the N . I I IIH BLI or any other mechanism of action or combination of mechanisms of action). The SMIC and BLI potentiation assay a N data Supports that the compounds of the invention (e.g. com O OSOH, pounds of Formula I, compounds of Formula A-I, compounds 10 of Formula II, compounds of Formula A-II) potentiate (i.e. inhibits 3-lactamases selected from class A, class C or class D make more potent) the activity of the B-lactam antibiotic B-lactamases. Class A B-lactamases for example, include, but against B-lactamase producing strains by inhibiting the B-lac are not limited to, TEM, SHV, CTX-M, KPC, GES, VEB, tamase. SME, and GEX. In a preferred aspect of the invention, the In one embodiment, the BLI activity is measured by growth compounds of the invention (e.g. compounds of Formula I, 15 inhibition of a B-lactamase producing bacterial strains in a Synergy MIC (sMIC) assay. Preferably, the sMIC value for compounds of Formula A-I, compounds of Formula II, com the compounds of the invention (e.g. compounds of Formula pounds of Formula A-II) inhibit KPC B-lactamases. In a pre I, compounds of Formula A-I, compounds of Formula II, ferred aspect of the invention, the compounds of Formula I compounds of Formula A-II) is 8 g/mL or less. In a more inhibit KPC B-lactamases. In a preferred aspect of the inven preferred aspect of the invention, the sMIC value for the tion, the compounds of Formula A-I inhibit KPC B-lacta compounds of the invention (e.g. compounds of Formula I, mases. In a preferred aspect of the invention, the compounds compounds of Formula A-I, compounds of Formula II, com of Formula II inhibit KPC 5-lactamases. In a preferred aspect pounds of Formula A-II) is 4 ug/mL to 8 ug/mL. In an even of the invention, the compounds of Formula A-II inhibit KPC more preferred aspect of the invention, the SMIC value for the B-lactamases. More preferably the compounds of the inven 25 compounds of the invention (e.g. compounds of Formula I, tion (e.g. compounds of Formula I, compounds of Formula compounds of Formula A-I, compounds of Formula II, com A-I, compounds of Formula II, compounds of Formula A-II) pounds of Formula A-II) is 1 to 2 ug/mL. In a still more inhibit KPC-2 or KPC-3 f-lactamases. More preferably the preferred aspect of the invention, the sMIC value for the compounds of Formula I inhibit KPC-2 or KPC-3 B-lacta compounds of the invention (e.g. compounds of Formula I, mases. More preferably the compounds of Formula A-I 30 compounds of Formula A-I, compounds of Formula II, com inhibit KPC-2 or KPC-3 f-lactamases. More preferably the pounds of Formula A-II) is 0.2 to 0.5 g/mL. Synergy MICs compounds of Formula II inhibit KPC-2 or KPC-3 B-lacta for representative compounds of the invention are described mases. More preferably the compounds of Formula A-II in Table III (See FIG.3). It will be understood by one of skill inhibit KPC-2 or KPC-3 B-lactamases. In one aspect of the in the art that the growth inhibition off-lactamase producing invention, the compounds of the invention (e.g. compounds 35 strains can also be measured by a checkerboard synergy assay of Formula I, compounds of Formula A-I, compounds of like that disclosed in International Patent Application Num Formula II, compounds of Formula A-II) inhibit KPC-2 or ber WO 2008/039420 or a standard BLI potentiation assay KPC-3 B-lactamases in clinical strains (FIG. 2, Table II and using a fixed concentration of BLI. FIGS. 6-9, Tables VI-IX). In one aspect of the invention, the In one embodiment, the BLI activity is measured by growth compounds of Formula I inhibit KPC-2 or KPC-3 B-lacta 40 inhibition of a B-lactamase producing bacterial strains in a mases in clinical strains (FIG. 2, Table II and FIGS. 6-9. standard BLI potentiation assay using a fixed concentration Tables VI-IX). In one aspect of the invention, the compounds of BLI. Preferably, the MIC value for the compounds of the of Formula A-I inhibit KPC-2 or KPC-3 f-lactamases in invention (e.g. compounds of Formula I, compounds of For clinical strains (FIG. 2, Table II and FIGS. 6-9, Tables VI-IX). mula A-I, compounds of Formula II, compounds of Formula In one aspect of the invention, the compounds of Formula II 45 A-II) is 8 g/mL or less. In a more preferred aspect of the inhibit KPC-2 or KPC-3?-lactamases in clinical strains (FIG. invention, the MIC value for the compounds of the invention 2, Table II and FIGS. 6-9, Tables VI-IX). In one aspect of the (e.g. compounds of Formula I, compounds of Formula A-I. invention, the compounds of Formula A-II inhibit KPC-2 or compounds of Formula II, compounds of Formula A-II) is 4 to KPC-3 B-lactamases in clinical strains (FIG. 2, Table II and 8 ug/mL. In an even more preferred aspect of the invention, FIGS. 6-9, Tables VI-IX). Class C B-lactamases for example, 50 the MIC value for the compounds of the invention (e.g. com include, but are not limited to chromosomal AmpCs, and pounds of Formula I, compounds of Formula A-I, compounds plasmid based ACC, DHA, CMY FOX, ACT, MIR, LAT, of Formula II, compounds of Formula A-II) is 1 to 2 ug/mL. MOX f-lactamases. Class D B-lactamase enzymes, for In a still more preferred aspect of the invention, the MIC value example, include, but are not limited to oxacillinases or OXA for the compounds of the invention (e.g. compounds of For B-lactamases. In a preferred aspect of the invention, the com 55 mula I, compounds of Formula A-I, compounds of Formula pounds of the invention (e.g. compounds of Formula I, com II, compounds of Formula A-II) is 0.2 ug/mL to 0.5ug/mL. pounds of Formula A-I, compounds of Formula II, com The compounds of the present invention (e.g. compounds pounds of Formula A-II) inhibit OXA-15 B-lactamases. In a of Formula I, compounds of Formula A-I, compounds of preferred aspect of the invention, the compounds of Formula Formula II, compounds of Formula A-II) have a broad spec I inhibit OXA-15 B-lactamases. In a preferred aspect of the 60 trum of activity across a wide variety off-lactamase produc invention, the compounds of Formula A-I inhibit OXA-15 ing bacteria. It was surprisingly found that the compounds of B-lactamases. In a preferred aspect of the invention, the com the present invention (e.g. compounds of Formula I, com pounds of Formula II inhibit OXA-15 B-lactamases. In a pounds of Formula A-I, compounds of Formula II, com preferred aspect of the invention, the compounds of Formula pounds of Formula A-II) are active in potentiating activity of A-II inhibit OXA-15 B-lactamases. 65 B-lactam antibiotics, in particular, Ceftolozane, against Unless otherwise indicated, the activity of the BLI com strains expressing class D B-lactamase OXA-15 B-lactamase. pounds can be described by the MIC value obtained from a Currently marketed BLIs inhibit most of the class A B-lacta US 8,933,232 B2 31 32 mases, but poorly inhibit class A KPCB-lactamases and class binding affinity of 100-999 mM's'. Compounds that have C B-lactamases and have variable Success in inhibiting peni a binding affinity of 100-999 mM s' are, for example, cillinase and carbapenemase-type class D B-lactamases. The compounds 701,702, 703,705, 706,707, 709,711, 712,713, compounds of the present invention (e.g. compounds of For 714, 720, 730, 740, 741, 742, 743, 745, 746, 748, 749, 752, mula I, compounds of Formula A-I, compounds of Formula 5 753, 767,770, 775, 779, 781, 782,785,794,798 (Table IV). II, compounds of Formula A-II) are active against a wide variety of bacterial strains that express class A and C 3-lac In one embodiment the compounds of the invention (e.g. tamases and also, Surprisingly are active against bacterial compounds of Formula I, compounds of Formula A-I, com strains that express the class D cephalosporinase OXA-15 pounds of Formula II, compounds of Formula A-II) have a (Tables II and III). This increased activity against the class D 10 binding affinity of 1-99 mM's'. Compounds that have a B-lactamase is critical because differential effectiveness binding affinity of 1-99 mM's are, for example, com against different types of B-lactamase producing bacteria is pounds 704, 706, 708, 710,715,716,717,718,719,721,722, necessary in order to effectively use f-lactam antibiotics to 723, 724, 725,726, 727, 728, 729, 731, 732,733, 734, 735, treat resistant strains of bacteria (vide infra). 736, 737, 738, 739, 744, 747, 750, 751, 754, 755, 756, 757, In one embodiment, the compounds the invention (e.g. 15 758, 759, 760, 761, 762, 763, 764, 765, 766, 769, 771, 772, compounds of Formula I, compounds of Formula A-I, com 773,774,776,777,778,780,783,784,786, 792,796, and 797 pounds of Formula II, compounds of Formula A-II) are unex (Table IV). pectedly more active against bacterial strains that express It was surprisingly found that the compounds of the present OXA-15 -lactamases than the most structurally similar com pound, Avibactam (comparator compound CCC). Com invention have a higher binding affinity for the B-lactamase pounds that are more active than Avibactam against bacterial enzyme than the closest structural comparator Avibactam strains that express the class D cephalosporinase OXA-15 are, (Table IV. See FIG. 4). for example, compounds 701, 702, 703,704, 705, 706, 707, The compounds of the invention were also shown to be 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 719, 720, better BLIs than other comparator compounds as shown in 721, 722, 723, 724, 725, 726, 727, 728, 729,730, 731, 732, 25 FIG.S. 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, Pharmaceutical Compositions Comprising the Compounds 745, 746, 747, 748,749, 750, 751, 752, 753, 754, 755, 756, of the Invention and Use Thereof 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773,774, 776, 777,778, 779, 780, 781, Another object of the invention is pharmaceutical compo 782, 783, 784, 792, 794, 795 and 797. 30 sitions or formulations comprising compounds the invention In one embodiment, the compounds of the invention (e.g. (e.g. compounds of Formula I, compounds of Formula A-I. compounds of Formula I, compounds of Formula A-I, com compounds of Formula II, compounds of Formula A-II), or pounds of Formula II, compounds of Formula A-II) are unex salts thereof, preferably further comprising a B-lactam anti pectedly more active against and/or show broader spectrum biotic. In one embodiment of the invention is pharmaceutical of activity against bacterial strains that express KPC B-lacta 35 compositions or formulations comprising compounds of For mases than the most structurally similar compound, Avibac mula I, or salts thereof, preferably further comprising a 3-lac tam. Compounds that are more active than Avibactam for at tam antibiotic. In one embodiment of the invention is phar least one, bacterial strain that expresses KPC B-lactamase maceutical compositions or formulations comprising and/or show a better spectrum of activity against bacterial compounds of Formula A-I, or salts thereof, preferably fur strains that express KPC B-lactamases than Avibactam are, 40 ther comprising a 3-lactam antibiotic. In one embodiment of for example, compounds 701, 702, 703,705, 706, 708, 709, the invention is pharmaceutical compositions or formulations 710, 711, 712714, 720, 721, 722, 723, 724, 726, 727, 728, comprising compounds of Formula II, or salts thereof, pref 729, 730, 731, 732, 734, 735, 736, 737, 738,739, 740, 741, erably further comprising a B-lactam antibiotic. In one 742, 743, 744, 745, 746, 747, 749, 750, 751, 752, 753, 754, embodiment of the invention is pharmaceutical compositions 755, 756, 758, 759, 760, 761, 762, 763, 764,765, 766, 767, 45 768, 770, 771, 772, 774, 775, 776, 777, 779, 782, 783,784, or formulations comprising compounds of Formula A-II, or 786, 794, and 795. salts thereof, preferably further comprising a B-lactam anti In another aspect of the invention, the compounds of the biotic. In one embodiment of the invention is pharmaceutical invention (e.g. compounds of Formula I, compounds of For compositions or formulations comprising compounds of mula A-I, compounds of Formula II, compounds of Formula 50 Table I. In one embodiment of the invention is pharmaceutical A-II) have high binding affinity for the B-lactamase enzyme. compositions or formulations comprising compounds of For Consequently these compounds are better inhibitors of the mula B-lactamase enzyme. The inhibition kinetics of the com pounds of the invention (e.g. compounds of Formula I, com N-N pounds of Formula A-I, compounds of Formula II, com 55 pounds of Formula A-II) was measured according to the procedure outlined in Example 102. The compounds of the { O >. invention (e.g. compounds of Formula I, compounds of For mula A-I, compounds of Formula II, compounds of Formula N I H A-II) have a high binding affinity for the B-lactamase enzyme. 60 In one embodiment the compounds of the invention (e.g. O/ NOSOH, compounds of Formula I, compounds of Formula A-I, com pounds of Formula II, compounds of Formula A-II) have a binding affinity of 1000-5000 mMs. or salts thereof, preferably further comprising a B-lactam In one embodiment the compounds of the invention (e.g. 65 antibiotic. In one embodiment of the invention is pharmaceu compounds of Formula I, compounds of Formula A-I, com tical compositions or formulations comprising compounds of pounds of Formula II, compounds of Formula A-II) have a Formula US 8,933,232 B2 33 34 eliminate infection (see, e.g., Remington’s Pharmaceutical HN N-N Sciences, Mack Publishing Company, Easton, Pa. and Good Y-Null ly. man and Gilman’s “The Pharmaceutical Basis of Therapeu O tics.” Pergamon Press, New York, N.Y., the contents of which 5 are incorporated herein by reference, for a general description N H of the methods for administering various antimicrobial agents for human therapy). a N The pharmaceutical compositions can comprise one or O OSOH, more of the compounds disclosed herein (e.g. one or more 10 compounds of Formula I, compounds of Formula A-I, com or salts thereof, preferably further comprising a B-lactam pounds of Formula II, compounds of Formula A-II), prefer antibiotic. In one embodiment the invention is pharmaceuti ably a compound of Formula A-I or Formula A-II, in conjunc cal compositions or formulations comprising compounds of tion with a B-lactam antibiotic, in association with one or Formula more nontoxic, pharmaceutically-acceptable carriers and/or 15 diluents and/or adjuvants and/or excipients. As used herein, the phrase “pharmaceutically-acceptable carrier refers to N-N any and all solvents, dispersion media, coatings, antibacterial N and antifungal agents, isotonic and absorption delaying N H N-K y%. O agents, and the like, that are compatible with pharmaceutical 2O administration. The use of Such media and agents for phar N III I IIH maceutically active Substances is well known in the art. Non limiting examples of carriers and excipients include corn A N starch or gelatin, lactose, Sucrose, microcrystalline cellulose, O OSOH, kaolin, mannitol, dicalcium phosphate, sodium chloride and 25 alginic acid. The compositions may contain croScarmellose or salts thereof, preferably further comprising a B-lactam Sodium, microcrystalline cellulose, corn starch, sodium antibiotic. In one embodiment of the invention is pharmaceu starch glycolate and alginic acid. tical compositions or formulations comprising compounds of Tablet binders that can be included are acacia, methylcel Formula lulose, Sodium carboxymethylcellulose, polyvinylpyrroli 30 done (PoVidone), hydroxypropyl methylcellulose, Sucrose, starch and ethylcellulose. N-N Lubricants that can be used include magnesium stearate or other metallic Stearates, Stearic acid, silicone fluid, talc, waxes, oils and colloidal silica. /-y-. Flavoring agents such as peppermint, oil of wintergreen, NO- O N IH 35 cherry flavoring or the like can also be used. It may also be desirable to add a coloring agent to make the dosage form more aesthetic in appearance or to help identify the product. O/ NOSOH, For oral or parenteral administration, compounds of the 40 present invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of or salts thereof, preferably further comprising a B-lactam Formula A-II) preferably a compound of Formula A-I or antibiotic. In one embodiment of the invention is pharmaceu Formula A-II, in conjunction with a B-lactam antibiotic, can tical compositions or formulations comprising compounds of be mixed with conventional pharmaceutical carriers and Formula or 45 excipients and used in the form of tablets, capsules, elixirs, Suspensions, syrups, wafers and the like. The compositions comprising a compound of this invention may contain from N-N about 0.1% to about 99% by weight of the active compound, f y, such as from about 10% to about 30%. HN O 50 For oral use, Solid formulations such as tablets and cap Sules are useful. Sustained release or enterically coated prepa N H rations may also be devised. Forpediatric and geriatric appli cations, one embodiment provides Suspensions, syrups and O/ N OSOH, chewable tablets. For oral administration, the pharmaceutical 55 compositions are in the form of for example, a tablet, cap Sule, Suspension or liquid. or salts thereof, preferably further comprising a B-lactam The pharmaceutical compositions may be made in the form antibiotic. of a dosage unit containing a therapeutically-effective The pharmaceutical compositions can be formulated for amount of the active ingredient. Examples of Such dosage oral, intravenous, intramuscular, Subcutaneous or parenteral 60 units are tablets and capsules. For therapeutic purposes, the administration for the therapeutic or prophylactic treatment tablets and capsules which can contain, in addition to the of diseases, such as bacterial infections. Preferably, the phar active ingredient, conventional carriers such as binding maceutical composition is formulated for intravenous admin agents, for example, acacia gum, gelatin, polyvinylpyrroli istration. done, Sorbitol, or tragacanth; fillers, for example, calcium The pharmaceutical preparations disclosed herein may be 65 phosphate, glycine, lactose, maize-starch, Sorbitol, or prepared in accordance with standard procedures and are Sucrose; lubricants, for example, magnesium Stearate, poly administered at dosages that are selected to reduce, prevent or ethylene glycol, silica, or talc, disintegrants, for example, US 8,933,232 B2 35 36 potato starch, flavoring or coloring agents, or acceptable wet Solid dosage forms for oral administration include cap ting agents. Oral liquid preparations generally are in the form Sules, tablets, pills, powders, and granules. Such forms may of aqueous or oily Solutions, Suspensions, emulsions, syrups include forms that dissolve or disintegrate quickly in the oral or elixirs, preparations of the invention may contain conven environment. In Such solid dosage forms, the active com tional additives such as Suspending agents, emulsifying pound preferably a compound of Formula A-I or Formula agents, non-aqueous agents, preservatives, coloring agents A-II in conjunction with a B-lactam antibiotic, can be mixed and flavoring agents. Non-limiting examples of additives for with at least one inert, pharmaceutically-acceptable excipient liquid preparations include acacia, almond oil, ethyl alcohol, or carrier. Suitable excipients include, for example, (a) fillers fractionated coconut oil, gelatin, glucose syrup, glycerin, or extenders such as starches, lactose, Sucrose, glucose, man hydrogenated edible fats, lecithin, methyl cellulose, methyl 10 nitol, and silicic acid; (b) binders such as cellulose and cel or propyl para-hydroxybenzoate, propylene glycol, Sorbitol, lulose derivatives (such as hydroxypropylmethylcellulose, or Sorbic acid. hydroxypropylcellulose, and carboxymethylcellulose), algi For intravenous (IV) use, the pharmaceutical composition nates, gelatin, polyvinylpyrrolidone. Sucrose, and acacia; (c) (e.g. compounds of Formula I, compounds of Formula A-I. humectants such as glycerol; (d) disintegrating agents such as compounds of Formula II, compounds of Formula A-II) pref 15 Sodium starch glycolate, croscarmellose, agar-agar, calcium erably a compound of Formula A-I or Formula A-II, in con carbonate, potato or tapioca Starch, alginic acid, certain sili junction with a B-lactam antibiotic, can be dissolved or Sus cates, and sodium carbonate; (e) solution retarding agents pended in any of the commonly used intravenous fluids and Such as paraffin, (f) absorption accelerators such as quater administered by infusion. Intravenous fluids include, without nary ammonium compounds; (g)wetting agents, such ascetyl limitation, physiological saline or Ringer's solution. Intrave alcohol and glycerol monostearate, fatty acid esters of Sorbi nous administration may be accomplished by using, without tan, poloxamers, and polyethylene glycols: (h) absorbents limitation, Syringe, mini-pump or intravenous line. Such as kaolin and bentonite clay; (ii) lubricants such as talc, Pharmaceutical compositions of this invention (e.g. com calcium Stearate, magnesium Stearate, Solid polyethylene gly pounds of Formula I, compounds of Formula A-I, compounds cols, sodium lauryl Sulfate, and mixtures thereof, and () of Formula II, compounds of Formula A-II) preferably a 25 glidants such as talc, and silicone dioxide. Other Suitable compound of Formula A-I or Formula A-II, for parenteral excipients include, for example, Sodium citrate or dicalcium injection comprise pharmaceutically-acceptable aqueous or phosphate. The dosage forms may also comprise buffering non-aqueous solutions, dispersions, Suspensions or emul agents. sions as well as sterile powders for reconstitution into sterile Solid dosage forms, including those of tablets, dragees, injectable solutions or dispersions just prior to use. Examples 30 capsules, pills, and granules, can be prepared with coatings of Suitable aqueous and non-aqueous carriers, diluents, Sol and shells such as functional and aesthetic enteric coatings vents or vehicles include water, ethanol, benzyl alcohol, poly and other coatings well known in the pharmaceutical formu ols (such as glycerol, propylene glycol, and polyethylene lating art. They may optionally contain opacifying agents and glycol), and Suitable mixtures thereof, vegetable oils (such as colorants. They may also be in a form capable of controlled or corn oil or olive oil), and injectable organic esters such as 35 Sustained release. Examples of embedding compositions that ethyl oleate. Proper fluidity can be maintained, for example, can be used for Such purposes include polymeric Substances by the use of coating materials such as lecithin, by the main and waxes. tenance of the required particle size in the case of dispersions, The pharmaceutical compositions can be delivered using and by the use of Surfactants. The compositions can include controlled (e.g., capsules) or Sustained release (e.g., bioerod various buffers. 40 able matrices) delivery systems. Exemplary delayed release These compositions may also contain adjuvants such as delivery systems for drug delivery that are suitable for admin preservatives, wetting agents, emulsifying agents, and dis istering the pharmaceutical compositions are described in persing agents. They may also contain taggants or other anti U.S. Pat. No. 4,452,775 (issued to Kent), U.S. Pat. No. 5,039, counterfeiting agents, which are well known in the art. Pre 660 (issued to Leonard), and U.S. Pat. No. 3,854,480 (issued vention of the action of microorganisms may be ensured by 45 to Zaffaroni). the inclusion of various antibacterial and antifungal agents, In some cases, in order to prolong the effect of the drug, it for example, paraben, chlorobutanol, and phenol Sorbic acid. may be desirable to slow the absorption of the drug following It may also be desirable to include isotonic agents such as Subcutaneous or intramuscular injection. This may be accom Sugars and sodium chloride. Prolonged absorption of the plished by the use of a liquid Suspension of crystalline or injectable pharmaceutical form may be brought about by the 50 amorphous material with poor water Solubility. Amorphous inclusion of agents which delay absorption, Such as alumi material may be used alone or together with stabilizers as num monostearate and gelatin. necessary. The rate of absorption of the drug then depends Injectable depot forms can be made by forming microen upon its rate of dissolution, which in turn, may depend upon capsulating matrices of the drug in biodegradable polymers crystal size and crystalline form. Such as polylactide-polyglycolide. Depending upon the ratio 55 Alternatively, delayed absorption of a parenterally admin of drug to polymer and the nature of the particular polymer istered drug form can be accomplished by dissolving or Sus employed, the rate of drug release can be controlled. pending the drug in an oil vehicle. Examples of other biodegradable polymers include poly For intramuscular preparations, a sterile formulation of (orthoesters) and poly(anhydrides). Depot injectable formu compounds, preferably a compound of Formula A-I or For lations can also be prepared by entrapping the drug in lipo 60 mula A-II in conjunction with a B-lactam antibiotic, or Suit Somes or microemulsions, which are compatible with body able soluble salt forms thereof, for example hydrochloride tissues. salts, can be dissolved and administered in a pharmaceutical The injectable formulations can be sterilized, for example, diluent such as Water-for-Injection (WFI), physiological by filtration through a bacterial-retaining filter, or by incor saline or 5% glucose. A suitable insoluble form of the com porating sterilizing agents in the form of sterile solid compo 65 pound may be prepared and administered as a Suspension in sitions, which can be dissolved or dispersed insterile water or an aqueous base or a pharmaceutically acceptable oil base, other sterile injectable medium just prior to use. e.g., an ester of a long chain fatty acid such as ethyl oleate. US 8,933,232 B2 37 38 A dose of an intravenous, intramuscular, or parental for Exemplary procedures for delivering an antibacterial agent mulation of compounds, preferably a compound of Formula are described in U.S. Pat. Nos. 6,468,967; 6,852,689; and A-I or Formula A-II in conjunction with a B-lactamantibiotic, 5,041,567, issued to Rogers and in PCT patent application may be administered as a bolus or by slow infusion. A bolus number EP94/02552 (publication no. WO95/05384), the dis is a dose that is administered in less than 30 minutes. In one closures of which are incorporated herein by reference in their embodiment, a bolus is administered in less than 15 or less entirety. In one embodiment, one or more compounds of the than 10 minutes. In another embodiment, a bolus is adminis invention, preferably a compound of Formula A-I or Formula tered in less than 5 minutes. In yet another embodiment, a A-II in conjunction with a B-lactamantibiotic, or pharmaceu bolus is administered in one minute or less. An infusion is a tical compositions thereofare administered orally, rectally or dose that is administered at a rate of 30 minutes or greater. In 10 via injection (intravenous, intramuscular or Subcutaneous). In one embodiment, the infusion is one hour or greater. In another embodiment, one or more compounds of the inven another embodiment, the infusion is Substantially constant. tion, preferably a compound of Formula A-I or Formula A-II For topical use the pharmaceutical compositions, prefer in conjunction with a B-lactam antibiotic, or pharmaceutical ably a compound of Formula A-I or Formula A-II in conjunc compositions thereof are administered orally, rectally or via tion with a B-lactam antibiotic, can also be prepared in Suit 15 injection (intravenous, intramuscular or Subcutaneous) to able forms to be applied to the skin, or mucus membranes of treat an infection caused by B-lactam resistant bacteria. In the nose and throat, and can take the form of creams, oint another embodiment, one or more compounds of the inven ments, liquid sprays or inhalants, lozenges, or throat paints. tion, preferably a compound of Formula A-I or Formula A-II Such topical formulations further can include chemical com in conjunction with a B-lactam antibiotic, or pharmaceutical pounds such as dimethylsulfoxide (DMSO) to facilitate sur compositions thereofare administered orally to treat an infec face penetration of the active ingredient. tion caused by B-lactamase producing bacteria. For application to the eyes or ears, the pharmaceutical As used herein, the phrases “therapeutically-effective composition can be presented in liquid or semi-liquid form dose” and “therapeutically-effective amount” refer to an formulated in hydrophobic or hydrophilic bases as ointments, amount of a compound that prevents the onset, alleviates the creams, lotions, paints or powders. 25 symptoms, stops the progression of a bacterial infection, or For rectal administration, the pharmaceutical composi results in another desired biological outcome Such as, e.g., tions, preferably a compound of Formula A-I or Formula A-II improved clinical signs or reduced/elevated levels of lympho in conjunction with a B-lactamantibiotic, can be administered cytes and/or antibodies. in the form of Suppositories admixed with conventional car The term “treating” or “treatment' is defined as adminis riers such as cocoa butter, polyethylene glycol or a Supposi 30 tering, to a subject, a therapeutically-effective amount of one tory wax or other glyceride that are solid at room temperature or more compounds both to prevent the occurrence of an but liquid at body temperature and therefore melt in the rec infection and to control or eliminate an infection. Those in tum or vaginal cavity and release the active compound. need of treatment may include individuals already having a Alternatively, the pharmaceutical compositions can be in particular medical disease as well as those at risk for the powderform for reconstitution in the appropriate pharmaceu 35 disease (i.e., those who are likely to ultimately acquire the tically acceptable carrier at the time of delivery. In another disorder). embodiment, the unit dosage form of compounds, preferably The term “subject, as used herein, refers to a mammal, a a compound of Formula A-I or Formula A-II in conjunction plant, a lower animal, or a cell culture. In one embodiment, a with a B-lactam antibiotic, can be a solution of one or more Subject is a human or other animal patient in need of antibac compounds, or salts thereof, in a suitable diluent, in sterile 40 terial treatment. hermetically sealed ampoules or Sterile Syringes. The concen The term “administering or “administration' and the like, tration of the compounds, preferably a compound of Formula refers to providing the compound of the invention (e.g. com A-I or Formula A-II in conjunction with a B-lactamantibiotic, pounds of Formula I, compounds of Formula A-I, compounds in the unit dosage may vary, e.g. from about 1 percent to about of Formula II, compounds of Formula A-II) to the subject in 50 percent, depending on the compound used and its solubil 45 need of treatment. Preferably the subject is a mammal, more ity and the dose desired by the physician. If the compositions preferably a human. The present invention comprises admin contain dosage units, each dosage unit can contain from istering the compound of the invention (e.g. compounds of 1-500 mg of the active material. For adult human treatment, Formula I, compounds of Formula A-I, compounds of For the dosage employed can range from 5 mg to 10 g, per day, mula II, compounds of Formula A-II) in conjunction with a depending on the route and frequency of administration. 50 B-lactamantiobiotic. When a compound of the invention (e.g. The pharmaceutical compositions disclosed herein can be compounds of Formula I, compounds of Formula A-I, com placed in a pharmaceutically acceptable carrier and are deliv pounds of Formula II, compounds of Formula A-II) is admin ered to a recipient Subject (e.g., a human) in accordance with istered in conjunction with a B-lactam antiobiotic, the com known methods of drug delivery. In general, the methods of pound of the invention (e.g. compounds of Formula I, delivering the pharmaceutical compositions in vivo utilize 55 compounds of Formula A-I, compounds of Formula II, com art-recognized protocols for delivering the agent with the pounds of Formula A-II) and the B-lactam antiobiotic can be only substantial procedural modification being the Substitu administered at the same time or different times. When the tion of the compounds of the present invention for the drugs in compounds of the invention (e.g. compounds of Formula I, the art-recognized protocols. Likewise, methods for using the compounds of Formula A-I, compounds of Formula II, com claimed compositions for treating cells in culture, for 60 pounds of Formula A-II) and the B-lactam antiobiotic are example, to eliminate or reduce the level of bacterial contami administered at the same time, they can be administered as a nation of a cell culture, utilize art-recognized protocols for single composition or pharmaceutical composition or they treating cell cultures with antibacterial agent(s) with the only can be administered separately. It is understood that when a substantial procedural modification being the substitution of compound of the invention (e.g. compounds of Formula I, the compounds of the present invention, preferably in com 65 compounds of Formula A-I, compounds of Formula II, com bination with a B-lactam antibiotic for the drugs in the art pounds of Formula A-II) is administered in conjunction with recognized protocols. a 3-lactam antibiotic, that the active agents can be adminis US 8,933,232 B2 39 40 tered in a single combination or in multiple combinations. For preventing a bacterial infection comprising administering to a example, when administered by IV, the compound of the subject in need thereof, a therapeutically-effective amount of invention (e.g. compounds of Formula I, compounds of For a B-lactam antibiotic in conjunction with a compound of mula A-I, compounds of Formula II, compounds of Formula Formula A-II. In one embodiment of the invention, is pro A-II) can be dissolved or Suspended in any of the commonly vided a method of treating or preventing a bacterial infection used intravenous fluids and administered by infusion, then a comprising administering to a Subject in need thereofathera B-lactam antibiotic can be dissolved or Suspended in any of peutically-effective amount of a B-lactam antibiotic in con the commonly used intravenous fluids and administered by junction with a compound of Table I. In one embodiment of infusion. Conversely the B-lactam antibiotic can be dissolved the invention, is provided a method of treating or preventing or Suspended in any of the commonly used intravenous fluids 10 a bacterial infection comprising administering to a subject in and administered by infusion, then a compound of Formula I can be dissolved or Suspended in any of the commonly used need thereof, atherapeutically-effective amount of a B-lactam intravenous fluids and administered by infusion. Alterna antibiotic in conjunction with a compound of Formula tively, a pharmaceutical composition comprising a compound of the invention (e.g. compounds of Formula I, compounds of 15 Formula A-I, compounds of Formula II, compounds of For N-N mula A-II) and a B-lactam antibiotic can be dissolved or 4 y. Suspended in any of the commonly used intravenous fluids O and administered by infusion. N . I I IIH In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising A N administering to a subject in need thereof a therapeutically O OSOH. effective amount of the pharmaceutical composition compris ing a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, 25 In one embodiment of the invention, is provided a method of compounds of Formula A-II) and a B-lactamantibiotic. In one treating or preventing a bacterial infection comprising admin embodiment of the invention, is provided a method of treating istering to a subject in need thereof, a therapeutically-effec or preventing a bacterial infection comprising administering tive amount of a B-lactam antibiotic in conjunction with a to a subject in need thereofatherapeutically-effective amount compound of Formula of the pharmaceutical composition comprising a compound 30 of Formula I, and a B-lactamantibiotic. In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering to a subject in need thereofatherapeutically-effective amount of the phar HN N-N maceutical composition comprising a compound of Formula 35 A-I, and a B-lactam antibiotic. In one embodiment of the invention, is provided a method of treating or preventing a Y-Nuk O >. bacterial infection comprising administering to a Subject in N IIH need thereofatherapeutically-effective amount of the phar maceutical composition comprising a compound of Formula 40 A N II, and a B-lactamantibiotic. In one embodiment of the inven O OSOH. tion, is provided a method of treating or preventing a bacterial infection comprising administering to a Subject in need thereofatherapeutically-effective amount of the pharmaceu tical composition comprising a compound of Formula A-II, 45 and a B-lactam antibiotic. In one embodiment of the invention, is provided a method of In one embodiment of the invention, is provided a method treating or preventing a bacterial infection comprising admin of treating or preventing a bacterial infection comprising istering to a subject in need thereof, a therapeutically-effec administering to a subject in need thereof, a therapeutically tive amount of a B-lactam antibiotic in conjunction with a effective amount of a B-lactamantibiotic in conjunction with 50 a compound of the invention (e.g. compounds of Formula I, compound of Formula compounds of Formula A-I, compounds of Formula II, com pounds of Formula A-II). In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering to a Subject in need 55 H %. thereof, atherapeutically-effective amount of a B-lactamanti Y-Nu? O y biotic in conjunction with a compound of Formula I. In one N II I I IIH embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering a N to a subject in need thereof, a therapeutically-effective 60 O OSOH. amount of a B-lactam antibiotic in conjunction with a com pound of Formula A-I. In one embodiment of the invention, is provided a method of treating or preventing a bacterial infec In one embodiment of the invention, is provided a method of tion comprising administering to a subject in need thereof, a treating or preventing a bacterial infection comprising admin therapeutically-effective amount of a B-lactam antibiotic in 65 istering to a subject in need thereof, a therapeutically-effec conjunction with a compound of Formula II. In one embodi tive amount of a B-lactam antibiotic in conjunction with a ment of the invention, is provided a method of treating or compound of Formula US 8,933,232 B2 41 42 In one embodiment the compound in step a is a compound of Formula

NO- NH N . I I IIH N N-N

NN-K O >. O !. N OSOH. N II I I IIH 10 In one embodiment of the invention, is provided a method of / N treating or preventing a bacterial infection comprising admin O OSOH. istering to a Subject in need thereof, a therapeutically-effec tive amount of a B-lactam antibiotic in conjunction with a 15 In one embodiment the compound in step a is a compound of compound of Formula Formula N-Ny. O . HN N I IIH NO-M. N H / N O OSOH. 25 !' O OSOH. In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection in a subject In one embodiment the compound in step a is a compound of comprising the steps of Formula a. administering to the Subject a compound of the inven 30 tion; and b. administering to the subject a therapeutically-effective amount of a B-lactam antibiotic. In one embodiment the compound in Stepa is a compound of Formula I. In one embodiment the compound in Stepa is a 35 compound of Formula A-I. In one embodiment the compound in Stepa is a compound of Formula II. In one embodiment the compound in Stepa is a compound of Formula A-II. In one embodiment the compound in Stepa is a compound of Table I. In one embodiment the compound in Stepa is a compound 40 of Formula A-II. In one embodiment the compound in step a is a compound of Formula

45 N-N Cy. In one embodiment, the B-lactam antibiotic in step b is O * Ceftolozane, Ceftazidime, Aztreonam or Meropenem, or N IH more preferably, Ceftolozane or Ceftazidime. 50 In one embodiment the compound in Stepa is a compound 2 N of Formula O OSOH

In one embodiment the compound in step a is a compound of 55 Formula

60 O '.

N H O OSOH

O 1. Yoso,H. 65 and the B-lactam antibiotic in step b is Ceftolozane. In one embodiment the compound in step a is a compound of For mula US 8,933,232 B2 43 44 In one embodiment the compound in step b is a compound HN of Formula I. In one embodiment the compound in step b is a compound of Formula A-I. In one embodiment the compound in step b is a compound of Formula II. In one embodiment the compound in step b is a compound of Formula A-II. In one embodiment the compound in step b is a compound of For mula II. In one embodiment the compound in step b is a compound of Table I. In one embodiment the compound in step b is a compound of Formula 10 O OSOH NN N and the f-lactam antibiotic in step b is Ceftolozane. In one embodiment the compound in Stepa is a compound ( -- of Formula 15 N H

N O OSOH,

In one embodiment the compound in step b is a compound of Formula N II I I IIH 25 N HN O OSOH and the f-lactam antibiotic in step b is Ceftolozane. 30 In one embodiment the compound in Stepa is a compound of Formula N . I IIH

N 35 O OSOH.

In one embodiment the compound in step b is a compound of N . I IIH Formula 40

O OSOH and the f-lactam antibiotic in step b is Ceftolozane. 45 In one embodiment the compound in Stepa is a compound of Formula N II I I IIH

N 50 V O OSOH.

In one embodiment the compound in step b is a compound of Formula 55

O OSOH /NNN and the f-lactam antibiotic in step b is Ceftolozane. 60 In one embodiment of the invention, is provided a method NO- A { J%, of treating or preventing a bacterial infection in a subject N . . . H comprising the steps of a. administering to the Subject a therapeutically-effective O OSOH. amount of a B-lactam antibiotic; and 65 b. administering to the Subject a compound of the inven tion. US 8,933,232 B2 45 46 In one embodiment the compound in step b is a compound of In one embodiment the compound in step b is a compound Formula of Formula

OSOH. O OSOH

In one embodiment, the B-lactam antibiotic in step a is 15 and the B-lactam antibiotic in Stepa is Ceftolozane. Ceftolozane, Ceftazidime, Aztreonam or Meropenem, or In one embodiment the compound in step b is a compound more preferably, Ceftolozane or Ceftazidime. In one embodi of Formula ment the compound in step b is a compound of Formula

NN {jN O '. 25 N . . . H

O OSOH 30 and the B-lactam antibiotic in step a is Ceftolozane. In one embodiment the compound in step b is a compound of For mula 35

HN and the B-lactam antibiotic in Stepa is Ceftolozane. In one embodiment, the invention provides a method for treating an infection in a subject by administering a therapeu ( 40 tically-effective amount of one or more compounds of the l'', invention (e.g. compounds of Formula I, compounds of For mula A-I, compounds of Formula II, compounds of Formula N . I I IIH A-II), preferably a compound of Formula A-I or Formula A-II N in conjunction with a B-lactam antibiotic, or compositions 45 thereof. In one embodiment, the method comprises adminis O OSOH tering to a subject in need thereof a pharmaceutical compo sition comprising at least one of the compounds described and the B-lactam antibiotic in Stepa is Ceftolozane. herein, preferably a compound of Formula A-I or Formula 50 A-II in conjunction with a B-lactamantibiotic. In one embodi ment the compound is of Formula In one embodiment the compound in step b is a compound of Formula

N H

N 60 O OSOH,

O OSOH in conjunction with a B-lactamantibiotic, preferably Ceftolo 65 Zane, Ceftazidime, AZtreonam or Meropenem, or more pref erably, Ceftolozane or Ceftazidime, or compositions thereof. and the B-lactam antibiotic in Stepa is Ceftolozane. In one embodiment the compound is of Formula US 8,933,232 B2 47 48 pound, composition, or biological material. The compound HN may be administered orally, parenterally, by inhalation, topi cally, rectally, nasally, buccally, vaginally, or by an implanted reservoir, external pump or catheter. The compound may be prepared for opthalmic or aerosolized uses. The compounds of the present invention can be administered as an aerosol for the treatment of pneumonia or other lung-based infections. In one embodiment, the aerosol delivery vehicle is an anhydrous or dry powder inhaler. One or more compounds of the inven 10 tion (e.g. compounds of Formula I, compounds of Formula O OSOH, A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II in in conjunction with a B-lactamantibiotic, preferably Ceftolo conjunction with a B-lactam antibiotic, or pharmaceutical Zane, Ceftazidime, AZtreonam or Meropenem, or more pref compositions thereof also may be directly injected or admin erably, Ceftolozane or Ceftazidime, or compositions thereof. 15 istered into an abscess, Ventricle or joint. Parenteral admin In one embodiment the compound is of Formula istration includes Subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, cisternal, intrathecal, intrahe patic, intralesional and intracranial injection or infusion. In one embodiment, one or more compounds of the invention NN (e.g. compounds of Formula I, compounds of Formula A-I. Y N compounds of Formula II, compounds of Formula A-II), pref ( erably a compound of Formula A-I or Formula A-II in con o-1", junction with a B-lactam antibiotic, are administered intrave nously, Subcutaneously or orally. In one embodiment for N II I I IIH 25 administering one or more compounds according to the invention (e.g. compounds of Formula I, compounds of For N mula A-I, compounds of Formula II, compounds of Formula O OSOH, A-II), preferably a compound of Formula A-I or Formula A-II in conjunction with a B-lactamantibiotic to a cell culture, the 30 one or more compounds may be administered in a nutrient in conjunction with a B-lactamantibiotic, preferably Ceftolo medium. Zane, Ceftazidime, AZtreonam or Meropenem, or more pref In one embodiment, one or more compounds according to erably, Ceftolozane or Ceftazidime, or compositions thereof. the invention (e.g. compounds of Formula I, compounds of In one embodiment the compound is of Formula Formula A-I, compounds of Formula II, compounds of For 35 mula A-II), preferably a compound of Formula A-I or A-II in conjunction with a B-lactamantibiotic, may be used to treat a subject having a bacterial infection in which the infection is caused or exacerbated by any type of bacteria, Such as Gram negative bacteria. In one aspect of the invention, the bacterial 40 infection is caused by B-lactam resistant bacteria. In one N I I IIH aspect the bacterial infection is caused by B-lactamase pro N ducing bacteria. In another aspect the bacterial infection is caused by class A, class C or class D B-lactamase producing O OSOH, bacteria. In another aspect the bacterial infection is caused by 45 class A B-lactamase producing bacteria. In another aspect the in conjunction with a B-lactamantibiotic, preferably Ceftolo infection is caused by class C B-lactamase producing bacte Zane, Ceftazidime, AZtreonam or Meropenem, or more pref ria. In still another aspect the infection is caused by class D erably, Ceftolozane or Ceftazidime, or compositions thereof. B-lactamase producing bacteria. In still another aspect the In one embodiment the compound is of Formula infection is caused by KPC B-lactamase producing bacteria. 50 In still another aspect the infection is caused by OXA B-lac tamase producing bacteria. In still another aspect, the bacte rial infection is caused by a bacteria that produces multiple B-lactamases. Bacteria that produce multiple B-lactamases may produce B-lactamases of the same class or of different 55 classes (e.g. class A and class A or class A and class C or class A and class D etc). Representative Gram-negative pathogens known to express B-lactamases include, but are not limited to Acineto O OSOH, bacter spp. (including Acinetobacter baumannii), Citro 60 bacter spp., Escherichia spp. (including Escherichia coli), Haemophilus influenzae, Morganella morganii, Pseudomo in conjunction with a B-lactamantibiotic, preferably Ceftolo nas aeruginosa, Klebsiella spp. (including Klebsiella pneu Zane, Ceftazidime, AZtreonam or Meropenem, or more pref moniae), Enterobacter spp. (including Enterobacter cloacae erably, Ceftolozane or Ceftazidime, or compositions thereof. and Enterobacter aerogenes), Pasteurella spp., Proteus spp. In one embodiment, the pharmaceutical composition can 65 (including Proteus mirabilis), Serratia spp. (including Serra comprise any one of the compounds described herein as the tia marcescens), and Providencia spp. Bacterial infections sole active compound or in combination with another com can be caused or exacerbated by Gram-negative bacteria US 8,933,232 B2 49 50 including strains which express B-lactamases that may confer used to treat a bacterial infection of any organ or tissue in the resistance to penicillins, cephalosporins, monobactams and/ body caused by B-lactam resistant bacteria, preferably, Gram or carbapenems. The co-administration of a novel BLI that negative B-lactam resistant bacteria. These organs or tissue inhibits these B-lactamases with a B-lactam antibiotic could include, without limitation, skeletal muscle, skin, blood be used to treat infections caused by B-lactam resistant bac 5 stream, kidneys, heart, lung and bone. For example, a phar teria. maceutical composition comprising at least one compound of In one aspect of the invention the infection is caused by a the invention (e.g. compounds of Formula I, compounds of B-lactamase producing bacteria selected from Acinetobacter Formula A-I, compounds of Formula II, compounds of For spp., Citrobacter spp., Escherichia coli, Enterobacter cloa mula A-II), preferably a compound of Formula A-I or For cae), Haemophilus influenzae, Pseudomonas aeruginosa, 10 mula A-II in conjunction with a B-lactam antibiotic, can be Proteus mirabilis, Serratia marcescens, and Klebsiella pneu administered to a Subject to treat, without limitation, skin and moniae, Soft tissue infections (e.g., complex skin infections), bacter B-Lactam antibiotics that may be administered concur emia, intra-abdominal infections and urinary tract infections rently with compounds of the invention (e.g. compounds of (e.g., cUTI). In addition, a compound of the invention (e.g. Formula I, compounds of Formula A-I, compounds of For 15 compounds of Formula I, compounds of Formula A-I, com mula II, compounds of Formula A-II) include, but are not pounds of Formula II, compounds of Formula A-II) may be limited to cephalosporin, carbapenem, monobactam, penem used to treat community acquired respiratory infections, and penicillin classes of antibiotics. including, without limitation, otitis media, sinusitis, chronic In one embodiment of the invention, the B-lactam antibi bronchitis and pneumonia (including community-acquired otic is a cephalosporin. Examples of cephalosporins include, pneumonia, hospital-acquired pneumonia and ventilator but are not limited to, Cefacetrile (cephacetrile), Cefadroxil associated pneumonia), including pneumonia caused by (cefadroxyl), Cefalexin (cephalexin), Cefaloglycin (cepha drug-resistant Pseudomonas aeruginosa. At least one com loglycin), Cefalonium (cephalonium), Cefaloridine (cepha pound of the invention (e.g. compounds of Formula I, com loradine), Cefalotin (cephalothin), Cefapirin (cephapirin), pounds of Formula A-I, compounds of Formula II, com Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (cephazolin), 25 pounds of Formula A-II), preferably a compound of Formula Cefradine (cephradine), Cefroxadine, Ceftezole, Cefaclor, A-I or Formula A-II in conjunction with a B-lactamantibiotic, Cefamandole, Cefnmetazole, Cefonicid, Cefotetan, Cefoxitin, can be administered to a subject to treat mixed infections that Cefprozil (cefproxil), Cefuroxime, CefuZonam, Cefcapene, comprise different types of Gram-negative bacteria, or which Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, comprise both Gram-positive and Gram-negative bacteria. Cefnmenoxime, Cefodizime, Cefotaxime, Ce?pimizole, Cef 30 These types of infections include intra-abdominal infections podoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, and obstetrical/gynecological infections. At least one com Ceftizoxime, Ceftriaxone, CefoperaZone, Ceftazidime, Cef pound of the invention (e.g. compounds of Formula I, com clidine, Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cef pounds of Formula A-I, compounds of Formula II, com pirome, Cefquinome, CefaclomeZine, Cefaloram, Cefap pounds of Formula A-II), preferably a compound of Formula arole, Cefcanel, Cefedrolor, Cefenpidone, Cefetrizole, 35 A-I or Formula A-II in conjunction with a B-lactamantibiotic, Cefivitril, Cefnmatilen, Cefnepidium, Cefovecin, Cefox may also be administered to a Subject to treat an infection azole, Cefrotil, Cefsumide, Ceftaroline, Ceftioxide, Cefu including, without limitation, endocarditis, nephritis, septic racetime, cefbuperaZone, cefiminox, ceforanide, cefotiam, arthritis, intra-abdominal sepsis, bone and joint infections ce?piramide, cefsulodin, ceftobiprole latamoxef, loracarbef and osteomyelitis. At least one compound of the invention and Ceftolozane. In one embodiment the cephalosporin is 40 (e.g. compounds of Formula I, compounds of Formula A-I. Ceftolozane or Ceftazidime. compounds of Formula II, compounds of Formula A-II), pref In one embodiment of the invention, the B-lactam antibi erably compound of Formula A-I or Formula A-II in conjunc otic is a carbapenen. Examples of carbapenem antibiotics tion with a B-lactam antibiotic, or pharmaceutical composi include, but are not limited to, Imipenem, Imipenem/Cilasta tions thereof, may also be directly injected or administered tin, Biapenem, Doripenem, Meropenem, Ertapenem and 45 into an abscess, Ventricle or joint. Pharmaceutical composi Panipenem. In one embodiment the Carbapenem is Imi tions of the invention (e.g. compounds of Formula I, com penem/Cilastatin or Meropenem. pounds of Formula A-I, compounds of Formula II, com In one embodiment of the invention, the B-lactam antibi pounds of Formula A-II), preferably compound of Formula otic is a monobactam. Examples of monobactam antibiotics A-I or Formula A-II in conjunction with a B-lactamantibiotic, include, but are not limited to Aztreonam, Tigemonam, Caru 50 may be administered as an aerosol for the treatment of pneu monam, BAL30072 and Nocardicin A. monia or other lung-based infections. In one embodiment, the In one embodiment of the invention, the B-lactam antibi aerosol delivery vehicle is an anhydrous, liquid or dry powder otic is a penem. In one embodiment of the invention, the inhaler. B-lactam antibiotic is a penicillin. Examples of penicillin Actual dosage levels of active ingredients in the pharma antibiotics include, but are not limited to Amoxicillin, Ampi 55 ceutical compositions of one or more compounds according cillin, AZlocillin, Mezlocillin, Apalcillin, Hetacillin, to the invention (e.g. compounds of Formula I, compounds of Becampicillin, Carbenicillin, Sulbenicillin, Ticarcillin, Pip Formula A-I, compounds of Formula II, compounds of For eracillin, AZlocillin, Mecillinam, Pivmecillinam, Methicillin, mula A-II), preferably a compound of Formula A-I or For Ciclacillin, Talampicillin, Aspoxicillin, Oxacillin, Cloxacil mula A-II in conjunction with a 3-lactam antibiotic, may be lin, Dicloxacillin, Flucloxacillin, Nafcillin and Pivampicillin. 60 varied so as to obtain a therapeutically-effective amount of In one embodiment the cephalosporin is Ceftolozane, the active compound(s) to achieve the desired therapeutic Ceftazidime, Aztreonam or Meropenem, or more preferably, response for a particular patient, compositions, and mode of Ceftolozane or Ceftazidime. administration. The effective amount can be determined as The pharmaceutical compositions, preferably a compound described herein. The selected dosage level will depend upon of the invention (e.g. compounds of Formula I, compounds of 65 the activity of the particular compound, the route of admin Formula A-I, compounds of Formula II, compounds of For istration, the severity of the condition being treated, and the mula A-II) in conjunction with a 3-lactam antibiotic, can be condition and prior medical history of the patient being US 8,933,232 B2 51 52 treated. However, it is within the skill of the art to start doses pounds of Formula A-II). In one embodiment the ratio is 1:4. of the compound at levels lower than required to achieve the In another embodiment the ratio is 3:4. In another embodi desired therapeutic effect and to gradually increase the dos ment the ratio is 5:4. In another embodiment the ratio is 7:4. age until the desired effect is achieved. In one embodiment, In another embodiment the ratio is 1:2. In another embodi the data obtained from the assays can be used in formulating 5 ment the ratio is 3:2. In another embodiment the ratio is 5:2. a range of dosage for use in humans. It will be understood by In another embodiment the ratio is 7:2. In another embodi one of skill in the art that the when the composition comprises ment the ratio is 1:3. In another embodiment the ratio is 2:3. a compound of the invention (e.g. compounds of Formula I, In another embodiment the ratio is 4:3. In another embodi compounds of Formula A-I, compounds of Formula II, com ment the ratio is 5:3. In another embodiment the ratio is 7:3. pounds of Formula A-II) and a B-lactam antibiotic, both the 10 In another embodiment the ratio is 1:2. In another embodi compound of the invention (e.g. compounds of Formula I, ment the ratio is 3:2. In another embodiment the ratio is 5:2. compounds of Formula A-I, compounds of Formula II, com In another embodiment the ratio is 7:2. In another embodi pounds of Formula A-II) and the B-lactamantibiotic are active ment the ratio is 1:1. In another embodiment the ratio is 2:1. compounds. In another embodiment the ratio is 3:1. In another embodi The method comprises administering to the Subject an 15 ment the ratio is 4:1. In another embodiment the ratio is 5:1. effective dose of one or more compounds of the invention In another embodiment the ratio is 6:1. In another embodi (e.g. compounds of Formula I, compounds of Formula A-I. ment the ratio is 7:1. In another embodiment the ratio is 8:1. compounds of Formula II, compounds of Formula A-II), pref It will be understood by one of skill in the art that the B-lactam erably in conjunction with a Blactamantibiotic. An effective antibiotic and compound of the invention (e.g. compounds of dose of a compound of the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of For Formula I, compounds of Formula A-I, compounds of For mula II, compounds of Formula A-II) can be administered mula II, compounds of Formula A-II) is generally between within the range of ratios provided regardless of the method 125 mg/day to 2000 mg/day. In one embodiment, an effective of drug delivery. It will also be understood by one of skill in dose is from about 0.1 to about 100 mg/kg of one or more the art that the B-lactam antibiotic and compound of the compounds of the invention (e.g. compounds of Formula I, 25 invention (e.g. compounds of Formula I, compounds of For compounds of Formula A-I, compounds of Formula II, com mula A-I, compounds of Formula II, compounds of Formula pounds of Formula A-II) orpharmaceutically acceptable salts A-II) can be administered within the range of ratios provided thereof. In one embodiment, the dose is from about 0.1 to together, for example, in a pharmaceutical composition, or about 50 mg/kg of one or more compounds of the invention sequentially, i.e. the B-lactam antibiotic is administered, fol (e.g. compounds of Formula I, compounds of Formula A-I. 30 lowed by administration of a compound of the invention (e.g. compounds of Formula II, compounds of Formula A-II) or compounds of Formula I, compounds of Formula A-I, com pharmaceutically acceptable salts thereof. In another pounds of Formula II, compounds of Formula A-II) or vice embodiment, the dose is from about 1 to about 25 mg/kg of WSa. one or more compounds of the invention (e.g. compounds of One or more compounds of the invention (e.g. compounds Formula I, compounds of Formula A-I, compounds of For 35 of Formula I, compounds of Formula A-I, compounds of mula II, compounds of Formula A-II) or pharmaceutically Formula II, compounds of Formula A-II) may also be admin acceptable salts thereof. In another embodiment, the dose is istered in the diet or feed of a patient or animal. If adminis from about 1 to about 12 mg/kg of one or more compounds of tered as part of a total dietary intake, the amount of compound the invention (e.g. compounds of Formula I, compounds of employed can be less than 1% by weight of the diet, such as Formula A-I, compounds of Formula II, compounds of For 40 no more than 0.5% by weight. The diet for animals can be mula A-II). In another embodiment, the dose is about 1, 2, 3, normal foodstuffs to which the compound can be added or it 4, 5, 6, 7, 8, 9, 10, 11, or 12 mg/kg of one or more compounds can be added to a premix. of the invention (e.g. compounds of Formula I, compounds of One or more compounds of the invention (e.g. compounds Formula A-I, compounds of Formula II, compounds of For of Formula I, compounds of Formula A-I, compounds of mula A-II). In another embodiment, the compounds of the 45 Formula II, compounds of Formula A-II), preferably a com invention (e.g. compounds of Formula I, compounds of For pound of Formula A-I or Formula A-II in conjunction with a mula A-I, compounds of Formula II, compounds of Formula B-lactamantibiotic, can be administered as a single daily dose A-II) are administered to a human at a dose of 100 mg to 1000 or in multiple doses per day. In one embodiment, one or more mg per dose up to four times per day. In another embodiment, compounds of the invention (e.g. compounds of Formula I, the compounds of the invention (e.g. compounds of Formula 50 compounds of Formula A-I, compounds of Formula II, com I, compounds of Formula A-I, compounds of Formula II, pounds of Formula A-II), preferably a compound of Formula compounds of Formula A-II) are administered to a human at A-I or Formula A-II in conjunction with a B-lactamantibiotic, a dose of 125 mg to 750 mg per dose up to four times per day. is administered as a single dose per day. In another embodi In another embodiment, the compounds of the invention (e.g. ment, one or more compounds of the invention (e.g. com compounds of Formula I, compounds of Formula A-I, com 55 pounds of Formula I, compounds of Formula A-I, compounds pounds of Formula II, compounds of Formula A-II) are of Formula II, compounds of Formula A-II), preferably a administered to a human at a dose of 250 mg to 500 mg per compound of Formula A-I of Formula A-II in conjunction dose up to four times a day. An effective dose for cell culture with a B-lactam antibiotic is administered as two equal doses is usually between about 0.1 and about 1000 ug/mL. In one per day. In another embodiment, the compounds of the inven embodiment, the effect dose for cell culture is between about 60 tion (e.g. compounds of Formula I, compounds of Formula 0.1 and about 200 g/mL. A-I, compounds of Formula II, compounds of Formula A-II), In one embodiment, a B-lactam antibiotic and a compound preferably a compound of Formula A-I or Formula A-II in of the invention (e.g. compounds of Formula I, compounds of conjunction with a 3-lactam antibiotic is administered in Formula A-I, compounds of Formula II, compounds of For three equal doses per day. In another embodiment, the com mula A-II) are administered in ratio of 1:4 to 8:1 antibiotic: 65 pounds of the invention (e.g. compounds of Formula I, com compound of the invention (e.g. compounds of Formula I, pounds of Formula A-I, compounds of Formula II, com compounds of Formula A-I, compounds of Formula II, com pounds of Formula A-II), preferably a compound of Formula US 8,933,232 B2 53 54 A-I or Formula A-II in conjunction with a B-lactamantibiotic mula A-II), preferably a compound of Formula A-I or For is administered in four equal doses per day. The treatment mula A-II and at least 1 carbapenem antibiotic or a pharma regime may require administration over extended periods of ceutically acceptable salt thereof. time, e.g., for several days or for from two to four weeks. The A pharmaceutical composition comprising a compound of amount per administered dose or the total amount adminis the invention (e.g. compounds of Formula I, compounds of tered will depend on such factors as the nature and severity of Formula A-I, compounds of Formula II, compounds of For the infection, the age and general health of the patient, the mula A-II), preferably a compound of Formula A-I or For tolerance of the patient to the compound of the invention and mula A-II and at least 1 monobactam antibiotic or a pharma the B-lactam antibiotic and the microorganism or microor ceutically acceptable salt thereof. ganisms involved in the infection. The treatment regimen for 10 The embodiments described herein provide compounds of one type of infection may differ greatly from the treatment the invention (e.g. compounds of Formula I, compounds of regimen of another infection. For example, one type of infec Formula A-I, compounds of Formula II, compounds of For tion may require administration via intravenous administra mula A-II), preferably a compound of Formula A-I or For tion once daily, while another infection may require a treat mula A-II that are novel and active B-lactamase inhibitors. ment regimen of multiple dosing orally. 15 Other embodiments described herein provide novel com One or more compounds of the invention (e.g. compounds pounds of the invention (e.g. compounds of Formula I, com of Formula I, compounds of Formula A-I, compounds of pounds of Formula A-I, compounds of Formula II, com Formula II, compounds of Formula A-II), preferably a com pounds of Formula A-II), preferably a compound of Formula pound of Formula A-I or Formula A-II in conjunction with a A-I or Formula A-II in conjunction with B-lactam antibiotics B-lactam antibiotic, may be administered according to this for treatment of infections. Further embodiments described method until the bacterial infection is eradicated or reduced. herein provide novel compounds of the invention (e.g. com In one embodiment, one or more compounds of the invention pounds of Formula I, compounds of Formula A-I, compounds (e.g. compounds of Formula I, compounds of Formula A-I. of Formula II, compounds of Formula A-II), preferably a compounds of Formula II, compounds of Formula A-II), pref compound of Formula A-I or Formula A-II that show unex erably a compound of Formula A-I or Formula A-II in con 25 pected activity against B-lactamases that other compounds in junction with a B-lactam antibiotic, are administered for a the class do not have. period of time from 3 days to 6 months. In another embodi Preparation of Compounds of the Invention ment, one or more compounds of the invention (e.g. com A compound of the invention (e.g. compounds of Formula pounds of Formula I, compounds of Formula A-I, compounds I, compounds of Formula A-I, compounds of Formula II, of Formula II, compounds of Formula A-II), preferably a 30 compounds of Formula A-II) can be prepared by a variety of compound of Formula A-I or Formula A-II in conjunction synthetic routes, including synthetic schemes described with a B-lactam antibiotic, are administered for 7 to 56 days. herein. These synthetic routes can be applied to large scale In another embodiment, one or more compounds of the inven synthesis with appropriate adjustment of reaction sequence, tion (e.g. compounds of Formula I, compounds of Formula reaction conditions, isolation/purification methods and A-I, compounds of Formula II, compounds of Formula A-II), 35 choice of solvents which are environmentally friendly and preferably a compound of Formula A-I or Formula A-II in cost-effective. conjunction with a B-lactamantibiotic, are administered for 7 The following abbreviations have the following meanings to 28 days. In a further embodiment, one or more compounds unless otherwise indicated. Abbreviations not defined below of the invention (e.g. compounds of Formula I, compounds of have their generally accepted meaning Formula A-I, compounds of Formula II, compounds of For 40 Bn=benzyl mula A-II), preferably a compound of Formula A-I or For Boc=tert-butoxycarbonyl mula A-II in conjunction with a B-lactam antibiotic, are BocO-di-tert-butyldicarbonate administered for 7 to 14 days. Compounds of the present Burgess reagent-methyl N-triethylammoniumsulfonyl) invention may be administered for a longer or shorter time carbamate period if it is so desired. 45 CDI-carbonyldiimidazole Other embodiments of the invention include: CFU=colony-forming units A pharmaceutical composition comprising a compound of CLSI-Clinical Laboratory Standards Institute the invention (e.g. compounds of Formula I, compounds of cSSSI-complicated skin and skin structure infections Formula A-I, compounds of Formula II, compounds of For DBU=1,8-diazabicyclo[5.4.0]undec-7-ene mula A-II), preferably a compound of Formula A-I or For 50 DCM-dichloromethane mula A-II and at least 1 B-lactam antibiotic or a pharmaceu DEAD-diethylazodicarboxylate tically acceptable salt thereof. DIAD-diisopropyl azodicarboxylate A pharmaceutical composition comprising a compound of DIPEA-diisopropylethylamine the invention (e.g. compounds of Formula I, compounds of DMF=N,N-dimethylformamide Formula A-I, compounds of Formula II, compounds of For 55 DMAc-N,N-dimethylacetamide mula A-II), preferably a compound of Formula A-I or For DMSO-dimethyl sulfoxide mula A-II and at least 1 cephalosporin antibiotic or a phar EDCI=1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide maceutically acceptable salt thereof. ELSD evaporative light scattering detector A pharmaceutical composition comprising a compound of EtOAc-ethyl acetate the invention (e.g. compounds of Formula I, compounds of 60 ESI-MS-electrospray ionization mass spectrometry Formula A-I, compounds of Formula II, compounds of For Fmoc-Fluorenylmethyloxycarbonyl mula A-II), preferably a compound of Formula A-I or For HAP=Hospital-Acquired Pneumonia mula A-II and Ceftolozane or a pharmaceutically acceptable HATU-2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetram salt thereof. ethyluronium hexafluorophosphate A pharmaceutical composition comprising a compound of 65 HCl–hydrochloride the invention (e.g. compounds of Formula I, compounds of HOBt=1-hydroxybenzotrizole Formula A-I, compounds of Formula II, compounds of For Hrs=hours US 8,933,232 B2 55 56 HPLC-high performance liquid chromatography The compounds of the invention (e.g. compounds of For Hunig's base-N,N-Diisopropylethylamine mula I, compounds of Formula A-I, compounds of Formula Lawesson’s reagent 2.4-bis(4-methoxyphenyl)-1,3,2,4- II, compounds of Formula A-II) can be prepared from inter dithiadiphosphetane-2,4-disulfide mediate 1, according to the following reaction schemes and MIC-minimum inhibitory concentration 5 mL milliliter examples, or modifications thereof, using readily available MS-mass spectrometry starting materials, reagents and conventional synthetic pro MRSA methicillin-resistant Staphylococcus aureus cedures including, for example, procedures described in U.S. NMR nuclear magnetic resonance Pat. No. 7,112,592 and WO2009/091856. Ns-nitrobenzenesulfonyl As depicted in Scheme 1, compound 3 can be synthesized Pa Pseudomonas aeruginosa 10 following standard oxadiazole ring formation chemistry from Prep preparative the diacylhydrazide intermediate 2g (see, e.g., Jakopin, Z.; Ppm parts per million Dolenc, M. S. Curr. Org. Chem. 2008, 12, 850-898; Walker, Py-pyridine D. G.; Brodfuehrer, P. R.; Brundidge, S. P. Shih, K. M.: sat. Saturated rt-room temperature 15 Sapino, C.Jr. J. Org. Chem. 1988, 53,983-991 and references TBAF-tetrabutylammonium fluride cited therein). Diacylhydrazide intermediate 2g can be pre TBS-t-butyldimethylsilyl pared via Standard amide coupling reactions from acylhy TES-triethylsilyl drazide intermediate 2f. Alternatively, 2g can be made by an TEA triethylamine amide coupling reaction of acylhydrazine derivative 2h and TEMPO=2.2,6,6-tetramethyl-1-piperidinyloxy, free radi- 20 acid intermediate 2a, which can be prepared from ester inter cal mediate 1. It may be necessary to protect certain functional THF-tetrahydrofuran ities in the molecule depending on the nature of the R' group. TFA=trifluoroacetic acid Protecting these functionalities should be within the expertise TMS-trimethylsilyl of one skilled in the art. See, e.g. P. G. M. Wuts and T. W. TLC-thin layer chromatography 25 Greene, Protective Groups in Organic Synthesis, Fourth Edi VAP=Ventilator-Associated Pneumonia tion, John Wiley and Sons, 2006, hereafter Greene.

Scheme I

O 1n - '', 1. 1. NHNHBoc IN-1- LiOH HATU, DIPEA H N III I IIH Thro' N ... I IIH 2. TFA/DCM, 30% N ... I IIH N N or 4M HCl in dioxane O 4 YOBn O -- YOBn o2 N YOBn

1 2a 2f

R-COOH HATU, DIPEA DCM

O O O H %. R N '', NH2 HO HATU, DIPEA N R N -- H N . I I IIH DCM O N 1 IIH

2h N JY. N O YOBn O YOBn

2a 2g

N O W NN is rusu nN.1", R-(Jo-1. H (CFSO2)2O/Py H2, catalyst O N It iH --> N ... I I IIH Her or Burgess reagent or BCl3 or PPh3. I, NEt O Y YOB l Ou1 YOB l

3 US 8,933,232 B2 57 58 -continued i-Kj%,/ R-(/ SO3 pyridine protecting group N I I IIH -e-deprotection if necessary 2. N O YOSOH

N I I IIH

OuS YOSOH

The benzylic ether protecting group in 3 can be removed 30 via standard hydrogenolysis conditions, such as, but not lim Scheme 2 ited to, Pd/H, in MeOH or THF or by acid-catalysed hydroly sis, such as, but not limited to, BC1 in DCM to provide the hydroxy-urea intermediate 4, which can be used directly in 35 Lawesson's reagent the next step without further purification. Sulfation of 4 can be Hb achieved by treatment with a sulfating reagent, Sucha, but not N I IIH heat limited to, SO-pyridine complex, in an appropriate solvent, such as pyridine, DMF or DMAc at a temperature of 0-80°C., preferable at room temperature. Compound 5 can then be 40 isolated and purified via conventional methods. For example, 5 can be purified by standard reverse phase prep-HPLC using appropriate buffer system, i.e. ammonium formate buffer. In Some cases, 5 can be purified by normal phase silica gel chromatography after converting to an appropriate salt form, 45 such as sulfate tetrabutyl ammonium salt. The tetrabutyl ammonium salt can then be converted to a sodium salt by 'e, cation exchange. When a protecting group(s) is present in the N . I I IIH sidechain (i.e. Boc or Fmoc for amine and guanidine protec 50 tion, TBS or TES for alcohol protection, etc), a deprotection V step is needed to convert 5 to its final product 6, which can be O Jr. OB purified by prep-HPLC using the conditions mentioned 3a above. For example, for N-Boc deprotection, 5 can be treated 55 with an acid, such as TFA, in an appropriate solvent. Such as DCM at a temperature of 0-30°C., preferable at 0°C. tort to N NN give 6. For an O-TBS, or O-TES deprotection, a fluoride W reagent such as HF-pyridine, HF.NEts, or TBAF can be used. i-g l, For Fmoc deprotection, amines can be used, such as diethy 60 SO3 pyridine lamine, DBU, piperidine, etc can be used. Similarly, as shown in Scheme 2, thiadiazole derivative 3a N V can be synthesized from diacylhydrazide intermediate 2g by O OH treatment with Lawesson’s reagent underheating. 3a can then 65 be converted to the final product 6ausing similar chemistry as described previously. US 8,933,232 B2 59 60 -continued trile. Solvent system using NH-OH buffer: solvent A: water with 0.1% NHOH, solvent B: acetonitrile with 0.1% NHOH.

protecting group Example 1 -e- . I IIH deprotection if necessary Synthesis of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6- diazabicyclo[3.2.1]octane-2-carboxylate (Intermedi O OSOH ate Compound 1) 10 5a

N III I IIH

N V O OB

Step 1 EXAMPLES 25 Synthesis of (S)-1-tert-butyl 2-ethyl The specific examples which follow illustrate the synthesis 5-oxopiperidine-1,2-dicarboxylate of certain compounds. The methods disclosed may be Method A: adopted to variations in order to produce compounds of the 30 invention (e.g. compounds of Formula I, compounds of For mula A-I, compounds of Formula II, compounds of Formula A-II) but not otherwise specifically disclosed. Further, the poe O N O disclosure includes variations of the methods described A TMSCHN 35 : III -e- herein to produce the compounds of the invention (e.g. com n-BuLi pounds of Formula I, compounds of Formula A-I, compounds OEt of Formula II, compounds of Formula A-II) that would be s O understood by one skilled in the art based on the instant N HN ls Rh2(OAc)4 disclosure. n s OEt -e- All temperatures are understood to be in Centigrade (C) 40 when not specified. The nuclear magnetic resonance (NMR) O spectral characteristics refer to chemical shifts (y) expressed Boc O in parts per million (ppm) versus tetramethylsilane (TMS) as reference standard. The relative area reported for the various 45 OEt shifts in the proton NMR spectral data corresponds to the number of hydrogen atoms of a particular functional type in the molecule. The nature of the shifts as to multiplicity is reported as broad singlet (bris), broad doublet (br d), 50 n-BuLi was added dropwise to a solution of TMSCHN singlet (s), multiplet (m), doublet (d), quartet (q), doublet of (690 mL, 1.38 mol) in dry THF (3 L) (600 mL, 1.5 mol) at doublet (dd), doublet of triplet (dt), and doublet of quartet -78°C., and the mixture was stirred at -78°C. for 30 minutes. (dq). The solvents employed for taking NMR spectra are The mixture was then transferred to a solution of (S)-1-tert DMSO-d6 (perdeuterodimethysulfoxide), DO (deuterated butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate (300 g, 55 1.17 mol) in dry THF (3 L) via cannula, and the mixture was water), CDC1 (deuterochloroform) and other conventional stirred at -78° C. for 30 minutes. The reaction mixture was deuterated solvents. The prep-HPLC conditions are: Waters then quenched with sat. NHCl solution, and extracted with SunFireR C18 (30x100 mm, 5um OBD) column; flow rate: DCM three times. The combined organic layer was concen 30-80 mL/minute, ELSD or Mass-triggered fraction collec trated in vacuum and the crude product was purified by silica tion; sample loading: Each injection loading varied from 60 gel column chromatography (3:1 petroleum ether:EtOAc) to -300 mg for different crude samples depending on their solu afford (S)-ethyl 2-((tert-butoxycarbonyl)amino)-6-diazo-5- oxohexanoate (262 g, 75%) as a yellow solid. bility and purity profiles; Solvent system using ammonium A solution of (S)-ethyl 2-((tert-butoxycarbonyl)amino)-6- formate buffer: solvent A: water with 20 mM ammonium diazo-5-oxohexanoate (350 g, 1.18 mol) in DCM (1500 mL) formate, solvent B: 85% of acetonitrile in water with 20 mM 65 was added to a solution of Rh(OAc) (3.5 g, 7.9 mmol) in ammonium formate. Solvent system using NHHCO buffer: DCM (750 mL) at 0°C. The reaction was then stirred at 20° solvent A: water with 10 mM NHHCO, solvent B: acetoni C. overnight and then concentrated in vacuum. The crude US 8,933,232 B2 61 62 sample was purified by Silica gel column chromatography EtOAc) to afford (2S,5S)-1-tert-butyl 2-ethyl 5-hydroxypip (5:1 petroleum ether/EtOAc) to afford (S)-1-tert-butyl eridine-1,2-dicarboxylate (205 g, 80%) as a yellow oil. 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (175.9 g, 55%) as a yellow oil. Step 3: Synthesis of (2S,5R)-1-tert-butyl 2-ethyl Method B: 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperi dine-1,2-dicarboxylate Boc O M O Y2 N 1V I 10 C h) - t-BuOK\ OEt DMSO OSO Boc O O NO pyridine N/ . Bn -e- n HN w Ir(COD)Cl] 15 n NHHC + O OEt toluene, 80°-e- C. O - rt, 1 h

O Boc O

OEt Boc O

NilsOEt 25 t-BuOK (330g, 2.9 mol) was added to a solution of trim ethylsulfoxonium iodide (750 g, 3.5 mol) in dry DMSO (3 L) How O and the mixture was stirred at rt for 1 h. (S)-1-tert-Butyl PhP, DEAD, THF 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate (900 g, 3.5 mol) was added and the mixture was stirred at rt for 2-3 hrs. Water 30 was added to quench the reaction and the mixture was extracted with EtOAc 5 times. The combined organic layer was concentrated in vacuum and the crude sample was puri fied by silica gel column chromatography (1:1 petroleum ether/EtOAc then 1:10 MeOH/DCM) to afford sulfoxonium 35 ylide intermediate (977 g. 80%) as a white solid. A solution of sulfoxoniumylide intermediate (156g, 0.446 mol) and Ir(COD)Cl]- (3 g, 4.46 mmol) in toluene (4 L) was degassed by bubbling nitrogen through the solution for 10 minutes. The reaction mixture was heated to 80-90° C. for 2-3 40 hrs and then cooled to 20°C. Then toluene was concentrated in vacuum, the residue was purified by silica gel column ON chromatography (10:1 to 3:1 gradient petroleum ether/EtOA) to afford (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicar boxylate (140g, 57.8%) as a yellow oil. 45 Step 2: Synthesis of (2S,5S)-1-tert-butyl 2-ethyl A solution of 2-nitrobenzene-1-sulfonyl chloride (500 g, 5-hydroxypiperidine-1,2-dicarboxylate 2.26 mol) in pyridine (1500 mL) was added dropwise to a 50 solution of O-benzylhydroxylamine hydrochloride (400 g, 2.51 mol) in pyridine (1500 mL) at 0°C. The reaction mixture Boc O was then stirred at 20° C. overnight. The mixture was con centrated in vacuum, diluted with DCM and washed with HCl ls (10%) three times. The combined organic layer was concen 55 trated in vacuum and recrystallized with DCM to afford O * OEt N-(benzyloxy)-2-nitrobenzenesulfonamide (485 g, 62.6%) O Ho as a yellow solid. To a solution of N-(benzyloxy)-2-nitrobenzenesulfona NaBH (36 g, 1.0 mol) was added in portions to a solution 60 mide (212 g, 0.69 mol) in THF (1000 mL) was added (2S, of (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate 5S)-1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxy (250 g., 0.92 mol) in EtOH (1500 mL) at -40°C. The reaction late (171 g, 0.63 mol) and PPhs (275g, 1.05 mol), followed by mixture was then stirred at -40°C. for 0.5 hr then quenched dropwise addition of a solution of DEAD (195g, 1.12 mol) in with 10% HOAc solution. After diluting with water, the mix THF (500 mL). The mixture was then stirred at 20° C. over ture was extracted with DCM three times. The combined 65 night. The reaction mixture was then concentrated in vacuum organic layer was concentrated in vacuum and purified by and purified by silica gel column chromatography (3:1 petro silica gel column chromatography (1:1 petroleum ether/ leum ether/EtOAc) to afford (2S,5R)-1-tert-butyl 2-ethyl US 8,933,232 B2 63 64 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1, MeOH) to afford (2S,5R)-ethyl 5-((benzyloxy)amino)piperi 2-dicarboxylate (283.8 g., 80%) as a yellow oil. dine-2-carboxylate (184.9 g, 95%) as a yellow oil.

Step 4: Synthesis of (2S.5R)-1-tert-butyl 2-ethyl Step 6: Synthesis of (2S.5R)-ethyl 6-(benzyloxy)-7- 5-((benzyloxy)amino)piperidine-1,2-dicarboxylate OXO-1,6-diazabicyclo3.2.1]octane-2-carboxylate

H 10 NN CO2Et Triphosgene, DIPEA --- DCM O NN 1C H LiOH, HSCHCOOH 15 He DMF -- |'. N II I I IIH

Boc O2 NOB NN CO2Et

25 Triphosgene (21.3 g, 72 mmol) was added in portions to a O NN 1O solution of (2S,5R)-ethyl 5-((benzyloxy)amino)piperidine H 2-carboxylate (50 g., 0.18 mol) and DIPEA (128 mL, 0.72 mol) in DCM (2000 mL) at 0° C. After stirring at 20° C. LiOH.HO (95g, 2.3 mol) and 2-mercaptoacetic acid (124 overnight, the reaction mixture was washed with HPO. g, 1.3 mol) were added to a solution of (2S,5R)-1-tert-butyl 30 (10%), sat. NaHCO, and saturated NaCl. The combined 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperi organic layer was concentrated in vacuum and purified by dine-1,2-dicarboxylate (251 g, 0.45 mol) in DMF (1200 mL). silica gel column chromatography (3:1 petroleum ether/ The reaction mixture was then stirred at 20°C. overnight. The EtOAc) to afford (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-di reaction mixture was diluted with water and extracted with azabicyclo[3.2.1]octane-2-carboxylate (27.4 g. 50%) as a EtOAc (3x). The combined organic layer was washed with 35 yellow solid. "H NMR (400 Mz, CDC1): 8 7.43-7.36 (m, brine (3x), concentrated in vacuum and purified by silica gel 5H), 5.06 (d.J=11.4 Hz, 1H), 4.90 (d. J=11.4 Hz, 1H), 4.24(q, column chromatography (3:1 petroleum ether/EtOAc) to J=7.1 Hz, 2H), 4.11-4.08 (m, 1H), 3.32-3.31 (m. 1H), 3.08 afford (2S,5R)-1-tert-butyl 2-ethyl 5-((benzyloxy)amino)pi 3.05 (m, 1H), 2.93 (d. J=11.9 HZ, 1H), 2.14-2.05 (m, 2H), peridine-1,2-dicarboxylate (122.9 g, 85%) as a yellow solid. 40 2.05-2.00 (m. 1H), 1.71-1.63 (m. 1H), 1.29 (t, J=7.1 Hz, 3H). Step 5: Synthesis of (2S,5R)-ethyl Example 2 5-((benzyloxy)amino)piperidine-2-carboxylate

45 Synthesis of (2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaz abicyclo[3.2.1]octane-2-carboxylic acid (Intermedi s ate Compound 2a) N CO2Et s TFADCM -as 50 OS 2a

H NN CO2Et 55 O NN 1O H

TFA (600 mL) was added to a solution of (2S,5R)-1-tert butyl 2-ethyl 5-((benzyloxy)amino)piperidine-1,2-dicar boxylate (263 g, 0.7 mol) in DCM (600 mL) at 20° C. The mixture was stirred at rt overnight and then concentrated in vacuum. The crude product was adjusted to pH 10 with sat. NaHCO solution, and then extracted with DCM three times. 65 The combined organic layer was concentrated in vacuum and purified by silica gel column chromatography (20:1 DCM/ US 8,933,232 B2 65 -continued N -continued O

HO l aw ( -- SOPy, py, rt N III I IIH N II I I IIH '''PS'' - then BuNHSO4. Z N N NaH2PO4 O OB O? YOH N 10 LiOH (1.2g, 29.6 mmol) was added to a solution of (2S, 5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]oc ( - tane-2-carboxylate (9 g, 29.6 mmol) in THF/HO (3:1, 240 N III I IIH Dowex 50WX8 Na" mL). The mixture was then stirred at rt overnight. The reac -- tion mixture was washed with EtOAc twice, then the aqueous 15 resin exchange solution was adjusted pH 2-3 with 1N HC1. The resulting mixture was extracted with DCM three times, and the com bined organic layer was dried over Saturated Na2SO4 and concentrated in vacuum to provide (2S,5R)-6-(benzyloxy)-7- oXo-1,6-diazabicyclo3.2.1]octane-2-carboxylic acid (7.0 g, 77.7%), which was directly used in the next step without further purification. ESI-MS (EI", m/z): 277.31. H NMR (300 MHz, CDC1) & 7.49-7.29 (m, 5H), 5.06 (d. J=11.4 Hz, 1H), 4.91 (d. J=11.4 Hz, 1H), 4.15-4.10 (m, 1H), 3.36-3.34 (m. 1H), 3.15-3.11 (m. 1H), 2.83 (d. J=11.8 Hz, 1H), 2.32 25 2.15 (m. 1H), 2.11-2.01 (m, 2H), 1.74-1.56 (m. 1H). Step 1 Example 3 30 1,1'-Carbonyldiimidazole (5.8 g. 36.2 mmol) was added to Synthesis of (2S,5R)-2-(1,3,4-oxadiazol-2-yl)-7-oxo a 0°C. solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabi 1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate cyclo[3.2.1]octane-2-carboxylic acid (5.0 g, 18.1 mmol) in (Compound 701) dry THF (200 mL). The reaction mixture was allowed to warm to rt then was stirred at rt for 3 hrs. Formohydrazide (5.4 35 g, 90.5 mmol) was added in one portion, and the reaction mixture was stirred for additional 3 hrs. The mixture was then diluted with saturated sodium chloride and extracted with EtOAc (3x). The combined organic layer was washed with saturated Sodium chloride (2x), dried over NaSO4, and con 40 centrated to afford crude (2S,5R)-6-(benzyloxy)-N'-formyl 7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbohydrazide (~11 g), which was directly used in the next step. ESI-MS (EI", m/z): 319.1 M+H". lO 45 Step 2 HO . NH To a -10° C. solution of (2S,5R)-6-(benzyloxy)-N'- 2N1 N2 CDI formyl-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbohy N III I IIH -- ca N THF, rt drazide (11 g) in dry DCM (200 mL) was added pyridine (28 50 mL), followed by dropwise addition of (CFSO)O (28 mL). Ou1 YOBn The reaction mixture was allowed to warm to rt and was O stirred for 3 hrs. The reaction mixture was then cooled to -10° C. and quenched with sat. NaHCO. The organic layer was 's-NR. J. ... separated and the aqueous layer was extracted with EtOAc H 55 (3x). The combined organic layer was dried over NaSO, N H (3929 Py concentrated and purified by silica gel column chromatogra DCM, rt N phy (gradient elution 1:3 to 2:1 EtOAc/hexanes) to give (2S, O YOB 5R)-6-(benzyloxy)-2-(1,3,4-oxadiazol-2-yl)-1,6-diazabicy N clo3.2.1]octan-7-one (4.6 g., 86% for two steps) as a slightly ( 60 yellow solid. ESI-MS (EI", m/z): 301.0M+H". -- Step 3 Pol/C, H2 N III I IIH up - To a solution of (2S,5R)-6-(benzyloxy)-2-(1,3,4-oxadia 65 Zol-2-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (4.6 g. 15.3 mmol) in THF (150 mL) was added 10% Pd/C (1 g). The mixture was stirred under H atmosphere at rt for 3 hrs. The US 8,933,232 B2 67 68 reaction mixture was then filtered and concentrated to afford -continued (2S.5R)-6-hydroxy-2-(1,3,4-oxadiazol-2-yl)-1,6-diazabicy clo3.2.1]octan-7-one (2.9 g, 91%), which was used directly in the next step. ESI-MS (EI", m/z): 211.1 M+H".

Step 4 To a solution of (2S,5R)-6-hydroxy-2-(1,3,4-oxadiazol-2- 10 HATU, DIPEA yl)-1,6-diazabicyclo[3.2.1]octan-7-one (2.9 g, 13.8 mmol) in COOH -e- dry pyridine (60 mL) was added SOPy (11.0 g. 69.0 mmol). Boein 1N1 DCM The reaction mixture was stirred at rt for 8 hrs and then O concentrated under vacuum. The residue was re-dissolved in aqueous NaH2PO (1.5 M, 100 mL) then tetrabutylammo BocN Nn o 15 H Ho nium hydrogensulphate (5.88 g. 17.3 mmol) was added. The O N H mixture was stirred at rt for 20 minutes, then was extracted with EtOAc (4x). The combined organic layer was dried and N concentrated and the residue was purified by silica gel column OB chromatography (gradient elution 10:1 to 2:1 DCM/acetone) to afford tetrabutylammonium (2S,5R)-2-(1,3,4-oxadiazol 2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (4.1 g, 97%) as a white solid. ESI-MS (EI, m/z): 289.0 M-H. 'H NMR (400 MHz, CDC1): 8 8.48 (s, 1H), 4.75 (d. J=6.5 Hz, 1H), 4.40 (brs, 1H), 3.34-3.26 (m, 9H), 2.82 (d. J=12.0 Hz, 25 1H), 2.37-2.25 (m, 3H), 2.06-1.98 (m. 1H), 1.71-1.65 (m, 8H), 1.49-142 (m, 8H), 1.01 (t, J=7.5 Hz, 12H).

Step 5 30 then BuNHSO4. Resin Exchange NaH2PO4 Tetrabutylammonium (2S,5R)-2-(1,3,4-oxadiazol-2-yl)- 7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl sulfate (4.1 g, 7.72 35 BocN N-K \ '. mmol) was dissolved in a minimum amount of HPLC grade TFA, DCM water (~40 mL) and passed through a column of 80 g of -e- DOWEX 50WX 8 Nat resin (the resin was prewashed with N >4 L of HPLC grade water) and eluted with HPLC grade 40 OSONBu water to afford sodium (2S,5R)-2-(1,3,4-oxadiazol-2-yl)-7- oxo-1,6-diazabicyclo[3.2.1]octan-6-ylsulfate (2.2g.91%) as a white solid after lyophilization. ESI-MS (EI", m/z): 291.2 M+H". "H NMR (300 MHz, DO) & 8.92 (s, 1H), 4.84 (d. J=6.7 Hz, 1H), 4.20 (brs, 1H), 3.25-3.16 (m, 1H), 2.92 (d. 45 J=12.3 Hz, 1H), 2.41-2.26 (m. 1H), 2.26-2.11 (m, 2H), 2.04 1.89 (m, 1H).

Example 4 50 Step 1 Synthesis of (2S,5R)-2-(5-(2-aminoethyl)-1,3,4-oxa DIPEA (1.1 g, 8.3 mmol) was added to a 0°C. solution of diazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl (2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1 hydrogen sulfate (Compound 703) 55 ctane-2-carbohydrazide (1.0g, 2.48 mmol) in DMF (10 mL). 3-((tert-Butoxycarbonyl)amino)propanoic acid (0.39 g, 2.07 mmol) and HATU (0.90g, 2.48 mmol) were then added. The reaction mixture was stirred at 0°C. for 1 hand then quenched with saturated sodium chloride (50 mL). The organic layer HN -\uly was separated and the aqueous layer was exacted with EtOAc (3x). The combined organic layer was washed with saturated sodium chloride (2x), dried over NaSO, and concentrated. The residue was purified by silica gel column chromatogra phy (gradient elution 10:1 to 2:1 hexanes/EtOAc) to afford 65 tert-butyl (3-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabi cyclo3.2.1]octane-2-carbonyl)hydrazinyl)-3-oxopropyl) carbamate (1.0 g, 87%). ESI-MS (EI", m/z): 462 M+H".

US 8,933,232 B2 71 72 -continued 3.73 (brs, 1H), 3.32-3.26 (m, 2H), 2.96-2.92 (m, 2H), 2.85 2.81 (m, 2H), 2.19-2.15 (m, 1H), 2.05-1.99 (m, 2H), 1.86 1.83 (m, 1H), 1.34 (s, 9H). fiNN- y, .. Step 3 O SOPy, py, rt N H -> TFA (0.5 mL) was added to a 0°C. solution of tert-butyl then BuNHSO4. 2-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo NaH2PO O Y. NOH 2-4 3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)ethylcarbamate (85 NBoc mg, 0.192 mmol) in CHCl (2.0 mL). The reaction mixture 10 was stirred at 0°C. for 2 hrs and was then concentrated under Bein- N-N vacuum to provide (2S,5R)-2-(5-(2-aminoethyl)-1,3,4-oxa HN ( y A. O diazol-2-yl)-6-(benzyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7- TFA, DCM one TFA salt as a sticky oil, which was used directly in the N III IIH -e- next step. ESI-MS (EI", m/z): 344.2 M+H". a N. 15 O OSONBu Step 4 NBoc H - N-N TEA (77 mg, 0.764 mmol) was added to a 0°C. solution of 2 (2S.5R)-2-(5-(2-aminoethyl)-1,3,4-oxadiazol-2-yl)-6-(ben Zyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one TFA salt from O the previous step in MeOH (3.0 mL). Tert-Butyl (((tert-bu toxycarbonyl)amino)(1H-pyrazol-1-yl)methylene)carbam N III I IIH ate (65 mg, 0.209 mmol) was then added at 0°C. and the reaction mixture was stirred at 0°C. for 4 hrs. The reaction / N mixture was evaporated and the residue was purified by silica O OSOH 25 gel column chromatography (gradient elution 30%-50% EtOAc/petroleum ether) to give (2S,5R)-2-(5-((2,3-bis(tert butoxycarbonyl)guanidino)ethyl)-1,3,4-oxadiazol-2-yl)-6- Step 1 (benzyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one (80 mg. 30 70% in 2 steps) as a colorless oil. ESI-MS (EI", m/z): 586.3 DIPEA (1.1 g, 8.3 mmol) was added to a 0°C. solution of M+H". (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo3.2.1]oc tane-2-carbohydrazide (1.0g, 2.48 mmol) in DMF (10 mL). Step 5 3-((tert-Butoxycarbonyl)amino)propanoic acid (0.39 g, 2.07 To a solution of (2S,5R)-2-(5-((2,3-bis(tert-butoxycarbo mmol) and HATU (0.90g, 2.48 mmol) were then added. The 35 nyl)guanidino)ethyl)-1,3,4-oxadiazol-2-yl)-6-(benzyloxy)- reaction mixture was stirred at 0° C. for 1 h and then was 1,6-diaza-bicyclo[3.2.1]octan-7-one (80 mg, 0.136 mmol) in quenched with saturated sodium chloride (50 mL). The THF (15 mL) was added 10% Pd/C (45 mg) and the mixture organic layer was separated and the aqueous layer was was stirred under H atmosphere at rt for 1 h. The reaction exacted with EtOAc (3x). The combined organic layer was mixture was filtered and concentrated to afford (2S,5R)-6- washed with saturated sodium chloride (2x), dried over 40 hydroxy-2-(5-((2,3-bis(tert-butoxycarbonyl)guanidino) NaSO, and concentrated. The residue was purified by silica ethyl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo[3.2.1]octan gel column chromatography (gradient elution 10:1 to 2:1 7-one, which was directly used in the next step. ESI-MS (EI", hexanes/EtOAc) to afford tert-butyl (3-(2-((2S,5R)-6-(ben m/z): 496.2 M+H". Zyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbonyl) hydrazinyl)-3-oxopropyl)carbamate (1.0 g, 87%). ESI-MS 45 Step 6 (EI", m/z): 462 M+H". To a solution of (2S,5R)-6-hydroxy-2-(5-((2,3-bis(tert-bu Step 2 toxycarbonyl)guanidino)ethyl)-1,3,4-oxadiazol-2-yl)-1,6- diaza-bicyclo[3.2.1]octan-7-one from the previous step in (CFSO)O (1.0 mL) was slowly added to a -10° C. 50 dry pyridine (2.0 mL) was added SOPy (152 mg, 0.962 solution of tert-butyl (3-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1, mmol). The mixture was stirred at rt for 3 hrs and then con 6-diazabicyclo[3.2.1]octane-2-carbonyl)hydrazinyl)-3-oxo centrated under vacuum. The residue was re-dissolved in aqueous NaH2PO (1.5M, 15 mL) then tetrabutylammonium propyl)carbamate (1.0g, 2.17 mmol) and pyridine (1.0 mL) in hydrogensulphate (50 mg 0.15 mmol) was added. The mix dry DCM (10 mL). The reaction mixture was allowed to 55 ture was stirred at rt for 20 minutes, then extracted with warm to rt, was stirred at rt for 1 h then sat. NaHCO was EtOAc (4x). The combined organic layer was dried and con added at 0°C. very slowly. The organic layer was separated centrated and the residue was purified by silica gel column and the aqueous layer was exacted with EtOAc (3x). The chromatography (gradient elution 10:1 to 3:1 DCM/acetone) combined organic layer was dried over Na2SO4 and concen to afford tetrabutylammonium (2S,5R)-2-(5-(2-(2,3-bis(tert trated. The residue was purified by silica gel column chroma 60 butoxycarbonyl)guanidino)ethyl)-1,3,4-oxadiazol-2-yl)-7- tography (gradient elution 10:1 to 4:1 petroleum ether/ oxo-1,6-diaza-bicyclo[3.2.1]octan-6-ylsulfate (60 mg, 53%) EtOAc) to give tert-butyl (2-(5-((2S.5R)-6-(benzyloxy)-7- as a white solid. ESI-MS (EI, m/z): 574.1 M-HI. oXo-1,6-diazabicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2- yl)ethyl)carbamate (0.40 g, 42%) as a slight yellow solid. Step 7 ESI-MS (EI", m/z): 444 M+H". "H NMR (500 MHz, 65 DMSO-d) & 748-746 (m, 2H), 7.45-7.37 (m, 3H), 6.99 (t, TFA (0.23 mL) was added to a 0°C. solution of tetrabuty J=5.5 Hz, 1H), 4.99-4.94 (m, 2H), 4.59 (d. J=7.0 Hz, 1H), lammonium (2S,5R)-2-(5-(2-(2,3-bis(tert-butoxycarbonyl)

US 8,933,232 B2 77 78 -continued (19.6 g., 52.2 mmol) and DIPEA (16.6 g., 130.5 mmol). The BocN N-N mixture was allowed to warm tort, was stirred at rt for 12hrs then diluted with CHCl (300 mL), washed with water (2x) TFA, DCM and saturated sodium chloride (2x), dried over NaSO, and -e- 5 concentrated. The residue was purified by silica gel column chromatography (gradient elution 50-80% EtOAc/petroleum ether) to afford tert-butyl 4-(2-((2S.5R)-6-(benzyloxy)-7- O OSONBu OXO-1,6-diaza-bicyclo3.2.1]octane-2-carbonyl)hydrazinyl)- HN N-N 4-oxobutylcarbamate (19.3 g.93%) as a white solid. ESI-MS Y-\ul ya. 10 (EI", m/z): 476 M+H". O N H Step 5: Synthesis of tert-butyl 3-(5-((2S,5R)-6-(ben Zyloxy)-7-oxo-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1, 3,4-oxadiazol-2-yl)propylcarbamate Z N 15 O OSOH Method A: Tf.C (8.0 mL, 0.0474 mol) was added dropwise to a -78° Step 1: Synthesis of C. solution of tert-butyl 4-(2-((2S.5R)-6-(benzyloxy)-7-oxo 4-(tert-butoxycarbonylamino)butanoic acid 1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazinyl)-4- oxobutylcarbamate (7.5 g., 0.0158 mol) and Py (10.2 mL, To an aqueous solution of 4-aminobutanoic acid (25g, 242 0.126 mol) in dry DCM (120 mL). The reaction mixture was mmol) in HO (500 mL) at rt was added NaCO (75 g, 726 allowed to warm to 0°C. then the reaction mixture was stirred mmol), followed by Boc-O (95 g, 435 mmol) in THF (200 at 0°C. for 3 hrs. Sat. NaHCO, was added at 0°C. very slowly. mL). The reaction mixture was stirred at rt for 12 hrs then 25 The organic layer was separated and the water layer was concentrated under reduced pressure. The aqueous residue exacted with DCM (3x). The combined organic layer was was extracted with EtO, then the aqueous layer was acidified washed with water, saturated sodium chloride, dried over to pH 4-5 with citric acid and extracted with EtOAc. The NaSO, and concentrated. The residue was purified by silica combined organic layer was dried over NaSO, and concen gel column (gradient elution 0-25% EtOAc/petroleum ether) trated to afford 4-(tert-butoxycarbonylamino)butanoic acid 30 to afford tert-butyl 3-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6- (45 g, 90%) as a colorless oil. ESI-MS (EI", m/z): 226 diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)propy M+Na". lcarbamate (3.9 g, 54%) as a yellow solid. ESI-MS (EI", m/z): 458 IM+H". "H NMR (500 MHz, CDC1) & 7.45-7.37 (m, Step 2: Synthesis of methyl 5H), 5.08 (d. J=14.5 Hz, 1H), 4.93 (d. J=14.5 Hz, 1H), 4.70 4-(tert-butoxycarbonylamino)butanoate 35 4.66 (m, 1H), 3.37 (brs, 1H), 3.23-3.21 (m, 2H), 2.94-2.88 (m, 3H), 2.79 (d. J=14.5 Hz, 1H), 2.30-2.28 (m, 2H), 2.11 To a solution of 4-(tert-butoxycarbonylamino)butanoic 1.97 (m, 4H), 1.45 (s, 9H). acid (7.0 g, 34.5 mmol) and KCO (9.5 g. 68.9 mmol) in Method B: acetone (70 mL) was added Me (7.5g, 51.8 mmol) at rt. The reaction solution was stirred at 45° C. for 12 hrs. The mixture 40 To s solution of PPhs (2.6 g., 10.0 mmol) in dry DCM (60 was washed with water and saturated sodium chloride, dried mL) was added I. (2.6 g. 10.0 mmol). After I was dissolved over NaSO, and concentrated to afford methyl 4-(tert-bu completely, TEA (3.5 mL. 25.0 mmol) was added quickly at toxycarbonylamino)butanoate (6.2 g, 83%) as a yellow oil. rt. The mixture was stirred for 15 mins. Tert-butyl 4-(2-((2S, ESI-MS (EI", m/z): 240M+Na". 5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2- 45 carbonyl)hydrazinyl)-4-oxobutylcarbamate (2.4 g. 5.0 Step 3: Synthesis of tert-butyl mmol) was added. The mixture was stirred at rt for 1 hr. The 4-hydrazinyl-4-oxobutylcarbamate solvent was concentrated. EtOAc (250 mL) was added, and the solution was filtrated to remove POPh. The filtrate was To a solution of methyl 4-(tert-butoxycarbonylamino)bu concentrated. The resulting residue was purified by silica gel tanoate (21.0 g, 96.8 mmol) in MeCH (180 mL) was added 50 column chromatography (gradient elution 0-40% EtOAc/ NHNH2.H2O (28.0 g, 483 mmol) at rt. The mixture was petroleum ether) to afford tert-butyl 3-(5-((2S.5R)-6-(benzy stirred at 65° C. for 12 hrs then concentrated under reduced loxy)-7-OXO-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxa pressure. The crude material was dissolved in DCM (400 diazol-2-yl)propylcarbamate (2.0 g, 86%) as a white solid. mL). The organic layer was washed with water (2x), and ESI-MS (EI", m/z): 458 IM+H". saturated Sodium chloride (2x), dried over NaSO4, and con 55 centrated to afford tert-butyl 4-hydrazinyl-4-oxobutylcar Step 6-8 bamate (18.9 g, 90%) as a white solid. ESI-MS (EI", m/z): 240 M+Na". Following Steps 3-5 in Example 4, replacing tert-butyl (2-(5-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo Step 4: Synthesis of tert-butyl-4-(2-((2S,5R)-6-(ben 60 3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)ethyl)carbamate in Zyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2- Step 3 with tert-butyl B-(5-((2S.5R)-6-(benzyloxy)-7-oxo-1, carbonyl)hydrazinyl)-4-oxobutylcarbamate 6-diazabicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)pro pyl)carbamate; (2S,5R)-2-(5-(3-aminopropyl)-1,3,4-oxadia To a 0° C. solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6- Zol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen diaza-bicyclo[3.2.1]octane-2-carboxylic acid (12.0 g, 43.5 65 sulfate (1.48 g) was obtained as a white solid after prep-HPLC mmol) and tert-butyl 4-hydrazinyl-4-oxobutylcarbamate purification using ammonium formate buffer. ESI-MS (EI", (10.5 g, 47.8 mmol) in CHCl (360 mL) was added HATU m/z): 348.1. "H NMR (300 MHz, DO) & 4.74 (d. J=6.2 Hz,

US 8,933,232 B2 81 82 -continued -10°C. The reaction mixture was stirred at rt for 3 hrs. Sat. N-N NaHCO was added at -10°C. very slowly. The organic layer ?\, -K >. was separated and the aqueous layer was exacted with EtOAc HN O P (3x). The combined organic layer was dried over NaSO and N I I I I IIH concentrated. The crude product was purified by silica gel column chromatography (2:1 EtOAC/hexanes) to give tert A N butyl 4-(5-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo O OSOH 3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)piperazine-1-car 10 boxylate (723 mg, 83%) as a slight yellow solid. ESI-MS Step 1 (EI", m/z): 485.2 M+H". DIPEA (105 g, 0.81 mol) was added to a 0° C. solution of tert-butyl piperazine-1-carboxylate (25.0 g, 0.134 mol) in 15 Step 4 DCM (250 mL), followed by the addition of triphosgene (92 g, 0.27 mol) in portions over a 40 minute time period. The To a solution of tert-butyl 4-(5-((2S,5R)-6-hydroxy-7-oxo reaction mixture was allowed to warm tort then was stirred at 1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl) rt for 3 hrs, filtered and concentrated to afford 1-tert-butyl piperazine-1-carboxylate (50 mg, 0.1 mmol) in THF (5.0 mL) 4-trichloromethyl piperazine-1,4-dicarboxylate (50 g) as an was added 10% Pd/C (20 mg). The mixture was stirred under oil. He atmosphere at rt for 3 hrs then filtered and concentrated to afford tert-butyl 4-(5-((2S.5R)-6-hydroxy-7-oxo-1,6-diaza A solution of 1-tert-butyl 4-trichloromethyl piperazine-1, bicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)piperazine 4-dicarboxylate (50 g., 0.145 mol) in THF (50 mL) was added 25 1-carboxylate (30 mg, 75%), which was used directly in the dropwise over a 30 minute time period to a solution of hydra next step. ESI-MS (EI", m/z): 385 M+H". zine hydrate (18 mL, 0.434 mol) in THF (150 mL). The reaction mixture was stirred at rt for 2 hrs then diluted with saturated sodium chloride (50 mL) and exacted with EtOAc 30 Step 5 (3x). The combined organic layer was washed with saturated sodium chloride (2x), dried over NaSO, and concentrated. To a solution of tert-butyl 4-(5-((2S,5R)-6-hydroxy-7-oxo The residue was purified by crystallization (3:1 petroleum 1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl) ether/EtOAc) to provide tert-butyl 4-(hydrazinecarbonyl)pip 35 piperazine-1-carboxylate (30 mg, 0.077 mmol) in dry pyri erazine-1-carboxylate (13 g, 40% for two steps). ESI-MS dine (3 ml) was added SO.Py (97 mg, 0.61 mmol). The (EI", m/z): 245 M+H". H-NMR (500 MHz, CDC1): 85.92 mixture was stirred at rt for 3 hrs. The pyridine was evapo (s, 1H), 3.45-3.35 (m, 8H), 1.41 (s.9H). rated under vacuum at 25°C. The residue was re-dissolved in 40 aqueous NaH2PO (1.5M, 20 mL) and tetrabutylammonium Step 2 hydrogensulphate (300 mg) was added. The mixture was stirred at rt for 20 minutes, then extracted with EtOAc (4x). DIPEA (3.7g, 10 mmol) was added to a 0° C. solution of The combined organic layer was dried and concentrated and (2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]oc the residue was purified by Silica gel column chromatography tane-2-carboxylic acid (1.45 g, 5.3 mmol. 0.8 eq) and tert 45 (gradient elution 10:1 to 1:1 DCM/acetone) to give tetrabu butyl 4-(hydrazinecarbonyl)piperazine-1-carboxylate (1.6 g. tylammonium (2S,5R)-2-(5-(4-(tert-butoxycarbonyl)piper 6.6 mmol) in dry DMF (50 mL), followed by the addition of azin-1-yl)-1,3,4-oxadiazol-2-yl)-7-OXO-1,6-diazabicyclo HATU (1.45 g, 5.3 mmol). The reaction mixture was allowed 3.2.1]octan-6-yl sulfate (20 mg, 20%). ESI-MS (EI, m/z): to warm tort then was stirred at rt overnight. The mixture was 50 473 M-H. then diluted with water (200 mL) and the resulting precipi tated material was collected by filtration, rinsed with water, and then recrystallized (3:1 petroleum ether/EtOAc) to afford Step 6 tert-butyl 4-(2-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diazabi cyclo3.2.1]octane-2-carbonyl)hydrazinecarbonyl)pipera 55 TFA (0.30 mL) was added to a 0°C. mixture of tetrabuty zine-1-carboxylate (1.4g, 54%), ESI-MS (EI", m/z): 503 lammonium tert-butyl 4-(5-((2S.5R)-7-oxo-6-(sulfooxy)-1, M+H". 6-diazabicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)pip erazine-1-carboxylate (21 mg 0.03 mmol) in dry DCM (0.80 mL). The reaction mixture was stirred at 0°C. for 2-3 hrs then Step 3 60 diluted with ether (-15 mL). The precipitate was collected via centrifugation, washed with ether (3x) and dried under high To a solution of tert-butyl 4-(2-((2S,5R)-6-(benzyloxy)-7- vacuum to afford (2S.5R)-7-oxo-2-(5-(piperazin-1-yl)-1,3,4- oXo-1,6-diaza-bicyclo3.2.1]octane-2-carbonyl)hydrazine oxadiazol-2-yl)-1,6-diazabicyclo3.2.1]octan-6-yl hydrogen carbonyl)piperazine-1-carboxylate (903 mg, 1.8 mmol) in 65 sulfate (4 mg., 35%) as a light yellow solid after prep-HPLC DCM (200 mL) was added pyridine (2.8 mL, 36.0 mmol). purification using ammonium formate buffer. ESI-MS (EI", (CFSO)O (2.8 ml, 9.0 mmol) was then slowly added at m/z): 375.2. "H NMR (300 MHz, DO) & 4.65 (d. J=6.6 Hz,

US 8,933,232 B2 86 -continued 3.2.1]octane-2-carbonyl)hydrazinyl)-2-oxoacetamide (0.57 O g, 64%). ESI-MS (EI", m/z): 362 M+H". TFAHN nN.1", H Step 2 N II I I IIH -- (CFSO),O (0.58 g. 2.08 mmol) was slowly added to a -10° C. solution of 2-(2-((2S.5R)-6-(benzyloxy)-7-oxo-1,6- OA NOB O diazabicyclo3.2.1]octane-2-carbonyl)hydrazinyl)-2-oxoac HATU 10 etamide (0.30 g, 0.83 mmol) and pyridine (0.6 mL) in dry OH He DCM (5 mL). The reaction mixture was allowed to warm tort. HN DIPEADMF The reaction mixture was stirred at rt for 1 h then quenched O with sat. NaHCO very slowly. The organic layer was sepa O O rated and the aqueous layer was exacted with EtOAc (3x). 15 The combined organic layer was dried over NaSO, and HN Ruln N o, concentrated. The residue was purified by silica gel column (CFSO2)2O/py chromatography (gradient elution 1:10 to 2:1 EtOAc/hexane O N III I IIH -e- followed by 10:1 to 2:1 petroleum ether/EtOAC) to give DCM 5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo3.2.1]oc O NOB tan-2-yl)-1,3,4-oxadiazole-2-carboxamide (0.18g. 47%) as a N-N slight yellow solid. ESI-MS (EI", m/z): 344.0M+H". O f y o O Step 3 Pd/C, H2 25 HN N I I I I IIH -> THF To a solution of 5-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-di aZabicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazole-2-carboxam O N OB N-N ide (0.14g, 0.41 mmol) in THF (10 mL) was added 10% Pd/C 30 (0.14 g). The mixture was stirred under H atmosphere at rt , for 3 hrs then filtered and concentrated to provide 5-((2S,5R)- O 4. SO-Py, py, rt 6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4- N II I I IIH He oxadiazole-2-carboxamide (103 mg, 100%), which was then BuNHSO4. directly used in the next step. ESI-MS (EI", m/z): 254 N NaH2PO4 35 M+H". O OH N-N O () y Step 4 '... O Dowex50w)x8 40 To a solution of 5-((2S.5R)-6-hydroxy-7-oxo-1,6-diazabi HN N III I IIH He cyclo3.2.1]octan-2-yl)-1,3,4-oxadiazole-2-carboxamide a NN (103 mg, 0.41 mmol) in dry pyridine (2.0 mL) was added O OSONBu SO.Py (323 mg, 2.03 mmol). The mixture was stirred at rt for N-N 3 hrs and then concentrated under vacuum. The residue was 45 re-dissolved in aqueous NaH2PO (1.5M, 10 mL) then tet rabutylammonium hydrogen Sulphate (166 mg, 0.49 mmol) was added. The mixture was stirred at rt for 20 minutes, then HN extracted with EtOAc (4x). The combined organic layer was dried and concentrated and the residue was purified by silica 50 gel column chromatography (gradient elution 10:1 to 8:1 DCM/acetone) to afford tetrabutylammonium (2S,5R)-2-(1, 3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6- ylsulfate (110 mg, 36%). ESI-MS (EI, m/z): 333.0M-HI. Step 1 55 HATU (0.95g, 2.5 mmol) was added to a 0°C. solution of Step 5 Sodium Resin Exchange (2S.5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]oc tane-2-carbohydrazide (1.00 g, 2.5 mmol), 2-amino-2- tetrabutylammonium (2S,5R)-2-(1,3,4-oxadiazol-2-yl)-7- oxoacetic acid (0.18 g. 2.1 mmol) and DIPEA (1.08 g, 8.4 oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate (110 mg) was mmol) in DMF (10 mL). The reaction mixture was stirred at 60 dissolved in a minimum amount of HPLC grade water (-80 0° C. for 1 h then quenched with saturated sodium chloride mL) and passed through a column of 20 g of DOWEX 50WX (50 mL) and the organic layer was separated. The aqueous 8 Na" resin (the resin was pre-washed with >5 L of HPLC layer was exacted with EtOAc (3x). The combined organic grade water) and eluted with HPLC grade water to provide layer was washed with saturated sodium chloride (2x), dried sodium (2S,5R)-2-(1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diaz over NaSO, and concentrated. The residue was purified by 65 abicyclo[3.2.1]octan-6-yl sulfate (55 mg, 80%) as a white silica gel column chromatography (1:1 EtOAC/hexane) to solid after lyophilization. ESI-MS (EI, m/z): 332.0 M-HI. give 2-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo "H NMR (500 MHz, DO): 84.97-4.93 (m, 1H), 4.27 (brs,

US 8,933,232 B2 89 90 piperidine-1-carboxylate from the former step in dry pyridine -continued (3 mL) was added SOPy (266 mg, 1.1 mmol). The mixture N OH K2CO3, MeI was stirred at rt for 3 hrs and then concentrated under vacuum. 1. Hip The residue was re-dissolved in aqueous NaH2PO (1.5M, 20 DMF, rt mL) then tetrabutylammonium hydrogensulphate (150 mg. 92% 0.44 mmol) was added. The mixture was stirred at rt for 20 Boc minutes, then extracted with EtOAc (4x). The combined N. NHNH-HO organic layer was dried and concentrated and the residue was 1. N EtOH, reflux purified by silica gel column chromatography (gradient elu O 98% tion 10:1 to 1:1 DCM/acetone) to afford tetrabutylammonium 10 tert-butyl 4-(5-((2S,5R)-7-oxo-6-(sulfooxy)-1,6-diaza-bicy Boc clo3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)piperidine-1-car boxylate as a white solid (110 mg, 62%). ESI-MS (EI, m/z): 488.1 M-H. 15 Boc H N N Step 5 1. N- YNH, -- O Tetrabutylammonium tert-butyl 4-(5-((2S.5R)-7-oxo-6- O (sulfooxy)-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-thia diazol-2-yl)piperidine-1-carboxylate (110 mg 0.15 mmol) was dissolved in a minimum amount of HPLC grade water HATU (~15 mL) and passed through a column of 16 g of DOWEX N . I I IIH -e- 50WX8 Na" resin (the resin was pre-washed with >0.5 L of DMF, 0° C. 25 !Y 78% HPLC grade water) and eluted with HPLC grade water to O afford sodium (2S,5R)-2-(5-(1-(tert-butoxycarbonyl)piperi din-4-yl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo 3.2.1]octan-6-yl sulfate after lyophilization as a white solid (50 mg, 65%). ESI-MS (EI, m/z): 488.0 M-H. 'H NMR Tf,0/Py, (500 MHz, DO) & 4.88 (d. J–7.0 Hz, 1H), 4.15 (s, 1H), 4.06 -10° C.-O. C. (d. J=10.5 Hz, 2H), 3.39-3.35 (m. 1H), 3.13 (d, J-2 Hz, 1H), 5196 2.94-2.87 (n, 3H), 2.45-2.41 (m, 1H), 2.17-2.04 (m, 4H), -- 1.89-1.86 (m, 1H), 1.67-1.62 (m, 2H), 1.36 (s, 9H). or PPh3, I, NEt3, DCM, rt 35 859% Step 6 TFA (0.20 mL) was added to a 0° C. mixture of sodium (2S.5R)-2-(5-(1-(tert-butoxycarbonyl)piperidin-4-yl)-1,3,4- thiadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo3.2.1]octan-6-yl 40 sulfate (50 mg, 0.10 mmol) in dry DCM (0.60 mL). The reaction mixture was stirred at 0° C. for 2-3 hrs and then BocN diluted with ether (~10 mL). The precipitate was collected via centrifugation, washed with ether (3x) and dried under high vacuum to afford (2S,5R)-7-oxo-2-(5-(piperidin-4-yl)-1,3,4- 45 thiadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate (10 mg, 25%) after prep-HPLC using ammonium formate buffer. ESI-MS (EI", m/z): 390.12. 50 y'. S Example 19 O -e- then BuNHSO4. BocN N s Synthesis of (2S.5R)-2-(5-(2-(methylamino)ethyl)-1, 1 50% for two steps 3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1 N octan-6-yl hydrogen sulfate (Compound 722) 55 O

-N

60 ly. O 8. TFA, DCM BocN N H -es A N 65 O OSONBu US 8,933,232 B2 91 92 -continued Step 4 N-N Ky., Synthesis of tert-butyl 2-(5-((2S.5R)-6-(benzyloxy)- O 7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4- BocN N ... I I IIH oxadiazol-2-yl)ethyl(methyl)carbamate / N O OSOH Method A: Tf.C) (7.2 mL, 0.0423 mol) was added slowly, drop-wise to 10 a -78°C. solution of tert-butyl 3-(2-((2S.5R)-6-(benzyloxy)- Step 1 7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazi nyl)-3-oxopropyl(methyl)carbamate (6.7 g., 14.1 mmol) and Synthesis of methyl pyridine (9.1 mL, 0.113 mol) in dry DCM (110 mL). The 3-(tert-butoxycarbonyl(methyl)amino)propanoate 15 reaction mixture was allowed to warm to 0° C. then was stirred at 0°C. for 3 hrs. Aqueous NaHCO was added at 0°C. To a solution of 3-(tert-butoxycarbonyl(methyl)amino) very slowly. The organic layer was separated and the water propanoic acid (7.0 g, 0.032 mol) and KCO (13.3 g, 0.096 mol) in DMF (100 mL) was added MeI (9.0 g, 0.064 mol) at layer was washed with DCM (3x). The combined organic rt. The resultant solution was stirred at rt for 3 hrs. The layer was dried over NaSO, and concentrated. The crude reaction mixture was quenched with ice water and extracted product was purified by reverse-phase biotage (waterfaceto with ethyl acetate (2x). The combined organic layer was dried nitrile gradient) to afford tert-butyl 2-(5-((2S.5R)-6-(benzy over NaSO, and concentrated under reduced pressure. The loxy)-7-OXO-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxa residue was purified by silica gel column chromatography diazol-2-yl)ethyl(methyl)carbamate (3.3 g 51%) as a light (gradient elution 0%-10% EtOAc/petroleum ether) to afford 25 methyl 3-(tert-butoxycarbonyl(methyl)amino)propanoate yellow solid. ESI-MS (EI", m/z): 458.0 M+H"; "H NMR (6.9 g, 92%) as a white solid. ESI-MS (EI", m/z): 118 (500 MHz, CDC1): 8 7.45-7.36 (m, 5H), 5.09 (d. J=11.5 Hz, M-100+H". 1H), 4.94 (d. J=11.5 Hz, 1H), 4.71 (t, J=4.5 Hz, 1H), 3.67 3.59 (m, 2H), 3.37 (s, 1H), 3.31 (t, J=6.5 Hz, 2H), 2.94-2.86 Step 2 30 (m. 2H), 2.82 (s.3H), 2.32-2.28 (m. 2H), 2.15-2.12 (m. 1H), Synthesis of tert-butyl 2.00-1.95 (m. 1H), 1.45 (s, 9H). 3-hydrazinyl-3-oxopropyl(methyl) carbamate Method B: To a solution of PPhs (5.3.g. 20.0 mmol) in CHCl (250 To a solution of methyl 3-(tert-butoxycarbonyl(methyl) 35 mL) at rt was added I (5.2 g. 20.0 mmol). After I was amino)propanoate (6.9 g, 0.0317 mol) in EtOH (15 mL) was added hydrazine monohydrate (7.7 mL, 0.158 mol) at rt. The dissolved completely, the solution was cooled to 0°C. and reaction mixture was heated to 80° C. and stirred at 80° C. TEA (7.0 mL, 50.0 mmol) was added. Tert-butyl 3-(2-((2S, overnight. The reaction mixture was concentrated, and then 5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2- DCM (300 mL) was added. The organic layer was washed 40 carbonyl) hydrazinyl)-3-oxopropyl(methyl)carbamate (4.8g. with water and saturated sodium chloride, dried over NaSO, 10.0 mol) was added and the mixture was stirred at rt for 1 hr. and concentrated to afford tert-butyl 3-hydrazinyl-3-oxopro The mixture was concentrated, EtOAc (250 mL) was added, pyl(methyl)carbamate (6.8 g., 98%) as sticky oil, which was and the solution was filtered to remove POPh. The filtrate used directly in the next step. ESI-MS (EI", m/z): 118 45 was concentrated and the residue was purified by silica gel M-100+H". column chromatography (gradient elution 30 to 50% EtOAc/ Step 3 petroleum ether) to afford tert-butyl 2-(5-((2S.5R)-6-(benzy loxy)-7-OXO-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxa Synthesis of tert-butyl 3-(2-((2S,5R)-6-(benzyloxy)- diazol-2-yl)ethyl(methyl)carbamate (4.1 g, 85%) as a white 7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl) 50 hydrazinyl)-3-oxopropyl(methyl)carbamate solid. ESI-MS (EI", m/z): 458 M+H". To a 0°C. solution of tert-butyl 3-hydrazinyl-3-oxopropyl Step 5-7 (methyl)carbamate (4.3 g 19.8 mmol) and (2S,5R)-6-(ben Zyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylic 55 Following Steps 3-5 in Example 4, replacing tert-butyl acid (4.9 g, 0.0178 mol) in DMF (100 mL) was added HATU (2-(5-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo (8.2g, 0.0218 mol), followed by dropwise addition of DIPEA (9.6 mL, 0.0594 mol) slowly. The mixture was stirred at 0°C. 3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)ethyl)carbamate in for 1 h, and then quenched with ice water. The aqueous layer Step 3 with tert-butyl (2-(5-((2S.5R)-6-(benzyloxy)-7-oxo-1, was extracted with EtOAc (2x). The combined organic layer 60 6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl) was dried over NaSO filtered and concentrated. The resi ethyl)(methyl)carbamate; (2S,5R)-2-(5-(2-(methylamino) due was purified by silica gel column chromatography (gra ethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo3.2.1 dient elution 20-60% EtOAc/petroleum ether) to afford tert butyl 3-(2-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo octan-6-yl hydrogen Sulfate (1.2 g) was obtained as a white 3.2.1]octane-2-carbonyl)hydrazinyl)-3-oxopropyl(methyl) 65 Solid after prep-HPLC purification using ammonium formate carbamate (6.7 g. 78%) as a white solid. ESI-MS (EI", m/z): buffer. ESI-MS (EI", m/z): 348.2. "H NMR (300 MHz, DO) 476 M+H". & 4.76 (d. J=6.3 Hz, 1H), 4.18 (brs, 1H), 3.44 (t, J=7.1 Hz, US 8,933,232 B2 93 94 2H), 3.32 (t, J=6.6 Hz, 2H), 3.18 (brid, J=12.0 Hz, 1H), 2.93 -continued N-N (d. J=12.3 Hz, 1H), 2.68 (s.3H), 2.33-2.06 (m,3H), 1.98-1.86 NBoc f \ (m. 1H). BocN '. O SO3-Py, py, rt, 12h 5 N . . H. -- Example 20 then NBuHSO4. N NaH2PO4 Synthesis of (2S,5R)-2-(5-((azetidin-3-ylamino)me O OH 60% thyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo N -N 3.2.1]octan-6-yl hydrogen sulfate (Compound 726) 10 1, ...C-R-KO TFA, DCM -e- 65.9% CN N 15 O OSONBu N-N

NH N I I I I IIH HN N O OSOH O

BocN NH + inus or -MeCN, K2CO - 25 rt, 17 h Step 1 81% Synthesis of tert-butyl 3-(2-ethoxy-2-oxoethylamino) BocN NH O MeCN, K2CO3 aZetidine-1-carboxylate -> 30 A mixture of tert-butyl 3-aminoazetidine-1-carboxylate OEt 90% (10.0 g, 58 mmol), ethyl 2-bromoacetate (10.7 g. 64 mmol) and potassium carbonate (24.0 g, 174 mmol) in MeCN (200 mL) was stirred at rt for 13 hrs. The reaction mixture was be{O- NBOC O NHNH2-H2O 35 -3- filtered and concentrated. The crude material was purified by ( EtOH, reflux, 17 h silica gel column chromatography (gradient elution 0-66% OEt 94% EtOAc/petroleum ether) to afford tert-butyl 3-(2-ethoxy-2- oxoethylamino)aZetidine-1-carboxylate (12.1 g, 81%) as a beg)- NBOC O white solid. ESI-MS (EI", m/z): 259.0M+H". 40 \ { -- Step 2 NHNH O Synthesis of tert-butyl 3-(tert-butoxycarbonyl(2- ethoxy-2-oxoethyl)amino)aZetidine-1-carboxylate 45 HO !.*. HATU, DIPEA A mixture of tert-butyl 3-(2-ethoxy-2-oxoethylamino)aze N H -- tidine-1-carboxylate (10.3 g 40 mmol), di-tert-butyl dicar DCM, rt. 2 h bonate (10.4g, 48 mmol) and potassium carbonate (16.6 g. N 86% 120 mmol) in MeCN (200 mL) was stirred at rt for 6 hrs. The O OB 50 reaction mixture was then filtered and concentrated. The O crude product was purified by silica gel column chromatog Pey raphy (gradient elution 0-80% EtOAc/petroleum ether) to N nN.1" afford tert-butyl 3-(tert-butoxycarbonyl(2-ethoxy-2-oxoet Boc H I2, PPh3, TEA hyl)amino)azetidine-1-carboxylate (13.0 g, 90%) as a white O N . . H. --> 55 N DCM, rt, 2h solid. ESI-MS (EI", m/z): 359.0 M+H". 80% O YOBn Step 3 N-N NBoc \ Synthesis of tert-butyl 3-(tert-butoxycarbonyl(2- BocN l, 60 hydrazinyl-2-oxoethyl)amino)aZetidine-1-carboxy O Pol/C, H2 late N IIH He MeOH, rt, 2h A mixture of tert-butyl 3-(tert-butoxycarbonyl(2-ethoxy 2-oxoethyl)amino)aZetidine-1-carboxylate (7.2g, 20 mmol), 65 hydrazine (5.0 g, 100 mmol) and ethanol (50 mL) was stirred at 80° C. for 17 hrs. The reaction mixture was then concen trated to afford the crude tert-butyl 3-(tert-butoxycarbonyl(2-

US 8,933,232 B2 103 104 Step 1 Step 5 Synthesis of Synthesis of 3-(tert-butyldimethylsilyloxy)propanenitrile 2-(1-(tert-butoxycarbonylamino)cyclopropyl)acetic acid Imidazole (33.6 g. 495 mmol) and tert-butyldimethylsilyl chloride (27.2g, 180 mmol) were added to a 0°C. solution of RuCls.HO (124 mg. 0.6 mmol) was added to a solution of 3-hydroxypropanenitrile (10.6 g. 150 mmol) in DMF 50 mL). tert-butyl 1-(2-hydroxyethyl)cyclopropylcarbamate (6.1 g, The mixture was allowed to warm to rt, then was stirred at rt 30 mmol), and NaIO (19.0 g, 90 mmol) in DCM/HO/ for 12 hrs. The mixture was quenched with water (500 mL), 10 CHCN (20 mL/40 mL/20 mL). The mixture was stirred at rt and then extracted with EtOAc (3x). The combined organic for 3 hrs. The mixture was then diluted with H2O (100 mL), layer was dried over NaSO and concentrated. The residue and extracted with DCM (3x). The combined organic layer was purified by silica gel column chromatography (gradient was dried over NaSO and concentrated. The residue was elution 0-5% EtOAc/petroleum ether) to give 3-(tert-bu purified by silica gel column chromatography (0-30% gradi tyldimethylsilyloxy) propanenitrile (18g, 75%) as a colorless 15 ent elution EtOAc/petroleum ether) to afford 2-(1-(tert-bu oil. "H NMR (400 MHz, CDC1): 8 3.74 (t, J=6.0 Hz, 2H), toxycarbonylamino)cyclopropyl)acetic acid (6.0g, 90%) as a 2.43 (t, J=6.0 Hz, 2H), 0.81 (s, 9H), 0.00 (s, 6H). white solid. ESI-MS (EI", m/z): 238 M+Na". Step 6 Step 2 Synthesis of tert-butyl 1-(2-(2-((2S,5R)-6-(benzy Synthesis of 1-(2-(tert-butyldimethylsilyloxy)ethyl) loxy)-7-oxo-1,6-diaza-bicyclo3.2.1]octane-2-carbo cyclopropanamine nyl)hydrazinyl)-2-oxoethyl)cyclopropylcarbamate

To a solution of 3-(tert-butyldimethylsilyloxy)propaneni 25 A solution of 2-(1-(tert-butoxycarbonylamino)cyclopro trile (11.1 g, 60 mmol) in EtO (400 mL) was added titanium pyl)acetic acid (1.5 g. 6.9 mmol), (2S,5R)-6-(benzyloxy)-7- tetraisopropanolate (28.9 g, 102 mmol) under N2 atmosphere. oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbohydrazide (3.1 Ethylmagnesium bromide (3-M in EtO, 50 mL) was slowly g, 7.6 mmol), DIPEA (2.7 g. 21.0 mmol), and HATU (3.9 g, added drop-wise at 0°C. The mixture was stirred at rt for 1 hr. 10.45 mmol) in DMF (20 mL) was stirred at 0°C. for 1 hr. The Boron trifluoride etherate (17.0 g, 120 mmol) was slowly 30 mixture was quenched with saturated sodium chloride (150 added at 0°C. The mixture was stirred at rt for 1 hr. The mL) and EtOAc (150 mL). The organic layer was separated mixture was quenched with 10% aq. NaOH (300 mL) and and washed with saturated sodium chloride (2x), dried over then extracted with DCM (3x). The combined organic layer NaSO and concentrated. The residue was purified by silica was dried over NaSO and concentrated. The residue was gel column chromatography (0-80% gradient elution EtOAc/ purified by silica gel column chromatography (gradient elu 35 petroleum ether) to afford tert-butyl 1-(2-(2-((2S,5R)-6-(ben tion 5-50% EtOAc/petroleum ether) to afford 1-(2-(tert-bu Zyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl) tyldimethylsilyloxy)ethyl)cyclopropanamine (3.9 g, 30%) as hydrazinyl)-2-oxoethyl)cyclopropylcarbamate (2.4 g. 70%) a colorless oil. ESI-MS (EI", m/z): 216.2 M+H". as a white solid. ESI-MS (EI", m/z): 488 M+H". Step 3 40 Step 7 Synthesis of tert-butyl 1-(2-(tert-butyldimethylsily Synthesis of tert-butyl 1-((5-((2S,5R)-6-(benzyloxy)- loxy)ethyl)cyclopropylcarbamate 7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4- oxadiazol-2-yl)methyl)cyclopropylcarbamate A solution of 1-(2-(tert-butyldimethylsilyloxy)ethyl)cy 45 clopropanamine (1.9 g, 8.8 mmol), (Boc)O (2.9 g, 13.2 Method A: mmol), NaHCO, (1.5g, 17.6 mmol) in THF/HO (20 mL/20 Pyridine (5.2g, 65.6 mmol) was added to a 0°C. solution mL) was stirred at rt for 17 hrs. The mixture was extracted of tert-butyl 1-(2-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6- with EtOAc (3x). The combined organic layer was dried over diaza-bicyclo3.2.1]octane-2-carbonyl)hydrazinyl)-2-oxoet NaSO and concentrated. The residue was purified by silica 50 hyl)cyclopropylcarbamate (4.0 g, 8.2 mmol) in DCM (40 gel column chromatography (0-15% gradient elution EtOAc/ mL). Tf,C) (5.7g, 20.5 mmol) was added slowly at 0°C. The petroleum ether) to afford tert-butyl 1-(2-(tert-butyldimeth reaction mixture was stirred at 0°C. for 3.5 hrs. Sat. NaHCO ylsilyloxy)ethyl)cyclopropylcarbamate (2.5g, 90%) as a col was added very slowly at 0°C. The organic layer was sepa orless oil. ESI-MS (EI", m/z): 316 M+H". rated and washed with saturated sodium chloride (2x), dried 55 over NaSO, and concentrated. The residue was purified by Step 4 silica gel column chromatography (0-70% gradient elution EtOAc/petroleum ether) to afford tert-butyl 1-((5-((2S,5R)- Synthesis of tert-butyl 6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1, 1-(2-hydroxyethyl)cyclopropylcarbamate 3,4-oxadiazol-2-yl)methyl)cyclopropylcarbamate (2.6 g. 60 65%) as a yellow solid. ESI-MS (EI", m/z): 470M+H". A solution of tert-butyl 1-(2-(tert-butyldimethylsilyloxy) Method B: ethyl)cyclopropylcarbamate (11.9 g, 37.8 mmol) and To s solution of PPhs (5.2g, 20.0 mmol) in dry DCM (60 3HFTEA (22.0 g) in DCM (50 mL) was stirred atrt for 17 hrs. mL) was added I (5.1 g, 20.0 mmol). After I was dissolved The mixture was concentrated and the residue was purified by completely, TEA (7.0 mL, 50.0 mmol) was added quickly at silica gel column chromatography (0-20% gradient elution 65 rt. The mixture was stirred for 15 mins. Tert-butyl 1-(2-(2- EtOAc/petroleum ether) to afford tert-butyl 1-(2-hydroxy ((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]oc ethyl)cyclopropylcarbamate (4.6 g. 60%) as a white solid. tane-2-carbonyl)hydrazinyl)-2-oxoethyl)cyclopropyl-car

US 8,933,232 B2 109 110 -continued 4.8 mmol) in MeOH (10 mL) at 23°C. The reaction mixture H O was stirred under a H. balloon at rt for 1.5 hrs. The mixture Tf,0, Py, DCM, was filtered and concentrated. The residue was purified by H Oo C. silica gel column (10% EtOAc/petroleum ether) to afford O N . H Her BocHNY cric or 12, PPh3, NEt3, 5 ethyl 2-(3-(tert-butoxycarbonylamino)cyclobutyl)-acetate DCM O% NN OB (1.2g, 96%). ESI-MS (EI, m/z): 258 M+H". Step 3 10 Synthesis of tert-butyl Pd(C 3-(2-hydrazinyl-2-oxoethyl)cyclobutylcarbamate . I I IIH -e- THF, rt A solution of ethyl 2-(3-(tert-butoxycarbonylamino)cy O OB clobutyl)acetate (1.2g, 4.7 mmol) and hydrazine hydrate (1.4 15 g, 23.5 mmol) in EtOH (8 mL) was heated at 80° C. for 17 hrs. The reaction mixture was concentrated and the residue was dissolved in DCM (20 mL). The organic phase was washed SO-Py, py with saturated sodium chloride (10 mL) and water (10 mL). N H -- S then BuNHSO4. The water layer was exacted with EtOAc (3x). The combined BocN N NaH2PO4 organic layer was dried over Na2SO4 and concentrated to O YOH afford tert-butyl 3-(2-hydrazinyl-2-oxoethyl)cyclobutylcar N bamate (1.1 g, 97%). ESI-MS (EI", m/z): 244 M+H". ( NN l'... 25 Step 4 TFADCM Synthesis of tert-butyl 3-(2-(2-((2S,5R)-6-(benzy loxy)-7-oxo-1,6-diaza-bicyclo3.2.1]octane-2-carbo nyl)hydrazinyl)-2-oxoethyl)cyclobutylcarbamate 30 HATU (1.6 g., 4.3 mmol) and DIPEA (0.93 g, 7.2 mmol) were added to a 0° C. solution of (2S,5R)-6-(benzyloxy)-7- OXO-1,6-diaza-bicyclo3.2.1]octane-2-carboxylic acid (1.0 g, 3.6 mmol), tert-butyl 3-(2-hydrazinyl-2-oxoethyl)cyclobu 35 tylcarbamate (0.97g, 3.9 mmol) in DMF (10 mL) at. The reaction mixture was stirred at 0°C. for 1 hr. The mixture was quenched with saturated sodium chloride (50 mL) and the organic layer was separated. The water layer was exacted with EtOAc (3x). The combined organic layer was washed with 40 saturated sodium chloride (2x), dried over NaSO, and con centrated. The residue was purified by silica gel column chro matography (50% EtOAc/petroleum ether) to afford tert-bu tyl 3-(2-(2-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo 3.2.1]octane-2-carbonyl)hydrazinyl)-2-oxoethyl) 45 cyclobutylcarbamate (1.5 g., 88%). ESI-MS (EI", m/z): 502 M+H". Step 1 Step 5 Synthesis of ethyl 50 Synthesis of tert-butyl 3-((5-((2S,5R)-6-(benzyloxy)- 2-(3-(tert-butoxycarbonylamino)cyclobutylidene) 7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4- acetate oxadiazol-2-yl)methyl)cyclobutylcarbamate To a solution of tert-butyl 3-oxocyclobutylcarbamate (1.0 Method A: g, 5.4 mmol) in toluene (10 mL) was added ethyl 2-(triph 55 To a solution of tert-butyl 3-(2-(2-((2S,5R)-6-(benzyloxy)- enylphosphoranylidene)acetate (2.1 g, 5.9 mmol). The reac 7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbonyl)hydrazi tion mixture was heated at 100° C. for 2 hrs, and then con nyl)-2-oxoethyl)cyclobutylcarbamate (1.0 g, 1.99 mmol) in centrated. The residue was purified by Silica gel column DCM (20 mL) was added pyridine (1.5 mL). (CFSO)2O chromatography (10% EtOAc/petroleum ether) to afford (1.4g, 4.97 mmol) was added slowly at -10°C. The reaction ethyl 2-(3-(tert-butoxycarbonylamino)cyclobutylidene)ac 60 mixture was stirred at 0°C. for 1 h. Sat. NaHCO was added etate (1.2g, 89%). ESI-MS (EI", m/z): 256 M+H". at 0°C. very slowly. The organic layer was separated and the water layer was exacted with EtOAc (3x). The combined Step 2: Synthesis of ethyl organic layer was dried over NaSO and concentrated. The 2-(3-(tert-butoxycarbonylamino)cyclobutyl)acetate residue was purified by silica gel column chromatography 65 (gradient elution 10-50% EtOAc/petroleum ether) to afford 10% Pd/C (360 mg) was added to a solution of ethyl 2-(3- tert-butyl 3-((5-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bi (tert-butoxycarbonylamino)cyclobutylidene) acetate (1.2 g, cyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)methyl)cy