(12) United States Patent (10) Patent No.: US 8,962,843 B2 Gu Et Al

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(12) United States Patent (10) Patent No.: US 8,962,843 B2 Gu et al. (45) Date of Patent: *Feb. 24, 2015

(54) 1,3,4-OXADIAZOLE AND 1,3,4-THIADIAZOLE (58) Field of Classification Search BETA-LACTAMASE INHIBITORS USPC ...... 546/183: 514/359 (71) Applicant: Cubist Pharmaceuticals, Inc., See application file for complete search history. Lexington,eX1ngton MA (US(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. 7,112,592 B2 9, 2006 Lamoilampilas et al.1 de SatyMyst" 7,612,087 B2 11/2009 ASZodi et al. Metcalf, III, Needham, MA (US) (Continued) (73) Assignee: Cubist Pharmaceuticals, Inc., FOREIGN PATENT DOCUMENTS Lexington, MA (US) (*) Notice: Subject to any disclaimer, the term of this 5. E. Al '3. patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. (Continued) This patent is Subject to a terminal dis- OTHER PUBLICATIONS claimer. (21) Appl. No.: 13/853,327 Crompton, et al: Beta-Lactamase inhibitors, the inhibition of serine 1-1. beta-lactamases by specific boronic acids; Biochem J., 1988, vol. (22) Filed: Mar. 29, 2013 251, pp. 453-459. (65) Prior Publication Data (Continued) US 2013/O29629O A1 Nov. 7, 2013 O O Primary Examiner — John Mabry Related U.S. Application Data Assistant Examiner — Daniel Carcanague (60) Provisional application No. 61/790,579, filed on Mar. (74) Attorney, Agent, or Firm — Lathrop & Gage LLP: Brian 15, 2013, provisional application No. 61/618,131, C. Trinque filed on Mar. 30, 2012. (51) Int. Cl. (57) ABSTRACT CO7D 47/08 (2006.01) B-Lactamase inhibitor compounds (BLIS) are disclosed, A6 IK3I/546 (2006.01) including compounds that have activity against class A, class (Continued) C or class D B-lactamases. Methods of manufacturing the (52) U.S. Cl. BLIS, and uses of the compounds in the preparation of phar CPC A6 IK3I/546 (2013.01); A61 K3I/436 maceutical compositions and antibacterial applications are ------(2013.01). Asiki/39 (2013.01), also disclosed. (Continued) 5 Claims, 31 Drawing Sheets

Table Compounds of Formula A-II Cmpd. R No. 701 || O -: QSOH 702 O 41& -: OSOH 703 O 1N, –ose H 04 O --osch 7 1N1

705 O

706 O

707 O

708 O

H 709 O l

70 O US 8,962,843 B2 Page 2

(51) Int. Cl. WO WO 2011/042560 A1 4/2011 A6 IK3I/439 (2006.01) WO WO2011, 101710 A1 8, 2011 A6 IK3I/545 2006.O1 WO WO2012,086241 A1 6, 2012 (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) WO WO 2013/030735 A1 3, 2013 atter cool) W8 W58365A 3.38 CPC ...... A6 IK3I/545 (2013.01); A61K 45/06 OTHER PUBLICATIONS (2013.01); A61 K3I/427 (2013.01); A61 K 31/407 (2013.01);s C07D 471/08 (2013.01)s Internationalston, Search earcn Report,Kepo PCT/US2013/034562, dateda Jul.U. 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. Yoshizawa, H. et al.; “New broad-spectrum parenteral 8.478.288.553 102 5 B2 10,6, 20132012 P5A: talal 1 cephalosporins exhibiting potent activity against both methicillin J. eaniya et al. resistant Staphylococcus aureus (MRSA) and Pseudomonas 2010/O1979288.487,093 A1B2 7,8, 20132010 BlizzardPriour et etal. al. aeruginosa. Partart 2:. Svnthesisynthesis andan stucture activity re lationshipsationships ini 2011, 00461 O2 A1 2/2011 Ledoussal et al. the S-3578 series': Bioorganic and Medicinal Chemistry 2004, vol. 2012fOO16553 A1 2/2012 Abe et al. 12, pp. 4211-4219. 2012.0053350 A1 3/2012 Mangion et al. Yoshizawa, H. et al.; “New broad-spectrum parenteral 2012/0323.01.0 A1 12/2012 Ronsheim et al. cephalosporins exhibiting potent activity against both methicillin 2013, OO12712 A1 1/2013 Priour et al. resistant Staphylococcus aureus (MRSA) and Pseudomonas 2013/0059774 A1 3/2013 Patelet al. aeruginosa. Part 3: 7b-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2- 2013/0225.554 A1 8/2013 Maiti et al. ethoxyiminoacetamidocephalosporins bearing 4-3-(aminoalkyl)-

38.93. A. ck 1938. phyll Cal. et. . .al...... 514,203 ureido)-1-pyridinium at C-3”; Bioorganic and Medicinal Chemistry 2013,0296291 A1* 11/2013 Gu et al. .. 514,202 2004, Vol. 12, pp. 4221 4231. ck Yoshizawa, H. et al.: "S-3578, A New Broad Spectrum Parenteral 2013,0296,2932013,0296292 A1* 11, 2013 Gu et al. .. 514,202 Cephal alosporin Exhibitinghibiti Potent Activity Againstinst both MethicillinMethicilli 2013,0296555 A1* 11, 2013 Gu et al. .. 544f127 resistant Staphylococcus tes (MRSA) and Pseudomonas 2013/0303504 A1* 11, 2013 Gu et al. .. 514,202 aeruginosa Synthesis and Structure—activity Relationships': The 2013/0345190 A1* 12/2013 Gu et al...... 514,207 Journal of Antibiotics 2002, vol. 55, No. 11, pp.975-992. Ida, T. et al. “CP6679, a new injectable cephalosporin with broad FOREIGN PATENT DOCUMENTS spectrum and potent activities against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa; Journal of FR 2812 635 A 2, 2008 Infection and Chemotherapy 2002, vol. 8, pp. 138-144. FR 2.930 553 A1 10/2009 Blizzard et al. Side chain SAR of bicyclic beta-lactamase inhibitors FR 2951 171 A 4/2011 (BLIs). 1. discovery of a class CBLI for combination with imipinem; KR 2010 130176 A 12/2010 Bioorganic & Medicinal Chemistry Letters; 2010, vol. 20, pp. 918 WO WO O2, 10172 A1 T 2002 921. WO WO 03/063864 A2 T 2003 Coleman: Diazabicyclooctanes (DBOs): a potent new class of non WO WO2005,108391 A1 11, 2005 WO WO2006/125974 A1 11, 2006 beta-lactam beta-lactamase inhibitors; Current Opinion in WO WO2007/1291.76 A2 11, 2007 Microbiology; 2011, vol. 14, pp. 1-6. WO WO 2009/09.1856 A2 T 2009 Milleretal:Practical and Cost-Effective Manufacturing Route for the WO WO2009/133442 A1 11, 2009 Synthesis of a beta-Lactamase Inhibitor; Organic Letters, 2014, vol. WO WO 2010-118361 A1 1, 2010 16, No. 1, pp. 174-177. WO WO 2010-056827 A1 5, 2010 WO WO2010, 126820 A1 11, 2010 * cited by examiner U.S. Patent Feb. 24, 2015 Sheet 1 of 31 US 8,962,843 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 1nc, –0so

NH 704 O us –0so HN 1,n NH 705 O H2N ls -Nt-g –0so 3 H H 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 Feb. 24, 2015 Sheet 2 of 31 US 8,962,843 B2

FIGURE 1B

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

O HN –oso

72 of H2N ul NO)- –0so 713 O HN( );

714 O . N 1-1)* H OH 715 O nullf

H 716 O 1\-1'N-1s o 717 O or. NH2 718 S 1N- –0son 719 S "O) 720 O 1n 1 --OSOH

NH 721 S us --OSOH HN H 1.na 722 O -N 1su, --OSOH H U.S. Patent Feb. 24, 2015 Sheet 3 of 31 US 8,962,843 B2

FIGURE 1C

Table Compounds of Formula A-II Cmpd R R No. NH / 723 O H2N ul- r’s -o-OSOHoso,

HN 724 O v)- s –0so * 725 O Dr. --OSOH

Y 726 O Nui, --Osor HN 727 O to-O-- –0so

728 O HNIII II I III -o-OSOH

HN 729 O

OH H 730 O "' -- –0so NH

H 731 O ---, –0so NH

732 O "N-N-y –0so

U.S. Patent Feb. 24, 2015 Sheet 5 of 31 US 8,962,843 B2

FIGURE 1E

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

HN Y

76 o|| HN Afr’sN or 747 O turkO- –0so

NH 748 O HN ls it (O- –0so

NH HN ls -O-- –0so "OXO- –0so

to-O is na –0so

MH 752 O - C R –0so wY-3-6. 753 O -O –0so H2N

754 O –0son HN 1

Y 755 O N-li, –0so H2N 756 O O N Nui.Y –0so w U.S. Patent Feb. 24, 2015 Sheet 6 of 31 US 8,962,843 B2

FIGURE 1 F

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

H w Y 757 O 17 N-6. –0so

2 W1 758 O C): –0so 759 O C-- –0so 760 O C-- –0so N 761 O C) –0so

762 O C-- –0so N

763 O C. D

764 O "O o 765 O Or' –0so

766 O C-1 W D

767 O 5) D U.S. Patent Feb. 24, 2015 Sheet 7 of 31 US 8,962,843 B2

FIGURE 1G

Table Compounds of Formula A-II

771 O HN "N-1-

772 O HN N-1-1 773 O to-O-- -: 774 O -O- –0so 775 O to ( )--. 776 O / \1\- D

777 O HN N-s --OSOH

HN 778 O ()-- --OSOH

HN 779 O ( ); 780 O to-O-

U.S. Patent Feb. 24, 2015 Sheet 9 of 31 US 8,962,843 B2

FIGURE I

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

794

795 to-O--

796

797

798 U.S. Patent US 8,962,843 B2

U.S. Patent Feb. 24, 2015 Sheet 11 of 31 US 8,962,843 B2

-- )) ------w s e d s - -- ce

{{ZPIRITTOIH U.S. Patent Feb. 24, 2015 Sheet 12 of 31 US 8,962,843 B2

. U.S. Patent Feb. 24, 2015 Sheet 13 of 31 US 8,962,843 B2

--ee V W O

VVVVVV V O

v 0 OOO C)OO OC)

CIZHRH(10.IH 06.DIJN U.S. Patent Feb. 24, 2015 Sheet 14 of 31 US 8,962,843 B2

U.S. Patent Feb. 24, 2015 Sheet 15 of 31 US 8,962,843 B2

s ca 3 H. M a liff SM U.S. Patent Feb. 24, 2015 Sheet 16 of 31 US 8,962,843 B2

OOOy:punoduuooJo?eJedtuooSI O is as V y U.S. Patent Feb. 24, 2015 Sheet 17 of 31 US 8,962,843 B2

US 8,962,843 B2

N HIIIIIIIminiN N U.S. Patent Feb. 24, 2015 Sheet 20 of 31 US 8,962,843 B2

SOINÍ10dIWOO

U.S. Patent Feb. 24, 2015 Sheet 22 of 31 US 8,962,843 B2

punoduuoouoleleduuoos?OOO SI-VXO

U.S. Patent Feb. 24, 2015 Sheet 24 of 31 US 8,962,843 B2

punoduuoouoleueduuoos?OOO

U.S. Patent Sheet 26 of 31 US 8,962,843 B2

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{{SETRITOIH U.S. Patent Feb. 24, 2015 Sheet 28 of 31 US 8,962,843 B2

---- Hº?S?ÇpunodLuooJo?ereduuooSIOOO U.S. Patent Feb. 24, 2015 Sheet 29 Of 31 US 8,962,843 B2

- Arti

w s s m

H N HIIIIIII,11:N N © U.S. Patent Feb. 24, 2015 Sheet 30 of 31 US 8,962,843 B2

4. O 4. O 1. OO

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6ETHI)OIH US 8,962,843 B2 1. 2 1,3,4-OXADIAZOLE AND 1,3,4-THADIAZOLE R is selected from BETA-LACTAMASE INHIBITORS

RELATED APPLICATIONS 5 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 CH2CO2H, and CF2CO2H entire contents of these applications are incorporated herein by reference in their entireties. and, TECHNICAL FIELD R" is selected from: 15 This disclosure is directed to B-lactamase inhibitors (BLIS) a. which are effective as inhibitors of B-lactamases and, when used in combination with B-lactam antibiotics are useful in the treatment of bacterial infections. The compounds when b. combined with a B-lactam antibiotic are effective in treating 20 infections caused by bacteria that are resistant to B-lactam antibiotics due to the presence of B-lactamases. Pharmaceu tical compositions comprising Such compounds, methods of using Such compounds, and processes for preparing Such compounds are also disclosed. 25 W1herein R& 1Sis SCCOselected f

BACKGROUND NR Bacterial resistance to B-lactam antibiotics, especially in 30 l Gram-negative bacteria, is most commonly mediated by NHR, and N NHR', B-lactamases. B-lactamases are enzymes that catalyze the H hydrolysis of the B-lactam ring, which inactivates the anti bacterial activity of the B-lactam antibiotic and allows the bacteria to become resistant Inhibition of the B-lactamase 35 wherein each of R. RandR is independently selected with a BLI slows or prevents degradation of the B-lactam from hydrogen, (C-C)-alkyl, aminoalkyl, aminocy antibiotic and restores -lactam antibiotic susceptibility to cloalkyl, and hydroxyalkyl, and n is selected from 1, 2 B-lactamase producing bacteria. Many of these B-lactamases and 3; 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 C. for novel BLIs that inhibit B-lactamases that are not effec- O 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.

SUMMARY OF INVENTION 6 : wherein R is H. (C-C) alkylamino, and 50 The present invention provides, in one aspect, compounds of chemical formula (I), orpharmaceutically-acceptable salts NH thereof, which are BLIs and are useful in combination with B-lactam antibiotics for the treatment of bacterial infections. NH2: 55

N (I) d. / amino; R-K X 60 C. N H N NR7, n Ou- R pg 65 wherein X is selected from O and S; US 8,962,843 B2 3 4 wherein R is selected from H. (C-C)-unsubstituted wherein R is selected from alkyl, amino-(C-C)-alkyl, aminocycloalkyl, hydroxyalkyl, NR6" and 5 NHR', and R'R''N l % NH

NH2, 10 R" is selected from hydrogen, (C-C)-alkyl, ami noalkyl, aminocycloalkyl, hydroxyalkyl,

and each of p and q is independently selected from 1 and NH 2; and 15 l f. CH(R)CH-NH, HN in H.N N wherein R is selected from amino and hydroxyl. 8. s In another aspect, the invention provides compounds of chemical Formula (A-I) or a pharmaceutically acceptable salt 2O each of R", R. Rand R7" is independently thereof, which are BLIs and are useful in combination with selected from hydrogen or (C-C)-alkyl, aminoalkyl, B-lactam antibiotics for the treatment of bacterial infections. aminocycloalkyl, and hydroxyalkyl, provided that at least one of R", R. Rand R7 "is hydrogen, 25 n is selected from 1, 2, 3 and 4, and (A-I) m is selected from 1, 2 and 3; NN R"-( N X" C 30 O N H

Oul NR R8." s

wherein X* is selected from O and S; 35 wherein R 8 * is: selected from NH, R* is selected from NR6" 40 ors’. or RRSN l NRI I ck s OSOH, SOH, OPOH, wherein each of R. R. RandR is as described previously and each of R. R', and R'' is indepen 45 dently selected from hydrogen or (C-C)-alkyl, pro vided that at least one of R. R', and R'' is hydrogen; CH2CO2H and CF2COH:

and (R 18 y R" is selected from: Z (T),-(CH), 55

wherein Z is selected from CR'R' or NR'', hydrogen; each of R'' and R' is independently selected from H. b 60 NH, and US 8,962,843 B2 5 wherein each of R. R. RandR is as described previously, alternatively, R'' and R' together with the carbon to which they are attached, form a cycloalkyl or hetero cyclyl ring containing 4-6 ring members, R" is selected from Hand

NR17 wherein R is selected from NH, -NH(C-C)-alkyl and 10

wherein each of R', Rand R" is independently selected from hydrogen, (C-C)- 15 l, X alkyl, aminoalkyl, aminocycloalkyl, and hydroxy alkyl, provided that at least one of R', Rand R7 is wherein each of R. R. Rand R7" is as described hydrogen, previously, R" is selected from NH and s is selected from 0 and 1, and V is selected from 0, 1, 2, and 3;

NR7 ck 25 HN wherein each of R', R, Rand R7 is as described previously, each of p and q is independently selected from 0, 1, 2 and 3, 30 T is selected from NH and O M N-(CH2) s t is selected from 0, 1, 2, 3, and 4, and each of r and y is independently selected from 0 and 1: 35 wherein M is selected from NR, CR'R'' and O, wherein R’ is Hor sus-X 40 wherein R' is selected from NH and

45 wherein each of R', R' and R7 is as described previously, NR7 c. each of R and R is independently selected from H, NH and wherein each of 50 R. R. RandR is as described previously, R’ is selected from amino and hydroxyl, and w is selected from 0 and 1: l, X 55 9. wherein each of R. R. Rand R7 "is as described previously, and

60 u is selected from 0, 1 and 2:

65 wherein each of R. R. Rand R7" is as described K2 previously; US 8,962,843 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 HN ; and aSS FIG. 9 shows Table IX, Standard BLI Potentiation MIC Assay of Compounds Partnered with Meropenem Against a y Panel of Isogenic and Clinical Strains Expressing B-Lacta aSS

10 DETAILED DESCRIPTION s Definitions Molecular terms, when used in this application, have their In one embodiment, the invention provides use of a com common meaning unless otherwise specified. pound of Formula I for inhibiting B-lactamases. 15 The term “alkyl is defined as a linear or branched, satu In one embodiment, the invention provides use of a com rated radical having one to about twenty carbon atoms unless pound of Formula A-I for inhibiting B-lactamases. otherwise specified. Preferred alkyl radicals are “lower alkyl In one embodiment, the invention provides compounds of radicals having one to about five carbon atoms. Examples of Formula 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 invention provides compounds of “(C-C)-unsubstituted alkyl” which is defined as an alkyl Formula A-I with high binding affinity for B-lactamase group that bears no substituent groups. Examples of (C-C)- enzymes. 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 25 tuted that one or more hydrogen atoms is replaced by a mula I and at least one B-lactam antibiotic. substitutent. In one embodiment, the present invention also provides The term amino denotes a NH2 radical. antibacterial compositions comprising compounds of For The term “aminoalkyl denotes an alkyl in which one or mula A-I and at least one B-lactam antibiotic. In one embodiment, the present invention provides phar more of the alkyl hydrogen atoms has been replaced by an maceutical compositions comprising compounds of Formula 30 amino group. I and at least one ?-lactam antibiotic and methods of use The term "aminocycloalkyl denotes a cycloalkyl in which thereof. one of the cycloalkyl hydrogenatoms has been replaced by an In one embodiment, the present invention provides phar amino group. maceutical compositions comprising compounds of Formula The term “cycloalkyl or “cycloalkyl ring is defined as a A-I and at least one 3-lactam antibiotic and methods of use 35 saturated or partially unsaturated carbocyclic ring in a single thereof. or fused carbocyclic ring system having from three to twelve In one embodiment, the invention provides methods of use ring members. In a preferred embodiment, a cycloalkyl is a of the compounds of Formula Ito treat bacterial infections in ring system having three to seven ring members. Examples of a subject. a cycloalkyl group include, without limitation, cyclopropyl. In one embodiment, the invention provides methods of use 40 cyclobutyl, cyclohexyl, and cycloheptyl. of the compounds of Formula A-I to treat bacterial infections The term “hydroxyalkyl denotes an alkyl radical in which in a Subject. one or more of the alkyl hydrogenatoms has been replaced by a hydroxyl group. BRIEF DESCRIPTION OF THE DRAWINGS It will be understood by one of skill in the art that a 45 FIGS. 1A-1I show Table I, Representative Compounds of Formula A-II FIGS. 2A-2G show Table II, Standard BLI potentiation or MIC assay against a panel of isogenic and clinical strains expressing B-lactamases. 50 denote the point of attachment of a Substituent group where FIGS. 3A-3F show Table III, the synergy MIC of represen indicated. For example tative compounds of Formula II-A against a panel of isogenic and clinical Strains expressing 3-lactamases. 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 NHRs Compounds Against a Panel of Isogenic and Clinical Strains Expressing B-lactamases FIG. 6 shows Table VI, Standard BLI Potentiation MIC 60 represent that the point of attachment of the amide moiety is Assay of Compounds Partnered with CXA-101 Against a at the carbonyl carbon. Panel of Isogenic and Clinical Strains Expressing B-Lacta The functional classification of B-lactamases and terms aSS “Class A. “Class C, and “Class D B-lactamases are under FIG. 7 shows Table VII, Standard BLI Potentiation MIC stood by one of skill in the art and are described in “Updated Assay of Compounds Partnered with Ceftazidime Against a 65 Functinal Classification of B-Lactamases, Bush, K.: Jacoby, Panel of Isogenic and Clinical Strains Expressing B-Lacta G. A.; Antimicrob. Agents Chemother. 2010, 54, 969-976, aSS herein incorporated by reference. US 8,962,843 B2 9 10 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 Vivo half-life or reduced dosage requirements). In yet another tion salts of the compounds of the invention may be prepared embodiment, Substitution with positron emitting isotopes, from an inorganic acid or an organic acid. Examples of Such 10 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 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 15 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 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 25 organic salts made from N,N'-dibenzylethylenediamine, biological assays Such as those described herein. chloroprocaine, choline, diethanolamine, ethylenediamine, B-Lactamase Inhibitors (BLIs) N-methylglucamine, lysine and procaine. All of these salts 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 30 compound of the invention with the appropriate acid or base. The compounds of the invention can possess one or more (I) asymmetric carbon atoms and are thus capable of existing in NN the form of optical isomers as well as in the form of racemic N or non-racemic mixtures thereof. The compounds of the 35 R-( invention can be utilized in the present invention as a single X isomer or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposable stereochemical N H isomers, can be separated by conventional means such as N chromatography, distillation, crystallization or Sublimation. 40 A N The optical isomers can be obtained by resolution of the O R racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment The substituent X of Formula I is selected from O and S. In with an optically active acid or base. Examples of appropriate one aspect of the invention X is S. In another aspect of the acids include, without limitation, tartaric, diacetyltartaric, 45 invention X is O. dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. The mixture of diastereomers can be separated by crys Substituent R of Formula I is selected from tallization followed by liberation of the optically active bases from the optically active salts. An alternative process for separation of optical isomers includes the use of a chiral 50 chromatography column optimally chosen to maximize the OSOH, SOH, OPOH, separation of the enantiomers. Still another available method involves synthesis of covalent diastereoisomeric molecules by treating compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate. The 55 CHCO2H, and CF2CO2H. synthesized diastereoisomers can be separated by conven tional means such as chromatography, distillation, crystalli zation or sublimation, and then hydrolyzed to obtain the enantiomerically pure compound. The optically active com In a preferred embodiment, R is pounds of the invention can likewise be obtained by utilizing 60 optically active starting materials. These isomers may be in the form of a free acid, a free base, an ester or a salt. Compounds described herein also include isotopically-la beled compounds wherein one or more atoms is replaced by OSOH. an atom having the same atomic number, but an atomic mass 65 or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes Suit US 8,962,843 B2 11 12 The group R' of Formula I is selected from: In one aspect of the invention R' is selected from H. a hydrogen; —CH-NH, CHCH-NH – CONH, -NH, -CH (OH)CH-NH. -CH(NH)CH-NH. - CHCH-NHCHCH-NH2, b. 5

pi R2, NH 10 HN2 ~y H2 1. ry wherein R is selected from NH

NR H2 ----XH NHR, and l s 15 NH N NHR -- H HN s wherein each of R. RandR is independently selected 2O from hydrogen, (C-C)-alkyl, aminoalkyl, aminocy NH cloalkyl, and hydroxyalkyl, and n is selected from 1, 2 and 3; ls HN HN N s s

25 O HN s a NH wherein R is H., (C-C) alkylamino, and HN N , and

NH 35 HN N > NH2: In one embodiment of the invention R' is selected from H 40 and d. amino;

C. HN 45 NR7, p In one embodiment of the invention, the compounds of the invention are of the stereochemistry disclosed in Formula II. wherein R is selected from H. (C-C)-unsubstituted 50 alkyl, amino-(C-C)-alkyl, aminocycloalkyl, hydroxyalkyl, (II) N and NN 55 R-(x 1%,J NH N I I IIH

NH2 N N 60 O R and each of p and q is independently selected from 1 and 2; and In another embodiment of the invention, the compound is f. CH(R)CH-NH, of Formula II and X is O, Ris-OSOH and R' is H. 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. - CH-NH. US 8,962,843 B2 13 14 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, R is OSOH and R' is of Formula II and X is O, Ris-OSOH and R' is —CHCH-NH.

In another embodiment of the invention, the compound is 5 of Formula II and X is O, Ris-OSOH and R' is NH --O l NH2. 10 carsN NH.2 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 O, Ris-OSOH and R' is 15

NH x- 1.2 N 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 CONH. 25 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 -NH. In another embodiment of the invention, the compound is NH of Formula II and X is O, Ris-OSOH and R is 30 > --> -- 2 NH. 35 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. 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 O, R is OSOH and R' is 40 CHCH-NHCHCH-NH. In another embodiment of the invention, the compound is NH of Formula II and X is O, R is OSOH and R' is —CH (NH2)CH-NH. N l NH2. 45 In another embodiment of the invention, the compound 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 In another embodiment of the invention, the compound is of Formula II and X is S, R is OSOH and R' is of Formula II and X is O, Ris-OSOH and R' is 50

NH. NH. 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 60 of Formula II and X is O, Ris-OSOH and R is

NH.

65 US 8,962,843 B2 15 16 In another embodiment of the invention, the compound is In a preferred embodiment, R* is of Formula and II X is S. Ris-OSOH and R' is

NH OSOH. cars 1.2 The group R' of Formula I is selected from: 10 a. hydrogen; Preferred compounds of Formula I are the compounds:

b. N 15 ( -- R2" pi and N IIH R" is selected from ? N O R

? 25 HN o-1, - X N ... I I IIH.

N N 30 R is selected from hydrogen, (C-C)-alkyl, aminoalkyl, O R aminocycloalkyl, hydroxyalkyl,

It will be understood by one of skill in the art that depend ing on the nature of R' and R, compounds of Formula I may NH 35 exist in a salt or Zwitterionic form. HN iii. -- iii. In one aspect, the invention provides compounds of For and H s mula A-I or pharmaceutically-acceptable salts thereof:

40 each of R", R. Rand R7 is independently selected (A-I) from hydrogen or (C-C)-alkyl, aminoalkyl, aminocy NN N cloalkyl, and hydroxyalkyl, provided that at least one of R"-( R", R", Rand R7" is hydrogen, X" n is selected from 1, 2, 3 and 4, and 45 N H m is selected from 1, 2 and 3;

N OZ N.R

50 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. Substituent R* of Formula A-I is selected from 55 wherein R is selected from NH,

--ol - on --ol 60

wherein each of R. R. Rand R7 is as described --not and --Geo 65 previously 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 8,962,843 B2 17 18 wherein R' is selected from NH and d. amino;

C. R18 (Seeg S-a-citype s wherein each of R. R. RandR is as described previously, Z is selected from CR'R' or NR'', R’ is selected from amino and hydroxyl, and each of R'' and R' is independently selected from H, NH, w is selected from 0 and 1: and 15

h. wherein each of R. R. Rand R7 is as described previously, 25 alternatively, RandR' together with the carbon to which they are attached, form a cycloalkyl or heterocyclyl ring containing 4-6 ring members, R" is selected from Hand wherein each of R', Rand R7 is as described previ 30 ously; NR17

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' 40 wherein R is selected from NH, -NH(C-C)-alkyland and R'' is hydrogen, R' is selected from NH and

wherein each of R. R. Rand R7" is as described wherein each of R. R. 50 previously, Rand R is as described 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 8,962,843 B2 19 20 wherein M is selected from NR, CR'R'' and O, -continued wherein R’ is Hand NH

NR17 HN s to O-- NH

HN N s s s 10 \ / H wherein each of R', RandR'7 is as described previ OH ously, each of RandR is independently selected from H, NH, HN2 s HN1\1 n-g and 15

wherein each of R. R. Rand R7" is as described previously, and 25 u is selected from 0, 1 and 2: ry --X s 30 NH

1. NH ; and

OH 45 H2N, H n "N." s NH OH 50 H "N." s NH OH OH 55 "N."H s in-l-X. NH

US 8,962,843 B2 23 24 -continued In one embodiment of the invention R' is selected from

HN s

5 H, HN s s -N H

rx O2 10 HN s K)^-y HN-(O- , and HN

15 In one embodiment of the invention, the compounds of the 's-X s invention are of the stereochemistry disclosed in Formula NH A-II.

HN ls N -->{ s HN-(O-- 2O H (A-II) NN N DN s HN -- s R-(- 25 x -1. / \

30 In one embodiment of the invention R' is selected from In another embodiment of the invention, X*, R* and R' are chosen from the substituents listed in Table I (See FIG.I). Preferred compounds of Formula A-I are H, ~y 35

-N ~y 40 N I IIH HN Z N Yx 45 O OSOH,

HN N crx or x Y-N-( -- N H 55 a N O OSOH,

N 60 N l.

O) - 65 US 8,962,843 B2 25 26 -continued Enzyme Inhibition and Binding Affinity NH The compounds of the invention (e.g. comp ounds of For - NNN mula I, compounds of Formula A-I, compounds of Formula HN / 5 II, compounds of Formula A-II) are effective in inhibiting B-lactamase. In one aspect of the invention the compounds of X-(J%. Table I are effective f3-lactamase inhibitors. In one aspect the compound

10 O2. N OSOH, NNN HN / N O 4, 15 ( -- N . . . H N . IIH a N O OSOH, 2O u1 N O YOSOH W o-1, is effective in inhibiting B-lactamase. 25 In one aspect the compound N ... I I IIH HN

O N OSOH, HN N / 30 NH l, O- Y - . N . I IIH 35 N O OSOH, is effective in inhibiting B-lactamase. ZN In one aspect the compound C-o-1%, 40 N . I I IIH N N H OA N OSOH, 45 N / s -- 50 N H s 1 is effective in inhibiting B-lactamase. HN O OSOH, In one aspect the compound N HN / 55 o-1,

N II I I IIH 60

O N OSOH,

It will be understood by one of skill in the art that depend- 65 ing on the nature of R'' and R*, compounds of Formula I may exist in a salt or Zwitterionic form. is effective in inhibiting B-lactamase. US 8,962,843 B2 27 28 In one aspect the compound 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 N N-(7 l 10 is effective in inhibiting B-lactamase. OSOH, When used in combination with B-lactam antibiotics, the 15 compounds of the invention (e.g. compounds of Formula I, inhibits 3-lactamases selected from class A, class C or class D compounds of Formula A-I, compounds of Formula II, com B-lactamases. In one aspect of the invention the compound of pounds of Formula A-II) potentiate the activity of the B-lac the Formula tam antibiotic against microorganisms that are normally resistant to B-lactam antibiotics due to the presence of a B-lactamase or multiple B-lactamases. In one aspect of the invention the compounds of the inven tion (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) 25 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 OSOH, C or class D B-lactamases. In one aspect of the invention the compounds of Formula A-I inhibit B-lactamases selected 30 inhibits 3-lactamases selected from class A, class C or class D from class A, class C or class D3-lactamases. In one aspect of B-lactamases. In one aspect of the invention the compound of the invention the compounds of Formula II inhibit B-lacta the Formula mases selected from class A, class C or class D B-lactamases. In one aspect of the invention the compounds of Formula A-II inhibit B-lactamases selected from class A, class C or class D 35 B-lactamases. In one aspect of the invention the compounds 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 40

OSOH,

45 inhibits 3-lactamases selected from class A, class C or class D B-lactamases. Class A B-lactamases for example, include, but . I I IIH are not limited to, TEM, SHV, CTX-M, KPC, GES, VEB, SME, and GEX. In a preferred aspect of the invention, the Y. compounds of the invention (e.g. compounds of Formula I, O YOSOHs 50 compounds of Formula A-I, compounds of Formula II, com pounds of Formula A-II) inhibit KPC B-lactamases. In a pre inhibits 3-lactamases selected from class A, class C or class D ferred aspect of the invention, the compounds of Formula I B-lactamases. In one aspect of the invention the compound of inhibit KPC B-lactamases. In a preferred aspect of the inven the Formula 55 tion, the compounds of Formula A-I inhibit KPC B-lacta mases. In a preferred aspect of the invention, the compounds HN N of Formula II inhibit KPC 5-lactamases. In a preferred aspect of the invention, the compounds of Formula A-II inhibit KPC B-lactamases. More preferably the compounds of the inven Y-N-X -- 60 tion (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) N . I I IIH inhibit KPC-2 or KPC-3 f-lactamases. More preferably the Y compounds of Formula I inhibit KPC-2 or KPC-3 B-lacta O YOSO,H, 65 mases. More preferably the compounds of Formula A-I inhibit KPC-2 or KPC-3 f-lactamases. More preferably the compounds of Formula II inhibit KPC-2 or KPC-3 B-lacta US 8,962,843 B2 29 30 mases. More preferably the 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 B-lactamases. In one aspect of the preferred aspect of the invention, the sMIC value for the invention, the compounds of the invention (e.g. compounds compounds of the invention (e.g. compounds of Formula I, of Formula I, compounds of Formula A-I, compounds of compounds of Formula A-I, compounds of Formula II, com Formula II, compounds of Formula A-II) inhibit KPC-2 or 5 pounds of Formula A-II) is 0.2 to 0.5 g/mL. Synergy MICs KPC-3 B-lactamases in clinical strains (FIG. 2, Table II and for representative compounds of the invention are described FIGS. 6-9, Tables VI-IX). In one aspect of the invention, the in Table III (See FIG.3). It will be understood by one of skill compounds of Formula I inhibit KPC-2 or KPC-3 B-lacta in the art that the growth inhibition off-lactamase producing mases in clinical strains (FIG. 2, Table II and FIGS. 6-9. 10 strains can also be measured by a checkerboard synergy assay Tables VI-IX). In one aspect of the invention, the compounds like that disclosed in International Patent Application Num of Formula A-I inhibit KPC-2 or KPC-3 f-lactamases in ber WO 2008/039420 or a standard BLI potentiation assay clinical strains (FIG. 2, Table II and FIGS. 6-9, Tables VI-IX). using a fixed concentration of BLI. In one aspect of the invention, the compounds of Formula II In one embodiment, the BLI activity is measured by growth inhibit KPC-2 or KPC-3?-lactamases in clinical strains (FIG. 15 inhibition of a B-lactamase producing bacterial strains in a 2, Table II and FIGS. 6-9, Tables VI-IX). In one aspect of the standard BLI potentiation assay using a fixed concentration invention, the compounds of Formula A-II inhibit KPC-2 or of BLI. Preferably, the MIC value for the compounds of the KPC-3 B-lactamases in clinical strains (FIG. 2, Table II and invention (e.g. compounds of Formula I, compounds of For FIGS. 6-9, Tables VI-IX). Class C B-lactamases for example, mula A-I, compounds of Formula II, compounds of Formula include, but are not limited to chromosomal AmpCs, and A-II) is 8 g/mL or less. In a more preferred aspect of the plasmid based ACC, DHA, CMY FOX, ACT, MIR, LAT, invention, the MIC value for the compounds of the invention MOX f-lactamases. Class D B-lactamase enzymes, for (e.g. compounds of Formula I, compounds of Formula A-I. example, include, but are not limited to oxacillinases or OXA compounds of Formula II, compounds of Formula A-II) is 4 to B-lactamases. In a preferred aspect of the invention, the com 25 8 ug/mL. In an even more preferred aspect of the invention, pounds of the invention (e.g. compounds of Formula I, com the MIC value for the compounds of the invention (e.g. com pounds of Formula A-I, compounds of Formula II, com pounds of Formula I, compounds of Formula A-I, compounds pounds of Formula A-II) inhibit OXA-15 B-lactamases. In a of Formula II, compounds of Formula A-II) is 1 to 2 ug/mL. preferred aspect of the invention, the compounds of Formula 30 In a still more preferred aspect of the invention, the MIC value I inhibit OXA-15 B-lactamases. In a preferred aspect of the for the compounds of the invention (e.g. compounds of For invention, the compounds of Formula A-I inhibit OXA-15 mula I, compounds of Formula A-I, compounds of Formula B-lactamases. In a preferred aspect of the invention, the com II, compounds of Formula A-II) is 0.2 ug/mL to 0.5ug/mL. pounds of Formula II inhibit OXA-15 B-lactamases. In a The compounds of the present invention (e.g. compounds preferred aspect of the invention, the compounds of Formula 35 of Formula I, compounds of Formula A-I, compounds of A-II inhibit OXA-15 B-lactamases. Formula II, compounds of Formula A-II) have a broad spec Unless otherwise indicated, the activity of the BLI com trum of activity across a wide variety off-lactamase produc pounds can be described by the MIC value obtained from a ing bacteria. It was surprisingly found that the compounds of Synergy MIC assay or a BLI potentiation assay (e.g. as the present invention (e.g. compounds of Formula I, com described herein), both of which are run in the presence of a 40 pounds of Formula A-I, compounds of Formula II, com B-lactam. The lower the sMIC or MIC value the more active pounds of Formula A-II) are active in potentiating activity of the BLI, regardless of the mechanism of action of the BLI B-lactam antibiotics, in particular, Ceftolozane, against compound (e.g., including inhibition of B-lactamases by the strains expressing class D B-lactamase OXA-15 B-lactamase. BLI or any other mechanism of action or combination of 45 Currently marketed BLIs inhibit most of the class A B-lacta mechanisms of action). The SMIC and BLI potentiation assay mases, but poorly inhibit class A KPCB-lactamases and class data Supports that the compounds of the invention (e.g. com C B-lactamases and have variable Success in inhibiting peni pounds of Formula I, compounds of Formula A-I, compounds cillinase and carbapenemase-type class D B-lactamases. The of Formula II, compounds of Formula A-II) potentiate (i.e. compounds of the present invention (e.g. compounds of For make more potent) the activity of the B-lactam antibiotic 50 mula I, compounds of Formula A-I, compounds of Formula against B-lactamase producing strains by inhibiting the B-lac II, compounds of Formula A-II) are active against a wide tamase. variety of bacterial strains that express class A and C 3-lac In one embodiment, the BLI activity is measured by growth tamases and also, Surprisingly are active against bacterial inhibition of a B-lactamase producing bacterial strains in a 55 strains that express the class D cephalosporinase OXA-15 Synergy MIC (sMIC) assay. Preferably, the sMIC value for (Tables II and III). This increased activity against the class D the compounds of the invention (e.g. compounds of Formula B-lactamase is critical because differential effectiveness I, compounds of Formula A-I, compounds of Formula II, against different types of B-lactamase producing bacteria is compounds of Formula A-II) is 8 g/mL or less. In a more necessary in order to effectively use f-lactam antibiotics to preferred aspect of the invention, the sMIC value for the 60 treat resistant strains of bacteria (vide infra). compounds of the invention (e.g. compounds of Formula I, In one embodiment, the compounds the invention (e.g. compounds of Formula A-I, compounds of Formula II, com compounds of Formula I, compounds of Formula A-I, com pounds of Formula A-II) is 4 ug/mL to 8 ug/mL. In an even pounds of Formula II, compounds of Formula A-II) are unex more preferred aspect of the invention, the SMIC value for the 65 pectedly more active against bacterial strains that express compounds of the invention (e.g. compounds of Formula I, OXA-15 -lactamases than the most structurally similar com compounds of Formula A-I, compounds of Formula II, com pound, Avibactam (comparator compound CCC). Com US 8,962,843 B2 31 32 pounds that are more active than Avibactam against bacterial 759, 760, 761, 762, 763, 764, 765, 766, 769,771, 772, 773, strains that express the class D cephalosporinase OXA-15 are, 774, 776, 777, 778, 780, 783, 784, 786, 792, 796, and 797 for example, compounds 701, 702, 703,704, 705, 706, 707, (Table IV). 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 719, 720, It was surprisingly found that the compounds of the present 721, 722, 723, 724, 725, 726, 727, 728, 729,730, 731, 732, invention have a higher binding affinity for the B-lactamase 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, enzyme than the closest structural comparator Avibactam 745, 746, 747, 748,749, 750, 751, 752, 753, 754, 755, 756, (Table IV. See FIG. 4). 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, The compounds of the invention were also shown to be 769, 770, 771, 772, 773,774, 776, 777,778, 779, 780, 781, 10 better BLIs than other comparator compounds as shown in 782, 783, 784, 792, 794, 795 and 797. FIG.S. In one embodiment, the compounds of the invention (e.g. Pharmaceutical Compositions Comprising the Compounds compounds of Formula I, compounds of Formula A-I, com of The Invention and Use Thereof pounds of Formula II, compounds of Formula A-II) are unex 15 Another object of the invention is pharmaceutical compo pectedly more active against and/or show broader spectrum sitions or formulations comprising compounds the invention of activity against bacterial strains that express KPC B-lacta (e.g. compounds of Formula I, compounds of Formula A-I. mases than the most structurally similar compound, Avibac compounds of Formula II, compounds of Formula A-II), or tam. Compounds that are more active than Avibactam for at salts thereof, preferably further comprising a B-lactam anti least one, bacterial strain that expresses KPC B-lactamase biotic. In one embodiment of the invention is pharmaceutical and/or show a better spectrum of activity against bacterial compositions or formulations comprising compounds of For strains that express KPC B-lactamases than Avibactam are, mula I, or salts thereof, preferably further comprising a 3-lac for example, compounds 701, 702, 703,705, 706, 708, 709, tam antibiotic. In one embodiment of the invention is phar 25 maceutical compositions or formulations comprising 710, 711, 712714, 720, 721, 722, 723, 724, 726, 727, 728, compounds of Formula A-I, or salts thereof, preferably fur 729, 730, 731, 732, 734, 735, 736, 737, 738,739, 740, 741, ther comprising a 3-lactam antibiotic. In one embodiment of 742, 743, 744, 745, 746, 747, 749, 750, 751, 752, 753, 754, the invention is pharmaceutical compositions or formulations 755, 756, 758, 759, 760, 761, 762, 763, 764,765, 766, 767, comprising compounds of Formula II, or salts thereof, pref 768, 770, 771, 772, 774, 775, 776, 777, 779, 782, 783,784, 30 786, 794, and 795. erably further comprising a B-lactam antibiotic. In one In another aspect of the invention, the compounds of the embodiment of the invention is pharmaceutical compositions invention (e.g. compounds of Formula I, compounds of For or formulations comprising compounds of Formula A-II, or mula A-I, compounds of Formula II, compounds of Formula salts thereof, preferably further comprising a B-lactam anti A-II) have high binding affinity for the B-lactamase enzyme. 35 biotic. In one embodiment of the invention is pharmaceutical Consequently these compounds are better inhibitors of the compositions or formulations comprising compounds of B-lactamase enzyme. The inhibition kinetics of the com Table I. In one embodiment of the invention is pharmaceutical pounds of the invention (e.g. compounds of Formula I, com compositions or formulations comprising compounds of For pounds of Formula A-I, compounds of Formula II, com mula pounds of Formula A-II) was measured according to the 40 procedure outlined in Example 102. The compounds of the invention (e.g. compounds of Formula I, compounds of For mula A-I, compounds of Formula II, compounds of Formula A-II) have a high binding affinity for the B-lactamase enzyme. 45 In one embodiment the compounds of the invention (e.g. 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 mM's'. OSOH, In one embodiment the compounds of the invention (e.g. 50 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) have a antibiotic. In one embodiment of the invention is pharmaceu binding affinity of 100-999 mM's'. Compounds that have a tical compositions or formulations comprising compounds of Formula binding affinity of 100-999 mM's are, for example, com 55 pounds 701,702, 703,705,706,707,709,711,712,713,714, 720, 730, 740, 741, 742, 743, 745, 746, 748, 749, 752, 753, HN N 767,770, 775, 779, 781,782,785,794, 798 (Table IV). In one embodiment the compounds of the invention (e.g. compounds of Formula I, compounds of Formula A-I, com 60 pounds of Formula II, compounds of Formula A-II) have a binding affinity of 1-99 mM's'. Compounds that have a binding affinity of 1-99 mM's are, for example, compounds 704, 706, 708, 710, 715, 716, 717, 718, 719, 721, 722, 723, 65 724, 725,726, 727, 728, 729, 731, 732,733, 734, 735, 736, 737, 738, 739, 744, 747, 750, 751, 754, 755, 756, 757, 758, US 8,962,843 B2 33 34 or salts thereof, preferably further comprising a B-lactam The pharmaceutical compositions can comprise one or antibiotic. In one embodiment the invention is pharmaceuti more of the compounds disclosed herein (e.g. one or more cal compositions or formulations comprising compounds of compounds of Formula I, compounds of Formula A-I, com Formula pounds of Formula II, compounds of Formula A-II), prefer ably a compound of Formula A-I or Formula A-II, in conjunc tion with a B-lactam antibiotic, in association with one or N 7 more nontoxic, pharmaceutically-acceptable carriers and/or N N-( l. diluents and/or adjuvants and/or excipients. As used herein, 10 the phrase “pharmaceutically-acceptable carrier refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical OSOH, administration. The use of Such media and agents for phar 15 maceutically active Substances is well known in the art. Non or salts thereof, preferably further comprising a B-lactam limiting examples of carriers and excipients include corn antibiotic. In one embodiment of the invention is pharmaceu starch or gelatin, lactose, Sucrose, microcrystalline cellulose, tical compositions or formulations comprising compounds of kaolin, mannitol, dicalcium phosphate, sodium chloride and Formula alginic acid. The compositions may contain croScarmellose Sodium, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid. Tablet binders that can be included are acacia, methylcel lulose, Sodium carboxymethylcellulose, polyvinylpyrroli 25 done (PoVidone), hydroxypropyl methylcellulose, Sucrose, starch and ethylcellulose. N . I I IIH Lubricants that can be used include magnesium Stearate or - 1 other metallic Stearates, Stearic acid, silicone fluid, talc, O YOSOHs waxes, oils and colloidal silica. 30 Flavoring agents such as peppermint, oil of wintergreen, or salts thereof, preferably further comprising a B-lactam cherry flavoring or the like can also be used. It may also be antibiotic. In one embodiment of the invention is pharmaceu desirable to add a coloring agent to make the dosage form tical compositions or formulations comprising compounds of more aesthetic in appearance or to help identify the product. Formula 35 For oral or parenteral administration, compounds of the present invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) preferably a compound of Formula A-I or Formula A-II, in conjunction with a B-lactam antibiotic, can 40 be mixed with conventional pharmaceutical carriers and 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 O OSOH, 45 about 0.1% to about 99% by weight of the active compound, such as from about 10% to about 30%. or salts thereof, preferably further comprising a B-lactam For oral use, Solid formulations such as tablets and cap antibiotic. Sules are useful. Sustained release or enterically coated prepa rations may also be devised. Forpediatric and geriatric appli The pharmaceutical compositions can be formulated for 50 oral, intravenous, intramuscular, Subcutaneous or parenteral cations, one embodiment provides Suspensions, syrups and chewable tablets. For oral administration, the pharmaceutical administration for the therapeutic or prophylactic treatment compositions are in the form of for example, a tablet, cap of diseases, such as bacterial infections. Preferably, the phar Sule, Suspension or liquid. maceutical composition is formulated for intravenous admin 55 The pharmaceutical compositions may be made in the form istration. of a dosage unit containing a therapeutically-effective The pharmaceutical preparations disclosed herein may be amount of the active ingredient. Examples of Such dosage prepared in accordance with standard procedures and are units are tablets and capsules. For therapeutic purposes, the administered at dosages that are selected to reduce, prevent or tablets and capsules which can contain, in addition to the eliminate infection (see, e.g., Remington’s Pharmaceutical 60 active ingredient, conventional carriers such as binding Sciences, Mack Publishing Company, Easton, Pa. and Good agents, for example, acacia gum, gelatin, polyvinylpyrroli man and Gilman’s “The Pharmaceutical Basis of Therapeu done, Sorbitol, or tragacanth; fillers, for example, calcium tics.” Pergamon Press, New York, N.Y., the contents of which phosphate, glycine, lactose, maize-starch, Sorbitol, or are incorporated herein by reference, for a general description 65 Sucrose; lubricants, for example, magnesium Stearate, poly of the methods for administering various antimicrobial agents ethylene glycol, silica, or talc, disintegrants, for example, for human therapy). potato starch, flavoring or coloring agents, or acceptable wet US 8,962,843 B2 35 36 ting agents. Oral liquid preparations generally are in the form The injectable formulations can be sterilized, for example, of aqueous or oily Solutions, Suspensions, emulsions, syrups by filtration through a bacterial-retaining filter, or by incor or elixirs, preparations of the invention may contain conven porating sterilizing agents in the form of sterile solid compo tional additives such as Suspending agents, emulsifying sitions, which can be dissolved or dispersed insterile water or agents, non-aqueous agents, preservatives, coloring agents other sterile injectable medium just prior to use. and flavoring agents. Non-limiting examples of additives for Solid dosage forms for oral administration include cap liquid preparations include acacia, almond oil, ethyl alcohol, Sules, tablets, pills, powders, and granules. Such forms may fractionated coconut oil, gelatin, glucose syrup, glycerin, include forms that dissolve or disintegrate quickly in the oral hydrogenated edible fats, lecithin, methyl cellulose, methyl 10 environment. In Such solid dosage forms, the active com or propyl para-hydroxybenzoate, propylene glycol, Sorbitol, pound preferably a compound of Formula A-I or Formula or Sorbic acid. For intravenous (IV) use, the pharmaceutical composition A-II in conjunction with a B-lactam antibiotic, can be mixed (e.g. compounds of Formula I, compounds of Formula A-I. with at least one inert, pharmaceutically-acceptable excipient compounds of Formula II, compounds of Formula A-II) pref 15 or carrier. Suitable excipients include, for example, (a) fillers erably a compound of Formula A-I or Formula A-II, in con or extenders such as starches, lactose, Sucrose, glucose, man junction with a B-lactam antibiotic, can be dissolved or Sus nitol, and silicic acid; (b) binders such as cellulose and cel pended in any of the commonly used intravenous fluids and lulose derivatives (such as hydroxypropylmethylcellulose, administered by infusion. Intravenous fluids include, without hydroxypropylcellulose, and carboxymethylcellulose), algi limitation, physiological saline or Ringer's solution. Intrave nates, gelatin, polyvinylpyrrolidone. Sucrose, and acacia; (c) nous administration may be accomplished by using, without humectants such as glycerol; (d) disintegrating agents such as limitation, Syringe, mini-pump or intravenous line. Sodium starch glycolate, croscarmellose, agar-agar, calcium Pharmaceutical compositions of this invention (e.g. com carbonate, potato or tapioca Starch, alginic acid, certain sili pounds of Formula I, compounds of Formula A-I, compounds 25 cates, and sodium carbonate; (e) solution retarding agents of Formula II, compounds of Formula A-II) preferably a Such as paraffin, (f) absorption accelerators such as quater compound of Formula A-I or Formula A-II, for parenteral nary ammonium compounds; (g)wetting agents, such ascetyl injection comprise pharmaceutically-acceptable aqueous or alcohol and glycerol monostearate, fatty acid esters of Sorbi non-aqueous solutions, dispersions, Suspensions or emul 30 tan, poloxamers, and polyethylene glycols: (h) absorbents sions as well as sterile powders for reconstitution into sterile Such as kaolin and bentonite clay; (ii) lubricants such as talc, injectable solutions or dispersions just prior to use. Examples calcium stearate, magnesium Stearate, solid polyethylene gly of Suitable aqueous and non-aqueous carriers, diluents, Sol cols, sodium lauryl Sulfate, and mixtures thereof, and () vents or vehicles include water, ethanol, benzyl alcohol, poly glidants such as talc, and silicone dioxide. Other Suitable ols (such as glycerol, propylene glycol, and polyethylene 35 excipients include, for example, Sodium citrate or dicalcium glycol), and Suitable mixtures thereof, vegetable oils (such as phosphate. The dosage forms may also comprise buffering corn oil or olive oil), and injectable organic esters such as agents. ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the main Solid dosage forms, including those of tablets, dragees, tenance of the required particle size in the case of dispersions, 40 capsules, pills, and granules, can be prepared with coatings and by the use of Surfactants. The compositions can include and shells such as functional and aesthetic enteric coatings various buffers. and other coatings well known in the pharmaceutical formu These compositions may also contain adjuvants such as lating art. They may optionally contain opacifying agents and preservatives, wetting agents, emulsifying agents, and dis 45 colorants. They may also be in a form capable of controlled or persing agents. They may also contain taggants or other anti Sustained release. Examples of embedding compositions that counterfeiting agents, which are well known in the art. Pre can be used for Such purposes include polymeric Substances vention of the action of microorganisms may be ensured by and waxes. the inclusion of various antibacterial and antifungal agents, The pharmaceutical compositions can be delivered using for example, paraben, chlorobutanol, and phenol Sorbic acid. 50 controlled (e.g., capsules) or Sustained release (e.g., bioerod It may also be desirable to include isotonic agents such as able matrices) delivery systems. Exemplary delayed release Sugars and sodium chloride. Prolonged absorption of the delivery systems for drug delivery that are suitable for admin injectable pharmaceutical form may be brought about by the istering the pharmaceutical compositions are described in inclusion of agents which delay absorption, Such as alumi 55 U.S. Pat. No. 4,452,775 (issued to Kent), U.S. Pat. No. 5,039, num monostearate and gelatin. 660 (issued to Leonard), and U.S. Pat. No. 3,854,480 (issued Injectable depot forms can be made by forming microen to Zaffaroni). capsulating matrices of the drug in biodegradable polymers In some cases, in order to prolong the effect of the drug, it Such as polylactide-polyglycolide. Depending upon the ratio may be desirable to slow the absorption of the drug following of drug to polymer and the nature of the particular polymer 60 Subcutaneous or intramuscular injection. This may be accom employed, the rate of drug release can be controlled. plished by the use of a liquid Suspension of crystalline or Examples of other biodegradable polymers include poly amorphous material with poor water Solubility. Amorphous (orthoesters) and poly(anhydrides). Depot injectable formu material may be used alone or together with stabilizers as lations can also be prepared by entrapping the drug in lipo 65 necessary. The rate of absorption of the drug then depends Somes or microemulsions, which are compatible with body upon its rate of dissolution, which in turn, may depend upon tissues. crystal size and crystalline form. US 8,962,843 B2 37 38 Alternatively, delayed absorption of a parenterally admin the dosage employed can range from 5 mg to 10 g, per day, istered drug form can be accomplished by dissolving or Sus depending on the route and frequency of administration. pending the drug in an oil vehicle. The pharmaceutical compositions disclosed herein can be For intramuscular preparations, a sterile formulation of placed in a pharmaceutically acceptable carrier and are deliv compounds, preferably a compound of Formula A-I or For 5 ered to a recipient Subject (e.g., a human) in accordance with mula A-II in conjunction with a B-lactam antibiotic, or Suit known methods of drug delivery. In general, the methods of able soluble salt forms thereof, for example hydrochloride delivering the pharmaceutical compositions in vivo utilize salts, can be dissolved and administered in a pharmaceutical art-recognized protocols for delivering the agent with the diluent such as Water-for-Injection (WFI), physiological 10 only substantial procedural modification being the Substitu saline or 5% glucose. A suitable insoluble form of the com tion of the compounds of the present invention for the drugs in pound may be prepared and administered as a Suspension in the art-recognized protocols. Likewise, methods for using the an aqueous base or a pharmaceutically acceptable oil base, claimed compositions for treating cells in culture, for e.g., an ester of a long chain fatty acid Such as ethyl oleate. example, to eliminate or reduce the level of bacterial contami A dose of an intravenous, intramuscular, or parental for 15 nation of a cell culture, utilize art-recognized protocols for mulation of compounds, preferably a compound of Formula treating cell cultures with antibacterial agent(s) with the only A-I or Formula A-II in conjunction with a B-lactamantibiotic, substantial procedural modification being the substitution of may be administered as a bolus or by slow infusion. A bolus the compounds of the present invention, preferably in com is a dose that is administered in less than 30 minutes. In one bination with a B-lactam antibiotic for the drugs in the art embodiment, a bolus is administered in less than 15 or less recognized protocols. than 10 minutes. In another embodiment, a bolus is adminis Exemplary procedures for delivering an antibacterial agent tered in less than 5 minutes. In yet another embodiment, a are described in U.S. Pat. Nos. 6,468,967; 6,852,689; and bolus is administered in one minute or less. An infusion is a 5,041,567, issued to Rogers and in PCT patent application dose that is administered at a rate of 30 minutes or greater. In 25 number EP94/02552 (publication no. WO95/05384), the dis one embodiment, the infusion is one hour or greater. In closures of which are incorporated herein by reference in their another embodiment, the infusion is Substantially constant. entirety. In one embodiment, one or more compounds of the For topical use the pharmaceutical compositions, prefer invention, preferably a compound of Formula A-I or Formula ably a compound of Formula A-I or Formula A-II in conjunc 30 A-II in conjunction with a B-lactamantibiotic, or pharmaceu tion with a B-lactam antibiotic, can also be prepared in Suit tical compositions thereofare administered orally, rectally or able forms to be applied to the skin, or mucus membranes of via injection (intravenous, intramuscular or subcutaneous). 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 35 in conjunction with a B-lactam antibiotic, or pharmaceutical pounds such as dimethylsulfoxide (DMSO) to facilitate sur compositions thereof are administered orally, rectally or via face penetration of the active ingredient. injection (intravenous, intramuscular or Subcutaneous) to For application to the eyes or ears, the pharmaceutical treat an infection caused by B-lactam resistant bacteria. In composition can be presented in liquid or semi-liquid form another embodiment, one or more compounds of the inven formulated in hydrophobic or hydrophilic bases as ointments, 40 tion, preferably a compound of Formula A-I or Formula A-II creams, lotions, paints or powders. in conjunction with a B-lactam antibiotic, or pharmaceutical For rectal administration, the pharmaceutical composi compositions thereofare administered orally to treat an infec tions, preferably a compound of Formula A-I or Formula A-II tion caused by B-lactamase producing bacteria. in conjunction with a B-lactamantibiotic, can be administered 45 As used herein, the phrases “therapeutically-effective in the form of Suppositories admixed with conventional car dose” and “therapeutically-effective amount” refer to an riers such as cocoa butter, polyethylene glycol or a Supposi amount of a compound that prevents the onset, alleviates the tory wax or other glyceride that are solid at room temperature symptoms, stops the progression of a bacterial infection, or but liquid at body temperature and therefore melt in the rec results in another desired biological outcome Such as, e.g., tum or vaginal cavity and release the active compound. 50 improved clinical signs or reduced/elevated levels of lympho Alternatively, the pharmaceutical compositions can be in cytes and/or antibodies. powderform for reconstitution in the appropriate pharmaceu The term “treating” or “treatment' is defined as adminis tically acceptable carrier at the time of delivery. In another tering, to a subject, a therapeutically-effective amount of one embodiment, the unit dosage form of compounds, preferably 55 or more compounds both to prevent the occurrence of an a compound of Formula A-I or Formula A-II in conjunction infection and to control or eliminate an infection. Those in with a B-lactam antibiotic, can be a solution of one or more need of treatment may include individuals already having a compounds, or salts thereof, in a suitable diluent, in sterile particular medical disease as well as those at risk for the hermetically sealed ampoules or Sterile Syringes. The concen disease (i.e., those who are likely to ultimately acquire the tration of the compounds, preferably a compound of Formula 60 disorder). A-I or Formula A-II in conjunction with a B-lactamantibiotic, The term “subject, as used herein, refers to a mammal, a in the unit dosage may vary, e.g. from about 1 percent to about plant, a lower animal, or a cell culture. In one embodiment, a 50 percent, depending on the compound used and its solubil Subject is a human or other animal patient in need of antibac ity and the dose desired by the physician. If the compositions 65 terial treatment. contain dosage units, each dosage unit can contain from The term “administering or “administration' and the like, 1-500 mg of the active material. For adult human treatment, refers to providing the compound of the invention (e.g. com US 8,962,843 B2 39 40 pounds of Formula I, compounds of Formula A-I, compounds maceutical composition comprising a compound of Formula of Formula II, compounds of Formula A-II) to the subject in A-I, and a B-lactam antibiotic. In one embodiment of the need of treatment. Preferably the subject is a mammal, more invention, is provided a method of treating or preventing a preferably a human. The present invention comprises admin 5 bacterial infection comprising administering to a subject in istering the compound of the invention (e.g. compounds of need thereofatherapeutically-effective amount of the phar Formula I, compounds of Formula A-I, compounds of For maceutical composition comprising a compound of Formula mula II, compounds of Formula A-II) in conjunction with a B-lactamantiobiotic. When a compound of the invention (e.g. II, and a B-lactamantibiotic. In one embodiment of the inven compounds of Formula I, compounds of Formula A-I, com 10 tion, is provided a method of treating or preventing a bacterial pounds of Formula II, compounds of Formula A-II) is admin infection comprising administering to a Subject in need istered in conjunction with a B-lactam antiobiotic, the com thereofatherapeutically-effective amount of the pharmaceu pound of the invention (e.g. compounds of Formula I, tical composition comprising a compound of Formula A-II, compounds of Formula A-I, compounds of Formula II, com 15 and a B-lactam antibiotic. pounds of Formula A-II) and the B-lactam antiobiotic can be In one embodiment of the invention, is provided a method administered at the same time or different times. When the of treating or preventing a bacterial infection comprising compounds of the invention (e.g. compounds of Formula I, administering to a subject in need thereof, a therapeutically compounds of Formula A-I, compounds of Formula II, com effective amount of a B-lactamantibiotic in conjunction with pounds of Formula A-II) and the B-lactam antiobiotic are a compound of the invention (e.g. compounds of Formula I, administered at the same time, they can be administered as a single composition or pharmaceutical composition or they compounds of Formula A-I, compounds of Formula II, com can be administered separately. It is understood that when a pounds of Formula A-II). In one embodiment of the invention, compound of the invention (e.g. compounds of Formula I, 25 is provided a method of treating or preventing a bacterial compounds of Formula A-I, compounds of Formula II, com infection comprising administering to a Subject in need pounds of Formula A-II) is administered in conjunction with thereof, atherapeutically-effective amount of a B-lactamanti a 3-lactam antibiotic, that the active agents can be adminis biotic in conjunction with a compound of Formula I. In one tered in a single combination or in multiple combinations. For 30 embodiment of the invention, is provided a method of treating example, when administered by IV, the compound of the or preventing a bacterial infection comprising administering invention (e.g. compounds of Formula I, compounds of For to a subject in need thereof, a therapeutically-effective mula A-I, compounds of Formula II, compounds of Formula A-II) can be dissolved or Suspended in any of the commonly amount of a B-lactam antibiotic in conjunction with a com pound of Formula A-I. In one embodiment of the invention, is used intravenous fluids and administered by infusion, then a 35 B-lactam antibiotic can be dissolved or Suspended in any of provided a method of treating or preventing a bacterial infec the commonly used intravenous fluids and administered by tion comprising administering to a Subject in need thereof, a infusion. Conversely the B-lactam antibiotic can be dissolved therapeutically-effective amount of a B-lactam antibiotic in or Suspended in any of the commonly used intravenous fluids conjunction with a compound of Formula II. In one embodi and administered by infusion, then a compound of Formula I 40 ment of the invention, is provided a method of treating or can be dissolved or Suspended in any of the commonly used preventing a bacterial infection comprising administering to a intravenous fluids and administered by infusion. Alterna subject in need thereof, a therapeutically-effective amount of tively, a pharmaceutical composition comprising a compound a B-lactam antibiotic in conjunction with a compound of of the invention (e.g. compounds of Formula I, compounds of 45 Formula A-I, compounds of Formula II, compounds of For Formula A-II. In one embodiment of the invention, is pro mula A-II) and a B-lactam antibiotic can be dissolved or vided a method of treating or preventing a bacterial infection Suspended in any of the commonly used intravenous fluids comprising administering to a Subject in need thereofathera and administered by infusion. peutically-effective amount of a B-lactam antibiotic in con In one embodiment of the invention, is provided a method 50 junction with a compound of Table I. In one embodiment of of treating or preventing a bacterial infection comprising the invention, is provided a method of treating or preventing administering to a subject in need thereof a therapeutically a bacterial infection comprising administering to a subject in effective amount of the pharmaceutical composition compris need thereof, atherapeutically-effective amount of a B-lactam ing a compound of the invention (e.g. compounds of Formula 55 antibiotic in conjunction with a compound of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II) and a B-lactamantibiotic. In one N embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering 60 ( to a subject in need thereofatherapeutically-effective amount -- of the pharmaceutical composition comprising a compound N . . H of Formula I, and a B-lactamantibiotic. In one embodiment of A N the invention, is provided a method of treating or preventing 65 O OSOH. a bacterial infection comprising administering to a subject in need thereofatherapeutically-effective amount of the phar US 8,962,843 B2 41 42 In one embodiment of the invention, is provided a method In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising of treating or preventing a bacterial infection in a subject administering to a subject in need thereof, a therapeutically comprising the steps of effective amount of a B-lactamantibiotic in conjunction with a. administering to the Subject a compound of the inven a compound of Formula tion; and b. administering to the subject a therapeutically-effective amount of a B-lactam antibiotic. HN N In one embodiment the compound in Stepa is a compound of Formula I. In one embodiment the compound in step a is a 2 Y-N-X 10 compound of Formula A-I. In one embodiment the compound -l in Stepa is a compound of Formula II. In one embodiment the compound in step a is a compound of Formula A-II. In one N . I IIH embodiment the compound in Stepa is a compound of Table I. In one embodiment the compound in Stepa is a compound a N 15 O OSOH. of Formula A-II. In one embodiment the compound in step a is a compound of Formula In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising administering to a subject in need thereof, a therapeutically effective amount of a B-lactamantibiotic in conjunction with a compound of Formula

N NNN 25 O OSOH.

N-(O %, In one embodiment the compound in Stepa is a compound of Formula N III I IIH 30 / N HN N O OSOH. Y-N-( l'. In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising 35 administering to a subject in need thereof, a therapeutically effective amount of a B-lactamantibiotic in conjunction with 2. NN a compound of Formula O OSOH. 40 In one embodiment the compound in Stepa is a compound of Formula

45 NN-(

O OSOH. 50 In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection comprising O OSOH. administering to a subject in need thereof, a therapeutically effective amount of a B-lactamantibiotic in conjunction with In one embodiment the compound in Stepa is a compound a compound of Formula 55 of Formula

65 US 8,962,843 B2 43 44 In one embodiment the compound in Stepa is a compound In one embodiment the compound in Stepa is a compound of Formula of Formula

N IIH 10 / N O OSOH and the B-lactam antibiotic in step b is Ceftolozane. In one embodiment the compound in Stepa is a compound 15 of Formula

In one embodiment, the B-lactam antibiotic in step b is Ceftolozane, Ceftazidime, Aztreonam or Meropenem, or more preferably, Ceftolozane or Ceftazidime. In one embodiment the compound in Stepa is a compound of Formula 25 and the ft-lactam antibiotic in step b is Ceftolozane. N In one embodiment of the invention, is provided a method of treating or preventing a bacterial infection in a subject ( comprising the steps of 30 a. administering to the Subject a therapeutically-effective N . I I IIH amount of a B-lactam antibiotic; and b. administering to the Subject a compound of the inven Z N tion. O OSOH In one embodiment the compound in step b is a compound 35 of Formula I. In one embodiment the compound in step b is a compound of Formula A-I. In one embodiment the compound and the B-lactam antibiotic in step b is Ceftolozane. In one in step b is a compound of Formula II. In one embodiment the embodiment the compound in Stepa is a compound of For compound in step b is a compound of Formula A-II. In one mula embodiment the compound in step b is a compound of For 40 mula II. In one embodiment the compound in step b is a HN N compound of Table I. In one embodiment the compound in step b is a compound of Formula 2 Y-N-X -- N NN 45 N . I I IIH M N O OSOH 50 V and the f-lactam antibiotic in step b is Ceftolozane. O OSOH, In one embodiment the compound in Stepa is a compound In one embodiment the compound in step b is a compound of Formula of Formula 55 N NNN HN N NN-( %, 2 Y-N-X 60 --

N . I I IIH

N O OSOH V 65 O OSOH. and the f-lactam antibiotic in step b is Ceftolozane. US 8,962,843 B2 45 46 In one embodiment the compound in step b is a compound and the B-lactam antibiotic in step a is Ceftolozane. In one of Formula embodiment the compound in step b is a compound of For mula

HN N N NNN NNN-( J o-1. 10 N H

N II I I IIH N

N O OSOH V O OSOH. 15 and the B-lactam antibiotic in Stepa is Ceftolozane. In one embodiment the compound in step b is a compound of Formula In one embodiment the compound in step b is a compound 20 of Formula N 7

NN 25 ( JN NO- NH O-1, O OSOH N . I I IIH 30

N and the B-lactam antibiotic in Stepa is Ceftolozane. V In one embodiment the compound in step b is a compound O OSOH. of Formula

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

40 NN- N

in-O-KO %, O OSOH

N H 45 and the B-lactam antibiotic in Stepa is Ceftolozane. In one embodiment the compound in step b is a compound Jr. of Formula O OSOH.

50 In one embodiment, the B-lactam antibiotic in step a is Ceftolozane, Ceftazidime, Aztreonam or Meropenem, or more preferably, Ceftolozane or Ceftazidime. In one embodi ment the compound in step b is a compound of Formula 55

N-N N O OSOH { le, O 2. 60 and the B-lactam antibiotic in Stepa is Ceftolozane. N . I I IIH In one embodiment, the invention provides a method for treating an infection in a subject by administering a therapeu Jr. V tically-effective amount of one or more compounds of the O OSOH 65 invention (e.g. compounds of Formula I, compounds of For mula A-I, compounds of Formula II, compounds of Formula A-II), preferably a compound of Formula A-I or Formula A-II US 8,962,843 B2 47 48 in conjunction with a B-lactam antibiotic, or compositions in conjunction with a B-lactamantibiotic, preferably Ceftolo thereof. In one embodiment, the method comprises adminis Zane, Ceftazidime, AZtreonam or Meropenem, or more pref tering to a subject in need thereof a pharmaceutical compo erably, Ceftolozane or Ceftazidime, or compositions thereof. sition comprising at least one of the compounds described In one embodiment the compound is of Formula herein, preferably a compound of Formula A-I or Formula A-II in conjunction with a B-lactamantibiotic. In one embodi ment the compound is of Formula

O 10

O OSOH, 15 in conjunction with a B-lactamantibiotic, preferably Ceftolo O OSOH, Zane, Ceftazidime, AZtreonam or Meropenem, or more pref erably, Ceftolozane or Ceftazidime, or compositions thereof. in conjunction with a B-lactamantibiotic, preferably Ceftolo In one embodiment, the pharmaceutical composition can Zane, Ceftazidime, AZtreonam or Meropenem, or more pref comprise any one of the compounds described herein as the erably, Ceftolozane or Ceftazidime, or compositions thereof. sole active compound or in combination with another com In one embodiment the compound is of Formula pound, composition, or biological material. The compound may be administered orally, parenterally, by inhalation, topi 25 cally, rectally, nasally, buccally, vaginally, or by an implanted HN N 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 30 one embodiment, the aerosol delivery vehicle is an anhydrous or dry powder inhaler. One or more compounds of the inven tion (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of Formula A-II), O OSOH, preferably a compound of Formula A-I or Formula A-II in 35 conjunction with a B-lactam antibiotic, or pharmaceutical in conjunction with a B-lactamantibiotic, preferably Ceftolo compositions thereof also may be directly injected or admin Zane, Ceftazidime, AZtreonam or Meropenem, or more pref istered into an abscess, Ventricle or joint. Parenteral admin erably, Ceftolozane or Ceftazidime, or compositions thereof. istration includes Subcutaneous, intravenous, intramuscular, In one embodiment the compound is of Formula intra-articular, intra-synovial, cisternal, intrathecal, intrahe 40 patic, intralesional and intracranial injection or infusion. In one embodiment, one or more compounds of the invention N NNN (e.g. compounds of Formula I, compounds of Formula A-I. compounds of Formula II, compounds of Formula A-II), pref O '', erably a compound of Formula A-I or Formula A-II in con 45 junction with a B-lactam antibiotic, are administered intrave N III I IIH nously, Subcutaneously or orally. In one embodiment for administering one or more compounds according to the N invention (e.g. compounds of Formula I, compounds of For V O OSOH, mula A-I, compounds of Formula II, compounds of Formula 50 A-II), preferably a compound of Formula A-I or Formula A-II in conjunction with a B-lactamantibiotic to a cell culture, the in conjunction with a B-lactamantibiotic, preferably Ceftolo one or more compounds may be administered in a nutrient Zane, Ceftazidime, AZtreonam or Meropenem, or more pref medium. erably, Ceftolozane or Ceftazidime, or compositions thereof. In one embodiment, one or more compounds according to In one embodiment the compound is of Formula 55 the invention (e.g. compounds of Formula I, compounds of Formula A-I, compounds of Formula II, compounds of For 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 60 caused or exacerbated by any type of bacteria, Such as Gram negative bacteria. In one aspect of the invention, the bacterial N . I I IIH infection is caused by B-lactam resistant bacteria. In one aspect the bacterial infection is caused by B-lactamase pro ducing bacteria. In another aspect the bacterial infection is O OSOH, 65 caused by class A, class C or class D B-lactamase producing bacteria. In another aspect the bacterial infection is caused by class A B-lactamase producing bacteria. In another aspect the US 8,962,843 B2 49 50 infection is caused by class C B-lactamase producing bacte Panipenem. In one embodiment the Carbapenem is Imi ria. In still another aspect the infection is caused by class D penem/Cilastatin or Meropenem. B-lactamase producing bacteria. In still another aspect the In one embodiment of the invention, the B-lactam antibi infection is caused by KPC B-lactamase producing bacteria. otic is a monobactam. Examples of monobactam antibiotics In still another aspect the infection is caused by OXA B-lac include, but are not limited to Aztreonam, Tigemonam, Caru tamase producing bacteria. In still another aspect, the bacte monam, BAL30072 and Nocardicin A. rial infection is caused by a bacteria that produces multiple In one embodiment of the invention, the B-lactam antibi B-lactamases. Bacteria that produce multiple B-lactamases otic is a penem. In one embodiment of the invention, the may produce B-lactamases of the same class or of different B-lactam antibiotic is a penicillin. Examples of penicillin classes (e.g. class A and class A or class A and class C or class 10 antibiotics include, but are not limited to Amoxicillin, Ampi A and class D etc). cillin, AZlocillin, Mezlocillin, Apalcillin, Hetacillin, Representative Gram-negative pathogens known to Becampicillin, Carbenicillin, Sulbenicillin, Ticarcillin, Pip express B-lactamases include, but are not limited to Acineto eracillin, AZlocillin, Mecillinam, Pivmecillinam, Methicillin, bacter spp. (including Acinetobacter baumannii), Citro Ciclacillin, Talampicillin, Aspoxicillin, Oxacillin, Cloxacil bacter spp., Escherichia spp. (including Escherichia coli), 15 lin, Dicloxacillin, Flucloxacillin, Nafcillin and Pivampicillin. Haemophilus influenzae, Morganella morganii, Pseudomo In one embodiment the cephalosporin is Ceftolozane, nas aeruginosa, Klebsiella spp. (including Klebsiella pneu Ceftazidime, Aztreonam or Meropenem, or more preferably, moniae), Enterobacter spp. (including Enterobacter cloacae Ceftolozane or Ceftazidime. and Enterobacter aerogenes), Pasteurella spp., Proteus spp. The pharmaceutical compositions, preferably a compound (including Proteus mirabilis), Serratia spp. (including Serra of the invention (e.g. compounds of Formula I, compounds of tia marcescens), and Providencia spp. Bacterial infections Formula A-I, compounds of Formula II, compounds of For can be caused or exacerbated by Gram-negative bacteria mula A-II) in conjunction with a 3-lactam antibiotic, can be 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 25 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 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 30 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, 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 35 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 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. 40 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 45 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), 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, Cefinetazole, Cefonicid, Cefotetan, Cefoxitin, can be administered to a subject to treat mixed infections that Cefprozil (cefproxil), Cefuroxime, CefuZonam, Cefcapene, 50 comprise different types of Gram-negative bacteria, or which Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, comprise both Gram-positive and Gram-negative bacteria. Cefinenoxime, Cefodizime, Cefotaxime, Ce?pimizole, Cef 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 55 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, A-I or Formula A-II in conjunction with a B-lactamantibiotic, Cefivitril, Cefnmatilen, Cefinepidium, Cefovecin, Cefoxazole, may also be administered to a Subject to treat an infection Cefirotil, Cefsumide, Ceftaroline, Ceftioxide, Cefuracetime, including, without limitation, endocarditis, nephritis, septic cefbuperaZone, cefiminox, ceforanide, cefotiam, ce?piramide, 60 arthritis, intra-abdominal sepsis, bone and joint infections cefsulodin, ceftobiprole latamoxefloracarbef and Ceftoloz and osteomyelitis. At least one compound of the invention ane. In one embodiment the cephalosporin is Ceftolozane or (e.g. compounds of Formula I, compounds of Formula A-I. 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 65 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 into an abscess, Ventricle or joint. Pharmaceutical composi US 8,962,843 B2 51 52 tions of the invention (e.g. compounds of Formula I, com invention (e.g. compounds of Formula I, compounds of For pounds of Formula A-I, compounds of Formula II, com mula A-I, compounds of Formula II, compounds of Formula pounds of Formula A-II), preferably compound of Formula A-II) are administered to a human at a dose of 100 mg to 1000 A-I or Formula A-II in conjunction with a B-lactamantibiotic, mg per dose up to four times per day. In another embodiment, may be administered as an aerosol for the treatment of pneu 5 the compounds of the invention (e.g. compounds of Formula monia or other lung-based infections. In one embodiment, the I, compounds of Formula A-I, compounds of Formula II, aerosol delivery vehicle is an anhydrous, liquid or dry powder compounds of Formula A-II) are administered to a human at inhaler. a dose of 125 mg to 750 mg per dose up to four times per day. Actual dosage levels of active ingredients in the pharma In another embodiment, the compounds of the invention (e.g. ceutical compositions of one or more compounds according 10 compounds of Formula I, compounds of Formula A-I, com to the invention (e.g. compounds of Formula I, compounds of pounds of Formula II, compounds of Formula A-II) are Formula A-I, compounds of Formula II, compounds of For administered to a human at a dose of 250 mg to 500 mg per mula A-II), preferably a compound of Formula A-I or For dose up to four times a day. An effective dose for cell culture mula A-II in conjunction with a 3-lactam antibiotic, may be is usually between about 0.1 and about 1000 ug/mL. In one varied so as to obtain a therapeutically-effective amount of 15 embodiment, the effect dose for cell culture is between about the active compound(s) to achieve the desired therapeutic 0.1 and about 200 ug/mL. response for a particular patient, compositions, and mode of In one embodiment, a B-lactam antibiotic and a compound administration. The effective amount can be determined as of the invention (e.g. compounds of Formula I, compounds of described herein. The selected dosage level will depend upon Formula A-I, compounds of Formula II, compounds of For the activity of the particular compound, the route of admin mula A-II) are administered in ratio of 1:4 to 8:1 antibiotic: istration, the severity of the condition being treated, and the compound of the invention (e.g. compounds of Formula I, condition and prior medical history of the patient being compounds of Formula A-I, compounds of Formula II, com 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 25 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 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, 30 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 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 35 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 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 40 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 45 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, 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 50 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. 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 55 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 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 60 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 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 65 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 Formula II, compounds of Formula A-II), preferably a com US 8,962,843 B2 53 54 pound of Formula A-I or Formula A-II in conjunction with a Other embodiments of the invention include: B-lactamantibiotic, can be administered as a single daily dose A pharmaceutical composition comprising a compound of or in multiple doses per day. In one embodiment, one or more the invention (e.g. compounds of Formula I, compounds of compounds of the invention (e.g. compounds of Formula I, Formula A-I, compounds of Formula II, compounds of For compounds of Formula A-I, compounds of Formula II, com mula A-II), preferably a compound of Formula A-I or For pounds of Formula A-II), preferably a compound of Formula mula A-II and at least 1 B-lactam antibiotic or a pharmaceu A-I or Formula A-II in conjunction with a B-lactamantibiotic, tically acceptable salt thereof. is administered as a single dose per day. In another embodi A pharmaceutical composition comprising a compound of ment, one or more compounds of the invention (e.g. com the invention (e.g. compounds of Formula I, compounds of pounds of Formula I, compounds of Formula A-I, compounds 10 Formula A-I, compounds of Formula II, compounds of For of Formula II, compounds of Formula A-II), preferably a mula A-II), preferably a compound of Formula A-I or For compound of Formula A-I of Formula A-II in conjunction mula A-II and at least 1 cephalosporin antibiotic or a phar with a B-lactam antibiotic is administered as two equal doses maceutically acceptable salt thereof. per day. In another embodiment, the compounds of the inven A pharmaceutical composition comprising a compound of tion (e.g. compounds of Formula I, compounds of Formula 15 the invention (e.g. compounds of Formula I, compounds of A-I, compounds of Formula II, compounds of Formula A-II), Formula A-I, compounds of Formula II, compounds of For preferably a compound of Formula A-I or Formula A-II in mula A-II), preferably a compound of Formula A-I or For conjunction with a 3-lactam antibiotic is administered in mula A-II and CeftoloZane or a pharmaceutically acceptable three equal doses per day. In another embodiment, the com salt thereof. pounds of the invention (e.g. compounds of Formula I, com A pharmaceutical composition comprising a compound of pounds of Formula A-I, compounds of Formula II, com the invention (e.g. compounds of Formula I, compounds of pounds of Formula A-II), preferably a compound of Formula Formula A-I, compounds of Formula II, compounds of For 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 25 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 30 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 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 35 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. 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 40 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. 45 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 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 50 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 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 55 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), 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 60 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 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 65 Burgess reagent-methyl N-triethylammoniumsulfonyl) invention may be administered for a longer or shorter time carbamate period if it is so desired. CDI-carbonyldiimidazole US 8,962,843 B2 55 56 CFU=colony-forming units rt-room temperature CLSI-Clinical Laboratory Standards Institute TBAF-tetrabutylammonium fluride cSSSI-complicated skin and skin structure infections TBS-t-butyldimethylsilyl DBU=1,8-diazabicyclo5.4.0]undec-7-ene TES-triethylsilyl DCM-dichloromethane TEA triethylamine DEAD-diethylazodicarboxylate TEMPO-2.2.6,6-tetramethyl-1-piperidinyloxy, free radi DIAD-diisopropyl azodicarboxylate cal DIPEA-diisopropylethylamine THF-tetrahydrofuran DMF=N,N-dimethylformamide TFA=trifluoroacetic acid DMAc=N,N-dimethylacetamide 10 TMS-trimethylsilyl DMSO-dimethylsulfoxide TLC-thin layer chromatography EDCI=1-ethyl-3-3'-dimethylaminopropyl)carbodiimide VAP=Ventilator-Associated Pneumonia ELSD evaporative light scattering detector The compounds of the invention (e.g. compounds of For EtOAc-ethyl acetate ESI-MS-electrospray ionization mula I, compounds of Formula A-I, compounds of Formula mass spectrometry 15 II, compounds of Formula A-II) can be prepared from inter Fmoc-Fluorenylmethyloxycarbonyl mediate 1, according to the following reaction schemes and HAP=Hospital-Acquired Pneumonia examples, or modifications thereof, using readily available HATU-2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetram starting materials, reagents and conventional synthetic pro ethyluronium hexafluorophosphate cedures including, for example, procedures described in U.S. HCl–hydrochloride Pat. No. 7,112,592 and WO2009/091856. HOBt=1-hydroxybenzotrizole As depicted in Scheme 1, compound 3 can be synthesized Hrs=hours following standard oxadiazole ring formation chemistry from HPLC-high performance liquid chromatography the diacylhydrazide intermediate 2 g (see, e.g., Jakopin, Z.; Hunig's base —N,N-Diisopropylethylamine Dolenc, M. S. Curr. Org. Chem. 2008, 12, 850-898; Walker, Lawesson’s reagent 2.4-bis(4-methoxyphenyl)-1,3,2,4- 25 D. G.; Brodfuehrer, P. R.; Brundidge, S. P. Shih, K. M.: dithiadiphosphetane-2,4-disulfide Sapino, C.Jr. J. Org. Chem. 1988, 53,983-991 and references MIC-minimum inhibitory concentration cited therein). Diacylhydrazide intermediate 2g can be pre mL milliliter pared via Standard amide coupling reactions from acylhy MS-mass spectrometry drazide intermediate 2f. Alternatively, 2g can be made by an MRSA methicillin-resistant Staphylococcus aureus 30 amide coupling reaction of acylhydrazine derivative 2h and NMR nuclear magnetic resonance acid intermediate 2a, which can be prepared from ester inter Ns-nitrobenzenesulfonyl mediate 1. It may be necessary to protect certain functional Pa Pseudomonas aeruginosa ities in the molecule depending on the nature of the R' group. Prep preparative Protecting these functionalities should be within the expertise Ppm parts per million 35 of one skilled in the art. See, e.g. P. G. M. Wuts and T. W. Py-pyridine Greene, Protective Groups in Organic Synthesis, Fourth Edi sat. Saturated tion, John Wiley and Sons, 2006, hereafter Greene.

Scheme I O -- '', - '... 1. NHNHBoc insul2n-1. LiOH HATU, DIPEA H N H THF/Ho N "IH -e-2. TFADCM. 30% N ... I I IIH or 4MHC in N N dioxane N O OB O OB O OB 1 2a 2f

R-COOH HATU, DIPEA DCM

O O

O HO ul'... is usua N '', HATU, DIPEA H NH + -e- R N1 N . I I IIH DCM O N . I I IIH H

O NOB O NOB 2h 2a 2g US 8,962,843 B2 57 58 -continued is usuNN '', H (CFSOO/Py H2, catalyst He O N ... H. -->or Burgess reagent N IH be, 1. or PPh3, I, NEt3 N O OB OM NOB 3 NN N

R-(-O %, R-(-o-1-, SO3 pyridine protecting group N - > N ... I IIH deprotection if N necessary N O 1. OH O OSOH 4 5 7'NN R J O '.

N . I I IIH A N O OSOH 6

The benzylic ether protecting group in 3 can be removed via standard hydrogenolysis conditions, such as, but not lim Scheme 2 35 ited to, Pd/H. in MeCH or THF or by acid-catalysed hydroly O sis, such as, but not limited to, BC1 in DCM to provide the hydroxy-urea intermediate 4, which can be used directly in RulNN '', the next step without further purification. Sulfation of 4 can be H achieved by treatment with a sulfating reagent, Sucha, but not 40 O N IIH limited to, SO-pyridine complex, in an appropriate solvent, such as pyridine, DMF or DMAc at a temperature of 0-80°C., Jr. V preferable at room temperature. Compound 5 can then be O OB isolated and purified via conventional methods. For example, 2g 5 can be purified by standard reverse phase prep-HPLC using 45 appropriate buffer system, i.e. ammonium formate buffer. In Some cases, 5 can be purified by normal phase silica gel N chromatography after converting to an appropriate salt form, 7'N such as sulfate tetrabutyl ammonium salt. The tetrabutyl R J 50 S le, ammonium salt can then be converted to a sodium salt by H2, catalyst cation exchange. When a protecting group(s) is present in the -- sidechain (i.e. Boc or Fmoc for amine and guanidine protec N . I I IIH or BC tion, TBS or TES for alcohol protection, etc), a deprotection step is needed to convert 5 to its final product 6, which can be Jr. V O OB purified by prep-HPLC using the conditions mentioned 55 above. For example, for N-Boc deprotection, 5 can be treated 3a 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 give 6. For an O-TBS, or O-TES deprotection, a fluoride 7'N reagent such as HF-pyridine, HF.NEts, or TBAF can be used. 60 R S-1%.J For Fmoc deprotection, amines can be used, such as diethy SO3 pyridine lamine, DBU, piperidine, etc can be used. N . I I IIH Similarly, as shown in Scheme 2, thiadiazole derivative 3a can be synthesized from diacylhydrazide intermediate 2g by treatment with Lawesson’s reagent underheating. 3a can then 65 O/ NOH be converted to the final product 6ausing similar chemistry as described previously. US 8,962,843 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 Her Example 1 . I I IIH deprotection if necessary N Synthesis of (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6- O OSOH 10 diazabicyclo[3.2.1]octane-2-carboxylate (Intermedi 5a ate Compound 1) NN N

R-(-S %, 15 N ... I I IIH r V O OSOH 6a

EXAMPLES 25 The specific examples which follow illustrate the synthesis Step 1: Synthesis of (S)-1-tert-butyl 2-ethyl of certain compounds. The methods disclosed may be 5-oxopiperidine-1,2-dicarboxylate adopted to variations in order to produce compounds of the Method A: invention (e.g. compounds of Formula I, compounds of For 30 mula A-I, compounds of Formula II, compounds of Formula A-II) but not otherwise specifically disclosed. Further, the Boc disclosure includes variations of the methods described O \ O TMSCHN herein to produce the compounds of the invention (e.g. com 35 w/ Hos pounds of Formula I, compounds of Formula A-I, compounds n-BuLi of Formula II, compounds of Formula A-II) that would be OEt understood by one skilled in the art based on the instant s O s O disclosure. N N HN v lsOEt Rh(OA2(OAc)4 N v ls OEt All temperatures are understood to be in Centigrade (C) 40 when not specified. The nuclear magnetic resonance (NMR) O O spectral characteristics refer to chemical shifts (y) expressed in parts per million (ppm) versus tetramethylsilane (TMS) as reference standard. The relative area reported for the various 45 n-BuLi was added dropwise to a solution of TMSCHN shifts in the proton NMR spectral data corresponds to the (690 mL, 1.38 mol) in dry THF (3 L) (600 mL, 1.5 mol) at number of hydrogen atoms of a particular functional type in -78°C., and the mixture was stirred at -78°C. for 30 minutes. The mixture was then transferred to a solution of (S)-1-tert the molecule. The nature of the shifts as to multiplicity is butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate (300 g, reported as broad singlet (bris), broad doublet (br d), 50 1.17 mol) in dry THF (3 L) via cannula, and the mixture was singlet (s), multiplet (m), doublet (d), quartet (q), doublet of stirred at -78° C. for 30 minutes. The reaction mixture was doublet (dd), doublet of triplet (dt), and doublet of quartet then quenched with sat. NHCl solution, and extracted with (dq). The solvents employed for taking NMR spectra are DCM three times. The combined organic layer was concen DMSO-d6 (perdeuterodimethysulfoxide), DO (deuterated trated in vacuum and the crude product was purified by silica water), CDC1 (deuterochloroform) and other conventional 55 gel column chromatography (3:1 petroleum ether:EtOAc) to deuterated solvents. The prep-HPLC conditions are: Waters afford (S)-ethyl 2-((tert-butoxycarbonyl)amino)-6-diazo-5- SunFireR C18 (30x100 mm, 5um OBD) column; flow rate: oxohexanoate (262 g, 75%) as a yellow solid. 30-80 mL/minute, ELSD or Mass-triggered fraction collec A solution of (S)-ethyl 2-((tert-butoxycarbonyl)amino)-6- tion; sample loading: Each injection loading varied from 60 diazo-5-oxohexanoate (350 g, 1.18 mol) in DCM (1500 mL) -300 mg for different crude samples depending on their solu was added to a solution of Rh(OAc) (3.5 g, 7.9 mmol) in bility and purity profiles; Solvent system using ammonium DCM (750 mL) at 0°C. The reaction was then stirred at 20° C. overnight and then concentrated in vacuum. The crude formate buffer: solvent A: water with 20 mM ammonium sample was purified by silica gel column chromatography formate, solvent B: 85% of acetonitrile in water with 20 mM 65 (5:1 petroleum ether/EtOAc) to afford (S)-1-tert-butyl ammonium formate. Solvent system using NHHCO buffer: 2-ethyl 5-oxopiperidine-1,2-dicarboxylate (175.9 g, 55%) as solvent A: water with 10 mM NHHCO, solvent B: acetoni a yellow oil. US 8,962,843 B2 61 62 Method B: EtOAc) to afford (2S,5S)-1-tert-butyl 2-ethyl 5-hydroxypip eridine-1,2-dicarboxylate (205 g, 80%) as a yellow oil.

M B OC N a' Step 3: Synthesis of (2S,5R)-1-tert-butyl 2-ethyl O N A O MNS I 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperi : I III t-BuOK dine-1,2-dicarboxylate OEt DMSO Boc O 10 > Yor Ir(COD)Cl] C toluene, 80° C. OESEO O pyridine Bn 1N NH, HCI -- NO -e-0-rt, 1 h Boc O 15 1C OEt s O 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) * OEt Bn-O and the mixture was stirred at rt for 1 h. (S)-1-tert-Butyl 25 w 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate (900 g, 3.5 mol) HN-S HO was added and the mixture was stirred at rt for 2-3 hrs. Water PhP, DEAD, THF O was added to quench the reaction and the mixture was extracted with EtOAc 5 times. The combined organic layer ON was concentrated in vacuum and the crude sample was puri 30 s fied by silica gel column chromatography (1:1 petroleum N CO2Et ether/EtOAc then 1:10 MeOH/DCM) to afford sulfoxonium ylide intermediate (977 g. 80%) as a white solid. BO 1C A solution of sulfoxoniumylide intermediate (156g, 0.446 35 s mol) and Ir(COD)Cl]- (3 g, 4.46 mmol) in toluene (4 L) was OESEO degassed by bubbling nitrogen through the solution for 10 ON minutes. The reaction mixture was heated to 80-90° C. for 2-3 hrs and then cooled to 20°C. Then toluene was concentrated 40 in vacuum, the residue was purified by silica gel column chromatography (10:1 to 3:1 gradient petroleum ether/EtOA) to afford (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicar A solution of 2-nitrobenzene-1-sulfonyl chloride (500 g, boxylate (140g, 57.8%) as a yellow oil. 45 2.26 mol) in pyridine (1500 mL) was added dropwise to a solution of O-benzylhydroxylamine hydrochloride (400 g, Step 2: Synthesis of (2S,55)-1-tert-butyl 2-ethyl 2.51 mol) in pyridine (1500 mL) at 0°C. The reaction mixture 5-hydroxypiperidine-1,2-dicarboxylate was then stirred at 20° C. overnight. The mixture was con centrated in vacuum, diluted with DCM and washed with HCl 50 (10%) three times. The combined organic layer was concen Boc O Boc O trated in vacuum and re-crystallized with DCM to afford N-(benzyloxy)-2-nitrobenzenesulfonamide (485 g, 62.6%)

N ls NaB N ls OEt as a yellow solid. 1C * OEt aBH4 O ax 55 To a solution of N-(benzyloxy)-2-nitrobenzenesulfona O How mide (212 g, 0.69 mol) in THF (1000 mL) was added (2S, 5S)-1-tert-butyl 2-ethyl 5-hydroxypiperidine-1,2-dicarboxy late (171 g, 0.63 mol) and PPhs (275g, 1.05 mol), followed by NaBH (36 g, 1.0 mol) was added in portions to a solution of (S)-1-tert-butyl 2-ethyl 5-oxopiperidine-1,2-dicarboxylate 60 dropwise addition of a solution of DEAD (195g, 1.12 mol) in (250 g., 0.92 mol) in EtOH (1500 mL) at -40°C. The reaction THF (500 mL). The mixture was then stirred at 20° C. over mixture was then stirred at -40°C. for 0.5 hr then quenched night. The reaction mixture was then concentrated in vacuum with 10% HOAc solution. After diluting with water, the mix and purified by silica gel column chromatography (3:1 petro ture was extracted with DCM three times. The combined 65 leum ether/EtOAc) to afford (2S,5R)-1-tert-butyl 2-ethyl organic layer was concentrated in vacuum and purified by 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperidine-1, silica gel column chromatography (1:1 petroleum ether/ 2-dicarboxylate (283.8 g., 80%) as a yellow oil. US 8,962,843 B2 63 64 Step 4: Synthesis of (2S.5R)-1-tert-butyl 2-ethyl MeOH) to afford (2S,5R)-ethyl 5-((benzyloxy)amino)piperi 5-((benzyloxy)amino)piperidine-1,2-dicarboxylate dine-2-carboxylate (184.9 g, 95%) as a yellow oil.

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

H 10 N CO2Et Triphosgene, LiOH, HSCHCOOH H -> O 1O DIPEADCM n N H 15 ---O Boc N I I I I IIH NN CO2Et

O l YOB l O NN 1O H Triphosgene (21.3 g, 72 mmol) was added in portions to a 25 solution of (2S,5R)-ethyl 5-((benzyloxy)amino)piperidine LiOH.H2O (95g, 2.3 mol) and 2-mercaptoacetic acid (124 2-carboxylate (50 g., 0.18 mol) and DIPEA (128 mL, 0.72 g, 1.3 mol) were added to a solution of (2S,5R)-1-tert-butyl mol) in DCM (2000 mL) at 0° C. After stirring at 20° C. 2-ethyl 5-(N-(benzyloxy)-2-nitrophenylsulfonamido)piperi overnight, the reaction mixture was washed with HPO. dine-1,2-dicarboxylate (251 g, 0.45 mol) in DMF (1200 mL). 30 (10%), sat. NaHCO and saturated NaCl. The combined The reaction mixture was then stirred at 20°C. overnight. The organic layer was concentrated in vacuum and purified by reaction mixture was diluted with water and extracted with silica gel column chromatography (3:1 petroleum ether/ EtOAc) to afford (2S,5R)-ethyl 6-(benzyloxy)-7-oxo-1,6-di EtOAc (3x). The combined organic layer was washed with azabicyclo[3.2.1]octane-2-carboxylate (27.4 g. 50%) as a brine (3x), concentrated in vacuum and purified by silica gel 35 yellow solid. "H NMR (400 Mz, CDC1): 8 7.43-7.36 (m, column chromatography (3:1 petroleum ether/EtOAc) to 5H), 5.06 (d.J=11.4 Hz, 1H), 4.90 (d. J=11.4 Hz, 1H), 4.24(q, afford (2S,5R)-1-tert-butyl 2-ethyl 5-((benzyloxy)amino)pi J=7.1 Hz, 2H), 4.11-4.08 (m, 1H), 3.32-3.31 (m. 1H), 3.08 peridine-1,2-dicarboxylate (122.9 g, 85%) as a yellow solid. 3.05 (m, 1H), 2.93 (d. J=11.9 HZ, 1H), 2.14-2.05 (m, 2H), 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 40 5-((benzyloxy)amino)piperidine-2-carboxylate Example 2

Synthesis of (2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaz 45 Boc abicyclo[3.2.1]octane-2-carboxylic acid (Intermedi ate Compound 2a) NN CO2Et TFADCM -e- O NN1C 50 H 2a H NN CO2Et

O NN -O 55 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 LiOH mixture was stirred at rt overnight and then concentrated in Her 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 8,962,843 B2 65 66 -continued -continued K./ N III I IIH SO3-Py, py, rt He then BuNHSO4. NaH2PO4 O !- N YOB l O NOH 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 Dowex 50WX8 Na" mL). The mixture was then stirred at rt overnight. The reac He 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 NaSO 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 O OSONa 2.15 (m. 1H), 2.11-2.01 (m, 2H), 1.74-1.56 (m. 1H). Example 3 Step 1: 1,1'-Carbonyldiimidazole (5.8 g., 36.2 mmol) was added to a 0°C. solution of (2S,5R)-6-(benzyloxy)-7-oxo-1, Synthesis of (2S,5R)-2-(1,3,4-oxadiazol-2-yl)-7-oxo 30 6-diazabicyclo[3.2.1]octane-2-carboxylic acid (5.0 g. 18.1 1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen sulfate mmol) in dry THF (200 mL). The reaction mixture was (Compound 701) allowed to warm to rt then was stirred at rt for 3 hrs. Formo hydrazide (5.4g, 90.5 mmol) was added in one portion, and the reaction mixture was stirred for additional 3 hrs. The 35 mixture was then diluted with saturated sodium chloride and exatracted with EtOAc (3x). The combined organic layer was washed with saturated sodium chloride (2x), dried over ( NaSO, and concentrated to afford crude (2S,5R)-6-(benzy loxy)-N'-formyl-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-car N I H 40 bohydrazide (~11 g), which was directly used in the next step. ESI-MS (EI", m/z): 319.1 M+H". Z N Step 2: To a -10° C. solution of (2S,5R)-6-(benzyloxy)- O OSOH N'-formyl-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbohy O drazide (11 g) in dry DCM (200 mL) was added pyridine (28 45 mL), followed by dropwise addition of (CFSO)O (28 mL). HO s The reaction mixture was allowed to warm to rt and was NH CD stirred for 3 hrs. The reaction mixture was then cooled to -10° N III I IIH - an 1 --> C. and quenched with sat. NaHCO. The organic layer was H THF, rt separated and the aqueous layer was extracted with EtOAc 50 O NOB (3x). The combined organic layer was dried over NaSO, O concentrated and purified by silica gel column chromatogra Os phy (gradient elution 1:3 to 2:1 EtOAc/hexanes) to give (2S, N1 n N ve, 5R)-6-(benzyloxy)-2-(1,3,4-oxadiazol-2-yl)-1,6-diazabicy (CFSO)2O, py clo3.2.1]octan-7-one (4.6 g., 86% for two steps) as a slightly H --> 55 yellow solid. ESI-MS (EI", m/z): 301.0M+H". N DCM, rt Step 3: To a solution of (2S,5R)-6-(benzyloxy)-2-(1,3,4- 2 N oxadiazol-2-yl)-1,6-diazabicyclo3.2.1]octan-7-one (4.6 g. O OB 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 60 reaction mixture was then filtered and concentrated to afford (2S.5R)-6-hydroxy-2-(1,3,4-oxadiazol-2-yl)-1,6-diazabicy Pol/C, H2 clo3.2.1]octan-7-one (2.9 g, 91%), which was used directly Her in the next step. ESI-MS (EI", m/z): 211.1 M+H". N H turn Step 4: To a solution of (2S.5R)-6-hydroxy-2-(1,3,4-oxa 65 diazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-7-one (2.9 g, 13.8 mmol) in dry pyridine (60 mL) was added SOPy (11.0 g, 69.0 mmol). The reaction mixture was stirred at rt for 8 hrs US 8,962,843 B2 67 and then concentrated under vacuum. The residue was re -continued dissolved in aqueous NaH2PO (1.5M, 100 mL) then tetrabu N-N tylammonium hydrogensulphate (5.88 g., 17.3 mmol) was Bein-Nuly le, added. The mixture was stirred at rt for 20 minutes, then was O ? Pd/C, H2 extracted with EtOAc (4x). The combined organic layer was 5 Her dried and concentrated and the residue was purified by silica N . I I IIH THF, rt gel column chromatography (gradient elution 10:1 to 2:1 DCM/acetone) to afford tetrabutylammonium (2S,5R)-2-(1, Ou1 YOBn 3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6- N-N yl sulfate (4.1 g, 97%) as a white solid. ESI-MS (EI, m/z): 10 BocN N-K y O 'o. SO3-Py, py, rt 289.0 M-H. 'H NMR (400 MHz, CDC1): 88.48 (s, 1H), Hos 4.75 (d. J=6.5 Hz, 1H), 4.40 (brs, 1H), 3.34-3.26 (m, 9H), N ..H then BuNHSO4. 2.82 (d. J=12.0 Hz, 1H), 2.37-2.25 (m, 3H), 2.06-1.98 (m, NaH2PO4 N 1H), 1.71-1.65 (m, 8H), 1.49-142 (m, 8H), 1.01 (t, J=7.5 Hz, 15 O YOH 12H). N-N Step 5: Resin Exchange: Tetrabutylammonium (2S,5R)-2- BocN N-K y,'... (1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan O TFA, DCM 6-yl Sulfate (4.1 g, 7.72 mmol) was dissolved in a minimum He amount of HPLC grade water (~40 mL) and passed through a N I IIH column of 80 g of DOWEX 50WX8 Na" resin (the resin was prewased with >4 L of HPLC grade water) and eluted with O ! Y. YOSONBus HPLC grade water to afford sodium (2S,5R)-2-(1,3,4-oxadia N-N Zol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate HN N-K >. (2.2g, 91%) as a white solid after lyophilization. ESI-MS 25 O 8. (EI", m/z): 291.2 M+H". "H NMR (300 MHz, DO) & 8.92 N . I I IIH (s, 1H), 4.84 (d. J=6.7 Hz, 1H), 4.20 (brs, 1H), 3.25-3.16 (m, 1H), 2.92 (d. J=12.3 Hz, 1H), 2.41-2.26 (m, 1H), 2.26-2.11 O Y YOSOH (m. 2H), 2.04-1.89 (m. 1H). 30 Example 4 Step 1: DIPEA (1.1 g, 8.3 mmol) was added to a 0° C. solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo 3.2.1ctane-2-carbohydrazide (1.0 g, 2.48 mmol) in DMF Synthesis of (2S,5R)-2-(5-(2-aminoethyl)-1,3,4-oxa 35 (10 mL). 3-((tert-Butoxycarbonyl)amino)propanoic acid diazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl (0.39 g, 2.07 mmol) and HATU (0.90g, 2.48 mmol) were then hydrogen sulfate (Compound 703) added. The reaction mixture was stirred at 0°C. for 1 h and then quenched with saturated sodium chloride (50 mL). The organic layer was separated and the aqueous layer was 40 exacted with EtOAc (3x). The combined organic layer was washed with saturated sodium chloride (2x), dried overNaSO, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 10:1 to 2:1 hexanes/EtOAc) to afford tert-butyl (3-(2-((2S.5R)-6- N IIH 45 (benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbo nyl)hydrazinyl)-3-oxopropyl)carbamate (1.0 g, 87%). ESI MS (EI", m/z): 462 M+H". O - 1 YOSOH Step 2: (CFSO)O (1.0 mL) was slowly added to a -10° O C. solution of tert-butyl (3-(2-((2S.5R)-6-(benzyloxy)-7- OXO-1,6-diazabicyclo3.2.1]octane-2-carbonyl)hydrazinyl)- TFAO H2HN u , 50 NN • 3-oxopropyl)carbamate (1.0g, 2.17 mmol) and pyridine (1.0 H -- mL) in dry DCM (10 mL). The reaction mixture was allowed N ... I IIH to warm tort and then was stirred at rt for 1 h. Then saturated NaHCO, was added at 0°C. very slowly. The organic layer O -1. YOBn 55 was separated and the aqueous layer was exacted with EtOAc (3x). The combined organic layer was dried over NaSO and COOH HATU, DIPEA He concentrated. The residue was purified by silica gel column Bein1 N1 DCM chromatography (gradient elution 10:1 to 4:1 petroleum O ether/EtOAc) to give tert-butyl (2-(5-((2S.5R)-6-(benzy H 60 loxy)-7-OXO-1,6-diazabicyclo3.2.1]octan-2-yl)-1,3,4-oxa BocN N diazol-2-yl)ethyl)carbamate (0.40 g, 42%) as a slight yellow n-n N Hip(CF3SO2)2O, py solid. ESI-MS (EI", m/z): 444 M+H". "H NMR (500 MHz, O N . I IIH DCM DMSO-d) & 7.48-746 (m, 2H), 7.45-7.37 (m, 3H), 6.99 (t, J=5.5 Hz, 1H), 4.99-4.94 (m, 2H), 4.59 (d. J=7.0 Hz, 1H), O Y. YOBn 65 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).

US 8,962,843 B2 71 72 added. The reaction mixture was stirred at 0°C. for 1 h and dino)ethyl)-1,3,4-oxadiazol-2-yl)-1,6-diaza-bicyclo3.2.1 then was quenched with saturated sodium chloride (50 mL). octan-7-one, which was directly used in the next step. ESI The organic layer was separated and the aqueous layer was MS (EI", m/z): 496.2 M+H". exacted with EtOAc (3x). The combined organic layer was 5 Step 6: To a solution of (2S,5R)-6-hydroxy-2-(5-((2,3-bis washed with saturated sodium chloride (2x), dried over (tert-butoxycarbonyl)guanidino)ethyl)-1,3,4-oxadiazol-2- NaSO, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 10:1 to 2:1 yl)-1,6-diaza-bicyclo[3.2.1]octan-7-one from the previous hexanes/EtOAc) to afford tert-butyl (3-(2-((2S,5R)-6-(ben step in dry pyridine (2.0 mL) was added SOPy (152 mg, 0.962 mmol). The mixture was stirred at rt for 3 hrs and then Zyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carbonyl) 10 hydrazinyl)-3-oxopropyl)carbamate (1.0 g, 87%). ESI-MS concentrated under vacuum. The residue was re-dissolved in (EI", m/z): 462 M+H". aqueous NaH2PO (1.5M, 15 mL) then tetrabutylammonium Step 2: (CFSO)O (1.0 mL) was slowly added to a -10° hydrogensulphate (50 mg 0.15 mmol) was added. The mix C. solution of tert-butyl (3-(2-((2S.5R)-6-(benzyloxy)-7- ture was stirred at rt for 20 minutes, then extracted with oXo-1,6-diazabicyclo3.2.1]octane-2-carbonyl)hydrazinyl)- 15 EtOAc (4x). The combined organic layer was dried and con 3-oxopropyl)carbamate (1.0g, 2.17 mmol) and pyridine (1.0 centrated and the residue was purified by silica gel column mL) in dry DCM (10 mL). The reaction mixture was allowed chromatography (gradient elution 10:1 to 3:1 DCM/acetone) to warm to rt, was stirred at rt for 1 h then sat. NaHCO was to afford tetrabutylammonium (2S,5R)-2-(5-(2-(2,3-bis(tert added at 0°C. very slowly. The organic layer was separated butoxycarbonyl)guanidino)ethyl)-1,3,4-oxadiazol-2-yl)-7- and the aqueous layer was exacted with EtOAc (3x). The oxo-1,6-diaza-bicyclo[3.2.1]octan-6-ylsulfate (60 mg, 53%) combined organic layer was dried over Na2SO4 and concen as a white solid. ESI-MS (Ef m/z): 574.1 M-HI. trated. The residue was purified by silica gel column chroma Step 7: TFA (0.23 mL) was added to a 0° C. solution of tography (gradient elution 10:1 to 4:1 petroleum ether/ tetrabutylammonium (2S,5R)-2-(5-(2-(2,3-bis(tert-butoxy EtOAc) to give tert-butyl (2-(5-((2S.5R)-6-(benzyloxy)-7- 25 carbonyl)guanidino)ethyl)-1,3,4-oxadiazol-2-yl)-7-OXO-1,6- oXo-1,6-diazabicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2- diaza-bicyclo[3.2.1]octan-6-yl sulfate (50 mg, 0.06 mmol) in yl)ethyl)carbamate (0.40 g, 42%) as a slight yellow solid. dry DCM (0.68 mL). The reaction mixture was stirred at 0°C. ESI-MS (EI, m/z): 444 M+H". "H NMR (500 MHz, for 2 hand then diluted with ether. The precipitate was col DMSO-d) & 748-746 (m, 2H), 7.45-7.37 (m, 3H), 6.99 (t, 30 lected via centrifugation, washed with ether (3x) and further J=5.5 Hz, 1H), 4.99-4.94 (m, 2H), 4.59 (d. J=7.0 Hz, 1H), dried under high vacuum to provide (2S,5R)-2-(5-(2-guani 3.73 (brs, 1H), 3.32-3.26 (m, 2H), 2.96-2.92 (m, 2H), 2.85 dinoethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo 2.81 (m, 2H), 2.19-2.15 (m, 1H), 2.05-1.99 (m, 2H), 1.86 3.2.1]octan-6-yl hydrogen sulfate as TFA salt (~12 mg). 1.83 (m, 1H), 1.34 (s, 9H). ESI-MS (EI", m/z): 376.18. 'H NMR (300 MHz, DO)4.77 Step 3: TFA (0.5 mL) was added to a 0° C. solution of 35 (d. J=6.7 Hz, 1H), 4.19 (brs, 1H), 3.62 (t, J=6.4 Hz, 2H), tert-butyl 2-(5-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bi 3.21-3.18 (m. 1H), 3.18 (t, J=6.4 Hz, 2H), 2.89 (d. J=12.3 Hz, cyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)ethylcarbam 1H), 2.32-2.20 (m. 1H), 2.20-2.11 (m, 2H), 1.98-1.86 (m, ate (85 mg, 0.192 mmol) in CHCl (2.0 mL). The reaction 1H). mixture was stirred at 0°C. for 2 hrs and was then concen 40 trated under vacuum to provide (2S,5R)-2-(5-(2-aminoet Example 6 hyl)-1,3,4-oxadiazol-2-yl)-6-(benzyloxy)-1,6-diaza-bicyclo 3.2.1]octan-7-one TFA salt as a sticky oil, which was used Synthesis of (2S,5R)-7-oxo-2-(5-(piperidin-4-yl)-1, directly in the next step. ESI-MS (EI", m/z): 344.2 M+H". 3,4-oxadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6- Step 4: TEA (77 mg, 0.764 mmol) was added to a 0° C. 45 solution of (2S,5R)-2-(5-(2-aminoethyl)-1,3,4-oxadiazol-2- yl hydrogen sulfate (Compound 711) yl)-6-(benzyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one TFA salt from the previous step in MeOH (3.0 mL). Tert-Butyl (((tert-butoxycarbonyl)amino)(1H-pyrazol-1-yl)methylene) carbamate (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 mixture was evaporated and the residue was purified by silica gel column chromatography (gradient elution 30%-50% EtOAc/petroleum ether) to give (2S,5R)-2-(5-((2,3-bis(tert 55 butoxycarbonyl)guanidino)ethyl)-1,3,4-oxadiazol-2-yl)-6- (benzyloxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one (80 mg. O OSOH 70% in 2 steps) as a colorless oil. ESI-MS (EI", m/z): 586.3 M+H". Following Steps 1-5 in Example 4, replacing 3-((tert-bu Step 5: To a solution of (2S,5R)-2-(5-((2,3-bis(tert-butoxy 60 toxycarbonyl)amino)propanoic acid in Step 1 with 1-(tert carbonyl)guanidino)ethyl)-1,3,4-oxadiazol-2-yl)-6-(benzy butoxycarbonyl)piperidine-4-carboxylic acid, (2S,5R)-7- loxy)-1,6-diaza-bicyclo[3.2.1]octan-7-one (80 mg, 0.136 oXo-2-(5-(piperidin-4-yl)-1,3,4-oxadiazol-2-yl)-1,6- mmol) in THF (15 mL) was added 10% Pd/C (45 mg) and the diazabicyclo3.2.1]octan-6-yl hydrogen sulfate (89 mg) was mixture was stirred under H atmosphere at rt for 1 h. The 65 obtained as a light yellow solid after prep-HPLC purification reaction mixture was filtered and concentrated to afford (2S, using ammonium formate buffer. ESI-MS (EI, m/z): 374.26 5R)-6-hydroxy-2-(5-((2,3-bis(tert-butoxycarbonyl)guani "H NMR (300 MHz, DO) & 4.77 (d. J=5.9 Hz, 1H), 4.20 (br

US 8,962,843 B2 75 Example 11 -continued O H Tf,0/Py, Synthesis of (2S,5R)-2-(5-(guanidinomethyl)-1,3,4- N , () () pen-n-n N. “ -e--10° C.-0° C. oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan- 5 O N ... I IIH O PPh3, I, 6-yl hydrogen sulfate (Compound 704) N NEt3, DCM, rt O YOBn BocNY-Null N-N ly. NN N 10 O s H2, Pd/C Her HN 7 J N ... I IH THF, rt y 4 Sl !- N HN N ... I IIH O YOBn 15 BocN N-N a N Y-Nuk o1ly, . SO-Py, py, rt O OSOH Her N . IIH then BuNHSO4. NaH2PO4 Following Steps 1-7 in Example 5, replacing 3-((tert-bu- 20 O Y YOH toxycarbonyl)amino)propanoic acid in Step 1 with 2-((tert BocN N-N butoxycarbonyl)amino)acetic acid, (2S,5R)-2-(5-(guanidi nomethyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo Y-\ul O ly, TFA, DCM 3.2.1]octan-6-yl hydrogen Sulfate (745 mg) was obtained as -e- a light yellow solid after prep-HPLC purification using N H ammonium formate buffer. ESI-MS (EI", m/z): 362.2. O ! Y. YOSO, NBu4 HN N-N Example 12 30 Y-\ul >. O Synthesis of (2S,5R)-2-(5-3-aminopropyl)-1,3,4- oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan 6-yl hydrogen sulfate (Compound 720) 35

Step 1: Synthesis of -Ncy. 4-(tert-butoxycarbonylamino)butanoic acid "N-Nui O .. 40 To an aqueous solution of 4-aminobutanoic acid (25g, 242 mmol) in H2O (500 mL) at rt was added NaCO (75 g, 726 N ... I IIH mmol), followed by BocO (95 g, 435 mmol) in THF (200 mL). The reaction mixture was stirred at rt for 12 hrs then Ou1 n OSOH concentrated under reduced pressure. The aqueous residue 45 was extracted with EtO, then the aqueous layer was acidified O Na2CO3 (aq) Hope to pH 4-5 with citric acid and extracted with EtOAc. The BocO, THF, rt combined organic layer was dried over NaSO4, and concen "N-N-N OH trated to afford 4-(tert-butoxycarbonylamino)butanoic acid O K2CO3, MeI (45 g, 90%) as a colorless oil. ESI-MS (EI", m/z): 226 He M+Na". bein-ul- DMF, rt 50 OH Step 2: Synthesis of methyl O NH-NH2-H2O 4-(tert-butoxycarbonylamino)butanoate -e- bein-ul- O 1. EtOH, reflux To a solution of 4-(tert-butoxycarbonylamino)butanoic 55 O acid (7.0 g, 34.5 mmol) and KCO (9.5 g. 68.9 mmol) in 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 ban-ul- N 1. NH2 -- H was washed with water and saturated sodium chloride, dried O over NaSO, and concentrated to afford methyl 4-(tert-bu 60 toxycarbonylamino)butanoate (6.2 g, 83%) as a yellow oil. ESI-MS (EI", m/z): 240M+Na". 1. HATU, DIPEA He CHCl2, rt Step 3: Synthesis of tert-butyl 4-hydrazinyl-4-oxobu lcarbamate 65 YOBn To a solution of methyl 4-(tert-butoxycarbonylamino)bu tanoate (21.0 g, 96.8 mmol) in MeCH (180 mL) was added

US 8,962,843 B2 79 80 obtained as TFA salt. ESI-MS (EI", m/z): 360.20. "H NMR -continued (300 MHz, DO) & 4.78 (d. J=6.5 Hz, 1H), 4.20 (brs, 1H), N N 4.05-3.94 (m. 1H), 3.80-3.73 (m, 1H), 3.66-3.60 (m, 1H), 3.53-3.38 (m, 2H), 3.20 (brid, J=12.5 Hz, 1H), 2.94 (d. J=12.3 -N - , y. HZ, 1H), 2.59-2.48 (m. 1H), 2.40-2.26 (m, 2H), 2.23-2.08 (m, 5 "Nu N H 2H), 2.00- 1.90 (m. 1H). O Y. YOSOH Example 15 10 Step 1: DIPEA (105 g, 0.81 mol) was added to a 0° C. Synthesis of (2S,5R)-7-oxo-2-(5-(piperazin-1-yl)-1, solution of tert-butyl piperazine-1-carboxylate (25.0 g, 0.134 3,4-oxadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6- mol) in DCM (250 mL), followed by the addition of triphos yl hydrogen sulfate (Compound 713) gene (92 g, 0.27 mol) in portions over a 40 minute time period. The reaction mixture was allowed to warm to rt then 15 was stirred at rt for 3 hrs, filtered and concentrated to afford N-N 1-tert-butyl 4-trichloromethyl piperazine-1,4-dicarboxylate (50 g) as an oil. A solution of 1-tert-butyl 4-trichloromethyl piperazine-1, -N -->'. 4-dicarboxylate (50 g., 0.145 mol) in THF (50 mL) was added dropwise over a 30 minute time period to a solution of hydra zine hydrate (18 mL, 0.434 mol) in THF (150 mL).The reac O -Y. YOSOH tion mixture was stirred at rt for 2 hrs then diluted with saturated sodium chloride (50 mL) and exacted with EtOAc NH triphosgene s (3x). The combined organic layer was washed with saturated -N - then NH-NH2 sodium chloride (2x), dried over NaSO, and concentrated. Boc The residue was purified by crystallization (3:1 petroleum O ether/EtOAc) to provide tert-butyl 4-(hydrazinecarbonyl)pip erazine-1-carboxylate (13 g, 40% for two steps). ESI-MS N-- -- 30 (EI", m/z): 245 M+H". H-NMR (500 MHz, CDC1): 85.92 (s, 1H), 3.45-3.35 (m, 8H), 1.41 (s, 9H). Boc-Nu H Step 2: DIPEA (3.7 g., 10 mmol) was added to a 0° C. O solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo l 3.2.1]octane-2-carboxylic acid (1.45 g, 5.3 mmol. 0.8 eq.) HO HATU 35 and tert-butyl 4-(hydrazinecarbonyl)piperazine-1-carboxy -- late (1.6 g. 6.6 mmol) in dry DMF (50 mL), followed by the N ... I IIH addition of HATU (1.45 g, 5.3 mmol). The reaction mixture was allowed to warm to rt then was stirred at rt overnight. The O - 1 YOBn Boc mixture was then diluted with water (200 mL) and the result 40 ing precipitated material was collected by filtration, rinsed s^ with water, and then recrystallized (3:1 petroleum ether/ EtOAc) to afford tert-butyl 4-(2-((2S.5R)-6-(benzyloxy)-7- N-N-r NNH - - OXO-1,6-diazabicyclo3.2.1]octane-2-carbonyl)hydrazin O ecarbonyl)piperazine-1-carboxylate (1.4g, 54%), ESI-MS NN 45 (EI", m/z): 503 M+H". OB Step 3: To a solution of tert-butyl 4-(2-((2S.5R)-6-(benzy N -N loxy)-7-oxo-1,6-diaza-bicyclo3.2.1]octane-2-carbonyl)hy drazine-carbonyl)piperazine-1-carboxylate (903 mg, 1.8 - 10% Pd/C, H, mmol) in DCM (200 mL) was added pyridine (2.8 mL, 36.0 Ho O THF, rt 50 mmol). (CFSO)O (2.8 ml, 9.0 mmol) was then slowly added at -10°C. The reaction mixture was stirred at rt for 3 hrs. Sat. NaHCO was added at -10° C. very slowly. The organic layer was separated and the aqueous layer was exacted with EtOAc (3x). The combined organic layer was 55 ?\-K O \ 17, SO3-Py, py, rt dried over NaSO and concentrated. The crude product was Boc-Nu -e- purified by silica gel column chromatography (2:1 EtOAC/ N ..H then Bu4NHSO4. hexanes) to give tert-butyl 4-(2S.5R)-6-(benzyloxy)-7-oxo 1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl) piperazine-1-carboxylate (723 mg, 83%) as a slight yellow 60 solid. ESI-MS (EI", m/z): 485.2 M+H". Step 4: To a solution of tert-butyl 4-(5-((2S,5R)-6-hy droxy-7-OXO-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxa Boc- NN- O --TFA, DCM diazol-2-yl)piperazine-1-carboxylate (50 mg, 0.1 mmol) in N . IH THF (5.0 mL) was added 10% Pd/C(20mg). The mixture was us 65 stirred under H atmosphere at rt for 3 hrs then filtered and O OSONBu concentrated to afford tert-butyl 4-(5-((2S.5R)-6-hydroxy-7- OXO-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2- US 8,962,843 B2 81 yl)piperazine-1-carboxylate (30 mg, 75%), which was used -continued directly in the next step. ESI-MS (EI", m/z): 385 M+H". N-N Step 5: To a solution of tert-butyl 4-(5-((2S.5R)-6-hy ( ), droxy-7-OXO-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-oxa pin-> ... Pd/C, H2 5 -es diazol-2-yl)piperazine-1-carboxylate (30 mg, 0.077 mmol) N . I I IIH MeOH, rt in dry pyridine (3 ml) was added SOPy (97 mg, 0.61 mmol). The mixture was stirred at rt for 3 hrs. The pyridine was OuY YOBn evaporated under vacuum at 25°C. The residue was re-dis N-N solved in aqueous NaH2PO (1.5 M, 20 mL) and tetrabuty 10 lammonium hydrogensulphate (300 mg) was added. The in- O y'',8. SO-Py, py, rt mixture was stirred at rt for 20 minutes, then extracted with Her EtOAc (4x). The combined organic layer was dried and con N IIH then BuNHSO4. centrated and the residue was purified by silica gel column NaH2PO4 chromatography (gradient elution 10:1 to 1:1 DCM/acetone) 15 O 1 YOH to give tetrabutylammonium (2S,5R)-2-(5-(4-(tert-butoxy N-N carbonyl)piperazin-1-yl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6- diazabicyclo[3.2.1]octan-6-yl sulfate (20 mg, 20%). ESI-MS in- O '. TFA (EI", m/z): 473 M-HI. He Step 6: TFA (0.30 mL) was added to a 0° C. mixture of N ... I I IIH DCM tetrabutylammonium tert-butyl 4-(5-((2S.5R)-7-oxo-6-(sul fooxy)-1,6-diazabicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol O -1. YOSONBu, 2-yl)piperazine-1-carboxylate (21 mg 0.03 mmol) in dry N-N DCM (0.80 mL). The reaction mixture was stirred at 0°C. for 2-3 hrs then diluted with ether (~15 mL). The precipitate was 25 HN - > collected via centrifugation, washed with ether (3x) and dried under high vacuum to afford (2S,5R)-7-oxo-2-(5-(piperazin N . I I IIH 1-yl)-1,3,4-oxadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6- yl hydrogen sulfate (4 mg., 35%) as a light yellow solid after O - 1 YOSOH prep-HPLC purification using ammonium formate buffer. 30 ESI-MS (EI", m/z): 375.2. "H NMR (300 MHz, DO) & 4.65 (d. J=6.6 Hz, 1H), 4.19 (brs, 1H), 3.80–3.65 (m, 2H), 3.38 Step 1: Saturated NaHCO (7.0 mL, 6.26 mmol) was added 3.28 (m, 2H), 3.20-3.16 (m, 1H), 2.99-2.95 (m, 1H), 2.32 to a solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicy 1.88 (m, 4H). clo3.2.1]octane-2-carbohydrazide TFA salt (2.3 g 5.69 35 mmol) in dioxane (11.4 mL) and the reaction mixture was Example 16 stirred at rt for 5 minutes. Cyanic bromide (3 M solution, 2.3 mL, 6.90 mmol) was added and the reaction mixture was stirred at rt for 30 minutes. The reaction mixture was then Synthesis of (2S,5R)-2-(5-amino-1,3,4-oxadiazol-2- 40 loaded on a silica gel cartridge and purified by silica gel yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl hydro column chromatography (gradient elution 0-10% MeOH/ gen sulfate (Compound 707) DCM) to give (2S.5R)-2-(5-amino-1,3,4-oxadiazol-2-yl)-6- (benzyloxy)-1,6-diazabicyclo[3.2.1]octan-7-one (0.57 g. 32%). ESI-MS (EI", m/z): 316.4 M+H". N-N 45 Step 2: N,N-Dimethylpyridin-4-amine (155 mg, 1.27 mmol) was added to a 0°C. solution of (2S,5R)-2-(5-amino 1,3,4-oxadiazol-2-yl)-6-(benzyloxy)-1,6-diazabicyclo - , '. 3.2.1]octan-7-one (0.20 g, 0.63 mmol), BocO (0.27g, 1.27 N ... I I IIH mmol), and triethylamine (0.18 mL, 1.27 mmol) in DMF. The 50 reaction mixture was allowed to warm tort and was stirred for u1 2-3 hrs. The reaction mixture was then concentrated, re O YOSOH dissolved in DCM (50 mL), washed with water (50 mL), dried O over NaSO, and concentrated. The residue was purified by silica gel column chromatography (gradient elution 0-50% TFA HN n l '., Br-CN N NaHCO 55 EtOAc/hexane) to give tert-butyl (5-((2S.5R)-6-(benzyloxy)- Hoss 7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol N . I I IIH Dioxane 2-yl)carbamate (0.16 g. 60.7%). ESI-MS (EI", m/z): 416.4 M+H". Ou1 YOBn Step 3-5: Following Steps 3-5 in Example 4, replacing N-N 60 tert-butyl (2-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabi cyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)ethyl)carbam HN -Ko1 >. BocO, DMAP ate in Step 3 with tert-butyl (5-((2S.5R)-6-(benzyloxy)-7- Her OXO-1,6-diazabicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2- N 1 IIH TEA, DMF yl)carbamate, (2S.5R)-2-(5-amino-1,3,4-oxadiazol-2-yl)-7- 65 OXO-1,6-diazabicyclo3.2.1]octan-6-yl hydrogen Sulfate was obtained (64 mg) after prep-HPLC using ammonium formate buffer. ESI-MS (EI", m/z): 306.25 M+H". "H NMR (300 US 8,962,843 B2 83 84 MHz, DO) & 4.61 (d. J=6.6 Hz, 1H), 4.19 (brs, 1H), 3.17 (br -continued d, J=12.2 Hz, 1H), 2.98 (d. J–12.3 Hz, 1H), 2.27-1.81 (m, N 4H). "S-C), Example 17 HN N IH

Synthesis of (2S.5R)-2-(5-carbamoyl-1,3,4-oxadia Ou1 n OSONa zol-2-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-6-yl 10 hydrogen sulfate (Compound 706) Step 1: HATU (0.95 g, 2.5 mmol) was added to a 0° C. solution of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo 3.2.1]octane-2-carbohydrazide (1.00 g, 2.5 mmol), N-N 2-amino-2-oxoacetic acid (0.18 g, 2.1 mmol) and DIPEA 15 (1.08 g, 8.4 mmol) in DMF (10 mL). The reaction mixture O y. was stirred at 0° C. for 1 h then quenched with saturated O w Sodium chloride (50 mL) and the organic layer was separated. HN N IIH The aqueous layer was exacted with EtOAc (3x). The com bined organic layer was washed with Saturated sodium chlo ride (2x), dried over NaSO, and concentrated. The residue Ou1 YOSOH was purified by silica gel column chromatography (1:1 EtOAc/hexane) to give 2-(2-((2S.5R)-6-(benzyloxy)-7-oxo 1,6-diazabicyclo[3.2.1]octane-2-carbonyl)hydrazinyl)-2- O oxoacetamide (0.57 g. 64%). ESI-MS (EI", m/z): 362 TFA HN !. 25 M+H". NN .. Step 2: (CFSO) O (0.58g, 2.08 mmol) was slowly added H -- to a -10°C. solution of 2-(2-((2S.5R)-6-(benzyloxy)-7-oxo N . I I IIH 1,6-diazabicyclo[3.2.1]octane-2-carbonyl)hydrazinyl)-2- oxoacetamide (0.30 g, 0.83 mmol) and pyridine (0.6 mL) in O - 1 YOBn 30 dry DCM (5 mL). The reaction mixture was allowed to warm to rt. The reaction mixture was stirred at rt for 1 h then O quenched with sat. NaHCO very slowly. The organic layer was separated and the aqueous layer was exacted with EtOAc OH HATU (3x). The combined organic layer was dried over NaSO, HN DIPEADMF 35 and concentrated. The residue was purified by silica gel col O umn chromatography (gradient elution 1:10 to 2:1 EtOAc/ O O hexane followed by 10:1 to 2:1 petroleum ether/EtOAC) to R. J. give 5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo HN N- N ,* (CFSO)2O/py 3.2.1]octan-2-yl)-1,3,4-oxadiazole-2-carboxamide (0.18 g. -- 40 47%) as a slight yellow solid. ESI-MS (EI", m/z): 344.0 O N . . . H DCM M+H". Step 3: To a solution of 5-((2S.5R)-6-(benzyloxy)-7-oxo 1,6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazole-2-car Ou1 YOBn N-N boxamide (0.14 g. 0.41 mmol) in THF (10 mL) was added O f y 45 10% Pd/C (0.14 g). The mixture was stirred under H atmo '', sphere at rt for 3 hrs then filtered and concentrated to provide O 8. Pd/C, H2 5-((2S,5R)-6-hydroxy-7-oxo-1,6-diazabicyclo[3.2.1]octan Her HN N I IIH THF 2-yl)-1,3,4-oxadiazole-2-carboxamide (103 mg, 100%), which was directly used in the next step. ESI-MS (EI", m/z): 50 254 M+H". O - 1 YOBn Step 4: To a solution of 5-((2S.5R)-6-hydroxy-7-oxo-1,6- N-N diazabicyclo3.2.1]octan-2-yl)-1,3,4-oxadiazole-2-carboxa O f y mide (103 mg, 0.41 mmol) in dry pyridine (2.0 mL) was O 'o, SO3-Py, py, rt added SOPy (323 mg, 2.03 mmol). The mixture was stirred -e- 55 at rt for 3 hrs and then concentrated under vacuum. The HN N H then BuNHSO4. residue was re-dissolved in aqueous NaH2PO (1.5 M, 10 NaH2PO4 mL) then tetrabutylammonium hydrogen Sulphate (166 mg, O 1 YOH 0.49 mmol) was added. The mixture was stirred at rt for 20 N-N minutes, then extracted with EtOAc (4x). The combined O W y 60 organic layer was dried and concentrated and the residue was '... purified by silica gel column chromatography (gradient elu O Dowex50w:x8 tion 10:1 to 8:1 DCM/acetone) to afford tetrabutylammonium -e- HN N . IIH (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): N 65 333.0 M-H. O YOSONHBuy Step 5 Sodium Resin Exchange: tetrabutylammonium (2S, 5R)-2-(1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1 US 8,962,843 B2 85 octan-6-yl Sulfate (110 mg) was dissolved in a minimum amount of HPLC grade water (-80 mL) and passed through a column of 20g of DOWEX 50WX8 Na" resin (the resin was pre-washed with >5 L of HPLC grade water) and eluted with HN S HPLC grade water to provide sodium (2S,5R)-2-(1,3,4-oxa diazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate N ... I IIH (55 mg, 80%) as a white solid after lyophilization. ESI-MS (EI, m/z): 332.0|M-H. 'H NMR (500 MHz, DO);84.97 O u n OSOH 4.93 (m, 1H), 4.27 (brs, 1H), 3.29 (d. J–11.0 Hz, 1H), 3.04 (d. 10 J=12.0 Hz, 1H), 2.43-2.39 (m. 1H), 2.31-2.23 (m, 2H), 2.04 Step 1: HATU (3.3 g, 8.7 mmol) and tert-butyl 4-(hydrazi 1.98 (m, 1H). necarbonyl)piperidine-1-carboxylate (2.29 g, 9.42 mmol) were added to a 0° C. solution of (2S,5R)-6-(benzyloxy)-7- Example 18 OXO-1,6-diaza-bicyclo3.2.1]octane-2-carboxylic acid (2 g, 15 7.25 mmol) in CHCl (50 mL), followed by the addition of DIPEA (2.8 g. 21.8 mmol). The reaction mixture was stirred Synthesis of (2S,5R)-7-oxo-2-(5-(piperidin-4-yl)-1, at 0° C. for 12 hrs then washed with water and saturated 3,4-thiadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6- sodium chloride, dried over NaSO, and concentrated. The yl hydrogen sulfate (Compound 719) residue was purified by silica gel column chromatography (gradient elution 1%-10% MeOH/CHCl) to give tert-butyl 4-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bicyclo 3.2.1]octane-2-carbonyl)hydrazinecarbonyl)piperidine-1- N-N carboxylate as a white solid. (2.2g, 62%). ESI-MS (EI", m/z): y. 502.2 M+H". 25 Step 2: Lawesson’s reagent (0.16 g. 6 mol) was added to a HN S solution of tert-butyl 4-(2-((2S.5R)-6-(benzyloxy)-7-oxo-1, N H 6-diaza-bicyclo3.2.1]octane-2-carbonyl)hydrazinecarbo nyl)piperidine-1-carboxylate (0.16 g. 0.319 mmol) in THF Ou1 YOSOH (20 mL). The reaction mixture was stirred at 70° C. for 0.5h. H 30 The solution was cooled to room temperature and sat. O O Nn NaHCO was added. The organic layer was separated and the NH2 HATU, aqueous layer was exacted with EtOAc (2x). The combined HO w DIPEA -- -- organic layer was dried over NaSO and concentrated. The N . I IIH DCM, rt residue was purified by silica gel column chromatography 35 (gradient elution 20%-50% EtOAc/petroleum ether) to give tert-butyl 4-(5-((2S.5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bi O - 1 YOBn See cyclo3.2.1]octan-2-yl)-1,3,4-thiadiazol-2-yl)piperidine-1- O carboxylate (90 mg, 50%) as a white solid. ESI-MS (EI", m/z): 500.2 M+H". "H NMR (500 MHz, DMSO-d): 8 os-N-NN 8. 40 748-7.37 (m, 5H), 4.99-4.94 (m, 2H), 4.77 (d. J=7.0 Hz, 1H), H Lawesson's reagent --- 3.99 (d. J=11 Hz, 2H), 3.69 (s, 1H), 3.39-3.35 (m, 1H), N H THF, 66° C. 2.88-2.85 (m,3H), 2.68 (d. J=12 Hz, 1H), 2.44 (dd, J-7.5, 5.5 HZ, 1H), 2.05-1.96 (m, 4H), 1.79-1.77 (m. 1H), 1.62-1.55 (m, O 1 YOBn 2H), 1.40 (s.9H). N 45 Step 3: To a solution of tert-butyl 4-(5-((2S.5R)-6-(benzy Boc loxy)-7-oxo-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1,3,4-thia N-N diazol-2-yl)piperidine-1-carboxylate (120 mg, 0.24 mmol) in y. THF (30 mL) was added 10% Pd(OH)/C (200 mg). The BocN S Pd(OH), THF mixture was stirred under H atmosphere at rt for 3 h, filtered N IH H2, rt 50 and concentrated to afford tert-butyl 4-(5-((2S,5R)-6-hy droxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-thia O - 1 YOBn diazol-2-yl)piperidine-1-carboxylate as a yellow solid, which N-N was directly used in the next step. ESI-MS (EI, m/z): 410.2 M+H". Cly. 55 Step 4: To a solution of tert-butyl 4-(5-((2S,5R)-6-hy BocN S SO3-Py, py, rt droxy-7-oxo-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-thia N . I I IIH then BuNHSO4. diazol-2-yl)piperidine-1-carboxylate from the former step in NaH2PO4 dry pyridine (3 mL) was added SOPy (266 mg, 1.1 mmol). O 1 NOH The mixture was stirred at rt for 3 hrs and then concentrated N-N 60 under vacuum. The residue was re-dissolved in aqueous y, NaH2PO (1.5 M, 20 mL) then tetrabutylammonium hydro BOCN S TFADCM gensulphate (150 mg, 0.44 mmol) was added. The mixture H was stirred at rt for 20 minutes, then extracted with EtOAc N ... I IIH (4x). The combined organic layer was dried and concentrated 65 and the residue was purified by silica gel column chromatog O uY YOSO,NBus raphy (gradient elution 10:1 to 1:1 DCM/acetone) to afford tetrabutylammonium tert-butyl 4-(5-((2S.5R)-7-oxo-6-(sul US 8,962,843 B2 87 88 fooxy)-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4-thiadiazol -continued 2-yl)piperidine-1-carboxylate as a white solid (110 mg. 62%). ESI-MS (EI, m/z): 488.1 M-H. Step 5: Tetrabutylammonium tert-butyl 4-(5-((2S,5R)-7- HATU -e- oxo-6-(sulfooxy)-1,6-diaza-bicyclo[3.2.1]octan-2-yl)-1,3,4- DMF, 0° C. thiadiazol-2-yl)piperidine-1-carboxylate (110 mg, 0.15 78% mmol) was dissolved in a minimum amount of HPLC grade water (~ 15 mL) and passed through a column of 16 g of O Tf,0/Py, DOWEX 50WX8 Na" resin (the resin was pre-washed with 10 -10° C.-0° C. N >0.5 L of HPLC grade water) and eluted with HPLC grade 1. l, 5196 water to afford sodium (2S,5R)-2-(5-(1-(tert-butoxycarbo N He nyl)piperidin-4-yl)-1,3,4-thiadiazol-2-yl)-7-OXO-1,6-diaza On-r . IIH or PPh3, I, NEt3, bicyclo[3.2.1]octan-6-yl sulfate after lyophilization as a DCM, rt 85% N n white solid (50 mg, 65%). ESI-MS (EI, m/z): 488.0M-HI. 15 O JC OB "H NMR (500 MHz, DO) & 4.88 (d. J–7.0 Hz, 1H), 4.15 (s, N 1H), 4.06 (d. J=10.5 Hz, 2H), 3.39-3.35 (m, 1H), 3.13 (d, J-2 HZ, 1H), 2.94-2.87(m,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). BocN H THF, rt

Step 6: TFA (0.20 mL) was added to a 0° C. mixture of N n sodium (2S,5R)-2-(5-(1-(tert-butoxycarbonyl)piperidin-4- O OB yl)-1,3,4-thiadiazol-2-yl)-7-oxo-1,6-diaza-bicyclo[3.2.1]oc N N C tan-6-yl 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 25 SO3-Py, py, rt diluted with ether (~10 mL). The precipitate was collected via He centrifugation, washed with ether (3x) and dried under high BocN then BuNHSO4. vacuum to afford (2S,5R)-7-oxo-2-(5-(piperidin-4-yl)-1,3,4- NaH2PO4 thiadiazol-2-yl)-1,6-diazabicyclo[3.2.1]octan-6-yl hydrogen N 50% for two steps N C sulfate (10 mg, 25%) after prep-HPLC using ammonium 30 N formate buffer. ESI-MS (EI", m/z): 390.12. TFA, DCM He Example 19 BocN ... I IIH 48% 35 Synthesis of (2S.5R)-2-(5-(2-(methylamino)ethyl)-1, O YOSONBu, 3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1 octan-6-yl hydrogen sulfate (Compound 722) ly. O 40 N . I I IIH / JY. O Soso H 45 / N H Step 1: Synthesis of methyl 3-(tert-butoxycarbonyl (methyl)amino)propanoate O Y n OSOH Boc 50 To a solution of 3-(tert-butoxycarbonyl(methyl)amino) 1N OH K2CO3, MeI Her propanoic acid (7.0 g, 0.032 mol) and KCO (13.3 g, 0.096 DMF, rt mol) in DMF (100 mL) was added Mel (9.0 g, 0.064 mol) at O 92% rt. The resultant solution was stirred at rt for 3 hrs. The reaction mixture was quenched with ice water and extracted Roe O 55 with ethyl acetate (2x). The combined organic layer was dried 1. n-r N EtOH, reflux over NaSO, and concentrated under reduced pressure. The O 98% residue was purified by silica gel column chromatography (gradient elution 0%-10% EtOAc/petroleum ether) to afford Boc methyl 3-(tert-butoxycarbonyl (methyl) amino)propanoate 60 (6.9 g, 92%) as a white solid. ESI-MS (EI", m/z): 118 M-100+H". Step 2: Synthesis of tert-butyl 3-hydrazinyl-3-oxopropyl (methyl)carbamate 65 To a solution of methyl 3-(tert-butoxycarbonyl (methyl) amino)propanoate (6.9 g, 0.0317 mol) in EtOH (15 mL) was

US 8,962,843 B2 92 -continued g, 94%) as a white solid, which was used directly in the next N-N NBoc f \ step. ESI-MS (EI", m/z): 345.0M+H". as? R-K O> Pd/C, H2 Step 4: Synthesis of tert-butyl 34(2-(2-((2S.5R)-6- -e- 5 N IIIH MeOH, rt, (benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2- 2.99% carbonyl)hydrazinyl)-2-oxoethyl)(tert-butoxycarbo N O YOBn nyl)amino)aZetidine-1-carboxylate N-N NBoc 10 DIPEA (14g, 0.11 mol) was added to a 0°C. solution of B.C R-K O y. SO3-Py,12h py, rt, -- tert-butyl 3-(tert-butoxycarbonyl(2-hydrazinyl-2-oxoethyl) N IH then NBuHSO4. amino)azetidine-1-carboxylate (15g, 0.044 mol), (5R)-6- N NaH2PO4 (benzyloxy)-7-oxo-1,6-diaza-bicyclo3.2.1]octane-2-car O YOH 60% boxylic acid (10g, 0.036 mol) and HATU (15g, 0.04 mol) in N-N 15 CHCl (250 mL). The mixture was allowed to warm to rt NBoc W \ then was stirred at rt for 2 hrs. The mixture was quenched with BNC Ü-K O cy. TFA, DCM -e- saturated Sodium chloride (50 mL) and the organic layer was N ... I IIH 65% separated. The water layer was extracted with EtOAc (3x). N The combined organic layer was washed with Saturated O OSONHBu sodium chloride (2x), dried over NaSO, and concentrated. The residue was purified by silica gel column chromatogra phy (gradient elution 0-50% EtOAc/petroleum ether) to afford tert-butyl 3-((2-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6- 25 diaza-bicyclo3.2.1]octane-2-carbonyl)hydrazinyl)-2-oxoet hyl)(tert-butoxycarbonyl)amino)azetidine-1-carboxylate (18.5g, 86%). ESI-MS (EI, m/z): 603.3 M+H".

Step 5: Synthesis of tert-butyl 3-(((5-(2S.5R)-6-(ben 30 Zyloxy)-7-oxo-1,6-diaza-bicyclo3.2.1]octan-2-yl)-1, Step 1: Synthesis of tert-butyl 3-(2-ethoxy-2-oxoet 3,4-oxadiazol-2-yl)methyl)(tert-butoxycarbonyl) hylamino)azetidine-1-carboxylate amino)aZetidine-1-carboxylate A mixture of tert-butyl 3-aminoazetidine-1-carboxylate (10.0 g, 58 mmol), ethyl 2-bromoacetate (10.7 g. 64 mmol) 35 EtN (8.3 g, 0.082 mol) was added to a 0° C. solution of and potassium carbonate (24.0 g, 174 mmol) in MeCN (200 I (10.43 g, 0.041 mol) and PPhs (10.76 g., 0.041 mol) in mL) was stirred at rt for 13 hrs. The reaction mixture was CHCl (250 mL). After the mixture was stirred at rt for 0.5 hr, filtered and concentrated. The crude material was purified by tert-butyl 3-((2-(2-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza silica gel column chromatography (gradient elution 0-66% bicyclo[3.2.1]octane-2-carbonyl)hydrazinyl)-2-oxoethyl) EtOAc/petroleum ether) to afford tert-butyl 3-(2-ethoxy-2- 40 (tert-butoxycarbonyl)amino)azetidine-1-carboxylate (12.34 oxoethylamino)aZetidine-1-carboxylate (12.1 g, 81%) as a g, 0.0205 mol) was added. The mixture was stirred for another white solid. ESI-MS (EI", m/z): 259.0M+1-1". 1 hr. The mixture was then concentrated and EtOAc (250 mL) Step 2: Synthesis of tert-butyl 3-(tert-butoxycarbonyl was added to the resulting residue. The residue, was stirred (2-ethoxy-2-oxoethyl)amino)aZetidine-1-carboxylate 45 and then filtrated to remove PhPO. The filtrate was concen trated and purified by Silica gel column chromatography (gra A mixture of tert-butyl 3-(2-ethoxy-2-oxoethylamino)aze dient elution 33-50% EtOAc/petroleum ether) to afford tert tidine-1-carboxylate (10.3 g 40 mmol), di-tert-butyl dicar butyl 3-(((5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diaza bonate (10.4 g. 48 mmol) and potassium carbonate (16.6 g. bicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)methyl)(tert 120 mmol) in MeCN (200 mL) was stirred at rt for 6 hrs. The 50 butoxycarbonyl)amino) aZetidine-1-carboxylate (9.6 g. reaction mixture wasthen filtered and concentrated. The 80%). ESI-MS (EI", m/z): 585.3 M+H". crude product was purified by silica gel column chromatog raphy (gradient elution 0-80% EtOAc/petroleum ether) to Step 6-8: Following Steps 3-5 in Example 4, replacing afford tert-butyl 3-(tert-butoxycarbonyl(2-ethoxy-2-oxoet tert-butyl (2-(5-((2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabi hyl)amino)azetidine-1-carboxylate (13.0 g, 90%) as a white 55 cyclo3.2.1]octan-2-yl)-1,3,4-oxadiazol-2-yl)ethyl)carbam solid. ESI-MS (EI", m/z): 359.0 M+H". ate in Step 3 with tert-butyl 3-(((5-((2S.5R)-6-(benzyloxy)- 7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-1,3,4-oxadiazol Step 3: Synthesis of tert-butyl 3-(tert-butoxycarbonyl 2-yl)methyl)(tert-butoxycarbonyl)amino)azetidine-1- (2-hydrazinyl-2-oxoethyl)amino)aZetidine-1-car carboxylate; (2S,5R)-2-(5-((azetidin-3-ylamino)methyl)-1, boxylate 60 3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6- A mixture of tert-butyl 3-(tert-butoxycarbonyl(2-ethoxy ylhydrogen sulfate (1.5 g) was obtained as a white solid after 2-oxoethyl)amino)aZetidine-1-carboxylate (7.2g, 20 mmol), prep-HPLC purification using ammonium formate buffer. hydrazine (5.0 g, 100 mmol) and ethanol (50 mL) was stirred ESI-MS (EI", m/z): 375.1. "H NMR (300 MHz, DO) & 5.00 at 80° C. for 17 hrs. The reaction mixture was then concen 65 (d. J=5.4 Hz, 1H), 4.39-4.34 (m, 3H), 4.25 (s. 2H), 4.21-4.08 trated to afford the crude tert-butyl 3-(tert-butoxycarbonyl(2- (m,3H), 3.41 (brd, J=13.1 Hz, 1H), 3.11 (d. J–12.3 Hz, 1H), hydrazinyl-2-oxoethyl)amino)aZetidine-1-carboxylate (6.4 2.57-2.28 (m, 3H), 2.19-2.08 (m, 1H).

US 8,962,843 B2 95 Example 22 -continued NBoc bein- N. Synthesis of (2S,5R)-2-(5-(1-(guanidinomethyl)cy HN W clopropyl)-1,3,4-oxadiazol-2-yl)-7-oxo-1,6-diazabi O-1”, Pol/C, H2 cyclo3.2.1]octan-6-yl hydrogen Sulfate (Compound CH3OH/THF, rt 723) N III IIH

Oa N. OB 10 NBoc NH pin- NSN HN M H - NNN O-1, SO3-Py, py 2 HN W J then O-1%, 15 N III IIH Bu-NHSO4. N NaH2PO4 N I I I I IIH O OH NBoc a N O OSOH bein- NSN HN W TFAf O-1/. DCM OH CDI, NH,NH-HO Ho N III IIH THF, rt 25 BOCHN O 84% Z N O OSONBu NH HN-NH -- e- NNN 30 HN 7 J BocN O O %

O N II I I IIH u. 35 Z N HO HATU, DIPEA O OSOH -e- N . I I IIH DCM, rt 859% N Step 1: Synthesis of tert-butyl (1-(hydrazinecarbonyl) O OB 40 cyclopropyl)methylcarbamate O CDI (12.5g, 0.077 mol) was added to a 0° C. solution of R.N J. , I2, PPh3, 1-((tert-butoxycarbonylamino)methyl)cyclopropanecar BocN N EtN H - T - 45 boxylic acid (15.0g, 0.07 mol) in THF (250 mL). The mixture O N ... I IIH DCM, rt was allowed to warm tort, and stirred at rt. for 2 hrs, and then 83% NHNH2.H2O (10.5 g., 0.21 mol) was added rapidly. The N mixture was stirred for another 1 hr. The reaction mixture was O OB concentrated, and then CHCl (500 mL) was added. The BocN N 50 combined organic layer was washed with HO (100 mL), W dried over NaSO, and concentrated to afford tert-butyl (1- 4, (hydrazinecarbonyl)cyclopropyl)methylcarbamate (13.5 g. O TFA -e- 84%). ESI-MS (EI", m/z): 230.2 M+H". DCM, rt N ... I I IIH 9996 55 Step 2: Synthesis of tert-butyl (1-(2-((5R)-6-(benzy loxy)-7-oxo-1,6-diaza-bicyclo3.2.1]octane-2-carbo OZ N OB nyl)hydrazinecarbonyl)cyclopropyl)methylcarbamate NBoc DIPEA (14g, 0.11 mol) was added to a solution of tert HN 60 butyl (1-(hydrazinecarbonyl)cyclopropyl)methylcarbamate (10.0 g, 0.044 mol), (5R)-6-(benzyloxy)-7-oxo-1,6-diaza-bi cyclo[3.2.1]octane-2-carboxylic acid (10g, 0.036 mol), and EtN, CHOH, rt HATU (15g, 0.04 mol) in CHCl (250 mL). The mixture was 86% stirred at rt for 2 hrs. The mixture was diluted with CH-Cl 65 (500 mL), washed with HO (300 mL), dried over NaSO, and concentrated. The crude product was washed with EtOAc (200 mL), and filtrated to afford tert-butyl(1-(2-((5R)-6-(ben

US 8,962,843 B2 100 -continued tetraisopropanolate (28.9 g, 102 mmol) under N2 atmosphere. NN Ethylmagnesium bromide (3M in Et2O, 50 mL) was slowly N added drop-wise at 0°C. The mixture was stirred at rt for 1 hr. BocN W J Boron trifluoride etherate (17.0 g, 120 mmol) was slowly o-1. Pol/C, H2 5 Hos added at 0°C. The mixture was stirred at rt for 1 hr. The THF, rt mixture was quenched with 10% aq. NaOH (300 mL) and N . . I IIH then extracted with DCM (3x). The combined organic layer Z N was dried over NaSO and concentrated. The residue was O OBN 10 purified by silica gel column chromatography (gradient elu tion 5-50% EtOAc/petroleum ether) to afford 1-(2-(tert-bu tyldimethylsilyloxy)ethyl)cyclopropanamine (3.9 g, 30%) as a colorless oil. ESI-MS (EI", m/z): 216.2 M+H". NNN BocN W J 15 Step 3: Synthesis of tert-butyl 1-(2-(tert-butyldimeth o-1, SO-Py, py ylsilyloxy)ethyl)cyclopropylcarbamate -- then BuNHSO4. N ... I I IIH NaH2PO4 A solution of 1-(2-(tert-butyldimethylsilyloxy)ethyl)cy clopropanamine (1.9 g, 8.8 mmol), (Boc)O (2.9 g, 13.2 Z NN 2O mmol), NaHCO, (1.5g, 17.6 mmol) in THF/HO (20 mL/20 O OH mL) was stirred at rt for 17 hrs. The mixture was extracted with EtOAc (3x). The combined organic layer was dried over NaSO and concentrated. The residue was purified by silica NN 25 gel column chromatography (0-15% gradient elution EtOAc/ BOCHN / N petroleum ether) to afford tert-butyl 1-(2-(tert-butyldimeth l, TFAf ylsilyloxy)ethyl)cyclopropylcarbamate (2.5g, 90%) as a col O .. DCM He orless oil. ESI-MS (EI", m/z): 316 M+H". N . I I IIH 30 Step 4: Synthesis of tert-butyl A N 1-(2-hydroxyethyl)cyclopropylcarbamate O OSONBu A solution of tert-butyl 1-(2-(tert-butyldimethylsilyloxy) ethyl)cyclopropylcarbamate (11.9 g, 37.8 mmol) and 35 3HFTEA (22.0 g) in DCM (50 mL) was stirred atrt for 17 hrs. N NN The mixture was concentrated and the residue was purified by HN / J silica gel column chromatography (0-20% gradient elution O %, EtOAc/petroleum ether) to afford tert-butyl 1-(2-hydroxy 40 ethyl)cyclopropylcarbamate (4.6 g. 60%) as a white solid. N ... I I IIH Step 5: Synthesis of Z N 2-(1-(tert-butoxycarbonylamino)cyclopropyl)acetic O OSOH acid 45 RuCl.HO (124 mg. 0.6 mmol) was added to a solution of Step 1: Synthesis of tert-butyl 1-(2-hydroxyethyl)cyclopropylcarbamate (6.1 g, 3-(tert-butyldimethylsilyloxy)propanenitrile 30 mmol), and NaIO (19.0 g, 90 mmol) in DCM/HO/ CHCN (20 mL/40 mL/20 mL). The mixture was stirred at rt Imidazole (33.6 g. 495 mmol) and tert-butyldimethylsilyl 50 for 3 hrs. The mixture was then diluted with H2O (100 mL), chloride (27.2g, 180 mmol) were added to a 0°C. solution of and extracted with DCM (3x). The combined organic layer 3-hydroxypropanenitrile (10.6 g. 150 mmol) in DMF 50 mL). was dried over NaSO and concentrated. The residue was The mixture was allowed to warm to rt, then was stirred at rt purified by silica gel column chromatography (0-30% gradi for 12 hrs. The mixture was quenched with water (500 mL), and then extracted with EtOAc (3x). The combined organic 55 ent elution EtOAc/petroleum ether) to afford 2-(1-(tert-bu layer was dried over NaSO and concentrated. The residue toxycarbonylamino)cyclopropyl)acetic acid (6.0g, 90%) as a was purified by silica gel column chromatography (gradient white solid. ESI-MS (EI", m/z): 238 M+Na". elution 0-5% EtOAc/petroleum ether) to give 3-(tert-bu tyldimethylsilyloxy) propanenitrile (18g, 75%) as a colorless Step 6: Synthesis of tert-butyl 1-(2-(2-((2S.5R)-6- (benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2- oil. "H NMR (400 MHz, CDC1): 8 3.74 (t, J=6.0 Hz, 2H), 60 carbonyl)hydrazinyl)-2-oxoethyl)cyclopropylcar 2.43 (t, J=6.0 Hz, 2H), 0.81 (s, 9H), 0.00 (s, 6H). bamate Step 2: Synthesis of 1-(2-(tert-butyldimethylsily loxy)ethyl)cyclopropanamine A solution of 2-(1-(tert-butoxycarbonylamino)cyclopro 65 pyl)acetic acid (1.5 g. 6.9 mmol), (2S,5R)-6-(benzyloxy)-7- To a solution of 3-(tert-butyldimethylsilyloxy)propaneni oxo-1,6-diaza-bicyclo[3.2.1]octane-2-carbohydrazide (3.1 trile (11.1 g, 60 mmol) in EtO (400 mL) was added titanium g, 7.6 mmol), DIPEA (2.7 g. 21.0 mmol), and HATU (3.9 g,

US 8,962,843 B2 106 -continued -continued O NN N / J O '. HN-NH2 5 N . I I IIH BochN O HNs O ! NOSOH ul. 10 HO . -- N ... I IIH Step 1: Synthesis of ethyl 2-(3-(tert-butoxycarbonylamino)cyclobulidene) acetate OZ NOB 15 To a solution of tert-butyl 3-oxocyclobutylcarbamate (1.0 O g, 5.4 mmol) in toluene (10 mL) was added ethyl 2-(triph ( HATU, DIPEA enylphosphoranylidene)acetate (2.1 g, 5.9 mmol). The reac -e- tion mixture was heated at 100° C. for 2 hrs, and then con HN-NH2 DMF, 0° C. centrated. The residue was purified by silica gel column chromatography (10% EtOAc/petroleum ether) to afford BochN ethyl 2-(3-(tert-butoxycarbonylamino)cyclobutylidene)ac O TfG), etate (1.2g, 89%). ESI-MS (EI", m/z): 256 M+H". Py 25 NH !. DCM,s Step 2: Synthesis of ethyl N Oo C 2-(3-(tert-butoxycarbonylamino) cyclobutyl)acetate H -e- BochN O N 1 IIH or 12, PPh3, 10% Pd/C (360 mg) was added to a solution of ethyl 2-(3- N NEt3, 30 (tert-butoxycarbonylamino)cyclobutylidene) acetate (1.2 g, O OB DCM 4.8 mmol) in MeOH (10 mL) at 23°C. The reaction mixture was stirred under a H. balloon at rt for 1.5 hrs. The mixture N was filtered and concentrated. The residue was purified by / silica gel column (10% EtOAc/petroleum ether) to afford 35 O l Po?C ethyl 2-(3-(tert-butoxycarbonylamino)cyclobutyl)-acetate -e- (1.2g, 96%). ESI-MS (EI, m/z): 258 M+H". N . . . H THF, rt Step 3: Synthesis of tert-butyl 3-(2-hydrazinyl-2-oxoethyl)cyclobutylcarbamate BocNs O !. N OB 40 N A solution of ethyl 2-(3-(tert-butoxycarbonylamino)cy clobutyl)acetate (1.2g, 4.7 mmol) and hydrazine hydrate (1.4 ? g, 23.5 mmol) in EtOH (8 mL) was heated at 80° C. for 17 hrs. O l w SO3-Py, py The reaction mixture was concentrated and the residue was -e- N . H then BuNHSO4. 45 dissolved in DCM (20 mL). The organic phase was washed NaH2PO4 with saturated sodium chloride (10 mL) and water (10 mL). S N The water layer was exacted with EtOAc (3x). The combined BocN O OH organic layer was dried over Na2SO4 and concentrated to N afford tert-butyl 3-(2-hydrazinyl-2-oxoethyl)cyclobutylcar W 50 bamate (1.1 g, 97%). ESI-MS (EI", m/z): 244 M+H". O l.'. Step 4: Synthesis of tert-butyl 3-(2-(2-((2S.5R)-6- TFADCM -e- (benzyloxy)-7-oxo-1,6-diaza-bicyclo[3.2.1]octane-2- N . I I IIH carbonyl)hydrazinyl)-2-oxoethyl)cyclobutylcarbam s !- N 55 ate BocN O OSONBu 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- NN OXO-1,6-diaza-bicyclo3.2.1]octane-2-carboxylic acid (1.0 g, 60 3.6 mmol), tert-butyl 3-(2-hydrazinyl-2-oxoethyl)cyclobu 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 65 EtOAc (3x). The combined organic layer was washed with saturated Sodium chloride (2x), dried over NaSO4, and con centrated. The residue was purified by silica gel column chro