EA044428B1 - BETA-LACTAMASE INHIBITOR COMPOUNDS - Google Patents

Description

Related applications

This application claims priority to U.S. Provisional Application No.

62/395464, filed September 16, 2016, and US Provisional Application No. 62/456423, filed February 8, 2017, the contents of each of which are incorporated herein by reference.

Field of technology

The present invention relates to new oral beta-lactamase inhibitors, pharmaceutical compositions containing them and methods of using them. In addition, the present invention relates to therapeutic methods for treating bacterial infections, including overcoming bacterial resistance to antibiotics.

State of the art

The international microbiological and infectious disease community continues to express serious concern that the continued development of antibacterial resistance may lead to the emergence of bacterial strains against which currently available antibacterial drugs are ineffective, which could result in significant morbidity and mortality. In general, bacterial pathogens can be classified as gram-positive or gram-negative pathogens. Antibiotics that are effective against both Gram-positive and Gram-negative pathogens are generally considered to have a broad spectrum of activity.

Beta-lactam antibiotics are needed to combat bacterial infection. Beta-lactams are a broad class of drugs, all of which contain a beta-lactam in the core molecular structure and generally demonstrate effectiveness against a wide range of gram-positive and gram-negative bacteria by inhibiting bacterial cell wall synthesis. Because the drug targets have no eukaryotic counterparts, their toxicity is low and they are generally well tolerated. They remain among the most widely prescribed, safe and effective drugs available to combat bacterial infection. However, their effectiveness is limited to highly resistant infectious strains such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant (MDR) strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae. Such resistant bacteria are major causes of morbidity and mortality in patients. Helfand, β-lactams Against Emerging 'Superbugs': Progress and Pitfalls, Expert Rev. Clin. Pharmacol. 1(4):559-571 (2008).

Beta-lactam antibiotics, alone and in combination with beta-lactamase inhibitors, continue to represent an important part of the antibacterial agents used to control disease. Beta-lactam resistance in Gram-negative infections is mainly due to beta-lactamase activity, and significant dependence on beta-lactam antibiotics has led to the diversification and increase in the prevalence of beta-lactamases. These beta-lactamases cause resistance to even the newest beta-lactam antibiotics. Llarrull, et al., The Future of Beta-Lactams, Current Opinion in Microbiology, 13:551-557 (2010).

The main threat to the effectiveness of these drugs is the increasing prevalence of extended spectrum betalactamase (ESBL). Beta-lactamases are enzymes produced by some bacteria that open the ring of the beta-lactam portion of the beta-lactam antibiotic, thereby deactivating it. There are currently four classes of beta-lactamases, designated as class A, class B, class C and class D. Class A, class C and class D beta-lactamases are serine beta-lactamases, and class B beta-lactamases are metallo-beta-lactamases (MBL). Bush & Jacoby, Updated Functional Classification of β-Lactamases, Antimicrobial Agents and Chemotherapy, 54(3):969–976 (March 2010).

To improve the effectiveness of beta-lactam antibiotics, several beta-lactamase inhibitors have been developed. However, currently available beta-lactamase inhibitors are in many cases insufficient to counteract the ever-increasing diversity of beta-lactamases. The three most commonly used agents currently used against serine beta-lactamases—clavulanic acid, tazobactam, and sulbactam—have activity only against certain class A enzymes, which seriously limits their practical value. In addition, new beta-lactamase inhibitors recently approved or currently in clinical trials, such as avibactam and MK-7655, are available only for intravenous use and act primarily on class A and C enzymes, with minimal effectiveness against beta-lactamase inhibitors. Class D lactamase Bebrone, et al., Current Challenges in Antimicrobial Chemotherapy: Focus on β-Lactamase Inhibition, Drugs, 70(6):651–679 (2010). Although these agents represent a significant improvement over currently available beta-lactamase inhibitors, to combat the significant beta-lactam resistance currently observed, agents that are effective against all three classes of serine beta-lactamases are desirable, with the added benefit of available an orally effective dosage form for use outside the hospital. Does not currently exist

- 1,044,428 approved beta-lactamase inhibitors that are administered orally and are effective against class C or class D beta-lactamases at a time when resistance levels to conventional antibiotics continue to rise.

Compounds similar to those described herein, also having broad beta-lactamase inhibition profiles (effective against most class A, class C and class D beta-lactamases), have been described in WO 2013/150296. This patent application covers compounds of the following formula:

While the compounds described in WO 2013/150296 represent a significant improvement in the range of beta-lactamase inhibitors currently available on the market or in clinical practice, the compounds described in this application can only be administered intravenously (i.v.). c) because they are not orally bioavailable. Moreover, the compounds disclosed therein cannot be made orally bioavailable by using a prodrug at the sulfate activating group in the molecule. Consequently, these effective beta-lactamase inhibitors are limited to intravenous or parenteral administration, which usually occurs only in hospital settings. Accordingly, for patients with serious persistent infections who are otherwise healthy and do not require hospitalization, or patients who could be discharged from the hospital but would benefit from antibiotic treatment in the outpatient setting (also known as step therapy with switching to the oral route), there are only limited treatment options. The compounds described in WO 2013/150296 may provide patients with more effective and broader beta-lactamase inhibition than any currently known drugs, but these compounds currently require intravenous administration in a hospital setting.

There is an urgent need for a new, oral, broad-spectrum beta-lactamase inhibitor that would provide significant benefit to patients infected with resistant pathogens who might otherwise be treated outside the hospital, or to hospitalized patients who may not have reliable venous access. These patients may have serious complicated infections with pathogens that produce one or more beta-lactamases but may not require hospital treatment, or they may be recovering from infections that were initially treated successfully with a fourth-generation beta-lactam/beta-lactamase inhibitor combination , but would benefit from further beta-lactam/beta-lactamase inhibitor combination therapy outside the hospital setting, which would only be possible with an orally active broad-spectrum beta-lactamase inhibitor such as the one described herein.

In addition, for patients with persistent bacterial infections that require hospitalization for initial treatment, the compounds of the present invention, as described in formulas (Ia), (IIa), (IIIa), (IVa) and (Va), can be administered intravenously into inpatient settings until the patient is stable enough to continue treatment on an outpatient basis. After discharge, the patient may continue therapy with the same drug by administering a compound according to any of formulas (I), (II), (III), (IV) or (V) in an outpatient setting, while continuing continuous treatment until the bacterial infection will not be cured. There are currently no class A, C, or D β-lactamase inhibitors that can be administered initially IV with the added benefit of oral administration once the patient is well enough to be discharged from the hospital. The ability for a physician to tailor treatment to a patient's needs would allow patients requiring hospitalization to be discharged earlier and significantly reduce overall treatment costs by avoiding long hospital stays.

There is an urgent need for new orally active beta-lactamase inhibitors that are effective against more than one of the class A, C and D beta-lactamases.

Brief description of the invention

The present invention relates to a process for preparing a compound of formula VI

R ²

he (VI);

or a salt thereof, wherein the variables R1, ^R2 , ^R3 , PG and PG' are as defined in the detailed description of the invention.

- 2 044428

Detailed Description of the Invention

The present invention relates to compounds that are orally available beta-lactamase inhibitors. The compounds and their pharmaceutically acceptable salts in combination with betalactam antibiotics are suitable for the treatment of bacterial infections, including infections caused by drug-resistant organisms, including multidrug-resistant organisms. More particularly, the invention relates to compounds of formula (I)

R ²

O or a pharmaceutically acceptable salt thereof, where

R ¹ represents -C(O)NR ⁷ R ⁸ , -CN, phenyl, 5-7 membered heteroaryl, -C(O)NR'NR'C(O)R ⁹ , -C(O)NR'OR ¹⁰ or a C1-C6 alkyl group, where the alkyl group is replaced by 1-3 groups consisting of halogen, C1-C3 alkoxy, -OH, -CN, -NR ⁷ R ⁸ , -NR ⁷ COR ⁹ , 5-7- membered heteroaryl and 5-7 membered heterocyclyl, and wherein the phenyl and heteroaryl represented among R ¹ are optionally and independently substituted with 1-3 groups selected from halogen, -OH, C1-C ₃ -alkoxy, -CN, -NR7R ⁸ and -CONR7R ⁸ ;

R ² and R ³ are independently selected from hydrogen, halogen, C ₁ -C ₃ alkyl and C ₃ -C ₆ cycloalkyl;

R ⁴ and R ⁵ are independently selected from hydrogen, halogen, -CN, -CO2R ⁹ , C ₁ -C ₃ alkyl and C1-C3 haloalkyl;

R ⁶ represents hydrogen, C1-C12-alkyl, C1-C4-alkyl-C1-C3-alkoxy-(NR'C1-C6-alkyl)-C1-C3-alkoxy, C1-C4-alkyl-C1-C3-alkoxy- C1-C3-alkoxy, C2-C12-alkenyl, C3-C10-cycloalkyl, 5-7 membered heteroaryl and 5-7 membered heterocyclyl, where alkyl, alkenyl, cycloalkyl, heteroaryl and heterocyclyl are optionally and independently substituted by 1-6 groups selected from carboxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl and phenyl. Alternatively, R ⁶ is C1-C ₁₂ alkyl, C ₁ -C ₄ alkyl-C ₁ -C ₃ alkoxy-(NR'C ₁ -C ₆ alkyl)-C ₁ -C ₃ alkoxy, C ₁ -C ₄ -alkyl-C ₁ -C ₃ -alkoxy-C ₁ -C ₃ -alkoxy, C ₂ -C ₁₂ -alkenyl, C3C ₁₀ -cycloalkyl, 5-7-membered heteroaryl and 5-7-membered heterocyclyl where alkyl, alkenyl, cycloalkyl, heteroaryl and heterocyclyl are optionally and independently substituted with 1-6 groups selected from carboxyl, halogen, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkyl and phenyl;

each R ⁷ and R ⁸ independently represents hydrogen, C1-C3 alkyl, C1-C3 alkoxy, phenyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or 5-7 membered heteroaryl, where alkyl, alkoxy, phenyl, cycloalkyl, heterocyclyl or heteroaryl represented by R ⁷ or R ⁸ is optionally and independently substituted with 16 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F atoms, carboxyl or -CO(OC1-6alkyl), 5- 6-membered heteroaryl, -CN, -OH, C ₁ -C ₃ -alkyl, optionally substituted -NH2 or -OH, C ₁ -C ₃ -haloalkyl, C ₁ -C ₃ -haloalkoxy, C ₁ -C ₃ -alkoxy NHCO(C ₁ -C ₃ -alkyl), -NHCO(C ₁ -C ₃ -alkoxy), -S(O) ₂ NR'R, -NHS(O) ₂ NR'R'', -NHS(O) ₂ (C ₁ -C ₃ -alkyl), -NR'R'' and -C(O)NR'R''; each R ⁹ is C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C 1 -C 6 -haloalkoxy or C ₁ -C ₆ -alkoxy;

each R ¹⁰ is C ₁ -C ₃ alkyl, optionally substituted with 1-6 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F atoms, carboxyl or -CO(OC _1-6 alkyl), C ₃ -C ₆ -cycloalkyl, 5-6-membered heteroaryl, -CN, -OH, -NHCO(C ₁ -C ₃ alkyl), -NHCO(C ₁ -C ₃ -alkoxy), -S(O) ₂ NR'R, -NHS(O) ₂ NR'R, -NHS(O) ₂ (C ₁ -C ₃ -alkyl), -NR'R'' or -C(O)NR'R''; and each R' and R''; independently represents hydrogen, methyl, ethyl or propyl; or R' and R together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclyl; with the proviso that at least one of R ² and R ³ is not hydrogen.

In one aspect, the invention relates to a compound of formula (I) that is an oral beta-lactamase inhibitor as described above.

In yet another aspect of the invention, R ¹ in formula (I) is -C(O)NR ⁷ R ⁸ , -CN, phenyl, 56-membered heteroaryl, -C(O)NR'NR'C(O)R ⁹ , -C(O)NR'OR ¹⁰ or C1-C6 alkyl group, where the alkyl group is replaced by 1-3 groups consisting of halogen, C1-C3 alkoxy, -OH, -CN, -NR ⁷ R ⁸ , -NR7COR ⁹ , 56-membered heteroaryl and 5-7-membered heterocyclyl, and wherein the phenyl and heteroaryl represented among R ¹ are optionally and independently substituted with 1-3 groups selected from halogen, -OH, C1-C ₃ -alkoxy, -CN, -NR ⁷ R ⁸ and -CONR ⁷ R ⁸ ;

R ⁶ is hydrogen, C1-C ₁₂ alkyl, C ₁ -C ₄ alkyl-C ₁ -C ₃ alkoxy-(NR'C ₁ -C ₆ alkyl)-C ₁ -C ₃ alkoxy, C ₁ -C ₄ -alkyl-C ₁ -C ₃ -alkoxy-C ₁ -C ₃ -alkoxy, C ₂ -C ₁₂ -alkenyl, C ₃ -C ₁₀ -cycloalkyl, 5-6-membered heteroaryl and 5-7-membered heterocyclyl, where alkyl, alkenyl, cycloalkyl, heteroaryl and heterocyclyl are optionally and independently substituted by 1-6 groups selected from carboxyl, halogen, C ₁ -C ₆ -alkoxy,

- 3 044428

C ₁ -C ₆ -alkyl and phenyl (alternatively, R ⁶ represents C1-C12-alkyl, C1-C4-αalkyl-C1-C3alkoxy-(NR'C1-C6-alkyl)-C1-C3-alkoxy, C1-C4-alkyl-C1-C3-alkoxy-C1-C3-alkoxy, C2-C12-alkenyl, C3C10-cycloalkyl, 5-6-membered heteroaryl and 5-7-membered heterocyclyl, where alkyl, alkenyl, cycloalkyl, heteroaryl and heterocyclyl are optionally and independently substituted with 1 to 6 groups selected from carboxyl, halogen, C1-C6 alkoxy, C1-C6 alkyl and phenyl); and each R ⁷ and R ⁸ independently represents hydrogen, C 1 -C ₃ -alkyl, C ₁ -C ₃ -alkoxy, phenyl, C ₃ -C ₆ -cycloalkyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, where alkyl, alkoxy, phenyl, cycloalkyl, heterocyclyl or heteroaryl represented by R ⁷ or R ⁸ is optionally and independently substituted with 1-6 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F, carboxyl or -CO atoms (OC1-6αalkyl), 5-6-membered heteroaryl, -CN, -OH, C1-C3-alkyl, optionally substituted -NH2 or -OH, C1-C3haloalkyl, C1-C3-haloalkoxy, C1-C3-alkoxy -NHCO(C1 -C3-alkyl), -NHCO(C1-C3-alkoxy), -S(O)2NR'R, -NHS(O)2NR'R”, -NHS(O)2(C1-C3-alkyl), - NR'R'' and -C(O)NR'R''; each R ⁹ is C1- _C6 alkyl, _C1 - _C6 haloalkyl, _C1 - _C6 haloalkoxy or _C1 - _C6 alkoxy;

In yet another aspect, the invention relates to a compound of formula (II)

R ²

R

R ⁶ (P) or a pharmaceutically acceptable salt thereof, wherein the variables R ¹ , R ² , R ⁴ , R ⁵ and R ⁶ are as defined for formula (I).

In one aspect, the invention relates to a compound of formula (III)

or a pharmaceutically acceptable salt thereof, wherein the variables R ¹ , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined for formula (I).

In yet another aspect, the invention relates to a compound of formula (IV)

or a pharmaceutically acceptable salt thereof, wherein the variables R ⁴ , R ⁵ and R ⁶ are as defined for formula (I).

In yet another aspect, the invention relates to a compound of formula (V) o

_H2N '

O (V) or a pharmaceutically acceptable salt thereof, wherein the variables R ⁴ , R ⁵ and R ⁶ are as defined for formula (I).

In yet another aspect, the invention relates to a compound of formula (Ia)

- 4 044428

R ²

O or a pharmaceutically acceptable salt thereof; Where

R ¹ represents -C(O)NR ⁷ R ⁸ , -CN, phenyl, 5-7 membered heteroaryl, -C(O)NR'NR'C(O)R ⁹ , -C(O)NR'OR ¹⁰ or C1-C6 alkyl group, where the alkyl group is replaced by 1-3 groups consisting of halogen, C1-C3 alkoxy, -OH, -CN, ^-NR7R8 , ^-NR7COR9 , ^5-7 -membered heteroaryl and 5-7 membered heterocyclyl, and wherein the phenyl and heteroaryl present among R ¹ are optionally and independently substituted with 1-3 groups selected from halogen, -OH, C1-C ₃ -alkoxy, -CN, -NR ⁷ R ⁸ and -CONR7R ⁸ ;

R ² and R ³ are independently selected from hydrogen, halogen, C ₁ -C ₃ alkyl and C ₃ -C ₆ cycloalkyl;

R ⁴ and R ⁵ are independently selected from hydrogen, halogen, -CN, -CO2R ⁹ , C ₁ -C ₃ alkyl and C ₁ -C ₃ haloalkyl;

each R ⁷ and R ⁸ independently represents hydrogen, C1-C3 alkyl, C1-C3 alkoxy, phenyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or 5-7 membered heteroaryl, where alkyl, alkoxy, phenyl, cycloalkyl, heterocyclyl or heteroaryl represented by R ⁷ or R ⁸ is optionally and independently substituted with 16 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F atoms, carboxyl or -CO(OC1-6 alkyl), 5 -6-membered heteroaryl, -CN, -OH, C ₁ -C ₃ -alkyl, optionally substituted -NH ₂ or -OH, C ₁ -C ₃ -haloalkal, C ₁ -C ₃ -haloalkoxy, C ₁ -C ₃ -alkoxyNHCO(C1-C3-alkyl), -NHCO(C1-C3-alkoxy), -S(O)2NR'R'', -NHS(O)2NR'R, -NHS(O)2(C1-C3 -alkyl), -NR'R'' and -C(O)NR'R;

each R ⁹ is C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C ₁ -C ₆ -haloalkoxy or C ₁ -C ₆ -alkoxy;

each R ¹⁰ is C ₁ -C ₃ alkyl, optionally substituted with 1-6 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F atoms, carboxyl or -CO(OC _1-6 alkyl), C ₃ -C ₆ cycloalkyl, 5-6-membered heteroaryl, -CN, -OH, -NHCO(C ₁ -C ₃ alkyl), -NHCO(C ₁ -C ₃ -alkoxy), -S(O) ₂ NR 'R, -NHS(O) ₂ NR'R'', -NHS(O) ₂ (C ₁ -C ₃ -alkyl), -NR'R” or -C(O)NR'R'';

and each R' and R'' independently represents hydrogen, methyl, ethyl or propyl; or R' and R'' together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclyl; with the proviso that at least one of R ² and R ³ is not hydrogen.

In yet another aspect, the invention relates to a compound of formula (Ia)

R ²

O or a pharmaceutically acceptable salt thereof; Where

R ¹ represents -C(O)NR ⁷ R ⁸ , -CN, phenyl, 5-6 membered heteroaryl, -C(O)NR'NR'C(O)R ⁹ , -C(O)NR'OR ¹⁰ or C1-C6 alkyl group, where the alkyl group is replaced by 1-3 groups consisting of halogen, C1-C3 alkoxy, -OH, -CN, -NR ⁷ R ⁸ , -NR ⁷ COR ⁹ , 5-6 membered heteroaryl and 5-7 membered heterocyclyl, and wherein the phenyl and heteroaryl represented among R ¹ are optionally and independently substituted with 1-3 groups selected from halogen, -OH, C1-C ₃ -alkoxy, -CN, -NR7R ⁸ and -CONR7R ⁸ ;

R ² and R ³ are independently selected from hydrogen, halogen, C ₁ -C ₃ alkyl and C ₃ -C ₆ cycloalkyl;

R ⁴ and R ⁵ are independently selected from hydrogen, halogen, -CN, -CO2R ⁹ , C ₁ -C ₃ alkyl and C ₁ -C ₃ haloalkyl;

each R ⁷ and R ⁸ independently represents hydrogen, C1-C3 alkyl, C1-C3 alkoxy, phenyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, where alkyl, alkoxy, phenyl, cycloalkyl, heterocyclyl or heteroaryl present among R ⁷ or R ⁸ is optionally and independently substituted with 1-6 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F atoms, carboxyl or -CO(OC1-6alkyl) , 5-6-membered heteroaryl, -CN, -OH, C ₁ -C ₃ alkyl, optionally substituted -NH ₂ or -OH, C ₁ -C ₃ -haloalkyl, C ₁ -C ₃ -haloalkoxy, C ₁ -C ₃ alkoxy -NHCO(C1-C3-alkyl), -NHCO(C1-Czalkoxy), -S(O)2NR'R'', -NHS(O)2NR'R'', -NHS(O)2(C1 -Nw

- 5 044428 alkyl), -NR’R’’ and -C(O)NR’R’’;

each R ⁹ is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy or C1-C6 alkoxy; each R ¹⁰ is C1- _C3 alkyl, optionally substituted with 1-6 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F, carboxyl or -CO(OC _{1 -6} alkyl) atoms, C ₃ -C ₆ -cycloalkyl, 5-6-membered heteroaryl, -CN, -OH, -NHCO(C1-C ₃ alkyl), -NHCO(C ₁ -C ₃ -alkoxy), -S(O) ₂ NR'R'', -NHS(O) ₂ NR'R'', -NHS(O) ₂ (C ₁ -C ₃ -alkyl), -NR'R'' or -C(O)NR'R”; and each R' and R'' independently represents hydrogen, methyl, ethyl or propyl; or R' and R'' together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclyl; with the proviso that at least one of R ² and R ³ is not hydrogen.

In yet another aspect, the invention relates to a compound of formula (IIa)

or a pharmaceutically acceptable salt thereof, wherein the variables R ¹ , R ² , R ⁴ and R ⁵ are as defined for formula (Ia).

In one aspect, the invention relates to a compound of formula (IIIa)

it (Sha) or a pharmaceutically acceptable salt thereof, wherein the variables R ¹ , R ³ , R ⁴ and R ⁵ are as defined for formula (Ia).

In yet another aspect, the invention relates to a compound of formula (IVa)

o (IVa) or a pharmaceutically acceptable salt thereof, wherein the variables R ⁴ and R ⁵ are as defined for formula (Ia).

In yet another aspect, the invention relates to a compound of formula (Va)

o (Va) or a pharmaceutically acceptable salt thereof, wherein the variables R ⁴ and R ⁵ are as defined for formula (Ia).

In one aspect of the inventions, for formulas (I), (Ia), (II) or (IIa), R ² represents Ci-C ₃ alkyl. In yet another aspect of the invention, for formulas (I), (Ia), (II) or (IIa), R ² represents methyl.

In one aspect of the invention, for formula (I), (Ia), (III) and (IIIa), R ³ represents C1-C3 alkyl. In yet another aspect of the invention, for formula (I), (Ia), (III) and (IIIa), R ³ is methyl.

In one aspect of the invention, for formula (I), (Ia), (II), (IIa), (III) and (IIIa), R ¹ is selected from oxadiazole, -C(O)NHNHC(O)(C1- C3-alkyl), -CH2NH2, -CH2NHCO(C1-C3-alkoxy), -CH2NHCO(C _r C3- 6 044428 alkyl) or -CH ₂ NHCO(C ₁ -C ₃ -haloalkyl), where oxadiazole R ¹ is optionally substituted -OH, Q-C3alkoxy,

-NR ⁷ R ⁸ or -CONR7R ⁸ . In one aspect of the invention for formula (I), (Ia), (II), (IIa), (III) and (IIIa), R ¹ is selected from -CH2NH2,

In yet another aspect of the invention for formula (I), (Ia), (IIa), (III) and (IIIa), R ¹ is

-CN, where R ¹¹ represents hydrogen or -C(O)NH2. In one aspect of the invention, for formula (I), (Ia), (II), (IIa), (III) and (IIIa), R ¹ is -CN or -C(O)NH2. In one aspect of the invention, for formula (I), (Ia), (II), (IIa), (III) and (IIIa), R ¹ represents -CN. In one aspect of the invention for formula (I), (Ia), (II), (IIa), (III) and (IIIa), R ¹ is -C(O)NH2. In one aspect of the invention for formula (I), (Ia), (II), (IIa), (III) and (IIIa), R ¹ is -C(O)NR ⁷ R ⁸ . In one aspect of the invention for formula (I), (Ia), (II), (IIa), (III) and (IIIa), when R ¹ is -C(O)NR ⁷ R ⁸ , R ⁷ is hydrogen and R ⁸ is 1) phenyl, optionally substituted with C1-C3 alkyl or C1-C3 alkyl-NH2, 2) C1-C3 alkyl or 3) C1-C3 alkoxy, wherein each alkyl or alkoxy of the R represented ⁸ is optionally and independently substituted with C3-C ₆ -cycloalkyl, -CN, -OH, -NH2, -SO2NH2, -NHSO2NH2, -C(O)NH ₂ , -NHC(O)(C ₁ -C ₃ -alkyl), pyrazinyl, oxytanyl, oxazolyl or pyrrolidinyl, optionally substituted with one or more carboxyl, fluorine or -C(O)O(C ₁ -C ₆ -alkyl). In one aspect of the invention for formula (I), (Ia), (II), (IIa), (III) and (IIIa), when R ¹ is -C(O)NR ⁷ R ⁸ , R ⁷ is hydrogen and R ⁸ selected from the group consisting of

In one aspect of the invention, for any of formulas (I), (II), (III), (IV) or (V), R ⁶ represents C1-C12 alkyl. In one aspect of the invention, for any of formulas (I), (II), (III), (IV) or (V), R ⁶ is ethyl, isopropyl, 2-butyl or isopentyl. In one aspect of the invention, for any of formulas (I), (II), (III), (IV) or (V), R ⁶ is isopropyl. In one aspect of the invention, for any of formulas (I), (II), (III), (IV) or (V), R ⁶ represents C1-C ₄ -alkyl-OS(O)-(NHC ₁ C ₆ -alkyl)-C(O)C ₁ -C3-alkoxy, C ₁ -C ₄ -alkyl-OS(O)-C ₁ -C ₄ -alkyl or C ₁ -C ₄ -alkyl-OC(O)- C ₁ -C ₃ -alkoxy. In one aspect of the invention, for any one of formulas (I), (II), (III), (IV) or (V), R ⁶ is selected from the group consisting of

methyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl or n-nonyl.

In one aspect of the invention, for any of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va), R ⁴ and R ⁵ are independently H, methyl or fluorine. In yet another aspect of the invention, for any of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va), one of R ⁴ and R ⁵ represents

- 7 044428 is hydrogen and the other is fluorine. In yet another aspect of the invention, for any of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va), R ⁴ represents fluorine and R ⁵ represents hydrogen. In yet another aspect of the invention, for any of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va), R ⁴ is hydrogen and R ⁵ is fluorine. In yet another aspect of the invention, for any of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va), R ⁴ and R ⁵ are both hydrogen. In yet another aspect of the invention, for any of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va), R ⁴ and R ⁵ are both fluorine.

Any embodiment described herein may be combined with any other suitable embodiment described herein to form additional embodiments. For example, when one embodiment separately or collectively describes possible groups for R ¹ and another embodiment describes possible groups for R ² , it is intended that these embodiments can be combined to produce a further embodiment in which any from the possible groups for R ¹ with any of the possible groups for R ² . Likewise, the invention covers any embodiments relating individually to R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ in combination with any separate embodiments relating to each of the remaining variables.

The compounds of formulas (I), (II), (III), (IV) and (V) and their pharmaceutically acceptable salts have an effective beta-lactamase inhibition spectrum and are suitable for oral administration. The compounds of formulas (Ia), (IIa), (IIIa), (IVa) and (Va) and their pharmaceutically acceptable salts have an effective beta-lactamase inhibition spectrum and are suitable for intravenous, intraperitoneal, intramuscular or subcutaneous administration, e.g. As such, the compounds of formulas (Ia), (IIa), (IIIa), (IVa) and (Va) and their pharmaceutically acceptable salts can be usefully used when the patient is unable to take antibiotics orally, for example, in a hospital, point-of-care setting emergency or nursing home settings. Once the patient's condition improves sufficiently to receive oral antibiotics, treatment can be changed so that a compound of formulas (I), (II), (III), (IV) and (V) or a pharmaceutically acceptable salt thereof is administered orally to the patient. In addition, compounds of formulas (Ia), (IIa), (IIIa), (IVa), (Va), (I), (II), (III), (IV) and (V) and pharmaceutically acceptable salts thereof may have beneficial active, metabolic, toxicological and/or pharmacodynamic properties.

One aspect of the invention includes a compound according to one example or a pharmaceutically acceptable salt thereof, namely

Example No. Structure Connection name 1 ⁰ 1 ,Μ g ⁰ °>°\^ O (I)-ethyl-2-((2S,5R)-2-carbamoyl-3 methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yloxy)-2-fluoroacetate 2 О 1 Η ₂ Α·Α d ^{0 0} О (8)-ethyl-2-((28.5R)-2-carbamoyl-3- methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yloxy)-2-fluoroacetate 3 I th “Kd o' o t (2S)-{[(2S,5R)-2-carbamoyl-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3 en-6-yl]oxy} (fluoro)ethanoic acid lithium salt 4 o I Ah g ⁰ °^y-OH O (2C)-{[(28.5R)-2-carbamoyl-3-methyl7-oxo-1,6-diazabicyclo[3.2.1 ]oct-3 en-6-yl]oxy} (fluoro)ethanoic acid lithium salt

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5 nDA M F A—h j o oA^on O {[(2 S, 5 R)-2-carbamoyl-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl]oxy } (fluoro)acetic acid lithium salt 6 o I nA-D M F ^{0 N} '°V- O ethyl {[(2S,5R)-2-κapbamoyl-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl]oxy} (fluoro)acetate 7 X th O %o O' ethyl {[(2S,5R)-2-κapbamoyl-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl]oxy} (difloro)acetate 8 X th O Ao T£>o'^O; =A^o I {[(2 S, 5R)-2-κapbamoyl-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl]oxy } (difluoro)acetic acid lithium salt 9 o 1 nL-D J---N ₄ ⁰ °Ίτ°- 0 ethyl {[(2S,5R)-2-κapbamoyl-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl]oxy} acetate 10 X th O >° Т£> O' Oh I {[(2 S, 5R)-2-κapbamoyl-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-ene- 6-yl]oxy}acetic acid lithium salt AND I th Z o Do Sh- o' 0¾^ ⁷¹ o t 2-{[(2 S, 5R)-2-κapbamoyl-3 -methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl]oxy } -2-fluoropropanoic acid lithium salt 12 o 1 nA-D An, ? ⁰ °Ύ° o / πpoπan-2-yl-(2R)-{[(2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl] oxy} (fluoro)ethanoate

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13 ⁰ I n ₂ lD J ⁰ 0 / nponaH-2-mi-(2S)-{[(2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy} (fluoro)ethanoate 14 ° 1 n ₂ I'·^ M F 2,4-dimethylpentan-3-yl-(2S)- {[(2 S, 5R)-2-κapbamoyl-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl]oxy } (fluoro)ethanoate 15 I z O. )=O Ti> O' 2,4-dimethylpentan-3-yl-(2R){[(2 S, 5R)-2-κapbamoyl-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl ]oxy } (fluoro)ethanoate 16 η _ζ ν ^Λ ·Α νΆ J---Ν F ° ^-° \ ⁰ 0 tetrahydro-2H-pyran-4-yl-{[(2S,5R)2-carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy} (fluoro )acetate 17 ΙΌ ζ ο. <=ο ϊ>ο' 0¾^ ⁷¹ / \ 2-methoxy-{[(2 S, 5R)-2-κapbamoyl-3 methyl-7-oxo-1,6- diazabicyclo[3.2.1 ]oct-3-en-6yl]oxy} (fluoro)acetate 18 I ΙΌ ζ ο. <=ο ϊοο' ο^·'^ / \ 2-MeTOKcn-(2R)-{[(2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy} (fluoro)ethanoate 19 ΙΌ ζ ο. <=ο Yuο' 0¾^ ⁷¹ / \ 2-MeTOKcn-(2S)-{[(2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy} (fluoro)ethanoate 20 ⁰ 1 η ₂ ν ^Λ Α An ? ⁰ °Ίτ° 0 (2R)-(S)-top-bytyl-2-(((2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl)oxy) -2-fluoroacetate

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21 0 ι A-A A» ; ° ° An ⁰ ) ⁴ (2S)-(S)-BTop-6yran-2-(((2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl)oxy) -2-fluoroacetate 22 o sh„,^О p a About z C.M. I (2R)-(R)-top-bytyl-2-(((2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl)oxy) -2-fluoroacetate 23 I z O. Ao A o' 0¾^ ⁷¹ o 7 (2S)-(R)-BTop-6y _T wi-2-(((2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl) hydroxy)-2-fluoroacetate 24 ⁰ 1 Η ₂ Ν ^Λ 'Α An - ^{f 0} °>° ° (R)-neHTaH-3 -yl-2-((2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yloxy)-2-fluoroacetate 25 Η ₂ Ν ^Λ Α AF J·---N / ° '° 7T° ° (S)-πpentan-3-yl-2-((2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yloxy)-2-fluoroacetate 26 A zi TZ About Ao Ti> o' oy^^ <° 3Tmi-2-(((2S,5R)-2-(2- acetylhydrazinecarbonyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl)oxy)-2-fluoroacetate 27 vaA ^{0 n} J j ⁰ θΑ_ ₀ Η 0 2-(((2S,5R)-2-(2- acetylhydrazinecarbonyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl)oxy)-2-fluoroacetic acid lithium salt

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28 O 1 P^^P HE (R)-2-((2S,5R)-2-(4- (aminomethyl)phenylcarbamoyl)-3 methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6yloxy)-2-fluoroacetic acid TFA salt 29 ^z \\ ιζ Οχ >° Τί>θ' (° ethyl-2-fluoro-2-(((2 S, 5R)-3 -methyl-7 oxo-2-((pyrazin-2ylmethyl)carbamoyl)-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl )oxy)acetate thirty ⁰ 1 yl ^l A ^Ν J ⁰ O'y-OH 0 2-fluoro-2-(((2 S, 5R)-3 -methyl-7-oxo-2((pyrazin-2-ylmethyl)carbamoyl)-1,6diazabicyclo[3.2.1]oct-3-en-6yl )hydroxy)acetic acid lithium salt 31 0 Λ η ₂ ν [ η) Μ F J---Ν. ? ° ° /Γ ⁰ ^ 0 (2th)-ethyl-2-(((2S,5R)-2-Kap6aMonn-4methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yl)oxy)-2-fluoroacetate 32 0 η ₂ ν Μ F Λ--- ^N S ] ^{0 0} /g ⁰ ^ o ⁷ (28)-ethyl-2-(((2S,5R)-2-Kap6aMonn-4methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yl)oxy)-2-fluoroacetate 33 O Μ F J----Ν _χ .- ⁰ O^y-OH 0 (2R)-2-(((2S,5R)-2- _K ap6aMomi-4methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetic acid lithium salt 34 MF J---N ₄ J ^{0 o} ^у-о ⁷ (2S)-2-(((2S,5R)-2- _K ap6aMomi-4methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetic acid lithium salt

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35 0 A h ₂ np MF J---NF ° ° /Г ⁰ ° 7^ (2R)-isoπpoπyl-2-(((2S,5R)-2carbamoyl-4-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetate 36 0 h ₂ n MF Λ--NJ ^{0 0} IT ⁰ ° 7^ (2S)-isoπpoπyl-2-(((2S,5R)-2carbamoyl-4-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetate 37 nD MF J—NF ⁰ '°Zr° ° 7^ (2R)-isoπpoπyl-2-(((2S,5R)-2carbamoyl-3-cyclopropyl-7-oxo1,6-diazabicyclo[3.2.1]oct-3-ene-6- yl)oxy)-2-fluoroacetate 38 ηΑ·Χ MF J---N / ⁰ '° /G ⁰ o K (2S)-isoπpoπyl-2-(((2S,5R)-2carbamoyl-3-cyclopropyl-7-oxo1,6-diazabicyclo[3.2.1]oct-3-ene-6- yl)oxy)-2-fluoroacetate 39 nA-X MF J---NF ⁰ '°Zr ⁰ ^ 0 (2R)-ethyl-2-(((2S,5R)-2-κcarbamoyl-3cyclopropyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetate 40 .AX MF J---N Г ^{0 0} /г ⁰ ^ 0 (28)-ethyl-2-(((2S,5R)-2-κapbamoyl-3cyclopropyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetate 41 I o yy z T (2R)-2-(((2S,5R)-2-κapbamoyl-3cyclopropyl-7-oxo-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl)oxy)-2-fluoroacetic acid lithium salt

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42 Η ₂ Ν^^*| Μ F o^oD^n O (2R)-2-(((2S,5R)-2-κapbamoyl-3cyclopropyl-7-oxo-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl)oxy)-2-fluoroacetic acid lithium salt 43 dL M F J—* A ⁰ O'y-OH 0 2-(((2S,5R)-2-κapbamoyl-3cyclopropyl-7-oxo-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl)oxy)-2-fluoroacetic acid lithium salt 44 I Mz o. Mo' o^V- (1-isopropyl-2-methylpropyl) 2[[(2S,5R)-2-κapbamoyl-3-methyl-7oxo-1,6-diazabicyclo[3.2.1]oct-3-ene- 6-yl]oxy]-2,2-difluoroacetate 45 ⁰ 1 N ₂ L ^ JMn ₄ G 0 °^r°\__ o ⁴ — okotil-(2R)-2-[[(2S,5R)-2-karbamoyl- 3-methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2-fluoroacetate 46 ⁰ 1 H ₂ |A-|A| JMl F ⁰ °' ^L )Г°\ 0 methyl-(2R)-2-[[(2S,5R)-2-karbamoyl3 -methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2-fluoro- acetate 47 ⁰ 1 h ₂ n ^A ·^ JMi ₄ Г ⁰ °>° 0 allyl-(2R)-2-[[(2S,5R)-2-κapbamoyl3 -methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2-fluoroacetate 48 =E ro X A Ϊ+ A πpopyl-(2R)-2-[[(2 S, 5R)-2-karbamoyl- 3-methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yl]oxy]-2-fluoroacetate

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49 I short circuit ο θ' ο^·^ H3o6yTwi-(2R)-2-[[(2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2-fluoro-acetate 50 ⁰ 1 η/Α ⁰ °>° 0 bytyl-(2R)-2-[[(2S,5R)-2-karbamoyl- 3-methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yl]oxy]-2-fluoroacetate 51 Η ₂ ΛΛ Μ F J---Ν ? ο °^Γ° ο ⁴ —\ pentyl-(2R)-2-[[(2S,5R)-2-carbamoyl- 3-methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yl]oxy]-2-fluoroacetate 52 M ζ Ο. ^=Ο Τί> ο' ο^·^ ο hecyl-(2R)-2-[[(2S,5R)-2-κcarbamoyl- 3-methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yl]oxy]-2-fluoroacetate 53 ⁰ 1 η ₂ ν ^Λ Α Dud, ι ⁰ °Άο ⁰ / У°у- ⁰ / 1 -isopropoxycarbonyloxyethyl(2R)-2- [ [(2 S, 5R)-2-κapbamoyl-3 methyl-7-oxo-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl] oxy] -2- fluoroacetate 54 τ Μ ζ ο. >Ο ζ (2R)-benzyl-2-(((2S,5R)-2-carbamoyl3 -methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-6- yl)oxy)-2-fluoroacetate 55 ,0 η ₂ ν--/ Γθ 1 ν-Д Μ F 0 2-[[(2S,5R)-2-(5-Kap6aMonn-l,3,4oxadiazol-2-yl)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene- 6yl]oxy]-2-fluoroacetic acid lithium salt

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56 I th Z O=cAyu p IZ o. Too' about 1 (28)-2-fluoro-2-[[(28.5K)-3-methyl-7oxo-2-(2- sulfamoylethoxycarbamoyl)-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid lithium salt 57 AA ^{H2N and} th _F 0 ^N Ay _0H 0 (28)-2-fluoro-2-[[(28.5K)-3-methyl-7oxo-2-(2- sulfamoylethylcarbamoyl)-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid lithium salt 58 0 ί n/a A. ' °°Ύ°νο Η 0 θVyA ο V ethyl-2-[[(2K)-2-[[(28.5K)-2carbamoyl-3 -methyl-7-oxo-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl]oxy]-2 -fluorine- acetyl]oxymethoxycarbonylamino]3-methylbutanoate 59 ⁴ Ν Α 0 ί 1“Α , ° ⁰ yo^. ο tert-butyl-(28)-2-((((28.5K)-6-((8)-2ethoxy-1-fluoro-2-oxoethoxy)-3 methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-ene-2carboxamido)oxy)methyl)pyrrolidium and-1-carboxylate 60 nA 0 ί Α - ⁰ ° ΑΑ 0 ethyl-(2 8)-2-fluoro-2-(((2 S, 5R)-3 -methyl7-oxo-2-((((8)-pyrrolidin-2yl)methoxy)carbamoyl)-1,6diazabicyclo[ 3.2.1 ]oct-3-en-6yl)oxy)acetate TFA salt 61 ΐζ' A ρΑ ΐζ' ο. Α° Μ- θ' (° ethyl-(2S)-2-(((2S,5R)-2-((((S)-4,4difluoropyrrolidine-2- yl)methoxy)carbamoyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl)oxy)-2-fluoroacetate TFA salt

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62 IZ J south. ο. M θ' ο τ (2S)-2-φτop-2-[[(2S,5R)-3-methyl-7oxo-2-[[(28)-pyrrolidine-2- yl]methoxycarbamoyl]-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid TFA salt 63 0 ο O'D^ON ο (2S)-2-(((2S,5R)-2-((((S)-4,4difluoropyrrolidine-2- yl)methoxy)carbamoyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl)oxy)-2-fluoroacetic acid TFA salt 64 Η ₂ Ν ^Α Α Μ F Д—Ν _χ -' ο θΊτ^ο О У\ ο [(2R)-2-[[(2S, 5R)-2-Kap6aMO^-3- methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2-fluoroacetyl]hydroxymethyl2,2-dimethylpropanoate 65 ⁰ ι n ₂ lD JMl, f o ^Z dUd indan-5-yl-(2R)-2-[[(2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2 -fluoroacetate 66 oh ooh-on 0 (2S)-2-φτop-2-[[(2S,5R)-3-methyl-2(oxetan-3-ylcarbamoyl)-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl ]oxy]acetic acid lithium salt 67 ΞΕ O r -a About ζι ΙΖ ω=ο ό'\ (2S)-2^Top-2-[[(2S,5R)-2-[2-( methanesulfonamido) ethylcarbamoyl]-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid lithium salt

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68 ιζ ABOUT M- O T (2 8)-2-fluoro-2-[ [(2 S, 5R)-3-methyl-2(oxazol-2-ylmethylcarbamoyl)-7oxo-1,6-diazabicyclo[3.2.1 ]oct-3-ene6 -yl]oxy]acetic acid lithium salt 69 ⁰ 1 (Ts% ^Ν F ⁰ ou.on 0 (28)-2-fluoro-2-[[(28^)-3-methyl-7oxo-2-(pyrazine-2- ylmethylcarbamoyl)-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid lithium salt 70 l ⁰ 1 _g N _s J o O^y-OH O (2S)-2-[[(2S,5R)-2- (cyclopropylmethoxycarbamoyl)-3methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2-fluoroacetic acid lithium salt 71 O O I J o ° /G OH ABOUT (2S)-2-[[(2S,5R)-2-[(3-aMHHO-3-OKCOpropyl)carbamoyl]-3-methyl-7-oxo1,6-diazabicyclo[3.2.1]oct-3-ene -6yl]oxy]-2-fluoroacetic acid lithium salt 72 o=\ 1 ^Ν Ί o I J--N _s J o O'^y-on O (2 8)-2-fluoro-2-[[(2 S, 5R)-3 -methyl-7oxo-2-[(5-oxopyrrolidin-2yl)methoxycarbamoyl]-1,6diazabicyclo[3.2.1]oct-3 -en-6yl]oxy]acetic acid lithium salt 73 ABOUT IZ ABOUT M- O' o t (2 8)-2-fluoro-2-[[(2S,5R)-3-methyl-7oxo-2-[2-(5-oxopyrrolidin-2yl)ethylcarbamoyl]-1,6- diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid lithium salt 74 o 1 L J—N ₄ J ⁰ O'^uon O (2 8)-2-fluoro-2- [ [(2 S, 5R)-3 -methyl-7oxo-2-(3- sulfamoylpropylcarbamoyl)-1,6diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid lithium salt

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75 ⁿ Cl _F A—n ₄ j ⁰ O'y-OH 0 (28)-2-fluoro-2-[[(28.5K)-3-methyl-7oxo-2-[2- (sulfamoylamino)ethylcarbamoyl]1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid lithium salt 76 A—N _s J o O'y-OH 0 (2S)-2-[[(2S,5R)-2-[( _T peT-butoxycarbonylamino)methyl]-3 methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl ]hydroxy]-2-fluoro-oxyhydric acid lithium salt 77 MF J----N ₄ J o O'A^OH 0 (28)-2-[[(28.5K)-2-(aminomethyl)-3methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2-fluoroacetic acid 78 IZ A o' Ο^^’Ή o ΞΕ (2 S)-2-[ [(2 S, 5R)-2-(acetamide methyl)3 -methyl-7-oxo-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl] oxy]-2 -fluoroacetic acid lithium salt 79 'AA _g A—n _s j ^{0 0} f ^0H O (28)-2-fluoro-2-[[(28.5K)-3-methyl-7oxo-2-[[(2,2,2trifluoroacetyl)amino]methyl]-1,6diazabicyclo[3.2.1]oct- 3-en-6yl] oxide] acetic acid lithium salt 80 'Ί IZ o Ao ϊ>o' 0^ ¹¹ (° ethyl-(28)-2-fluoro-2-[[(28.5K)-2(cyanomethylcarbamoyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1 ]oct-3-en6-yl] oxy]acetate 81 ⁰ 1 _F У—N^ F ⁰ °>°0 ethyl-(2K)-2-fluoro-2-[[(28.5K)-2(cyanomethylcarbamoyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1 ]oct-3-en6-yl] oxy]acetate

- 19 044428

82 ΞΕΖ o o' o t (2S)-2^to _P -2-[[(2S,5R)-2-(cyanomethylcarbamoyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl] hydroxy]acetic acid lithium salt 83 o> O oC o ZI IZ ω=Ο // \ About Ζ <ΝΙ t ethyl-(2S)-2-φτop-2-[[(2S,5R)-2(cyanomethylcarbamoyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1 ]oct-3-en6-yl] oxy]acetate 84 I th Ζ O X // O=(L za: ΙΖ o. <=o ΪΣ>o' O^ ⁷¹ f ethyl-(2R)-2-φτop-2-[[(2S,5R)-2(cyanomethylcarbamoyl)-3-methyl-7 oxo-1,6-diazabicyclo[3.2.1 ]oct-3-en6-yl] oxy]acetate 85 ⁰ 1 ^Ί Έ ρ Ν _χ J ° ⁰ ?τ ^ο \^ 0 ethyl-(2S)-2-φτop-2-[[(2S,5R)-2(hydroxymethylcarbamoyl)-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl]oxy ] acetate 86 I°> ΞΖ o >o ϊ> (° ethyl-(2R)-2-φτop-2-[[(2S,5R)-2(hydroxymethylcarbamoyl)-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl]oxy ] acetate 87 I ΞΖ oo'O;^ ⁷¹ O I (2S)-2^Top-2-[[(2S,5R)-2- (hydroxymethylcarbamoyl)-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl]oxy]acetic acid lithium salt 88 0 0 I Α-? ^ι ··Λ ^HH Μ F V~ ^Ns oA^oh 0 (2S)-2^Top-2-[[(2S,5R)-2- (acetamidomethylcarbamoyl)-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl]oxy]acetic acid lithium salt

- 20 044428

89 T th L xO ωζ ΙΖ > ΙΖ About Vo o' ABOUT ZE (28)-2-fluoro-2-[[(28.5K)-3-methyl-7oxo-2- [(sulfamoylamino)methylcarbamoyl]-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy]acetic acid lithium salt 90 O h ₂ n M F _c 0 ethyl 2-(((2 S, 5 R)-2-carbamoyl-4methyl-7-oxo-1,6diazabicyclo[3.2.1 ]oct-3-en-6yl)oxy)-2,2-difluoroacetate 91 Jw O 2-(((28.5R)-2-carbamoyl-4-methyl-7oxo-1,6-diazabicyclo[3.2.1]oct-3-ene- 6-yl)oxy)-2,2-difluoroacetic acid lithium salt 92 ABOUT J--N_o/ 0 0 ethyl 2-(((2 S, 5 R)-2-carbamoyl-4methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6yl)oxy)acetate 93 0 H ₂ N 0 2-(((28.5R)-2-carbamoyl-4-methyl-7oxo-1,6-diazabicyclo[3.2.1 ]oct-3-en6-yl)oxy)acetic acid lithium salt 94 exhaust gas F dU O / o °^|f O ethyl-(2K)-2-(((28.5K)-2-cyano-4methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetate 95 FROM 5 ο^~ ^Ν 'ο ^Λ γ ^ΟΗ 0 (2K)-2-(((28.5K)-2-cyano-4-methyl-7oxo-1,6-diazabicyclo[3.2.1 ]oct-3-en6-yl)oxy)-2-fluoroacetic acid lithium salt 96 0 h ₂ n ⁰ ° TI 0 1 isopropyl-2-(((2S,5R)-2-carbamoyl4-methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6yl)oxy)acetate

The compounds of the invention include compounds of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V), (Va) in free base form ( uncharged), as well as their pharmaceutically acceptable salts.

Alkyl—As used herein, the term alkyl refers to straight and branched chain saturated hydrocarbon radicals having the stated number of carbon atoms. References to individual alkyl groups, such as propyl, characterize only the straight chain variant, and references to individual branched chain alkyl groups, such as isopropyl and 3-pentyl, characterize only the branched chain variant. In one aspect, alkyl is methyl.

Halogen—As used herein, the term halogen is intended to include fluorine, chlorine, bromine, and iodine. In one aspect, halogen can refer to fluorine, chlorine and bromine. In yet another aspect, halogen may refer to fluorine or chlorine. In yet another aspect, the halo may refer to fluorine. In yet another aspect, halogen may refer to chlorine.

Haloalkyl, as used herein, is an alkyl moiety, as defined above, substituted with one or more halogen atoms. In one aspect, the haloalkyl may be -CHF2, -CH2F or -CF ₃ .

Cycloalkyl - In one aspect, cycloalkyl refers to a saturated monocyclic carbon ring, one or more -CH ₂ - groups of which may optionally be replaced by a corresponding number of -C(O)- groups. Illustrative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclopentenyl. In one aspect, the 3-5 membered carbocyclyl may be cyclopropyl.

5-7 membered heterocyclyl - the term 5-7 membered heterocyclyl refers to a saturated or partially saturated non-aromatic monocyclic ring containing 5 to 7 atoms

- 21 044428 rings, at least one of which is selected from nitrogen, sulfur and oxygen, and the -CH2- group of which may optionally be replaced by a -C(O)- group. Likewise, a 5- to 6-membered heterocyclyl refers to a saturated or partially saturated non-aromatic monocyclic ring containing from 5 to 6 ring atoms, at least one of which is selected from nitrogen, sulfur and oxygen, and the CH2- group of which may optionally be replaced by the group -S(O)-. Unless otherwise specified, 5-7 membered heterocyclyl and 5-6 membered heterocyclyl groups may be carbon or nitrogen bonded. The ring nitrogen atoms may optionally be oxidized to form an N-oxide. The ring sulfur atoms may optionally be oxidized to form S-oxides or sulfones. Illustrative examples of 5-7 membered heterocyclyl and 5-6 membered heterocyclyl include, but are not limited to, azetidinyl, dioxidotetrahydrothiophenyl, 2,4-dioxoimidazolidinyl, 3,5dioxopiperidinyl, furanyl, imidazolyl, isothiazolyl, isoxazolyl, morpholinyl, oxazolyl, oxetanyl, oxo imidazolidinyl , 3-oxo-1-piperazinyl, 2-oxopyrrolidinyl, 2-oxotetrahydrofuranyl, oxo1,3-thiazolidinyl, piperazinyl, piperidyl, 2H-pyranyl, pyrazolyl, pyridinyl, pyrrolyl, pyrrolidinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridazinyl, 4-pyridonyl , tetrahydrofuranyl, tetrahydropyranyl, thiazolyl, 1,3,4-thiadiazolyl, thiazolidinyl, thiomorpholinyl, thiophenyl, 4H-1,2,4-triazolyl, pyridine-N-oxidyl, tetrazolyl, oxadiazolyl, triazolyl, pyrazinyl, triazinyl and homopiperidinyl. In one embodiment, the terms 5-7 membered heterocyclyl and 5-6 membered heterocyclyl include 5-7 or 5-6 member siderophores containing at least one heteroatom.

5- or 6-membered heteroaryl - The term 5-6 membered heteroaryl refers to a monocyclic aromatic heterocyclyl ring containing 5 or 6 ring atoms, at least one of which is selected from nitrogen, sulfur and oxygen. Unless otherwise specified, 5-6 membered heteroaryl groups may be carbon or nitrogen bonded. The ring nitrogen atoms may optionally be oxidized to form an N-oxide. The ring sulfur atoms may optionally be oxidized to form S-oxides. Illustrative examples of 5-6 membered heteroaryl include furanyl, imidazolyl, isothiazolyl, isoxazole, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyridinyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thiophenyl and triazolyl.

Optionally Substituted—As used herein, the expression optionally substituted indicates that the substitution is optional and, therefore, the specified group may be substituted or unsubstituted. If substitution is necessary, the appropriate number of hydrogen atoms in a specified group may be replaced by a selection from the specified substituents, provided that the normal valency of the atoms in the particular substituent is not exceeded and that the substitution results in a stable compound.

In one aspect, when a particular group is designated as optionally substituted with one or more substituents, that particular group may be unsubstituted. In yet another aspect, a particular group may carry one substituent. In yet another aspect, a particular substituent may carry two substituents. In yet another aspect, a particular group may carry three substituents. In yet another aspect, a particular group may carry four substituents. In a further aspect, a particular group may bear one or two substituents. In yet another aspect, the particular group may be unsubstituted or may bear one or two substituents.

Pharmaceutically Acceptable - As used herein, the expression pharmaceutically acceptable refers to those compounds, substances, compositions and/or dosage forms that are medically suitable for use involving contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable risk/benefit ratio.

Prodrug—As used herein, the term prodrug refers to a chemically modified version of a pharmacologically active agent that undergoes conversion in vivo to release the active drug. (See Rautio, J., et al., Prodrugs: Design and Clinical Applications, Nature Rev., vol. 7, page 255 (March 2008)). In the present invention, prodrugs are used to make active beta-lactamase inhibitor compounds orally bioavailable after absorption from the gastrointestinal tract.

Effective Amount—As used herein, the expression effective amount means an amount of a compound or composition sufficient to produce a significant and beneficial change in the symptoms and/or conditions being treated (eg, to provide a positive clinical response). The effective amount of active ingredient for use in the pharmaceutical composition will vary depending on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concomitant therapy, the particular active ingredient(s) used, the particular pharmaceutically acceptable excipient(s)/carrier(s) used ) and similar factors within the knowledge and experience of the attending physician.

The compounds of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) and (Va) can form stable pharmaceutically acceptable salts with acids or bases, and in such cases it may be appropriate to administer the compound in salt form. Examples of acid addition salts include acetate, adipate, ascorbate, benzoate, benzenesulfonate, bicarbonate, bisulfate, butyrate, camphorate, camphor

- 22 044428 sulfonate, choline, citrate, cyclohexylsulfamate, diethylenediamine, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxyethylsulfonate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxyamalate, lactate, malate, maleate, methanesulfo nat, meglumine, 2 -naphthalene sulfonate, nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate, diphosphate, picrate, pivalate, propionate, quinate, salicylate, stearate, succinate, sulfamate, sulfanilate, sulfate, tartrate, tosylate (p-toluenesulfonate), trifluoroacetate and undecanoate. Examples of basic salts include ammonium salts; alkali metal salts such as sodium, lithium and potassium salts; alkaline earth metal salts such as aluminum, calcium and magnesium salts; salts with organic bases such as dicyclohexylamine and N-methylD-glucamine salts; and salts with amino acids such as arginine, lysine, ornithine and so on. Also, basic nitrogen-containing groups can be quaternized by such agents as: lower alkyl halides, such as methyl, ethyl, propyl and butyl halides; dialkyl sulfates such as dimethyl, diethyl, dibutyl; diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl halides; arylalkyl halides such as benzyl bromide and others. Non-toxic physiologically acceptable salts are preferred, although other salts may also find use, for example in product isolation or purification.

Salts can be prepared by conventional methods, such as reacting the product in free base form with one or more equivalents of the corresponding acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is removed in vacuo or by lyophilization, or by exchanging the anions of the existing salt for another anion on a suitable ion exchange resin.

When a compound disclosed herein is described by name or structure and has one or more chiral centers, and when the name or structure covers more than one stereoisomer, e.g., the stereochemistry of one or more chiral centers is not indicated, the name or structure is intended to cover all such stereoisomers and mixtures thereof.

Synthesis of optically active forms can be accomplished by standard methods of organic chemistry well known in the art, for example, by synthesis from optically active starting materials or resolution of the racemic form. Racemates can be separated into individual enantiomers using known methods (see, for example, Advanced Organic Chemistry: 3rd Edition: author J March, p104-107). A suitable method involves the formation of diastereomeric derivatives by reacting a racemic substance with a chiral auxiliary substance, followed by separation, for example chromatography, of the diastereomers and then elimination of the auxiliary substances. Likewise, the above activity can be assessed using standard laboratory techniques mentioned below.

Stereoisomers can be separated using conventional techniques such as chromatography or fractional crystallization. Enantiomers can be isolated by resolution of the racemate, for example by fractional crystallization, resolution or HPLC. Diastereoisomers can be isolated by resolution due to the different physical properties of the diastereoisomers, for example by fractional crystallization, HPLC or flash chromatography. Alternatively, specific stereoisomers can be prepared by chiral synthesis from chiral starting materials under conditions that do not cause racemization or epimerization, or by derivatization with a chiral reagent.

When a particular stereoisomer is designated, structurally or by name, it is preferably separated or substantially isolated from other stereoisomers of the same compound. In one aspect, a mixture containing a particular stereoisomer of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) , may contain less than 30% by weight, in particular less than 20% by weight and in particular less than 10% by weight of other stereoisomers of the same compound. In yet another aspect, a mixture containing a particular stereoisomer of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) , may contain less than 6 wt.%, in particular less than 3 wt.% and, in particular, less than 2 wt.% of other stereoisomers of this compound. In yet another aspect, a mixture containing a particular stereoisomer of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) , may contain less than 1 wt.%, in particular less than 0.5 wt.% and, in particular, less than 0.3 wt.%, in particular less than 0.1 wt.% of other stereoisomers of this compound.

In one aspect, the terms infection and bacterial infection may refer to gynecological infection. In yet another aspect, the terms infection and bacterial infection may refer to respiratory tract infection (RTI). In yet another aspect, the terms infection and bacterial infection may refer to a sexually transmitted disease. In yet another aspect, the terms infection and bacterial infection may refer to an uncomplicated urinary tract infection (UTI). In yet another aspect, the terms infection and bacterial infection may refer to complicated urinary tract infection (cUTI). In a further aspect, the terms infection and bacterial infection may refer to an acute attack of chronic bronchitis (ACB). In yet another additional aspect, the terms infection and bacterial infection may refer to acute otitis media. In one aspect, the terms infection and bacterial

- 23 044428 This infection may relate to acute sinusitis. In yet another aspect, the terms infection and bacterial infection may refer to infection caused by drug-resistant bacteria. In yet another aspect, the terms infection and bacterial infection may refer to catheter-associated sepsis. In yet another aspect, the terms infection and bacterial infection may refer to chancroid. In a further aspect, the terms infection and bacterial infection may refer to chlamydia. In yet another additional aspect, the terms infection and bacterial infection may refer to community-acquired pneumonia (CAP). In yet another additional aspect, the terms infection and bacterial infection can refer to a complicated infection of the skin and skin structures. In one aspect, the terms infection and bacterial infection may refer to uncomplicated infection of the skin and skin structures (USSI). In yet another aspect, the terms infection and bacterial infection may refer to endocarditis. In yet another aspect, the terms infection and bacterial infection may refer to febrile neutropenia. In yet another aspect, the terms infection and bacterial infection may refer to gonococcal cervicitis. In a further aspect, the terms infection and bacterial infection may refer to gonococcal urethritis. In yet another additional aspect, the terms infection and bacterial infection may refer to nosocomial pneumonia (NP). In yet another aspect, the terms infection and bacterial infection may refer to osteomyelitis. In a further aspect, the terms infection and bacterial infection may refer to sepsis. In yet another aspect, the terms infection and bacterial infection may refer to syphilis. In a further aspect, the terms infection and bacterial infection may refer to intra-abdominal infection (IAI). In one aspect of the invention, the terms infection and bacterial infection may refer to an infection selected from the group consisting of complicated urinary tract infection, uncomplicated urinary tract infection, kidney infection, lower respiratory tract infection, nosocomial bacterial pneumonia, pneumonia, acute bacterial prostatitis, acute bacterial skin and soft tissue infection, sepsis, intra-abdominal infection and diabetic foot infection.

In one embodiment, the terms infection and bacterial infection refer to infection caused by gram-negative bacteria, also called gram-negative infection. In one aspect of this embodiment, the gram-negative infection is an infection that is resistant to one or more antibiotics. In one aspect of this embodiment, the gram-negative infection is a multidrug-resistant infection. In one aspect of this embodiment, the gram-negative infection is caused by one or more Enterobacteriaceae spp. In one aspect of this embodiment, one or more Enterobacteriaceae spp. include one or more of E. coli, K. pneumoniae, K. oxytoca, C. freundii, C. koseri, E. cloacae, P. mirabilis, M. morganii and/or S. marcescens. In one aspect of this embodiment, the one or more Enterobacteriaceae spp pathogens include one or more E. coli or K. pneumoniae pathogens. In yet another aspect of this embodiment, the gram-negative infection is caused by one or more biothreat pathogens. In one aspect of this embodiment, the one or more biothreat pathogens are Burkholderia spp., Y. pestis and/or F. tularensis. In any of these aspects of the embodiment, one or more gram-negative pathogens may express one or more serine beta-lactamase enzymes. In one aspect of this embodiment, the one or more serine beta-lactamase enzymes include one or more class A, class C, and/or class D beta-lactamases.

All of the above infections can be caused by a variety of bacteria that could potentially be treated with the claimed agents alone or in combination with penicillin binding protein inhibitors. One embodiment of the invention relates to a method of treating one or more of the above infections, comprising administering to a subject suffering from a bacterial infection an effective amount of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa). ), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable salt thereof in combination with an additional antibiotic. In one aspect of this embodiment, the additional antibiotic is a beta-lactam antibiotic. In one aspect of this embodiment, the additional antibiotic is a penicillin binding protein inhibitor.

In one aspect, the use of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V), or (Va) or its a pharmaceutically acceptable salt for the manufacture of a medicament for achieving the effect of inhibiting bacterial peptidoglycan, alone or in combination with a penicillin binding protein inhibitor, in a warm-blooded animal such as a human.

In yet another aspect, the use of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V), or (Va) or thereof is provided. a pharmaceutically acceptable salt for the manufacture of a medicament for treating a bacterial infection in a warm-blooded animal such as a human. In one aspect, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or a pharmaceutical thereof

- 24 044428 a commercially acceptable salt is administered in combination with an additional antibiotic, such as a betalactam antibiotic. In one aspect of this embodiment, the additional antibiotic is a penicillin binding protein inhibitor. In one aspect of this embodiment, the additional antibiotic is a beta-lactam antibiotic. In one aspect of this embodiment, the beta-lactam antibiotic is selected from cefpodoxime, cefuroxime, tigemonam, loracarbef, cefixime, cephalexin, cefadroxil, cefetamet, cefprozil, ceftibuten, cefditorene, faropenem, tebipenem, amoxicillin, carbenicillin, cefdinir, ampicillin, cefditorene or their prodrugs. In one aspect of this embodiment, the betalactam antibiotic is cefpodoxime proxetil. In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime axetil. In one aspect of this embodiment, the beta-lactam antibiotic is cefpodoxime or a prodrug thereof. In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime or a prodrug thereof.

In another aspect, the use of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va), or thereof is provided. pharmaceutically acceptable salt, for the manufacture of a medicament for the treatment of complicated urinary tract infection, uncomplicated urinary tract infection, kidney infection, lower respiratory tract infection, nosocomial bacterial pneumonia, pneumonia, acute bacterial prostatitis, acute bacterial skin and soft tissue infection, sepsis, intra-abdominal infection and infections associated with diabetic foot in a warm-blooded animal such as a human. One aspect of the invention relates to the use of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or thereof a pharmaceutically acceptable salt for the manufacture of a medicament for the treatment of complicated urinary tract infections. In one aspect of the two preceding embodiments, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) is administered in combination with an additional antibiotic. In one aspect of this embodiment, the additional antibiotic is a penicillin binding protein inhibitor. In one aspect of this embodiment, the additional antibiotic is a beta-lactam antibiotic. In one aspect of this embodiment, the beta-lactam antibiotic is selected from cefpodoxime, cefuroxime, tigemonam, loracarbef, cefixime, cephalexin, cefadroxil, cefetamet, cefprozil, ceftibuten, cefditorene, faropenem, tebipenem, amoxicillin, carbenicillin, cefdinir, ampicillin, cefditorene and their prodrugs In one aspect of this embodiment, the beta-lactam antibiotic is cefpodoxime proxetil. In one aspect of this embodiment, the betalactam antibiotic is cefuroxime axetil. In one aspect of this embodiment, the beta-lactam antibiotic is cefpodoxime or a prodrug thereof. In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime or a prodrug thereof.

In yet another aspect, there is provided a method of achieving a bacterial peptidoglycan inhibitory effect, alone or in combination with a penicillin binding protein inhibitor, in a warm-blooded animal such as a human, wherein said method comprises administering to said animal an effective amount of a compound of formulas (I), (Ia) , (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable salt thereof.

In an additional aspect, there is provided a method of treating a bacterial infection in a warm-blooded animal, such as a human, comprising administering to said animal an effective amount of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV) ), (IVa), (V) or (Va) or a pharmaceutically acceptable salt thereof. In one aspect of this embodiment, the compound is as described for formulas (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof. In one aspect of this embodiment, for a compound of formulas (I), (II), (III), (IV) or (V), the compound is administered orally. In yet another aspect of this embodiment, a compound of formulas (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is administered in combination with an additional antibiotic. In one aspect of this embodiment, the additional antibiotic is a penicillin binding protein inhibitor. In one aspect, the additional antibiotic is a beta-lactam antibiotic. In one aspect of this embodiment, the beta-lactam antibiotic is selected from cefpodoxime, cefuroxime, tigemonam, loracarbef, cefixime, cephalexin, cefadroxil, cefetamet, cefprozil, ceftibuten, cefditorene, faropenem, tebipenem, amoxicillin, carbenicillin, cefdinir, ampicillin, cefditorene and their prodrugs In one aspect of this embodiment, the beta-lactam antibiotic is cefpodoxime proxetil. In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime axetil. In one aspect of this embodiment, the betalactam antibiotic is cefpodoxime or a prodrug thereof. In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime or a prodrug thereof.

In a further embodiment, a method of treating a bacterial infection in

- 25 044428 to a subject in need thereof, comprising administering to said subject an effective amount of a compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) or a pharmaceutically acceptable salt thereof intravenously, intraperitoneally, intramuscularly or subcutaneously, preferably intravenously . As such, a compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) or a pharmaceutically acceptable salt thereof is advantageously used when the patient is unable to take medications orally, for example in a hospital setting (eg, in a intensive care, emergency department, cardiology department, etc.), emergency room setting, and nursing home setting. In one aspect of this embodiment, a compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) or a pharmaceutically acceptable salt thereof is administered intravenously (IV). In one aspect of this embodiment, a compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) or a pharmaceutically acceptable salt thereof is administered until the patient is able to take the drugs orally, e.g. duration of stay in a hospital or emergency room, until the subject can be discharged from the hospital or emergency room, or until the patient's condition improves enough to allow him to take oral medications, e.g. until the patient can take medications orally in a nursing home setting. In one aspect of this embodiment, a compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) or a pharmaceutically acceptable salt thereof is administered with a beta-lactam antibiotic. In one aspect of this embodiment, the beta-lactam antibiotic is selected from cefpodoxime, cefuroxime, tigemonam, loracarbef, cefixime, cephalexin, cefadroxil, cefetamet, cefprozil, ceftibuten, cefditorene, faropenem, tebipenem, amoxicillin, carbenicillin, cefdinir, ampicillin, cefditorene and their prodrugs. In one aspect of this embodiment, the beta-lactam antibiotic is cefpodoxime or a prodrug thereof. In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime or a prodrug thereof. In yet another aspect of this embodiment, the method further comprises administering to the subject an effective amount of a compound of formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, in an outpatient setting, upon discharge from a hospital or hospital. emergency care, or when the patient's condition has improved enough to allow the patient to take medications orally, such as in a nursing home setting. In one aspect of this embodiment, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is administered orally in an outpatient setting. In one aspect of this embodiment, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is administered in combination with the same beta-lactam antibiotic with which the compound of formulas ( Ia), (IIa), (IIIa), (IVa), or (Va) in a hospital, emergency room, or nursing home setting. In one aspect of this embodiment, after administration of a compound of formulas (Ia), (IIa), (IIIa), (IVa), or (Va), the compound of formula (I), (II), (III), (IV) is administered orally. or (V) or a pharmaceutically acceptable salt thereof, as soon as the patient is able to take the drug orally, for example, after the patient has been discharged from a hospital or emergency room and is in an outpatient setting, or as soon as the patient's condition in a nursing home setting has improved enough so that he can take medications orally. It is preferable to switch from intravenous, intraperitoneal, intramuscular or subcutaneous (compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va)) to oral administration (compound of formulas (I), (II), (III) , (IV) or (V)) occurs without any interruption in the patient's treatment.

In yet another additional aspect, a method is provided for the treatment of complicated urinary tract infection, uncomplicated urinary tract infection, kidney infection, lower respiratory tract infection, nosocomial bacterial pneumonia (NBP), pneumonia, acute bacterial prostatitis, acute bacterial skin and soft tissue infection, sepsis, intra-abdominal infections and infections associated with diabetic foot in a warm-blooded animal such as a human, wherein said method comprises administering to said animal an effective amount of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa ), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable salt thereof. In yet another further aspect, there is provided a method of treating complicated urinary tract infections in a warm-blooded animal, such as a human, comprising administering to said animal an effective amount of a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa). ), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable salt thereof. In one aspect of any of the preceding embodiments, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable salt thereof is administered in combination with an additional antibiotic. In one aspect of this embodiment, the additional antibiotic is a penicillin binding protein inhibitor. In one aspect, the additional antibiotic is a beta-lactam antibiotic. In one aspect of this embodiment, the beta-lactam antibiotic is selected from cefpodoxime, cefuroxime, tigemonam, loracarbef, cefixime, cephalexin, cefadroxil, cefetamet, cefprozil, ceftibuten, cefditorene, faropenem, tebipenem, amoxicillin, carbenicillin, cefdinir, ampicillin, cefditoren and their prodrugs In one aspect

- 26 044428 of this embodiment, the beta-lactam antibiotic is cefpodoxime proxetil.

In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime axetil. In one aspect of this embodiment, the beta-lactam antibiotic is cefpodoxime or a prodrug thereof. In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime or a prodrug thereof.

In yet another additional aspect, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V), or (Va) or thereof is provided a pharmaceutically acceptable salt for use in achieving the effect of inhibiting bacterial peptidoglycan, alone or in combination with a penicillin binding protein inhibitor, in a warm-blooded animal such as a human. In one aspect, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable form thereof is provided. salt for use in the treatment of gram-negative bacterial infections, alone or in combination with a beta-lactam antibiotic. In one aspect of this embodiment, the beta-lactam antibiotic is selected from cefpodoxime, cefuroxime, tigemonam, loracarbef, cefixime, cephalexin, cefadroxil, cefetamet, cefprozil, ceftibuten, cefditorene, faropenem, tebipenem, amoxicillin, carbenicillin, cefdinir, ampicillin, cefditorene and their prodrugs In one aspect of this embodiment, the beta-lactam antibiotic is cefpodoxime proxetil. In one aspect of this embodiment, the betalactam antibiotic is cefuroxime axetil. In one aspect of this embodiment, the beta-lactam antibiotic is cefpodoxime or a prodrug thereof. In one aspect of this embodiment, the beta-lactam antibiotic is cefuroxime or a prodrug thereof.

In one aspect of the invention there is provided a method of inhibiting one or more beta-lactamase enzymes, comprising administering a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa ), (V) or (Va) or a pharmaceutically acceptable salt thereof to an animal in need thereof. In a further aspect, the one or more beta-lactamase enzymes are serine beta-lactamase enzymes. In a further aspect, the one or more beta-lactamase enzymes are selected from the group consisting of class A, class C, and class D. In a further aspect, the one or more beta-lactamase enzymes are a class A enzyme. In a further aspect, the one or more beta-lactamase enzymes are is a class C enzyme. In a further aspect, the one or more beta-lactamase enzymes are a class D enzyme. In a further aspect, the one or more beta-lactamase enzymes are a class D enzyme and one or more class A and C enzymes. In a further aspect, one or more beta-lactamase enzymes are enzymes of all three classes A, C and D.

Beta-lactamase inhibitors of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) can be administered in combination with any beta-lactam antibiotic belonging to, but not limited to, the classes of clavams, carbapenems, monobactams, penems, penicillins and/or cephalosporins, or any other compound susceptible to serine beta-lactamases. In one aspect of the invention, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) is combined with one or more of penicillin, methicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temocillin, amoxicillin, ampicillin, co-amoxiclav, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, cephalexin, cephalothin, SXA-101, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cefoxitin, ceftriaxone, cefotaxime, cefpodoxime, cefixime, ceftazidime, ceftobiprole medocaril, cefepime, cefpirome, ceftaroline, imipenem, meropenem, ertapenem, faropenem, sulopenem, doripenem, PZ-601 (Protez Pharmaceuticals), ME1036 (Forest Labs) , BAL30072, MC-1, tomopenem, tebipenem, aztreonam, tigemonam, nocardicin A or tabtoxin beta-lactam. In one aspect of the invention, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) is combined with cefpodoxime, cefuroxime, tigemonam, cefixime or faropenem. In one aspect of the invention, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) is combined with an antibacterial compound from the group consisting of penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, amoxicillin, ampicillin, bacampicillin, amoxicillin-clavulanate, carbenicillin, cefadroxil, cephalexin, cephradine, cefaclor, cefprozil, cefuroxime axetil, ce fdinir, loracarbef, cefixime, cefpodoxime and ceftibuten , or a prodrug or salt thereof. In one aspect of the invention, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) is combined with an antibacterial compound from the group consisting of cefpodoxime, cefuroxime, tigemonam, loracarbef, cefixime, cephalexin, cefadroxil, cefetamet, cefprozil, ceftibuten, cefditorene, faropenem, tebipenem, amoxicillin, carbenicillin, cefdinir, ampicillin, cefdi toren and their prodrugs. In one aspect of the invention, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) is combined with cefpodoxime or its prodrug such as cefpodoxime proxetil. In one aspect of the invention, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) is combined with cefuroxime or its prodrug such as cefuroxime axetil.

- 27 044428

In yet another aspect of the invention, a compound of formulas (I), (II), (III) or (IV) is administered in combination with a beta-lactam antibiotic and an additional antibiotic and/or an additional beta-lactamase inhibitor. In one aspect of the invention, the additional antibiotic is selected from one of the classes of aminoglycosides, spectinomycins, macrolides, ketolides, streptogramins, oxazolidinones, tetracyclines, fluoroquinolones, quinolones, coumarin antibiotics, glycopeptides, lipoglycopeptides, nitroimidazoles, ansamycins, phenicols, mupirocin, fosfomycin, tobram tsina, linezolid, daptomycin, vancomycin, beta-lactams and classes mentioned in ANTIMICROBIAL AGENTS (ASM Press, Ed: A. Bryskier (2005)).

In one aspect of the invention, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) is administered in combination with a beta-lactam antibiotic and a second agent designed to overcome beta-lactam resistance. In one aspect of the present invention, the second agent for overcoming beta-lactam resistance may be a metallo-beta-lactamase (MBL) inhibitor, also known as a class B inhibitor.

In one aspect, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V), or (Va) or pharmaceutical thereof is provided an acceptable salt for use in treating a bacterial infection in a warm-blooded animal such as a human.

In yet another aspect, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable form thereof is provided. salt for use in the treatment of complicated urinary tract infection, uncomplicated urinary tract infection, kidney infection, lower respiratory tract infection, nosocomial bacterial pneumonia, pneumonia, acute bacterial prostatitis, acute bacterial skin and soft tissue infection, sepsis, intra-abdominal infection and infections associated with diabetic foot, in a warm-blooded animal such as a human. In yet another aspect, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V), or (Va) or pharmaceutical thereof is provided. an acceptable salt for use in the treatment of complicated urinary tract infections in a warm-blooded animal such as a human.

In another aspect, a pharmaceutical composition is provided containing a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent or excipient. In one aspect of this embodiment, the pharmaceutical composition further comprises a betalactam antibiotic. In one aspect of this embodiment, the beta-lactam antibiotic is selected from cefpodoxime, cefuroxime, tigemonam, loracarbef, cefixime, cephalexin, cefadroxil, cefetamet, cefprozil, ceftibuten, cefditorene, faropenem, tebipenem, amoxicillin, carbenicillin, cefdinir, ampicillin, cefditorene and their prodrugs

The compositions of the invention may be presented in a form suitable for oral administration (for example, tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical administration ( for example, in the form of creams, ointments, gels or aqueous or oily solutions or suspensions), for administration by inhalation (for example, as a fine powder or liquid aerosol), for administration by insufflation (for example, as a fine powder) or for parenteral administration (eg, as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, or intramuscular administration, or as a suppository for rectal administration). In one aspect of the invention, a compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) or a pharmaceutically acceptable salt thereof is administered intravenously. In yet another aspect of the invention, a compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) or a pharmaceutically acceptable salt thereof is administered intravenously in combination with one or more other antibacterial drugs. In one aspect of the invention, a compound of formulas (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, is administered orally. In yet another aspect of the invention, a compound of formulas (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, is administered orally in combination with one or more other antibacterial drugs. In one aspect of any of these embodiments, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or a pharmaceutically acceptable salt thereof is administered concomitantly with one or more other antibacterial drugs. In yet another aspect of this embodiment, a compound of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) or thereof the pharmaceutically acceptable salt is administered sequentially with one or more other antibacterial drugs, such as a beta-lactam antibiotic.

One embodiment of the invention relates to a method of treating a bacterial infection in a person in need thereof, comprising administering to said person an effective amount of a compound of one of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) intravenously in combination with one or more additional antibacterial drugs in an outpatient, emergency room, or nursing home setting, followed by administering to the individual an effective amount of a compound of formula (I), (II), (III), (IV), or (V) orally at combinations with one or more additional antibacterial drugs outside, for example, an outpatient, emergency room, or nursing home setting, once the patient is able to take oral medications again.

One embodiment of the invention relates to a method of treating a bacterial infection in a person in need thereof, comprising orally administering to said person an effective amount of a compound of one of formulas (I), (II), (III), (IV) or (V) in combination with one or more additional antibacterial drugs as step-down therapy, transitioning to the oral route of administration after administering to a specified person an effective amount of one or more antibacterial drugs administered intravenously, intraperitoneally, intramuscularly or subcutaneously, for example, a compound of formulas (Ia), (IIa), ( IIIa), (IVa) or (Va) or a pharmaceutically acceptable salt thereof. The nature, dose, and duration of antibiotic therapy and the timing of switching from intravenous, intraperitoneal, intramuscular, or subcutaneous routes of administration to an oral drug are usually selected by the physician and may depend on the patient's health status, his or her ability to receive oral treatment, and the type of infections from which the patient is suffering. Human. The patient may be switched from intravenous, intraperitoneal, intramuscular, or subcutaneous therapy to oral treatment when the patient is no longer symptomatic and has no or only a mild fever (eg, less than 100.5°F (approximately 38°C) , 100°F (about 37.8°C), 99.5°F (about 37.5°C), etc.), has been removed from the ventilator or no longer requires intravenous fluids.

The compositions of the invention can be prepared by conventional methods using conventional pharmaceutical excipients well known in the art. Suitable pharmaceutically acceptable excipients for the preparation of tablets include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch; lubricants such as magnesium stearate, stearic acid or talc; preservatives such as ethyl or propyl p-hydroxybenzoate; and antioxidants such as ascorbic acid. Tablet formulations may be uncoated or coated to modify their disintegration and subsequent absorption of the active ingredient in the gastrointestinal tract, or to improve their stability and/or appearance, in either case using conventional coating agents and methods , well known in the art.

The amount of active ingredient combined with one or more excipients to form a unit dosage form will vary as necessary depending on the recipient being treated and the particular route of administration. For example, a preparation intended for oral administration to humans will typically contain, for example, the active agent mixed with a suitable and convenient amount of excipients, which may vary from about 5 to about 98 wt.% based on the weight of the total composition. Dosage units typically contain from about 100 to about 4000 mg of active ingredient. For oral administration of, for example, a compound of formulas (I), (II), (III), (IV) or (V) or pharmaceutically acceptable salts thereof, 0.1 to 10 g-eq of active ingredient per day is suitable; and for intravenous administration of, for example, a compound of formulas (Ia), (IIa), (IIIa), (IVa) or (Va) or pharmaceutically acceptable salts thereof, 0.5 to 8 g-eq of active ingredient per day is suitable.

In addition to the compounds of the present invention, the pharmaceutical composition of the present invention may also contain or be co-administered with (simultaneously, sequentially or separately) one or more known drugs selected from other clinically useful classes of antibacterial drugs (for example, macrolides, quinolones, betalactams or aminoglycosides) and/or other anti-infective drugs (eg, antifungal triazole or amphotericin). These may include carbapenems, such as meropenem or imipenem, to enhance therapeutic efficacy. The compounds of the present invention may also contain or be co-administered with bactericidal/permeability enhancing (BPI) protein products or efflux pump inhibitors to improve activity against Gram-negative bacteria and antimicrobial resistant bacteria.

As stated above, the dose size required for therapeutic or prophylactic treatment of a particular pathological condition will vary as needed depending on the recipient being treated, the route of administration and the severity of the disease being treated. Accordingly, the optimal dosage can be determined by the practitioner treating any particular patient.

Compounds of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va) can be prepared in several ways. The processes shown below illustrate a method for the synthesis of compounds of formula (I) (wherein R ¹ , R ² and R ³ , unless otherwise stated, are as defined above). Reactions are carried out in solvents appropriate to the reagents and substances used and suitable for the transformations being carried out. In addition, when describing the synthesis methods given below, it is understood that all proposed reaction conditions, including the choice of solvents, the atmosphere in which

- 29 044428 reaction temperature, reaction time, experimental duration and processing methods are chosen as conditions standard for this reaction, which should be obvious to one skilled in the art. One skilled in the art of organic synthesis will appreciate that the functional groups present in the various parts of the molecule must be compatible with the proposed reagents and reactions. Such limitations on substituents compatible with the reaction conditions will be apparent to one skilled in the art and alternative methods must be used. The schemes and processes are not intended to cover an exhaustive list of methods for preparing compounds of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (IVa), (V) or (Va ); conversely, additional techniques known to the skilled chemist may also be used to synthesize the compounds. The claims are not limited to the structures shown in the diagrams and processes.

It should also be understood that in some of the reactions shown in the schemes and in the processes mentioned herein, it may be necessary/desirable to protect any sensitive groups in the compounds. Those skilled in the art will know when protection is necessary or desirable, as well as suitable methods for such protection. Conventional protecting groups can be used in accordance with standard practice (for an example, see T. W. Greene, Protective Groups in Organic Synthesis, published by John Wiley and Sons, (1991)) and as described above herein.

A skilled chemist will be able to use and adapt the information contained in the above references, as well as the information referenced therein and in the accompanying examples and the examples and diagrams given herein, to obtain the necessary starting materials. substances and products.

The necessary starting materials for methods such as those described herein, if not commercially available, can be prepared using methods selected from standard organic chemistry techniques, techniques similar to the synthesis of known, structurally similar compounds, or techniques similar to those described the method or methods described in the examples.

It is noted that many of the starting materials for the synthetic methods as described herein are commercially available and/or widely described in the scientific literature, or can be prepared from commercially available compounds using adaptations of processes described in the scientific literature. Next, the reader can refer to the source Advanced Organic Chemistry, ^5th Edition, for author. Jerry March and Michael Smith, published by John Wiley & Sons (2001), for general guidance on reaction conditions and reagents.

General methods and schemes

In one aspect, the compounds of formulas (I) and (Ia) or pharmaceutically acceptable salts thereof can be prepared by the method described in Scheme 1. From the Weinreb amide, the introduction of substituents at the R ³ position of formulas (I) and (Ia) can be accomplished using Grignard reactions. Ester moieties can be introduced by palladium-catalyzed deallylation followed by alkylation with bromoacetates. As a result of hydrolysis of esters, acids are obtained. Alternatively, other R ¹ groups can be obtained by modification of the primary alcohol.

Scheme 1

[C ₃ H ₅ PdCI] ₂ ligand (RR), O phthalimide,

Na ₂ CQ ₃ DCM,

Ome

TBSCI, .N. TBSO-^^

.......... - hydrazine, Br O MeOH, 65 C _TBS0 -^x^. Oh _Vos

NH ₂ Boc^O, DMF primary amine Weinreb amide

generation, toluene, 65 C catalyst

Hoveyda-Grubbs tbso*

Vos'

TBSO

CeCI ₃ NaBH *0 MeOH, OS

AIIONH-Ns, PPh ₃ DIAD, _TBS0 toluene, room temp. □

.OAN

Ns

Alternative methods for the synthesis of compounds with substitution at R ³ are shown in Scheme 2, where the key steps are the Diels-Alder reaction and the nitrosoene reaction.

Scheme 2

EY

Diels-Alder .OTBS nitroso-ene

Vos

R ³ CPI, NH ₄ OAc h ₂ n

BUT

BocNHOH, O _{2 R} 3 or air

ZnBr _{2 f} triphosgene, H ₂ N^'''-|^4y ^{R3 HN} x^A _N 'OTBS N.^

H J--N

ΟOTBS

Acetate and ester moieties can be introduced in one or two steps according to Scheme 3.

Scheme 3

In yet another aspect, the compounds of formulas (I) and (Ia) or pharmaceutically acceptable salts thereof can be prepared by the method described in Scheme 4, wherein substitution at R ² can be introduced by Michael addition to enone 1 followed by oxidation to yield the enone 2, after which the synthesis is similar to that described in Scheme 1.

Scheme 4

TBSO '‘Ύ χΝ Vos

1. R ² Li, Cui

2. TMSCI, DIEA TBSO

OTMS

Pd(OAc) ₂ TBSO

5.

enone 1

Alternatively, amide R ¹ can be introduced after enone 2 cyclization of urea according to the scheme

Scheme 5

Compounds with substitution at R ² can also be synthesized from Harner's aldehyde, as shown in Scheme 6. The path from the primary amine to compounds of formulas (I) or (Ia) is similar to Scheme 1.

Scheme 6 °l ? , °l I ⁿ °l /I _D °^\ / 11 11 ^R Dess-Martin^Ts^ V, Witti \r2 1)TsOH g TBSO^^y^R ² ZnBr2^ TBSO'^'^r'R ²

Vos Vos Vos Βο^ ¹⁴ ^ ^η 2

o) I doGI

In addition, compounds with substitution at R ² can be synthesized according to Scheme 7 below, where the key step is the nitrosoene reaction. The amide can also be introduced at an earlier stage of the synthesis from a carboxylic acid and retained until the end of the synthesis.

- 31 044428

Scheme 7

Alkyl bromoacetates for the introduction of carboxylic acids and ester moieties can be obtained by transesterification according to Scheme 8.

Scheme 8

Chiral bromofluoroacetates for the introduction of R- and S-fluorocarboxylic acid and ester moieties can be obtained by recrystallization of bromofluoroacetic acid with chiral phenylethylamine, followed by esterification according to Scheme 9.

Scheme 9

Any of the above-mentioned pharmaceutical compositions, processes, methods, applications, medicinal products and manufacturing features of the present invention are also applicable to any alternative embodiments of the compounds of the invention described herein. For example, further details and method of carrying out the nitrosoene reaction on various substrates are as described below in the examples section. These details and methods include, for example, a first method for preparing a compound of formula VI

or its salts, where

R ¹ represents -C(O)NR ⁷ R ⁸ , -C(O)OR ⁷ , -CH2OR ⁷ , -CN, phenyl, 5-7 membered heteroaryl, -C(O)NR'NR'C(O )R ⁹ , -C(O)NR'OR ¹⁰ or C1-C6 alkyl group, where the alkyl group is replaced by 1-3 groups consisting of halogen, C1-C3-αalkoxy, -OH, -CN, -NR ⁷ R ⁸ , -NR7COR ⁹ , 5-7 membered heteroaryl and 5-7 membered heterocyclyl, and wherein phenyl and heteroaryl represented by R ¹ are optionally and independently substituted with 1-3 groups selected from halogen, -OH, C1-C ₃ - alkoxy, -CN, -NR7R ⁸ and -CONR7R ⁸ ;

each of R ² and R ³ is independently selected from hydrogen, halogen, C ₁ -C ₃ -alkylα and C ₃ -C ₆ -cycloalkyl, provided that at least one of R ² and R ³ is not hydrogen;

each R ⁷ and R ⁸ independently represents hydrogen, C1-C3 alkyl, C1-C3 alkoxy, phenyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, where alkyl, alkoxy, phenyl, cycloalkyl, heterocyclyl or heteroaryl represented by R ⁷ or R ⁸ is optionally and independently substituted with 16 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F atoms, carboxyl or -CO( _OC1.6 αlkyl), 5 -6-membered heteroaryl, -CN, -OH, C ₁ -C ₃ -alkyl, optionally substituted -NH2 or -OH, C ₁ -C ₃ -haloalkyl, C ₁ -C ₃ -haloalkoxy, C ₁ -C ₃ - alkoxy -NHCO(C ₁ -C ₃ -alkylα), -NHCO(C ₁ -C ₃ -alkoxy), -S(O)2NR'R'', -NHS(O)2NR'R'', -NHS( O) ₂ (C ₁ -C ₃ -αalkyl), -NR'R'' and -C(O)NR'R'';

each R ⁹ is C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C ₁ -C ₆ -haloalkoxy or C1-C6 alkoxy;

R' and R'' are each independently hydrogen, methyl, ethyl or propyl; or

- 32 044428

R' and R'', together with the nitrogen atom to which they are attached, form a 5-6-membered heterocyclyl;

and each of PG and PG' independently represents a protecting group for an amino group;

wherein the method involves reacting a compound of formula XI

or a salt thereof with PG'NHOH in the presence of an oxidizing agent to form a compound of formula VI; A second method for preparing the compound of formula VI is also proposed.

or its salts, where

R1 is -C(O)NR ⁷ R ⁸ , -C(O)OR ⁷ , -CH2OR7, -CN, phenyl, 5-7 membered heteroaryl, -C(O)NR'NR'C(O)R ⁹ , -C(O)NR'OR ¹⁰ or C1-C6 alkyl group, where the alkyl group is replaced by 1-3 groups consisting of halogen, C1-C3 alkoxy, -OH, -CN, -NR ⁷ R ⁸ , - NR7COR ⁹ , 5-7 membered heteroaryl and 5-7 membered heterocyclyl, and wherein the phenyl and heteroaryl represented by ^R1 are optionally and independently substituted with 1-3 groups selected from halogen, -OH, C1- _C3 alkoxy, -CN, -NR7R ⁸ and -CONR7R ⁸ ;

each of R ² and R ³ is independently selected from hydrogen, halogen, C ₁ -C ₃ alkyl and C ₃ -C ₆ cycloalkyl, provided that at least one of R ² and R ³ is not hydrogen;

each R ⁷ and R ⁸ independently represents hydrogen, C1-C3 alkyl, C1-C3 alkoxy, phenyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, where alkyl, alkoxy, phenyl, cycloalkyl, heterocyclyl or heteroaryl represented by R ⁷ or R ⁸ is optionally and independently substituted with 16 groups selected from 5-6 membered heterocyclyl, optionally substituted with one or two -F atoms, carboxyl or -CO(OC1-6alkyl), 5- 6-membered heteroaryl, -CN, -OH, C ₁ -C ₃ -alkyl, optionally substituted -NH ₂ or -OH, C ₁ -C ₃ -haloalkyl, C ₁ -C ₃ -haloalkoxy, C ₁ -C ₃ - alkoxyNHCO(C1-C3-alkyl), -NHCO(C1-C3-alkoxy), -S(O)2NR'R, -NHS(O)2NR'R, -NHS(O)2(C1-C3-alkyl) , -NR'R” and -C(O)NR’R”;

each R ⁹ is C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ haloalkoxy or C1 -C6 alkoxy;

R' and R'' are each independently hydrogen, methyl, ethyl or propyl; or

R' and R'', together with the nitrogen atom to which they are attached, form a 5-6-membered heterocyclyl;

and each of PG and PG' independently represents a protecting group for an amino group; wherein the method involves reacting a compound of formula XI

or its salt with PG'N=O to form a compound of formula VI.

In a first aspect, the compound of formula XI in the first or second method has the formula

or its salt.

In a second aspect, the compound of formula VI in the first or second method has formula VII

or its salt.

In the third aspect, R ² in the first or second method or formula VII is C1-C3 alkyl, with other features being as described in the first or second method and the first or second aspect. Alternatively, R ² in the first or second method or formula VII is methyl, the remaining features being as described in the first or second method and the first or second aspect.

In a fourth aspect, the compound of formula VI in the first or second method has formula VIII:

- 33 044428

Rk.

/N. X* /PG'

PG N

I

OH (VIII);

or a salt thereof, the remaining features being those described in the first or second method and the first or second aspect.

In a fifth aspect, R ³ in the first or second method or formula VII is C ₁ -C ₃ -alkyl, the remaining features being as described in the first or second method and the first or second aspect. Alternatively, R ³ in the first or second method or formula VIII is methyl, the remaining features being as described in the first or second method and the first or second aspect.

In the sixth aspect, R ¹ in the first or second method is selected from oxadiazole, -C(O)NHNHC(O)(C1-C3alkyl), -CH2NH2, -CH2NHCO(C1-C3-alkoxy), -CH2NHCO(C1-C3-alkyl ) or -CH2NHCO(C1-C3haloalkyl), wherein the oxadiazole R ¹ is optionally substituted with -OH, C1- _C3 alkoxy, -NR7R ⁸ or -CONR7R ⁸ ; and wherein the remaining features are as described in the first or second method and the first, second, third, fourth or fifth aspect.

In the seventh aspect, R ¹ in the first or second method is selected from -CH2NH2

and wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth or sixth aspect.

In the eighth aspect, R ¹ in the first or second method is -CN,

where R ¹¹ represents hydrogen or -C(O)NH2; and wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth or seventh aspect.

In the ninth aspect, R ¹ in the first or second method is -C(O)NR ⁷ R ⁸ , -C(O)OR ⁷ or CN; and wherein the remaining features are as described in the first or second method and in the first, second, third, fourth, fifth, sixth, seventh or eighth aspect. Alternatively, R ¹ in the first or second method is -C(O)NH2, -C(O)OH, -CN or -C(O)OC1-C6-alkyl; wherein the remaining features are as described in the first or second method and in the first, second, third, fourth, fifth, sixth, seventh or eighth method. In yet another alternative, R ¹ in the first or second method is -CN or -C(O)NH2; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh or eighth aspect. In yet another alternative, R ¹ in the first or second method is -CN; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh or eighth aspect. In yet another alternative, R ¹ in the first or second method is -C(O)NR ⁷ R ⁸ ; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh or eighth aspect.

In the tenth aspect, R ⁷ and R ⁸ in the first or second method are both hydrogen; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspect. Alternatively, R ⁷ in the first or second method is hydrogen, and R ⁸ is 1) phenyl, optionally substituted with C1-C3 alkyl or C1-C3 alkyl-NH2, 2) C1-C3 alkyl, or 3) C1-C3-alkoxy, where each alkyl or alkoxy of the presented R ⁸ is optionally and independently substituted with C3-C ₆ -cycloalkyl, -CN, -OH, -NH2, -SO2NH2, -NHSO2NH2, -C(O)NH ₂ , - NHC(O)(C ₁ -C ₃ -alkyl), pyrazinyl, oxytanyl, oxazolyl or pyrrolidinyl, optionally substituted with one or more carboxyl, fluorine atom or -C(O)O(C ₁ -C ₆ -alkyl); wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspect. In yet another alternative, R ⁷ in the first or second method is hydrogen and R ⁸ is selected from the group consisting of

- 34 044428

-CH2CN and -CH2OH;

wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspect.

In an eleventh aspect, the compound of formula VI in the first or second method has formula IX

OH (IX);

or its salt.

In the twelfth aspect, the compound of formula VI in the first or second method has formula X

OH (X);

or its salt.

In a thirteenth aspect, each of PG and PG' in the first or second method, together with the amine nitrogen atom they protect, independently forms a carbamate, amide or N-benzyl or N-aryl; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh or twelfth aspect. Alternatively, each of PG and PG' in the first or second method is independently selected from tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), 2,2,2trichloroethoxycarbonyl (Troc), CF ₃ CO, acetyl ( Ac), para-toluenesulfonamide (Ts) and methylsulfonyl (Ms); wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh or twelfth aspect. In yet another alternative, each of PG and PG' in the first or second method is the same; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh or twelfth aspect. In yet another alternative, each of PG and PG' in the first or second method is tert-butoxycarbonyl; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh or twelfth aspect.

In a fourteenth aspect, the first method further comprises reacting a compound of formula XI with PG'NHOH in the presence of a metal catalyst; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or thirteenth aspect. Alternatively, the metal catalyst is selected from CuCl, CuBr, CuI, CuCN, CuSCN, CuBrMe ₂ S, Cu(OAc) ₂ and CuOTf; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or thirteenth aspect. In yet another alternative, the metal catalyst includes a copper salt; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or thirteenth aspect. In yet another alternative, the metal catalyst comprises a copper halide salt; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or thirteenth aspect. In yet another alternative, the metal catalyst is CuCl or CuBrMe ₂ S; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or thirteenth aspect.

In a fifteenth aspect, the first method further comprises reacting a compound of formula XI with PG'NHOH in the presence of an amine additive; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth aspect. In one aspect, the amine additive is pyridine, (1R,2R)-cyclohexane-1,2-diamine, N,N'dimethylethane-1,2-diamine, 2,6-di-tert-butyl-4-methylpyridine, 1,10-phenanthroline, trans-cyclohexane-1,2-diamine, N1-(2-(diethylamino)ethyl)-N2,N2-diethylethan-1,2-diamine, cis-cyclohexane-1,2-diamine or N1,N1 ,N2,N2-tetramethylethane-1,2-diamine; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth aspect. Alternatively, the amine is selected from pyridine, 2,6-lutidine, 4-dimethylaminopyridine, picoline, 1,8diazabicyclo[5.4.0]undec-7-ene and N,N-diisopropylethylamine; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth aspect. In yet another alternative, the amine is pyridine; wherein the remaining features are as described in the first or second method and in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth aspect.

In the sixteenth aspect, the oxidizing agent in the first or second method is O2, air, _FeCl3 , _MnO2 , meta-chloroperoxybenzoic acid (mCPBA), _NaIO4 , 2-iodoxybenzoic acid (IBX), (2,2,6,6tetramethylpiperidin-1- yl)oxyl (TEMPO), benzoyl peroxide (BPO), HIO ₃ , urea-H ₂ O ₂ , I ₂ , Nchlorosuccinimide (NCS), Dess-Martin periodinane (DMP), H2O2 or N-methylmorpholine N-oxide (NMMO); wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth aspect. Alternatively, the oxidizing agent is urea - H2O2, H2O2 or O2; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth aspect.

In the seventeenth aspect, the reaction in the first or second method is carried out in a polar solvent; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth or sixteenth aspect. Alternatively, the reaction is carried out in DCM, THF, MTBE, EtOAc, iPrOAc, MeCN, H2O, MeOH, EtOH, i-PrOH, t-BuOH, nBuOH, 2-methyl-2-butanol, DMF, DMSO, ethylene glycol, polyethylene glycol, sulfolane, mixtures of sulfolane/H ₂ O, DMF/H2O, NMP/H2O, DCM/ _H2O , MeOH/H ₂ O, EtOH/H 2 O, iPrOH/H ₂ O or n-BuOH/H ₂ O; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth or sixteenth aspect. In yet another alternative, the reaction is carried out in methylene chloride or sulfolane; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth or sixteenth aspect.

In an eighteenth aspect, the reaction in the first or second method further comprises adding water; wherein the remaining features are as described in the first or second method and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth or seventeenth aspect.

Examples

The invention will now be further described with reference to the following illustrative examples, in which, unless otherwise indicated:

(i) temperature is expressed in degrees Celsius (°C);

operations are carried out at room temperature or ambient temperature, that is, in the range of 18-25°C;

(ii) the organic solutions were dried over anhydrous magnesium sulfate;

evaporation of the organic solvent was carried out using a rotary evaporator at reduced pressure (4.5-30 mmHg) and at a bath temperature of up to 60°C;

- 36 044428 (iii) chromatography means flash chromatography on silica gel;

thin layer chromatography (TLC) was performed on silica gel plates;

(iv) reaction progress was typically monitored by TLC or liquid chromatography/mass spectroscopy (LC/MS), and reaction times are given as examples only;

(v) the final products have satisfactory proton nuclear magnetic resonance (NMR) spectra and/or mass spectral data;

(vi) the yields are for illustrative purposes only and do not necessarily represent yields that could be obtained by diligent development of the process;

the preparation was repeated if more starting materials were required;

(vii) NMR data, where reported, are presented as delta values for the major protons used for identification, given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard, determined at 300 MHz in DMSO- d ₆ unless otherwise stated;

(viii) chemical symbols have their usual meanings;

(ix) solvent ratios are given in volume:volume (v/v) units;

(x) ISCO Combiflash refers to silica gel flash chromatography using the Isco Combiflash® separation system: RediSep normal phase flash column, flow rate 30-40 ml/min;

(xi) the following abbreviations may be used:

ACN - acetonitrile

BINAF - 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl

Boc ₂ O - tert-butyloxycarbonyl anhydride KDI - N,N-carbonyldiimidazole

DAST - diethylaminosulfur trifluoride DCM - dichloromethane

DIPEA/DIEA - N,N-diisopropylethylamine

DMAc - N,N-dimethylacetamide

DMF - N,N-dimethylformamide

DMAP - 4-dimethylaminopyridine

DMSO - dimethyl sulfoxide ee - enantiomeric excess EtOAc/EA - ethyl acetate Et ₂ O - diethyl ether GC - gas chromatography HATU - O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate Hex - hexanes

HPLC - high performance liquid chromatography h/h - hours

KOtBu - potassium tert-butoxide

LCMS - liquid chromatography-mass spectrometry

LDA - lithium diisopropylamide

MeCN - acetonitrile

MeOH - methanol min - minutes

MTBE - methyl tert-butyl ether in flow. nights - during the night

Pd ₂ (dba) ₃ - tris(dibenzylideneacetone)dipalladium(0)

PE - petroleum ether iPrOH - isopropyl alcohol rac. - racemic

TBAF - tetra-n-butylammonium fluoride

TEA - triethylamine

TFA - trifluoroacetic acid

THF - tetrahydrofuran

TMS - trimethylsilyl

Tosyl, Ts para-toluenesulfonyl

UHPLC-MS - ultra-high performance liquid chromatography-mass spectrometry

Conditions for carrying out and testing the nitrosoene reaction

In some aspects, the nitrosoene reaction is used to introduce the desired allylic hydroxylamine functionality. (Waldemar Adam and Oliver Krebs, Chem. Rev., 2003,103, 4131-4146; Charles P. Frazier, Jarred R. Engelking, and Javier Read de Alaniz, J. Am. Chem. Soc, 2011, 133 (27) , 10430-10433; Leoni I. Palmer, Charles P. Frazier, Javier Read de Alaniz, Synthesis 2014, 46, 269-280). In the presence of an oxidizing agent, hydroxylamines are oxidized to form highly reactive nits

- 37 044428 rose compounds that react with an allylic substrate as shown in Scheme 10. The oxidizing agent can be an organic oxidizing agent or a combination of an oxidizing agent, a metal catalyst and, optionally, an amine additive. Nitroso compounds can be formed in situ (as in the reaction to form a compound of formula VI) from hydroxylamine or prepared separately and then added to a substrate, for example, a compound of formula XI. In the case of a 1,2,3,6-tetrahydropyridine backbone with an Ncarbamate functionality, it is believed that the 1,3-allylic strain on the N-Boc functionality causes the R ¹ substituent to adopt a pseudoaxial orientation. It is believed that it blocks the approach of the nitroso reagent from this side of the double bond. Consequently, the nitrosoreactant reacts regio- and diastereoselectively from the opposite side of R ¹ to form the target product with high diastereoselectivity and regioselectivity. Enantioselectivity was measured and found to be high in all cases, greater than 99% ee, when analyzed by chiral HPLC.

Scheme 10 pg ₂

NHOH

Oxidizing agent, solvent or catalyst, additive, oxidizing agent, solvent

Reaction parameters such as oxidant, catalyst, ligand, solvent, stoichiometry of reactants, temperature and reaction time can influence the reaction outcome and have been tested and optimized. Some results are summarized in the following tables (N/A = not determined).

Oxidizer check

Record Substrate Reagents and conditions Oxidizer Conversion (as determined by % HPLC peak area) Product Ref. VVA 1 PGi=PG ₂ =Boc Ri=CONH ₂ R ₂ =Me, R ₃ =H BocNHOH(l.5 eq.) CuCl (0.05 eq.) Pyridine (0.013 eq.) DCM (10 vol.), 17-25 °C, 20-60 O _2.1 atm 84.5 5.9 2 FeCh, 1.2 eq. 15.3 43.2 3 MnO ₂ , 1.2 eq. 48.0 10.28 4 NaIO4, 2.2 eq. 43.8 21.3 5 t-CPBA, 2.2 eq. 10.7 30.9 6 VPO, 1.0 eq. TEMPO, 1.5 eq. 32.3 19.6 7 NUS, 1.2 eq. 6.4 3.0 8 1 ₂ , 1.2 eq. 5.8 24.6 9 H ₂ O ₂ , 3.0 eq. 78 8.9 10 MnO ₂ , 1.2 eq. H ₂ O ₂ , 1 drop 71.6 5.0 AND IBX, 1.2 eq. BUT 12 PH(OAc) ₂ , 1.2 eq. BUT 13 hydroperite, 1.2 eq. 63.9 12.1 14 NCS, 1.0 eq. 59.9 12.5 15 DMP, 1.0 eq. BUT

- 38 044428

16 3%H ₂ O2, 1.2 eq. 70 3.1 17 1.5%H ₂ O ₂ , 1.2 eq. 64.4 4.1 18 6%Η ₂ Ο ₂ , 1.2 eq 73.6 5.5 19 ΝΜΜ0, 1.0 eq. 37.8 38.6 20 PGi=PG2=Boc Ri=CO2Me R ₂ =CH ₂ OPMB, R ₃ =H BocNHOH (1.5 EQ.) CuCl (0.05 eq.) Pyridine (0.013 eq.) DCM (10 vol.), 17-25 °C, 20-60 h Og, 1 atm. 36% allocated product 21 PGi=PG2=Boc Ri=CONH ₂ R ₂ =H, R ₃ =Me BocNHOH (1.5 eq.) CuCl (0.05 eq.) Pyridine (0.013 eq.) DCM (10 vol.), 20-30 °C, 20 60 h O2, 1 atm. 46.08 10.28 22 FeCl ₃ (1.2 eq.) 1.33 71.43 23 MnOg (1.2 eq.) 40.79 16.85 24 NaIO ₄ (1.2 eq.) 15.2 35.58 25 m-SRV A (1.2 eq.) 3.04 89.1 26 NU ₄ (1.2 eq.) 0.52 46.47 27 30%H ₂ O ₂ (1.2 eq.) 4.69 7.9 28 1g (1.2 eq.) 1.39 52.85 29 CaO ₂ (1.2 eq.) 19.66 33.73 thirty BocNHOH (3.0 eq.), CuCl (0.05 eq.), sulfolane (5 vol), H ₂ O (5 vol.), Ru (0.13 eq.), 0-5 °C, H2O2 (3%, 3 eq.), 16 h H ₂ O ₂ (3%, 3 eq.), 96 h 54.3 38.8 30.5 0.24 31 BocNHOH (2.0 eq.), CuBrMe ₂ S (0.05 eq.), sulfolane (5 vol.), H ₂ O (0.5 vol.), Ru (0.13 eq.), 25-30 °C H2O2 (3%, 2 eq.), 64 h 70.0 4.1

- 39 044428

Solvent check

Record Substrate Reagents and conditions Solvent, time Conversion (as determined by % HPLC peak area) Product Ref. VVA 1 PGi=PG ₂ =Boc Ri=CONH ₂ R ₂ =Me, R ₃ =H BocNHOH(l.5 eq.) CuCl (0.05 eq.) Pyridine (0.013 eq.), 15 °C, air 5% THF in DCM (10 vol), 90 h 77.6 5.0 2 10% THF in DCM (10 vol), 90 h 77.5 4.7 3 20% THF in DCM (10 vol), 90 h 68.8 19.4 4 DXM (10 vol), 68 h 65.8 20.4 5 THF (10 vol.), 20 h 81.9 7.8 6 EtOAc (10 vol), 68 h 69.5 19.0 7 Acetone (10 vol.), 68 87.3 3.6 8 PGi=PG ₂ =Boc Ri=CONH ₂ R ₂ =H, R ₃ =Me BocNHOH(l,5 eq.) CuCl (0.05 eq.) Pyridine (0.013 eq.), 20-30 °C, air, 60 h DXM (10 vol.) 35.2 20.6 9 DXM/N ₂ O 1:1 (10 vol.) 38.0 18.7 10 MeON(Uob) 50.0 21.8 AND MeOH/H ₂ O 1:1 (10 vol.) 45.7 28.3 12 EYUN(Yoob) 47.5 15.9 13 z-PrOH (10 vol.) 43.3 10.0 14 2-methyl-2-butanol (10 vol.) 48.0 16.6 15 Ethylene glycol (10 vol.) 14.1 76.4 16 Polyethylene glycol 2000/N ₂ 0 (5 g/5 vol.) 9.3 96.9 17 sulfolane/H ₂ O 1:1 (10 vol.) 57.0 14.7 18 sulfolane/H ₂ O 9:1 (10 vol.) 55.58 17.5 19 sulfolane/H ₂ O 3:7 (10 vol.) 23.3% 66.7 20 sulfolane/H ₂ O 1:1 (5 vol.) 62.3 5.9 21 BocNHOH(l.5 eq.) CuCl (0.05 eq.) Pyridine (0.013 eq.), 15-30 °C, O ₂ (1 atm.), 48 h dmfa/n ₂ o 5 vol./5 vol. 58.11 1.71 22 nmp/h ₂ o 5 rev./5 rev. 44.18 0.43

- 40 044428

Checking the catalyst

Record Substrate Reagents and conditions Catalyst Conversion (as determined by % HPLC peak area) Product Ref. VVA 1 PGi=PG2=Boc Ri=CONH ₂ R ₂ =H, R ₃ =Me BocNHOH(l.5 eq.) CuCl (0.05 eq.) Pyridine (0.013 eq.) DCM (10 vol.), 20-30 °C, O ₂ (1 atm), 65 h CuCl, 0.05 eq. 35.2 20.6 2 CuBr, 0.05 eq. 30.7 25.2 3 Cui, 0.05 eq. 21.5 43.1 4 Cu(OAc) ₂ , 0.05 eq 33.0 28.5 5 sulfolane/H ₂ O 5 vol./5 vol., 24 h 58.49 1.30 6 sulfolane/H ₂ O 5 vol./5 vol., 96 h 38.83 0.24 7 HOAc/N ₂ O 5 vol./5 vol., 96 h 7.95 71.02 8 BocNHOH (l.5 eq.), pyridine (0.013 eq.), sulfolane (5 vol.), H ₂ O (5 vol.), O ₂ , 20-30 ° C, CuCl, 0.05 eq., 24 h 58.49 1.30 9 CuCl, 0.05 eq., 96 h 38.83 0.24 10 CuBr, 0.05 eq. 72 h 63.42 7.31 AND Cui, 0.05 eq. 67.49 7.35 12 CuSCN (0.05 eq), 72 h 32.98 47.05 13 Cu(OAc)2 (0.05 eq.), 72 h 59.69 15.94 14 CuBr-SMe ₂ (0.05 eq.), 72 h 68.71 3.36 15 CuCN (0.05 eq), 84 h 5.60 5.32 16 Cu(CH ₃ CN) ₄ pf ₆ (0.05 eq.), 84 h 61.56 14.07 17 Cu(OT1)-toluene (0.05 eq.), 84 h 66.24 9.31 18 CuCl (0.05 eq.) 2-ethyloxazoline (0.05 eq.), 84 h 59.03 15.20 19 CuCl (0.05 eq) TBAI (0.05 eq), 89 h 49.9 26.26

- 41 044428

Checking Additives

Record Substrate Reagents and conditions Ligand Transformation (as defined by 1 2 3 4 5 6 7 8 9 10 I 12 BocNHO H (1.5 eq.) CuCl (0.05 eq.) DCM (10 vol.), 20-30 °C, O ₂ (1 atm), 65 h Pyridine (0.013 eq.) Ethane-1,2-diamine (0.013 eq.) (17?,27?)-cyclohexane-1,2-diamine (0.013 eq.) N1, A7-dimethylethane-1,2-diamine ( 0.013 eq.) 2,6-di-tert-butyl-4-methylpyridine (0.013 eq.) 1,10-o-phenanthroline (0.013 eq.) Without ligand Η ₂ Ν^\ _Μμ ^νη 2.0.013 eq., 72 h Η ₂ Ν^ _ν χ 1 0.013 eq., 72 h 1,10-o-phenanthroline (0.013 eq.), 72 h hXXk 1 I 0.013 eq. 96 h % VE peak plot Product 35.2 31.8 36.3 35.0 31.5 But 24.8 37.73 5.60 64.45 69.09 69.11 adi LC) Ref. in-va 20.6 29.0 30.1 21.5 34.0 47.4 27.4 9 5.32 5.14 0.94 0.93 13 14 15 16 17 PGi=PG ₂ = Vos Ri=CONH 2 r ₂ =h, R ₃ =Me BocNHO H (1.5 eq.), CuCl (0.05 eq.), sulfolane (5 vol.), H ₂ O (5 vol.), O ₂ (1 atm), 2030 °C A^ _N || ₂ trans - 0.013 eq. 96 h Pyridine (0.013 eq.), 24 h Pyridine (0.013 eq.), 96 h Pyridine (0.065 eq.), 84 h Pyridine (0.13 eq.), 24 h NH ₂ Nl 1 ₂ trans- 0.013 eq., 65 h 58.49 38.83 74.1 73.80 66.63 1.30 0.24 7.1 5.57 5.09 18 nh ₂ trans- 0.13 eq, 48 h 9.48 86.4 7 19 1 ⁿ 0.013 eq. 72 h 62.64 10.4 5

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20 0.013 eq., 72 h 64.43 9.24 21 0.013 eq., 72 h 66.21 7.52 22 NH- Cis-0.013 eq., 72 h 65.09 6.79 23 0.013 eq., 130 h 71.2 10.0 24 0.013 eq., 130 h 66.3 12.0 25 0.013 eq., 130 h 15.0 61.8 26 DMAP, 0.03 eq., 60 h 70.88 5.91 0.013 eq., 60 h 71.56 4.93 27 0.013 eq., 89 h 57.34 I,8 5 28 0.013 eq., 89 h 61.04 14.6 5 29 18-crown-6 (0.001 eq), KS1 (0.05 eq), 36 h 73.67 5.53 thirty Sodium L-ascorbate (0.1 eq.), 36 hours Trace quantity 98.7 2 31 18-crown-6 (0.001 eq.), KS1 (0.05 eq.), Ru (0.013 eq.), 86 h 75.8 4.8 32 0.013 eq, 18-crown-6 (0.001 eq), KC1 (0.05 eq), 86 h 74.0 10.6

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33 A-0 0.013 eq., 18-crown -6 (0.001 eq.), KS1 (0.05 eq.), 86 h 60.6 22.5 34 BocNHO H (1.5 eq.), CuCl (0.05 eq.), sulfolane (5 vol.), H ₂ O (5 vol.), air, 20-30 °C Ru (0.013 eq.), 85 h 46.74 24.5 5 35 N H H ₂ N — — N — NH ₂ Η 0.013 eq., 85 h 54.08 16.8 1 36 O O A A η ₂ ν ν νη ₂ η 0.013 eq., 85 h 51.32 18.7 8 37 Η Η 0.013 eq., 85 h 53.35 17D 38 \—/ 0.013 eq., 85 h 51.11 18.2 1 39 Ά 0.013 eq., 85 h 55.61 14.3 9 40 BocNHO H (1.5 eq.), CuBrMe ₂ S (0.05 eq.), sulfol n (5 vol.), H ₂ O (5 vol.), O ₂ , 20-30 °C Pyridine (0.013 eq.), 72 h 68.71 3.36 41 DMAP (0.013 eq.), 86 h 66.99 10.8 8 42 1 A— ^N - 1 0.013 eq., 86 g 3.01 96.5 7 43 nh ₂ AA> _N || ₂ trans - 0.013 eq., 86 h 71.53 6.53 44 BocNHO Η (3 eq.), CuCl (0.05 eq.), sulfol n (5 vol.), H ₂ O (5 vol.), H ₂ O ₂ (3%, 3 nh ₂ AA> _N h ₂ trans- 0.013 eq, 66 h 74.3 8.7 eq.), 20-30 °C

After testing, the best conditions for the substrate with PG1=PG2=Boc, R1=CONH2, R2=Me, R3=H were: BocNHOH (1.5 eq.), CuCl (0.05 eq.), Ru (0.013 eq.) , DCM (10 vol.), O ₂ (1 atm.), 15-25°C. For the substrate with PG1=PG2=Boc, R1=CONH2, R ₂ =H, R ₃ =Me, the best conditions were: BocNHOH (1.5 eq.), CuCl (0.05 eq.), Ru (0.013 eq. ), sulfolane (5 vol.), H ₂ O (5 vol.), O ₂ (1 atm.), 15-25 ° C or BocNHOH (2 eq.), CuBr-Me ₂ S (0.05 eq. ), Ru (0.13 eq.), sulfolane (5 vol.), H ₂ O (0.5 vol.), H ₂ O ₂ (3% in water, 2-3 eq.), 15-25 ° C.

The following experimental procedures are provided for illustrative purposes.

BocNHOH (2 eq.),

CuBr-Me ₂ S (0.05 eq.), Ru (0.13 eq.), sulfolane (5 vol.),

H ₂ O (0.5 vol.)

H ₂ O ₂ (3% in water, 2 eq.), 15-25 °C, 29 h

Sulfolane (5 vol.) and _{H 2} O (0.5 vol.) _, pyridine ( 0.13 eq.). The mixture was stirred for 30-40 minutes at 15-25°C. 3% H ₂ O ₂ (2 eq.) was added dropwise over 24-30 hours. After the reaction was considered complete, a solution of EDTA-2Na (0.31-0.32 wt. eq.) in water was added ( 3-3.2 times the weight) and MTBE (7.7 times the weight). The resulting mixture was stirred for 20-30 minutes and allowed to stand for 20-30 minutes. The two phases were separated. The aqueous phase was extracted three times with MTBE (4x the mass). The combined organic solution was dried over Na ₂ SO ₄ and filtered, concentrated and analyzed by assay. Yield 47.5%, purity 74.12% as determined by HPLC peak area %.

O _Ξ θ I

BocNHOH (1.5 eq.) CuCl (0.05 eq.) Η ₂ Ν^'''·ρ^4 Βοο ^Ν ^ Ru (0.013 eq.), O ₂ (g) Boc-'I'^^Н' ⁶⁰⁰

DXM HE

To the crude solution of the substrate from the previous step in DCM was added CuCl (0.05 eq.), Boc

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NHOH (1.5 eq.) and Ru (0.013 eq.). The mixture was stirred under O2 atmosphere at 20±5°C until the amount of starting material was <5%, as determined by % HPLC peak area. The EDTA-2Na solution (5.0 vol) was charged and the resulting mixture was stirred for at least 4 hours at 25±5°C. The two phases were separated. The aqueous phase was extracted twice with DCM (3.0 vol). The organic phases were combined and washed once with water (5.0 vol.), concentrated in vacuo at <40°C to about 3.0 original volumes. The reactor was charged with i-PrOAc (5.0 vol) and the mixture was concentrated in vacuo at <40°C to about 4.0 original volumes. This process was repeated one more time. n-heptane (5.0 vol.) was added to the reactor at 40±5°C. The resulting mixture was gradually cooled to 20±5°C. The solid was obtained by centrifugation and washed with i-PrOAc/n-heptane (1:1.2 vol.), and dried in vacuum at 35±5°C for at least 12 hours. Yield in 2 stages: 71.68 %, purity 99.7% as determined by HPLC peak area %.

Intermediate 1: (S)-2-(1-hydroxybut-3-en-2-yl)isoindoline-1,3-dione

A 2 L reaction flask containing a magnetic stir bar and sodium carbonate (1.981 g, 18.69 mmol) was placed under high vacuum and dried using a jet air dryer for ten minutes. After cooling, the flask was filled with nitrogen. Allylpalladium chloride dimer (0.553 g, 1.53 mmol), (1R,2R)-(+)-1,2-diaminocyclohexane-N,N'-bis(2-diphenylphosphino-1naphthoyl) (CAS 174810-09) was added to this -4) (3.36 g, 4.25 mmol) and phthalimide (50 g, 339.83 mmol). The flask was then purged with nitrogen for 10 min. Then 1.4 liters of methylene chloride, which had been pre-degassed with nitrogen for ten minutes, was added. This suspension was placed under a nitrogen atmosphere; it was alternately stirred and sonicated for ten minutes to facilitate solvation. At this point it was a yellow or light orange solution containing a white solid. To this mixture was added 2-vinyloxirane (24.06 g, 343.23 mmol). The resulting mixture was stirred under nitrogen at ambient temperature for approximately 48 hours. Analysis by LCMS and TLC (1:1 hexanes:ethyl acetate) during this process indicated progress of the reaction, and final analyzes using these methods indicated complete conversion starting material into one main product. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. A yellow viscous liquid was injected into a 330 g silica gel column: a minimal volume of methylene chloride was used to dilute the crude material. Silica gel chromatography (15-75% ethyl acetate in hexanes, 40 min, 330 g column) was used to isolate the title product as a viscous yellow liquid, which became a pale yellowish-white solid (69.6 g, 94%) within several hours at reduced pressure. Optical rotation: (2.02 g/100 ml, methylene chloride) literature value = -72.2, obtained value = -71.

Intermediate 2: (S)-2-(1-(tert-butyldimethylsilyloxy)but-3-en-2-yl)isoindoline1,3-dione

4630^---^4

To a stirred solution of (S)-2-(1-hydroxybut-3-en-2-yl)isoindolin-1,3-dione (Intermediate 1, 69.4 g, 319.49 mmol) and imidazole (26.1 g, 383.39 mmol) in methylene chloride (160 ml) at ambient temperature under nitrogen atmosphere was added tert-butyldimethylchlorosilane (55.4 g, 367.41 mmol) as a solid. This addition was carried out over approximately ten minutes. During this addition, heating of the mixture was observed. After stirring for two hours, the solution was poured into a saturated aqueous sodium bicarbonate solution (approximately 150 ml); this two-phase mixture was shaken and the organic layer was separated. The aqueous layer was stripped three times, each time using 200 ml of methylene chloride. The organic layers were combined, dried over magnesium sulfate, filtered and concentrated in vacuo. The title product was obtained as a pale yellow solid after drying overnight under high vacuum (107 g, 101%).

Intermediate 3: (S)-1-(tert-butyldimethylsilyloxy)but-3-en-2-amine

NH ₂

To a stirred solution of (S)-2-(1-(tert-butyldimethylsilyloxy)but-3-en-2-yl)isoindoline1,3-dione (Intermediate 2, 108.28 g, 326.65 mmol) in methanol ( 1 L) at ambient temperature under nitrogen atmosphere, hydrazine (35.9 mL, 1143.29 mmol) was added. The yellow solution was heated to 65°C. 30 minutes after reaching the reaction temperature, a white precipitate was observed in the reaction mixture; this solid quickly became the bulk of the mixture, and at this time

- 45 044428 water (about 150 ml) was added to the reaction mixture. Stirring of the reaction mixture was continued without interruption, and within a few minutes the solid dissolved. After complete conversion, as determined by LCMS analysis (both starting material and product give intense

UV signals and easily identified by LCMS), heating was stopped and more water was added (total amount of water 600 ml). The mixture was allowed to reach ambient temperature.

Methanol was removed in vacuo at 35°C (moderately low pressure); the vacuum was removed and the aqueous solution was heated to approximately 50°C and then extracted with 4x200 ml methylene chloride. The organic extracts were combined, washed with saturated sodium bicarbonate (aq), washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo at a temperature not exceeding 30°C. The target product was obtained as a yellow liquid (58.5 g, 94%).

Intermediate 4: 2-bromo-N-methoxy-N-methylacetamide

Ome

A stirred solution of potassium carbonate (343 g, 2.48 mol) in water (about 800 ml) was prepared and cooled in an ice bath for 15 min under a nitrogen atmosphere. O,Ndimethylhydroxylamine hydrochloride (110 g, 1.13 mol) and diethyl ether (about 800 ml) were added to it. Bromoacetyl bromide (273 g, 1.35 mol) was then added to this mixture using a dropping funnel over 20 minutes. The ice bath was removed and the mixture was stirred under nitrogen for 2 hours. The layers were separated and the aqueous layer was extracted with ether (about 350 ml). The organic layers were combined, dried over magnesium sulfate, filtered and concentrated in vacuo. The target product was obtained as a yellow liquid (143 g, 70%).

Intermediate 5: (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)but-3-en-2-yl(2(methoxy(methyl)amino)-2-oxoethyl)carbamate k -N boc

ABOUT IN

I

A suspension of (S)-1-(tert-butyldimethylsilyloxy)but-3-en-2-amine (Intermediate 3, 60.4 g, 300 mmol) and cesium carbonate (103 g, 315 mmol) in acetonitrile (about 700 mmol) was prepared. ml) and water (about 120 ml) and it was stirred in an ice bath under nitrogen atmosphere for 5 min. The mixture was biphasic and remained so during the reaction. 2-Bromo-N-methoxy-Nmethylacetamide (Intermediate 4, 57.0 g, 285 mmol) was then added to this mixture using a dropping funnel over 10 minutes. The mixture was stirred for two days, maintaining the temperature around 0°C. The mixture was kept in the freezer overnight. Another 0.05 equiv of electrophile was added. Di-tert-butyl dicarbonate (165 ml, 2 M solution in THF) was added to the mixture. The organic layer was separated from the aqueous layer (TLC indicated that no product remained in the aqueous layer) and the organic layer was concentrated in vacuo. Chromatography on silica gel (5-55% ethyl acetate in hexanes) divided into 3 batches provided the desired product as a pale yellow oil (80 g, 66%).

Intermediate 6: (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)but-3-en-2-yl(2oxopent-3-enyl)carbamate

TBSO^^^ k -N bos

ABOUT

To a solution of (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)but-3-en-2-yl(2-(methoxy-(methyl)amino)-2-oxoethyl)carbamate (Intermediate 5, 32, 5 g, 80.73 mmol) in THF (400 ml) in a nitrogen atmosphere at 0°C was added dropwise prop-1-enylmagnesium bromide (323 ml, 161.45 mmol). The reaction mixture was stirred at 0°C for 1 hour, then the reaction was stopped by adding 400 ml of 10% citric acid, further diluted with 100 ml of water and extracted with ether. The organic substances were concentrated and the resulting oil was dissolved in ether, and washed with water and brine. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (5-20% ethyl acetate/hexanes) provided the desired product as a colorless oil (27 g, 87%).

MS: 384 ES+ (C20H37NO4SO ^1H NMR (300 MHz, CDCl ₃ ) δ: 0.05 (2, 6H); 0.88 (s, 9H); 1.39-1.47 (m, 9H); 1 .90 (m, 3H); 3.80 (m, 2H); 4.05-4.18 (m, 2H); 4.43-4.76 (m, 1H); 5.22 (m, 2H ); 5.86 (m, 1H); 6.21 (m, 1H); 6.91 (m, 1H).

Intermediate 7: (S)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-5-oxo-5,6dihydropyridine-1(2H)-carboxylate

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(S)-tert-Butyl-1-(tert-butyldimethylsilyloxy)but-3-en-2-yl(2-oxopent-3-enyl)carbamate (Intermediate 6, 27.0 g, 70.39 mmol) was dissolved in toluene (650 ml). The solution was purged with nitrogen for 15 min before adding 2nd generation Hoveyda-Grubbs catalyst (0.885 g, 1.41 mmol). The reaction mixture was heated under a nitrogen atmosphere at 65°C. The reaction mixture was concentrated under reduced pressure. Chromatography on silica gel (10-35% ethyl acetate/hexanes) gave the desired product as a solid (17.0 g, 70%). Optical rotation: 0.1 g/dl, methylene chloride = -175

Intermediate 8: (68)-tert-butyl-6-((tert-butyldimethylsilyloxy)methyl)-5-methyl-3(trimethylsilyloxy)-5,6-dihydropyridine-1(2H)-carboxylate tbso^'-A,

VoSA-A^

To a suspension of copper(I) iodide (22.31 g, 117.12 mmol) in diethyl ether (250 ml) at 0°C, methyl lithium (1.6 M in ether) (146 ml, 234.25 mmol) was added via cannula ). The suspension was stirred for 45 min at 0°C. A solution of (B)-tert-butyl-2-(((tertbutyldimethylsilyl)oxy)methyl)-5-oxo-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate 7, 20 g) was added dropwise to the suspension. 58.56 mmol) in diethyl ether (50 ml) at 0°C. After addition was complete, the reaction mixture was stirred for 45 minutes at 0°C. Chlorotrimethylsilane (1 M in THF) (117 mL, 117.12 mmol) was then added dropwise to the reaction mixture, followed by triethylamine (16.28 mL, 117.12 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was then diluted with ethyl acetate and washed three times with ice-cold saturated sodium bicarbonate (added with great care) and then with brine. The organics were dried over sodium sulfate, filtered and concentrated to give a brown oil.

Intermediate 9: (8)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-3-methyl-5oxo-5,6-dihydropyridine-1(2H)-carboxylate

Boc'' ^N —

To a solution of (68)-tert-butyl-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-methyl-3-((trimethylsilyl)oxy)-5,6-dihydropyridine-1(2H)-carboxylate ( Intermediate 8, 24.1 g, 56.08 mmol) in acetonitrile (280 ml) was added palladium(II) acetate (12.59 g, 56.08 mmol) at room temperature. The reaction mixture was stirred at room temperature for about 40 hours, then diluted with ethyl acetate and filtered through celite. The filtrate was concentrated on silica gel. Chromatography on silica gel (0-20% ethyl acetate/hexanes) gave (8)-tert-butyl-2-(((tert-butyldimethylsilyl)oxy)methyl)-3-methyl-5-oxo-5,6-dihydropyridine-1 (2H)-carboxylate (12.95 g, 65%) as a yellow solid.

Intermediate 10: (28.58)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-5hydroxy-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate

7630^-(^% Vos^^^^'^OH

To a suspension of cerium(III) chloride (8.98 g, 36.42 mmol) and (8)-tert-butyl-2-(((tertbutyldimethylsilyl)oxy)methyl)-3-methyl-5-oxo-5,6 -dihydropyridine-1(2H)-carboxylate (Intermediate 9, 12.95 g, 36.42 mmol) in methanol (200 ml) at 0°C, sodium borohydride (1.387 g, 36.42 mmol) was added portionwise. After 15 min, the reaction mixture was diluted with saturated ammonium chloride (100 ml) and water (100 ml), then extracted twice with ether. Organic extracts were washed with brine, dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-20% ethyl acetate/hexanes) gave (2S,5S)-tert-butyl-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-hydroxy-3-methyl-5,6-dihydropyridine -1(2H)-carboxylate (9.79 g, 75%) as a colorless oil.

Intermediate 11: (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)2-((tert-butyldimethylsilyloxy)methyl)-3-methyl-5,6-dihydropyridine-1( 2H)-carboxylate

- 47 044428

To a solution of (2S,5S)-tert-butyl-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-hydroxy-3-methyl-5,6dihydropyridine-1(2H)-carboxylate (Intermediate 10, 9 .79 g, 27.38 mmol) in toluene (100 ml) at room temperature was added triphenylphosphine (8.58 g, 32.86 mmol), N-(αlyloxy)-2nitrobenzenesulfonamide (7.07 g, 27.38 mmol) and diisopropyl azodicarboxylate (6.47 ml, 32.86 mmol). The reaction mixture was stirred overnight, then filtered and concentrated. The resulting oil was purified twice by washing with hexanes and filtered. Chromatography on silica gel (0-20% ethyl acetate/hexanes) gave (2S,5R)-tert-butyl-5-(N-(allyloxy)-2nitrophenylsulfonamido)-2-(((tert-butyldimethylsilyl)oxy)methyl) -3-methyl-5,6-dihydropyridine 1(2H)-carboxylate (10.75 g, 66%) as a light yellow foam.

Intermediate 12: (2S,5R)-tert-butyl-5-(N-(αlyloxy)-2-nitrophenylsulfonamido)2-(hydroxymethyl)-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate ^x A Boc^^N' ⁰ '

Ns

To a solution of (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-2-(((tertbutyldimethylsilyl)oxy)methyl)-3-methyl-5,6-dihydropyridine-1( 2H)-carboxylate (Intermediate 11, 10.75 g, 17.98 mmol) in THF (100 ml) was added tetrabutylammonium fluoride (1 M in THF) (23.38 ml, 23.38 mmol) at 0°C. The reaction mixture turned from yellow to greenish-brown. The reaction mixture was stirred for approximately 2 hours, then concentrated on silica gel. Chromatography on silica gel (0-70% ethyl acetate/hexanes) gave (2S,5R)-tert-butyl-5-(N-(αlyloxy)-2-nitrophenylsulfonamido)-2-(hydroxymethyl)-3-methyl-5,6 -dihydropyridine 1(2H)-carboxylate (7.72 g, 89%) as a brownish-beige foam.

Intermediate 13: (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)-3-methyl-1,2,5,6-tetrahydropyridine-2-carboxylic acid ⁰ I "aA o ζ ^Ν κΑ .-OAN Vos Ν I Ns

Chromium(VI) oxide (0.019 g, 0.19 mmol) was added to a solution of periodic acid (1.588 g, 8.27 mmol) in wet acetonitrile (20 ml) (0.75 vol.% water) at room temperature. The mixture was stirred until completely dissolved. To a solution of (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-2(hydroxymethyl)-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate 12.2 g, 4.14 mmol) in wet acetonitrile (20 ml) (0.75 vol.%) at 0°C, the previously obtained periodic acid/chromium oxide solution (20 ml, 2 eq.) was added dropwise and stirred within 15 min. The reaction mixture was diluted with DCM (100 ml) and washed with 10% citric acid (50 ml) and twice with brine. The organics were dried over sodium sulfate, filtered and concentrated to give a brownish-beige foam (1.9 g, 92%).

Intermediate 14: (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)2-carbamoyl-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate

To a solution of (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)-3methyl-1,2,5,6-tetrahydropyridine-2-carboxylic acid (Intermediate 13 , 1.9 g, 3.82 mmol) in DMF (9.5 ml) at 0°C was added dropwise HATU (2.178 g, 5.73 mmol), ammonium chloride (0.613 g, 11.46 mmol) and DIEA (2.67 ml, 15.28 mmol). The reaction mixture was warmed to room temperature and stirred for 15 minutes. The reaction mixture was diluted with ethyl acetate and washed with saturated sodium bicarbonate and a 1:1 mixture of brine and water. Chromatography on silica gel (0-70% ethyl acetate/hexanes) gave (2S,5R)-tert-butyl-5-(N-(allyloxy)-2nitrophenylsulfonamido)-2-(hydroxymethyl)-3-methyl-5,6 -dihydropyridine-1(2H)-carboxylate (1.270 g, 67%) as a light orange foam.

Intermediate 15: (2S,5R)-tert-butyl-5-((αlyloxy)amino)-2-carbamoyl-3-methyl-5,6dihydropyridine-1(2P)-carboxylate

To a solution of (2S,5R)-tert-butyl-5-(N-(αlyloxy)-2-nitrophenylsulfonamido)-2-carbamoyl-3methyl-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate 14, 3 .63 g, 7.31 mmol) in

- 48 044428 Potassium carbonate (5.05 g, 36.55 mmol) and thiophenol (3.75 mL, 36.55 mmol) were added to acetonitrile (100 ml) at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated and the resulting residue was purified by washing with DCM and filtered to remove solids. The filtrate was concentrated on silica and purified. Chromatography on silica gel (0-90% ethyl acetate/hexanes) gave (2S,5R)-tert-butyl-5-((allyloxy)amino)-2-carbamoyl-3-methyl-5,6-dihydropyridine-1(2H) -carboxylate (1.49 g, 65%) as a yellow oil.

Intermediate 16: (2S,5R)-tert-butyl-5-(N-(allyloxy)-1H-imidazole-1carboxamido)-2-carbamoyl-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate

To a solution of (2S,5R)-tert-butyl-5-((allyloxy)amino)-2-carbamoyl-3-methyl-5,6-dihydropyridine1(2H)-carboxylate (Intermediate 15, 1.49 g, 4 .79 mmol) in THF (30 ml) were added N,N-diisopropylethylamine (2.5 ml, 14.36 mmol) and N,N-carbonyldiimidazole (2.328 g, 14.36 mmol). The reaction mixture was stirred for about 2 hours at room temperature. Another equivalent of CDI was added and the reaction mixture was stirred at room temperature for 1 hour, then another equivalent of CDI was added and the reaction mixture was stirred for another hour. The reaction mixture was diluted with DCM and washed four times with 1:1 brine/water, then dried over magnesium sulfate, filtered and concentrated to give an off-white foam, 1.86 g.

Intermediate 17: (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct3-ene-2-carboxamide

To a solution of (2S,5R)-tert-butyl-5-(N-(allyloxy)-1H-imidazole-1-carboxamido)-2-carbamoyl-3methyl-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate compound 16, 1.86 g, 4.59 mmol) trifluoroacetic acid (3.53 mL, 45.88 mmol) was added to DCM (20 ml) at 0°C. The reaction mixture was allowed to warm slowly to room temperature and stirred overnight. The reaction mixture was concentrated. The oil was redissolved in DCM and washed with saturated sodium bicarbonate solution. The aqueous layer was extracted once with about 10% MeOH/DCM. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-30% acetone/dichloromethane) gave (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2 -carboxamide (0.83 g, 76%) as a light yellow oil.

Example 1. (R)-Ethyl-2-((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yloxy)-2 -fluoroacetate

Example 2. (S)-Ethyl-2-((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yloxy)-2 -fluoroacetate

Example 3. (2S)-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}( fluoroethanoic acid lithium salt

Example 4. (2R)-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}( fluoroethanoic acid lithium salt

- 49 044428

Example 5. {[(2S,5R)-2-Carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}(fluoro)acetic acid lithium salt (mixture of diastereomers)

Examples 1-2.

To a solution of (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 17, 100 mg, 0 .42 mmol) in methanol (3 ml) at room temperature, 1,3-dimethylbarbituric acid (132 mg, 0.84 mmol) and tetrakis(triphenylphosphine)palladium(0) (48.7 mg, 0.04 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated to give an orange film. The orange film was dissolved in DMF (3 ml) and potassium carbonate (175 mg, 1.26 mmol) and ethyl bromofluoroacetate (0.299 ml, 2.53 mmol) were added. The reaction mixture was stirred overnight at room temperature before being diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed twice with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-70% ethyl acetate/hexanes) gave ethyl 2-(((2S,5R)-2-carbamoyl-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl)oxy)-2-fluoroacetate (97 mg, 76%) as a mixture of the diastereomers of Example 1 and Example 2, as an orange foam. MS: 198 ES+ (C8H11N3O3)

Examples 3-5.

To a solution of ethyl 2-(((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetate (Examples 1-2, 97 mg, 0.32 mmol) in THF (3 ml) and water (1 ml) at -5°C was added lithium hydroxide (9.25 mg, 0.39 mmol) in the form of an aqueous solution ( 0.5 ml). The mixture was stirred at 0°C for 60 min, allowed to warm to room temperature and stirred for 15 min. The pH of the reaction mixture was adjusted to pH=7 using 0.5 M HCl. The THF was evaporated, after which the remaining aqueous phase was frozen and lyophilized.

Cleaning up examples 1-5.

The mixture resulting from the reaction described in examples 3-5 was purified using reverse phase HPLC (Synergi Polar RP 21.2 mm x 100 mm, 4 µm, coupled to YMC C30 20 mm x 150 mm, 5 µm; 0 -50% acetonitrile in water, 10 min; 20 ml/min) to obtain:

Example 1: ethyl {[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy}(fluoro)acetate ( first ester eluted): 4.4 mg, 4.5% MS: 302 ES+ (C ₁₂ H ₁₆ FN ₃ O ₅ ) ¹ H NMR (300 MHz, DMSO-d6) δ: 1.21 (t, 3H) ; 1.62(s, 3H); 3.05 (m, 1H); 3.75 (m, 1H); 4.03 (m, 1H); 4.20 (m, 3H); 6.01 (m, 1H); 6.13-6.31(d, 1H); 7.36 (bs, 1H); 7.81 (bs, 1H).

Example 2: (S)-ethyl-2-((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yloxy)-2 -fluoroacetate (second ester eluted): 4.2 mg, 4.3% MS: 302 ES+ (C ₁₂ H ₁₆ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 1.26 (t, 3H); 1.63(s, 3H); 3.08 (m, 1H); 3.75 (m, 1H); 3.94 (m, 1H); 4.20 (m, 1H); 4.27(q,2H); 6.03 (m, 1H); 6.24-6.50 (d, 1H); 7.37 (bs, 1H); 7.83 (bs, 1H).

Example 3: (2S)-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy}(fluoro) ethanoic acid (first eluting acid): 7.7 mg, 8.8%. MS: 274 ES+ (C ₁₀ H ₁₂ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 1.61 (s, 3H); 3.05 (m, 1H); 3.68(m, 1H); 3.96(m, 1H); 4.13(m, 1H); 5.12-5.33 (d, 1H); 6.03 (m, 1H); 7.31 (bs, 1H); 7.80 (bs, 1H).

Example 4: (2R)-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy}(fluoro) ethanoic acid (second eluting acid): 9.9 mg, 11% MS: 274 ES+ (C ₁₀ H ₁₂ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 1.61 (s, 3H); 3.05 (m, 1H); 3.70 (m, 1H); 4.00 (m, 1H); 4.13(m, 1H); 5.15-5.37 (d, 1H); 6.01 (m, 1H); 7.31 (bs, 1H); 7.78 (bs, 1H).

Example 5: {[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}(fluoro)acetic acid (mixture of diastereomers): 20.4 mg, 23% MS: 274 ES+ (C ₁₀ H ₁₂ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 1.62 (s, 6H); 3.06 (m, 2H); 3.70 (m, 2H); 4.01 (m, 2H); 4.14 (m, 2H); 5.14-5.35 (d, 1H); 5.18-5.40 (d, 1H); 6.03 (m, 2H); 7.32 (bs, 2H); 7.80 (bs, 2H).

Absolute stereochemistry for all compounds was determined by characterizing the cocrystal structure of Example 4 in complex with AmpC. The second diastereomer, example 3, was assigned the opposite absolute stereochemistry at the fluoroacetate carbon atom. The stereochemistry of each of the esters was assigned by hydrolyzing each of the esters to the corresponding acid and comparing UHPLC retention times with those of Examples 3 and 4.

Example 6. Ethyl-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}(fluorine) acetate (mixture of diastereomers)

According to the procedure for Examples 1 and 2, (2S,5R)-6-(αlyloxy)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (intermediate 17, 0.506 g, 2.13 mmol) was converted to 70 mg of Example 6 after HPLC (Synergi Polar RP 21.2 mm x 100 mm, 4 µm, coupled to YMC C30 20 mm x 150 mm, 5 µm; 20-60% acetonitrile in water, 10 min; 20 ml/min) and lyophilization. MS: 302 ES+ (C ₁₂ H ₁₆ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 1.22 (t, 3H); 1.27 (t, 3H); 1.63(s, 6H); 3.07 (m, 2H); 3.76 (m, 2H); 3.94 (m, 1H); 4.05 (m, 1H); 4.20 (m, 2H); 4.26 (m, 4H); 6.03 (m, 2H); 6.06-6.24 (d, 1H); 6.14-6.32 (d, 1H); 7.37 (bs, 1H); 7.83 (bs, 1H).

Example 7 Ethyl-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy} (difluoro) acetate

(2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (intermediate 17, 204.2 mg, 0 .86 mmol) was converted according to the procedure for Examples 1 and 2 using ethyl bromodifluoroacetate (0.441 mL, 3.44 mmol) to give 130 mg (47%) of the title compound as an orange foam. As a result of reverse phase chromatography on 40 mg (Synergi Polar RP 21.2 mm x 100 mm, 4 µm, coupled with YMC C30 20 mm x 150 mm, 5 µm; 20-40% acetonitrile in water, 10 min; 20 ml /min) gave the title compound as a white solid (21 mg). MS: 320 ES+ (C ₁₂ H ₁₅ F ₂ N ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 1.30 (t, 3H); 1.65(s, 3H); 3.19 (m, 1H); 3.84 (m, 1H); 4.06 (m, 1H); 4.26 (m, 1H); 4.39(q, 2H); 6.05 (m, 1H); 7.42 (bs, 1H); 7.87 (bs, 1H).

Example 8 {[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}(difluoro)acetic acid lithium salt

Ethyl 2-(((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)-2,2- difluoroacetate (example 7, 113.7 mg, 0.36 mmol) was converted according to the procedure for examples 3-5. After HPLC (Synergi Polar RP 21.2 mm x 100 mm, 4 µm, coupled with YMC C30 20 mm x 150 mm, 5 µm; 100% water, 10 min; 20 ml/min) and lyophilization, a white solid was obtained, 36.7 mg. MS: 292 ES+ (C10H11F2N3O5)

1H NMR (300 MHz, DMSO-di) δ: 1.63 (s, 3H); 3.13(m, 1H); 3.75 (m, 1H); 3.95 (m, 1H); 4.18(m, 1H); 6.04 (m, 1H); 7.34 (bs, 1H); 7.83 (bs, 1H).

Example 9 Ethyl {[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}acetate

(2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (intermediate 17, 0.198 g, 0.83 mmol) was converted according to the procedure for Examples 1-2 using ethyl bromoacetate (0.592 ml) to give 147 mg (62%) as an orange solid. As a result of reverse phase chromatography on 41.5 mg (Synergi Polar RP 21.2 mm x 100 mm, 4 µm, coupled with YMC C30 20 mm x 150 mm, 5 µm; 10%-50% acetonitrile in water, 10 min ; 20 ml/min) gave the title compound as a white solid (32 mg). MS: 284 ES+ (C12H17N3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.22 (t, 3H); 1.61(s, 3H); 3.00 (m, 1H); 3.68(m, 1H); 4.05 (m, 1H); 4.13(m, 1H); 4.16(q,2H); 4.37-4.65 (m, 2H); 6.05 (m, 1H); 7.33 (bs, 1H); 7.77 (bs, 1H).

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Example 10. {[(2S,5R)-2-Carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}acetic acid lithium salt

Ethyl 2-(((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)acetate (example 9, 105.5 mg, 0.37 mmol) was hydrolyzed according to the procedure for Examples 3-5 to obtain 34 mg (37%) of the title compound after HPLC (Synergi Polar RP 21.2 mm x 100 mm, 4 µm; 0-20% acetonitrile in water, 10 min; 20 ml/min) and lyophilization. MS: 256 ES+ (C10H13N3O5) ¹ H NMR (300 MHz, DMSOD6) δ: 1.60 (s, 3H); 2.96 (m, 1H); 3.58 (m, 1H); 3.87 (m, 2H); 4.05 (m, 1H); 4.27 (m, 1H); 6.08 (m, 1H); 7.27 (bs, 1H); 7.74 (bs, 1H).

Example 11 2-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy}-2fluoropropanoic acid lithium salt (mixture of diastereomers)

(2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (intermediate 17, 0.101 g, 0.43 mmol) was converted according to the procedure for Examples 1-2 using methyl 2-bromo-2-fluoropropanoate (0.315 g, 1.70 mmol) to give methyl 2-(((2S,5R)-2carbamoyl-3 -methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)-2-fluoropropanoate (0.105 g, 82%) as an orange foam. As a result of reverse phase chromatography (Synergi Polar RP 21.2 mm x 100 mm, 4 µm, coupled with YMC C30 20 mm x 150 mm, 5 µm; 100% water, 10 min; 20 ml/min) the title was obtained compound as a white solid (19.1 mg, 19%). MS: 302 ES+ (C12H16FN3O5) ¹ H NMR (300 MHz, DMSO^) δ: 1.67 (m, 6H); 3.04 (m, 1H); 3.72 (m, 4H); 3.80 (m, 1H); 4.17 (m, 1H); 6.02 (m, 1H); 7.36 (bs, 1H); 7.81 (bs, 1H).

Hydrolysis according to the procedure for Examples 3-5 yielded 19.1 mg of Example 11 as a white solid after purification by reverse phase chromatography (Synergi Polar RP 21.2 mm x 100 mm, 4 µm, coupled with YMC C30 20 mm x 150 mm, 5 µm; 100% water, 10 min; 20 ml/min). MS: 288 ES+ (C11H14FN3O5) ¹ H NMR (300 MHz, DMSO^) δ: 1.45 (m, 3H); 1.61(s, 3H); 3.02 (m, 1H); 3.65 (m, 1H); 3.92-4.09 (m, 1H); 4.11(m, 1H); 6.04 (m, 1H); 7.32 (m, 1H); 7.80 (m, 1H).

Intermediate 18: Isopropyl-2-bromo-2-fluoroacetate

F

iPrOH, tBuOK, hexane, 0 C

Vg

ABOUT

Vg

ABOUT

F

Potassium tert-butoxide (0.091 g, 0.81 mmol) was added to a solution of ethyl 2-bromo-2-fluoroacetate (0.639 ml, 5.41 mmol) in hexanes (70 ml) and isopropanol (7 ml) at 0°C. in two equal parts, with an interval of 5 minutes. The reaction mixture was stirred for 1 hour at 0°C. The reaction was stopped by adding concentrated HCl (7 ml) and the layers were separated. The organics were washed twice with water, dried over magnesium sulfate, filtered and concentrated at 0°C to give a colorless oil (0.88 g, 4.42 mmol, 82%). The product containing trace amounts of hexanes was identified by NMR. The product was used in the next step in its original form.

^1H NMR (300 MHz, CDCl3-d) δ: 1.34 (m, 6H); 5.18 (m, 1H); 6.45-6.62 (d, 1H). Link: Tet. Lett. (2000) 791.

Example 12. Propan-2-yl-(2R)-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl ]oxy} (fluoro)ethanoate

Example 13. Propan-2-yl-(2S)-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl ]oxy} (fluoro)ethanoate

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Examples 12-13.

(2S,5R)-6-(Allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 17, 0.103 g, 0.43 mmol) was converted according to the procedure for Examples 1-2 using isopropyl-2-bromo-2-fluoroacetate (Intermediate 18, 0.518 g, 2.60 mmol). As a result of HPLC and lyophilization, 34 of each diastereomer were obtained (Synergi Polar RP

21.2 mm x 100 mm, 4 µm, paired with YMC C30 20 mm x 150 mm, 5 µm; 25%-50% acetonitrile in water, 10 min; 20 ml/min). Ester stereochemistry was assigned by hydrolyzing each ester to the corresponding acid and comparing UHPLC retention times to those of Examples 3 and 4.

Example 12: (first ester eluted) 33.4 mg, 24%

MS: 316 ES+ (C13H18FN3O5)

1H NMR (300 MHz, DMSO-d6) δ: 1.22 (m, 6H); 1.63(s, 3H); 3.03 (m, 1H); 3.75 (m, 1H); 4.01 (m, 1H); 4.19 (m, 1H); 5.00 (m, 1H); 6.01 (m, 1H); 6.11-6.29 (d, 1H); 7.37 (bs, 1H); 7.81 (bs, 1H).

Example 13: (second ester eluted) 33.6 mg, 25%

MS: 316 ES+ (C13H18FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.27 (m, 6H); 1.63(s, 3H); 3.07 (m, 1H); 3.75 (m, 1H); 3.94 (m, 1H); 4.20 (m, 1H); 5.05 (m, 1H); 6.03 (m, 1H); 6.02-6.21(d, 1H); 7.37 (bs, 1H); 7.82 (bs, 1H).

Intermediate 19: 2,4-dimethylpentan-3-yl-2-bromo-2-fluoroacetate FI ⁰

By the method given for intermediate 18, using ethyl 2-bromo-2-fluoroacetate (0.639 ml, 5.41 mmol) and 2,4-dimethyl-3-pentanol (7.05 ml, 50.27 mmol), the title compound as a colorless oil (0.988 g, 3.87 mmol, 71.6%). 1H NMR (300 MHz, CDCl ₃ -d) δ: 0.94 (m, 12H); 2.01 (m, 2H); 4.73 (m, 1H); 6.52-6.69 (d, 1H).

Example 14 2,4-Dimethylpentan-3-yl-(2S)-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl]oxy}(fluoro)ethanoate ⁰ I n/a

F ⁰ Ay

Example 15 -en-6-yl]oxy} (fluoro)ethanoate

Examples 14-15.

(2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 17, 0.097 g, 0.41 mmol) was converted according to the procedure for Examples 1-2 using 2,4-dimethylpentan-3-yl-2-bromo-2-fluoroacetate (0.626 g, 2.45 mmol) to give 35 mg of each diastereomer (23 % for each, 46% overall) after HPLC (Synergi Polar RP 21.2 mm x 100 mm, 4 µm, coupled to YMC C30 20 mm x 150 mm, 5 µm; 40-70% acetonitrile in water, 10 min ; 20 ml/min) and lyophilization. Ester stereochemistry was assigned by hydrolyzing each ester to the corresponding acid and comparing UHPLC retention times to those of Examples 3 and 4.

Example 14: (second eluted peak) 34.5 mg, 23%

MS: 372 ES+ (C17H26FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.86 (m, 12H); 1.63(s, 3H); 1.94 (m, 2H); 3.07 (m, 1H); 3.78 (m, 1H); 3.98 (m, 1H); 4.23 (m, 1H); 4.62 (m, 1H); 6.04 (m, 1H); 6.15-6.34 (d, 1H); 7.38 (bs, 1H); 7.81 (bs, 1H).

Example 15: (first eluted peak) 35.2 mg, 23%

MS: 372 ES+ (C17H26FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.84 (m, 12H); 1.63(s, 3H); 1.93 (m, 2H); 3.03 (m, 1H); 3.78 (m, 1H);

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4.07 (m, 1H); 4.21(m, 1H); 4.60 (m, 1H); 5.99 (m, 1H); 6.27-6.45 (d, 1H); 7.37 (bs, 1H); 7.83 (bs, 1H).

Intermediate 20: Tetrahydro-2H-pyran-4-yl-2-bromo-2-fluoroacetate

The method given for Intermediate 18 using ethyl 2-bromo-2-fluoroacetate (0.319 ml, 2.70 mmol) and tetrahydro-2H-pyran-4-ol (2.333 ml, 24.33 mmol) gave the following: title compound as a colorless oil (0.15 g, 0.62 mmol, 29%).

MS: ES+ 241.2 for SuNuVgBOz

1H NMR (300 MHz, chloroform-d) δ ppm: 1.47-1.81 (m, 2H) 1.84-2.13 (m, 2H) 3.39-3.68 (m, 2H) 3.754 .07 (m, 2H) 4.37 (q, J=7.18 Hz, 1H) 6.37-6.74 (m, 1H)

Example 16. Tetrahydro-2H-pyran-4-yl-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-6 -yl]oxy}(fluoro)acetate (mixture of diastereomers)

By the method given for Examples 1-2 using (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 17, 40 mg, 0.17 mmol) and tetrahydro-2H-pyran-4-yl-2-bromo-2-fluoroacetate (Intermediate 20, 312 mg, 1.29 mmol), giving the title compound after purification in reverse -phase ISCO (15.5 g C18 Gold, 0-80% acetonitrile/water), which was a light orange solid, in the form of a mixture of diastereomers. (5.5 mg, 9%) MS: ES+ 358.1 for C ₁₅ H ₂₀ FN ₃ O ₆

1H NMR (300 MHz, chloroform-d) δ ppm 1.15-1.42 (m, 2H) 1.60 (br. s., 2H) 1.92 (s, 3H) 3.14-3.45 (m, 2H) 3.56 (d, J=11.52 Hz, 1H) 3.88-4.13 (m, 2H) 4.20-4.50 (m, 2H) 5.09 (br. s., 1H) 5.35-5.58 (m, 1H) 5.63-5.96 (m, 1H) 6.09 (br. s., 1H)

Intermediate 21: 2-methoxyethyl-2-bromo-2-fluoroacetate

Following the method given for Intermediate 18 using ethyl 2bromo-2-fluoroacetate (1.278 mL, 10.81 mmol) and 2-methoxyethanol (13.94 mL, 183.79 mmol) afforded the title compound in as a colorless oil (1.04 g, 4.84 mmol, 44.7%). The NMR spectrum indicated a 3:1 mixture of product and starting material, which was used as is in the next step.

1H NMR (300 MHz, DMSO-d ₆ ) δ: 3.27 (s, 3H); 3.57 (m, 2H); 4.35 (m, 2H); 7.22-7.38 (d, 1H).

Example 17 2-Methoxyethyl-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl]oxy}(fluoro) acetate (mixture of diastereomers)

Example 18 2-Methoxyethyl-(2R)-{[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct3-en-6-yl]oxy } (fluoro)ethanoate

Example 19 } (fluoro)ethanoate

Example 17-19.

According to the procedure for Examples 1-2, (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 17, 0.101 g, 0.43 mmol) was converted to the title compound using 2-methoxyethyl-2-bromo-2-fluoroacetate (Intermediate 21, 0.549 g, 2.55 mmol). As a result of HPLC (Synergi Polar RP 21.2 mm x

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100 mm, 4 µm, paired with YMC C30 20 mm x 150 mm, 5 µm; 30-50% acetonitrile in water, 10 min; 20 ml/min) and lyophilization yielded the following products (where the stereochemistry of the esters was assigned by hydrolyzing each ester to the corresponding acid and comparing UHPLC retention times with those of Examples 3 and 4):

Example 17: diastereomer mixture: 14.3 mg, 10.1% MS: 332 ES+ (C ₁₃ H ₁₈ FN ₃ O ₅ ) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 1.63 (s, 6H ); 3.07 (m, 2H); 3.26(s, 3H); 3.27(s, 3H); 3.54 (m, 2H); 3.59 (m, 2H); 3.73 (m, 1H); 3.77 (m, 1H); 3.98 (m, 1H); 4.03 (m, 1H); 4.19 (m, 1H); 4.32 (m, 4H); 6.02 (m, 2H); 6.11-6.30 (d, 1H); 6.17-6.35 (d, 1H); 7.37 (bs, 2H); 7.81 (bs, 2H).

Example 18: (first ester eluted): 22 mg, 15.6%,

MS: 332 ES+ (C13H18FN3O5)

1H NMR (300 MHz, DMSO- _db ) δ: 1.63 (s, 3H); 3.06 (m, 1H); 3.27(s, 3H); 3.55 (m, 2H); 3.77 (m, 1H); 4.05 (m, 1H); 4.20 (m, 1H); 4.30 (m, 2H); 6.02 (m, 1H); 6.18-6.36(d, 1H); 7.37 (bs, 1H); 7.82 (bs, 1H).

Example 19: (second ester eluted): 22.8 mg, 16.1%

MS: 332 ES+ (C13H18FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.62 (s, 3H); 3.07 (m, 1H); 3.27(s, 3H); 3.59 (m, 2H); 3.75 (m, 1H);

3.99 (m, 1H); 4.20 (m, 1H); 4.34 (m, 2H); 6.03 (m, 1H); 6.11-6.30 (d, 1H); 7.37 (bs, 1H); 7.83 (bs, 1H).

Intermediate 22: (8)-sec-butyl-2-bromo-2-fluoroacetate

To a solution of ethyl 2-bromo-2-fluoroacetate (0.319 ml, 2.70 mmol) and (S)-butan-2-ol (3.72 ml, 40.54 mmol) in hexane (35 ml) at 0° C, potassium tert-butoxide (0.061 g, 0.54 mmol) was added in two equal parts at 5 min intervals. The reaction mixture was stirred for 2 hours at 0°C, then overnight at room temperature. The reaction was stopped by adding a saturated ammonium chloride solution and the layers were separated. The organics were washed with water three times, dried over magnesium sulfate, filtered and concentrated at 0°C to give a colorless oil, 0.550 g, 96%.

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.88 (m, 3H); 1.25 (m, 3H); 1.61 (m, 2H); 4.91(m, 1H); 7.16-7.33 (m, 1H).

Intermediate 23: (R)-sec-butyl-2-bromo-2-fluoroacetate

To a solution of ethyl 2-bromo-2-fluoroacetate (0.319 ml, 2.70 mmol) and (R)-butan-2-ol (3.72 ml, 40.54 mmol) in hexane (35 ml) at 0° C, potassium tert-butoxide (0.061 g, 0.54 mmol) was added in two equal parts at 5 min intervals. The reaction mixture was stirred for 2 hours at 0°C, then at room temperature for 2 hours. Then (R)-butan-2-ol (3.72 ml, 40.54 mmol) was added again, followed by 0. 1 eq. potassium tert-butoxide. The reaction mixture was stirred at room temperature overnight. Every 2 hours over 6 hours, 0.1 equiv was added. potassium tert-butoxide. The reaction was stopped by adding a saturated ammonium chloride solution and the layers were separated. The organics were washed with water four times, dried over magnesium sulfate, filtered and concentrated at 0°C to give a colorless oil, 422 mg, 73%.

1H NMR (300 MHz, DMSO-de) δ: 0.88 (m, 3H); 1.25 (m, 3H); 1.61 (m, 2H); 4.91(m, 1H); 7.16-7.33 (m, 1H).

Example 20. (2R)-(S)-sec-butyl-2-(((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl)oxy)-2-fluoroacetate

Example 21 -en-6-yl)oxy)-2-fluoroacetate

Example 20-21.

Received in accordance with the procedure for examples 1-2. To a solution of (2S,5R)-6-(allyloxy)-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 17, 0.196 g, 0.83 mmol) in methanol (5 ml) at room temperature, 1,3-dimethylbarbituric acid (0.258 g, 1.65 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.095 g, 0.08 mmol) were added. Reaction mixture

- 55 044428 was stirred at room temperature for 2 hours, then concentrated to obtain an orange film. The orange film was dissolved in DMF (5 ml) and potassium carbonate (0.343 g, 2.48 mmol) and (S)-sec-butyl-2-bromo-2-fluoroacetate (Intermediate 22, 0.528 g, 2.48 mmol) were added ). The reaction mixture was stirred for about 5 hours at room temperature, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed three times with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-65% ethyl acetate/hexanes) gave (S)-sec-butyl-2-(((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo [3.2.1]oct-3-en-6-yl)oxy)-2-fluoroacetate (0.218 g, 80%) as a light yellow foam, a mixture of diastereomers with a ratio of about 1:1. Separation of diastereomers was carried out using reverse phase HPLC (Atlantis T3 19 mm x 150 mm, 30-50% acetonitrile in water, 20 ml/min, 15 min). Stereochemistry of the esters was assigned by hydrolyzing each ester to the corresponding acid and comparing UHPLC retention times with those of Examples 3 and 4.

Example 20: (first eluted peak) 84 mg, 31%

MS: 330 ES+ (C14H20FN3O5) ¹ H NMR (300 MHz, DMSO-di) δ: 0.84 (t, 3H); 1.22(d,3H); 1.55 (m, 2H); 1.63(s, 3H); 3.03 (m, 1H); 3.76(d, 1H); 4.04 (m, 1H); 4.19(s, 1H); 4.86(m, 1H); 6.01 (m, 1H); 6.13-6.31 (m, 1H); 7.37 (bs, 1H); 7.82 (bs, 1H).

Example 21: (second eluted peak) 85 mg, 31%.

MS: 330 ES+ (C14H20FN3O5) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 0.84 (t, 3H); 1.22(d,3H); 1.55 (m, 2H); 1.63(s, 3H); 3.03 (m, 1H); 3.76(d, 1H); 4.04 (m, 1H); 4.19(s, 1H); 4.86(m, 1H); 6.01 (m, 1H); 6.13-6.31 (m, 1H); 7.37 (bs, 1H); 7.82 (bs, 1H).

Example 22. (2R)-(R)-sec-Butyl-2-(((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl)oxy)-2-fluoroacetate

Example 23. (2S)-(R)-sec-Butyl-2-(((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl)oxy)-2-fluoroacetate

Example 22-23.

Examples 22-23 were prepared in accordance with the procedure for examples 1-2. To a solution of (2S,5R)-6(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 17, 0.202 g, 0. 85 mmol) in methanol (5 ml) at room temperature, 1,3dimethylbarbituric acid (0.266 g, 1.70 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.098 g, 0.09 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated to give an orange film. The orange film was dissolved in DMF (5 ml) and potassium carbonate (0.353 g, 2.55 mmol) and (R)-sec-butyl-2-bromo-2-fluoroacetate (Intermediate 23, 0.421 g, 1.98 mmol) were added ). The reaction mixture was stirred for 3 hours at room temperature, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed three times with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-65% ethyl acetate/hexanes) gave (R)-sec-butyl-2-(((2S,5R)-2carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2 .1]oct-3-en-6-yl)oxy)-2-fluoroacetate (0.220 g, 78%) in the form of an orange foam, a mixture of diastereomers in a 1:1 ratio. Separation of diastereomers was carried out using reverse phase HPLC (Atlantis T3 19 mm x 150 mm, 30-50% acetonitrile in water, 20 ml/min, 15 min). Stereochemistry of the esters was assigned by hydrolyzing each ester to the corresponding acid and comparing UHPLC retention times with those of Examples 3 and 4.

Example 22: (first eluted peak) 90 mg, 32%

MS: 330 ES+ (C14H20FN3O5) ¹ H NMR (300 MHz, DMSO-di) δ: 0.87 (t, 3H); 1.17(d,3H); 1.59 (m, 2H); 1.63(s, 3H); 3.03 (m, 1H); 3.76(d, 1H); 4.02 (m, 1H); 4.19(s, 1H); 4.86(m, 1H); 6.02 (m, 1H); 6.16-6.34 (m, 1H); 7.37 (bs, 1H); 7.82 (bs, 1H).

Example 23: (second eluted peak) 87 mg, 31%

MS: 330 ES+ (C14H20FN3O5) ¹ H NMR (300 MHz, DMSO-di) δ: 0.88 (t, 3H); 1.24(d,3H); 1.61 (m, 2H); 1.63(s, 3H); 3.08 (m, 1H); 3.76

- 56 044428 (d, 1H); 3.94 (m, 1H); 4.21(s, 1H); 4.89 (m, 1H); 6.04 (m, 1H); 6.03-6.22 (m, 1H); 7.38 (bs, 1H); 7.82 (bs, 1H).

Intermediate 24: pentan-3-yl-2-bromo-2-fluoroacetate

F

Г в _г ^у ₀

BrA. ^but 1 —o o \

Ethyl 2-bromo-2-fluoroacetate (0.639 ml, 5.41 mmol) was added to a mixture of dry pentan-3-ol (4.68 ml, 43.25 mmol) and hexane (20 ml). The resulting mixture was cooled to 0°C. KOtBu (0.091 g, 0.81 mmol) was added and the mixture was left to stir for 16 h at 25°C. Then the reaction was stopped by adding 1N. HCl (30 ml), washed with water (50 ml) and brine (50 ml), dried with Na ₂ SO ₄ and concentrated. The crude material was purified by flash chromatography (20 g silica gel, 0100% Et ₂ O in hexanes, 25 min) to give pentan-3-yl-2-bromo-2-fluoroacetate (0.754 g, 61.4%) as colorless oil.

1H NMR (300 MHz, chloroform-d) δ ppm 0.96 (td, J=7.46, 2.08 Hz, 6H) 1.60-1.76 (m, 4H) 4.93 (dt, J =12.28, 6.33 Hz, 1H) 6.50 (s, 0.5H) 6.67 (s, 0.5H)

Example 24 yloxy)-2-fluoroacetate

J-A ?L F

A--- ^N s?

° O%O

Example 25 yloxy)-2-fluoroacetate

Example 24-25.

Examples 24-25 were prepared in accordance with the procedure for examples 1-2. To a solution of (2S,5R)-6(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 17, 272 mg, 1. 15 mmol) in methanol (5 ml) at room temperature, 1,3dimethylbarbituric acid (358 mg, 2.29 mmol) and Pd(Ph ₃ P) ₄ (132 mg, 0.11 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. The mass of the target product was monitored using UHPLC; no starting material was detected. The reaction mixture was concentrated to give an orange film. The orange film was dissolved in DMF (5 ml) and K ₂ CO ₃ (475 mg, 3.44 mmol) and pentan-3-yl-2-bromo-2-fluoroacetate (Intermediate 24, 751 mg, 3.31 mmol) were added ). The reaction mixture was stirred overnight at room temperature, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed twice with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-70% ethyl acetate/hexanes) gave pentαn-3-yl-2-(((2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2 .1]oct3-en-6-yl)oxy)-2-fluoroacetate (320 mg, 81%) as a light yellow foam. UHPLC and NMR confirmed that it was a 1:1 mixture of diastereomers. Diastereomers were separated using reverse phase HPLC (Atlantis T3 4.6 mm x 50 mm 5 μm column, 30-50% ACN in water for 5 min, flow rate 1 ml/min).

Example 24: 125 mg, 32%

UHPLC LCMS retention time, method 2min_Acid_CV10, acidic conditions: 0.86 min, 344 (M+H)+ ^1H NMR (300 MHz, chloroform-d) δ ppm 0.92 (td, J=7.46, 3, 21 Hz, 6H) 1.61-1.75 (m, 4H) 1.93 (s, 3H) 3.21-3.35 (m, 2H) 4.07 (dd, J=4.91, 2 ,64 Hz, 1H) 4.36 (s, 1H) 4.91 (quin, J=6.18 Hz, 1H) 5.47 (br. s., 1H) 5.77 (s, 0.5H) 5.94 (s, 0.5H) 6.07-6.12 (m, 1H) 6.59 (br. s., 1H)

Example 25: 125 mg, 32%

UHPLC LCMS retention time, method 2min_Acid_CV10, acidic conditions: 0.91 min, 344 (M+H)+

1H NMR (300 MHz, chloroform-d) δ ppm 0.93 (t, J=7.46 Hz, 3H) 0.95 (t, J=7.46 Hz, 3H) 1.63-1.74 ( m, 4H) 1.93 (s, 3H) 3.20-3.38 (m, 2H) 4.02 (dd, J=5.00, 2.55 Hz, 1H) 4.35 (s, 1H ) 4.92 (quin, J=6.18 Hz, 1H) 5.56 (br. s., 1H) 5.69 (s, 0.5H) 5.89 (s, 0.5H) 6.07 -6.14 (m, 1H) 6.64 (br. s., 1H)

Intermediate 25: N-[(3R,6S)-6-[[(tert-butyldimethylsilyl)oxy]methyl]-5-methyl1,2,3,6-tetrahydropyridin-3-yl]-2-nitro-N- (prop-2-en-1-yloxy)benzene-1-sulfonamide

- 57 044428

A solution of tert-butyl-(3R,6S)-6-[[(tert-butyldimethylsilyl)oxy]methyl]-5-methyl-3-[N( prop-2-en-1-yloxy)(2-nitrobenzene)sulfonamido]-1,2,3,6-tetrahydropyridine-1-carboxylate (Intermediate 11, 13.6 g, 22.75 mmol, 1 eq.) in dichloromethane (100 ml). ZnBr ₂ (10.2 g, 45.29 mmol, 2 eq.) was then added in several batches. The resulting solution was stirred overnight at room temperature. The resulting solution was diluted with 500 ml of dichloromethane. The resulting mixture was washed with 2x200 ml sodium bicarbonate (aq) and 2x200 ml NH4Cl (aq). The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. This resulted in 12 g (crude product) of the title compound as a yellow oil. MS: 498 ES+ (C ₂₂ H ₃₅ N ₃ O ₆ SSi)

Intermediate 26: (3R,6S)-6-[[(tert-butyldimethylsilyl)oxy]methyl]-5-methyl-N(prop-2-en-1-yloxy)-1,2,3,6-tetrahydropyridine -3-amine

A solution of N-[(3R,6S)-6-[[(tert-butyldimethylsilyl)oxy]methyl]-5-methyl-1,2,3, 6tetrahydropyridin-3-yl]-2-nitro-N-(prop-2-en-1-yloxy)benzene-1-sulfonamide (Intermediate 25, 12 g, 24.11 mmol, 1 eq.) in N,N -dimethylformamide (100 ml), 2-sulfanylacetic acid (4.4 g, 47.77 mmol, 2 eq.). LiOH (5.8 g, 242.17 mmol, 10 eq.) was then added in portions. The resulting solution was stirred for 2 hours at room temperature, then diluted with 500 ml water and extracted with 5x200 ml ethyl acetate, and the organic layers were combined. The organic mixture was washed with 3x200 ml brine and 2x200 ml sodium bicarbonate (aq). The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. This resulted in 8.4 g (crude product) of the title compound as a yellow oil. MS 313 ES+ (C ₁₆ H ₃₂ N ₂ O ₂ Si)

Intermediate 27: (2S,5R)-2-[[(tert-butyldimethylsilyl)oxy]methyl]-3-methyl-6-(prop2-en-1-yloxy)-1,6-diazabicyclo[3.2.1] okt-3-en-7-on

A solution of (3R,6S)-6-[[(tert-butyldimethylsilyl)oxy]methyl]-5-methyl-N-(pron-2en-1) was placed in a 2-liter three-neck round-bottom flask, purged and maintained in an inert atmosphere of nitrogen. -yloxy)-1,2,3,6-tetrahydropyridin-3-amine (Intermediate 26, 8.4 g, 26.88 mmol, 1 eq.) in acetonitrile (1.6 L) and N,N-diisopropylethylamine (14.2 g, 109.87 mmol, 4 eq.). A solution of ditrichloromethyl carbonate (2.9 g, 9.77 mmol, 0.4 eq) in acetonitrile (100 ml) was then added dropwise with stirring at -15°C over 3 hours. The resulting solution was stirred overnight at room temperature . The resulting mixture was concentrated in vacuo. The crude product was diluted with 500 ml of ethyl acetate. The resulting mixture was washed with 2x400 ml NH4Cl (aq) and 2x400 ml brine, then concentrated in vacuo. The residue was eluted on a silica gel column with ethyl acetate/petroleum ether (1:10). The result was 3.9 g (43%) of the title compound as a yellow oil. MS: 339 ES+ (C17H30N2O3SO

Intermediate 28: (2S,5R)-2-(hydroxymethyl)-3-methyl-6-(pron-2-en-1-yloxy)-1,6diazabicyclo[3.2.1]oct-3-ene-7- He

Tetrahydrofuran (30 ml) and (2S,5R)-2-[[(tertbutyldimethylsilyl)oxy]methyl]-3-methyl-6-(prop-2-en-1-yloxy)-1 were placed in a 100 ml round bottom flask ,6-diazabicyclo[3.2.1]oct-3-en-7-one (Intermediate 27, 3.2 g, 9.45 mmol, 1 eq.) and cool the solution to 0°C, then add TBAF dropwise (14.2 ml 1N in THF, 1.5 eq.). The reaction mixture was stirred for 1 hour at 0°C in a water/ice bath. The resulting mixture was concentrated in vacuo. The residue was eluted on a silica gel column with ethyl acetate/petroleum ether (1:5-1:2). The result was 1.6 g (75%) of the title compound as a light yellow solid.

MS: 225 ES+ (ScH^Oz)

- 58 044428

1H NMR (300 MHz, CDCl ₃ ) δ 1.63 (3H, d), 3.20 (2H, d), 3.62-3.84 (2H, m), 3.85-3.90 (2H , m), 4.35-4.48 (2H, m), 5.28-5.39 (2H, m), 5.95-6.08 (2H, m).

Intermediate 29: (2S,5R)-3-methyl-7-oxo-6-(prop-2-en-1-yloxy)-1,6-diazabicyclo[3.2.1]oct-3-ene-2- carboxylic acid ⁰ ι

J--N

O 'O\^

To a solution of H ₅ IO ₆ (12.93 g, 56.71 mmol) in wet CH3CN (150 ml, 0.75 vol.% H ₂ O) at room. pace. CrO ₃ (128 mg, 1.28 mmol) was added. The mixture was stirred until completely dissolved. Wet acetonitrile (35 ml) and (2S,5R)-2-(hydroxymethyl)-3-methyl-6(prop-2-en-1-yloxy)-1,6-diazabicyclo[ 3.2.1]oct-3-en-7-one (Intermediate 28, 740 mg, 3.30 mmol, 1 eq.) and cooled to 0°C. The above oxidizing agent solution (35 mL, 3 eq.) was then added dropwise over 30 min at 0°C. The resulting solution was stirred for 1 hour at 0°C in a water/ice bath. Then the reaction mixture was diluted with 200 ml of chloroform and 50 ml of citric acid solution (25%). The organic layer was separated and then washed with 3x50 ml brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The result was 0.70 g of the crude title compound as a yellow oil. MS: 239 ES+ (C11H16N2O4)

Intermediate 30: (2S,5R)-N'-acetyl-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carbohydrazide

To a solution of (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid (Intermediate 29, 195.7 mg, 0.82 mmol) in DCM (10 ml) at room temperature was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (236 mg, 1.23 mmol), 1-hydroxybenzotriazole hydrate (219 mg, 1.23 mmol) and monoacetylhydrazine (101 mg, 1.23 mmol). The mixture was cooled to 0°C and DIEA (0.715 ml, 4.11 mmol) was added. The reaction mixture was allowed to warm to room. pace. and stirred at room temperature overnight, then diluted with ethyl acetate and washed twice with a 1:1 mixture of brine and water. The aqueous wash solutions contained some product and were strip-extracted twice with ethyl acetate and once with about 5% methanol in dichloromethane. The combined organics were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0%-5% methanol/dichloromethane) gave the title compound as a white foam (45.5 mg, 19%, about 50% purity). MS: 295 ES+ (C ₁₃ H ₁₈ N ₄ O ₄ )

II NMR (300 MHz, DMSO-de) δ: 1.62 (s, 3H); 1.86 (m, 3H); 2.88(m, 1H); 3.40 (m, 1H); 3.81(m, 1H); 3.95 (m, 1H); 4.38 (m, 2H); 5.32 (m, 2H); 5.94 (m, 1H); 6.11 (m, 1H); 9.86(s, 1H); 10.24 (bs, 1H).

Example 26 )oxy)-2-fluoroacetate

O

To a solution of (2S,5R)-N'-acetyl-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2carbohydrazide (Intermediate 30, 22 .75 mg, 0.08 mmol) in methanol (3 ml) at room temperature, 1,3-dimethylbarbituric acid (48.3 mg, 0.31 mmol) and tetrakis(triphenylphosphine) palladium(0) (17.87 mg) were added , 0.02 mmol). The reaction mixture was stirred at room temperature for 2 hours, then concentrated to give an orange film. The orange film was dissolved in DMF (3 ml). Potassium chloride (64.1 mg, 0.46 mmol) and ethyl 2-bromo-2fluoroacetate (0.055 ml, 0.46 mmol) were added. The reaction mixture was stirred for about 5 hours at room temperature, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed three times with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-65% ethyl acetate/hexanes) gave the title compound as an orange foam (25.9 mg, 94%). The two diastereomers were present in a 1:1 ratio. The substance was dissolved in ethyl acetate and washed three times with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Elution was performed again on a silica gel column (0-30% acetone/dichloromethane) to obtain pure title compound (16.6 mg, 60%).

- 59 044428

MS: 359 ES+ (C ₁₄ H ₁₉ FN ₄ O6) ¹ H NMR (300 MHz, DMSO-d6) δ: 1.24 (m, 3H); 1.65(s, 3H); 1.86 (m, 3H); 3.09 (m, 1H); 3.89(d, 1H); 4.01 (m, 1H); 4.25 (m, 3H); 6.09 (m, 1H); 6.20 (m, 1H); 9.90(s, 1H); 10.30(d, 1H).

Example 27 2-fluoroacetic acid lithium salt

To a solution of ethyl 2-(((2S,5R)-2-(2-acetylhydrazinecarbonyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl) hydroxy)-2-fluoroacetate (Example 26, 16.6 mg, 0.05 mmol) in THF (1 ml) and water (0.33 ml) at 0°C added lithium hydroxide (1 M) (0.05 ml , 0.05 mmol) and stirred for 10 min at 0°C. Another 0.2 equiv was added. lithium hydroxide. After 30 minutes, another 0.2 equiv was added. lithium hydroxide. The reaction mixture was stirred for another 30 minutes. HCl (0.5M) (0.046 mL, 0.02 mmol) was added to adjust the pH to about 4-5. The reaction mixture was extracted twice with ethyl acetate. The aqueous layer was frozen and lyophilized. 15 mg of the title compound was obtained as an orange solid. The two diastereomers were present in a 1:1 ratio. MS: 331 ES+ (C12H15FN4O6) ¹ H NMR (300 MHz, DMSO-d6) δ: 1.65 (s, 3H); 1.87 (m, 3H); 3.08 (m, 1H); 3.84 (m, 1H); 4.03 (m, 1H); 4.23(s, 1H); 5.23 (m, 1H); 6.10 (m, 1H); 9.88(s, 1H); 10.26 (bs, 1H).

Intermediate 31: tert-butyl 4-((28.5K)-6-(allyloxy)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-enecarboxamido)benzylcarbamate

To a solution of (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid (Intermediate 29, 130.5 mg, 0.55 mmol) in DMF (5 ml) at room temperature was added tert-butyl-4-aminobenzylcarbamate (146 mg, 0.66 mmol), Hunig's base (0.287 ml, 1.64 mmol) and cyclic anhydride 1- propanephosphonic acid (50 wt.% in DMF) (0.326 ml, 1.10 mmol). The mixture was stirred at room temperature for 1 hour. Water (10 ml) and 10% MeOH in DCM (20 ml) were added. The organic layer was separated and concentrated to obtain the crude product. Purification by flash chromatography (20 g silica gel, 0-10% MeOH in DCM, 20 min) gave the title compound (178 mg, 73.4% yield, about 50% purity) as a yellow oil. MS: 443 ES+ (C23H30N4O5) ¹ H NMR (300 MHz, chloroform-d) δ ppm 1.47 (m, 12 H) 3.10-3.23 (m, 1H) 3.25-3.39 (m , 1H) 4.34-4.54 (m, 3H) 4.36-4.49 (m, 3H) 5.25-5.45 (m, 2H) 6.03 (d, J=6.61 Hz, 1H) 6.09-6.17 (m, 1H) 7.24-7.29 (m, 2H) 7.427.62 (m, 2H) 8.64 (s, 1H)

Intermediate 32: (R)-ethyl-2-((2S,5R)-2-(4-((tert-butoxycarbonylamino)methyl)phenyl-carbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo [3.2.1]oct-3-en-6-yloxy)-2-fluoroacetate

To a solution of tert-butyl-4-((2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo-[3.2.1]oct-3-ene-2carboxamido)benzylcarbamate ( Intermediate 31, 178 mg, 0.40 mmol) in methanol (10 ml) was added Pd(Ph ₃ P) ₄ (465 mg, 0.40 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated to give the crude intermediate tert-butyl-4-((2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2. 1]oct-3-enecarboxamido)benzylcarbamate in the form of an orange film. The crude substance was dissolved in DMF (1 ml). K ₂ CO ₃ (167 mg, 1.21 mmol) and ethyl 2-bromo-2-fluoroacetate (0.052 ml, 0.44 mmol) were added. The reaction mixture was stirred overnight at room temperature. EtOAc (20 ml) was added and the reaction mixture was washed with water (10 ml). The organic layer was concentrated to give the crude product, which was purified by flash chromatography (12 g silica gel, 0-100% EtOAc in hexanes, 20 min; then 5% MeOH in DCM, 10 min) to give the title compound (11 mg, yield 5.4%) as an orange solid. MS: 507 ES+ (C24H31FN4O7)

- 60 044428

1H NMR (300 MHz, chloroform-d) δ ppm 1.36 (t, 3H) 1.48 (s, 9 H) 2.00 (s, 3H) 3.21-3.27 (m, 1H) 3 .36(d,

J=1.70 Hz, 1H) 4.08 (dd, J=5.00, 2.74 Hz, 1H) 4.24-4.42 (m, 5 N) 4.47 (s, 1H) 5 .78-5.95 (m, 1H) 6.12-6.17 (m,

1H) 7.38-7.55 (m, 4H) 8.47 (s, 1H)

Intermediate 33: (R)-2-((2S,5R)-2-(4-((tert-butoxycarbonylamino)methyl)phenylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1 ] oct-3-en-6-yloxy)-2-fluoroacetic acid lithium salt

To a solution of (2R)-ethyl-2-(((2S,5R)-2-((4-(((tert-butoxycarbonyl)amino)methyl)phenyl)-carbamoyl)-3methyl-7-oxo-1,6 -diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)-2-fluoroacetate (Intermediate 32, 11 mg, 0.02 mmol) in THF (1 ml) and water (0.5 ml) at 0°C lithium hydroxide (1M) (0.02 ml, 0.02 mmol) was added. The mixture was stirred for 5 min at 0°C and DCM (10 ml) was added. Hydrochloric acid (0.5N) was carefully added to adjust the pH to about 5-6. The organic layer was separated and concentrated to give the title compound (8 mg, 77%) as a yellow solid. UHPLC, acidic conditions, retention time: 0.80 min, 479 (M+H)+ *H NMR (300 MHz, chloroform-d) δ ppm 1.47 (s, 9 H) 1.98 (s, 3 H ) 3.19-3.44 (m, 1H) 4.03-4.55 (m, 6H) 5.68-6.05 (m, 1H) 7.44-7.56 (m, 4H) 8 .49 (br. s., 1H)

Example 28 -6-yloxy)-2-fluoroacetic acid TFA salt

To a solution of lithium salt (2R)-2-(((2S,5R)-2-((4-(((tert-butoxycarbonyl)amino)methyl)phenyl)carbamoyl)-3-methyl-7-oxo-1, 6-Diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)-2-fluoroacetic acid (Intermediate 33, 8 mg, 0.02 mmol) in DCM (2 ml) at 0°C added TFA (0.128 ml, 1.67 mmol). The reaction mixture was stirred at 0°C for 6 hours and the solvent was removed. Toluene (2x1 ml) was added and concentrated to remove excess TFA. Et ₂ O (2 ml) was added and the resulting precipitate was added and filtered, washed with Et ₂ O and dried to give the title compound (8 mg, 97%) as the TFA salt. MS: 379 ES+ (C ₁₇ H ₁₉ FN ₄ O ₅ ) *H NMR (300 MHz, D ₂ O) δ ppm 1.75 (s, 3H) 3.23-3.61 (m, 2H) 4.12 -4.25 (m, 3H) 4.51-4.60 (m, 1H) 5.535.98 (m, 1H) 6.29 (br. s., 1H) 7.45-7.57 (m, 4H)

Intermediate 34: (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-N-(pyrazin-2-ylmethyl)-1,6diazabicyclo[3.2.1]oct-3-ene-2- carboxamide

To a solution of (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid (Intermediate 29, 199 mg, 0.84 mmol) in DMF (5 ml) at 0°C was added pyrazin-2-ylmethanamine (91 mg, 0.84 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N', N'-tetramethyluronium hexafluorophosphate (635 mg, 1.67 mmol) and DIEA (0.582 ml, 3.34 mmol). The reaction mixture was stirred for 30 minutes at room temperature, then diluted with ethyl acetate and washed once with saturated sodium bicarbonate solution and three times with a 1:1 mixture of brine and water. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-2.5% methanol/dichloromethane) gave the title compound (147 mg, 53.5%) as an orange oil. MS: 330 ES+ (C ₁₆ H ₁₉ N ₅ O ₃ ) *H NMR (300 MHz, DMSO-d6) δ: 1.60 (s, 3H); 3.14 (m, 2H); 3.64 (m, 1H); 3.94 (m, 1H); 4.35 (m, 2H); 4.46 (m, 2H); 5.29 (m, 2H); 5.96(m, 1H); 6.07 (m, 1H); 8.56 (m, 3H); 9.05 (m, 1H).

Example 29 3-en-6-yl)oxy)acetate

- 61 044428

To a solution of (2S,5R)-6-(allyloxy)-3-methyl-7-oxo-N-(pyrazin-2-ylmethyl)-1,6-diazabicyclo[3.2.1]oct-3-ene-2- carboxamide (Intermediate 34, 153.8 mg, 0.47 mmol) in methanol (3 ml) add 1,3-dimethylbarbituric acid (146 mg, 0.93 mmol) and tetrakis(triphenylphosphine)palladium(0) at room temperature (54.0 mg, 0.05 mmol). The reaction mixture was stirred at room temperature for 2 hours. Tetrakis(triphenylphosphine)palladium(0) (54.0 mg, 0.05 mmol) was added again, as well as 2 ml of methanol, and the mixture was stirred at room temperature for another 2 hours The reaction mixture was concentrated to give an orange oil. The oil was dissolved in DMF (3 ml) and potassium carbonate (194 mg, 1.40 mmol) and ethyl 2-bromo-2-fluoroacetate (0.166 ml, 1.40 mmol) were added. The reaction mixture was stirred overnight at room temperature, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed three times with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-100% ethyl acetate/hexanes) provided the title compound (95 mg, 52%) as a light orange foam. The compound was a mixture of diastereomers in a 1:1 ratio.

MS: 394 ES+ (C17H20FN5O5) ¹ H NMR (300 MHz, DMSO-d6) δ: 1.22 (m, 3H); 1.63(s, 3H); 2.71 (m, 1H); 3.10 (m, 1H); 3.98(d, 1H); 4.02 (m, 1H); 4.26 (m, 2H); 4.47 (m, 2H); 6.05 (m, 1H); 6.20 (m, 1H); 8.56 (m, 3H); 9.10 (m, 1H).

Example 30 3-en-6-yl)oxy)acetic acid lithium salt

O

To a solution of ethyl 2-fluoro-2-(((2S,5R)-3-methyl-7-oxo-2-((pyrazin-2-ylmethyl)-carbamoyl)-1,6diazabicyclo[3.2.1]oct- 3-en-6-yl)oxy)acetate (Example 29, 95.3 mg, 0.24 mmol) in THF (2 ml) and water (0.66 ml) at 0°C added lithium hydroxide (1 M) (0.254 ml, 0.25 mmol) and stirred for 25 min at 0°C. Another 0.1 eq was added. lithium hydroxide. After 15 min, hydrochloric acid (0.5N) (0.242 ml, 0.12 mmol) was added to adjust the pH to about 5-6. The reaction mixture was frozen and lyophilized. 90 mg of light yellow solid was purified by reverse phase HPLC (YMC Carotenoid C30, 21.2 mm x 150 mm, 4 µm, coupled to Synergi Polar RP, 21.2 mm x 100 mm, 4 µm, 0-30 % acetonitrile in water, 20 ml/min, 15 min). 23.6 mg (27%) of the title compound was obtained as a white solid. The compound was a mixture of diastereomers in a 1:1 ratio. MS: 366 ES+ (C15H16FN5O5) ¹ H NMR (300 MHz, DMSO-a ₆ ) δ: 1.63 (s, 3H); 3.09 (m, 1H); 3.68(m, 1H); 4.02 (m, 1H); 4.29(s, 1H); 4.48(d,2H); 5.22 (m, 1H); 6.05 (m, 1H); 8.57 (m, 3H); 9.07 (m, 1H).

Intermediate 35: (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)but-3-en-2-yl(3methyl-2-oxobut-3-enyl)carbamate

TBSO^^Y^ to boc η

To a solution of (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)but-3-en-2-yl(2-(methoxy(methyl)amino)2-oxoethyl)carbamate (Intermediate 5, 30.79 g , 76.48 mmol) in THF (200 ml) at 0°C was added prop-1-en-2-ylmagnesium bromide (0.5 M in THF) (300 ml, 149.90 mmol) and stirred at 0°C for 1 hour. The reaction was stopped by adding 200 ml of 10% citric acid, further diluted with 100 ml of water and extracted with ether. The organic substances were concentrated and the resulting oil was dissolved in ether and washed with water and brine. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0%-20% ethyl acetate/hexanes) gave the desired product (26.2 g, 89%) as a colorless oil. MS: 384 ES+ (C20H37NO4SO

1H NMR (300 MHz, DMSO-d6) δ: 0.02 (d, 6H); 0.83(s,9H); 1.27-1.38 (m, 9H); 1.80 (m, 3H); 3.71 (m, 2H); 4.34 (m, 2H); 4.61(m, 1H); 5.17 (m, 2H); 5.77 (m, 1H); 5.85 (m, 1H); 6.03 (m, 1H).

Intermediate 36: (S)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-4-methyl-5oxo-5,6-dihydropyridine-1(2H)-carboxylate

TBSO ^/, '''^Y ^X '

BoD' ^h/ i

(S)-tert-Butyl-1-(tert-butyldimethylsilyloxy)but-3-en-2-yl(3-methyl-2-oxobut-3enyl)carbamate solution (Intermediate 35, 26.18 g, 68.25 mmol) in toluene (600 ml) was purged with nitrogen for 15 min. (1,3-Bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)-62 044428 dichloro(o-isopropoxyphenylmethylene)ruthenium (0.987 g, 1.57 mmol) was then added. The reaction mixture was at 65°C for 1.5 hours. The reaction mixture was concentrated on silica gel. Chromatography on silica gel (0-15% ethyl acetate/hexanes) gave the desired product (21.18 g, 87%) as a colorless oil.

MS: 356 ES+ (C18H33NO4SO

1H NMR (300 MHz, DMSO-46) δ: 0.01 (d, 6H); 0.81(s,9H); 1.42(s,9H); 1.75 (m, 3H); 3.74-3.89 (m, 3H); 4.04-4.32 (m, 1H); 4.67 (m, 1H); 6.88 (m, 1H).

Intermediate 37: (2S,5S)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-5hydroxy-4-methyl-5,6-dihydroniridine-1(2H)-carboxylate

Uh,,

Bos 'ON

To a solution of cerium (III) chloride (14.68 g, 59.57 mmol) and (S)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-4-methyl-5-oxo-5,6-dihydropyridine- 1(2H)-carboxylate (Intermediate 36, 21.18 g, 59.57 mmol) in methanol (300 mL), sodium borohydride (2.254 g, 59.57 mmol) was added portionwise at 0°C. After 15 minutes, the reaction mixture was diluted with saturated ammonium chloride solution (100 ml) and water (100 ml), then extracted twice with diethyl ether.

Organic extracts were washed with brine, dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-20% ethyl acetate/hexanes) gave the desired product (19.45 g, 91%) as a colorless oil.

MS: 358 ES+ (C18H35NO4S1)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.02 (s, 6H); 0.86(s,9H); 1.39(s,9H); 1.69 (m, 3H); 2.63-2.72 (m, 1H); 3.59 (m, 2H); 3.82(m, 1H); 4.03 (m, 1H); 4.21(m, 1H); 5.04 (d, 1H); 5.38 (m, 1H).

Intermediate 38: N-(allyloxy)-2-nitrobenzenesulfonamide

To a stirred solution of O-allylhydroxylamine hydrochloride (147.05 g, 1341.59 mmol) in DCM (2.5 L) at 0°C was added pyridine (318 ml, 3948 mmol), followed by portions of 2-nitrobenzene-1sulfonyl chloride ( 250 g, 1128.05 mmol) in solid form. The reaction mixture was then stirred at the same temperature for 1 hour. The completion of the reaction was monitored by TLC. The reaction was stopped by adding 1.5N. HCl (1 l). The organic layer was separated, washed with water (250 ml), brine (250 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to leave a residue. The crude material was purified by crystallization using a mixture of EtOAc and petroleum ether (1:3) (800 ml) to obtain 202 g of the title compound as a light brown solid.

The mother liquor was concentrated and purified by silica gel column chromatography (60-120 mesh) using a mixture of EtOAc and petroleum ether (7:3) to obtain an additional 19.1 g of the title compound as a yellow solid. The overall yield was 76%.

UHPLC: 257 (M-1) for C9H10N2O5S ^1H NMR (400 MHz, DMSO-d6): δ 4.36-4.38 (m, 2H), 5.22-5.32 (m, 2H), 5 .84-5.91 (m, 1H), 7.92-7.96 (m, 2H), 8.02-8.05 (m, 2H), 11.07 (s, 1H).

Intermediate 39: (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)2-((tert-butyldimethylsilyloxy)methyl)-4-methyl-5,6-dihydropyridine-1( 2H)-carboxylate

To a solution of (2S,5S)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-5-hydroxy-4-methyl-5,6dihydropyridine-1(2H)-carboxylate (Intermediate 37, 19.45 g , 54.40 mmol) in toluene (300 ml) at room temperature was added triphenylphosphine (17.06 g, 65.28 mmol), N-(allyloxy)2-nitrobenzenesulfonamide (Intermediate 38, 14.05 g, 54.40 mmol) and diisopropyl azodicarboxylate (12.85 ml, 65.28 mmol). After 2 hours, the reaction mixture was concentrated on silica gel and purified. Chromatography on silica gel (0-50% ethyl acetate/hexanes) gave the desired product (25.2 g, 78%) as a yellow oil. MS: 598 ES+ (C ₂₇ H ₄₃ N ₃ O ₈ SSi)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.00 (s, 6H); 0.83(s,9H); 1.31 (m, 9H); 1.34 (m, 3H); 3.10-3.25 (m, 1H); 3.59 (m, 2H); 3.99-4.41 (m, 5H); 5.17 (m, 2H); 5.72 (m, 2H); 7.93-8.16 (m, 4H).

Intermediate 40: (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)2-(hydroxymethyl)-4-methyl-5,6-dihydropyridine-1(2H)-carboxylate

- 63 044428

To a solution of (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-2-((tert-butyldimethylsilyloxy)methyl)-4-methyl-5,6-dihydropyridine-1(2H )-carboxylate (Intermediate 39, 1 g, 1.67 mmol) in THF (11 ml) was added tetrabutylammonium fluoride (1 M in THF) (2.175 ml, 2.17 mmol) at 0°C. After 90 minutes, the reaction mixture was concentrated on silica gel. Chromatography on silica gel (0-70% ethyl acetate/hexanes) gave the desired product (0.732 g, 90%) as a brownish-beige foam. MS: 484 ES+ (C ₂₁ H ₂₉ N ₃ O ₈ S) ¹ H NMR (300 MHz, DMSO-de) δ: 1.31 (m, 9H); 1.35 (m, 3H); 3.20 (m, 1H); 3.41(m, 2H); 3.96-4.37 (m, 5H); 4.76 (m, 1H); 5.19 (m, 2H); 5.66-5.84 (m, 2H); 7.94-8.18 (m, 4H).

Intermediate 41: (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)-4-methyl-1,2,5,6-tetrahydropyridine-2-carboxylic acid o l ^^ but ru boc^ ^N

Ns

Chromium(VI) oxide (10 mg, 0.10 mmol) was added to a solution of periodic acid (6 g, 31.26 mmol) in wet acetonitrile (60 ml) (0.75 vol.% water) at room temperature. The mixture was stirred until completely dissolved. To a solution of (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-2(hydroxymethyl)-4-methyl-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate 40.5 g, 10.34 mmol) in wet acetonitrile (60 ml) (0.75 vol.%) at 0°C, the previously prepared periodic acid/chromium oxide solution (60 ml, 3 eq.) was added dropwise. After 30 minutes, the reaction mixture was diluted with ether and washed with 10% citric acid, saturated sodium bicarbonate solution and brine. The organics were dried over magnesium sulfate, filtered and concentrated to give an orange foam (4.16 g, 81%). MS: 498 ES+ (C ₂₁ H ₂₇ N ₃ O ₉ S)

1H NMR (300 MHz, DMSO-de) δ: 1.26 (m, 9H); 1.31(m, 3H); 3.02-3.25 (m, 1H); 3.90 (m, 1H); 4.17 (m, 3H); 4.65-4.77 (m, 1H); 5.12-5.21 (m, 2H); 5.68(m, 1H); 5.88(m, 1H); 7.92-8.17 (m, 4H).

Intermediate 42: (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)2-carbamoyl-4-methyl-5,6-dihydropyridine-1(2H)-carboxylate o

L h ₂ n уу boc ^{x Ν} ι Ns

To a solution of (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)-4methyl-1,2,5,6-tetrahydropyridine-2-carboxylic acid (Intermediate 41 , 4.16 g, 8.36 mmol) in DMF (35 ml) at room temperature was added ammonium chloride (0.895 g, 16.72 mmol), HATU (4.77 g, 12.54 mmol) and DIEA (5, 84 ml, 33.45 mmol). After 15 minutes, the reaction mixture was diluted with ethyl acetate, washed with saturated sodium bicarbonate solution and twice with a 1:1 mixture of brine and water. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel twice (0-80% ethyl acetate/hexanes) gave the desired product (2.16 g, 52%) as a yellow foam. MS: 497 ES+ (C21H28N4O8S)

1H NMR (300 MHz, DMSO-de) δ: 1.26 (m, 9H); 1.37 (m, 3H); 3.12-3.35 (m, 1H); 3.80 (m, 1H); 4.18 (m, 3H); 4.64-4.79 (m, 1H); 5.13-5.22 (m, 2H); 5.68(m, 1H); 5.88(m, 1H); 7.04 (m, 1H); 7.45 (bs, 1H); 7.90-8.18 (m, 4H).

Intermediate 43: (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-4-methyl1,2,5,6-tetrahydropyridine-2-carboxamide

To a solution of (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-2-carbamoyl-4methyl-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate 42, 2 .16 g, 4.35 mmol) in DCM (20 ml) was added zinc bromide (0.700 ml, 13.05 mmol) at room temperature. After stirring overnight at room temperature, the reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate solution and brine. The organics were dried over magnesium sulfate, filtered and concentrated to give the title product (1.450 g, 84%) as a yellow foam.

MS: 397 ES+ (C16H20N4O6S)

1H NMR (300 MHz, DMSO-de) δ: 1.65 (m, 3H); 2.71 (m, 3H); 3.76 (m, 1H); 3.95 (m, 1H); 4.18-4.42 (m, 2H); 5.23 (m, 2H); 5.82 (m, 1H); 6.02 (m, 1H); 7.05 (bs, 1H); 7.30 (bs, 1H); 7.93-8.18 (m, 4H).

Intermediate 44: (2S,5R)-5-(allyloxyamino)-4-methyl-1,2,5,6-tetrahydropyridine-2carboxamide and (2R,5R)-5-(allyloxyamino)-4-methyl-1, 2,5,6-tetrahydropyridine-2-carboxamide

- 64 044428

To a solution of (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-4-methyl-1,2,5,6-tetrahydropyridine-2-carboxamide (Intermediate 43, 1.4 g, 3 .53 mmol) and cesium carbonate (9.21 g, 28.25 mmol) in THF (100 ml) at room temperature, PS-thiophenol (3-(3mercaptophenyl)propanamidomethylpolystyrene) (1.55 mmol/g) was added (9, 12 g, 14.13 mmol). After stirring overnight at room temperature, the reaction mixture was filtered through a porous glass funnel and the resin was washed twice with DCM. The filtrate was concentrated to give a yellow oil. Chromatography on silica gel (0-5% methanol/dichloromethane) gave a 3:1 mixture of trans and cis isomers (0.473 g, 63.4%) as a light yellow oil. The mixture was used further without separation. MS: 212 ES+ (C ₁₀ H ₁₇ N ₃ O ₂ )

1H NMR (300 MHz, DMSO-d6) δ: 1.73 (m, 3H); 2.63 (m, 1H); 2.97 (m, 1H); 3.01 (m, 1H); 3.60 (m, 1H); 4.12 (m, 2H); 5.11-5.26 (m, 2H); 5.92(m, 1H); 6.45 (m, 1H); 7.00 (m, 1H); 7.33 (bs, 1H).

Intermediate 45: (2S,5R)-6-(allyloxy)-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct3-ene-2-carboxamide

To a solution of (2S,5R)-5-(allyloxyamino)-4-methyl-1,2,5,6-tetrahydropyridine-2-carboxamide and (2R,5R)-5-(allyloxyamino)-4-methyl-1, 2,5,6-tetrahydropyridine-2-carboxamide (Intermediate 44, 0.429 g, 2.03 mmol) and DIEA (1.415 ml, 8.12 mmol) in acetonitrile (170 ml) was added triphosgene (0.241 g) at 0°C , 0.81 mmol) as a solution in acetonitrile (1.5 ml) at a rate of 0.1 ml/min. After addition was complete, the reaction mixture was warmed to room temperature and stirred for two days. The reaction mixture was diluted with ethyl acetate, washed with saturated sodium bicarbonate and brine, dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-20% ethyl acetate/hexanes) provided the product (0.312 g, 64.8%) as a light yellow oil. MS: 238 ES+ (C _n H ₁₅ N ₃ O ₃ ) 'H NMR (300 MHz, DMSO-d6) δ: 1.79 (m, 3H); 3.19 (m, 2H); 3.81(m, 1H); 4.12 (m, 1H); 4.36 (m, 2H); 5.24-5.45 (m, 3H); 5.89-6.00 (m, 1H); 7.28 (bs, 1H); 7.49 (bs, 1H).

Example 31: (2R)-ethyl-2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy )-2-fluoroacetate o

Λ η ₂ ν [ ηρ

Μ F

Λ---Ν. F ^{0 0} ^V ⁰ N-Ο

Example 32: (2S)-ethyl-2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy )-2-fluoroacetate o l η ₂ ν r

O

Examples 31-32.

To a solution of (2S,5R)-6-(allyloxy)-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 45, 0.2972 g , 1.25 mmol) in methanol (6 ml) at room temperature, 1,3-dimethylbarbituric acid (0.391 g, 2.51 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.145 g, 0.13 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, then concentrated to give an orange film. The orange film was dissolved in DMF (6 ml). Potassium carbonate (0.519 g, 3.76 mmol) and ethyl bromofluoroacetate (0.592 mL, 5.01 mmol) were added. The reaction mixture was stirred overnight at room temperature, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed twice with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. After chromatography on silica gel (0-65% ethyl acetate/hexanes) a mixture of diastereomers was obtained in a ratio of 1:1, 372 mg, 99%. Separation of diastereomers was carried out using reverse phase HPLC (Atlantis T3, 19 mm x 150 mm, 5 µm, 20-40% acetonitrile in water, 20 ml/min, 15 min). After lyophilization, both diastereomers were obtained as white solids. The following products were obtained:

- 65 044428

Example 31: (first diastereomer to elute): 108.8 mg, 29% MS: 302 ES+ (C ₁₂ H ₁₆ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 1.23 (t, 3H); 1.82 (m, 3H); 3.19 (m, 1H); 3.29 (m, 1H); 3.96(m, 1H);

4.23 (m, 1H); 4.24(q,2H); 5.52 (m, 1H); 6.28(m, 1H); 7.32(br s, 1H); 7.56 (br s, 1H).

Example 32: (second diastereomer eluting): 103.3 mg, 27%

MS: 302 ES+ (C _P H^3O ₅ )

1H NMR (300 MHz, DMSO-d^δ: 1.27 (t, 3H); 1.81 (m, 3H); 3.21 (m, 1H); 3.31 (m, 1H); 3. 82 (m, 1H); 4.24 (m, 1H); 4.28 (q, 2H); 5.52 (m, 1H); 6.17 (m, 1H); 7.32 (br s, 1H); 7.55 (br s, 1H).

The stereochemistry of the two diastereomers was determined based on their order of elution as well as the inhibitory activity of the corresponding carboxylic acids (Examples 33 and 34): the more active acid corresponding to the first diastereomer to elute was designated the R-isomer.

Example 33 )-2-fluoroacetic acid lithium salt

To a solution of (2R)-ethyl-2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo-[3.2.1]oct-3-en-6yl)oxy )-2-fluoroacetate (Example 31, 96.6 mg, 0.32 mmol) in THF (3 ml) and water (1 ml) was added lithium hydroxide (0.337 ml, 0.34 mmol) at 0°C. The reaction mixture was kept in an ice bath and stirred for 15 minutes. Another 0.2 equiv was added. lithium hydroxide. After 15 minutes, the pH of the reaction mixture was adjusted to pH=7 using 0.5N. HCl. The THF was evaporated and the remaining aqueous solution was frozen and lyophilized to give a pale yellow solid. After reverse phase HPLC and lyophilization (YMC Carotenoid C30, 19 mm x 150 mm, 5 µm, coupled with Synergi Polar RP 21.2 mm x 100 mm, 4 µm, 0-40% acetonitrile in water, 20 ml/min , 15 min) gave the title compound as a white solid, 34.8 mg, 40%. MS: 274 ES+ (C10H12FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.83 (m, 3H); 3.21 (m, 2H); 3.91(m, 1H); 4.16(m, 1H); 5.33 (m, 1H); 5.44 (m, 1H); 7.27(br s, 1H); 7.53 (br s, 1H).

Example 34. (2S)-2-(((2S,5R)-2-Carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)- 2-fluoroacetic acid lithium salt

The title compound was prepared from (2S)-ethyl-2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo1,6-diazabicyclo[3.2.1]oct-3-ene-6- yl)oxy)-2-fluoroacetate (example 32, 91.8 mg, 0.30 mmol) according to the procedure for example 33. Purification conditions were similar; lyophilization yielded an off-white solid, 11.7 mg, 14%.

MS: 274 ES+ (C ₁₀ H ₁₂ FN ₃ O ₅ ) ¹ H NMR (300 MHz, DMSO-di) δ: 1.81 (m, 3H); 3.21(m, 2H); 3.87 (m, 1H); 4.16(m, 1H); 5.25 (m, 1H); 5.45 (m, 1H); 7.28(br s, 1H); 7.54 (br s, 1H).

Example 35 )-2-fluoroacetate

Example 36 yl)oxy)-2-fluoroacetate

To a solution of (2S,5R)-6-(allyloxy)-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 45, 0.15 g , 0.63 mmol) in methanol (3 ml) at room temperature, 1,3-dimethylbarbituric acid (0.197 g, 1.26 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.073 g, 0.06 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, then

- 66 044428 was concentrated to obtain an orange film. The orange film was dissolved in DMF (4 ml). Potassium carbonate (0.175 g, 1.26 mmol) and isopropyl 2-bromo-2-fluoroacetate (Intermediate 18, 0.377 g, 1.90 mmol) were added. The reaction mixture was stirred at room temperature for 4.5 hours, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed twice with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. After chromatography on silica gel (0-65% ethyl acetate/hexanes) a mixture of diastereomers was obtained in a ratio of 1:1, 196 mg, 98%. Separation of diastereomers was carried out using reverse phase HPLC (Atlantis T3, 19 mm x 150 mm, 5 μm, 20-40% acetonitrile in water, 20 ml/min, 15 min).

After lyophilization, both diastereomers were obtained as white solids.

Example 35: (first diastereomer to elute): 58.6 mg, 31%.

MS: 316 ES+ (C13H18FN3O5) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 1.22 (m, 6H); 1.81 (m, 3H); 3.17 (m, 1H); 3.34 (m, 1H); 3.93 (m, 1H); 4.22 (m, 1H); 5.01 (q, 2H); 5.51 (m, 1H); 6.23 (m, 1H); 7.31 (br s, 1H); 7.54 (br s, 1H).

Example 36: (second diastereomer eluting): 53.8 mg, 29%.

MS: 316 ES+ (C13H18FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.28 (m, 6H); 1.81 (m, 3H); 3.19 (m, 1H); 3.29 (m, 1H); 3.82(m, 1H); 4.24 (m, 1H); 5.06 (m, 1H); 5.52 (m, 1H); 6.14 (m, 1H); 7.32(br s, 1H); 7.55 (br s, 1H).

Intermediates 46-50 were deliberately omitted.

Intermediate 51: (R)-tert-butyl-4-(cyclopropyl(hydroxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate

To a solution of (R)-tert-butyl-4-formyl-2,2-dimethyloxazolidine-3-carboxylate (Aldrich, 12.44 g, 54.26 mmol) in THF (150 ml) at -78°C was added cyclopropylmagnesium bromide (217 ml, 108.52 mmol), drop by drop. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was stopped by adding water and diluted with ethyl acetate and brine. The resulting emulsion was filtered through celite and the layers were separated. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-20% ethyl acetate/hexanes) provided the title compound as a light yellow oil (12.47 g, 85%).

1H NMR (300 MHz, DMSO-d6) δ: 0.16 (m, 2H); 0.37 (m, 2H); 0.82 (m, 1H); 1.45 (m, 15H); 2.87 (m, 1H); 3.86(m, 2H); 3.97 (m, 1H); 4.74 (m, 1H).

Intermediate 52: (R)-tert-butyl-4-(cyclopropanecarbonyl)-2.,2-dimethyloxazolidine 3-carboxylate

To a solution of (R)-tert-butyl-4-(cyclopropyl(hydroxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate (Intermediate 51, 12.47 g, 45.95 mmol) in DCM (300 ml) Dess-Martin periodate (29.2 g, 68.93 mmol) was added at room temperature. The reaction mixture was stirred overnight, then diluted with ethyl acetate and washed with saturated sodium bicarbonate solution. An emulsion was formed, which was filtered through celite. The layers were separated and the organic matter was washed with brine. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-20% ethyl acetate/hexanes) gave the title compound as a colorless oil (11.15 g, 90%).

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.90 (m, 4H); 1.38 (m, 12H); 1.54 (m, 3H); 2.12(m, 1H); 3.94 (m, 1H); 4.18(m, 1H); 4.56 (m, 1H).

Intermediate 53: (S)-tert-butyl 4-(1-cyclopropylvinyl)-2,2-dimethyloxazolidine-3carboxylate

To a suspension of potassium tert-butoxide (9.29 g, 82.80 mmol) in ether (250 ml) was added methyltriphenylphosphonium bromide (29.6 g, 82.80 mmol) at room temperature. The mixture turned bright yellow in color and was heated to 40°C for 1 hour. The mixture was cooled to room temperature and a solution of (R)-tert-butyl-4-(cyclopropanecarbonyl)-2,2-dimethyloxazolidine-3-carboxylate (Intermediate) was added 52, 11.15 g, 41.40 mmol) in ether (30 ml), and stirred, the reaction mixture was stirred for 2 hours. The reaction was stopped by adding water (10 ml) and the layers were separated. The aqueous layer was extracted once with ether. The combined organic extracts were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-15% ethyl acetate/hexanes) provided the title compound as a colorless oil (9.84 g, 89%).

^1H NMR (300 MHz, DMSO-d6) δ: 0.42 (m, 2H); 0.65 (m, 2H); 1.43 (m, 16H); 3.76 (m, 1H); 4.09 (m, 1H); 4.27 (m, 1H); 4.66 (m, 2H).

Intermediate 54: (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)-3-cyclopropylbut-3-en-2-ylcarbamate

To a solution of (S)-tert-butyl-4-(1-cyclopropylvinyl)-2,2-dimethyloxazolidine-3-carboxylate (Intermediate 53, 8.25 g, 30.86 mmol) in methanol (100 ml) at room temperature, p-toluenesulfonic acid monohydrate (1.174 g, 6.17 mmol) was added. The reaction mixture was heated to 80°C overnight. Another 0.2 equiv was added. p-toluenesulfonic acid monohydrate was added and heated at 80°C for another 2 hours. The reaction mixture was cooled to room temperature. Triethylamine (4.29 mL, 30.86 mmol) and di-tert-butyl dicarbonate (3.37 g, 15.43 mmol) were added. The reaction mixture was stirred for two days and then concentrated. The residue was dissolved in ethyl acetate and washed once with saturated sodium bicarbonate solution. The combined organic extracts were dried over magnesium sulfate, filtered and concentrated. The resulting oil was dissolved in DCM (100 ml). Imidazole (2.73 g, 40.11 mmol), 4-dimethylaminopyridine (0.754 g, 6.17 mmol) and tert-butyldimethylsilyl chloride (4.65 g, 30.86 mmol) were added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was filtered to remove solids and washed twice with brine. The organic layer was dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-10% ethyl acetate/hexanes) gave the title compound as a colorless oil (6.77 g, 64%). MS: 342 ES+ (C ₁₈ H ₃₅ NO ₃ Si) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 0.04 (s, 6H); 0.39 (m, 2H); 0.63 (m, 2H); 0.85(s,9H); 1.32(m, 1H); 1.37 (m, 9H); 3.55 (m, 1H); 3.67 (m, 1H); 3.99 (m, 1H); 4.63(s, 1H); 4.78(s, 1H); 6.80 (m, 1H).

Intermediate 55: (S)-1-(tert-butyldimethylsilyloxy)-3-cyclopropylbut-3-ene-2 amine nh ₂ ^v

To a solution of (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)-3-cyclopropylbut-3-en-2-ylcarbamate (Intermediate 54, 6.77 g, 19.82 mmol) in DCM (100 ml) Zinc bromide (17.86 g, 79.28 mmol) was added at room temperature. The reaction mixture was stirred overnight at room temperature. Added another 1 eq. zinc bromide. After several hours, the reaction mixture was filtered and washed with saturated sodium bicarbonate solution. The resulting emulsion was filtered through a nylon filter and the layers were separated. The organics were dried over magnesium sulfate, filtered and concentrated to give the title compound as a yellow oil (4.61 g, 96%).

¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 0.04 (s, 6H); 0.39 (m, 2H); 0.63 (m, 2H); 0.87(s,9H); 1.35 (m, 1H); 1.81 (m, 2H); 3.33 (m, 1H); 3.45 (m, 1H); 3.67 (m, 1H); 4.59(s, 1H); 4.83 (m, 1H).

Intermediate 56: (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)-3-cyclopropylbut-3-en-2-yl(2-(methoxy(methyl)amino)-2-oxoethyl)carbamate bocA oA I

The title compound was prepared from (S)-1-(tert-butyldimethylsilyloxy)-3cyclopropylbut-3-en-2-amine (Intermediate 55, 4.61 g, 19.09 mmol) and 2-bromo-N-methoxyN -methylacetamide (Intermediate 4, 3.16 g, 17.36 mmol) following the same procedure as Intermediate 5. The title product was obtained as a light yellow oil (4.94 g, 64%). MS: 443 ES+ ^H^OsSi) ¹ H NMR (300 MHz, DMSO-d6) δ: 0.03 (m, 6H); 0.35 (m, 1H); 0.48 (m, 1H); 0.61 (m, 2H); 0.83 (m, 9H); 1.35 (m, 9H); 3.07 (m, 3H); 3.65 (m, 3H); 3.84 (m, 2H); 4.02 (m, 2H); 4.54 (m, 1H); 4.83 (m, 2H).

Intermediate 57: (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)-3-cyclopropylbut-3-en-2-yl(2-oxopent-3-enyl)carbamate

- 68 044428

A suspension of cerium(III) chloride (27.8 g, 112.95 mmol) in THF (100 ml) was stirred vigorously at room temperature for 2 hours. The suspension was cooled to -78°C and (E)prop-1 was added dropwise -enylmagnesium bromide (0.5 M in THF) (226 ml, 112.95 mmol). The mixture was stirred at -78°C for 1.5 hours. Then (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)-3cyclopropylbut-3-en-2-yl(2-(methoxy(methyl) )amino)-2-oxoethyl)carbamate (Intermediate 56. 5 g, 11.30 mmol) in THF (20 ml) at -78°C. The reaction mixture was stirred at -78°C for 30 minutes and then warmed to 0°C for 15 minutes. The reaction was stopped by adding 10% citric acid, further diluted with water and extracted twice with ether. Organic substances were washed once with brine, dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-20% ethyl acetate/hexanes) provided the title compound as a light yellow oil (4.0 g, 84%). MS: 424 ES+ (C ₂₃ H ₄₁ NO ₄ Si)

1H NMR (300 MHz, DMSO-06) δ: 0.03 (m, 6H); 0.43 (m, 2H); 0.61 (m, 2H); 0.83 (m, 9H); 1.34 (m, 10H); 1.84 (m, 2H); 2.04 (m, 1H); 3.74 (m, 1H); 3.84 (m, 2H); 4.03 (m, 1H); 4.57 (m, 1H); 4.79 (m, 2H); 6.28(m, 1H); 6.84 (m, 1H).

Intermediate 58: (S)-tert-butyl 2-((tert-butyldimethylsilyloxy)methyl)-3cyclopropyl-5-oxo-5,6-dihydropyridine-1(2H)-carboxylate

L tbso ''γη boc'' ^N ' ^x ^^O

The title compound was prepared from (S)-tert-butyl-1-(tert-butyldimethylsilyloxy)-3cyclopropylbut-3-en-2-yl(2-oxopent-3-enyl)carbamate (Intermediate 57, 4 g, 9 .44 mmol) following the same procedure as for Intermediate 7, except that the reaction mixture was heated at 110°C overnight. The target product was obtained as a light brown oil (2.97 g, 82%).

MS: 382 ES+ (C ₂₀ H ₃₅ NO ₄ Si) ¹ H NMR (300 MHz, DMSO-de) δ: 0.01 (m, 6H); 0.62 (m, 1H); 0.80(s,9H); 1.00 (m, 3H); 1.42(s,9H); 1.61(m, 1H); 3.80 (m, 1H); 3.95 (m, 2H); 4.19 (m, 1H); 4.75 (m, 1H); 5.72(s, 1H).

Intermediate 59: (2S,5S)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-3cyclopropyl-5-hydroxy-5,6-dihydropyridine-1(2H)-carboxylate

L tbso '''<η boc'' ^N ' ⁴ ^ 'ΌΗ

The title compound was prepared from (S)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-3-cyclopropyl-5-oxo-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate 58, 2.97 g, 7.78 mmol) following the procedure described for Intermediate 10. The title product was obtained as a brownish-beige oil (2.74 g, 92%). MS: 384 ES+ (C20H37NO4SO

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.02 (m, 6H); 0.34 (m, 1H); 0.47 (m, 1H); 0.64 (m, 2H); 0.85 (m, 9H); 1.26(m, 1H); 1.39(s,9H); 2.65 (m, 1H); 3.89(m, 3H); 4.05 (m, 1H); 4.95 (m, 1H); 5.34 (m, 1H).

Intermediate 60: (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)2-((tert-butyldimethylsilyloxy)methyl)-3-cyclopropyl-5,6-dihydropyridine-1( 2H)-carboxylate

L TBSO ''Λ boc/ ^Ν Ν '°

Ns

The title compound was prepared from (2S,5S)-tert-butyl-2-((tert-butyldimethylsilyloxy)methyl)-3-cyclopropyl-5-hydroxy-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate 59, 2.74 g, 7.14 mmol) and X-(allyloxy)-2-nitrobenzenesulfonamide (1.85 g, 7.14 mmol) according to the procedure given for Intermediate 11. The title product was obtained as light yellow oils (3.19 g, 71%).

MS: 624 ES+ (C ₂₉ H ₄₅ N ₃ O ₈ SSi)

1H NMR (300 MHz, DMSO-ds) δ: 0.00 (m, 6H); 0.34 (m, 1H); 0.63 (m, 2H); 0.83 (m, 9H); 1.37 (m, 9H); 3.30 (m, 1H); 3.84 (m, 2H); 4.30 (m, 4H); 5.18 (m, 2H); 5.75 (m, 1H); 8.04 (m, 4H).

Intermediate 61: (2S,5R)-tert-butyl-5-(N-(allyloxy)-2-nitrophenylsulfonamido)3-cyclopropyl-2-(hydroxymethyl)-5,6-dihydropyridine-1(2H)-carboxylate

- 69 044428

The title compound was prepared from (2S,5R)-tert-butyl-5-(N-(allyloxy)-2nitrophenylsulfonamido)-2-((tert-butyldimethylsilyloxy)methyl)-3-cyclopropyl-5,6-dihydropyridine-1 (2H)-carboxylate (Intermediate 60, 3.19 g, 5.11 mmol) following the same procedure as Intermediate 12. The title product was obtained as a brownish-beige foam (2.35 g, 90%). MS: 510 ES+ (C23H31N3O8S)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.32 (m, 2H); 0.62 (m, 2H); 1.35 (m, 9H); 3.30 (m, 1H); 3.67 (m, 2H); 4.27 (m, 4H); 4.71 (m, 1H); 5.19 (m, 2H); 5.71 (m, 1H); 8.04 (m, 4H).

Intermediate 62: (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)-3-cyclopropyl-1,2,5,6-tetrahydropyridine-2-carboxylic acid

The title compound was prepared from (2S,5R)-tert-butyl-5-(N-(allyloxy)-2nitrophenylsulfonamido)-3-cyclopropyl-2-(hydroxymethyl)-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate 61, 2.35 g, 4.61 mmol) following the same procedure as Intermediate 13. The title product was obtained as an orange foam (2.28 g, 94%).

MS: 524 ES+ (C ₂₃ H ₂₉ N ₃ O ₉ S)

Intermediate 63: (2S,5R)-tert-butyl 5-(N-(allyloxy)-2-nitrophenylsulfonamido)2-carbamoyl-3-cyclopropyl-5,6-dihydropyridine-1(2H)-carboxylate

The title compound was prepared from (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-1-(tert-butoxycarbonyl)-3-cyclopropyl-1,2,5,6-tetrahydropyridine-2- carboxylic acid (Intermediate 62, 2.28 g, 4.35 mmol) following the same procedure as Intermediate 14. The title product was obtained as an orange foam (1.07 g, 47%). MS: 523 ES+ (C23H30N4O8S)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.23 (m, 2H); 0.59 (m, 2H); 1.35 (m, 9H); 3.58 (m, 1H); 4.23 (m, 3H); 4.72 (m, 1H); 5.19 (m, 2H); 5.71 (m, 1H); 7.18 (m, 1H); 7.59 (m, 1H); 8.04 (m, 4H).

Intermediate 64: (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-3cyclopropyl-1,2,5,6-tetrahydropyridine-2-carboxamide

The title compound was prepared from (2S,5R)-tert-butyl-5-(N-(allyloxy)-2nitrophenylsulfonamido)-2-carbamoyl-3-cyclopropyl-5,6-dihydropyridine-1(2H)-carboxylate (Intermediate compound 63, 0.932 g, 1.78 mmol) following the procedure described for Intermediate 25. The title product was obtained as an orange foam (0.518 g, 68%).

MS: 423 ES+ (C ₁₈ H ₂₂ N ₄ O ₆ S)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.18 (m, 2H); 0.53 (m, 2H); 1.29 (m, 1H); 2.30 (m, 1H); 2.58 (m, 1H); 2.95 (m, 1H); 3.72 (m, 1H); 4.22 (m, 1H); 4.36 (m, 2H); 4.96 (m, 1H); 5.24 (m, 2H); 5.80 (m, 1H); 7.07 (bs, 1H); 7.39 (bs, 1H); 8.04 (m, 4H).

Intermediate 65: (R)-5-(allyloxyamino)-3-cyclopropyl-1,2,5,6-tetrahydropyridine-2-carboxamide

The title compound was prepared from (2S,5R)-5-(N-(allyloxy)-2-nitrophenylsulfonamido)-3-cyclopropyl-1,2,5,6-tetrahydropyridine-2-carboxamide (Intermediate 64, 0.518 g , 1.23 mmol) following the procedure described for Intermediate 26. The title product was obtained as a light yellow oil (0.171 g, 59%). The product was a mixture of diastereomers. MS: 238 ES+ (C ₁₂ H ₁₉ N ₃ O ₂ ) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 0.28 (m, 2H); 0.41 (m, 2H); 0.54 (m, 2H); 1.33 (m, 1H); 2.49 (m, 1H); 2.64 (m, 1H); 2.93 (m, 1H); 3.23 (m, 1H); 3.65 (m, 1H); 4.07 (m, 2H); 5.19 (m, 3H); 5.89 (m, 1H); 6.26(m, 1H); 6.97 (bs, 1H); 7.34 (bs, 1H).

- 70 044428

Bicyclo[3.2.1]oct-3-ene-2-carboxamide intermediate

66: (2S,5R)-6-(allyloxy)-3-cyclopropyl-7-oxo-1,6-diaza-

The title compound was prepared from (C)-5-(allyloxyamino)-3-cyclopropyl-1,2,5,6tetrahydropyridine-2-carboxamide (Intermediate 65, 0.316 g, 1.33 mmol) according to the procedure given for Intermediate compound 27. The target product was obtained as a colorless oil (0.261 g, 74%). MS: 264 ES+ (C ₁₃ H ₁₇ N ₃ O ₃ )

1H NMR (300 MHz, DMSO-do) δ: 0.37 (m, 2H); 0.60 (m, 2H); 1.20 (m, 1H); 2.98(m, 1H); 3.79 (m, 1H); 3.92(m, 1H); 4.20 (m, 1H); 4.33 (m, 2H); 5.28 (m, 2H); 5.93 (m, 2H); 7.30 (bs, 1H); 7.86 (bs, 1H).

Example 37 )oxy)-2-fluoroacetate lH M F 7---N ?

ο 7^

Example 38 )oxy)-2-fluoroacetate nD M F J--NJ ⁰ ° /G ⁰ o K

To a solution of (2S,5R)-6-(allyloxy)-3-cyclopropyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 66, 0.15 g , 0.57 mmol) in methanol (3 ml) at room temperature, 1,3-dimethylbarbituric acid (0.178 g, 1.14 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.066 g, 0.06 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, then concentrated to give an orange film. The orange film was dissolved in DMF (4 ml). Potassium carbonate (0.157 g, 1.14 mmol) and isopropyl 2-bromo2-fluoroacetate (Intermediate 18, 0.340 g, 1.71 mmol) were added. The reaction mixture was stirred at room temperature overnight, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed twice with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-65% ethyl acetate/hexanes) gave a 1:1 mixture of diastereomers, 166.3 mg, 86%. Separation of diastereomers was carried out using reverse phase HPLC (Atlantis T3, 19 mm x 150 mm, 5 μm, 20-40% acetonitrile in water, 20 ml/min, 15 min). After lyophilization, both diastereomers were obtained as white solids.

Example 37: (first diastereomer to elute): 47.3 mg, 24%.

MS: 342 ES+ (C ₁₅ H ₂₀ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 0.39 (m, 2H); 0.61 (m, 1H); 1.19(d,3H); 1.21(m, 1H); 1.24(d,3H); 2.99 (m, 1H); 3.88(m, 1H); 4.01 (m, 1H); 4.29 (m, 1H), 5.00 (m, 1H); 5.87 (m, 1H); 6.20 (m, 1H); 7.36(br s, 1H); 7.90 (br s, 1H).

Example 38: (second diastereomer eluting): 49.8 mg, 26%.

MS: 342 ES+ (C15H20FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.41 (m, 2H); 0.62 (m, 1H); 1.22(m, 1H); 1.27(d,3H); 1.29(d, 3H); 3.03 (m, 1H); 3.91(m, 1H); 3.94 (m, 1H); 4.31 (m, 1H), 5.05 (m, 1H); 5.91(m, 1H); 6.12 (m, 1H); 7.37(br s, 1H); 7.93 (br s, 1H).

Example 39 -2-fluoroacetate

Example 40 )-2-fluoroacetate

- 71 044428

To a solution of (2S,5R)-6-(allyloxy)-3-cyclopropyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2carboxamide (Intermediate 66, 0.2972 g, 1 .13 mmol) in methanol (6 ml) at room temperature, 1,3-dimethylbarbituric acid (0.352 g, 2.26 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.130 g, 0.11 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, then concentrated to give an orange film. The orange film was dissolved in DMF (6 ml). Potassium carbonate (0.468 g, 3.39 mmol) and ethyl bromofluoroacetate (0.534 mL, 4.52 mmol) were added. The reaction mixture was stirred overnight at room temperature, then diluted with ethyl acetate and filtered through a 0.45 μm filter to remove solid potassium carbonate. The filtrate was washed twice with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. After chromatography on silica gel (0-65% ethyl acetate/hexanes), a mixture of diastereomers was obtained in a ratio of 1:1, 303.7 mg,

82%. Separation of diastereomers was carried out using reverse phase HPLC (Atlantis T3, 19 mm x 150 mm, 5 μm, 20-40% acetonitrile in water, 20 ml/min, 15 min). After lyophilization, both diastereomers were obtained as white solids.

Example 39: (first diastereomer to elute): 107 mg, 29%

MS: 328 ES+ (C14H18FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.39 (m, 2H); 0.61 (m, 2H); 1.21 (m, 4H); 3.01 (m, 1H); 3.89 (m, 1H); 4.02 (m, 1H); 4.19 (m, 2H); 4.29(s, 1H); 5.88(m, 1H); 6.22 (m, 1H); 7.36(br s, 1H); 7.91 (brs, 1H).

Example 40: (second diastereomer eluting): 110.9 mg, 30%.

MS: 328 ES+ (C14H18FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.40 (m, 2H); 0.61 (m, 2H); 1.26 (m, 4H); 3.04 (m, 1H); 3.90 (m, 1H); 3.94 (m, 1H); 4.26(m, 3H); 5.90 (m, 1H); 6.24 (m, 1H); 7.36(br s, 1H); 7.92 (br s, 1H).

Example 41 (2R)-2-(((2S,5R)-2-carbamoyl-3-cyclopropyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)- 2-fluoroacetic acid lithium salt

To a solution of (2R)-ethyl-2-(((2S,5R)-2-carbamoyl-3-cyclopropyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3en-6-yl)oxy) -2-fluoroacetate (example 39, 96.6 mg, 0.30 mmol) in THF (3 ml) and water (1 ml) at 0°C added lithium hydroxide (1 M) (0.310 ml, 0.31 mmol) . The reaction mixture was kept in an ice bath and stirred for 15 minutes. Another 0.2 equiv was added. lithium hydroxide. After 15 minutes, the pH of the reaction mixture was adjusted to pH = 7 using 0.5N. HCl. The mixture was frozen and lyophilized to give a pale yellow solid, 90.4 mg. After reverse phase HPLC (YMC Carotenoid 30, 19 mm x 150 mm, 5 µm, coupled to Synergi Polar RP, 21.2 mm x 100 mm, 4 µm, 025% acetonitrile in water, 20 ml/min, 5 min ) gave the title compound as a white solid, 45.9 mg, 52%. MS: 300 ES+ (C12H14FN3O5) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 0.38 (m, 2H); 0.59 (m, 2H); 1.20 (m, 1H); 3.02 (m, 1H); 3.83 (m, 1H); 4.00 (m, 1H); 4.25 (m, 1H); 5.24 (m, 1H); 5.89 (m, 1H); 7.30 (br s, 1H); 7.88(br s, 1H).

Example 42 (2R)-2-(((2S,5R)-2-carbamoyl-3-cyclopropyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)- 2-fluoroacetic acid lithium salt

To a solution of (2 S)-2-(((2S,5R)-2-carbamoyl-3-cyclopropyl-7-oxo-1,6-diazabicyclo[3.2.1] oct-3-en-6yl)oxy)- 2-fluoroacetic acid (example 40, 96.6 mg, 0.30 mmol) in THF (3 ml) and water (1) at 0°C was added lithium hydroxide (1 M) (0.310 ml, 0.31 mmol). The reaction mixture was kept in an ice bath and stirred for 15 minutes. Another 0.2 equiv was added. lithium hydroxide. After 15 minutes, the pH of the reaction mixture was adjusted to pH = 7 using 0.5N. HCl. The mixture was frozen and lyophilized to give a pale yellow solid, 90.6 mg. After performing reverse phase

- 72 044428

HPLC (YMC Carotenoid 30, 19 mm x 150 mm, 5 µm, coupled to Synergi Polar RP, 21.2 mm x 100 mm, 4 µm, 0-25% acetonitrile in water, 20 ml/min, 5 min) obtained the indicated title compound as a white solid, 41.2 mg, 45%. MS: 300 ES+ (C ₁₂ H ₁₄ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-di) δ: 0.39 (m, 2H); 0.59 (m, 2H); 1.20 (m, 1H); 3.02 (m, 1H); 3.82(m, 1H); 3.98 (m, 1H); 4.24 (m, 1H); 5.24 (m, 1H); 5.90 (m, 1H); 7.31(br s, 1H); 7.90 (br s, 1H).

Example 43 2-(((2S,5R)-2-Carbamoyl-3-cyclopropyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetic acid lithium salt

(2R)-2-(((2S,5R)-2-carbamoyl-3-cyclopropyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)-2fluoroacetic acid (example 41, 8 mg, 0.03 mmol) and (2S)-2-(((2S,5R)-2-carbamoyl-3-cyclopropyl-7oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl)oxy)-2-fluoroacetic acid (Example 42, 8 mg, 0.03 mmol) was combined in an amber tube. Water (1.5 ml) was added. The mixture was frozen and lyophilized to give a white solid, 16 mg. MS: 300 ES+ (C ₁₂ H ₁₄ FN ₃ O ₅ ) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 0.39 (m, 2H); 0.58 (m, 2H); 1.19 (m, 1H); 3.00 (m, 1H); 3.81(m, 1H); 3.98 (m, 1H); 4.24 (m, 1H); 5.19 (m, 1H); 5.89 (m, 1H); 7.29(br s, 1H); 7.88(br s, 1H).

Intermediate 67: (1-isopropyl-2-methylpropyl)-2-bromo-2,2-difluoroacetate

To a solution of 2,4-dimethylpentan-3-ol (0.72 ml, 5.17 mmol) and N,N-diisopropylethylamine (1.81 ml, 10.34 mmol) in DCM (20 ml) at 0°C at 2-bromo-2,2-difluoroacetyl chloride (0.49 mL, 5.17 mmol) was added dropwise. The reaction mixture was stirred at 35°C overnight. The reaction was stopped by adding 10 ml of 1N. of hydrochloric acid. The layers were separated. The organics were washed twice with water, once with brine, then dried over magnesium sulfate, filtered and concentrated to give the title compound as a dark orange oil, 1.95 g, qty. The crude material was used in the next step.

1H NMR (300 MHz, CDCl ₃ -d) δ: 0.95 (m, 12H); 2.06 (m, 2H); 4.74 (m, 1H).

Example 44 (1-Isopropyl-2-methylpropyl)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -6-yl]oxy]-2,2-difluoroacetate

To a solution of (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 150 mg, 0.76 mmol) in DMF (5 ml) at room temperature was added potassium carbonate (210.27 mg, 1.52 mmol) and (1-isopropyl-2-methylpropyl)2-bromo-2,2-difluoroacetate (Intermediate 67, 623 .28 mg, 2.28 mmol). The reaction mixture was stirred for 4 hours at room temperature. Added another 2 eq. (1-isopropyl-2-methylpropyl)2-bromo-2,2difluoroacetate and stirred the reaction mixture overnight at room temperature. The reaction mixture was diluted with ethyl acetate and filtered to remove potassium carbonate. The filtrate was washed three times with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-15% acetone/dichloromethane) and lyophilization gave the title compound as a light orange sticky film, 44.6 mg, 13%. MS: 390 ES+ (C ₁₇ H ₂₅ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-di) δ: 0.86 (m, 12H); 1.64(s, 3H); 1.99 (m, 2H); 3.15 (m, 1H); 3.85 (m, 1H); 4.07 (m, 1H); 4.28(s, 1H); 4.69 (m, 1H); 6.03 (m, 1H); 7.42(s, 1H); 7.85(s, 1H).

Intermediate 68: Octyl-(2R)-2-bromo-2-fluoroacetate

To a suspension of (2R)-2-bromo-2-fluoroacetic acid; (1S)-1-phenylethanamine (Intermediate 168, 642.8 mg, 2.31 mmol) and 1-octanol (554 mg, 5.78 mmol) in DCM (9 ml) was added dropwise at room temperature with chlorotrimethylsilane (1 .19 ml, 9.39 mmol). The suspension turned into solution and was stirred overnight at room temperature. The reaction mixture was washed three times

- 73 044428 with water. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-10% ethyl acetate/hexanes) gave the title compound as a colorless liquid, 554.9 mg, 89%.

'H NMR (300 MHz, CDCl ₃ -d) δ: 0.84 (m, 3H); 1.24 (m, 10H); 1.61 (m, 2H); 4.22 (m, 2H); 7.25 (d, 1H).

Example 45 Octyl-(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy ]-2-fluoroacetate

To a solution of (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 150 mg, 0.76 mmol) in 1,4-dioxane (4 ml) and DMF (0.5 ml), octyl-(2R)-2-bromo-2-fluoroacetate (Intermediate 68, 0.09 ml, 2.06 mmol) was added. The reaction mixture was cooled to 0°C and DBU (0.11 mL, 0.76 mmol) was added dropwise. The reaction mixture was stirred for 10 min, then diluted with ethyl acetate and washed three times with a 1:1 mixture of brine and water. The organics were dried over magnesium sulfate, filtered and concentrated to give an orange oil. Chromatography on silica gel (0-60% ethyl acetate/hexanes) provided the title compound as a white solid, 247.9 mg, 84%. MS: 386 ES+ (C!8H28FNsO5) ‘H NMR (300 MHz, DMSO-di) δ: 0.86 (m, 3H); 1.25 (m, 10H); 1.59 (m, 5H); 3.04 (m, 1H); 3.78 (m, 1H); 4.04 (m, 1H); 4.16 (m, 3H); 6.00 (m, 1H); 6.24(d, 1H); 7.37(s, 1H); 7.81(s, 1H).

Intermediate 69: Octyl-(2R)-2-bromo-2-fluoroacetate

The title compound was prepared from (2R)-2-bromo-2-fluoroacetic acid; (1S)-phenylethanamine (Intermediate 168, 526.2 mg, 1.89 mmol) and methanol (260 mg, 5.68 mmol) following the procedure for Intermediate 68 to give a white oil/solid, 260 mg, 80 %. 'H NMR (300 MHz, CDCl ₃ -d) δ: 3.84 (s, 3H); 6.52(d, 1H).

Example 46 Methyl-(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy ]-2-fluoroacetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 120 mg, 0 .61 mmol) and methyl (2R)-2-bromo-2-fluoroacetate (Intermediate 69, 0.09 mL, 1.52 mmol) according to the procedure for Example 45 as a white foam, 81.2 mg, 46% . MS: 288 ES+ (CnHi ₄ FN ₃ O ₅ ) 'H NMR (300 MHz, DMSO-d6) δ: 1.63 (s, 3H); 3.07 (m, 1H); 3.75 (m, 1H); 3.78(s, 3H); 4.05 (m, 1H); 4.19(s, 1H); 6.02 (m, 1H); 6.24 (m, 1H); 7.37(s, 1H); 7.81(s, 1H).

Intermediate 70: Allyl-(2R)-2-bromo-2-fluoroacetate

The title compound was prepared from (2R)-2-bromo-2-fluoroacetic acid; (1S)-1phenylethanamine (Intermediate 168, 510 mg, 1.83 mmol) and allylic alcohol (0.37 mL, 5.5 mmol) following the procedure for Intermediate 68 to give a colorless liquid, 100 mg, 28%.

'H NMR (300 MHz, CDCl ₃ -d) δ: 4.71 (m, 2H); 5.31 (m, 2H); 5.86(m, 1H); 6.55 (d, 1H).

Example 47 Allyl-(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy ]-2-fluoroacetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 50 mg, 0 .25 mmol) and al-74 044428 lyl-(2R)-2-bromo-2-fluoroacetate (Intermediate 70, 100 mg, 0.51 mmol) according to the procedure for Example 45 to give a white foam, 61.3 mg, 77%.

MS: 314 ES+ (C13H16FN3O5) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 1.63 (s, 3H); 3.55 (m, 1H); 3.76 (m, 1H); 4.05 (m, 1H); 4.19(s, 1H);

4.70 (m, 2H); 5.36 (m, 2H); 5.90 (m, 1H); 6.01 (m, 1H); 6.28(d, 1H); 7.37(s, 1H); 7.82(s, 1H).

Intermediate 71: propyl-(2R)-2-bromo-2-fluoroacetate

The title compound was prepared from (2R)-2-bromo-2-fluoroacetic acid; (1S)-1phenylethanamine (Intermediate 168, 444.9 mg, 1.6 mmol) and 1-propanol (0.3 mL, 4 mmol) following the procedure for Intermediate 68 to give a colorless liquid, 318 mg, 100%.

1H NMR (300 MHz, CDCl3-d) δ: 0.92 (t, 3H); 1.67 (m, 2H); 4.19 (m, 2H); 6.51(d, 1H).

Example 48 Propyl-(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl]oxy ]-2-fluoroacetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 150 mg, 0 .76 mmol) and propyl-(2R)-2-bromo-2-fluoroacetate (Intermediate 71, 302.78 mg, 1.52 mmol) according to the procedure for Example 45 to give a white sticky foam, 156 mg, 65%.

MS: 316 ES+ (C13H18FN3O5)

Ή NMR (300 MHz, DMSO-d6) δ: 0.89 (t, 3H); 1.61 (m, 5H); 3.05 (m, 1H); 3.76 (m, 1H); 4.04 (m, 1H); 4.13 (m, 2H); 4.19 (m, 1H); 6.01 (m, 1H); 6.25(d, 1H); 7.37(s, 1H); 7.82(s, 1H).

Intermediate 72: Isobutyl-(2K)-2-bromo-2-fluoroacetate

The title compound was prepared from (2R)-2-bromo-2-fluoroacetic acid; (1S)-1phenylethanamine (Intermediate 168, 487.5 mg, 1.75 mmol) and 2-methyl-1-propanol (0.4 mL, 4.38 mmol) following the procedure for Intermediate 68 to obtain a colorless liquid, 373 mg, 100%.

¹ H NMR (300 MHz, CDCl ₃ -d) δ: 0.91 (d, 6H); 1.97 (m, 1H); 4.00 (m, 2H); 6.51(d, 1H).

Example 49. Isobutyl-(2R)-2-[[(2 S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 en-6-yl ]oxy]-2-fluoroacetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 150 mg, 0 .76 mmol) and isobutyl-(2R)-2-bromo-2-fluoroacetate (Intermediate 72, 372.73 mg, 1.75 mmol) according to the procedure for Example 45 to give a white sticky foam, 161 mg, 64%.

MS: 330 ES+ (C ₁₄ H ₂₀ FN ₃ O ₅ ) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 0.90 (d, 6H); 1.63(s, 3H); 1.90 (m, 1H); 3.05 (m, 1H); 3.76 (m, 1H); 3.97 (m, 2H); 4.04 (m, 1H); 4.20(s, 1H); 6.00 (m, 1H); 6.27(d, 1H); 7.37(s, 1H); 7.82(s, 1H).

Intermediate 73: Butyl-(2R)-2-bromo-2-fluoroacetate

The title compound was prepared from (2R)-2-bromo-2-fluoroacetic acid; (1S)-1phenylethanamine (Intermediate 168, 400 mg, 1.44 mmol) and 1-butanol (0.33 mL, 3.6 mmol) following the procedure for Intermediate 68 to give a colorless liquid, 322 mg, qt.

¹ H NMR (300 MHz, CDCl ₃ -d) δ: 0.88 (t, 3H); 1.33 (m, 2H); 1.65 (m, 2H); 4.23 (m, 2H); 6.50(d, 1H).

- 75 044428

Example 50 Butyl-(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy ]-2-fluoroacetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 150 mg, 0 .76 mmol) and butyl (2R)-2-bromo-2-fluoroacetate (Intermediate 73, 322.49 mg, 1.51 mmol) according to the procedure for Example 45 to give a white sticky foam, 198 mg, 79%.

MS: 330 ES+ (C ₁₄ H ₂₀ FN ₃ O ₅ )

1H NMR (300 MHz, DMSO-d6) δ: 0.88 (t, 3H); 1.35 (m, 2H); 1.59 (m, 2H); 1.63(s, 3H); 3.06 (m, 1H); 3.77 (m, 1H); 4.04 (m, 1H); 4.20 (m, 2H); 4.21(s, 1H); 6.00 (m, 1H); 6.24(d, 1H); 7.37(s, 1H); 7.82(s, 1H).

Intermediate 74: Pentyl-(2R)-2-bromo-2-fluoroacetate

The title compound was prepared from (2R)-2-bromo-2-fluoroacetic acid; (1S)-1phenylethanamine (Intermediate 168, 474.2 mg, 1.71 mmol) and 1-pentanol (0.46 mL, 4.26 mmol) following the procedure for Intermediate 68 to give a colorless liquid, 363 mg, 93 %.

1H NMR (300 MHz, CDCl ₃ -d) δ: 0.88 (m, 3H); 1.28 (m, 4H); 1.60 (m, 2H); 4.17 (m, 2H); 6.45(d, 1H).

Example 51 Pentyl-(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en6-yl]oxy ]-2-fluoroacetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 150 mg, 0 .76 mmol) and pentyl-(2R)-2-bromo-2-fluoroacetate (Intermediate 74, 362.73 mg, 1.6 mmol) following the procedure for Example 45 to give a sticky white foam, 225 mg, 86%.

MS: 344 ES+ (C15H22FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.87 (m, 3H); 1.30 (m, 4H); 1.58 (m, 2H); 1.63(s, 3H); 3.04 (m, 1H); 3.77 (m, 1H); 4.04 (m, 1H); 4.20 (m, 2H); 4.21(s, 1H); 6.00 (m, 1H); 6.24(d, 1H); 7.37(s, 1H); 7.82(s, 1H).

Intermediate 75: Hexyl-(2R)-2-bromo-2-fluoroacetate

The title compound was prepared from (2R)-2-bromo-2-fluoroacetic acid; (1S)-1phenylethanamine (Intermediate 168, 400 mg, 1.44 mmol) and hexyl alcohol (0.45 mL, 3.6 mmol) following the procedure for Intermediate 68 to give a colorless liquid, 313 mg, 90%.

1H NMR (300 MHz, CDCl3-d) δ: 0.86 (m, 3H); 1.22 (m, 6H); 1.59 (m, 2H); 4.17 (m, 2H); 6.45(d, 1H).

Example 52 Hexyl-(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy ]-2-fluoroacetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 193, 150 mg, 0 .76 mmol) and hexyl-(2R)-2-bromo-2-fluoroacetate (Intermediate 75, 313.62 mg, 1.3 mmol) according to the procedure for Example 45 to give a sticky white foam, 224 mg, 82%.

MS: 358 ES+ (C16H24FN3O5)

1H NMR (300 MHz, DMSO-d6) δ: 0.87 (m, 3H); 1.30 (m, 6H); 1.57 (m, 2H); 1.62(s, 3H); 3.04 (m, 1H); 3.77 (m, 1H); 4.04 (m, 1H); 4.19 (m, 2H); 4.20(s, 1H); 6.00 (m, 1H); 6.24(d, 1H); 7.37(s, 1H); 7.82(s, 1H).

Intermediate 76: 1-chloroethyl isopropyl carbonate

- 76 044428

To a solution of 2-propanol (0.64 ml, 8.39 mmol) and pyridine (0.79 ml, 9.79 mmol) at -78°C, 1-chloroethyl chloroformate (0.76 ml, 6.99 mmol) was added dropwise ). The reaction mixture was allowed to warm slowly to room temperature and stirred overnight. The reaction mixture turned into a white solid and was sonicated in dichloromethane. The resulting suspension was concentrated and the white solid was dissolved in ethyl acetate, and washed with water and brine. The organics were dried over magnesium sulfate, filtered and concentrated to give the title compound as a colorless liquid, 1.29 g, 99%.

1H NMR (300 MHz, CDCl ₃ -d) δ: 1.35 (m, 6H); 1.84(d,3H); 4.96 (m, 1H); 4.44 (m, 1H).

Example 53 ]hydroxy]-2-fluoroacetate

To a solution of (2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy] -2-fluoroacetic acid (example 4, 194.46 mg, 0.71 mmol), Ν,N-diisopropylethylamine (0.12 ml, 0.71 mmol) and 1-chloroethyl isopropyl carbonate (Intermediate 76, 237.15 mg, 1.42 mmol) in DMF (5 ml) was added tetrabutyl ammonium chloride (162.22 mg, 0.71 mmol) at room temperature. The reaction mixture was heated at 40°C for about 4 hours, then diluted with ethyl acetate and washed twice with a 1:1 mixture of brine and water. The organics were dried over magnesium sulfate, filtered and concentrated to give an orange oil. Chromatography on silica gel (0-50% ethyl acetate/hexanes) provided the title compound as a 1:1 mixture of diastereomers as a white foam, 33.2 mg, 11%. MS: 404 ES+ (C ₁₆ H ₂₂ FN ₃ O ₈ )

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.23 (m, 6H); 1.46 (m, 3H); 1.62 (m, 3H); 3.05 (m, 1H); 3.79 (m, 1H); 4.00 (m, 1H); 4.19(s, 1H); 4.80 (m, 1H); 6.00 (m, 1H); 6.33 (dd, 1H); 7.37(s, 1H); 7.80(d, 1H).

Example 54 )-2-fluoroacetate

DBU (8.83 mL, 58.57 mmol) in DMF (30 mL) was added dropwise to a solution of (2S,5R)-6-hydroxy-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct -3-ene-2-carboxamide (Intermediate 193, 10.5 g, 53.25 mmol) and (R)-benzyl-2-bromo-2-fluoroacetate (Intermediate 171, 14.47 g, 58.57 mmol) in DMF (100 ml) at -40°C for 30 min under nitrogen. The resulting solution was stirred at -40°C for 30 min, then the reaction was stopped by adding water (15 ml). The reaction mixture was extracted with ethyl acetate (3x20 ml). The combined organics were washed with brine (3x20 ml), dried over sodium sulfate, filtered and concentrated. Chromatography on silica gel (0-40% ethyl acetate/petroleum ether) gave the title compound as a white solid, 8.8 g, 45%. MS: 364 ES+ (C ₁₇ H ₁₈ FN ₃ O ₅ )

1H NMR (400 MHz, DMSO-d6) δ: 1.62 (s, 3H); 2.92(d, 1H); 3.75(d, 1H); 3.99 (m, 1H); 4.02(s, 1H); 5.25(s, 2H); 5.76(s, 1H); 6.33(d, 1H); 7.38 (m, 5H); 7.39(s, 1H); 7.85(s, 1H).

Intermediate 77: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid

To a solution of methyl (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylate (Intermediate 185, 538.5 mg, 1.65 mmol) Trimethyltin hydroxide (477.17 mg, 2.64 mmol) was added to DCE (3 mL) in a 20 mL microwave-safe tube. The reaction was carried out in a microwave oven for 2 hours at 80°C. The solvent was removed and the resulting residue was dissolved in ethyl acetate and washed three times with 0.01N. potassium bisulfate solution and once with brine. Organics were dried over sodium sulfate, filtered and concentrated to give an orange foam, 649 mg, 100%. MS: 313 ES+ (C ₁₄ H ₂₄ N2O ₄ Si)

- 77 044428 ¹ H NMR (300 MHz, DMSO-d6) δ: 0.00 (s, 6H); 0.78(s,9H); 1.50(s, 3H); 2.92 (m, 1H); 3.47 (m, 1H); 3.53 (m, 1H); 3.77 (m, 1H); 5.85 (m, 1H).

Intermediate 78: 2-[2-[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo1,6-diazabicyclo[3.2.1]oct-3-ene -2-carbonyl]hydrazino]-2-oxoacetamide

To a solution of (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct3-ene-2-carboxylic acid (Intermediate 77, 397.5 mg, 1.02 mmol) in DMF (6 ml) at 0°C was added oxamic acid hydrazide (209.83 mg, 2.04 mmol), HATU (387 mg, 1.02 mmol) and N ,N-diisopropylethylamine (0.57 ml, 3.26 mmol). The reaction mixture was stirred for 1 hour, then diluted with ethyl acetate and washed once with saturated sodium bicarbonate solution. The aqueous solution contained part of the product and was subjected to a single extraction with ethyl acetate. The combined organics were dried over magnesium sulfate, filtered and concentrated to give a yellow oil. Chromatography on silica gel (0-5% methanol) provided the title compound as a light yellow solid, 142 mg, 35%. MS: 398 ES+ (C16H27N5O5Si)

1H NMR (300 MHz, DMSOL) δ: 0.00 (s, 6H); 0.78(s,9H); 1.53(s, 3H); 2.95 (m, 1H); 3.60 (m, 1H); 3.67 (m, 1H); 4.04(s, 1H); 5.98(m, 1H); 7.75(s, 1H); 8.07(s, 1H); 10.30(s, 1H); 10.48(s, 1H).

Intermediate 79: 5-[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2- yl]-1,3,4-oxadiazol-2-carboxamide

To a solution of 2-[2-[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene 4-nitrobenzenesulfonyl chloride (79.17 mg, 0.36 mmol) and N ,N-diisopropylethylamine (0.19 ml, 1.07 mmol). The reaction mixture turned yellow and was stirred for 30 minutes, then diluted with dichloromethane and washed with brine. The organics were dried over magnesium sulfate, filtered and concentrated to give an orange oil. Chromatography on silica gel (0-5% methanol/dichloromethane) gave the title compound as an orange foam, 93.8 mg, 69%.

MS: 380 ES+ (C ₁₆ H ₂₅ N ₅ O ₄ Si)

1H NMR (300 MHz, DMSOL) δ: 0.00 (s, 6H); 0.77(s,9H); 1.53(s, 3H); 3.02 (m, 2H); 3.70 (m, 1H); 5.01(s, 1H); 6.14 (m, 1H); 8.13(s, 1H); 8.53(s, 1H).

Intermediate 80: 5-[(2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct3-en-2-yl]-1,3,4- Oxadiazole-2-carboxamide

To a solution of 5-[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2- yl]-1,3,4-oxadiazol-2-carboxamide (Intermediate 79, 267.6 mg, 0.71 mmol) in ethyl acetate (6 ml) was added HF-pyridine (0.04 ml, 1.41 mmol) . The reaction mixture was stirred for 2.5 hours, then another 2 eq. was added. HF-pyridine and stirred the reaction mixture for an additional 1 hour. The reaction mixture was concentrated to give the title compound as a brownish beige solid, 245 mg, 99%. MS: 266 ES+ (C ₁₀ H _n N ₅ O ₄ )

1H NMR (300 MHz, DMSO-d6) δ: 1.52 (s, 3H); 3.01 (m, 2H); 3.66(m, 1H); 4.95(s, 1H); 6.18 (m, 1H); 8.09(s, 1H); 8.45 (m, 1H).

Intermediate 81: Ethyl-(2S)-2-[[(2S,5R)-2-(5-carbamoyl-1,3,4-oxadiazol-2-yl)-3methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]-2-fluoroacetate

- 78 044428

To a solution of 5-[(2 S,5R)-6-hydroxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-2-yl]-1,3,4oxadiazole -2-carboxamide (Intermediate 80, 176.8 mg, 0.67 mmol) in DMF (5 ml) was added ethyl (2S)-2-bromo-2-fluoroacetate (0.16 ml, 1.33 mmol) and potassium carbonate (276.39 mg, 2 mmol). The reaction mixture was stirred for 2 hours, then diluted with ethyl acetate, filtered and washed three times with a 1:1 mixture of brine and water. The organics were dried over magnesium sulfate, filtered and concentrated to give a yellow oil. Chromatography on silica gel (0-70% ethyl acetate/hexanes) gave the title compound as a light yellow film, 66.8 mg, 27%. The compound was a mixture of diastereomers in a ratio of 7:3. MS: 370 ES+ (C14H16FN5O6)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.25 (m, 3H); 1.72(s, 3H); 3.20 (m, 2H); 4.19 (m, 1H); 5.30 (m, 1H); 6.25 (m, 1H); 6.26(m, 1H); 8.26(s, 1H); 8.65(s, 1H).

Example 55 ]oct-3-en-6-yl]oxy]-2-fluoroacetic acid lithium salt

To a solution of ethyl-(2S)-2-[[(2S,5R)-2-(5-carbamoyl-1,3,4-oxadiazol-2-yl)-3-methyl-7-oxo-1,6diazabicyclo[ 3.2.1]oct-3-en-6-yl]oxy]-2-fluoroacetate (Intermediate 81, 66.8 mg, 0.18 mmol) in THF (1 ml) and water (0.5 ml) at At 0°C, lithium hydroxide (0.18 mL, 0.18 mmol) was added. The reaction mixture was stirred for 10 minutes. Another 0.5 equiv was added. lithium hydroxide. After 10 min, the reaction mixture was treated with an additional 0.5 eq. lithium hydroxide. The reaction mixture was stirred for 20 minutes, neutralized by adding 0.5N. HCl, frozen and lyophilized to give a yellow solid. Reverse phase ISCO (50 g RediSep Gold C18, 100% water, 4 min; then 0-50% acetonitrile/water) gave the title compound as an off-white solid, 26 mg, 36%. The compound was a mixture of diastereomers in a ratio of 7:3. MS: 342 ES+ (C12H12FN5O6)

1H NMR (300 MHz, DMSO-d6) δ: 1.70 (s, 3H); 3.20 (m, 2H); 4.15 (m, 1H); 5.20(s, 1H); 5.28 (m, 1H); 6.27 (m, 1H); 8.25(s, 1H); 8.65(s, 1H).

Intermediate 82: 2-(1,3-Dioxoisoindolin-2-yl)oxyethanesulfonamide

To a suspension of 2-hydroxyethanesulfonamide (Enamine, 1.92 ml, 7.19 mmol), N-hydroxyphthalimide (1.41 g, 8.63 mmol) and triphenylphosphine (2.26 g, 8.63 mmol) at 0°C diisopropyl azodicarboxylate (1.7 mL, 8.63 mmol) was added dropwise. The reaction mixture turned dark orange and then pale yellow. After stirring for about 3 hours, the reaction mixture was concentrated to give a viscous pale yellow oil, which was purified by washing with ethyl acetate/hexanes. The white solid was collected by filtration and was the title compound, 1.8 g, 70%. MS: 271 ES+ (C ₁₀ H ₁₀ N ₂ O ₅ S)

1H NMR (300 MHz, DMSO-d6) δ: 3.52 (m, 2H); 4.50 (m, 2H); 6.96(s, 2H); 7.87(s,4H).

Intermediate 83: 2-aminooxyethanesulfonamide

To a solution of 2-(1,3-dioxoisoindolin-2-yl)oxyethanesulfonamide (Intermediate 82, 740 mg, 2.05 mmol) in DCM (20 mL) was added methylhydrazine (0.11 mL, 2.05 mmol) at room temperature ). A precipitate immediately formed. The suspension was stirred at room temperature for about 2 hours and then concentrated. The solid was purified by washing with DCM and collected by filtration. The solid was purified by washing with methanol, and the resulting white solid was filtered. An NMR spectrum of the solid showed that it was a by-product. The filtrate was concentrated to give the title compound as an off-white solid, 228.2 mg, 58%. The NMR spectrum showed that 73% of the target was obtained

- 79 044428 product.

Ή NMR (300 MHz, DMSO-d^δ: 3.27 (m, 2H); 3.85 (m, 2H); 6.11 (bs, 2H); 6.79 (s, 2H).

Intermediate 84: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-N-(2sulfamoylethoxy)-1,6-diazabicyclo[3.2.1]oct-3 -ene-2-carboxamide o ^z 'otbs

To a solution of (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct3-ene-2-carboxylic acid (Intermediate 77, 465.74 mg, 1.19 mmol) in DMF (8 ml) at 0°C was added 2-aminooxyethanesulfonamide (Intermediate 83, 228.96 mg, 1.19 mmol), HATU (453.43 mg, 1 .19 mmol) and N,N-diisopropylethylamine (0.57 ml, 3.26 mmol). The reaction mixture was then stirred for 1 hour at 0°C, then diluted with ethyl acetate and washed twice with a 1:1 mixture of brine and water. The organics were dried over magnesium sulfate, filtered and concentrated to give a yellow oil. Chromatography on silica gel (080% ethyl acetate/hexanes) gave the title compound as a colorless oil, 343 mg, 66%. MS: 435 ES+ (C16H ₃ 0N4O6SiS)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.00 (s, 6H); 0.78(s,9H); 1.45(s, 3H); 2.97 (m, 1H); 3.20 (m, 2H); 3.57 (m, 2H); 3.84 (m, 1H); 4.02 (m, 2H); 6.01 (m, 1H); 6.80(s, 2H); 11.74(s, 1H).

Intermediate 85: (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-(2-sulfamoylethoxy)-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

To a solution of (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-N-(2-sulfamoylethoxy)-1,6 diazabicyclo[3.2.1]oct-3- HF-pyridine (0.04 mL, 1.58 mmol) was added to ene-2-carboxamide (Intermediate 84, 343 mg, 0.79 mmol) in ethyl acetate (4 mL). The reaction mixture was stirred for 30 minutes. Added another 2 eq. HF-pyridine and stirred the reaction mixture for another hour. After 6 hours and a total of 7 eq. The HF-pyridine reaction was completed. The reaction mixture was filtered to collect an off-white solid. The solid became viscous and stuck to the filter. Ethyl acetate and a small amount of methanol were used to rinse the filter and transfer the solid to the flask. The solvent was removed in vacuo to give the title compound as an off-white solid, 337 mg, 100%. MS: 321 ES+ (C ₁₀ H ₁₆ N ₄ O ₆ S)

Intermediate 86: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-(2-sulfamoylethoxycarbamoyl)-1,6-diazabicyclo [3.2.1] oct-3-en-6-yl]oxy]acetate ^11

O ^N M _F °

O

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-(2sulfamoylethoxy)-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide ( Intermediate 85, 279.32 mg, 0.65 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.08 ml, 0.65 mmol) following the procedure for Example 45 to give light yellow oil, 26.7 mg, 10%. MS: 425 ES+ (C14H21FN4O8S)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.25 (m, 3H); 1.60 (m, 3H); 3.15 (m, 1H); 3.36 (m, 1H); 3.78 (m, 1H); 4.02 (m, 3H); 4.07 (m, 2H); 4.28 (m, 2H); 6.13 (m, 1H); 6.16(d, 1H); 6.95(s, 2H); 11.91(s,1H).

Example 56 -6-yl]oxy]acetic acid lithium salt

To a solution of ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-(2-sulfamoylethoxy-carbamoyl)-1,6diazabicyclo[3.2.1]oct- 3-en-6-yl]oxy]acetate (Intermediate 86, 26.7 mg, 0.06 mmol) in THF (1 ml) and water (0.5 ml) at 0°C was added LiOH (0.06 ml, 0.06 mmol). The reaction mixture was stirred for 15 minutes and another 0.5 equivalent of lithium hydroxide was added. After 15 minutes added

- 80 044428 another 0.5 eq. lithium hydroxide was added. After 30 minutes and after some heating, the reaction was complete. The reaction mixture was neutralized by adding 0.5N. HCl, frozen and lyophilized to give a yellow oil. The compound was purified by reverse phase ISCO (5.5 g RediSep Gold C18,

100% water). The title compound was obtained as an off-white solid, 11.3 mg, 29%. MS: 397 ES+ (C12H17FN4O8S) 'H NMR (300 MHz, DMSO-d ₆ ) δ: 1.56 (s, 3H); 3.02 (m, 1H); 3.26(m, 1H); 3.98 (m, 5H); 5.20(d, 1H); 6.03 (m, 1H); 7.06(s, 2H).

Intermediate 87: (2S,5R)-6-allyloxy-3-methyl-7-oxo-N-(2-sulfamoylethyl)-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-allyloxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid (Intermediate 29, 208.5 mg, 0.88 mmol) and 2-amino-ethylsulfonamide hydrochloride (281.1 mg, 1.75 mmol) following the procedure for Intermediate 84 to give a pale yellow oil, 86.6, 29%.

MS: 345 ES+ (C13H20N4O5S)

1H NMR (300 MHz, DMSO-d6) δ: 1.63 (s, 3H); 3.04 (m, 1H); 3.14 (m, 2H); 3.26(m, 1H); 3.51 (m, 2H); 3.94 (m, 1H); 4.09 (m, 1H); 4.36 (m, 2H); 5.27 (m, 2H); 5.95 (m, 1H); 6.07 (m, 1H); 6.89(s, 2H); 8.52 (m, 1H).

Intermediate 88: (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-(2-sulfamoylethyl)-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

To a solution of (2S,5R)-6-allyloxy-3-methyl-7-oxo-N-(2-sulfamoylethyl)-1,6-diazabicyclo[3.2.1]oct3-ene-2-carboxamide (Intermediate 87, 86.6 mg, 0.25 mmol) in methanol (3 ml) at room temperature, 1,3-dimethylbarbituric acid (78.53 mg, 0.5 mmol) and tetrakis(triphenylphosphine) palladium(0) (58.12 mg, 0.05 mmol). The reaction mixture was stirred for 1 hour at room temperature, then concentrated to give a dark orange oil, 76.5 mg, 100%. MS: 305 ES+ (C ₁₀ H ₁₆ N ₄ O ₅ S)

Intermediate 89: Ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-(2-sulfamoylethylcarbamoyl)-1,6-diazabicyclo[3.2.1] oct-3-en-6-yl]oxy]acetate

To a solution of (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-(2-sulfamoylethyl)-1,6-diazabicyclo[3.2.1]oct-3ene-2-carboxamide (Intermediate 88, 76.5 mg, 0.25 mmol) in 1,4-dioxane (2 ml) and DMF (0.25 ml) was added ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.09 ml, 0.75 mmol). The reaction mixture was cooled to 0°C and DBU (0.04 mL, 0.25 mmol) was added dropwise. Ethyl (2S)-2-bromo-2-fluoroacetate (0.09 mL, 0.75 mmol) was added again. DBU (0.04 mL, 0.25 mmol) was then added again. After another 15 min at 0°C with periodic heating, another 0.5 eq was added. DBU. The reaction mixture was stirred for another 15 minutes at room temperature. The reaction mixture was diluted with ethyl acetate and washed three times with a 1:1 mixture of brine and water. The organics were dried over magnesium sulfate, filtered and concentrated to give an orange oil. Chromatography on silica gel (0-25% acetone/dichloromethane) gave the title compound with some triphenylphosphine oxide as an orange film, 77.3 mg, 75%. MS: 409 ES+ (C ₁₄ H ₂₁ FN ₄ O ₇ S)

Example 57 -en-6-yl]oxy]acetic acid lithium salt

To a solution of ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-(2-sulfamoylethyl-carbamoyl)-1,6- 81 044428 diazabicyclo[3.2. 1]oct-3-en-6-yl]oxy]acetate (Intermediate 89, 72.3 mg, 0.18 mmol) in THF (2 ml) and water (1 ml) at 0°C added 1 M hydroxide lithium (0.18 ml, 0.18 mmol). The reaction mixture was stirred for 10 minutes at 0°C, then neutralized by adding 0.5N. hydrochloric acid, frozen and lyophilized to give a yellow solid. Reverse phase ISCO (100% water) provided the title compound as a light yellow solid, 14.3 mg, 21%. MS: 381 ES+ (C ₁₂ H ₁₇ FN ₄ O ₇ S) ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: 1.64 (s, 3H); 3.13(m, 3H); 3.52 (m, 3H); 3.99 (m, 1H); 4.14 (m, 1H); 5.24(d, 1H); 6.04 (m, 1H); 6.90 (m, 2H); 8.57 (m, 1H).

Example 58 Ethyl 2-[[(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -yl]oxy]-2-fluoroacetyl]oxymethoxycarbonylamino]-3-methylbutanoate

To a solution of (2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2 -fluoroacetic acid (example 4, 100 mg, 0.37 mmol), Hunig's base (0.06 ml, 0.37 mmol) and ethyl 2-(chloromethoxycarbonylamino)-3-methylbutanoate (0.05 ml, 0.73 mmol) tetrabutyl ammonium chloride (83.42 mg, 0.37 mmol) was added to DMF (1.5 ml) at room temperature. The reaction mixture was stirred at 40°C for 2 hours. It was then diluted with ethyl acetate and washed twice with brine/water (1:1). The organics were dried over anhydrous magnesium sulfate, filtered and concentrated to give an orange oil. Chromatography on silica gel (080% ethyl acetate/hexanes) gave the title compound (7 mg, 3.63%) as a white solid. MS: 475 ES+ (C ₁₉ H ₂₇ FN ₄ O ₉ )

1H NMR (300 MHz, DMSO-d ₆ ) δ: 0.90 (m, 6H); 1.21(m, 3H); 1.61(s, 3H); 2.15 (m, 1H); 3.78 (m, 1H); 3.95 (m, 1H); 4.01 (m, 1H); 4.15 (m, 2H); 4.22 (m, 1H); 5.80 (m, 2H); 5.95 (m, 1H); 6.30(d, 1H); 7.40(s, 1H); 7.80(s, 1H); 8.10(d,1H).

Intermediate 90: tert-butyl-(2S)-2-[(1,3-dioxoisoindolin-2-yl)oxymethyl]pyrrolidine-1-carboxylate

To a solution of diethyl azodicarboxylate (14.21 ml, 12.48 mmol) in THF (10 ml) at -10°C, a solution of triphenylphosphine (3273.22 mg, 12.48 mmol) in THF (20 ml) was added dropwise. The suspension was stirred at -10°C. After 20 minutes, the suspension became a solid and another 40 ml of THF was added. After 1 hour, a solution of Boc-L-prolinol (448.18 ml, 5.94 mmol) in THF (10 ml) was added, followed by a solution of Nhydroxyphthalimide (969.41 mg, 5.94 mmol) in THF (10 ml). The reaction mixture was allowed to warm to room temperature and stirred overnight. It was concentrated and the resulting oil was purified by washing with ethyl acetate/hexanes. The residue was removed by filtration and the filtrate was concentrated on silica gel. Chromatography on silica gel (0-40% EtOAc/hexanes) provided the title compound (2.37 g, quant.) as a light yellow solid. MS: 347 ES+ (C ₁₈ H ₂₂ N ₂ O ₅ )

Intermediate 91: tert-butyl-(2S)-2-(aminooxymethyl)pyrrolidine-1-carboxylate

To a solution of tert-butyl-(2S)-2-[(1,3-dioxoisoindolin-2-yl)oxymethyl]pyrrolidine-1-carboxylate (Intermediate 90, 2.06 g, 5.95 mmol) in DCM (20 ml) hydrazine monohydrate (2.14 ml, 17.84 mmol) was added at room temperature. A white precipitate immediately formed. The suspension was stirred at room temperature for 1 hour, then filtered through celite. The filtrate was washed twice with brine/water (1:1), dried over anhydrous magnesium sulfate, filtered and concentrated to give the title compound (1.35 g, quant.) as a viscous oil.

¹ H NMR (300 MHz, CDCl ₃ ) δ: 1.50 (s, 9H); 1.85 (m, 4H); 3.32 (m, 2H); 3.63 (m, 1H); 4.25 (m, 2H).

Intermediate 92: tert-butyl-(2S)-2-[[[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3methyl-7-oxo-1,6-diazabicyclo [3.2. 1] oct-3-ene-2-carbonyl] amino] hydroxymethyl] pyrrolidine-1-carboxylate

- 82 044428

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 1.85 g, 5.92 mmol) and tert-butyl (2S)-2-(aminooxymethyl)pyrrolidine-1-carboxylate (Intermediate 91, 1.28 g, 5.92 mmol) following the procedure for Intermediate 84 to obtain (585 mg, 19%) as a white sticky foam. MS: 511 ES+ (C ₂₄ H ₄₂ N ₄ O ₆ Si)

Intermediate 93: tert-butyl-(2S)-2-[[[(2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2 -carbonyl]amino]oxymethyl]pyrrolidine-1-carboxylate

To a solution of tert-butyl-(2S)-2-[[[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1] oct-3-ene-2-carbonyl]amino]oxymethyl]pyrrolidine-1-carboxylate (Intermediate 92, 401 mg, 0.79 mmol) in ethyl acetate (20 ml) at 0°C under nitrogen was added HFpyridine (24 µl , 0.94 mmol). The reaction mixture was stirred at room temperature for 3 hours and then concentrated. The crude material was partitioned between DCM (100 ml) and brine (50 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated to give the title compound (309 mg, 84%) as a white sticky solid.

MS: 396 ES+ (C' ₈ H28N4O6)

Example 59. tert-Butyl-(2S)-2-((((2S,5R)-6-((S)-2-ethoxy-1-fluoro-2-oxoethoxy)-3-methyl-7-oxo- 1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamido)oxy)methyl)pyrrolidine-1-carboxylate

To a suspension of tert-butyl-(2S)-2-[[[(2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3ene-2-carbonyl ]amino]oxymethyl]pyrrolidine-1-carboxylate (Intermediate 93, 309 mg, 0.78 mmol) and cesium carbonate (304.75 mg, 0.94 mmol) in THF (15 ml) at 0°C was added ethyl- (2S)-2bromo-2-fluoroacetate (Intermediate 174, 0.14 ml, 1.17 mmol). The reaction mixture was stirred at 0°C for 8 hours. Water (50 ml) and EtOAc (100 ml) were added. The organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by flash chromatography (20 g silica gel, 0-100% EtOAc/hexanes) to give the title compound (164 mg, 40%) as a sticky solid.

MS: 501 ES+ (C22H33FN4O8) 'H NMR (300 MHz, CDCl ₃ ) δ: 1.38 (m, 3H); 1.45(s,9H); 1.80(s, 3H); 1.95 (m, 4H); 3.35 (m, 3H); 3.60 (m, 2H); 3.95 (m, 1H); 4.01 (m, 1H); 4.28 (m, 2H); 4.35 (m, 2H); 5.75(d, 1H); 6.10(s, 1H).

Example 60 Ethyl-(2S)-2-fluoro-2-(((2S,5R)-3-methyl-7-oxo-2-((((S)-pyrrolidin-2-yl)methoxy)carbamoyl) -1,6-diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)acetate TFA salt

To a solution of tert-butyl-(2S)-2-((((2S,5R)-6-((S)-2-ethoxy-1-fluoro-2-oxoethoxy)-3-methyl-7-oxo-1 ,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamido)oxy)methyl)pyrrolidine-1-carboxylate (example 59, 76 mg, 0.15 mmol) in DCM (10 ml) at 0°C dropwise TFA (0.58 mL, 7.59 mmol) was added. The reaction mixture was stirred at 0°C for 2 hours and then concentrated. The residue was purified by washing with diethyl ether to give the title compound (53 mg, 83%) as the TFA salt.

MS: 401 ES+ (C ₁₇ H ₂₅ FN ₄ O ₆ ) 'H NMR (300 MHz, CDCl ₃ ) δ: 1.45 (m, 3H); 1.86 (m, 4H); 2.15 (m, 3H); 3.55 (m, 4H); 4.01 (m, 2H); 4.32 (m, 5H); 5.85(d, 1H); 6.13(s, 1H).

- 83 044428

Intermediate 94: tert-butyl-(S)-2-(((1,3-dioxoisoindolin-2-yl)oxy)methyl)-4,4difluoropyrrolidine-1-carboxylate

The title compound was prepared from tert-butyl(8)-4,4-difluoro-2-(hydroxymethyl)pyrrolidine-1-carboxylate (2.8 g, 11.8 mmol) according to the procedure for Intermediate 90 to give ( 4.3 g, 95%) pale yellow sticky solid. MS: 383 ES+ (C ₁₈ H ₂₀ F ₂ N ₂ O ₅ )

Intermediate 95: tert-butyl-(S)-2-((aminooxy)methyl)-4,4-difluoropyrrolidine-1carboxylate

The title compound was prepared from tert-butyl(8)-2-(((1,3-dioxoisoindolin-2yl)oxy)methyl)-4,4-difluoropyrrolidine-1-carboxylate (Intermediate 94, 1.9 g , 4.97 mmol) following the procedure for Intermediate 91 to obtain (1.25 g, 99%) as an orange viscous oil.

1H NMR (300 MHz, CDCl ₃ ) δ: 1.51 (s, 9H); 2.42(m, 2H); 3.63 (m, 2H); 3.85 (m, 2H); 4.97 (m, 1H).

Intermediate 96: tert-butyl-(2S)-2-((((2S,5R)-6-((tert-butyldimethylsilyl)oxy)-3methyl-7-oxo-1,6-diazabicyclo[3.2.1] Oct-3-ene-2-carboxamido)oxy)methyl)-4,4-difluoropyrrolidine-1carboxylate

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 1.3 g, 4.16 mmol) and tert-butyl-(S)-2-((aminooxy)methyl)-4,4-difluoropyrrolidine-1-carboxylate (Intermediate 95, 1, 26 g, 4.99 mmol) following the procedure for Intermediate 84 to obtain (489 mg, 18%) as a sticky white foam. MS: 547 ES+ (C ₂₄ H ₄₀ F ₂ N ₄ O ₆ Si)

Intermediate 97: tert-butyl-(2S)-4,4-difluoro-2-((((2S,5R)-6-hydroxy-3-methyl-7oxo-1,6-diazabicyclo[3.2.1]oct -3-ene-2-carboxamido)oxy)methyl)pyrrolidine-1-carboxylate

The title compound was prepared from tert-butyl-(2S)-2-((((2S,5R)-6-((tertbutyldimethylsilyl)oxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2. 1]oct-3-ene-2-carboxamido)oxy)methyl)4,4-difluoropyrrolidine-1-carboxylate (Intermediate 96, 489 mg, 0.89 mmol) following the procedure for Intermediate 93 to give (330 mg, 72%) as a white sticky solid. mС: 433 ES+ (C ₁₈ H ₂₆ F ₂ N ₄ O ₆ )

Intermediate 98: tert-butyl-(2S)-2-((((2S,5R)-6-((S)-2-ethoxy-1-fluoro-2-oxoethoxy)3-methyl-7-oxo- 1,6-Diazabicyclo[3.2.1]oct-3-ene-2-carboxamido)oxy)methyl)-4,4-difluoropyrrolidine-1carboxylate

To a suspension of tert-butyl-(2S)-4,4-difluoro-2-((((2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct -3-ene-2-carboxamido)oxy)methyl)pyrrolidine-1-carboxylate (Intermediate 97, 300 mg, 0.69 mmol) and cesium carbonate (339 mg, 1.04 mmol) in ethyl acetate (15 ml) at At 0°C, ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.1 mL, 0.83 mmol) was added. The reaction mixture was stirred at 10°C for 1 hour. Water (50 ml) and EtOAc (100 ml) were added. The organic layer was separated, concentrated and purified by flash silica gel chromatography (0-100%,

EtOAc/hexane) to give the title compound (70 mg, 18%) as a sticky white foam.

MS: 537 ES+ (C22H31F3N4O8)

Example 61 Ethyl-(2S)-2-(((2S,5R)-2-((((S)-4,4-difluoropyrrolidin-2-yl)methoxy)carbamoyl)-3methyl-7-oxo-1 ,6-diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)-2-fluoroacetate TFA salt

The title compound was prepared from tert-butyl-(2S)-2-((((2S,5R)-6-((S)-2-ethoxy-1-fluoro2-oxoethoxy)-3-methyl-7-oxo -1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamido)oxy)methyl)-4,4difluoropyrrolidine-1-carboxylate (Intermediate 98, 26 mg, 0.05 mmol) according to the procedure for Example 60 to obtain (20 mg, 85%) as TFA salt. MS: 437 ES+ (C ₁₇ H ₂₃ F ₃ N ₄ O ₆ ) 'H NMR (300 MHz, CDCI3) δ: 1.42 (m, 3H); 1.80(s, 3H); 2.65 (m, 2H); 3.45 (m, 2H); 3.82(m, 2H); 4.10 (m, 1H); 4.38 (m, 6H); 5.80(d, 1H); 6.18(s, 1H).

Intermediate 99: (2S)-2-[[(2S,5R)-2-[[(2S)-1-tert-butoxycarbonylpyrrolidin-2yl]methoxycarbamoyl]-3-methyl-7-oxo-1,6-diazabicyclo [3.2.1]oct-3-en-6-yl]oxy]-2-fluoroacetic acid

To a solution of tert-butyl-(2S)-2-((((2S,5R)-6-((S)-2-ethoxy-1-fluoro-2-oxoethoxy)-3-methyl-7-oxo-1 ,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamido)oxy)methyl)pyrrolidine-1-carboxylate (example 59, 81 mg, 0.16 mmol) in THF (1 ml) and water (0.50 ml) at 0°C, lithium hydroxide (1N, 0.01 ml, 0.40 mmol) was added. The reaction mixture was stirred for 30 minutes. A dilute solution of HCl (0.5N) was added to adjust the pH to 2. The reaction mixture was extracted with EtOAc (20 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain the title compound (50 mg, 65%) as a gum. MS: 471 ES- (C20H29FN4O8)

Example 62 [3.2.1]oct-3-en-6-yl]oxy]acetic acid TFA salt

The title compound was prepared from (2S)-2-[[(2S,5R)-2-[[(2S)-1-tert-butoxycarbonylpyrrolidin-2-yl]methoxycarbamoyl]-3-methyl-7-oxo-1 ,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]-2fluoroacetic acid (Intermediate 99, 65 mg, 0.14 mmol) following the procedure for Example 60 to give (40 mg, 70% ) as TFA salt.

MS: 473 ES+ (C20H29FN4O8) ‘H NMR (300 MHz, D2O) δ: 1.65 (s, 3H); 1.82(m, 1H); 2.23 (m, 2H); 2.35 (m, 1H); 3.40 (m, 3H); 3.58 (m, 1H); 3.82(m, 1H); 4.02 (m, 1H); 4.18(m, 1H); 4.33 (m, 2H); 5.70(d, 1H); 6.28(s, 1H).

Intermediate 100: (2S)-2-(((2S,5R)-2-((((S)-1-(tert-butoxycarbonyl)-4,4difluoropyrrolidin-2-yl)methoxy)carbamoyl)-3- methyl 7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetic acid

- 85 044428

The title compound was prepared from tert-butyl-(2S)-2-((((2S,5R)-6-((S)-2-ethoxy-1-fluoro2-oxoethoxy)-3-methyl-7-oxo -1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamido)oxy)methyl)-4,4-difluoropyrrolidine-1-carboxylate (Intermediate 98, 37 mg, 0.07 mmol) according to method for Intermediate 99 to obtain (35 mg, 90%) as a gum. MS: 507 ES(C20H27F3N4O8)

Example 63 -diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)-2-fluoroacetic acid TFA salt

The title compound was prepared from (2S)-2-(((2S,5R)-2-((((S)-l-(tert-butoxycarbonyl)4,4-difluoropyrrolidin-2-yl)methoxy)carbamoyl) -3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2-fluoroacetic acid (Intermediate 100, 35 mg, 0.07 mmol) according to the method for example 60 to obtain (22 mg, 70%) as the TFA salt. MS: 409 ES+ (C ₁₅ H ₁₉ F ₃ N ₄ O ₆ )

1H NMR (300 MHz, D2O) δ: 1.78 (s, 3H); 2.62(m, 1H); 2.95 (m, 1H); 3.13(m, 1H); 3.42(m, 1H); 3.58 (m, 1H); 3.96 (m, 2H); 4.61(m, 1H); 4.40 (m, 3H); 5.82(d, 1H); 6.33(s, 1H).

Example 64: [(2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy] -2-fluoroacetyl]oxymethyl-2,2-dimethylpropanoate

The title compound was prepared from (2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-6-yl] hydroxy]-2-fluoroacetic acid (example 4, 47 mg, 0.17 mmol) and chloromethyl pivalate (0.05 ml, 0.34 mmol) according to the procedure for example 53 to obtain (33.2 mg, 49.8%) as a white solid.

MS: 388 ES+ (C ₁₆ H ₂₂ FN ₃ O ₇ )

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.12 (s, 9H); 1.62(s, 3H); 3.01 (m, 1H); 3.82(m, 1H); 4.02 (m, 1H); 4.19 (m, 1H); 5.76(m, 1H); 5.87 (m, 1H); 5.91(m, 1H); 6.25-6.43 (d, 1H); 7.41 (bs, 1H); 7.86 (bs, 1H).

Example 65 -yl]oxy]-2-fluoroacetate

To a solution of (2R)-2-[[(2S,5R)-2-carbamoyl-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl]oxy]-2 -fluoroacetic acid (example 4, 100 mg, 0.37 mmol) and 5-indanol (58.9 mg, 0.44 mmol) in DCM (3 ml) and THF (3 ml) at 0°C, N,N was added '-dicyclohexylcarbodiimide (113.3 mg, 0.55 mmol) and DMAP (5.0 mg). The reaction mixture was stirred at room. pace. for 1 hour. The reaction mixture was concentrated, then dissolved in EtOAc. The resulting white solid was filtered off. The filtrate was concentrated. Chromatography on silica gel (0-80% ethyl acetate/hexanes) provided the title compound (72 mg, 48%) as a white solid. MS: 390 ES+ (C19H20FN3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.64 (s, 3H); 2.05 (m, 2H); 2.85 (m, 4H); 3.10 (m, 1H); 3.79 (m, 1H); 4.06 (m, 1H); 4.22 (m, 1H); 6.06 (m, 1H); 6.44-6.51 (d, 1H); 6.86(m, 1H); 6.97 (m, 1H); 7.28 (m, 1H); 7.43 (bs, 1H); 7.87 (bs, 1H).

Intermediate 101: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-N-(oxetan-3yl)-7-oxo-1,6-diazabicyclo[3.2.1]oct -3-ene-2-carboxamide

To a solution of (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]okt- 86 044428

3-ene-2-carboxylic acid (Intermediate 77, 100 mg, 0.32 mmol) in DMF (1.5 ml) at 0°C was added HATU (35 mg, 0.48 mmol) and N,N'- diisopropylethylamine (0.167 ml, 0.96 mmol). The reaction mixture was stirred for 30 minutes at room temperature, then diluted with ethyl acetate and washed once with saturated sodium bicarbonate and three times with a 1:1 mixture of brine and water. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-2.5% methanol/dichloromethane) gave the title compound (80 mg, 68%) as a yellow oil. MS: 368 ES+ (C ₁₇ H ₂₉ N ₃ O ₄ Si)

Intermediate 102: (2S,5R)-6-hydroxy-3-methyl-N-(oxetan-3-yl)-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3methyl-N-(oxetan-3-yl)-7-oxo-1,6-diazabicyclo [3.2. 1]oct-3-ene-2-carboxamide (Intermediate 101, 0.10 g, 0.22 mmol) in ethyl acetate (0.5 ml) at room temperature under nitrogen. HF-pyridine (0.015 mL, 0.33 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed. MS: 254 ES+ (C11H15N3O4)

Intermediate 103: Ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-2-(oxetan-3-ylcarbamoyl)7-oxo-1,6-diazabicyclo[3.2.1 ]oct-3-en-6-yl]oxy]acetate

O

To a solution of (2S,5R)-6-hydroxy-3-methyl-N-(oxetan-3-yl)-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2carboxamide (Intermediate 102, 50 mg, 0.20 mmol) in 1,4-dioxane (2 ml) and DMF (0.25 ml) was added ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.047 ml, 0.39 mmol). The reaction mixture was cooled to 0°C and DBU (0.089 mL, 0.59 mmol) was added dropwise.

The reaction mixture was stirred at 0°C for 15 min, then diluted with ethyl acetate and washed three times with a 1:1 mixture of brine and water. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (080% EtOAc/hexane) provided the title compound (58 mg, 82.2%) as a colorless oil. The compound was a mixture of diastereomers in a ratio of 2:8. MS: 358 ES+ (C ₁₅ H ₂₀ FN ₃ O ₆ )

Example 66 -3-en-6-yl]oxy]acetic acid lithium salt

O

To a solution of ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-2-(oxetan-3-ylcarbamoyl)-7-oxo-1,6diazabicyclo[3.2.1]oct- 3-en-6-yl]oxy]acetate (Intermediate 103, 56.3 mg, 0.16 mmol) in THF (1.0 ml) and water (0.5 ml) at 0°C was added lithium hydroxide ( 1M) (0.50 ml, 0.50 mmol). The reaction mixture was stirred for 1 hour. HCl solution (1N) was added to adjust the pH to about 5-6. The solvent was removed. After elution with water (0-5% ACN/water) on a sepabead column (first saturated with water, then ACN, then washed with water) and lyophilization, the title compound (10 mg, 17.3%) was obtained as a white solid. MS: 330 ES+ (C ₁₃ H ₁₆ FN ₃ O ₆ )

1H NMR (300 MHz, D2O) δ: 1.63 (s, 3H); 3.22(m, 1H); 3.37 (m, 1H); 4.04 (m, 1H); 4.30(s, 1H); 4.58 (m, 2H); 4.86(m, 3H); 5.15-5.56(d, 1H); 6.15 (m, 1H).

Intermediate 104: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-N-[2(methanesulfonamido)ethyl]-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1 ] oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 300 mg, 0.96 mmol) in DMF (1.5 ml) and N-(2-aminoethyl)-methanesulfonamide hydrochloride (251.5 mg, 1.44 mmol) according to the procedure for Intermediate 101 to obtain (220 mg, 53%) as a white solid.

MS: 433 ES+ (C ₁₇ H ₃₂ N ₄ O ₅ SiS)

Intermediate 105: (2S,5R)-6-hydroxy-N-[2-(methanesulfonamido)ethyl]-3-methyl-7oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-N-[2(methanesulfonamido)ethyl]-3-methyl-7-oxo-1,6-diazabicyclo [3.2 .1] oct-3-ene-2-carboxamide (Intermediate 104, 0.12 g, 0.28 mmol) following the procedure for Intermediate 102 to give a brownish-beige residue. MS: 319 ES+ (C11H18N4O5S)

Intermediate 106: Ethyl-(2S)-2-fluoro-2-[[(2S,5R)-2-[2-(methanesulfonamido)ethylcarbamoyl]-3-methyl-7-oxo-1,6-diazabicyclo[3.2 .1]oct-3-en-6-yl]oxy]acetate

ABOUT

The title compound was prepared from (2S,5R)-6-hydroxy-N-[2-(methanesulfonamido)ethyl]-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -2-carboxamide (Intermediate 105, 80 mg, 0.25 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.089 mL, 0.75 mmol) according to the procedure for Intermediate 103 to give (25 mg, 18.8%) a white solid. The compound was a mixture of diastereomers in a ratio of 1:4. MS: 423 ES+ (C15H23FN4O7S)

Example 67 3-en-6-yl]oxy]acetic acid lithium salt

O

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-2-(3methylsulfonylpropylcarbamoyl)-7-oxo-1,6-diazabicyclo[3.2.1] oct-3-en-6-yl]oxy]acetate (Intermediate 106.25 mg, 0.06 mmol) following the procedure for Example 66 to give (4.0 mg, 15.4%) after lyophilization of a white solid.

MS: 395 ES+ (C13H19FN4O7S)

1H NMR (300 MHz, D2O) δ: 1.63 (s, 3H); 2.95(s, 3H); 3.18-3.38 (m, 6H); 4.02 (m, 1H); 4.27(s, 1H); 5.56-5.74 (d, 1H); 6.13 (m, 1H).

Intermediate 107: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-N-(oxazol-2ylmethyl)-7-oxo-1,6-diazabicyclo[3.2.1]oct -3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 400 mg, 1.28 mmol) and oxazol-2-yl-methylamine hydrochloride (258.4 mg, 1.92 mmol) following the procedure for Intermediate 101 to give (120 mg, 23.9% ) white solid.

MS: 393 ES+ (C^N^Si)

Intermediate 108: (2S,5R)-6-hydroxy-3-methyl-N-(oxazol-2-ylmethyl)-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methylX-(oxazol-2-ylmethyl)-7-oxo-1,6-diazabicyclo[3.2.1 ]oct-3-ene-2-carboxamide (Intermediate 107, 0.12 g, 0.31 mmol) following the procedure for Intermediate 102 to give

- 88 044428 balance.

MS: 279 ES+ (C12H14N4O4)

Intermediate 109: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-2-(oxazol-2ylmethylcarbamoyl)-7-oxo-1,6-diazabicyclo[3.2.1] oct-3-en-6-yl]oxy]acetate

O

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-N-(oxazol-2-ylmethyl)7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2 -carboxamide (Intermediate 108, 80 mg, 0.29 mmol) and ethyl-(2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.10 mL, 0.86 mmol) according to the procedure for Intermediate 103 to obtain (15 mg, 13.6%) a colorless oil. The compound was a mixture of diastereomers in a ratio of 15:85. MS: 383 ES+ (C ₁₆ H ₁₉ FN ₄ O ₆ )

Example 68 -3-en-6-yl]oxy]acetic acid lithium salt

O

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-2-(oxazol-2ylmethylcarbamoyl)-7-oxo-1,6-diazabicyclo[3.2. 1]oct-3-en-6-yl]oxy]acetate (Intermediate 109, 15 mg, 0.039 mmol) following the procedure for Example 66 to give (4.0 mg, 25.9%) after lyophilization of a white solid.

MS: 355 ES+ (C ₁₄ H ₁₅ FN ₄ O ₆ ) ¹ H NMR (300 MHz, D ₂ O) δ: 1.62 (s, 3H); 3.28(m, 2H); 4.03 (m, 1H); 4.36(s, 1H); 4.47 (m, 2H); 5.565.74(d, 1H); 6.13 (m, 1H); 7.01(d, 1H); 7.71 (d, 1H).

Intermediate 110: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-N(pyrazin-2-ylmethyl)-1,6-diazabicyclo[3.2.1] oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 200 mg, 0.64 mmol) and pyrazin-2-yl-methylamine oxalate (191.2 mg, 0.96 mmol) following the procedure for Intermediate 101 to give (120 mg, 46.4% ) white solid.

MS: 404 ES+ (Q9H29N5O3SO

Intermediate 111: (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-(pyrazin-2-ylmethyl)-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-N-(pyrazin-2-ylmethyl)-1,6-diazabicyclo[3.2.1 ]oct-3-ene-2-carboxamide (Intermediate 110, 0.10 g, 0.26 mmol) following the procedure for Intermediate 102 to provide a residue.

MS: 290 ES+ (C13H15N5O3)

Intermediate 112: Ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-(pyrazin-2ylmethylcarbamoyl)-1,6-diazabicyclo[3.2.1] oct-3-en-6-yl]oxy]acetate

O

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-(pyrazin-2ylmethyl)-1,6-diazabicyclo[3.2.1]oct-3-ene-2- carboxamide (Intermediate 111, 70 mg, 0.24 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.086 mL, 0.73 mmol) following the procedure for Intermediate 103 to give ( 50 mg, 52.5%) colorless oil.

- 89 044428

The compound was a mixture of diastereomers in a ratio of 15:85.

MS: 394 ES+ (C17H20FN5O5)

Example 69 -3-en-6-yl]oxy]acetic acid lithium salt

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2(pyrazin-2-ylmethylcarbamoyl)-1,6-diazabicyclo[3.2 .1]oct-3-en-6-yl]oxy]acetate (Intermediate 112, 50 mg, 0.13 mmol) following the procedure for Example 66 to give (4.0 mg, 8.2%) after lyophilization of white solid matter.

MS: 366 ES+ (C15H16FN5O5) ¹ H NMR (300 MHz, D2O) δ: 1.62 (s, 3H); 3.28(m, 2H); 4.03 (m, 1H); 4.36(s, 1H); 4.53 (m, 2H); 5.565.75(d, 1H); 6.13 (m, 1H); 8.49 (m, 3H).

Intermediate 113: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-N-(cyclopropylmethoxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxu-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 200 mg, 0.64 mmol) and O-(cyclopropylmethyl)hydroxylamine (83.6 mg, 0.96 mmol) following the procedure for Intermediate 101 to give (100 mg, 40.9%) colorless oils MS: 382 ES+ (C18H31N3O ₄ Si)

Intermediate 114: (2S,5R)-N-(cyclopropylmethoxy)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-N(cyclopropylmethoxy)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct- 3-ene-2-carboxamide (Intermediate 113, 0.1 g, 0.26 mmol) following the procedure for Intermediate 102 to provide a residue.

MS: 268 ES+ (C ₁₂ H ₁₇ N ₃ O ₄ )

Intermediate 115: Ethyl-(2S)-2-[[(2S,5R)-2-(cyclopropylmethoxycarbamoyl)-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-6- yl]oxy]-2-fluoroacetate

The title compound was prepared from (2S,5R)-N-(cyclopropylmethoxy)-6-hydroxy-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 114, 60 mg, 0.22 mmol) and ethyl-(2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.08 mL, 0.67 mmol) following the procedure for Intermediate 103 to give (15 mg , 18%) colorless oil. The compound was a mixture of diastereomers in a ratio of 1:9. MS: 372 ES+ (C ₁₆ H ₂₂ FN ₃ O ₆ )

Example 70 ]oxy]-2-fluoroacetic acid lithium salt

The title compound was prepared from ethyl-(2S)-2-[[(2S,5R)-2-(cyclopropylmethoxycarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3- en-6-yl]oxy]-2-fluoroacetate (Intermediate 115, 15 mg, 0.04 mmol) following the procedure for Example 66 to give (4.5 mg, 32.5%) at

- 90 044428 after lyophilization of a white solid.

MS: 344 ES+ (C ₁₄ H ₁₈ FN ₃ O ₆ )

1H NMR (300 MHz, D2O) δ: 0.00 (m, 2H); 0.28 (m, 2H); 0.81 (m, 1H); 1.38(s, 3H); 3.02 (m, 1H); 3.35(m,

1H); 3.42(m, 2H); 3.82(m, 1H); 3.92(s, 1H); 5.37-5.55 (d, 1H); 5.95 (m, 1H).

Intermediate 116: (2S,5R)-N-(3-amino-3-oxopropyl)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2 .1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 200 mg, 0.64 mmol) and alanine amide hydrochloride (119.6 mg, 0.96 mmol) following the procedure for Intermediate 101 to give (140 mg, 57.2%) a white solid.

MS: 383 ES+ (C17H30N4O4S1)

Intermediate 117: (2S,5R)-N-(3-amino-3-oxopronyl)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2- carboxamide

The title compound was prepared from (2S,5R)-N-(3-amino-3-oxopropyl)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2 .1] oct-3-ene-2-carboxamide (Intermediate 116, 140 mg, 0.37 mmol) following the procedure for Intermediate 102 to provide a residue. MS: 269 ES+ (C11H16N4O4)

Intermediate 118: ethyl-(2S)-2-[[(2S,5R)-2-[(3-amino-3-oxopropyl)carbamoyl]-3methyl-7-oxo-1,6-diazabicyclo [3.2.1 ]oct-3-en-6-yl]oxy]-2-fluoroacetate

The title compound was prepared from (2S,5R)-N-(3-amino-3-oxopropyl)-6-hydroxy-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene- 2-carboxamide (Intermediate 117, 90 mg, 0.34 mmol), K ₂ CO ₃ (139.1 mg, 1.01 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174 , 0.12 mL, 1.01 mmol) following the procedure for Intermediate 103 to give (26.0 mg, 20.8%) a white solid. The compound was a mixture of diastereomers in a ratio of 1:9. MS: 373 ES+ (C ₁₅ H ₂₁ FN ₄ O ₆ )

Example 71 Oct-3-en-6-yl]oxy]-2-fluoroacetic acid lithium salt

The title compound was prepared from ethyl-(2S)-2-[[(2S,5R)-2-[(3-amino-3-oxopropyl)carbamoyl]-3-methyl-7-oxo-1,6- diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]-2-fluoroacetate (Intermediate 118, 25 mg, 0.07 mmol) following the procedure for Example 66 to give (8 mg, 34.6% ) after lyophilization of a white solid.

MS: 345 ES+ (C13H17FN4O6).

^1H NMR (300 MHz, D2O) δ: 1.62 (s, 3H); 2.49 (m, 2H); 3.29 (m, 1H); 3.42(m, 1H); 3.50 (m, 2H); 4.10 (m, 1H); 4.30(s, 1H); 5.65-5.82(d, 1H); 6.22 (m, 1H).

Intermediate 119: 2-[[(2S)-5-oxopyrrolidin-2-yl]methoxy]isoindoline-1,3-dione

To a solution of diethyl azodicarboxylate (16.6 ml, 14.6 mmol, 40 wt%) in THF (10 ml) at -10°C was added dropwise a solution of triphenylphosphine (3.83 g, 14.6 mmol) in THF (20 ml). The suspension was stirred at -10°C. After 1 hour, a solution of (S)-(+)-5-(hydroxymethyl)-2-pyrrolidinone) (0.80 g, 6.95 mmol) in THF (10 ml) was added dropwise, followed by a solution of N-hydroxyphthalimide ( 1.13 g, 6.95 mmol) in THF (10 ml). The reaction mixture was allowed to warm to room temperature and stirred for 2 days. The reaction mixture was concentrated. Chromatography on silica gel (0-100% EtOAc/hexane then 10% MeOH/DCM) provided the title compound (1.5 g, 83%) as a pale yellow solid. MS: 261 ES+ (C ₁₃ H ₁₂ N ₂ O4)

Intermediate 120: (5S)-5-(aminooxymethyl)pyrrolidin-2-one

O- _NH2

To a solution of 2-[[(2S)-5-oxopyrrolidin-2-yl]methoxy]isoindoline-1,3-dione (Intermediate 119, 1.5 g, 5.76 mmol) in DCM (70 mL) at room temperature temperature, hydrazine monohydrate (0.84 mL, 17.3 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, then washed with water (3x20 ml). The aqueous layer was concentrated. After elution on a sepabead column, the title compound (0.60 g, 79.9%) was obtained as a white solid. MS: 163 ES+ (C8H6N2O2)

Intermediate 121: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-N-[(5oxopyrrolidin-2-yl)methoxy]-1,6-diazabicyclo[ 3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 250 mg, 0.80 mmol) and 5-(aminooxymethyl)pyrrolidin-2-one (Intermediate 120, 156.2 mg, 1.2 mmol) following the procedure for Intermediate 101 to give (55 mg, 16.2%) white solid.

MS: 425 ES+ (C ₁₉ H ₃₂ N ₄ O ₅ Si)

Intermediate 122: (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-[(5-oxopyrrolidin-2yl)methoxy]-1,6-diazabicyclo[3.2.1]oct-3-ene -2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-N-[[(2S)-5-oxopyrrolidin-2-yl]methoxy]- 1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 121, 55 mg, 0.13 mmol) following the procedure for Intermediate 102 to give a light yellow resin.

MC: 311 ES+ (C ₁₃ H ₁₈ N ₄ O ₅ )

Intermediate 123: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-[(5-oxopyrrolidin-2-yl)methoxycarbamoyl]-1,6 -diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]acetate

O

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-[(5oxopyrrolidin-2-yl)methoxy]-1,6-diazabicyclo[3.2.1]oct-3 -ene-2-carboxamide (Intermediate 122, 35 mg, 0.11 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.04 ml, 0.34 mmol) according to the method for Intermediate 103 to give (15 mg, 32.1%) a sticky solid. The compound was a mixture of diastereomers in a ratio of 1:4. MS: 415 ES+ (C17H23FN4O7)

Example 72 .1]oct-3-en-6-yl]oxy]acetic acid lithium salt

- 92 044428

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-[(5oxopyrrolidin-2-yl)methoxycarbamoyl]-1,6 -diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]acetate (Intermediate 123, 15 mg, 0.036 mmol) following the procedure for Example 66 to give (7.0 mg, 40%) after lyophilization of white solid matter.

MS: 387 ES+ (C ₁₅ H ₁₉ FN ₄ O ₇ ).

1H NMR (300 MHz, D ₂ O) δ: 1.64 (s, 3H); 1.82(m, 1H); 2.25 (m, 1H); 2.38 (m, 2H); 3.24 (m, 1H); 3.76 (m, 2H); 3.96(m, 2H); 4.09 (m, 2H); 5.63-5.82(d, 1H); 6.17 (m, 1H).

Intermediate 124: [(28)-5-oxopyrrolidin-2-yl]methyl-4-methylbenzenesulfonate

To a stirred solution of (S)-(+)-5-(hydroxymethyl)-2-pyrrolinone (2.0 g, 17.4 mmol) and paratoluenesulfonyl chloride (4.17 g, 21.9 mmol) in CH ₂ Cl ₂ ( 50 ml) at 0°C, dimethylaminopyridine (111.4 mg, 0.91 mmol) and triethylamine (3.05 ml, 21.9 mmol) were added. The resulting mixture was allowed to warm to room. pace. and stirred for 12 hours. The reaction was then stopped by adding water and the aqueous layer was extracted with CH ₂ Cl ₂ . The combined organic extracts were washed with 1N. HCl solution and dried over anhydrous Na ₂ SO ₄ . Removal of the solvent under reduced pressure and flash chromatography (2.5% MeOH in DCM) afforded the title compound (4.58 g, 93.2%) as a white solid. MS: 270 ES+ (C ₁₂ H ₁₅ NO ₄ S).

Intermediate 125: 2-[(2S)-5-oxopyrrolidin-2-yl]acetonitrile

KCN (2.76 g, 41 .8 mmol). The solution was heated at 85°C for 18 hours. The solution was then diluted with acetonitrile (200 ml), filtered through celite and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (9:1 DCM/MeOH) to give the title compound (1.8 g, 86.8%) as a white solid. MS: 125 ES+ (C6H8N2O).

Intermediate 126: tert-butyl-N-[2-[(2S)-5-oxopyrrolidin-2-yl]ethyl]carbamate

To a stirred solution of 2-[(2S)-5-oxopyrrolidin-2-yl]acetonitrile (Intermediate 125, 300 mg, 2.42 mmol) in methanol (15 ml) at 0°C was added di-tert-butyl dicarbonate (1.05 g, 4.83 mmol) and NiCl, 6 H ₂ O (57.4 mg, 0.24 mmol). NaBH4 (0.64 g, 16.9 mmol) was then added over 30 min. The reaction mixture was heated to room. pace. and stirred for 1 hour. Hunig's base (0.42 mL, 2.42 mmol) was added, then stirred for 30 minutes. The solvent was removed. The residue was dissolved in EtOAc and washed with sat. NaHCO ₃ solution, brine, dried over MgSO ₄ , filtered and concentrated to give the title compound (0.50 g, 90.6%) as a white solid. MS: 229 ES+ (C11H ₂₀ N ₂ O ₃ )

Intermediate 127: (5S)-5-(2-aminoethyl)pyrrolidin-2-one

To a solution of tert-butyl-N-[2-[(2S)-5-oxopyrrolidin-2-yl]ethyl]carbamate (Intermediate 126, 500 mg, 2.19 mmol) in DCM (2.5 mL) was added trifluoroacetic acid. acid (1.15 g, 10.9 mmol). The reaction mixture was then stirred at room temperature. for 2 hours. The solvent was removed to obtain the title compound as the TFA salt. MS: 129 ES+ (C ₆ H ₁₂ N ₂ O)

Intermediate 128: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-N-[2(5-oxopyrrolidin-2-yl)ethyl]-1,6 -diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

- 93 044428

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 300 mg,

0.96 mmol) and TFA salt of 5-(2-aminoethyl)pyrrolidin-2-one (Intermediate 127, 0.35 g, 1.44 mmol) following the procedure for Intermediate 101 to give (120 mg, 29.6 %) white

solid matter.

MS: 423 ES+ (C ₂₀ H ₃₄ N ₄ O ₄ Si)

Intermediate 129: (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-[2-(5-oxopyrrolidin-2yl)ethyl]-1,6-diazabicyclo[3.2.1]oct-3 -ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-U-[2-(5-oxopyrrolidin-2-yl)ethyl]-1, 6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 128, 120 mg, 0.28 mmol) following the procedure for Intermediate 102 to give a white gum.

MS: 309 ES+ (C14H20N4O4)

Intermediate 130: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-[(5-oxopyrrolidin-2-yl)methoxycarbamoyl]-1.6 -diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]acetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-[2-(5oxopyrrolidin-2-yl)ethyl]-1,6-diazabicyclo[3.2.1]oct -3-ene-2-carboxamide (Intermediate 129, 80 mg, 0.26 mmol), K ₂ CO ₃ (179.3 mg, 1.3 mmol) and ethyl-(2S)-2-bromo-2- fluoroacetate (Intermediate 174, 0.12 mL, 0.78 mmol) following the procedure for Intermediate 103 to give (30 mg, 28%) a white solid. The compound was a mixture of diastereomers in a ratio of 1:4. MS: 413 ES+ (C18H25FN4O6)

Example 73 [3.2.1]oct-3-en-6-yl]oxy]acetic acid lithium salt

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-[2-(5oxopyrrolidin-2-yl)ethylcarbamoyl]-1 ,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]acetate (Intermediate 130, 30 mg, 0.07 mmol) following the procedure for Example 66 to obtain after lyophilization (8.0 mg, 25.8%) white solid.

MS: 385 ES+ (C16H21FN4O6)

1H NMR (300 MHz, D2O) δ: 1.69 (s, 3H); 1.77 (m, 3H); 2.36(m, 2H); 3.30 (m, 3H); 3.44 (m, 1H); 3.76 (m, 2H); 4.11(m, 1H); 4.31(m, 1H); 5.63-5.82(d, 1H); 6.23 (m, 1H).

Intermediate 131: ((2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-N-(3sulfamoylpropyl)-1,6-diazabicyclo[3.2.1]oct- 3-ene-2-carboxamide

- 94 044428

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 174, 300 mg,

0.96 mmol) and 3-aminopropane-1-sulfonamide hydrochloride (251 mg, 1.44 mmol) according to the method for

Intermediate 101 to give (198 mg, 47.7%) a white solid.

MS: 433 ES+ (C17H32N4O5S1S)

Intermediate 132: (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-(3-sulfamoylpropyl)-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-N-(3-sulfamoylpropyl)-1,6-diazabicyclo[3.2.1]oct -3-ene-2-carboxamide (Intermediate 131, 200 mg, 0.46 mmol) following the procedure for Intermediate 102 to give a white gum.

MS 319 ES+ (C11H18N4O5S)

Intermediate 133: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-(3-sulfamoylpropylcarbamoyl)-1,6-diazabicyclo[3.2.1] oct-3-en-6-yl]oxy]acetate

O

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-(3sulfamoylpropyl)-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide ( Intermediate 132, 100 mg, 0.31 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.11 mL, 0.94 mmol) following the procedure for Intermediate 103 to give ( 22 mg, 16.7%) white solid. The compound was a mixture of diastereomers in a ratio of 1:4.

MS: 423 ES+ (C15H23FN4O7S)

Example 74: (2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-(3-sulfamoylpropylcarbamoyl)-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl]oxy]acetic acid lithium salt

O

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-(3sulfamoylpropylcarbamoyl)-1,6-diazabicyclo[3.2.1] oct-3-en-6-yl]oxy]acetate (Intermediate 133, 22 mg, 0.05 mmol) according to the procedure for Example 66 to obtain after lyophilization (8.0 mg, 37%) a white solid.

MS: 395 ES+ (C ₁₃ H ₁₉ FN ₄ O ₇ S)

1H NMR (300 MHz, D ₂ O) δ: 1.69 (s, 3H); 2.04 (m, 2H); 3.35 (m, 6H); 4.11(m, 1H); 4.34 (m, 1H); 5.655.83(d, 1H); 6.22 (m, 1H).

Intermediate 134: tert-butyl-N-[2-(sulfamoylamino)ethyl]carbamate

A solution of tert-butyl-N-(2-aminoethyl)carbamate (2.0 g, 12.5 mmol) and sulfamide (2.0 g, 24.9 mmol) in dioxane (10 ml) was stirred at 90°C for 5 hours. The mixture was then filtered to remove insoluble matter and the filtrate was concentrated under reduced pressure. The residue was then dissolved in EtOAc, washed three times with dilute HCl, then brine, dried over MgSO ₄ , filtered and concentrated to give the title compound (1.2 g, 40.2%) as a yellow oil.

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.39 (s, 9H); 2.92 (m, 2H); 3.04 (m, 6H); 6.50 (m, 3H); 6.75 (m, 1H).

Intermediate 135: 1-amino-2-(sulfamoylamino)ethane TFA salt n ⁿ °V ^N

_H2N %

NH ₂

To a solution of tert-butyl-N-[2-(sulfamoylamino)ethyl]carbamate (Intermediate 134, 1.2 g, 5.01 mmol) in DCM (5 mL) was added trifluoroacetic acid (5.72 g, 50.1 mmol). Reactionary

- 95 044428 the mixture was stirred at room temperature for 2 hours. The solvent was removed to obtain the title compound as TFA yellow salt. MS: 140 ES+ (C ₂ H9N3O ₂ S)

Intermediate 136: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-N-[2(sulfamoylamino)ethyl]-1,6-diazabicyclo[3.2.1 ] oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 300 mg, 0.96 mmol) and TFA salt of 1-amino-2-(sulfamoylamino)ethane (Intermediate 135, 365 mg, 1.44 mmol) following the procedure for Intermediate 101 to give (117 mg, 28.1%) white solid.

MS: 434 ES+ (C ₁₆ H ₃₁ N ₅ O ₅ SiS)

Intermediate 137: (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-[2-(sulfamoylamino)ethyl]1,6-diazabicyclo[3.2.1]oct-3-ene-2- carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-N-[2-(sulfamoylamino)ethyl]-1,6-diazabicyclo [3.2. 1] oct-3-ene-2-carboxamide (Intermediate 136, 117 mg, 0.27 mmol) following the procedure for Intermediate 102 to obtain the title compound as a white gum. MS: 320 ES+ (C ₁₀ H ₁₇ N ₅ O ₅ S)

Intermediate 138: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-[2-(sulfamoylamino)ethylcarbamoyl]-1,6-diazabicyclo [3.2 .1]oct-3-en-6-yl]oxy]acetate

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-[2(sulfamoylamino)ethyl]-1,6-diazabicyclo[3.2.1]oct-3-ene- 2-carboxamide (Intermediate 137, 80 mg, 0.25 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.089 ml, 0.75 mmol) according to the procedure for Intermediate 103 with obtaining (22.0 mg, 16.5%) as a white solid. The compound was a mixture of diastereomers in a ratio of 3:7.

MS: 424 ES+ (C14H22FN5O7)

Example 75 3-en-6-yl]oxy]acetic acid lithium salt

O

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-[2(sulfamoylamino)ethylcarbamoyl]-1,6-diazabicyclo[ 3.2.1]oct-3-en-6-yl]oxy]acetate (Intermediate 138, 30 mg, 0.071 mmol) following the procedure for Example 66 to give (13.0 mg, 44.1%) a white solid.

MS: 396 ES+ (C ₁₂ H ₁₈ FN ₅ O ₇ S) ¹ H NMR (300 MHz, D2O) δ: 1.71 (s, 3H); 3.21-3.47 (m, 6H); 4.11(m, 1H); 4.35 (m, 1H); 5.64-5.84 (d, 1H); 6.22 (m, 1H).

Intermediate 139: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carbonitrile

To a solution of (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct- 96 044428

3-ene-2-carboxamide (Intermediate 192, 3.0 g, 9.63 mmol) in DCM (50 ml) at room temperature, add Burgess' reagent (3.44 g, 14.4 mmol) portionwise over 2 h. The reaction mixture was stirred for another 16 hours, then washed twice with a mixture of brine and water in a 1:1 ratio. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-25% ethyl acetate/hexanes) provided the title compound (2.3 g, 81.3%) as a white solid. MS: 294 ES+ (C ₁₄ H ₂₃ N ₃ O ₂ Si)

Intermediate 140: tert-butyl-N-[[(2S)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -2-yl]methyl]carbamate

To a stirred solution of (2S)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carbonitrile (Intermediate 139, 1.2 g, 4.09 mmol) in methanol (100 ml) at 0°C, di-tert-butyl dicarbonate (1.78 g, 8.18 mmol) and NiCl _2.6 H ₂ O (97, 2 mg, 0.41 mmol). NaBH ₄ (1.08 g, 28.6 mmol) was then added over 30 minutes. The reaction mixture was then warmed to room temp. and stirred for 1 hour. Hunig's base (0.71 mL, 4.09 mmol) was added, then stirred for 30 minutes. The solvent was removed. The residue was dissolved in EtOAc and washed with sat. NaHCO ₃ solution, brine, dried over MgSO ₄ , filtered and concentrated. The residue was purified by silica gel chromatography (40 g, 0-35% EtOAc/hexane) to give the title compound (0.58 g, 35.7%) as a white solid. MS: 398 ES+ (C19H35N3O ₄ Si)

Intermediate 141: tert-butyl-N-[[(2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-2-yl]methyl] carbamate

The title compound was prepared from tert-butyl-N-[[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo [3.2.1 ] oct-3-en-2-yl]methyl] carbamate (Intermediate 140, 80 mg, 0.20 mmol) following the procedure for Intermediate 102 to obtain a white gum. MS: 284 ES+ (C ₁₃ H ₂₁ N ₃ O ₄ )

Intermediate 142: ethyl-(2S)-2-(((2S,5R)-2-(((tert-butoxycarbonyl)amino)methyl)-3methyl-7-oxo-1,6-diazabicyclo [3.2.1] Oct-3-en-6-yl)oxy)-2-fluoroacetate

The title compound was prepared from tert-butyl-N-[[(2S,5R)-6-hydroxy-3-methyl-7-oxo1,6-diazabicyclo[3.2.1]oct-3-en-2-yl] methyl]carbamate (Intermediate 141, 50 mg, 0.18 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.063 mL, 0.53 mmol) according to the procedure for Intermediate 103 with yielding (50 mg, 73.1%) a white solid. The compound was a mixture of diastereomers in a ratio of 1:9. MS: 388 ES+ (C17H26FN3O6)

Example 76 en-6-yl]oxy]-2-fluoroacetic acid lithium salt

The title compound was prepared from ethyl-(2S)-2-(((2S,5R)-2-(((tert-butoxycarbonyl)amino)methyl)-3-methyl-7-oxo-1,6-diazabicyclo[ 3.2.1]oct-3-en-6-yl)oxy)-2-fluoroacetate (Intermediate 142, 50 mg, 0.13 mmol) following the procedure for Example 66 to obtain after lyophilization (35 mg, 67.9% ) white solid.

MS: 360 ES+ (C15H22FN3O6)

1H NMR (300 MHz, D2O) δ: 1.43 (s, 9H); 1.65(s, 3H); 3.17-3.36 (m, 3H); 3.49 (m, 1H); 3.74 (m, 1H); 4.05 (m, 1H); 5.62-5.81(d, 1H); 6.08 (m, 1H).

- 97 044428

Example 77 ]-2-fluoroacetic acid

To a solution of (2S)-2-[[(2S,5R)-2-[(tert-butoxycarbonylamino)methyl]-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -6-yl]oxy]-2-fluoroacetic acid (Example 76, 25 mg, 0.07 mmol) in DCM (1 ml) was added trifluoroacetic acid (0.79 g, 6.96 mmol) at 0°C. The reaction mixture was stirred for 2 hours and then concentrated. The residue was dissolved in pH 7 buffer, then loaded onto a sepabead column (first saturated with water, then ACN, then washed with water) and eluted (02.5% ACN/water) to give, after lyophilization, the title compound (8 mg, 37 .7%) as a white solid.

MS: 260 ES+ (C10H14FN3O4)

1H NMR (300 MHz, D2O) δ: 1.64 (s, 3H); 3.19-3.30 (m, 3H); 3.44 (m, 1H); 3.96(m, 1H); 4.09 (m, 1H); 5.64-5.84 (d, 1H); 6.16 (m, 1H).

Intermediate 143: (2S,5R)-2-(aminomethyl)-6-[tert-butyl(dimethyl)silyl]oxy-3methyl-1,6-diazabicyclo[3.2.1]oct-3-en-7-one

To a solution of tert-butyl-N-[[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene -2-yl]methyl]carbamate (Intermediate 140, 100 mg, 0.25 mmol) in DCM (5 ml) at 0°C was added ZnBr ₂ (170 mg, 0.75 mmol). The reaction mixture was stirred at room. pace. for 16 h, then concentrated and used in the next step without purification.

MS: 298 ES+ (C^N^Si)

Intermediate 144: N-[[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo1,6-diazabicyclo[3.2.1]oct-3-ene-2 -yl]methyl]acetamide

To a solution of (2S,5R)-2-(aminomethyl)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-1,6-diazabicyclo[3.2.1]oct-3-en-7-one (Intermediate 143, 74 mg, 0.25 mmol) Ac ₂ O (168 mg, 0.75 mmol) was added to pyridine (1 ml) at 0°C. The reaction mixture was stirred at room. pace. for 30 minutes, then washed with water, sat. NaHCO ₃ solution, brine, dried over MgSO ₄ , filtered and concentrated. The residue was purified by silica gel chromatography (0-100% EtOAc/hexane) to give the title compound (70 mg, 78.7%) as a white solid. MS: 340 ES+ (C16H29N3O3SO.

Intermediate 145: N-[[(2S,5R)-6-hydroxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-2-yl]methyl]acetamide

The title compound was prepared from N-[[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -2-yl]methyl]acetamide (Intermediate 144, 70 mg, 0.21 mmol) following the procedure for Intermediate 102 to give a white gum. MS: 226 ES+ (C ₁₀ H ₁₅ N ₃ O ₃ )

Intermediate 146: ethyl-(2S)-2-[[(2S,5R)-2-(acetamidomethyl)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-6- yl]oxy]-2-fluoroacetate

O

- 98 044428

The title compound was prepared from M-[[(28.5E)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-en-2-yl]methyl]acetamide ( Intermediate 145, 40 mg, 0.18 mmol) and ethyl (28)-2-bromo-2-fluoroacetate (Intermediate 174, 0.063 ml, 0.53 mmol) following the procedure for Intermediate 103 to obtain after lyophilization ( 15 mg, 25.6%) white solid. The compound was a mixture of diastereomers in a ratio of 15:85. MS: 330 ES+ (Ci ₄ H ₂₀ FN ₃ O ₅ )

Example 78 ]-2-fluoroacetic acid lithium salt

O

The title compound was prepared from ethyl-(28)-2-[[(28.5K)-2-(acetamidomethyl)-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene- 6-yl]oxy]-2-fluoroacetate (Intermediate 146, 15 mg, 0.05 mmol) according to the procedure for Example 66 to obtain after lyophilization (10 mg, 65.6%) a white solid.

MS: 302 ES+ (Ci ₂ H ₁₆ FN ₃ O ₅ )

II NMR (300 MHz, D ₂ O) δ: 1.64 (s, ZN); 1.99 (s, ZN); 3.19 (m, 1H); 3.34 (m, 2H); 3.64 (m, 1H); 3.79 (m, 1H); 4.05 (m, 1H); 5.63-5.81(d, 1H); 6.09 (m, 1H).

Intermediate 147: ethyl-(28)-2-[[(28.5K)-2-(aminomethyl)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-6- yl]oxy]-2-fluoroacetate TFA salt

To a solution of ethyl-(28)-2-[[(2S,5R)-2-[(tert-butoxycarbonylamino)methyl]-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene -6-yl]oxy]-2-fluoroacetate (Intermediate 142, 97.4 mg, 0.25 mmol) in DCM (2 ml) was added trifluoroacetic acid (2.87 g, 25.2 mmol) at 0°C. . The reaction mixture was stirred at 0°C for 2 hours, then concentrated to obtain the title compound as the TFA salt. MS: 288 ES+ (Ci ₂ H ₁₈ FN ₃ O ₄ )

Intermediate 148: tert-butyl-M-chlorosulfonylcarbamate

I o o •Sh, n o

To a stirred solution of tert-butanol (1.9 ml, 20 mmol) in CH ₂ C1 ₂ (12 ml) at 0°C, chlorosulfonylisocyanate (1.4 ml, 15 mmol) was added dropwise over 10 minutes. After stirring at 0°C for 5 min, the reaction mixture was warmed to room. temp, and stirred for 20 minutes. The reaction mixture was concentrated in vacuo to one third of the original volume. The flask was placed back into the 0°C bath and the product crystallized out of solution. After 50 minutes, the product was collected by filtration and washed with hexanes to give the title compound (2.8 g, 80.7%) as a white solid.

Intermediate 149: ethyl-(2S)-2-φτop-2-[[(2S,5R)-3-methyl-7-oκco-2-[[(2,2,2trifluoroacetyl)amino]methyl]-1, 6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]acetate

O

To a solution of TFA salt e-gyl-(28)-2-[[(28.5E)-2-(aminoms gyl)-3-me gyl-7-oxo-1,6-diazabi cyclo[3.2.1]oct- 3-en-6-yl]oxy]-2-fluoroacetate (Intermediate 147, 72 mg, 0.25 mmol) in DCM (2 ml) was added at 0° C. tert-butyl-M-chlorosulfonylcarbamate (Intermediate 148, 54.0 mg, 0.25 mmol) and triethylamine (63.4 mg, 0.63 mmol). The reaction mixture was stirred at room. temp for 30 minutes, then diluted with DCM, washed with water, brine, dried over MgSO ₄ , filtered and concentrated. The residue was purified by flash chromatography (0-100% EtOAc/hexanes) to give the title compound (38 mg, 39.6%) as a white solid. MS: 384 ES+ (C ₁₄ H ₁₇ F ₄ N ₃ O ₅ )

Example 79 6diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]acetic acid lithium salt

-99044428

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2[[(2,2,2-trifluoroacetyl)amino]methyl] -1,6-diazabicyclo[3.2.1]oct-3-en-6-yl]oxy]acetate (Intermediate 149, 38 mg, 0.10 mmol) following the procedure for Example 66 to obtain after lyophilization (25 mg, 60.3%) white solid.

MS: 356 ES+ (C12H13FN3O5)

1H NMR (300 MHz, D ₂ O) δ: 1.66 (s, 3H); 3.22(m, 1H); 3.36 (m, 1H); 3.59 (m, 1H); 3.73 (m, 1H); 3.86(m, 1H); 4.08 (m, 1H); 5.62-5.83 (d, 1H); 6.11 (m, 1H).

Intermediate 150: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-N-(cyanomethyl)-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 700 mg, 2.24 mmol) and aminoacetonitrile hydrochloride (207 mg, 2.24 mmol) following the procedure for Intermediate 101 to give (302 mg, 38.4%) a white solid.

MS: 351 ES+ (C ₁₆ H ₂₆ N ₄ O ₃ Si)

Intermediate 151: (2S,5R)-N-(cyanomethyl)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-N(cyanomethyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct- 3-ene-2-carboxamide (Intermediate 150, 300 mg, 0.86 mmol) following the procedure for Intermediate 102 to give the residue.

MS: 237 ES+ (C10H12N4O3)

Example 80 Ethyl-(2S)-2-fluoro-2-[[(2S,5R)-2-(cyanomethylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl]oxy]acetate

To a solution of (2S,5R)-N-(cyanomethyl)-6-hydroxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2carboxamide (Intermediate 151, 239 mg , 1.01 mmol) in THF (3 ml) and DMF (0.3 ml) at 40°C was added DBU (0.18 ml, 1.22 mmol) and ethyl-(2S)-2-bromo-2- fluoroacetate (Intermediate 174, 0.18 ml, 1.52 mmol). The reaction mixture was stirred at -40°C for 30 min, then diluted with ethyl acetate and washed three times with a 1:1 mixture of brine and water. Organic substances were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash chromatography (0-100% EtOAc/hexane) to give a 1:4 mixture of diastereomers. Diastereomers were separated by reverse phase HPLC (T3 column, 20-50% ACN/water, 10 min) to give Example 80 (64 mg, 18.2%) and Example 81 (8 mg, 2.3%) as white solids. MS: 341 ES+ (C14H17FN4O5)

1H NMR (300 MHz, DMSO-de) δ: 1.27 (m, 3H); 1.64(s, 3H); 3.13(m, 1H); 3.61(m, 1H); 3.98 (m, 1H); 4.21 (m, 5H); 6.06-6.26 (m, 1H); 6.10 (m, 1H); 9.18 (m, 1H).

Example 81 Ethyl-(2R)-2-fluoro-2-[[(2S,5R)-2-(cyanomethylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl]oxy]acetate

- 100 044428

MS: 341 ES+ (C ₁₄ H ₁₇ FN ₄ O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.21 (m, 3H); 1.65(s, 3H); 3.12(m, 1H); 3.60 (m, 1H); 4.07 (m, 1H);

4.22 (m, 5H); 6.08 (m, 1H); 6.15-6.33 (m, 1H); 9.16 (m, 1H).

Example 82 -6-yl]oxy]acetic acid lithium salt

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-2-(cyanomethylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1] oct-3-en-6-yl]oxy]acetate (Example 80, 50 mg, 0.15 mmol) according to the procedure for Example 66 to obtain after lyophilization (32 mg, 66.3%) a white solid.

MS: 313 ES+ (C ₁₂ H' ₃ FN ₄ O ₅ ) 'H NMR (300 MHz, D ₂ O) δ: 1.74 (s, 3H); 3.35 (m, 2H); 4.12 (m, 1H); 4.25 (m, 2H); 4.42 (m, 1H); 5.635.83(d, 1H); 6.23 (m, 1H).

Example 83 -en-6-yl]oxy]acetate

Intermediate 138 (230 mg) was separated by reverse phase preparative HPLC (T3 column, ACN/water 20-50% for 10 min) to give Example 83 (104 mg) and Example 84 (7 mg) as white solids substances. MS: 424 ES+ (C ₁₄ H ₂₂ FN ₅ O ₇ S)

1H NMR (300 MHz, DMSO-di) δ: 1.27 (m, 3H); 1.63(s, 3H); 2.97 (m, 2H); 3.10 (m, 1H); 3.26(m, 2H); 3.73 (m, 1H); 3.96(m, 1H); 4.21(m, 3H); 6.06-6.26 (m, 2H); 6.56 (m, 3H); 8.45 (m, 1H).

Example 84 Ethyl-(2R)-2-fluoro-2-[[(2S,5R)-2-(cyanomethylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl]oxy]acetate

MS: 424 ES+ (C ₁₄ H ₂₂ FN ₅ O ₇ S) 'H NMR (300 MHz, DMSO-di) δ: 1.21 (m, 3H); 1.62(s, 3H); 2.95 (m, 2H); 3.07 (m, 1H); 3.24 (m, 2H); 3.71 (m, 1H); 4.06 (m, 1H); 4.21(m, 3H); 6.03 (m, 1H); 6.14-6.31 (m, 1H); 6.55 (m, 3H); 8.42 (m, 1H).

Intermediate 152: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-N-(hydroxymethyl)-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -2-carboxamide

To a solution of (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct3-ene-2-carboxamide (Intermediate 192 .1.0 g, 3.21 mmol) paraformaldehyde (1.45 g, 16.1 mmol) in 1,4-dioxane (10 ml) was added. The reaction mixture was heated to 90°C for 16 h in a microwave oven. The solvent was removed. Chromatography on silica gel (0-80% EtOAc/hexane) provided the title compound (0.62 g, 56.5%) as a white solid. MS: 342 ES+ (C‘5H27N3O4Si)

Intermediate 153: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-2-(hydroxymethylcarbamoyl)-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 - en-6-yl]oxy]acetate

To a solution of (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-N-(hydroxymethyl)-3-methyl-7-oxo-1,6- 101 044428 diazabicyclo[3.2.1]oct-3 -ene-2-carboxamide (Intermediate 152, 230 mg, 0.67 mmol) and cerium(III) chloride (166 mg, 0.67 mmol) in THF (3 ml) at -78°C was added TBAF (0. 67 ml, 0.67 mmol) (1 M in THF). The mixture was stirred for about 10 minutes. Ethyl (2S)-2bromo-2-fluoroacetate (Intermediate 174, 24.4 mg, 0.67 mmol) was added to the reaction mixture. After 5 min, the reaction mixture was partitioned between water and EtOAc. The organic layer was collected, washed with water, brine, dried over MgSO ₄ , filtered and concentrated. Chromatography on silica gel (080% EtOAc/hexane) gave a 1:3 mixture of the two diastereomers, 180 mg.

Example 85 Ethyl-(2S)-2-fluoro-2-[[(2S,5R)-2-(hydroxymethylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl]oxy]acetate

Intermediate 153 (180 mg) was separated by reverse phase preparative HPLC (T3 column, ACN/water 20-50% for 10 min) to give Example 85 (43 mg) and Example 86 (2.6 mg) as white solids.

MS: 332 ES+ (C ₁₃ H ₁₈ FN ₃ O ₆ )

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.28 (m, 3H); 1.61(s, 3H); 3.09 (m, 1H); 3.79 (m, 1H); 3.96(m, 1H); 4.25 (m, 3H); 4.54 (m, 2H); 5.68(m, 1H); 6.06-6.24 (m, 2H); 8.99 (m, 1H).

Example 86 Ethyl-(2R)-2-fluoro-2-[[(2S,5R)-2-(hydroxymethylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -en-6-yl]oxy]acetate

MS: 332 ES+ (C13H18FN3O6)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.24 (m, 3H); 1.61(s, 3H); 2.97 (m, 1H); 3.35 (m, 1H); 4.06 (m, 1H); 4.26(m, 3H); 4.59 (m, 2H); 5.52 (m, 1H); 6.03 (m, 1H); 6.06-6.24 (m, 1H); 8.99 (m, 1H).

Example 87 -6-yl]oxy]acetic acid lithium salt

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-2(hydroxymethylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct -3-en-6-yl]oxy]acetate (Example 85, 15 mg, 0.05 mmol) according to the procedure for Example 66 to obtain after lyophilization (8 mg, 55.3%) a white solid.

MS: 304 ES+ (C11H14FN3O6)

1H NMR (300 MHz, D2O) δ: 1.77 (s, 3H); 3.42(m, 2H); 4.17 (m, 1H); 4.41 (m, 1H); 4.79 (m, 2H); 5.705.89 (d, 1H); 6.28 (m, 1H).

Intermediate 154: tert-butyl-N-(isocyanatomethyl)carbamate

To a stirred solution of BOC-GLY-OH (10 g, 57.08 mmol) in THF (200 mL) at 0°C, methyl chloroformate (5.29 mL, 68.5 mmol) was added dropwise, followed by triethylamine ( 9.55 ml, 68.5 mmol). A white precipitate immediately formed. The mixture was stirred for 45 min before adding NaN ₃ (5.58 g, 85.6 mmol) in water (10 ml) at 0°C. The resulting mixture was stirred at 0°C for 1 hour, then diluted with water. The acyl azide was extracted four times with toluene (4x25 ml) and the combined organic extracts were washed successively with saturated sodium bicarbonate (2x30 ml) and water (50 ml). The organics were dried over MgSO ₄ at 0°C, filtered and then heated slowly with stirring until evolution of nitrogen gas was observed, which continued at 59°C for 20 minutes. The temperature was increased and maintained at 64°C for 1.5 h, then slowly increased to 70°C over 20 min. The solution was concentrated under reduced pressure to give the title compound (8.6 g, 87.5%) as a colorless oil.

- 102 044428

Intermediate 155: tert-butyl-N-(benzyloxycarbonylaminomethyl)carbamate ί o o

To a solution of tert-butyl-N-(isocyanatomethyl)carbamate (Intermediate 154, 8.3 g, 48.2 mmol) in DCE (5 ml) at 0°C was added benzyl alcohol (7.48 ml, 72.3 mmol ) and triethylamine (0.67 ml, 4.82 mmol), dropwise. The reaction mixture was heated to room. pace. and stirred for 30 minutes. The resulting white solid was collected by filtration and washed with hexane to give the title compound (5.2 g, 38.5%) as a white solid. MS: 303 ES+Na (C14H20N2O4).

Intermediate 156: tert-butyl-Shaminomethyl)carbamate

A solution of tert-butyl-N-(benzyloxycarbonylaminomethyl)carbamate (Intermediate 155, 1.5 g, 5.35 mmol) in methanol (20 ml) was bubbled with nitrogen gas. Pd/C (10%) (150 mg) was added. The reaction mixture was degassed and then placed under a hydrogen atmosphere from a balloon for 30 minutes. The reaction mixture was filtered through celite. The filtrate was concentrated to give the title compound as a colorless oil.

MS: 147 ES+ (C6H14N2O2)

Intermediate 157: tert-butyl-N-[[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 -ene-2-carbonyl]amino]methyl]carbamate

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 350 mg, 1.12 mmol) and tert-butyl-N-(aminomethyl)carbamate (Intermediate 156, 246 mg, 1.68 mmol) following the procedure for Intermediate 101 to give (170 mg, 34.4%) white solid. MS: 441 ES+ (C ₂₀ H ₃₆ N ₄ O ₅ Si)

Intermediate 158: (2S,5R)-N-(aminomethyl)-6-[tert-butyl(dimethyl)silyl]oxy-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -2-carboxamide

To a solution of tert-butyl-N-[[[(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3- ene-2-carbonyl]amino]methyl]carbamate (Intermediate 157, 170 mg, 0.39 mmol) in DCM (3 ml) at 0°C was added ZnBr ₂ (261 mg, 1.16 mmol). The reaction mixture was stirred at room. pace. for 16 hours, then concentrated to give the title compound. MS: 341 ES+ (C ₁₅ H ₂₈ N ₄ O ₃ Si)

Intermediate 159: (2S,5R)-N-(acetamidomethyl)-6-[tert-butyl(dimethyl)silyl]oxy-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -2-carboxamide

To a solution of (2S,5R)-N-(aminomethyl)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2 -carboxamide (Intermediate 158, 131 mg, 0.39 mmol) in pyridine (3 ml) was added acetic anhydride (394 mg, 3.86 mmol) at 0°C. The reaction mixture was stirred at room. pace. For 30 min, then partition between EtOAc and water. The organic layer was washed with water, sat. NaHCO ₃ solution, brine, dried over MgSO ₄ , filtered and concentrated. Chromatography on silica gel (0-80% EtOAc/hexane) provided the title compound (75 mg, 50.8%) as a white solid. MS: 383 ES+ (C ₁₇ H ₃₀ N ₄ O ₄ Si)

Intermediate 160: (2S,5R)-N-(acetamidomethyl)-6-hydroxy-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

- 103 044428

The connection specified in the header

prepared from (2S,5R)-N-(acetamidomethyl)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene- 2-carboxamide (Intermediate 159, 75 mg, 0.20 mmol) according to the procedure for Intermediate 102 to obtain a white gum. MS: 269 ES+ (C11H16N4O4)

Intermediate 161: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-2-(acetamidomethylcarbamoyl)-3methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3 - en-6-yl]oxy]acetate

The title compound was prepared from (2S,5R)-N-(acetamidomethyl)-6-hydroxy-3-methyl-7oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 160, 50 mg, 0.19 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.067 mL, 0.56 mmol) following the procedure for Intermediate 103 to give a white solid after elution (0-15% ACN/water) on a sepabead column (first saturated with water, then ACN, then eluting with water) and lyophilize, 21 mg, 27%. The compound was a mixture of diastereomers in a ratio of 1:4. MS: 373 ES+ (C ₁₅ H ₂₁ FN ₄ O ₆ )

Example 88: (2S)-2-fluoro-2-[[(2S,5R)-2-(acetamidomethylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene -6-yl]oxy]acetic acid lithium salt

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-2-(acetamido methylcarbamoyl)-3-methyl-7-oxo-1,6-diazabicyclo[3.2.1 ]oct-3-en-6-yl]oxy]acetate (Intermediate 161, 21 mg, 0.056 mmol) in water (1 ml) according to the procedure for Example 66 to obtain after lyophilization (6.0 mg, 29.3% ) white solid.

MS: 345 ES+ (C13H17FN4O6) ¹ H NMR (300 MHz, D2O) δ: 1.75 (s, 3H); 2.02(s, 3H); 3.42(m, 2H); 4.15 (m, 1H); 4.37 (m, 1H); 4.64 (m, 2H); 5.70-5.89 (d, 1H); 6.27 (m, 1H).

Intermediate 162: tert-butyl-N-[(tert-butoxycarbonylamino)methylsulfamoyl]carbamate

To a solution of tert-butyl-N-(aminomethyl)carbamate (Intermediate 156, 700 mg, 4.79 mmol) in DCM (20 mL) at 0°C was added triethylamine (0.67 mL, 4.79 mmol). Tert-Butyl-N-chlorosulfonylcarbamate (Intermediate 148.1.03 g, 4.79 mmol) in DCM (5 ml) was then added dropwise. The reaction mixture was stirred at 0°C for 30 minutes, then diluted with DCM, washed with water, brine, dried over MgSO ₄ , filtered and concentrated. The residue was purified by washing with DCM/hexane to give the title compound (650 mg, 41.7%) as a white solid. MS: 324 ES- (C11H23N3O6S)

Intermediate 163: amino(sulfamoylamino)methane TFA salt

V

H ₂ N ^x N^NHo

Η

Trifluoroacetic acid (2.28 g, 19.9 mmol). The reaction mixture was stirred at room. pace. for 30 minutes, then concentrated to give the title compound as a light yellow resin.

Intermediate 164: (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo-N[(sulfamoylamino)methyl-1,6-diazabicyclo[3.2.1]oct- 3-ene-2-carboxamide

- 104 044428

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylic acid acid (Intermediate 77, 350 mg, 1.12 mmol) following the procedure for Intermediate 101 to give (125 mg, 26.6%) a white solid. MS: 420 ES+ (C ₁₅ H ₂₉ N ₅ O ₅ SiS)

Intermediate 165: (2S,5R)-6-hydroxy-3-methyl-7-oxo-N-[(sulfamoylamino)methyl]1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

The title compound was prepared from (2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl7-oxo-N-[(sulfamoylamino)methyl]-1,6-diazabicyclo[3.2.1] oct-3-ene-2-carboxamide (Intermediate 164, 125 mg, 0.30 mmol) following the procedure for Intermediate 102 to give a light yellow resin. MS: 306 ES+ (C9H15N5O5S)

Intermediate 166: ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2-[(sulfamoylamino)methylcarbamoyl]-1,6-diazabicyclo [3.2.1 ]oct-3-en-6-yl]oxy]acetate

O

The title compound was prepared from (2S,5R)-6-hydroxy-3-methyl-7-oxo-N[(sulfamoylamino)methyl]-1,6-diazabicyclo[3.2.1]oct-3-ene-2- carboxamide (Intermediate 165, 88 mg, 0.29 mmol) and ethyl (2S)-2-bromo-2-fluoroacetate (Intermediate 174, 0.10 mL, 0.86 mmol) according to the procedure for Intermediate 103 with yielding (18 mg, 12.2%) a white solid. The compound was a mixture of diastereomers in a ratio of 17:83. MS: 410 ES+ (C13H20FN5O7S)

Example 89 -en-6-yl]oxy]acetic acid lithium salt

O

The title compound was prepared from ethyl-(2S)-2-fluoro-2-[[(2S,5R)-3-methyl-7-oxo-2[(sulfamoylamino)methylcarbamoyl]-1,6-diazabicyclo[3.2. 1]oct-3-en-6-yl]oxy]acetate (Intermediate 166, 18 mg, 0.040 mmol) according to the procedure for Example 66 to obtain after lyophilization (5.0 mg, 25.3%) a white solid.

MS: 382 ES+ (C _n H ₁₆ F5 ₄ O ₇ S) ¹ H NMR (300 MHz, D ₂ O) δ: 1.76 (s, 3H); 3.40 (m, 2H); 4.16(m, 1H); 4.41 (m, 1H); 4.63 (m, 2H); 5.705.89 (d, 1H); 6.28 (m, 1H).

Intermediate 167: Racemic 2-bromo-2-fluoroacetic acid

F

ABOUT

A solution of ethyl 2-bromo-2-fluoroacetate (3.5 kg) in tetrahydrofuran (7 l, 2 vol.) and a solution of sodium hydroxide (830 g) in water were loaded dropwise into a reactor with a volume of 50 l at 0-5°C (7 L, 2 vol.) for 1 hour. The resulting solution was stirred at 0-5°C for 1 hour. HCl (160 ml) was added dropwise at 0-5°C. Water and tetrahydrofuran were removed by concentration in vacuo. The residue was suspended in tetrahydrofuran (35 L, 10 vol.) and conc. HCl (1.57 L, 1.0 eq.).

Anhydrous sodium sulfate was added and the resulting mixture was stirred for 2 hours. The solid was filtered and washed with THF (1 L x 2). The filtrate was concentrated in vacuo to give 2-bromo-2-fluoroacetic acid (2.2 kg) as a yellow oil, which was combined with the previous batch obtained by the same method (940 g, purity: 72%) and distilled in vacuo (65-70°C

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100 Pa) to obtain 2-bromo-2-fluoroacetic acid (2.55 kg, overall yield 67%) as a colorless oil.

1H NMR (400 MHz, CDCl3): δ 11.15 (s, 1H), 6.66 (d, 1H, J=68 Hz).

Intermediate 168: (S)-1-phenylethan-1-amine-(R)-2-bromo-2-fluoroacetate

A solution of 2-bromo-2-fluoroacetic acid (Intermediate 167, 2.0 kg) in 1 L of chloroform (1 vol.) was loaded into a 10 L reactor at 0-5°C, to which solution (S) was added dropwise -1 phenylethanamine (1.39 kg) in 1 liter of chloroform (1 vol.). The mixture was stirred at room temperature overnight and the resulting white solid was collected by filtration to give (S)-1phenylethanamine 2-bromo-2-fluoroacetate salt (2.5 kg; ee: 6%), which was charged into a 10 L reactor. and then chloroform (5 L, 2 vol.) was added. The resulting mixture was stirred for 2 hours at 50°C (the solid was partially dissolved in chloroform), cooled to 0°C and left for 2 hours. The solid was collected by filtration and washed with cooled chloroform (500 ml, 0.2 vol). The recrystallization procedure was repeated 4 times to obtain 1.09 kg (97% ee) of the title compound as a white solid with an overall yield of 31% (2 steps).

Intermediate 169: Pentan-3-yl-(R)-2-bromo-2-fluoroacetate

A 2 L reactor at room temperature was charged with (S)-1-phenylethanamine (S)-2-bromo-2fluoroacetate (Intermediate 168, 450 g), dichloromethane (900 ml, 2 vol.) and iPrOH (2.0 eq. ). Chlorotrimethylsilane (1.12 L) was added slowly and a white precipitate formed. The resulting mixture was stirred at room temperature overnight. The white precipitate was filtered off and the filter cake was washed with hexane (450 ml, 1 vol.). The combined filtrate was washed with water (3x100 ml). The organic solution was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was subjected to distillation (54-60°C, 100 Pa) to obtain the title compound as a colorless oil (290 g, 79% yield, 95% purity).

^1H NMR (CDCl3, 400 MHz): δ 6.53 (d, J=51.2 Hz, 1H), 5.17 (m, 1H), 1.32 (m, 6H).

Intermediate 170: Ethyl (R)-2-bromo-2-fluoroacetate

(1R)-1-phenylethan-1-amine was placed in a 50 mL three-neck round-bottom flask, purged and maintained under an inert nitrogen atmosphere; (2S)-2-bromo-2-fluoroacetic acid (Intermediate 168, 30 g, 107.9 mmol) and ethanol (34.7 g, 755.3 mmol). Chlorotrimethylsilane (82 g, 755.3 mmol) was then added dropwise with stirring at room temperature. The resulting solution was stirred for 4 h at room temperature, then the reaction was stopped by adding 10 ml of water/ice. The resulting solution was extracted with 3x20 ml petroleum ether (30-60 degrees) and the organic layers were combined. The resulting mixture was washed with 2x20 ml brine. The mixture was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was eluted with petroleum ether (30-60 degrees) on a silica gel column. The result was 10 g (50%) of the title compound as a colorless oil.

1H NMR (300 MHz, CDCl ₃ ): δ 6.58 (d, 1H, J=51 Hz), 4.38 (q, 2H, J=6 Hz), 1.38 (t, 3H, J=6 Hz).

Intermediate 171: (R)-benzyl-2-bromo-2-fluoroacetate

Chlorotrimethylsilane (60 ml, 719.12 mmol) was added portionwise to (S)-1-phenylethanamine (S)-2-bromo2-fluoroacetate (Intermediate 168, 20 g, 71.91 mmol) and phenylmethanol (60 ml, 71. 91 mmol) at 25°C for 3 min under nitrogen. The resulting solution was stirred at 25°C for 4 hours. The reaction mixture was diluted with heptane (500 ml), then washed with water and brine. Organic substances were dried over sodium sulfate, filtered and concentrated. Chromatography on silica gel (0-10% ethyl acetate/petroleum ether) gave the title compound as a yellow oil, 17.5 g, 98%.

1H NMR (300 MHz, CDCl3-d, 30°C) δ: 5.30 (s, 2H); 6.60(d, 1H); 7.40 (m, 5H).

Intermediate 172: (1R)-1-phenylethan-1-amine; (2S)-2-bromo-2-fluoroacetic acid

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A solution of (1R)-1-phenylethan-1-amine (107.7 g, 0.89 mol) in methanol (325 ml) was placed in a 100 mL round bottom flask. A solution of 2-bromo-2-fluoroacetic acid (Intermediate 167, 140 g, 0.89 mol) in methanol (420 ml) was then added dropwise with stirring at 0° C. for 30 minutes. The resulting solution was stirred overnight at room temperature, then concentrated in vacuo. The residue was diluted with CHCl ₃ (3 vol.). Solids were collected by filtration. The solid was dried in vacuo, then suspended in CHCl ₃ and heated to 60°C for 2 hours. The mixture was then cooled to 0°C and the solid was filtered. This process was repeated 6 times. The result was 80 g (32%) of the title compound as a white solid.

1H NMR (300 MHz, d6-DMSO): δ 8.56 (brs, 3H), 7.49-7.46 (m, 2H), 7.42-7.38 (m, 2H), 7.36 -7.34 (m, 1H), 6.48 (d, 1H, J=56 Hz), 4.39-4.34 (m, 1H), 1.48 (d, 3H, J=6.8 Hz).

Intermediate 173: (S)-isopropyl-2-bromo-2-fluoroacetate

F

(R)-1-phenylethanamine (S)-2-bromo-2-fluoroacetate (Intermediate 172, 32.0 g, 116 mmol) and isopropanol were placed in a 250 mL three-neck round bottom flask, purged and maintained under an inert nitrogen atmosphere. (13.9 g, 232 mmol) in DCM (64 ml). Chlorotrimethylsilane (56.4 g, 519 mmol) was then added dropwise with stirring at room temperature. The resulting solution was stirred overnight at room temperature, then the reaction was stopped by adding 100 ml of water/ice. The resulting solution was extracted with 3x100 ml of petroleum ether (30-60 degrees) and the organic layers were combined. The resulting mixture was washed with 3x70 ml brine. The mixture was dried over anhydrous sodium sulfate and concentrated.

The residue was eluted with petroleum ether (30-60°) on a silica gel column.

The result was 19 g (83%) of the title compound as a colorless oil.

1H NMR (CDCI3, 400 MHz): δ 6.53 (d, J=50.8 Hz, 1H), 5.17 (m, 1H), 1.32 (m, 6H).

Intermediate 174: Ethyl (2S)-2-bromo-2-fluoroacetate

F

Br^°^ O

(1R)-1-phenylethan-1-amine (2S)-2-bromo-2-fluoroacetic acid (Intermediate 172, 20 g, 72 mmol) was added to a 50 mL three-neck round bottom flask, purged and maintained under an inert nitrogen atmosphere. ) and ethanol (23.2 g, 504 mmol). Chlorotrimethylsilane (54.8 g, 504 mmol) was then added dropwise with stirring at room temperature. The resulting solution was stirred for 4 h at room temperature, then the reaction was stopped by adding 10 ml of water/ice. The resulting solution was extracted with 3x20 ml petroleum ether (30-60 degree) and the organic layers were combined. The resulting mixture was washed with 2x20 ml brine. The mixture was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was eluted with petroleum ether (30-60 degrees) on a silica gel column. The result was 6 g (45%) of the title compound as a colorless oil.

1H NMR (300 MHz, d6-DMSO): δ 6.57 (d, 1H, J=56 Hz), 4.37 (q, J=7.2 Hz, 2H), 1.33 (t, 3H, J=7.2 Hz).

Intermediate 175: (E)-But-2-enylboronic acid

A=\OH

HE

To a solution of (E)-but-2-en-1-ol (59.5 g, 826 mmol) in MeOH (360 ml) was added H ₂ PdCl ₄ (2.06 g, 8.34 mmol) at room temperature. then B ₂ (OH) ₄ (81.8 g, 919 mmol) was added to the mixture in parts at 30-40°C. The resulting solution was stirred at 30-40°C for 20 minutes, then filtered through celite.

1H-NMR (DMSO-d6, 400 MHz): δ 5.39-5.49 (m, 1H), 5.16-5.26 (m, 1H), 3.38 (s, 2H), 1. 38-1.58 (m, 5H)

Intermediate 176: (S,E)-2-(tert-butylsulfinylimino)acetic acid

To type 4A molecular sieves (500 g) in DCM (600 ml) at room temperature were added (S)2-methylpropane-2-sulfinamide (100 g, 819 mmol) and 2-oxoacetic acid hydrate (91.2 g, 991 mmol ).

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The resulting solution was stirred at room temperature overnight, then filtered through celite.

1H-NMR (DMSO-d6, 400 MHz): δ 7.77 (s, 1H), 5.74 (s, 1H), 1.13 (s, 9H)

Intermediate 177: (2S,3R)-2-((S)-1,1-dimethylethylsulfinamido)-3-methylpent-4enoic acid

To a solution of crude (S,E)-2-(tert-butylsulfinylimino)acetic acid (Intermediate 176) in DCM (600 ml) at 0-15°C was added dropwise a solution of (E)-but-2-enylboronic acid ( Intermediate 175) in MeOH (360 ml). The resulting solution was stirred at 015°C for 1 hour. The molecular sieves were removed by filtration and washed with DCM. The filtrate was removed by vacuum distillation to obtain the crude product. H2O (600 ml), petroleum ether (240 ml) and methyl tert-butyl ether (120 ml) were added to the crude product, stirred at room temperature for 1 hour, then filtered and the solid was collected and dried in vacuum at 25° C to give product (110 g, 58%) as a white solid.

1H-NMR (DMSO-f, 400 MHz): δ 5.71-5.80 (m, 1H), 5.01-5.07 (m, 2H), 4.94 (d, J=8 Hz , 1H), 3.58-3.62 (m, 1H), 2.56-2.61 (m, 1H), 1.15 (s, 9H), 0.975 (d, J=4 Hz, 3H)

Intermediate 178: (2S,3R)-methyl-2-amino-3-methylpent-4-enoate

To a solution of (2S,3R)-2-((S)-1,1-dimethylethylsulfinamido)-3-methylpent-4-enoic acid (Intermediate 177, 44 g, 189 mmol) in MeOH (200 ml) was added dropwise SOCl ₂ (68.6 ml, 944 mmol) at 0°C. The reaction mixture was stirred at 0°C for 1 hour, then heated to 70°C and stirred overnight. The solvent was removed and the residue was diluted with water. NaHCO ₃ was added to the aqueous phase to neutralize the pH to about 8, extracted with DCM, dried over Na ₂ SO ₄ , filtered and concentrated to give 23 g of crude product as a yellow liquid.

1H-NMR (DMSO- _d6 , 400 MHz): δ 5.71-5.82 (m, 1H), 4.97-5.05 (m, 2H), 3.61 (s, 3H), 3 .27 (d, J=8 Hz, 1H), 2.33-2.45(m, 1H), 1.79 (s, 2H), 0.96 (d, J=8 Hz, 3H)

LCMS: retention time = 0.682, [M+H]+ 144.2

Intermediate 179: (2S,3R)-methyl-2-(allylamino)-3-methylpent-4-enoate

To a solution of (2S,3R)-methyl-2-amino-3-methylpent-4-enoate (Intermediate 178, 23 g, 161 mmol) in DMF (90 mL) at 0°C was added LiOH (4.248 g, 177 mmol ). The mixture was stirred at 0°C for 30 minutes. A solution of 4-bromobut-1-ene (17.5 g, 145 mmol) in DMF (15 ml) was then added dropwise. The reaction mixture was stirred at -7°C for 20 minutes, then slowly warmed to room temperature and stirred overnight. The reaction was stopped by adding water and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na ₂ SO ₄ , filtered and concentrated to give 28 g of crude product as a yellow liquid.

1H-NMR (DMSO-d6, 400 MHz): δ 5.65-5.83 (m, 2H), 4.93-5.16 (m, 4H), 3.60 (s, 3H), 3. 14-3.22 (m, 1H), 3.06 (d, J=8 Hz, 1H), 2.93-3.01 (m, 1H), 2.34-2.41 (m, 1H) , 0.99 (d, J=8 Hz, 3H)

LCMS: retention time = 0.461, m/z: 184 [M+H]

Intermediate 180: (2S,3R)-methyl-2-(allyl(tert-butoxycarbonyl)amino)-3methylpent-4-enoate

To a solution of (2S,3R)-methyl-2-(allylamino)-3-methylpent-4-enoate (Intermediate 179, 28g, 153 mmol) in t-BuOH (150 ml) was added (Boc) _2O (33, 4 g, 153 mmol). The reaction mixture was stirred at room temperature for 10 minutes, then heated to 90°C and stirred overnight. The reaction mixture was concentrated. The crude product was purified by flash chromatography on silica gel with a gradient of 0 to 6% EtOAc in petroleum ether. The pure fractions were evaporated to give the product (24 g, 56%) as a light yellow liquid.

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1H-NMR (DMSO-d ₆ , 400 MHz): δ 5.71-5.84 (m, 2H), 5.00-5.11 (m, 4H), 3.66-4.43 (m, 3H), 3.58 (s, 3H),

2.81 (s, 1H), 1.39 (s, 9H), 0.93 (d, J=8 Hz, 3H)

LCMS: retention time = 1.096, 184 [M-Voc+H]

Intermediate 181: (2S,3R)-1-tert-butyl-2-methyl-3-methyl-2,3-dihydropyridine 1,2(6H)-dicarboxylate

Vos

To a solution of (2S,3R)-methyl-2-(allyl(tert-butoxycarbonyl)amino)-3-methylpent-4-enoate (Intermediate 180, 24 g, 84.8 mmol) in DCM (250 ml) in three batches Add first generation Grubbs catalyst (886 mg 1.06 mmol) at 0°C and stir for 4 hours. Add first generation Grubbs catalyst (886 mg 1.06 mmol) again at 0°C in three batches, then warm to 25° C. The resulting solution was stirred at room temperature overnight. LCMS: retention time = 0.987, 156.2 [M-Boc+H], 295 [M+K]

Intermediate 182: (2S,5R)-1-tert-butyl-2-methyl-5-(tert-butoxycarbonyl(hydroxy)amino)-3-methyl-5,6-dihydropyridine-1,2(2H)-dicarboxylate

O I

Boc' ^N %w ^B ° ^C

HE

To a solution of (2S,3R)-1-tert-butyl-2-methyl-3-methyl-2,3-dihydropyridine-1,2(6H)-dicarboxylate (Intermediate 181, 21.6 g, 84.8 mmol ) in DCM (250 ml) was added BocNHOH (16.9 g, 127 mmol), CuCl (0.419 g, 4.24 mmol) and pyridine (87 mg, 1.1 mmol), and degassed with oxygen. The resulting solution was stirred at room temperature for 44 hours in an oxygen atmosphere. The solid was removed by filtration. The filtrate was washed with water (3x200 ml) and brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by flash chromatography on silica gel, eluting with 0-50% DCM in petroleum ether to give the product (29 g, 88%) as a brown oil.

1H-NMR (CDCl3, 400 MHz): δ 5.66 (d, J=16 Hz, 1H), 5.31 (s, 1H), 4.90 (d, 1H), 4.51 (d, 1H ), 4.11 (t, J=20 Hz, 1H), 3.75 (s, 3H), 3.50-3.61 (m, 1H), 1.90 (d, J=8 Hz, 3H ), 1.43-1.51 (m, 18N)

LCMS: retention time = 0.713, 279 [M-Boc+Na]

Intermediate 183: (2S,5R)-1-tert-butyl-2-methyl-5-(tert-butoxycarbonyl(tertbutyldimethylsilyloxy)amino)-3-methyl-5,6-dihydropyridine-1,2(2H)-dicarboxylate

To a solution of (2S,5R)-1-tert-butyl-2-methyl-5-(tert-butoxycarbonyl(hydroxy)amino)-3-methyl-5,6dihydropyridine-1,2(2H)-dicarboxylate (Intermediate 182 , 23 g 59.5 mmol) imidazole (8.09 g, 119 mmol) was added to DCM (180 ml). The resulting solution was stirred at room temperature for 10 min, then a solution of TBS-Cl (11.6 g, 77.4 mmol) in DCM (20 ml) was added dropwise at 0-5°C. The reaction mixture was stirred at 0°C for another 18 hours. The organic phase was washed with water and brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by flash silica gel chromatography, eluted with petroleum ether and DCM to give the product (20 g, 67%) as a brown oil.

¹ H-NMR (CDCl3, 400 MHz): δ 5.7(s, 1H), 4.45-4.85 (m, 2H), 4.09 (d, J=20 Hz, 1H), 3, 76 (s, 3H), 3.533.58 (m, 1H), 1.9 (s, 3H), 1.45-1.54 (m, 18H), 0.92 (d, J=16 Hz, 9H ), 0.10 (d, J=20 Hz, 6H).

LCMS: retention time = 1.467, 523.55 [M+Na]

Intermediate 184: (2S,5R)-methyl-5-(tert-butyldimethylsilyloxyamino)-3-methyl1,2,5,6-tetrahydropyridine-2-carboxylate

To a solution of (2S,5R)-1-tert-butyl-2-methyl-5-(tert-butoxycarbonyl(tert-butyldimethylsilyloxy)amino)-3-methyl-5,6-dihydropyridine-1,2(2H)-dicarboxylate (Intermediate 183, 20 g, 40 mmol) ZnBr ₂ (35.5 g, 160 mmol) was added to DCM (200 mL) at 0°C. The resulting solution was stirred at room temperature overnight. The solid was removed by filtration. The filtrate was washed with saturated NaHCO ₃ solution to neutralize the pH to about 8, extracted with DCM, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by flash chromatography on silica gel, eluting with 0-1% MeOH in DCM to give the product (8.4 g, 70%) as a brown oil.

1H-NMR ( _CDCl3 , 400 MHz): δ 5.59-5.62 (m, 1H), 3.82 (s, 1H), 3.75 (s, 3H), 3.14-3.23 (m, 2H), 2.92-2.97 (m, 1H), 1.79 (s, 3H), 0.90 (s, 9H), 0.10 (s, 6H).

LCMS: retention time = 1.073, 301.2 [M+H]

Intermediate 185: Methyl-(2S,5R)-6-[tert-butyl(dimethyl)silyl]oxy-3-methyl-7-oxo1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxylate

To a solution of (2S,5R)-methyl-5-(tert-butyldimethylsilyloxyamino)-3-methyl-1,2,5,6-tetrahydropyridine-2-carboxylate (Intermediate 184, 42 g, 140 mmol) in MeCN (840 ml) DIEA (72.2 g, 560 mmol) was added and degassed with nitrogen, then a solution of triphosgene (16.408 g, 56 mmol) in MeCN (120 ml) was added dropwise at 0±5°C under nitrogen. The resulting solution was stirred at room temperature under nitrogen atmosphere overnight. The reaction mixture was concentrated, then EtOAc was added and washed with 1N. citric acid, saturated NaHCO ₃ solution and brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by flash chromatography on a C-18 stationary phase, eluting with 0-38% MeCN in H2O to give the product (22 g, 49%) as an orange solid.

1H-NMR (CDCl ₃ , 400 MHz): δ 6.15 (t, J=1.6 Hz, 1H), 4.412 (s, 1H), 3.807 (s, 3H), 3.610-3.629 (m, 1H) , 3.506 (d, J=11 Hz, 1H), 3.290-3.322 (m, 1H), 1.722 (s, 3H), 0.962 (s, 9H), 0.195 (s, 3H), 0.172 (s, 3H)

LCMS: retention time = 1.585, 327.35 [M+H]

Intermediate 186: (2S,3R)-2-amino-3-methylpent-4-enamide hydrochloride

O s

Η ₂ Ν y

NH ₂ HCI

CDI (61.9 kg, 382.1 mol, 1.2 eq) was added in batches at 20±10°C. The mixture was stirred for 2 hours at 20±10°C. The mixture was cooled to -40±5°C. NH3-H2O (43.55 kg, 640.4 mol, 2.0 eq, 25 wt%) was added dropwise at -40±5°C. The mixture was stirred for 10 minutes at -40±5°C. After completion of the reaction, the mixture was heated to -10~0°C, then concentrated in vacuo to about 3.0 of the original volumes. THF (2.0 vol) was added and concentrated in vacuo to about 3.0 vol. THF was replaced twice with EtOAc (2.0 vol). EtOAc (6.0 vol) was added to the solution and cooled to 5±5°C. MeSO ₃ H (148.3 kg, 1543.0 mol, 2.4 eq) was added dropwise at <10°C and stirred for 1 hour at 5±5°C. The mixture was centrifuged and the resulting solid was washed twice with EtOAc (1.0 vol). The mother liquor was collected to give the compound in solution in EtOAc, which was directly used in the next step. HCl (gas) was bubbled through the solution at 0±5°C for 17 hours (about 4 kg/h). After completion of the reaction, the mixture was centrifuged and the resulting solid was washed twice with EtOAc (1.0 vol). The residue was dried in vacuo at 25±5°C for at least 12 hours to obtain the title compound as a light yellow solid (98 kg, total yield of two steps 90%).

1H NMR (400 MHz, DMSO): δ 8.24 (s, 2H), 8.00 (s, 1H), 7.56 (s, 1H), 5.84-5.76 (m, 1H), 5.17-5.09 (m, 2H), 3.70-3.68 (m, 1H), 2.77-2.72 (m, 1H), 1.04 (d, J=6.8 Hz, 3H).

Intermediates 187 and 188: allyl ((2S,3R)-1-amino-3-methyl-1-oxopent-4-en-2-yl) and tert-butyl-allyl ((2S,3R)-1-amino -3-methyl-1-oxopent-4-en-2-yl)carbamate

To a solution of LiOH (27.55 kg, 1147.9 mol, 2.0 eq) in DMF (285 L) was added Intermediate 186 (95 kg, 564.67 mol, 1.0 eq) in batches at 0 ± 5 °C. The mixture was stirred for 0.5 h. Allyl bromide (76.76 kg, 634.5 mol, 1.1 eq.) was added dropwise over 9 h at 0±5°C. The reaction mixture was heated to 20±5°C and stirred for at least 8 hours. The reaction mixture was cooled to 10±5°C and softened water (3.0 vol.) was added. The mixture was extracted three times with DCM (3.0 vol). The combined organics were washed with brine (2.0 vol). The brine layer was extracted with DCM (3.0 vol). The combined organic layers were concentrated at atmospheric pressure at <

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60°C to about 5.0 original volumes. Tert-butyl alcohol (2.0 vol) was added to the solution and concentrated in vacuo at <60°C to about 5.0 of the original volumes. Concentration with t-butyl alcohol (2.0 vol) was repeated one more time until the water content was <0.2% to yield Intermediate 187. Tert-butyl alcohol (8.0 vol) was added to the concentrated solution. Boc ₂ O (138.07 kg, 633.3 mol, 1.1 eq.) was added at 20±5°C. The mixture was heated to 70±5°C and stirred for at least 15 hours.

After completion of the reaction, the mixture was cooled to 40±5°C, then concentrated in vacuo at <60°C to about 5 original volumes. The concentrated mixture was cooled to 25±5°C and softened water (5.0 vol.) was added. The mixture was extracted twice with methyl tert-butyl ether (4.0 vol). The combined organics were washed once with 0.5 M HCl (2.0 vol), three times with brine (1.0 vol), and concentrated in vacuo at <50°C to about 2.0 of the original volumes. Nheptane (1.0 vol) was added to the reactor and concentrated in vacuo at <50°C to about 2.0 of the original volumes. This procedure was repeated one more time. n-heptane (1.0 vol.) was added to the concentrated solution, cooled to 10 ± 5°C and stirred for at least 2 hours. The mixture was centrifuged and the resulting solid was washed with cooled n-heptane (0.5 vol.) . The residue was dried in vacuo at 25 ± 5°C for at least 12 hours to obtain Intermediate 188 as a white solid (84.2 kg, 100% purity, total yield of two steps 54.4%).

^1H NMR (400 MHz, DMSO): δ 7.42 (s, 1H), 6.95 (s, 1H), 5.78-5.70 (m, 2H), 5.11-4.98 ( m, 4H), 4.33 (d, J=10.8 Hz, 1H), 3.92-3.78 (m, 2H), 2.70 (br, 1H), 1.41 (s, 9H ), 0.88 (d, J=6.4 Hz, 3H). LC/MS (ES ⁺ ) m/z 169.2 (M+H)

Intermediates 189: (2S,5R)-tert-butyl-5-(tert-butoxycarbonyl(hydroxy)amino)-2carbamoyl-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate

O I

A 1 h ₂ np η oh

To a solution of Intermediate 188 (81.0 kg, 301.8 mol, 1.0 eq) in DCM (810 L), first generation Grubbs catalyst (1.215 kg, 1.5 mol, 0.005 eq) was added in three batches at 0 ±5°C. The reaction mixture was stirred for 1 hour at 0±5°C, then heated to 25±5°C and stirred for 1 hour. The mixture was cooled to 0±5°C and the catalyst was added again (1.215 kg, 1.5 mol, 0.005 eq.) in three batches at 0±5°C. The mixture was heated to 25±5°C and stirred for at least 8 hours. After completion of the reaction, CuCl (1.46 kg, 14.8 mol, 0.05 eq.), BocNHOH (59.94 kg, 450.7 mol, 1.5 eq.) and pyridine (0.31 kg, 3.9 mol, 0.013 eq.) at 25±5°C. The mixture was stirred for at least 43 hours at 25±5°C in an oxygen atmosphere. After completion of the reaction, EDTA ₂ Na solution (5.0 vol) was added to the reactor and stirred for at least 4 hours at 25±5°C. The layers were separated and the aqueous layer was extracted twice with DCM (3.0 vol). Organic substances were combined and concentrated in vacuum at <40°C to about 3.0 of the original volumes. Methyl tertiary butyl ether (MTBE) (2.0 vol) was added to the reactor and concentrated in vacuo at <40°C to about 3.0 original volumes. This procedure was repeated one more time. MTBE (2.5 vol), n-heptane (2.5 vol) and softened water (5.0 vol) were added to the concentrated solution and the mixture was suspended at 20±5°C for at least 2 hours. The mixture was centrifuged and the precipitate was washed with MTBE/n-heptane (0.5 vol, 1:1). The residue was dried in vacuo at 35±5°C for at least 12 hours to <1% water content to give the title compound as a light brown solid (70.3 kg, 98% purity, total yield of two steps 61.3%).

1H NMR (400 MHz, DMSO): δ 8.93-8.78 (m, 1H), 7.49 (s, 1H), 7.07 (s, 1H), 5.57 (s, 1H), 4.62-4.41 (m, 2H), 3.76-3.50 (m, 2H), 1.79 (s, 3H), 1.43 (s, 1H), 1.40 (s, 9H). LC/MS (ES ⁺ ) m/z 272.2 (M+H)

Intermediates 190 and 191: tert-butyl-(3R,6S)-3-((tert-butoxycarbonyl)((tert-butyldimethylsilyl)oxy)amino)-6-carbamoyl-5-methyl-3,6-dihydropyridine-1(2H )-carboxylate and (2S,5R)-5-(tert-butyldimethylsilyloxyamino)-3-methyl-1,2,5,6-tetrahydropyridine-2-carboxamide

TBS- Cl (30.5 kg, 202.0 mol, 1.5 eq) in solution in DCM (1.5 vol) at 0±5°C for 4.5 hours. The mixture was warmed to 20±5°C and stirred for at least 5 hours. After completion of the reaction, softened water (250 L, 5.0 vol.) was added. The layers were separated and the aqueous layer was extracted with DCM (3.0 vol). The combined organic layers were washed twice with softened water (3.0 vol), then concentrated by atmospheric distillation at <50°C to ca.

- 111 044428

2.0 initial volumes. DCM (200 L, 4.0 vol) was added to the solution and concentrated by atmospheric distillation at <50°C to about 2.0 original volumes, <1% water content, to yield Intermediate 190. Added to the solution with stirring DCM (750 L, 15.0 vol.) at 20±5°C under nitrogen atmosphere. ZnBr ₂ (60.5 kg, 268.9 mol, 2.0 eq.) was added to the solution and stirred for 6 hours at 20±5°C. ZnBr ₂ (30.5 kg, 135.6 mol, 1.0 eq.) was added back to the reaction mixture every 6-8 hours until the reaction was complete (about 5 eq. ZnBr ₂ total). The reaction was stopped by adding a solution (18.0 vol.) of NaHCO ₃ (113.0 kg, 1345.2 mol, 10.0 eq.) at a temperature less than 20°C. The mixture was stirred for at least 1 hour at 20±5°C, then centrifuged and the centrifuge collected. The resulting solid precipitate was suspended with dichloromethane (5.0 vol.) at 20±5°C for 1 hour, then centrifuged. The centrifugate was combined with the previously collected centrifugate and the layers were separated. The aqueous layer was extracted twice with DCM (3.0 vol). The combined organics were washed four times with softened water (4.0 vol), then concentrated under normal pressure at <50°C to about 2.0 original volumes. CH3CN (2.0 vol) was added to the solution and concentrated in vacuo at <50°C to about 4.0 original volumes to give Intermediate 191 in solution.

LC/MS: (ES+) m/z 282.2 (M+H)

Intermediate 192: (2S,5R)-6-(tert-butyldimethylsilyloxy)-3-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide °I n/A m

About OTBS

To a solution of Intermediate 191 (38.43 kg (in theory), 134.6 mol, 1.0 eq.) in CH ₃ CN (1036 L, 27 vol.) was added DIEA (69.56 kg, 539.2 mol , 4.0 eq.) at 20±5°C. The mixture was cooled to 0±5°C and triphosgene (13.07 kg, 44.0 mol, 0.33 eq) in CH3CN (115.2 L, 3.0 vol) was added dropwise. The mixture was heated to 25±5°C and stirred for at least 8 hours, then cooled to 10±5°C and quenched by adding softened water (24.19 kg, 1343.9 mol, 10.0 eq). The mixture was stirred for at least 1 hour, then concentrated in vacuo at <40°C to about 5.0 original volumes. The solution was cooled to 10±5°C and MTBE (384 L, 10 vol.) and brine (4.0 vol.) were added. The layers were separated and the organics were washed with brine (4.0 vol.), and concentrated in vacuo at <40°C to about 2.0 original vol. MTBE was replaced with n-heptane (1.0 vol) and the solid was suspended for 1 h at 20 ± 5°C. The mixture was centrifuged and the precipitate was washed twice with n-heptane (0.5 vol.). The residue was suspended in MTBE (67.5 L) for at least 3 hours at 20 ± 5°C, then n-heptane (336 L) was added with stirring for at least 1 hour. The mixture was centrifuged and the resulting precipitate was washed with n -heptane (1.0 vol.). The residue was dried in vacuo at 30±5°C for 12 hours to obtain the title compound as a light brown solid (21.1 kg, purity 99.6%, total yield of three steps 49.6%).

1H NMR (400 MHz, DMSO): δ 7.80 (s, 1H), 7.33 (s, 1H), 6.06 (t, J=2 Hz, 1H), 4.10 (s, 1H) , 3.71-3.69 (m, 1H), 3.65 (d, J=10.8 Hz, 1H), 3.10 (dd, J=10.8 Hz, 2.0 Hz, 1H) .1.63 (s, 3H), 0.92 (s, 9H), 0.14 (d, J=0.8 Hz, 1H). LC/MS (ES ⁺ ) m/z 312.2 (M+H)

Intermediate 193: (2S,5R)-tert-butyl-5-(tert-butoxycarbonyl(hydroxy)amino)-2carbamoyl-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate

To a solution of Intermediate 192 (18.5 kg, 59.4 mol, 1.0 eq) in EtOAc (92.5 L, 5 vol) was added HF-Py (2.04 kg, 71.4 mol, 1 .2 eq., 70 wt.%) at 0±5°C. The mixture was stirred for at least 10 hours at 20±5°C, then cooled to 0±5°C and HF-Py (0.33 kg, 11.6 mol, 0.2 eq., 70 wt.) was added again. %). The mixture was stirred for at least 3 hours at 20±5°C, then MTBE (27.8 L, 1.5 vol) was added and stirred for 3 hours at 10±5°C. The mixture was centrifuged and the precipitate was washed with EtOAc (0.5 vol). The pellet was suspended in EtOAc (2.0 vol) for at least 1 hour at 20 ± 5°C, then centrifuged. The residue was suspended in EtOAc (0.5 vol) then dried in vacuo at 25±5°C for 12 hours to give the title compound as a yellow solid (11.0 kg, 99.5% purity, yield 91%).

^1H NMR (400 MHz, DMSO): δ 9.56 (s, 1H), 7.79 (s, 1H), 7.32 (s, 1H), 6.10 (d, J=3.2 Hz , 1H), 4.06 (s, 1H), 3.68-3.62 (m, 2H), 3.07 (d, J= 8.4 Hz, 1H), 1.61 (s, 3H) . LC/MS (ES ⁺ ) m/z 198.1 (M+H)

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Intermediate 194: Ethyl (S,E)-2-((tert-butylsulfinyl)imino)acetate

To a solution of ethyl 2-oxoacetate (66 ml, 321 mmol, 50% in toluene) in DCM (1 L) at 0°C was added (S)-2-methylpropane-2-sulfinamide (30 g, 248 mmol) and molecular sieves (4A, 500 g). The resulting solution was stirred at room temperature for 18 hours. The molecular sieves were removed by filtration; The filtrate was concentrated by vacuum distillation to give the crude product, which was purified by flash silica gel chromatography (0-5% EtOAc in petroleum ether) to give a colorless oil, 45 g, 88%.

1HNMR (400 MHz, CDC13): δ 1.28 (s, 9H), 1.39 (t, J=12 Hz, 3H), 4.38 (q, J=12 Hz, 2H), 8.01 ( s, 1H).

Tetrahydropyridine-2-carboxylate intermediate

195: ethyl-(S)-1-((S)-tert-butylsulfinyl)-4-methyl-1,2,3,6-

To a solution of (S,E)-ethyl-2-(tert-butylsulfinylimino)acetate (Intermediate 194, 50 g, 244 mmol) in DCM (600 ml) at -78°C was added isoprene (97.21 ml, 971, 91 mmol) and then TMSOTf (97.42 ml, 416.54 mmol). The resulting solution was stirred at -78°C for 3 hours and the reaction was slowly stopped at -78°C by adding a phosphate buffer solution (pH=7.4, 1 L). After warming to room temperature, the mixture was extracted with DCM (3x500 ml). The combined organic extracts were washed with water (2x500 ml) and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated to give 60 g of crude product as a brown oil. The product was used in the next step without further purification.

1H NMR (400 MHz, d6-DMSO): δ 1.08 (s, 9H), 1.18 (t, J=12 Hz, 3H), 1.64 (q, J=4 Hz, 3H), 3 .59 (m, 2H), 4.11 (dq, J=12.4 Hz, 2H), 4.30 (dd, J=8.4 Hz, 1H), 5.39 (ddd, J=4, 84 Hz, 1H);

LCMS: (ES ⁺ ) [M+H] ⁺ = 274; HPLC retention time = 1.78 min.

Intermediate 196: 1-(tert-butyl)-2-ethyl-(S)-4-methyl-3,6-dihydropyridine-1,2(2H)dicarboxylate

ABOUT

Eto Υ|Α g, JJ Vos

To a solution of crude (S)-ethyl-1-((S)-tert-butylsulfinyl)-4-methyl-1,2,3,6-tetrahydropyridine-2-carboxylate (Intermediate 195, 100 g) in MeOH (1 l) Hydrogen chloride (100 ml, 4 M in dioxane) was added at 0°C. The resulting solution was stirred at room temperature for 18 hours. MeOH and HCl/dioxane were removed by vacuum distillation to give the crude product, which was dissolved in water (1 L) and extracted with EtOAc (3x500 ml). The pH of the aqueous solution was adjusted to 7 using solid NaHCO ₃ . The aqueous solution was extracted with EtOAc until the product was no longer detectable by LCMS. The organic phases were combined and dried over Na ₂ SO ₄ , filtered and concentrated to give the crude product (30 g, 177 mmol) as a light yellow oil. The oil was dissolved in THF (500 ml) and cooled with an ice water bath. To the cooled solution was added a solution of sodium bicarbonate (22.3 g, 265.5 mmol) in water (500 ml), followed by di-tert-butyl dicarbonate (57.8 g, 265.5 mmol). The resulting solution was stirred at room temperature for 18 hours. The two layers were separated. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash chromatography (0-30% EtOAc in PE) to give the title compound 47.5 g, 43% yield from Intermediate 194.

1H NMR (400 MHz, CDC13): δ 1.24 (t, 3H), 1.50 (m, 9H), 1.71 (s, 3H), 2.46 (m, 2H), 3.73 ( m, 1H), 4.10 (m, 3H), 4.95 (m, 1H); LC-MS: (ES ⁺ ) [M+Na] ⁺ = 292; HPLC retention time = 1.71 min.

Intermediate 197: tert-butyl (S)-2-carbamoyl-4-methyl-3,6-dihydropyridine-1(2H)carboxylate

To a solution of 1-(tert-butyl) 2-ethyl-(S)-4-methyl-3,6-dihydropyridine-1,2(2H)-dicarboxylate (Pro- 113 044428 intermediate 196, 47.5 g, 176 mmol) in THF (1000 ml) and water (500 ml) at 0°C, lithium hydroxide (1 M, 440 ml, 440 mmol) was added dropwise.

The reaction mixture was warmed to room temperature and stirred for 16 hours. The solvent was removed; the residue was diluted with water. The pH of the solution was adjusted to about 3 using HCl solution (1N). The mixture was extracted with EtOAc (3x300 ml).

The organic layers were combined, washed with water and brine, dried over MgSO ₄ , filtered and concentrated to give a colorless oil, 40.3 g.

¹ H NMR (300 MHz, d ₆ -DMSO): δ 1.38 (m, 9H), 1.64 (s, 3H), 2.53 (m, 2H), 3.68 (m, 3H), 4.73 (m, 1H), 5.35 (dd, J=3.15 Hz, 1H), 12.45 (s, 1H);

LCMS: (ES ⁺ ) [M+Na] ⁺ = 264; HPLC retention time = 1.01 min.

Intermediate 198: tert-butyl (S)-2-carbamoyl-4-methyl-3,6-dihydropyridine-1(2H)carboxylate

To a solution of ^)-1-(tert-butoxycarbonyl)-4-methyl-1,2,3,6-tetrahydropyridine-2-carboxylic acid (Intermediate 197, 40.3 g, 167.2 mmol) in THF (500 ml) at 0°C, N,N'-carbonyldiimidazole (32.5 g, 200.6 mmol) was added portionwise. The crude material was stirred at 0°C for 5 hours. Ammonium acetate (38.2 g, 502.9 mmol) was then added. The reaction mixture was stirred at room temperature for another 18 hours, the reaction was stopped by adding water and the reaction mixture was extracted with EtOAc. The combined organic layers were washed with water and brine, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by flash chromatography on silica gel (0%-30% EtOAc in PE) to give a white solid, 25 g, 62%.

^1H NMR (400 MHz, d6-DMSO): δ 1.41 (s, 9H), 1.66 (s, 3H), 2.35 (s, 2H), 3.84 (m, 2H), 4 .66 (m, 1H), 5.35 (m, 1H), 6.96 (s, 1H), 7.19 (s, 1H);

LCMS: (ES ⁺ ) [M+Na] ⁺ = 263; HPLC retention time = 0.86 min.

Intermediate 199: tert-butyl-(3R,6S)-3-((tert-butoxycarbonyl)(hydroxy)amino)-6carbamoyl-4-methyl-3,6-dihydropyridine-1(2H)-carboxylate

To a solution of tert-butyl-(S)-2-carbamoyl-4-methyl-3,6-dihydropyridine-1(2H)-carboxylate (Intermediate 198, 25 g, 104.1 mmol) in DCM (250 ml) was added BocNHOH (70.6 g, 530.9 mmol), CuCl (6.1 g, 62.5 mmol) and pyridine (106.9 mg, 1.3 mmol). The resulting solution was stirred at room temperature for 44 hours in an oxygen atmosphere. Solids were removed by filtration. The filtrate was washed with water (6x500 ml) and brine, dried over Na2SO4, filtered and concentrated. The crude product was purified by silica gel flash chromatography (0-50% EtOAc in PE) to give the title compound as a white solid, 40% yield. The starting material was isolated (10 g). This procedure was repeated three times to obtain a total of 15 g of product.

LCMS: (ES ⁺ ) [M+Na] ⁺ = 394 (C17H29N3O6)

Intermediate 200: tert-butyl-(3R,6S)-3-((tert-butoxycarbonyl)((tert-butyldimethylsilyl)oxy)amino)-6-carbamoyl-4-methyl-3,6-dihydropyridine-1 (2H )-carboxylate

To a solution of tert-butyl-(3R,6S)-3-((tert-butoxycarbonyl)(hydroxy)amino)-6-carbamoyl-4-methyl3,6-dihydropyridine-1(2H)-carboxylate (Intermediate 199, 12 g, 32.3 mmol) imidazole (4.4 g, 64.6 mmol) was added to DCM (96 ml) at 0±5°C. The resulting solution was stirred at room temperature for 10 min, then TBS-Cl (4.8 g, 32.3 mmol) in DCM (10 ml) was added dropwise.

The reaction mixture was stirred at 0°C for another 18 hours, washed with water and brine, dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography on silica gel (0-20% EtOAc in PE) to give the title compound as a white solid, 10 g, 63%.

LCMS: (ES ⁺ ) 486 (C23H43N3O6SO

Intermediate 201:

1,2,5,6-tetrahydropyridine-2-carboxamide (2S,5R)-5-(((tert-butyldimethylsilyl)oxy)amino)-4-methyl- 114 044428

To a solution of tert-butyl-(3R,6S)-3-[tert-butoxycarbonyl-[tert-butyl(dimethyl)silyl]oxyamino]-6carbamoyl-4-methyl-3,6-dihydro-2H-pyridine-1-carboxylate (Intermediate 200, 21.7 g, 44.68 mmol) Zinc bromide (40.24 g, 178.71 mmol) was added to DCM (250 mL) at 0°C. The resulting suspension was allowed to warm to room temperature and stirred for about 66 hours. The reaction mixture was cooled using an ice water bath to which was added a suspension of NaHCO ₃ (38.23 g, 10 eq.) in water (300 ml). The resulting mixture was stirred for 1 hour. The solid was removed by filtration and washed 3-4 times with DCM until no more product was detected in the wash solution. The two layers of filtrate were separated. The aqueous layer was extracted with DCM three times (until no more product was detected in the aqueous solution). The combined DCM solution was concentrated to remove most of the solvent. The residue was partially dissolved in 10% MeOH in DCM and loaded onto a thin layer of silica gel and eluted with 10% MeOH in DCM. The filtrate was evaporated and dried in vacuo to give a solid yellow foam (9.9 g crude, 77%). MS: 286 ES+ (C ₁₃ H ₂₇ N ₃ O ₂ Si)

Intermediate 202: (2S,5R)-6-((tert-butyldimethylsilyl)oxy)-4-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carboxamide

In a clear solution of (3R,6S)-3-[[tert-butyl(dimethyl)silyl]oxyamino]-4-methyl-1,2,3,6tetrahydropyridine-6-carboxamide (Intermediate 201, 7.66 g, 26 .83 mmol) in MeCN (150 ml) and DCM (200 ml) at 0°C was added N,N'-diisopropylethylamine (19.11 ml, 107.34 mmol), followed by a solution of triphosgene (2.71 g, 9 .12 mmol) in MeCN (50 ml) dropwise (2 ml/hour using a syringe pump). After addition, the solution was allowed to warm to room temperature and stirred overnight. The reaction mixture was concentrated to dryness. The resulting residue was diluted with EtOAc and washed with brine. The aqueous layer was extracted with EtOAc. The combined organic extracts were dried over MgSO ₄ , filtered and concentrated. The crude product was purified by silica gel chromatography (0-100% EtOAc/hexane) to give the title compound as a white solid, 4.36 g, 52%.

MS: 312 ES+ (C14H25N3O3S0

Intermediate 203: (2S,5R)-6-hydroxy-4-methyl-7-oxo-1,6-diazaαbicyclo[3.2.1]oct-3ene-2-carboxamide

To a solution of (2S,5R)-5-(((tert-butyldimethylsilyl)oxy)amino)-4-methyl-1,2,5,6-tetrahydropyridine2-carboxamide (Intermediate 202, 165 mg, 0.53 mmol) HF-pyridine (0.02 mL, 0.64 mmol) was added in ethyl acetate (4 mL) at 0°C. The reaction mixture was warmed to room temperature and stirred for 1 hour. Only a small amount of product was observed. Another equivalent of HF-pyridine was added and the reaction mixture was stirred for 3 hours. The reaction mixture was concentrated to give an orange solid.

MS: 198 ES+ (C8H11N3O3)

Example 35 )-2-fluoroacetate

To a solution of (2S,5R)-6-hydroxy-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 203, 582 mg, 2.95 mmol) in 1,4-dioxane (16 ml) and DMF (2 ml) was added isopropyl-(2R)-2-bromo-2-fluoroacetate (Intermediate 169, 881.1 mg, 4.43 mmol). The reaction mixture was cooled to 0°C and DBU (0.44 mL, 2.95 mmol) was added dropwise. The reaction mixture was stirred for 10 minutes, then diluted with ethyl acetate and washed twice with a 1:1 mixture of brine and water. Organic substances were dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0-90% ethyl acetate/hexanes) gave a white foam. The foam was dissolved in a 1:1 mixture of acetonitrile and water, frozen and lyophilized to give a white solid, 614 mg, 66%. 6% S-diastereomer was present.

MS 316 ES+ (C13H18FN3O5)

1H NMR (300 MHz, DMSO-d6) δ: 1.22 (m, 6H); 1.81 (m, 3H); 3.17 (m, 1H); 3.34 (m, 1H); 3.93(m, 1H);

4.22 (m, 1H); 5.01 (m, 2H); 5.51 (m, 1H); 6.23 (m, 1H); 7.31(s, 1H); 7.55(s, 1H).

Example 90. Ethyl 2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2, 2-difluoroacetate

To a solution of (2S,5R)-6-hydroxy-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 203, 64.5 mg, 0.33 mmol) and ethyl bromodifluoroacetate (0.13 ml, 0.98 mmol) in DMF (3 ml) was added potassium carbonate (135.62 mg, 0.98 mmol) at room temperature. The mixture was stirred for about 3 hours, then diluted with ethyl acetate and filtered. The filtrate was combined with the previous small batch, washed twice with a 1:1 mixture of brine and water, dried over magnesium sulfate, filtered and concentrated. Silica gel chromatography (0-80% ethyl acetate/hexanes) and lyophilization provided the title compound (52.1 mg, 34%) as an orange solid.

MS: 320 ES+ (C12H15F2N3O5)

1H NMR (300 MHz, DMSOC) δ: 1.29 (t, 3H); 1.82 (m, 3H); 3.36 (m, 2H); 3.94 (m, 1H); 4.31(m, 1H); 4.39 (m, 2H); 5.57 (m, 1H); 7.36(s, 1H); 7.59(s, 1H).

Example 91 -difluoroacetic acid lithium salt

To a solution of ethyl 2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)-2,2 -difluoroacetate (Example 90, 46 mg, 0.14 mmol) in THF (2 ml) and water (0.50 ml) at 0°C was added 1 M lithium hydroxide (0.14 ml, 0.14 mmol). The reaction mixture was stirred at 0°C for 10 min. Another 0.2 equiv was added. lithium hydroxide and after 5 minutes the reaction was neutralized by adding 0.5N. hydrochloric acid, and THF was evaporated. The resulting solution was frozen and lyophilized. After purification in a Gilson system (Synergi Polar RP 21.2 mm χ 100 mm, 4 µm, coupled with YMC C30 20 mm χ 150 mm, 5 µm, 0-16% acetonitrile/water, 6 min) the title was obtained compound (27.2 mg, 64.8%) as an off-white solid. MS: 292 ES+ (C10H11F2N3O5)

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.81 (m, 3H); 3.30 (m, 2H); 3.84 (m, 1H); 4.20 (m, 1H); 5.48 (m, 1H); 7.29(s, 1H); 7.55(s, 1H).

Example 92 Ethyl 2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)acetate

To a solution of (2S,5R)-6-((tert-butyldimethylsilyl)oxy)-4-methyl-7-oxo-1,6-diazabicyclo-[3.2.1]oct3-ene-2-carboxamide (Intermediate 202, 152 mg, 0.49 mmol) in THF (4 ml) at 0°C was added TBAF (0.49 ml, 0.49 mmol). The mixture was stirred for about 10 minutes. Ethyl bromoacetate (0.05 mL, 0.49 mmol) was added to the reaction mixture and stirred for 10 minutes. Ethyl bromoacetate (0.05 mL, 0.49 mmol) was added again and stirred for 30 minutes. Ethyl bromoacetate (0.05 mL, 0.49 mmol) was added again and the reaction mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was concentrated on silica gel and purified (0%-90% ethyl acetate/hexanes) to give a colorless oil. . The oil was dissolved in a 1:1 mixture of acetonitrile and water, frozen and lyophilized to give the title compound as a white solid, 78.5 mg, 56%. MS: 284 ES+ (C ₁₂ H ₁₇ N ₃ O ₅ )

1H NMR (300 MHz, DMSO-d ₆ ) δ: 1.23 (t, 3H); 1.84 (m, 3H); 3.19 (m, 2H); 3.97 (m, 1H); 4.17 (m, 3H); 4.53 (m, 2H); 5.46 (m, 1H); 7.29(s, 1H); 7.50(s, 1H).

- 116 044428

Example 93. 2-(((2S,5R)-2-Carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6-yl)oxy)acetic acid lithium salt

To a solution of ethyl 2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)acetate (example 92 , 57.4 mg, 0.2 mmol) in THF (2 ml) and water (1 ml) at 0°C was added 1 M lithium hydroxide (0.66 ml, 0.66 mmol). The reaction mixture was stirred at 0°C for 10 min. Another 0.2 equiv was added. Lithium hydroxide was added and after 10 minutes the reaction was complete. The reaction mixture was neutralized by adding 0.5N. hydrochloric acid and added 1 eq. sodium bicarbonate in water at 0°C. The resulting solution was frozen and lyophilized. Purification on a Gilson system (0-16%, 6 min) provided the title compound as a white solid, 18.3 mg, 35%.

MS: 256 ES+ (C10H13N3O5)

1H NMR (300 MHz, DMSOL) δ: 1.85 (m, 3H); 3.11(m, 2H); 3.88(m, 2H); 4.07 (m, 1H); 4.24 (m, 1H); 5.39 (m, 1H); 7.24(s, 1H); 7.48(s, 1H).

Intermediate 204: (2S,5R)-6-((tert-butyldimethylsilyl)oxy)-4-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-2-carbonitrile

N^.

J—N _s /L O O' ^Sl \ \

The title compound was prepared from (2S,5R)-6-((tert-butyldimethylsilyl)oxy)-4-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate compound 202, 0.205 g, 0.66 mmol) following the procedure for Intermediate 139 to obtain the title compound (159 mg, 82%) as a white solid. MS: 294 ES+ (C ₁₄ H ₂₃ N ₃ O ₂ Si)

Example 94 Ethyl-(2R)-2-(((2S,5R)-2-cyano-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy )-2-fluoroacetate

F L— ^N 4 A o / o o

The title compound was prepared from (2S,5R)-6-((tert-butyldimethylsilyl)oxy)-4-methyl7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carbonitrile (Intermediate compound 204, 152 mg, 0.49 mmol) according to an alternative procedure for example 35 to obtain (26.9 mg, 19%) a colorless oil. Approximately 9% S-diastereomer was present.

MS: 284 ES+ (C12H14FN3O4)

1H NMR (300 MHz, DMSO-06) δ: 1.09 (t, 3H); 1.72 (m, 3H); 3.26 (m, 2H); 3.97 (m, 1H); 4.11(m, 2H); 4.94 (m, 1H); 5.30 (m, 1H); 6.16 (m, 1H).

Example 95 )-2fluoroacetic acid lithium salt (ΓΆγ o

The title compound was prepared from ethyl-(2R)-2-(((2S,5R)-2-cyano-4-methyl-7-oxo-1,6diazabicyclo[3.2.1]oct-3-ene-6- yl)oxy)-2-fluoroacetate (Example 94, 22 mg, 0.08 mmol) according to the procedure for Example 91 to give (3.8 mg, 15%) a light yellow solid. MS: 256 ES+ (C10H10FN3O4)

1H NMR (300 MHz, DMSO-d6) δ: 1.81 (m, 3H); 3.30 (m, 2H); 4.00 (m, 1H); 4.92(m, 1H); 5.27 (m, 1H); 5.28 (m, 1H).

Example 96 Isopropyl 2-(((2S,5R)-2-carbamoyl-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-en-6yl)oxy)acetate

- 117 044428

To a solution of (2S,5R)-6-hydroxy-4-methyl-7-oxo-1,6-diazabicyclo[3.2.1]oct-3-ene-2-carboxamide (Intermediate 203, 93.38 mg, 0 .47 mmol) and isopropyl bromoacetate (0.18 ml, 1.42 mmol) in DMF (4 ml) was added potassium carbonate (196.35 mg, 1.42 mmol) at room temperature. The reaction mixture was stirred for about 3 hours, then diluted with ethyl acetate and filtered. The filtrate was washed twice with a 1:1 mixture of brine and water, dried over magnesium sulfate, filtered and concentrated. Chromatography on silica gel (0%-80% ethyl acetate/hexanes) and lyophilization in acetonitrile gave the title compound as an off-white solid, 106.6 mg, 72%. MS: 298 ES+ (C13H19N3O5)

1H NMR (300 MHz, DMSO-de) δ: 1.23 (m, 6H); 1.84 (m, 3H); 3.19 (m, 2H); 3.97 (m, 1H); 4.16(m, 1H); 4.35 (m, 1H); 4.62 (m, 1H); 5.00 (m, 1H); 5.46 (m, 1H); 7.29(s, 1H); 7.50(s, 1H).

Biological examples

Example 102 Inhibition of beta-lactamase enzymes

For all enzymes, a buffer consisting of 0.1 M sodium phosphate (pH 7.0), 10 mM NaHCO ₃ and 0.005% Triton X-100 was used. The chromogenic substrate nitrocefin (SynGene, Bangalore, India) was used at 100 μM. Enzyme activity was monitored by the increase in absorbance at 490 nm resulting from the hydrolysis of nitrocefin. Assays were performed in clear 384-well polystyrene plates (Greiner Bio-One, Monroe, NC). Absorbance was measured over 1 h at 30 second intervals using a Spectramax absorbance plate reader (Molecular Devices, Sunnyvale, CA). When measuring beta-lactamase inhibition by the inhibitor, serial 3-fold dilutions of the inhibitor in assay buffer were used, ranging from 100 μM to 62.7 pM. The background curve characterizing the absorption process for the control containing no enzyme and inhibitor was subtracted from each process curve. The complete set of process curves for one enzyme with all inhibitor concentrations was numerically integrated using Kintek Global Kinetic Explorer (Kintek Corp, Snow Shoe, PA) to obtain the best fit to the mechanism shown in Scheme 10.

Scheme 10

where E, S, ES, P, I and EI respectively represent the concentrations of the enzyme, nitrocefin, enzyme-nitrocefin complex, nitrocefin hydrolysis product, inhibitor and enzyme inhibitor complex. The measured value of K _m (nitrocefin) was used to determine the fixed values k ₊₁ , k -1 and k ₊₂ , where k ₊₁ = k -1 = 1 and k ₊₂ = K _m -1 . The values of k ₊₂ , k ₊₃ and k _-3 were selected. Concentration-biased runs were used to correct for minor differences in background absorbance between wells in absorbance measurements. The parameter k ₊₃ is equivalent to the second order rate constant k _inact /Ki. In some cases, inhibition was in rapid equilibrium on the experimental time scale, so that only the ratio k _-3 /k ₊₃ = Ki could be determined. Although k _-3 represents the reversal of inhibitor binding in Scheme 10, hydrolysis of the enzyme-inhibitor complex cannot be ruled out based on kinetic measurements.

The best-fit absorbance values obtained from the above procedure at each time point for each compound concentration were exported to Excel. To calculate the IC ₅₀ at 60 min, the % inhibition for each inhibitor concentration at 60 min was calculated based on the best-fitting absorbance values at that time point, using the equation % inhibition = 100 X (1 - Ainhih/Amax), where A is the best-fit the absorbance value without the inhibitor, and A _inhlb is the most closely corresponding absorbance value in the presence of the inhibitor. IC ₅₀ was calculated from a set of % inhibition values using nonlinear least squares regression using the equation % inhibition = 100 [I] ⁿ /(ICso + [I] ⁿ ) where [I] is the inhibitor concentration and n is the Hill coefficient. For nonlinear regression, we used the Excel XLfit add-in (ID Business Solutions). In table Table 1 shows the IC ₅₀ values of the compound samples (µM).

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Table 1

Example Class A TEM-1 60 min ICso (µM) Class C ASHRS 60 min IC50 (µM) Class D OXA-48 60 min IC50 (µM) 3 0.019 0.071 0.015 4 0.00135 0.011 0.015 5 0.0044 0.02 0.017 8 0.0015 0.012 0.014 10 0.43 1.2 0.051 AND 0.23 0.4 22 27 0.047 0.0083 0.018 thirty 0.081 0.0018 0.14 33 0.0032 0.16 0.077 34 0.026 o, s 0.019 41 0.0035 0.022 0.0064 42 0.024 0.095 0.0052 55 0.024 0.18 0.0073 56 0.093 0.14 0.22 57 0.057 0.0041 0.41 62 0.21 0.41 0.87 63 oh and 0.04 0.23 66 0.66 0.054 0.012 67 0.064 0.0073 0.34 68 0.31 0.0093 0.078 69 0.18 0.002 oh and 70 0.026 0.13 0.087 71 oh and 0.0044 0.075 72 0.077 0.053 oh and 73 0.73 0.01 0.53 74 0.28 0.022 0.59 75 0.083 0.014 0.42 76 0.94 44 0.22 77 >100 >100 >100 78 3.6 >100 0.69 79 4.2 >100 5.7 82 0.085 0.014 0.028 87 0.15 0.017 0.011 88 0.054 0.0031 0.045 89 0.033 0.0021 0.15 91 0.00047 0.036 0.018 93 0.15 4.6 0.053 95 0.035 0.59 0.0013 o «/η М FJ—Ν ₄ ? ⁰ o^\^on O Comparative compound 98 0.0013 2 2.6 ην^ o A oh he O Comparative compound 99 0.03 1.6 82

Example 103. Restoration of activity of cefpodoxime in the presence of a fixed concentration of 4 μg/ml beta-lactamase inhibitor

Minimum inhibitory concentration (MIC) values for each organism and drug combination were determined using the broth microdilution technique from the Clinical and Laboratory Standards Institute (CLSI) Manual (CLSI M07-A10). Quality control (QC) recommended bacterial strains E. coli ATCC 25922, E. coli ATCC 35218 and Klebsiella pneumoniae ATCC 700603 were included in each test in accordance with CLSI guidelines to ensure there was no variation between testing dates (CLSI M100-S25) . The MICs of these QC strains were within the QC range in all tests performed. Drug containing plates were prepared using the replica method. A solution of cefpodoxime with a 20-fold concentration and serial 2-fold dilutions were carried out in Mueller-Hinton broth with a standardized content of cations. Equal volumes of compound samples at a fixed 20-fold concentration were added to the original plate. 10 μl was printed on daughter plates with

- 119 044428 using the Tecan EVO robot. Suspensions of organisms were adjusted to the McFarland standard of 0.5 and further diluted to obtain 3x10 ⁵ to 7x10 ⁵ colony forming units (CFU)/ml of finished inoculum. Bacterial inoculum was prepared in sterile Mueller Hinton broth with a standardized content of cations (Beckton Dickinson). 90 µL of inoculum at 1.1× concentration was added to the wells (using a Tecan EVO robot). All seeded microdilution plates were incubated in ambient air at 35°C for 18-24 hours. After incubation, the lowest drug concentration that prevented visible growth, determined by absorbance at 600 nm, was recorded as the MIC (Table 2, all MICs are given in µg/ml, and all beta-lactamase inhibitors were tested at a fixed concentration of 4 µg/ml).

table 2

Compound Beta-lactamase content: Cefpodoxime alone + 3 + 4 + 5 + 10 + 11 + 27 + 28 + 30 + 33 + 34 + 41 + 42 freundui AmpC, TEM-1, CMY65 >64 <1 <0.125 <0.03125 2 16 <0.03125 32 0.5 <0.03125 <0.03125 <0.00012 4 E. Wild type (ATCC 25922) 1 <0.5 <0.06 <0.03125 0.5 0.5 <0.03125 1 0.03125 <0.03125 <0.03125 <0.00012 0.5 ?oli AmpC, OXA-1, STX-M- 15, TEM-1 >64 1 0.06 <0.03125 4 2 <0.03125 4 0.5 <0.03125 <0.03125 0.00024 2 Klebsiella / SHV-18, OXA-1, OKR-6 (ATCC 700603) 16 2 0.25 0.0625 2 2 0.25 1 2 <0.0625 <0.125 0.25 2 neumoniae KRS-2, SHV-11, TEM-1 OXA-9 >64 0.5 0.06 <0.03125 1 4 <0.03125 2 1 <0.03125 <0.0625 0.01117 1 + 43 0.13 0.016 0.0039 1 0.063 + 55 >32 0.0625 2 2 32 + 56 8 <0.03125 <0.03125 0.125 0.0625 + 57 <0.0625 <0.0625 <0.0625 1 0.0625 + 62 <0.03125 <0.03125 <0.03125 <0.0625 <0.03125 + 63 >32 0.25 32 8 >32 + 66 16 0.5 >32 4 8 + 67 0.5 <0.03125 <0.03125 4 1 + 68 >32 0.5 >32 4 8 + 69 >32 0.25 16 4 8 + 70 >32 0.5 16 4 >32 + 71 0.25 0.5 2 2 1 + 72 >32 <0.03125 4 4 8 + 73 >32 0.25 32 But 16 + 74 0.5 <0.03125 <0.03125 2 0.03125 + 75 <0.03125 <0.03125 <0.03125 0.125 <0.03125 + 76 >32 1 >32 16 >32 + 77 <0.03125 <0.03125 <0.03125 <0.03125 <0.03125 + 78 >32 1 >32 8 >32 + 79 >32 1 >32 16 >32 + 82 <0.03125 <0.03125 <0.03125 0.0625 <0.03125 + 87 <0.03125 <0.03125 <0.03125 0.125 <0.03125 + 88 <0.03125 <0.03125 <0.03125 2 <0.03125 + 89 <0.03125 <0.03125 <0.03125 0.0625 <0.03125 + 91 <0.03 <0.03 <0.03 <0.03 <0.03 + 93 <0.03 <0.03 <0.03 0.125 <0.03 + 95 32 0.25 1 2 2 o D H ₂ N f ] M F A—n _x / ⁰ ° f ^0H + o Comparative compound 97 >32 1 2 2 2 O M F A— N _x F ⁰ °A-on + o Comparative compound 98 16 0.5 2 2 0.5 ΗνΆ O A ^{0 0} U ^he + O Comparative compound 99 >32 0.5 8 2 4

- 120 044428

Example 104: Restoration of activity of various oral beta-lactams in the presence of a fixed concentration of 4 μg/ml beta-lactamase inhibitor

Following the procedure used in Example 103, the MICs for several beta-lactams were determined in the presence of a fixed concentration (4 μg/ml) of sample compounds (Table 3) against 3 Enterobacteriaceae strains.

Table 3

Compound S. freundui E. coll Klebsiella pneumoniae AshrS, TEM-1, CMY65 Wild type (ATCC 25922) SHV-18, OXA-2, OKR-6 (ATCC 700603) Cefpodoxime >64 1 16 Cefpodoxime + Pr. 3 (4 µg/ml) 0.5 0.125 2 Cefpodoxime + Pr. 4 (4 µg/ml) <0.125 <0.06 0.25 Cefpodoxime + Pr. 33 (4 µg/ml) <0.03125 <0.03125 0.25 Cefpodoxime + Pr. 34 (4 µg/ml) <0.03125 <0.03125 0.25 Cefpodoxime + Pr. 93 (4 µg/ml) <0.06 <0.06 0.25 Cefuroxime >64 4 32 Cefuroxime + Pr. 3 (4 µg/ml) 1 0.02 But Cefuroxime + Pr. 4 (4 µg/ml) 0.02 0.02 But Cefuroxime + Pr. 33 (4 µg/ml) <0.03125 <0.03125 4 Cefuroxime + Pr. 34 (4 µg/ml) <0.03125 <0.03125 1 Tigemons 4 1 32 Tigemonam + Ave. 3 (4 µg/ml) 0.125 1 2 Tigemonam + Ave. 4 (4 µg/ml) <0.06 <0.06 0.5 Tigemonam + Ave. 33 (4 µg/ml) <0.06 <0.06 0.5 Tigemonam + Ave. 34 (4 µg/ml) <0.06 <0.06 0.5

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Tigemonam + Ave. 93 (4 µg/ml) 0.125 0.125 1 Tebipenem 0.125 <0.06 0.125 Tebipenem + Ave. 3 (4 µg/ml) <0.06 <0.06 0.125 Tebipenem + Ave. 4 (4 µg/ml) <0.06 <0.06 <0.06 Tebipenem + Ave. 33 (4 µg/ml) <0.06 <0.06 0.125 Tebipenem + Ave. 34 (4 µg/ml) <0.06 <0.06 <0.06 Tebipenem + Ave. 93 (4 µg/ml) <0.06 <0.06 <0.06 Faropenem 8 0.5 8 Faropenem + Pr. 3 (4 µg/ml) 0.25 0.25 2 Faropenem + Pr. 4 (4 µg/ml) <0.06 <0.06 1 Faropenem + Pr. 33 (4 µg/ml) <0.06 <0.06 0.5 Faropenem + Pr. 34 (4 µg/ml) <0.06 <0.06 0.5 Faropenem + Pr. 93 (4 µg/ml) <0.06 <0.06 1 Cefixime >64 0.5 8 Cefixime + Pr. 3 (4 µg/ml) 1 0.125 0.5 Cefixime + Pr. 4 (4 µg/ml) <0.06 <0.06 <0.06 Cefixime + Pr. 33 (4 µg/ml) <0.06 <0.06 <0.06 Cefixime + Pr. 34 (4 µg/ml) <0.06 <0.06 <0.06 Cefixime + Pr. 93 (4 µg/ml) <0.06 <0.06 0.125 Loracarbef >64 2 32 Loracarbef + Pr. 3 (4 µg/ml) 1 0.5 0.5 Loracarbef + Pr. 4 (4 µg/ml) <0.06 <0.06 0.125 Loracarbef + Pr. 33 (4 µg/ml) <0.06 <0.06 0.125 Loracarbef + Pr. 34 (4 µg/ml) <0.06 <0.06 0.125 Loracarbef + Pr. 93 (4 µg/ml) <0.06 <0.06 0.25

Example 105 Stability/conversion in the absence or presence of metabolizing enzymes

Preparations of human intestinal fraction S9 in the absence of phenylmethylsulfonyl fluoride (PMSF), human liver fraction S9, rat intestinal fraction S9, and rat liver fraction S9 were obtained from Xenotech (Lenexa, KS).

The 500 μl incubation solution contained 0.8 mg/ml enzyme (or no enzyme for stability studies in buffer), 10 μM test compounds in 100 mM HEPES buffer, pH 7.4. Hydrolysis reactions were performed in a 1-mL glass insert (Analytical Sales, Pompton Plains, NJ) in a shaking water bath at 37°C. At 0, 2, 5, 10, 15, 30, and 60 min, the reaction was stopped by pipetting 50 μl of incubate into a 96-well DeepWell plate (Thermo Fisher Scientific, Rochester, NY) containing 100 μl of acetonitrile at 250 ng/ml carbutamide (Sigma-Aldrich, St. Louis, MO) as internal standard. The precipitated solutions are then well

- 122 044428 was mixed by vortex method and then centrifuged at 4000 rpm for 15 minutes at

4°C. The extract was transferred to a new DeepWell 96-well plate for analysis by LC-MS/MS.

LC-MS/MS analysis was performed using a Sciex Qtrap 6500 AB mass spectrometer in positive ionization mode coupled to a Schimadzu Nexera LC system.

A Waters Atlantis T3 column (3 µm, 3.0x50 mm) was used for separation.

The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 1.2 ml/min. For prodrug analysis, compound-specific multiple reaction monitoring (MRM) was set up so that both the prodrug and its hydrolyzed active substance could be monitored simultaneously.

In table Table 4 lists the first order half-lives of the sample compounds (T _1/2 in minutes). The half-lives given are based on the disappearance of the parent ester. As can be seen from table. 4, There is a good correlation between rat and human enzymes. Data in table. 4 indicate that overall there is a faster conversion of the ester to the active compound by human and rat liver S9 enzymes, whereas in the presence of buffer and intestinal S9 the esters are predominantly stable.

Table 4

Example Buffer, pH 7.4 Fraction S9 of rat intestine Rat liver fraction S9 Fraction S9 of human intestine Fraction S9 of human liver 1 41 39 4.4 38 6.5 2 29 24 1.4 21 1.9 7 3.9 3.9 0.5 2.4 0.8 9 18.3 27.3 1.4 17.8 2.9 12 236 233 14 132 16 13 55 BUT BUT 44 1 15 98 >100 1.5 >100 12.4 18 18 16 7 15 9.2 20 39 54 6.1 40 5.1 22 38 56 9.2 43 4.6 24 >100 BUT BUT 34 9 31 44 42 20 35 27 32 22 18 2.4 6.9 2.4 35 186 240 32 163 39 36 91 68 3 36 8.5 45 33 17 0.22 2 0.39 47 12 eleven 1.8 8.2 2.3 48 62 50 3 42 6.1 49 68 26 1.8 32 6.9 50 120 thirty 2.8 73 3.5 51 75 4.1 1.7 1.5 6.4 52 76 39 0.45 5.7 0.84 53 4.8 4.2 2 <1.0 1.7 54 22 19 0.9 5 1.9 60 29 21 10 22 9.9 64 1.2 1.1 <1.0 <1.0 <1.0 65 1.7 1.3 <1.0 <1.0 <1.0 85 26 21 3.8 20 3.5 86 39 43 23 36 24 90 2.9 3.3 0.7 1 0.7 92 160.9 397 eleven 193.1 24.8 94 46.9 25.9 1.3 10.6 0 96 >100 210.7 10.3 1475.6 57

N/A = not defined

Example 106 Pharmacokinetics in rats

Pharmacokinetics in rats following intravenous administration of sample compounds were determined in jugular vein cannulated Sprague-Dawley rats (n=3) (Harlan Laboratories, Indianapolis, IN) at a dose of 10 mg/kg. The compounds were dissolved and administered intravenously in 0.9% saline, pH 6.5 at a dose of 2 ml/kg. Blood samples (about 100 μl) were obtained through a jugular vein catheter before administration and at 0.08, 0.17, 0.25, 0.5, 1, 2, 4 and 8 hours after administration and prepared for plasma isolation in tubes Microtainer with K2EDTA. Oral pharmacokinetic studies were conducted with samples of compounds at 10 mg/kg carboxylic acid equivalents. Doses were dissolved and administered orally in a 25:75 mixture of PEG400 and water for injection, pH 4.5, at a dose volume of 10 ml/kg. Blood samples were obtained through a jugular venous catheter before administration and at 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration and prepared for plasma isolation in Microtainer tubes with K ₂ EDTA. Plasma samples were stored at -80°C until biological analysis.

Sample preparation for LC/MS/MS analysis

Plasma samples were thawed on ice before processing. Samples (30 µl) were diluted and proteins were precipitated using 180 µl acetonitrile containing 0.1% formic acid and 250 ng/ml carbutamide (N1-(butylcarbamoyl)sulfonamide, Sigma-Aldrich cat. no. S385433) as internal standard. Samples were vortex-mixed for 30 s, centrifuged at 3400 g for min, and the supernatant was transferred into injection vials.

LC/MS/MS conditions

Rapid hydrolysis prevented satisfactory analysis of circulating concentrations of Examples 12 and 35. LC/MS/MS analysis was performed using a Sciex QTrap 6500 AB mass spectrometer in positive ionization mode coupled to a Schimadzu Nexera LC system.

A Waters Atlantis T3 column (3 μm, 3.0x50 mm) was used for chromatographic separation. The volume of the injected sample was 1 μl. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with a gradient of 2–98% B:A over 2 min.

Pharmacokinetic analysis

Plasma concentration-time profiles following intravenous (IV) and oral (PO) administration of compound samples were analyzed by non-compartmental analysis using WinNonLin 6.4. The average AUC and F% values are given in table. 5. The absolute oral bioavailability (F%) of the test compounds after administration of their respective esters was determined as:

F% = 100 * (AuC^o*Dose _in /v)/(AuDv/v*DoseA·

In general, sample compounds demonstrate increased oral AUC compared to sulfate-derived beta-lactamase inhibitors, resulting in favorable F%.

Table 5

Input Example Method of administration Analyte compound AUC, µM*h F% 4 IV 4 19.1 3 IV 3 34.9 13 By h 18.3 53 12 By 4 8.2 38 2 By h 18.5 58 1 By 4 8.4 45 60 By 62 2.2 6.3 33 IV 33 26.3 34 IV 34 16.1 36 By 34 13.5 84 35 By 33 25.8 98 32 By 34 11.5 71 31 By 33 19.8 75 53 By 4 2.2 eleven 65 By 4 2.8 15 AA m Λ--N O OSO ₃ H ETX2514 By ETX2514 0.44 ABOUT % About OSO3H Relebaktam By Relebaktam 0.29 Cl L—h About OSO3H WCK 4234 By WCK 4234 0.45

Example 106. Oral efficacy of cefpodoxime proxetil in vivo in combination with Example 35 against E. coli (beta-lactamase content: AmpC, CTX-M-14)

The in vivo oral efficacy of Example 35 was assessed in combination with cefpodoxime proxetil in a murine model of neutropenic thigh infection against a corresponding clinical strain. The strain, E. coli ARC2687, expresses the beta-lactamases AmpC and CTX-M-14, both of which can readily hydrolyze cefpodoxime, resulting in insensitive MICs greater than 512 μg/ml. When combined with Example 33 (4 μg/ml), the MIC of cefpodoxime was reduced to <0.03 μg/ml. The study dosage was adjusted to achieve cefpodoxime exposure above the MIC of 0.03 μg/mL for at least 50% of the dosing interval for all treatment groups while gradually introducing increasing doses of the compound from Example 35. CD-I mice (Charles River, Wilmington, USA) were housed in shoebox cages with contact bedding and allowed to acclimate to the housing conditions for a minimum of 2 days before use. The animal room was maintained at 70°F, 50 ± 10% relative humidity, and a 12-hour light/dark cycle. The study was conducted using an IACUC-approved protocol in accordance with Title 9 of the Code of Federal Regulations. Neutropenia was induced in animals with two intraperitoneal doses of cyclo-124

Claims (19)

phosphamide (150 mg/kg 4 days and 100 mg/kg 1 day before infection (Gerber et al. (1983) J Infect Dis 147(5); 910-917). Animals were infected by intramuscular injection of approximately 1x1x 10 ⁶ CFU in 100 μl of 0.9% saline. The inoculum was prepared from 25 ml of an overnight culture of E. coli ARC 2687 in trypticase soy broth. After determining the optical density at 600 nm, the inoculum was diluted in 0.9% saline to a concentration of approximately 1.0x10 ⁶ CFU/ml before inoculation into the left and right thighs. Oral therapy with cefpodoxime proxetil alone and in combination with example 35 was started 2 hours after bacterial challenge. Doses were suspended in 0.5% HPMC/0.1% Tween 80 and administered orally via gavage at a dose volume of 10 mL/kg. The end point was reached at 24 hours to determine the bacterial count in the thigh tissue (CFU/g). Animals were ethically euthanized, and femurs were aseptically removed, weighed, and homogenized in 1 ml saline. Enumeration of the bacterial load of the tissue homogenate was performed by serial dilution in trypticase soy agar plates, which were incubated overnight at 37°C until colony counts (CFUs) were obtained. The lower limit of detection was approximately 2.6 log ₁₀ CFU/g tissue. As shown in table. 6, the bacterial load in the thighs of mice treated with cefpodoxime proxetil alone at a dose of 50 mg/kg every 6 hours or Example 35 at a dose of 10 mg/kg every 6 hours showed an increase of more than 3 log ₁₀ CFU/g after 24 hours of treatment. In combination with cefpodoxime proxetil, increasing the dose of the compound from Example 35 led to a dose-dependent reduction in bacterial load, with a maximum logarithmic reduction of -0.75 log ₁₀ CFU/g (relative to the start of therapy) being achieved at 50 mg/kg cefpodoxime proxetil + 100 mg/kg of Example 35 every 6 hours. Meropenem, used as a positive control, provided a logarithmic reduction of CFU just above 1-log ₁₀ relative to the beginning of therapy at a dose of 600 mg/kg every 6 hours administered subcutaneously. Table 6 Group Dose (mg/kg) Method/Mode logioKOE/g femoral tissue SK ABOUT Changes in eCFU after 2 hours Changes in eCFU after 26 hours Start therapy Not apply MO not applicable 6.27 0.35 -4.59 26 h growth control carrier p/o/every 6 hours 10.86 0.19 +4.59 — Cefpodoxime and proxetil alone 50 p/o/every 6 hours 10.24 0.15 +3.97 -0.62 Example 35 separately 10 p/o/every 6 hours 9.56 0.29 +3.29 -1.30 Example 35 + cefpodoxime a proxetil 10+50 p/o/every 6 hours 5.77 0.14 -0.50 -5.09 Example 35 + cefpodoxime a proxetil 25+50 p/o/every 6 hours 5.59 0.17 -0.68 -5.27 Example 35 + cefpodoxime a proxetil 100+50 p/o/every 6 hours 5.52 0.20 -0.75 -5.34 Meropenem 600 s/c/every 6 hours 5.17 0.27 -1.10 -5.69 CLAIM 1. A method for preparing a compound of formula VI or its salt, where Rk.^^^/R ³ A. /PG' PG N 1 he (VI); - 125 044428 R ¹ is selected from -CN, -CH ₂ NH ₂ , -C(O)NH ₂ , where R ¹¹ represents hydrogen or -C(O)NH ₂ ; or R ¹ is selected from -C(O)NR ⁷ R ⁸ where R ⁷ is hydrogen and R ⁸ is: 1) phenyl, optionally substituted with C1-C3 alkyl or C1-C3 alkyl-NH2, 2) C1- C3-alkyl or 3) C1-C3-alkoxy, wherein each alkyl or alkoxy represented by ^R8 is optionally and independently substituted with Q-Qcycloalkyl, -CN, -OH, -NH2, -SO2NH2, -NHSO2NH2, -C(O) NH2, -NHC(O)(C1-C3-alkyl), pyrazinyl, oxetanyl, oxazolyl, or pyrrolidinyl, optionally substituted with one or more carboxyl, fluorine atom or -C(O)O(C1- _C6 -alkyl); each of R ² and R ³ independently represents C ₁ -C ₃ -αalkyl; each of PG and PG' is independently selected from tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), 2,2,2-trichloroethoxycarbonyl (Troc), CF ₃ CO, acetyl (Ac), para-toluenesulfoyl (Ts) and methylsulfonyl (Ms); wherein the method involves reacting a compound of formula XI or its salts with PG'NHOH in the presence of an oxidizing agent. 2. Method for preparing the compound of formula VI or its salts, where R ¹ is selected from -CN, -CH2NH2, -C(O)NH2, where R ¹¹ represents hydrogen or -C(O)NH ₂ ; or R ¹ is selected from -C(O)NR ⁷ R ⁸ where R ⁷ is hydrogen and R ⁸ is: 1) phenyl, optionally substituted with C1-C3 alkyl or C1-C3 alkyl-NH2, 2) C1- C3-alkyl or 3) C1-C3-alkoxy, where each alkyl or alkoxy represented by R ⁸ is optionally and independently substituted with Q-Qcycloalkyl, -CN, -OH, -NH2, -SO ₂ NH ₂ , -NHSO ₂ NH ₂ , -C(O)NH ₂ , -NHC(O)(C ₁ -C ₃ -alkyl), pyrazinyl, oxetanyl, oxazolyl or pyrrolidinyl, optionally substituted with one or more carboxyl, fluorine atom or -C(O)O(C ₁ -C ₆ -alkyl); R ² and R ³ are each independently C ₁ -C ₃ alkyl; each of PG and PG' is independently selected from tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), 2,2,2-trichloroethoxycarbonyl (Troc), CF ₃ CO, acetyl (Ac), para-toluenesulfoyl (Ts) and methylsulfonyl (Ms); wherein the method involves reacting a compound of formula XI or its salts with PG'N=O. 3. Method according to claim 1 or 2, wherein the compound of formula XI is characterized by the formula or a salt thereof. 4. Method according to any one of claims 1 to 3, wherein the compound of formula VI is characterized by formula VII - 126 044428 (VII); or its salt. 5. Method according to any one of claims 1 to 4, where R ² represents methyl. 6. Method according to any one of claims 1 to 3, wherein the compound of formula VI is characterized by formula VIII I he (VIII); or its salt. 7. Method according to any one of claims 1-3 and 6, where R ³ represents methyl. 8. Method according to any one of claims 1 to 7, where R ¹ represents -C(O)NH ₂ . 9. Method according to claim 1, wherein the compound of formula VI is characterized by formula IX I OH (IX); or its salt. 10. Method according to claim 1, wherein the compound of formula VI is characterized by formula X I ^he (X); or its salt. 11. Method according to any one of claims 1 to 10, wherein each of PG and PG' represents tert-butyloxycarbonyl. 12. The method according to any one of claims 1 and 3-11, further comprising reacting a compound of formula XI with PG'NHOH in the presence of a metal catalyst. 13. The method according to claim 12, where the metal catalyst is selected from CuCl, CuBr, Cui, CuCN, CuSCN, CuBr-Me ₂ S, Cu(OAc) ₂ and CuOTf. 14. The method according to any one of claims 1 and 3-13, further comprising reacting a compound of formula XI with PG'NHOH in the presence of an amine additive. 15. The method according to claim 14, where the amine is pyridine, (1E,2E)-cyclohexane-1,2-diamine, N,N'dimethylethane-1,2-diamine, 2,6-di-tert-butyl- 4-methylpyridine, 1,10-phenanthroline, trans-cyclohexane-1,2-diamine, X1-(2-(diethylamino)ethyl)-X2,X2-diethylethane-1,2-diamine, cis-cyclohexane-1,2-diamine or N1,N1,X2,X2-tetramethylethane-1,2-diamine. 16. The method according to any one of claims 1 and 3-15, where the oxidizing agent is O ₂ , air, FeCl ₃ , MnO ₂ , meta-chloroperoxybenzoic acid (mCPBA), NaIO ₄ , 2-iodoxybenzoic acid (IBX), (2 ,2,6,6tetramethylpiperidin-1-yl)oxyl (TEMPO), benzoyl peroxide (BPO), HNu ₃ , urea-H ₂ O ₂ , I ₂ , Nchlorosuccinimide (NCS), Dess-Martin periodinane (DMP), H ₂ O ₂ or N-methylmorpholine N-oxide (ΝΜΜ0). 17. Method according to any one of claims 1 to 16, where the reaction is carried out in a polar solvent. 18. Method according to any one of claims 1-17, where the reaction is carried out in DCM, THF, MTBE, EtOAc, iPrOAc, MeCN, H ₂ O, MeOH, EtOH, i-PrOH, t-BuOH, n-BuOH, 2- methyl-2-butanol, DMF, DMSO, ethylene glycol, polyethylene glycol, sulfolane, sulfolane/H ₂ O mixtures, DMF/H 2 O, NMP/H ₂ O, DCM/H ₂ O, MeOH/ _{H 2 O} , EtOH/H ₂ O, iPrOH/H ₂ O or n-BuOH/H ₂ O. 19. The method according to any one of claims 1 to 18, further comprising adding water. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2 - 127-