Discovery of 2-Sulfinyl-Diazabicyclooctane Derivatives, Potential Oral β-Lactamase Inhibitors for Infections Caused by Serine β-Lactamase-Producing Enterobacterales

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Discovery of 2‑Sulﬁnyl-Diazabicyclooctane Derivatives, Potential

Oral β‑Lactamase Inhibitors for Infections Caused by Serine

β‑Lactamase-Producing Enterobacterales

Motohiro Fujiu,* Katsuki Yokoo, Jun Sato, Satoru Shibuya, Kazuo Komano, Hiroki Kusano,

Soichiro Sato, Toshiaki Aoki, Naoki Kohira, Sachi Kanazawa, Ryosuke Watari, Tomoyuki Kawachi,

Yuya Hirakawa, Daiki Nagamatsu, Emi Kashiwagi, Hideki Maki, and Kenji Yamawaki

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ABSTRACT: Coadministration of β-lactam and β-lactamase inhibitor

(BLI) is one of the well-established therapeutic measures for bacterial

infections caused by β-lactam-resistant Gram-negative bacteria, whereas

we have only two options for orally active BLI, clavulanic acid and

sulbactam. Furthermore, these BLIs are losing their clinical usefulness

because of the spread of new β-lactamases, including extended-spectrum

β-lactamases (ESBLs) belonging to class A β-lactamases, class C and D

β-lactamases, and carbapenemases, which are hardly or not inhibited by these classical BLIs. From the viewpoints of medical cost

and burden of healthcare personnel, oral therapy oﬀers many advantages. In our search for novel diazabicyclooctane (DBO) BLIs

possessing a thio-functional group at the C2 position, we discovered a 2-sulﬁnyl-DBO derivative (2), which restores the antibacterial

activities of an orally available third-generation cephalosporin, ceftibuten (CTB), against various serine β-lactamase-producing

strains including carbapenem-resistant Enterobacteriaceae (CRE). It can be orally absorbed via the ester prodrug modiﬁcation and

exhibits in vivo eﬃcacy in a combination with CTB.

relatively stable covalent bonds between active site serine. On

the other hand, the boronates are competitive inhibitors which

mimic the intermediate in the hydrolysis of β-lactam by β-

lactamase. Avibactam (AVI) is the ﬁrst clinically used DBO

compound, which was approved by the FDA in 2015 in

combination with ceftazidime as an intravenous agent.⁷There

are currently many derivatives in the market or under clinical

development.⁸⁻¹³However, many of these inhibitors are

intravenously administered, and orally available DBOs and

bicyclic boronates do not exist in the market. At present, there

are only the following four compounds being developed as oral

agents (Figure 1b): ARX-1796, a prodrug of AVI (with AV-006,

phase I);¹⁴ETX0282 (with cefpodoxime proxetil, phase I);¹⁵

QPX7728 (preclinical);¹⁶and VNRX-7145 [with ceftibuten

(CTB), preclinical].¹⁷Oral administration can oﬀer the

opportunity of home-care, which reduces the cost of medical

care and the burden of healthcare personnel. In this respect, it is

necessary to prepare eﬃcacious oral BLIs due to the rapid

emergence of β-lactamase-producing pathogens.

INTRODUCTION

■

β-Lactam antibiotics, commonly called β-lactams, have been

playing an important role for the treatment of bacterial

infections, but pathogens resistant to them have been increasing

in many countries around the world.¹The key resistance

mechanism of Gram-negative bacteria is primarily the

production of β-lactamases which hydrolyze and deactivate β-

lactams. In order to combat β-lactamase-producing pathogens,

β-lactamase inhibitors (BLIs) have been used in combination

with β-lactam.²Clavulanic acid, the ﬁrst clinically introduced

BLI, has been used with amoxicillin as an oral agent in the US

since 1982. Sulbactam has also been used for the purpose of oral

administration as a mutual prodrug with ampicillin, or

sultamicillin (Figure 1a). Unfortunately these inhibitors have

been losing their clinical usefulness due to the worldwide spread

of extended-spectrum β-lactamases (ESBLs) belonging to class

A β-lactamases and class C and D β-lactamases, which are hardly

or not inhibited by these classical BLIs.^3,4Furthermore, the

emergence of carbapenem-resistant Enterobacteriaceae (CRE)

producing carbapenemase, such as KPC, has recently become a

threatening issue.⁵

Received: May 3, 2021

Published: June 18, 2021

The spread of strains producing such new β-lactamases has

triggered the research and development of new BLIs, and we

now have two novel classes of BLI in hand, 1,6-

diazabicyclo[3.2.1]octane (DBO) and bicyclic boronates.^2,6

Mechanistically, DBOs inhibit serine β-lactamases via forming

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Figure 1. (a) Oral BLIs in the market. (b) AVI and oral BLIs in clinical development.

Table 1. Enzymatic Inhibition and Clearance of Reported Combination of a Thio-Functional Group at the C2 Position (R²) and

an Acidic Moiety at the 6-Position (R¹)

a

b

IC₅₀against CTX-M-15. The values of IC₅₀were determined without enzyme-inhibitor preincubation. N = 1 determination. Total clearance in

c

d

rats. Remaining amount in pH 7.0 phosphate buﬀer at 40 °C after 16 h. CL could not be determined due to its low stability in serum.

We have recently reported that a thio-functional group, such

as sulfone, sulfoxide, or sulfonamide, at the C2 position of DBO

enhances the ability to restore antimicrobial activities of β-

lactam against pathogens producing various serine β-lactamases

and expands structural tolerance for β-lactamase inhibition at

the 6-position.¹⁸In designing oral agents, carboxylic acid ester

prodrugs are preferred to sulfate esters because they have been a

proven strategy for oral administration. Although we have

already ascertained that replacing the sulfate at the 6-position

with ﬂuoroacetic acid maintains β-lactamase inhibition

potential, they were unstable in serum and showed high

clearance in rats. Herein, we report our exploratory research for

further combinations of thio-substituents at the C2 position and

ﬂuoroacetic acid at the 6-position, which lead us to a 2-sulﬁnyl-

diazabicyclooctane derivative with high potential as an oral BLI,

that not only exhibits high inhibition potential against various

serine β-lactamases but also is stable in serum and can be orally

absorbed as an ester prodrug.

RESULTS AND DISCUSSION

■

Design. We have recently reported the fundamental

structure−activity relationship (SAR) of thio-functionalized

DBOs.¹⁸The representative compounds and their proﬁles are

summarized in Table 1. The sulfone and sulfoxide derivatives

exhibited better inhibition against β-lactamases. However, their

clearance in rats was high due to instability in serum. On the

other hand, the sulfonamide derivative showed a better

pharmacokinetic (PK) proﬁle although the enzymatic inhib-

itions were weaker than those of the others. These results imply

that inhibition activities and clearance could be controllable by

the combination of substitutions at the C2 and 6-position. If

solution stability can be regarded as an indicator for predicting

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Table 2. Combination of Thio-Functional Groups at the C2 Position and Fluoroacetic Acids at the 6-Position

a

IC₅₀(μM)

b

c

d

compound

KPC-2

CTX-M-15

CMY-2

CL_tot(mL/min/kg)

serum stability (%)

solution stability at pH 7.0 (%)

1

2

3

4

0.008

0.014

0.052

0.977

0.005

0.012

0.104

0.249

0.026

0.028

1.34

289

no data

85

39

17

95

55

27.0

84.5

19.8

1.11

78

91

a

b

c

The values of IC₅₀were determined without enzyme-inhibitor preincubation. N = 1 determination. Total clearance in rats. Remaining amount in

rat serum after 30 min incubation. Remaining amount in pH 7.0 phosphate buﬀer at 40 °C after 16 h.

d

serum stability, the activity and serum stability can be balanced

by a combination of monoﬂuoroacetic acid with sulfoxide or

sulfonamide. Therefore, we proceeded with further exploration

of unadministered combinations to ﬁnd the best balance.

Exploratory Study to Find a Lead Compound

Exhibiting Both Robust Activities and Low Clearance.

Our exploratory investigation began with combinations of

sulfoxide or sulfonamide with ﬂuoroacetic acid (Table 2). A

combination of sulfoxide with diﬂuoroacetic acid (1) or R-

isomer of monoﬂuoroacetic acid (2) showed robust inhibition.

From our previous study,¹⁸it was revealed that S-isomer of

monoﬂuoroacetic acid had a negative impact on the inhibitory

activities, and a similar trend was observed. In contrast to

sulfoxide, the inhibitory activities of the sulfonamide derivatives

(3 and 4) were not satisfactory. We also found a trend of

improved clearance by replacing diﬂuoroacetic acid (1 and 3)

with monoﬂuoroacetic acid (2 and 4). These compounds

showing a good PK proﬁle were stable in both the serum of rat

and a pH 7.0 aqueous solution of phosphate buﬀer. This

indicates that the physical stability is related to serum stability.

The combination of sulfoxide with monoﬂuoroacetic acid

turned out to be the best, and our SAR study focused on the

lead optimization of 2.

Synthesis of Sulfoxide Derivatives. We have recently

reported the synthetic routes and methods of DBO derivatives

possessing a thio-functional group, involving the photo-induced

decarboxylative thiolation of carboxylic acid 23.¹⁹We applied

this methodology to the synthesis of sulfoxide derivatives

bearing various side chains. The scope and limitation of the

photo-induced decarboxylative thiolation and the selectivity of

the oxidation to sulfoxide are important components in the

synthesis. We summarized the yields of sulfoxide 27a-n from the

corresponding carboxylic acid 23−25 and the diastereoselec-

tivity of oxidation to sulfoxide in Table 3. The sulﬁde

intermediate 26 was unstable on silica gel, and there were

cases when it was diﬃcult to isolate them in a pure form. Thus,

two-step yields of sulfoxide 27a-n from the corresponding

carboxylic acid 23−25 were presented.

disulﬁdes bearing thiophenyl (SPh) group as a leaving group

are useful for improving the yield and reducing the required

amount of the thiolating agent (3−5 equiv). The decarbox-

ylative thiolations stereoselectively proceeded, and no stereo-

isomer at the C2 position was observed because the radical

intermediate reacted with the thiolating agent from the convex

side. Steric hindrance of the agent had an impact on yield of the

reaction. The yield decreased with the increasing bulkiness of

the side chain (27a vs 27e, 27g, and 27i), and the tertiary thio-

group could not be introduced (27m). We observed that the

yield of the targeted compound was inversely related to that of

the byproduct 29, to which the radical intermediates from the

active ester recombined after decarboxylation (Scheme 1). This

means that the recombination interferes with the reaction

between the radical intermediate and the thiolating agent. The

electronic eﬀect also seems to be associated with the yield. The

electron-withdrawing side chain gave better results (27e, 27g,

and 27i vs 27c and 27j) except for an amide (27n). This result

suggests that the reactivity of thiolating agents would be

enhanced because the energy level of antibonding orbital (σ*) of

the S−S bond in the thiolating agent was lowered by an

inductive eﬀect, and the energy level gap between singly

occupied molecular orbital (SOMO) of the radical intermediate

and antibonding orbital (σ*) of S−S bond was reduced.

We observed that many cases of oxidation to sulfoxide 27

proceeded with good diastereoselectivity. The relative conﬁg-

uration of 27a as a representative was established by X-ray

crystallography,¹⁹and other analogues presumably have the

same conﬁguration on the analogy of ¹H NMR spectra

comparison between 27a and its diastereomer.³⁷The

diastereoselectivity can be explained by the conformation of

sulﬁde 26. Stable conformation of 26 would be the same as

shown in Figure 2 by steric repulsion of the side chain and

electric repulsion between the lone pair on sulfur and that on

nitrogen at the bridge-head. The sterically less hindered lone

pair on sulfur would be preferentially oxidized to give sulfoxide

27 with high diastereoselectivity.

The two-step yields were moderate to low, which is attributed

to undesired side reactions including the recombination of the

radical intermediates to give 29 and the instability of sulﬁde 26,

but we could obtain the various compounds in adequate

amounts for SAR.

We prepared thio-introduced DBOs 2 and 6−18 via two

synthetic routes. In the ﬁrst route, thio-group is introduced

before ﬂuoroacetic acid at the 6-position (Scheme 2). On the

other hand, alkyl ﬂuoroacetate was introduced at ﬁrst, then the

Construction of a sulﬁde from a photo-active N-hydroxy-2-

thiopyridone ester was originally reported by Barton and his co-

workers.³⁵In spite of its unique transformation of carboxylic

acid into sulﬁde, there are limited examples to introduce an alkyl

thio-group, and a large excess amount of disulﬁde (≥45 equiv) is

required to obtain a product.^35,36In order to synthesize sulﬁdes

with various alkyl side chains, we explored sources of the alkyl

thio-group to ﬁnd that thiosulfonates and unsymmetrical

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Table 3. Substrate Scope and Limitation of Photo-Induced Decarboxylative Thiolation and Diastereoselectivity of Oxidation to

a

Sulfoxides

a

b

1

Yields in the table are two-step yields from the corresponding carboxylic acid (23, 24, or 25). Diastereomeric ratios were determined by H

c

NMR. The oxime was obtained as a single geometrical isomer, but the geometry (E/Z) is unknown.

Scheme 1. Mechanism for the Formation of Byproduct 29

intermediate underwent the photo-induced thiolation in the

second route (Scheme 3). The ﬁrst route is straightforward to

obtain a single targeted compound, and the second one beneﬁts

eﬃcient diversiﬁcation of side chains at the C2 position.

After the construction of sulfoxide, as summarized in Table 3,

removal of the tert-butyl group from 27c followed by

condensation with ammonia gave 31 (Scheme 2). Hydro-

genolysis of the benzyl group in the presence of Pd(OH)₂and

DABCO^19,38and following alkylation using chiral (R)-ﬂuoro-

acetic acid ester prepared via the analogous optical resolution, as

reported,^15,20gave 32−39 from 27a, 27e, 27g−j, and 31,

respectively. The chiral ﬂuoroacetate could successfully be

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Figure 2. Proposed mechanism of diastereoselective oxidation.

Scheme 2. Synthesis of Compounds 2, 7, 9, and 11−16

a

Reagents and conditions: (a) Conditions are shown in Table 3; (b) TFA, 0 °C, 86%; (c) EDC HCl, HOSu, CH₂Cl₂, −50 °C, then aq NH₃, rt,

80%; (d) (i) Pd(OH)₂on carbon, DABCO, H₂(1 atm), rt; (ii) (R)-BrCHFCO₂Bzh (65), DBU, DMF, 0 °C, 9−78% over two steps for 32−34, 36,

37, and 39; (e) (i) Pd(OH)₂on carbon, DABCO, H₂(1 atm), rt; (ii) (R)-BrCHFCO₂R, R = Me (66), Et (67), K₂CO₃, DMF, −40 to −20 °C,

20−68% over two steps for 35 and 38; (f) TBAF, AcOH, MS3A, THF, rt, 10%; (g) CSI, THF, −30 °C, then aq NaHCO₃, 84%; (h) AlCl₃, anisole,

MeNO₂/CH₂Cl₂, −30 °C, 78%; (i) (i) TASF, DMF, rt; (ii) isobutyl chloroformate, Et₃N, CH₂Cl₂, 0 °C, then aq NH₃, 0 °C, 64% over two steps;

(j) (i) TFA, CH₂Cl₂, 0 °C; (ii) Ac₂O, Et₃N, CH₂Cl₂, −78 °C to rt, quant. over two steps; (k) NaOH, THF/H₂O, 0 °C, 43−78% for 2, 7, 12, 15,

and 16; (l) AlCl₃, (anisole), MeNO₂/CH₂Cl₂, −30 °C, then aq NaHCO₃49−79% for 9 (from 34), 11, and 13; (m) TFA, CH₂Cl₂, 0 °C, quant. for

14 (from 37). ^bCompound 14 is not Na salt but TFA salt of amine on the side chain. ^dThe oxime was obtained as a single geometrical isomer, but

the geometry (E/Z) is unknown.

introduced by using both DBU and K₂CO₃as a base, but the

reaction is faster without noticeable epimerization in the case of

DBU. It was essential to keep reaction temperature at ≤ −20 °C

for suppression of epimerization at the α-position of ﬂuoroacetic

acid ester when K₂CO₃was used. After the introduction of

ﬂuoroacetate, corresponding functional group transformations

took place. Desilylation of 34 followed by the introduction of the

carbamoyl group using chlorosulfonyl isocyanate (CSI) gave 41.

Acetonide in 35 was deprotected by AlCl₃to aﬀord diol 42.

Selective deprotection of 2-(trimethylsilyl)ethyl ester of 36 in

the presence of benzhydryl (Bzh) ﬂuoroacetate at the 6-position

was successfully achieved by tris(dimethylamino)sulfonium

diﬂuorotrimethylsilicate (TASF), and following condensation

with ammonia gave 43. Deprotection of the Boc group from 38

and acetylation aﬀorded 44. Hydrolysis of both ethyl and Bzh

ester by NaOH gave targeted compound 2, 7, 12, 15, and 16.

Bzh ester can be deprotected by Lewis acid as well (9, 11, and

13). Both Bzh and Boc were simultaneously deprotected by

triﬂuoroacetic acid (TFA) to give 14 as TFA salt.

In the second route, tert-butyl ester 45 was prepared via

t

condensation carboxylic acid 23 with BuOH (Scheme 3).

Debenzylation of 45 followed by alkylation gave methyl ester 46

or ethyl ester 47. The common key intermediate 24 or 25 was

obtained after removal of tert-butyl ester by TiCl₄. After the

construction of sulfoxide, as summarized in Table 3, tert-

butyldimethylsilyl (TBS) (27f) or Boc (27k and 27l) group on

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a

Scheme 3. Synthesis of Compound 6, 8, 10, 17, and 18

a

t

Reagents and conditions: (a) BuOH, pyridine, EDC HCl, DMAP, CH₂Cl₂, rt, 66%; (b) (i) Pd(OH)₂on carbon or Pd/C, H₂(1 atm),

(DABCO), MeOH or DMF, rt; (ii) (R)-BrCHFCO₂R, R = Me (66), Et (67), K₂CO₃, DMF, −40 to −30 °C, 21−55% over two steps; (c) TiCl₄,

MeNO₂/CH₂Cl₂, −30 °C, 78−89%; (d) Conditions are shown in Table 3; (e) AlCl₃, MeNO₂/CH₂Cl₂, −30 °C, 66−72%; (f) TFA, CH₂Cl₂, rt,

quant.; (g) NaOH, THF/H₂O, 0 °C, 50−66%.

the side chain was deprotected by AlCl₃or TFA. The following

hydrolysis gave targeted compounds 6, 8, 10, 17, and 18.

Lead Optimization. The SAR studies on the side chain of

sulfoxide are summarized in Table 4. At ﬁrst, we evaluated their

biochemical inhibition (IC₅₀) against KPC-2, CTX-M-15,

CMY-2, and OXA-48. In addition to IC₅₀, we used the minimum

potentiating concentration (MPC), which is the minimum

concentration of BLI needed to reduce the minimum inhibitory

concentration (MIC) of β-lactam to a PK-PD breakpoint based

on human dosing, as a cell-based indicator of the potential for

BLI. In this study, we selected CTB as a β-lactam breakpoint of

which is 1 μg/mL for Enterobacterales (400 mg, QD, by

EUCAST).²¹The MICs of CTB and individual inhibitor against

all the strains are also presented in Table 4.

Gram-negative bacteria are characteristically surrounded by

an outer membrane which serves as a selective permeation

barrier.²²Some hydrophilic antibacterial drugs go across an

outer membrane through a porin, and AVI uses some porins to

penetrate into bacterial cells.²³It has been reported that

diﬀusion rates of β-lactams through a porin depend on their

lipophilicity and molecular size, and more polar and small

molecules are preﬀered.²⁴Therefore, we focused our SAR study

on the simple and polar side chains both neutral (7−13 and 15)

and cationic (14). DBOs covalently inhibit β-lactamases, and

enhancement of reactivity by an electron inductive eﬀect would

improve the inhibition potential. We also designed compounds

bearing an electron-withdrawing group such as ﬂuorine (6),

amide (7), nitrile (8), and oxime (13) on the side chain.

Heteroaromatic compounds (16−18) were tested as well.

Compound 2 showed slightly lower IC₅₀values than AVI

against KPC-2, CTX-M-15, and CMY-2, and this was translated

into MPC₁against strains producing these β-lactamases (ATCC

BAA-1705, SR34100, and SR09613). Furthermore, it notably

has >10-fold lower IC₅₀against OXA-48 compared to AVI

although it was not reﬂected in MPC₁against SR201218 (2 μg/

mL for 2 vs 1 μg/mL for AVI). We speculated that the

contradiction is due to stability of CTB to OXA-48. MICs of

CTB against isogenic Escherichia coli strains overexpressing

individual β-lactamases are summarized in Table 5. The MIC for

OXA-48 producer did not shift from the one for parent, and this

suggests that inhibition against OXA-48 by BLI does not

contribute to reduce MIC of CTB. The sulfoxide epimer 5,

which was derived from a minor isomer of stereoselective

oxidation of the sulﬁde,²⁵showed >10-fold higher IC₅₀s than 2

for all β-lactamases tested. In spite of its lower biochemical

activities, MPC₁s of 5 against three in four strains (ATCC BAA-

1705, SR34100, and SR202118) were comparable to 2, and 4-

fold increase in MPC₁was observed for only SR09613,

expressing CMY-2. This would be also explained by the

susceptibility of CTB to β-lactamase. The MIC of CTB for a

CMY-2 producing isogenic strain was shifted 256-fold from the

parent, which was the largest shift, as listed in Table 5. This

suggests that MIC of CTB for a CMY-2 producer is more

variable than that for KPC-2 or CTX-M-15 producer depending

on the inhibition potential of BLI. Thus, the increase in IC₅₀of 5

was notably translated into MPC₁for SR09613 expressing

CMY-2.

On the whole, a remarkable shift of IC₅₀was not observed

after structural diversiﬁcation on the side chain (6−18). More

than 5-fold increase in IC₅₀compared to 2 was detected only in

two compounds, 13 and 14. The introduction of oxime 13

resulted in >5-fold increase in IC₅₀s against KPC-2, CTX-M-15,

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Table 4. SAR of Side Chain of Sulfoxide

a

b

c

IC₅₀(μM)

MPC₁(μg/mL) /MIC (μg/mL)

K. pneumoniae ATCC

E. coli

d

BAA-1705

e

SR34100

E. coli

K. pneumoniae

d

e

d

e

M_w

c log P KPC-2 CTX-M-15 CMY-2 OXA-48

8 μg/mL

> 32 μg/mL SR09613 > 32 μg/mL SR201218 > 32 μg/mL

2

5

6

7

8

9

10

11

12

13

14

15

16

17

18

AVI

280

298

323

305

310

324

353

340

366

309

351

362

346

378

265

−0.30

−0.13

−1.04

−0.52

−0.55

−0.30

−0.51

−0.45

−0.86

−2.75

−0.59

−1.88

−0.77

−0.51

−1.63

0.014

0.443

0.090

0.035

0.039

0.036

0.038

0.027

0.056

0.132

0.069

0.007

0.016

0.078

0.088

0.072

0.012

0.445

0.006

0.050

0.007

0.016

0.010

0.014

0.010

0.070

0.159

0.101

0.024

0.008

0.010

0.013

0.028

0.655

0.113

0.057

0.096

0.057

0.039

0.064

0.052

0.143

0.431

0.101

0.036

0.059

0.041

0.059

0.443

32.6

0.063/32

0.125 [≤0.031] /32

0.5/32

0.125/32

0.125/16

0.25/32

0.25/>32

0.5/>32

1/>32

2/>32

0.125/>32

0.5/>32

1/>32

1/32

1/>32

0.125/32

0.5/32

0.25/32

2/>32

4/>32

2/>32

4/>32

8/>32

f

no data

0.981

0.063/32

≤0.031/>32

1/>32

0.5/>32

1/>32

0.25/>32

2/>32

≤0.031/>32

0.25/>32

1/>32

0.125/32

0.125/>32

0.063/>32

0.125/>32

0.25/>32

0.5/>32

0.063/>32

0.125/>32

0.125/32

0.5/>32

f

32 [8] />32

f

4 [1] />32

8/no data

4/>32

1/>32

1/32

0.25/>32

0.125/32

5.70

0.125/16

0.5/16

a

b

N = 1 determination. The values of IC₅₀were determined without enzyme-inhibitor preincubation. N = 1 determination. MPC₁to reduce the

MIC of CTB to 1 μg/mL. N = 1 determination. MIC of inhibitor. Following are the expressed β-lactamases and the deletion of porin on each

strain: ATCC BAA-1705, KPC-2, and ΔompK35; SR34100, CTX-M-15; SR09613, CMY-2 type; and SR201218, OXA-48, and CTX-M-15. The

MIC of CTB alone. MPC₁did not converge on an identical value. “X [Y]” means a compound reduced MIC of CTB to 1 μg/mL at its

c

d

e

f

concentration of X and Y skipping values between them.

Table 5. MICs of CTB against Isogenic Strains of E. coli

Expressing β-Lactamase

pounds. Compound 14 also has similar preferable physical

properties and exhibited the good cell-based activities except for

against SR09613 producing CMY-2, IC₅₀of 14 for which was

inferior to others. All compounds with molecular weight of >350

showed ≥4-fold increase in MPC₁against both ATCC BAA-

1705 and SR34100 despite their good enzymatic inhibitions

(11, 13, 15, 16, and 18).

We have previously reported that some thio-functionalized

DBO analogues show intrinsic antibacterial activities based on

multi-penicillin-binding protein inhibition.¹⁸However, all the

sulfoxide derivatives shown in the table did not display

signiﬁcant antibacterial activities. This indicates that they

potentiate CTB not by their intrinsic antibacterial activity but

by β-lactamase inhibition.

Following the result that a compound with low-molecular

weight is advantageous to cell-based activity, we selected 2 for

further evaluation. Microbial activities of 2 and AVI against

additional clinical isolates are summarized in Table 6. In this

study, 2 successfully restored the antibiotic activities of CTB

against broad spectrum β-lactamase-producing strains including

a

β-lactamases

MIC (μg/mL)

parent

KPC-2

CTX-M-15

CMY-2

OXA-48

0.031

0.25

8

0.031

a

N = 1 determination.

and CMY-2, and >10-fold increase in IC₅₀s against CTX-M-15

and CMY-2 was observed on 14 possessing positively charged

amino group. In spite of comparable IC₅₀s to compound 2,

almost all compounds exhibited ≥4-fold increase in MPC₁

against at least one of the four strains tested. This may be due

to decline in the outer membrane permeability resulting from

the increased molecular weight. We could see MPC₁comparable

to 2 against the all four strains on 7 and 8, which have lower

molecular weight and lipophilicity among the tested com-

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Table 6. MPC₁and MIC of 2 and AVI against a Set of Clinical Isolates

b

MPC₁(μg/mL) /

c

MIC (μg/mL)

MIC of CTB

fold reduction in MPC₁

d

a

c

species

strain ID

characteristics

(μg/mL)

2

AVI

16/32

compared to AVI

4

Enterobacter

cloacae

SR200030 PER-2, ACT, ΔompF

>32

4/>32

K. pneumoniae

Enterobacter

cloacae

SR100758 KPC-2 (D179Y)

SR201325 KPC-3

16

32

>32

0.25/>32

2/>32

2/32

4/32

8

1

2

SR36276

AmpC

Enterobacter

cloacae

SR100108 AmpC

>32

1/>32

8/32

8

K. pneumoniae

SR09635

SR09603

DHA-1

CMY-8 type

>32

2/32

0.5/>32

8/>32

0.063/>32

4/>32

0.25

64

0.5

8

0.125/32

0.5/>32

SR200263 OXA-48, CTX-M-1 group

SR200487 VEB-8, CTX-M-15, SHV-110, CMY-16,

OXA-48, OXA-10

a

b

c

The expressed β-lactamases and the deletion of porin on each strain. N = 1 determination. MPC₁to reduce the MIC of CTB to 1 μg/mL. N = 1

d

determination. MIC of inhibitor. Calculated as (MPC₁of AVI)/(MPC₁of 2).

a

Table 7. PK Parameter of 2 after Intravenous Administration

animal

dose (mg/kg)

AUC (μg·h/mL)

T

_1/2(h)

CL_tot(mL/min/kg)

34.7 6.8

PB (% unbound)

mouse

rat

dog

10

1

5

4.92 0.94

0.4 0.2

0.2

0.9 0.1

0.5 0.1

74

100

86

b

0.62

27.0

4.30 0.24

6.15 1.06

19.4 1.1

13.8 2.2

monkey

5

84

a

N = 3 determination. AUC, area under the plasma concentration−time curve from time zero to inﬁnity. T_1/2, apparent terminal elimination half-

b

life. CL_tot, total clearance. PB, serum protein binding. N = 2 determination.

a b

,

Table 8. PK Parameter of 2 after Oral Administration of Prodrugs

rat (fed, 5 mg/kg, N = 2)

dog (fasted, 10 mg/kg, N = 2)

compound

C

_max(μg/mL)

AUC (μg·h/mL)

T

_max(h)

F (%)

C

_max(mg/mL)

AUC (μg·h/mL)

T

_max(h)

F (%)

68.3

48.1

19

20

21

2.15

1.70

0.62

2.30

2.09

1.17

0.5

74.4

67.6

37.8

10.6

7.39

15.8 1.4

26.0

18.4

41.2 3.8

1.0

0.8

1.0 0.0

106.1 7.4

monkey (fasted, 10 mg/kg, N = 3)

c

compound

C_max(μg/mL)

AUC (μg·h/mL)

T_max(h)

F (%)

solution stability at pH 7.4 (%)

19

20

21

4.51 0.33

4.38 1.10

7.94 2.74

8.32 1.96

7.06 1.26

15.2 2.7

1.0 0.0

29.9 2.8

25.5 0.7

54.8 3.7

11

56

a

C_max, peak concentration. AUC, area under the plasma concentration−time curve from time zero to inﬁnity. T_max, time at the peak concentration.

b

c

F, bioavailability calculated from AUC_poand AUC_ivof 2. N = 3 determination. Remaining amount in pH 7.4 phosphate buﬀer at 40 °C after 16 h.

class C and D producers. In particular, ≥8-fold reduction in

MPC₁compared to AVI was observed for SR100758,

SR100108, SR09603, and SR200487. The strain SR100758

expresses KPC-2 with amino acid substitution (D179Y). It has

been reported that AVI shows impaired inhibition for this β-

lactamase,²⁶and 16-fold deterioration of MPC₁compared to

that against ATCC BAA-1705 expressing KPC-2 was actually

observed in our experiment. On the other hand, MPC₁of 2

showed only 4-fold increase. This result suggests that 2 is less

susceptible to the D179Y substitution compared to AVI.

Compound 2 also exhibited the obvious superior activity against

some class C β-lactamase (AmpC and CMY-8) producing

strains (SR100108 and SR09603). It is remarkable that 2

successfully rescued MICs of CTB (≤1 μg/mL) against all

highly resistant strains at ≤ 4 μg/mL.

Pharmacokinetics. The PK parameters after intravenous

administration of 2 in mouse, rat, dog, and monkey are shown in

Table 7. Compound 2 showed moderate total clearance (CL_tot)

and short half-life (T_1/2), and these PK properties are

comparable with other DBO derivatives including AVI.^11,13,15

As expected from its low lipophilicity (c log P = −0.30), 2 was

hardly absorbed after oral administration (bioavailability in rat:

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Figure 3. In vivo eﬃcacy in a murine urinary tract infection model. ^aChange in log CFU/kidney compared to the untreated control at the start of

treatment. N = 5 determination.

1.4%). The serum protein binding (PB, % unbound) of 2 was

high for all animals tested including humans (81% unbound).

To identify an orally available BLI, we explored ester

prodrugs. We prepared several esters consisting of an alcohol

employed as a part of ester prodrugs in the market.²⁷In addition

to alkyl esters 19−21, we attempted to synthesize the pivoxil 22

which is utilized for the oral prodrugs of several β-lactam

antibiotics,²⁸but 22 was quite unstable and diﬃcult to isolate.

The proﬁles of the prodrugs are summarized in Table 8. In all

cases, the prodrug modiﬁcations eﬀectively enhanced the

bioavailability of 2, and intact prodrugs were not detected in

the blood. This observation indicates that the prodrugs are

immediately converted into the corresponding carboxylic acid 2

treatment, and 30 mg/kg of both achieved >1 log reduction

although 30 mg/kg of CTB alone was not eﬀective.

Safety. We conducted a 2 week toxicology study of 21 in rats

following daily oral administration. Doses of 600 mg/kg/day

and >2000 mg/kg/day were, respectively, established as the no

observed adverse eﬀect level and the lethal dose.

CONCLUSIONS

■

We have presented the discovery of 2-sulﬁnyl-diazabicyclooc-

tane derivatives, which are highly potent oral BLIs. A thio-

functional group, such as a sulfone, at the C2 position of DBO

enhances the potential for BLI, whereas it destabilizes a

molecule against rat serum. In our continuous research, we

found that the combination of sulfoxide at the C2 position and

ﬂuoroacetic acid at the 6-position could balance the β-lactamase

inhibition potential with serum stability. We established the

stereoselective synthesis of the sulfoxides with various side

chains and conducted exploratory SAR studies. Simple methyl

sulfoxide 2 was the best compound to restore the antibacterial

activities of CTB and was orally absorbed by an ester prodrug

modiﬁcation. Coadministration of CTB with 21 (cyclohexyl

ester prodrug of 2) showed eﬃcacy in a murine urinary tract

infection model, and 21 showed appropriate safety in a rat 2

week toxicology study. These results indicate that 21 is one of

the best orally active BLIs for infections caused by β-lactamase-

producing Enterobacterales.

after absorption. The structure of the ester did not aﬀect T_max

.

Although these ester prodrugs were hydrolyzed to carboxylic

acid 2 in pH 7.4 phosphate buﬀer, branched alkyl esters (20, 21)

were more stable than linear ones (19). Therefore, we presumed

that branched ester prodrugs would be preferable as drug

candidates. Prodrug 21 showed better bioavailability in dogs and

monkeys compared to the others, and we selected 21 for further

studies.

In Vivo Eﬃcacy in an Infection Model by Oral

Administration. With orally available BLIs in hand, prodrug

21 was evaluated in a murine urinary tract infection model

against Klebsiella pneumoniae strains SR08667, which produces

KPC (Figure 3). The MIC of CTB alone is 32 μg/mL, and the

MPC₁of 2 is 1 μg/mL. After oral administration, plasma

exposure of 2 showed dose linearity up to 100 mg/kg in mice

(Table 9). The coadministration of 10 mg/kg of both CTB and

21 gave >0.5 log reduction in cfu/kidney from the start of

EXPERIMENTAL SECTION

■

General. Unless otherwise noted, reactions were performed under a

nitrogen atmosphere. Solvents and commercial reagents were used

without puriﬁcation. Carboxylic acid 23 was purchased from Pharma-

Block R&D Co. AVI was synthesized via the analogous method, as

reported.²⁹Analytical thin-layer chromatography was run on silica gel

F254 precoated plates. Visualization of the developed chromatogram

was performed by ﬂuorescence quenching or Vaughn’s reagent.

Column chromatography was carried out using silica gel. Reverse

phase column chromatography was performed using HP20SS and

Table 9. PK Parameter of 2 after Oral Administration of 21 in

a

Mice

dose (mg/kg)

C_max(μg/mL)

AUC (μg·h/mL)

F (%)

10

30

100

5.6

21.4

52.0

3.9

18.1

50.2

80

122

102

octadecylsilyl (ODS) silica gel (Yamazen Ultra Pack). ¹H, ¹³C, and ¹⁹

F

NMR spectra were recorded on a Bruker AV400 spectrometer.

Chemical shifts (δ) are reported in parts per million from

tetramethylsilane (in CD₃Cl, DMSO-d₆, methanol-d₄), sodium 2,2-

dimethyl-2-silapentane-5-sulfonate (DSS-d₆) (in D₂O), or the solvent

residual peak (in D₂O δ 4.79) as an internal reference. High-resolution

a

N = 3 determination. C_max, peak concentration. AUC, area under the

plasma concentration−time curve from time zero to inﬁnity. F,

bioavailability calculated from AUC_poand AUC_ivof 2.

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mass spectral data were acquired on Orbitrap Q Exactive Plus (ESI).

Low-resolution mass spectral data were collected on a Waters ZQ mass

detector (ESI), a Shimadzu LCMS-8030 (ESI) or a Shimadzu LCMS-

2020 (ESI). Elemental analysis was performed using Micro Corder

JM11 (J-SCIENCE LAB CO., Ltd.). Light promoted reactions were

carried out using a Kessil A 160WE Tuna Blue 40 W lamp with the

white light mode. LED lamps were placed 2−10 cm away from the

reaction vessel without any ﬁlters.³⁰Purities for all tested compounds

were determined by high-performance liquid chromatography (HPLC)

analysis and described along with other characteristic data. All tested

compounds were isolated with ≥95% purity except for compound 1.

Purity was determined on a Shimadzu Prominence HPLC system using

a COSMOSIL 5C18-AR-II column. Mobile phase A was 0.1%

triﬂuoroacetic acid in water, and mobile phase B was 0.1%

triﬂuoroacetic acid in acetonitrile. One of the following methods (A−

D) was used: method-A, a gradient from 2 to 100% B in 10 min and held

at 100% B for 5 min; method-B, a gradient from 10 to 100% B in 10 min

and held at 100% B for 5 min; method-C, an isocratic 5% B; and

method-D, an isocratic 7% B.

pound 27d (172 mg, 23%, dr = 7/1 over two steps) was prepared

from carboxylic acid 24 and thiolating agent 54 in a similar manner to

27a. ¹H NMR (400 MHz, CDCl₃): δ 5.89 (1H, d, J = 52.1 Hz), 4.42

(1H, t, J = 6.9 Hz), 4.12−4.10 (1H, m), 3.98 (1H, d, J = 16.1 Hz), 3.88

(3H, s), 3.74 (1H, d, J = 16.2 Hz), 3.29−3.26 (1H, m), 3.22 (1H, d, J =

12.0 Hz), 2.41−2.24 (3H, m), 2.05−1.98 (1H, m). MS-ESI (m/z): 320

[M + H]⁺.

(2R,5R)-6-(Benzyloxy)-2-((R)-(2-((tert-butyldimethylsilyl)oxy)-

ethyl)sulﬁnyl)-1,6-diazabicyclo[3.2.1]octan-7-one (27e). Compound

27e (16.2 g, 24%, dr = >10/1 over two steps) was prepared from

carboxylic acid 23 and thiolating agent 55 in a similar manner to 27a.

¹H NMR (400 MHz, CDCl₃): δ 7.42−7.37 (5H, m), 5.05 (1H, d, J =

11.4 Hz), 4.90 (1H, d, J = 11.5 Hz), 4.18 (1H, dd, J = 7.7, 3.9 Hz),

4.12−4.00 (2H, m), 3.37−3.36 (1H, br m), 3.24 (1H, d, J = 11.8 Hz),

3.19−3.16 (1H, m), 3.02 (1H, d, J = 10.9 Hz), 2.77 (1H, dt, J = 13.1, 3.9

Hz), 2.39−2.33 (1H, m), 2.17−2.07 (2H, m), 1.83−1.77 (1H, m), 0.90

(9H, s), 0.10 (3H, s), 0.09 (3H, s). MS-ESI (m/z): 439 [M + H]⁺.

Ethyl (2R)-2-(((2R,5R)-2-((R)-(3-((tert-butyldimethylsilyl)oxy)-

propyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂu-

oroacetate (27f). Compound 27f (216 mg, 12%, dr = >10/1 over two

steps) was prepared from carboxylic acid 25 and thiolating agent 56 in a

All studies with animals were approved by the Institutional Animal

Care and Use Committee of Shionogi & Co., Ltd.

1

(2R,5R)-6-(Benzyloxy)-2-((R)-methylsulﬁnyl)-1,6-diazabicyclo-

[3.2.1]octan-7-one (27a).¹⁹To a solution of carboxylic acid 23 (10.0 g,

36.2 mmol) and 2-mercaptopyridine 1-oxide 28 (4.60 g, 36.2 mmol,

1.00 equiv) in CH₂Cl₂(100 mL) was added EDC HCl (6.94 g, 36.2

mmol, 1.00 equiv). The solution was stirred for 1 h at room temperature

in the dark (solution A). To a solution of PhSSMe³²51 (17.0 g, 109

mmol, 3.00 equiv) in CH₂Cl₂(100 mL) was added solution A over 10

min under white light irradiation at 0 °C. The mixture was stirred under

white light irradiation for 30 min at 0 °C. The reaction mixture was

poured into water, and the layers were separated. The organic layer was

dried over MgSO₄and concentrated under reduced pressure. The crude

product was puriﬁed by ﬂash column chromatography (10−30%

EtOAc/hexane) to give sulﬁde (3.46 g) as a colorless oil. To a solution

of the sulﬁde in CH₂Cl₂(30 mL) was added 72 wt % m-CPBA (3.13 g,

13.1 mmol, 1.05 equiv) at −78 °C. After being stirred for 2 h at −78 °C,

the mixture was poured into aq Na₂S₂O₃, and the layers were separated.

The aqueous layer was extracted with EtOAc three times, and the

combined organic layers were dried over Na₂SO₄. After the solvent was

removed under reduced pressure, the crude product was puriﬁed by

ﬂash column chromatography (0−10% MeOH/EtOAc) to give 27a

(3.06 g, 29%, dr = >10/1 over steps) as a white solid. ¹H NMR (400

MHz, CDCl₃): δ 7.43−7.37 (5H, m), 5.04 (1H, d, J = 11.3 Hz), 4.90

(1H, d, J = 11.5 Hz), 4.02 (1H, dd, J = 7.5, 4.3 Hz), 3.39−3.39 (1H, m),

3.18 (1H, d, J = 11.8 Hz), 3.03 (1H, d, J = 11.8 Hz), 2.68 (3H, s), 2.41−

2.34 (1H, m), 2.19−2.09 (2H, m), 1.83−1.74 (1H, m). ¹³C{¹H} NMR

(100 MHz, CDCl₃): δ 166.9, 135.5, 129.3, 128.9, 128.6, 78.4, 76.3, 57.5,

45.7, 37.2, 19.6, 15.9. HRMS (ESI) m/z: [M + H]⁺calcd for

C₁₄H₁₉N₂O₃S, 295.1111; found, 295.1108. mp 96−99 °C.

similar manner to 27a. H NMR (400 MHz, CDCl₃): δ 5.87 (1H,

d,³J_H−F= 52.3 Hz), 4.38−4.26 (2H, m), 4.18−4.15 (1H, m), 4.05−4.03

(1H, br m), 3.81−3.70 (2H, m), 3.41 (1H, d, J = 12.1 Hz), 3.20−3.15

(1H, m), 3.05 (1H, dt, J = 15.1, 6.6 Hz), 2.79−2.72 (1H, m), 2.45−2.37

(1H, m), 2.26−2.21 (1H, m), 2.14 (1H, td, J = 14.9, 7.4 Hz), 2.04−1.93

(3H, m), 1.33 (3H, t, J = 7.2 Hz), 0.88 (9H, s), 0.05 (6H, s). MS-ESI

(m/z): 467 [M + H]⁺.

(2R,5R)-6-(Benzyloxy)-2-((R)-(((S)-2,2-dimethyl-1,3-dioxolan-4-

yl)methyl)sulﬁnyl)-1,6-diazabicyclo[3.2.1]octan-7-one (27g). Com-

pound 27g (3.48 g, 22%, dr = >10/1 over two steps) was prepared from

carboxylic acid 23 and thiolating agent 57 in a similar manner to 27a.

¹H NMR (400 MHz, CDCl₃): δ 7.44−7.37 (5H, m), 5.04 (1H, d, J =

11.5 Hz), 4.90 (1H, d, J = 11.5 Hz), 4.64−4.57 (1H, m), 4.18 (1H, dd, J

= 8.5, 6.1 Hz), 4.13 (1H, dd, J = 7.3, 4.6 Hz), 3.73 (1H, dd, J = 8.5, 6.2

Hz), 3.39−3.39 (1H, br m), 3.26 (1H, dd, J = 13.1, 9.0 Hz), 3.19 (1H, d,

J = 11.8 Hz), 3.04−3.02 (1H, br m), 2.80 (1H, dd, J = 13.1, 3.8 Hz),

2.40−2.32 (1H, m), 2.20−2.10 (2H, m), 1.82−1.76 (1H, m), 1.43 (3H,

s), 1.38 (3H, s). MS-ESI (m/z): 395 [M + H]⁺.

2-(Trimethylsilyl)ethyl 3-((R)-((2R,5R)-6-(benzyloxy)-7-oxo-1,6-

diazabicyclo[3.2.1]octan-2-yl)sulﬁnyl)-2-(((4-methoxybenzyl)oxy)-

imino)propanoate (27h). Compound 27h (1.93 g, 21%, dr = >10/1

over two steps) was prepared from carboxylic acid 23 and thiolating

agent 58 in a similar manner to 27a. The oxime was obtained as a single

1

geometrical isomer, but the geometry (E/Z) is unknown. H NMR

(400 MHz, CDCl₃): δ 7.42−7.37 (5H, m), 7.32 (2H, d, J = 8.6 Hz),

6.88 (2H, d, J = 8.6 Hz), 5.30 (1H, d, J = 11.4 Hz), 5.26 (1H, d, J = 11.4

Hz), 5.03 (1H, d, J = 11.4 Hz), 4.87 (1H, d, J = 11.4 Hz), 4.39−4.35

(2H, m), 4.18−4.15 (2H, m), 4.01 (1H, d, J = 12.1 Hz), 3.76 (3H, s),

3.28−3.27 (1H, br m), 2.98 (1H, d, J = 11.9 Hz), 2.85−2.82 (1H, br m),

2.35−2.29 (1H, m), 2.04−1.96 (2H, m), 1.71−1.63 (1H, m), 1.13−

1.09 (2H, m), 0.06 (9H, s). MS-ESI (m/z): 602 [M + H]⁺.

tert-Butyl (2-((R)-((2R,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo-

[3.2.1]octan-2-yl)sulﬁnyl)ethyl)carbamate (27i). Compound 27i

(834 mg, 16%, dr = >10/1 over two steps) was prepared from

carboxylic acid 23 and thiolating agent 59 in a similar manner to 27a.

¹H NMR (400 MHz, CDCl₃): δ 7.41−7.37 (5H, m), 5.12 (1H, br s),

5.04 (1H, d, J = 11.4 Hz), 4.89 (1H, d, J = 11.4 Hz), 4.16−4.13 (1H, m),

3.67−3.65 (2H, br m), 3.39−3.38 (1H, br m), 3.27−3.20 (1H, m), 3.17

(1H, d, J = 11.8 Hz), 3.03 (1H, d, J = 12.0 Hz), 2.85 (1H, dt, J = 13.3, 5.5

Hz), 2.38−2.28 (1H, m), 2.19−2.10 (2H, m), 1.81−1.74 (1H, m), 1.44

(9H, s). MS-ESI (m/z): 424 [M + H]⁺.

Ethyl (2R)-2-ﬂuoro-2-(((2R,5R)-2-((S)-(ﬂuoromethyl)sulﬁnyl)-7-

oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (27b). Com-

pound 27b (333 mg, 26%, dr = >10/1 over two steps) was prepared

from carboxylic acid 25 and thiolating agent 52³¹in a similar manner to

27a. ¹H NMR (400 MHz, CDCl₃): δ 5.87 (1H, d,³J_H−F= 52.3 Hz), 5.55

(1H, dd, J = 46.0, 8.8 Hz), 5.33 (1H, dd, J = 45.6, 8.8 Hz), 4.55 (1H, t, J

= 6.3 Hz), 4.36−4.28 (2H, m), 4.10−4.07 (1H, br m), 3.35 (1H, d, J =

12.4 Hz), 3.24 (1H, d, J = 11.9 Hz), 2.43−2.35 (1H, m), 2.31−2.14

(2H, m), 2.05−1.96 (1H, m), 1.34 (3H, t, J = 7.2 Hz). MS-ESI (m/z):

327 [M + H]⁺.

tert-Butyl 2-((R)-((2R,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo-

[3.2.1]octan-2-yl)sulﬁnyl)acetate (27c).¹⁸Compound 27c (21.5 g,

36%, dr = 7/1 over two steps) was prepared from carboxylic acid 23 and

1

thiolating agent 53 in a similar manner to 27a. H NMR (400 MHz,

(2R,5R)-6-(Benzyloxy)-2-((R)-((5-methyl-1,3,4-oxadiazol-2-yl)-

methyl)sulﬁnyl)-1,6-diazabicyclo[3.2.1]octan-7-one (27j). Com-

pound 27j (340 mg, 59%, dr = 7/1 over two steps) was prepared

from carboxylic acid 23 and thiolating agent 60 in a similar manner to

27a. ¹H NMR (400 MHz, CDCl₃): δ 7.41−7.39 (5H, m), 5.04 (1H, d, J

= 11.4 Hz), 4.90 (1H, d, J = 11.4 Hz), 4.54 (1H, d, J = 14.2 Hz), 4.39

(1H, t, J = 6.1 Hz), 4.22 (1H, d, J = 14.2 Hz), 3.42−3.41 (1H, m), 3.13

CDCl₃): δ 7.42−7.37 (5H, m), 5.03 (1H, d, J = 11.4 Hz), 4.89 (1H, d, J

= 11.4 Hz), 4.31−4.28 (1H, m), 3.89 (1H, d, J = 14.7 Hz), 3.59 (1H, d, J

= 14.4 Hz), 3.40−3.39 (1H, m), 3.17 (1H, d, J = 11.9 Hz), 3.03−3.00

(1H, m), 2.39−2.32 (1H, m), 2.19−2.10 (2H, m), 1.83−1.74 (1H, m),

1.49 (9H, s). MS-ESI (m/z): 395 [M + H]⁺.

Methyl (2R)-2-(((2R,5R)-2-((R)-(cyanomethyl)sulﬁnyl)-7-oxo-1,6-

diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (27d). Com-

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(1H, d, J = 11.9 Hz), 3.05 (1H, d, J = 11.8 Hz), 2.56 (3H, s), 2.36−2.27

(1H, m), 2.24−2.14 (2H, m), 1.83−1.74 (1H, m).

δ −134.1 (d,³J_H−F= 53.1 Hz). HRMS (ESI) m/z: [M + H]⁺Calcd for

C₂₂H₂₄N₂O₅FS, 447.1384; found, 447.1385.

Benzhydryl (2R)-2-(((2R,5R)-2-((R)-(2-amino-2-oxoethyl)sulﬁnyl)-

7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (33).

Compound 33 (510 mg, 78% over two steps) was prepared from 31

in a similar manner to 32. ¹H NMR (400 MHz, CDCl₃): δ 7.41−7.29

(10H, m), 6.95 (1H, s), 6.53 (1H, br s), 5.98 (1H, d,³J_H−F= 53.2 Hz),

5.56 (1H, br s), 4.35 (1H, t, J = 6.3 Hz), 4.00−3.97 (1H, br m), 3.80

(1H, d, J = 14.1 Hz), 3.53 (1H, d, J = 14.1 Hz), 3.09 (1H, d, J = 12.3

Hz), 2.67 (1H, d, J = 12.0 Hz), 2.35−2.28 (1H, m), 2.24−2.15 (2H, m),

1.97−1.90 (1H, m).

tert-Butyl 4-(((R)-((2R,5R)-6-((R)-2-ethoxy-1-ﬂuoro-2-oxoethoxy)-

7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)sulﬁnyl)methyl)-1H-imida-

zole-1-carboxylate (27k). Compound 27k (95.0 mg, 14%, dr = 7/1

over two steps) was prepared from carboxylic acid 25 and thiolating

agent 61 in a similar manner to 27a. ¹H NMR (400 MHz, CDCl₃): δ

8.06 (1H, d, J = 1.0 Hz), 7.45 (1H, s), 5.87 (1H, d,³J_H−F= 52.5 Hz),

4.40 (1H, dd, J = 7.2, 5.0 Hz), 4.36−4.27 (2H, m), 4.20 (1H, d, J = 13.7

Hz), 4.06−4.04 (1H, br m), 3.92 (1H, d, J = 13.8 Hz), 3.44 (1H, d, J =

12.2 Hz), 3.23 (1H, d, J = 12.2 Hz), 2.45−2.36 (1H, m), 2.29−2.11

(2H, m), 2.03−1.94 (1H, m), 1.62 (9H, s), 1.33 (3H, t, J = 7.2 Hz).

Methyl (2R)-2-(((2R,5R)-2-((R)-((2-((tert-butoxycarbonyl)amino)-

thiazol-5-yl)methyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-

yl)oxy)-2-ﬂuoroacetate (27l). Compound 27l (78.2 mg, 36%, dr = 5/1

over two steps) was prepared from carboxylic acid 23 and thiolating

agent 62 in a similar manner to 27a. ¹H NMR (400 MHz, CDCl₃): δ

8.07 (1H, br s), 6.93 (1H, s), 5.89 (1H, d,³J_H−F= 52.5 Hz), 4.54 (1H, t, J

= 6.2 Hz), 4.20 (1H, d, J = 13.6 Hz), 4.06−4.03 (1H, br m), 4.01 (1H, d,

J = 13.7 Hz), 3.87 (3H, s), 3.42 (1H, d, J = 12.0 Hz), 3.24 (1H, d, J =

12.2 Hz), 2.40−2.31 (1H, m), 2.27−2.22 (1H, m), 2.15−1.93 (2H, m),

1.54 (9H, s). MS-ESI (m/z): 493 [M + H]⁺.

Benzhydryl (2R)-2-(((2R,5R)-2-((R)-(2-((tert-butyldimethylsilyl)-

oxy)ethyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-

ﬂuoroacetate (34). Compound 34 (3.46 g, 64% over two steps) was

1

prepared from 27e in a similar manner to 32. H NMR (400 MHz,

CDCl₃): δ 7.42−7.28 (10H, m), 6.95 (1H, s), 5.98 (1H, d,³J_H−F= 53.3

Hz), 4.27 (1H, dd, J = 7.5, 4.7 Hz), 4.13−4.01 (2H, m), 3.96−3.94 (1H,

br m), 3.26 (1H, d, J = 12.0 Hz), 3.16−3.09 (1H, m), 2.77 (1H, dt, J =

13.1, 3.8 Hz), 2.66 (1H, d, J = 12.2 Hz), 2.40−2.33 (1H, m), 2.22−2.07

(2H, m), 1.97−1.91 (1H, m), 0.91 (9H, s), 0.12 (3H, s), 0.10 (3H, s).

MS-ESI (m/z): 591 [M + H]⁺.

Ethyl (2R)-2-(((2R,5R)-2-((R)-(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-

methyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂu-

oroacetate (35). To a solution of 27g (3.48 g, 8.82 mmol) in THF/

MeOH (v/v = 1/1, 35 mL) were added 10% Pd(OH)₂on carbon (619

mg, 0.18 wt equiv) and DABCO (20 mg, 0.18 mmol, 0.02 equiv). After

being stirred under a H₂atmosphere (1 atm) for 30 min, the mixture

was ﬁltered, and the solvent was removed under reduced pressure.

Trituration with diisopropyl ether (IPE) gave the debenzylated product

(2.09 g, 78%). To a solution of the debenzylated product (2.09 g, 6.87

mmol) in DMF (20 mL) were added (R)-ethyl bromoﬂuoroacetate 67

(61.5 wt %, 2.48 g, 8.24 mmol, 1.20 equiv) and K₂CO₃(1.04 g, 7.55

mmol, 1.10 equiv) at −40 °C. After being stirred overnight at −20 °C,

the mixture was poured into 10% aq citric acid, and the aqueous layer

was extracted with EtOAc twice. The combined organic layers were

washed with water and dried over Na₂SO₄. After the solvent was

removed under reduced pressure, the crude was puriﬁed by ﬂash

column chromatography (0−10%, MeOH/EtOAc) to give 35 (2.45 g,

68% over two steps) as a colorless gum. ¹H NMR (400 MHz, CDCl₃): δ

5.88 (1H, d,³J_H−F= 52.3 Hz), 4.61−4.59 (1H, m), 4.37−4.29 (2H, m),

4.24−4.21 (2H, m), 4.07−4.05 (1H, br m), 3.73 (1H, dd, J = 8.5, 6.2

Hz), 3.38 (1H, d, J = 12.1 Hz), 3.25−3.19 (2H, m), 2.82 (1H, dd, J =

13.0, 3.7 Hz), 2.46−2.38 (1H, m), 2.29−2.12 (2H, m), 2.03−1.94 (1H,

m), 1.44 (3H, s), 1.38 (3H, s), 1.35 (3H, q, J = 7.2 Hz). MS-ESI (m/z):

409 [M + H]⁺

2-((R)-((2R,5R)-6-(Benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]-

octan-2-yl)sulﬁnyl)acetic acid (30). To 27c (21.5 g, 54.6 mmol) was

added TFA (150 mL) at 0 °C. After being stirred for 3 h at 0 °C, the

solvent was removed under reduced pressure. The crude was triturated

with EtOAc to give 30 (15.9 g, 86%) as a white solid. ¹H NMR (400

MHz, DMSO-d₆): δ 13.21 (1H, br s), 7.45−7.35 (5H, m), 4.94 (1H, d,

J = 12.0 Hz), 4.91 (1H, d, J = 11.6 Hz), 4.30 (1H, t, J = 6.4 Hz), 3.97

(1H, d, J = 14.9 Hz), 3.78−3.75 (1H, br m), 3.60 (1H, d, J = 14.7 Hz),

3.27 (1H, d, J = 11.9 Hz), 2.98 (1H, d, J = 11.6 Hz), 2.30−2.20 (1H, m),

1.95−1.80 (3H, m). MS (ESI) m/z: [M + H]⁺Calcd for C₁₅H₁₉N₂O₅S,

339.10; found, 339.30.

2-((R)-((2R,5R)-6-(Benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]-

octan-2-yl)sulﬁnyl)acetamide (31). To a solution of 30 (15.6 g, 46.2

mmol) in CH₂Cl₂(109 mL) were added HOSu (6.40 g, 55.5 mmol,

1.20 equiv) and EDC HCl (10.6 g, 55.5 mmol, 1.20 equiv) at 0 °C. After

being stirred for 2.5 h at room temperature, the mixture was cooled to

−50 °C (solution A). To another solution of 28% aq ammonia (9.4 mL,

139 mmol, 3.00 equiv) in CH₂Cl₂(47 mL) was added the solution A at

−50 °C. After being stirred for 1 h at room temperature, the mixture

was poured into 10% aq citric acid, which was saturated with NaCl, and

the aqueous layer was extracted with CH₂Cl₂three times. The

combined organic layers were washed with aq NaHCO₃, which was

saturated with NaCl, before being dried over MgSO₄. After the solvent

was removed under reduced pressure, the crude was puriﬁed by ﬂash

column chromatography (0−10%, MeOH/EtOAc) to give 31 (12.5 g,

80%) as a white solid.

Benzhydryl (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-methylsulﬁnyl)-7-

oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (32). To a solu-

tion of 27a (15.1 g, 51.1 mmol) in MeOH (150 mL) were added 10%

Pd(OH)₂on carbon (3.59 g, 0.24 wt equiv) and DABCO (229 mg, 2.05

mmol, 0.04 equiv). After being stirred at room temperature under a H₂

atmosphere (1 atm) for 1 h, the mixture was ﬁltered. The solvent was

removed in vacuo to aﬀord the debenzylated product as a white solid.

To a solution of the debenzylated product and DBU (8.48 mL, 56.2

mmol, 1.1 equiv) in DMF (50 mL) was added a solution of 65 (19.8 g,

61.3 mmol, 1.20 equiv) in DMF (30 mL) at 0 °C. After being stirred for

1 h at 0 °C, the mixture was poured into 5% aq citric acid, and the

aqueous layer was extracted with EtOAc twice. The combined organic

layers were washed with brine and dried over MgSO₄. The solvent was

removed under reduced pressure to give a yellow solid. The solid was

washed with ⁱPrOAc to give 32 (13.1 g, 57% over two steps) as a white

solid. ¹H NMR (400 MHz, CDCl₃): δ 7.37−7.31 (10H, m), 6.95 (1H,

s), 5.98 (1H, d,³J_H−F= 53.2 Hz), 4.08 (1H, t, J = 6.1 Hz), 3.97−3.95

(1H, br m), 3.19 (1H, d, J = 12.0 Hz), 2.68−2.65 (1H, m), 2.65 (3H, s),

2.41−2.33 (1H, m), 2.23−2.07 (2H, m), 1.97−1.89 (1H, m). ¹³C{¹H}

NMR (100 MHz, CDCl₃): δ 167.0, 162.1 (d,³J_C−F= 35.2 Hz), 138.6,

138.5, 128.7, 128.5, 128.4, 128.4, 127.6, 127.1, 105.4 (d,²J_C−F= 238.4

Hz), 79.7, 76.7, 60.8, 44.5, 37.0, 19.1, 15.8. ¹⁹F NMR (377 Hz, CDCl₃):

2-(Trimethylsilyl)ethyl 3-((R)-((2R,5R)-6-((R)-2-(benzhydryloxy)-1-

ﬂuoro-2-oxoethoxy)-7-oxo-1,6-diazabicyclo[3.2.1]octan-2-yl)-

sulﬁnyl)-2-(((4-methoxybenzyl)oxy)imino)propanoate (36). Com-

pound 36 (930 mg, 39% over two steps) was prepared from 27h in a

similar manner to 32. The oxime was obtained as a single geometrical

1

isomer, but the geometry (E/Z) is unknown. H NMR (400 MHz,

CDCl₃): δ 7.40−7.29 (12H, m), 6.94 (1H, s), 6.89 (2H, d, J = 8.6 Hz),

5.96 (1H, d,³J_H−F= 53.3 Hz), 5.32 (1H, d, J = 11.4 Hz), 5.28 (1H, d, J =

11.4 Hz), 4.38 (2H, dd, J = 9.6, 7.3 Hz), 4.24−4.20 (1H, m), 4.12 (1H,

d, J = 12.1 Hz), 3.98 (1H, d, J = 12.1 Hz), 3.87−3.84 (1H, br m), 3.80

(3H, s), 3.09 (1H, d, J = 12.1 Hz), 2.55 (1H, d, J = 11.9 Hz), 2.38−2.31

(1H, m), 2.09−1.94 (2H, m), 1.86−1.81 (1H, m), 1.12 (2H, t, J = 8.7

Hz), 0.06 (9H, s). MS-ESI (m/z): 755 [M + H]⁺.

Benzhydryl (2R)-2-(((2R,5R)-2-((R)-(2-((tert-butoxycarbonyl)-

amino)ethyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-

2-ﬂuoroacetate (37). Compound 37 (164 mg, 20% over two steps)

was prepared from 27i in a similar manner to 32. ¹H NMR (400 MHz,

CDCl₃): δ 7.41−7.29 (10H, m), 6.95 (1H, s), 5.98 (1H, d,³J_H−F= 53.3

Hz), 5.10 (1H, br s), 4.19 (1H, t, J = 6.2 Hz), 3.97−3.94 (1H, br m),

3.66−3.65 (2H, br m), 3.24−3.15 (2H, m), 2.83 (1H, dt, J = 13.2, 5.6

Hz), 2.65 (1H, d, J = 12.4 Hz), 2.37−2.30 (1H, m), 2.23−2.07 (2H, m),

1.96−1.88 (1H, m), 1.45 (9H, s). MS-ESI (m/z): 576 [M + H]⁺

Methyl (2R)-2-(((2R,5R)-2-((R)-(2-((tert-butoxycarbonyl)amino)-

ethyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuo-

roacetate (38). Compound 38 (286 mg, 69% over two steps) was

1

prepared from 27i in a similar manner to 35. H NMR (400 MHz,

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CDCl₃): δ 5.89 (1H, d,³J_H−F= 52.3 Hz), 5.10 (1H, br s), 4.22 (1H, dd, J

= 7.3, 5.0 Hz), 4.06−4.04 (1H, br m), 3.87 (3H, s), 3.67−3.66 (2H, m),

3.35 (1H, d, J = 12.2 Hz), 3.27−3.20 (2H, m), 2.85 (1H, dt, J = 13.3, 5.5

Hz), 2.46−2.36 (1H, m), 2.29−2.12 (2H, m), 2.02−1.94 (1H, m), 1.44

(9H, s). MS-ESI (m/z): 424 [M + H]⁺.

mL, 3.69 mmol, 3.00 equiv) was added, and the mixture was stirred for

15 min at 0 °C. The mixture was poured into water, and the aqueous

layer was extracted with CH₂Cl₂twice. The combined organic layers

were dried over MgSO₄, and the solvent was removed under reduced

pressure. The crude was puriﬁed by ﬂash column chromatography

(50−100%, EtOAc/hexane) to give 43 (517 mg, 64% over two steps) as

a white amorphous solid. The oxime was obtained as a single

Benzhydryl (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-((5-methyl-1,3,4-oxa-

diazol-2-yl)methyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-

yl)oxy)acetate (39). Compound 39 (43.0 mg, 9.0% over two steps) was

1

geometrical isomer, but the geometry (E/Z) is unknown. H NMR

1

prepared from 27j in a similar manner to 32. H NMR (400 MHz,

(400 MHz, CDCl₃): δ 7.41−7.27 (12H, m), 6.94 (1H, s), 6.91 (2H, d, J

= 8.7 Hz), 6.59 (1H, br s), 5.97 (1H, d,³J_H−F= 53.3 Hz), 5.29 (1H, br s),

5.23 (2H, s), 4.24 (1H, t, J = 6.4 Hz), 4.12 (1H, d, J = 11.9 Hz), 4.03

(1H, d, J = 11.9 Hz), 3.91−3.88 (1H, br m), 3.81 (3H, s), 3.22 (1H, d, J

= 12.2 Hz), 2.63 (1H, d, J = 12.2 Hz), 2.42−2.35 (1H, m), 2.14−2.00

(2H, m), 1.90−1.83 (1H, m). MS-ESI (m/z): 653 [M + H]⁺.

Methyl (2R)-2-(((2R,5R)-2-((R)-(2-acetamidoethyl)sulﬁnyl)-7-oxo-

1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (44). To a

solution of 38 (164 mg, 0.387 mmol) in CH₂Cl₂(2 mL) was added

TFA (2 mL) at 0 °C. After the mixture was stirred for 15 min at 0 °C,

the solvent was removed under reduced pressure to give a crude

product. To a solution of the crude product in CH₂Cl₂(2 mL) were

added Et₃N (0.268 mL, 1.93 mmol, 5.00 equiv) and Ac₂O (0.037 mL,

0.387 mmol, 1.00 equiv) at −78 °C. After the mixture was stirred at

from −78 °C to room temperature, the mixture was directly puriﬁed by

ﬂash column chromatography (0−10%, MeOH/CHCl₃) to give 44

(142 mg, quant. over two steps) as a white gum. ¹H NMR (400 MHz,

CDCl₃): δ 6.29 (1H, br s), 5.89 (1H, d,³J_H−F= 52.2 Hz), 4.25 (1H, t, J =

6.2 Hz), 4.08−4.05 (1H, br m), 3.91−3.72 (2H, m), 3.87 (3H, s), 3.32

(1H, d, J = 12.2 Hz), 3.28−3.20 (2H, m), 2.94−2.88 (1H, m), 2.42−

2.34 (1H, m), 2.29−2.13 (2H, m), 2.03−1.97 (1H, m), 1.99 (3H, s).

MS-ESI (m/z): 366 [M + H]⁺.

tert-Butyl (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]-

octane-2-carboxylate (45). To a solution of 23 (25.0 g, 90.0 mmol)

in CH₂Cl₂(250 mL) were added tert-butanol (86.0 mL, 905 mmol, 10.0

equiv), pyridine (14.6 mL, 181 mmol, 2.00 equiv), DMAP (1.10 g, 9.1

mmol, 0.10 equiv), and EDC HCl (20.8 g, 109 mmol, 1.20 equiv) at 0

°C. After being stirred for 2 h, at room temperature, the mixture was

washed with 1 N aq HCl, water, aq NaHCO₃, and brine. After the

solvent was removed under reduced pressure, the crude product was

puriﬁed by ﬂash column chromatography (0−30% EtOAc/hexane) to

give 45 (19.9 g, 66%) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ

7.42 (2H, d, J = 6.3 Hz), 7.39−7.32 (3H, m), 5.05 (1H, d, J = 11.4 Hz),

4.90 (1H, d, J = 11.4 Hz), 4.01−3.99 (1H, br m), 3.31−3.29 (1H, br m),

3.04 (1H, d, J = 12.1 Hz), 2.98 (1H, d, J = 11.6 Hz), 2.09−2.02 (3H, m),

1.71−1.62 (1H, m), 1.48 (9H, s). MS-ESI (m/z): 333 [M + H]⁺.

tert-Butyl (2S,5R)-6-((R)-2-methoxy-1-ﬂuoro-2-oxoethoxy)-7-

oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate (46). Compound

46 (5.80 g, 55% over two steps) was prepared from 45 in a similar

manner to 35. ¹H NMR (400 MHz, CDCl₃): δ 5.90 (1H, d,³J_H−F= 52.5

Hz), 4.09−4.08 (1H, br m), 3.96−3.94 (1H, br m), 3.86 (3H, s), 3.21

(1H, d, J = 12.1 Hz), 3.13 (1H, d, J = 12.1 Hz), 2.18−2.06 (3H, m),

1.88−1.81 (1H, m), 1.50 (9H, s).

tert-Butyl (2S,5R)-6-((R)-2-ethoxy-1-ﬂuoro-2-oxoethoxy)-7-oxo-

1,6-diazabicyclo[3.2.1]octane-2-carboxylate (47). Compound 47

(17.3 g, 21% over two steps) was prepared from 45 in a similar

manner to 35. ¹H NMR (400 MHz, CDCl₃): δ 5.89 (1H, d,³J_H−F= 52.5

Hz), 4.35−4.26 (2H, m), 4.08 (1H, d, J = 6.3 Hz), 3.97−3.95 (1H, br

m), 3.20 (1H, d, J = 12.1 Hz), 3.13 (1H, d, J = 11.9 Hz), 2.19−2.04 (3H,

m), 1.89−1.81 (1H, m), 1.50 (9H, s), 1.33 (3H, t, J = 7.2 Hz). MS-ESI

(m/z): 347 [M + H]⁺.

(2S,5R)-6-((R)-2-Methoxy-1-ﬂuoro-2-oxoethoxy)-7-oxo-1,6-

diazabicyclo[3.2.1]octane-2-carboxylic acid (24). Compound 24

(2.25 g, 78%) was prepared from 46 in a similar manner to 25. ¹H NMR

(400 MHz, DMSO-d₆): δ 6.30 (1H, d,³J_H−F= 55.5 Hz), 3.98−3.96 (2H,

m), 3.78 (3H, s), 3.06−3.03 (2H, br m), 2.04−1.76 (4H, m).

(2S,5R)-6-((R)-2-Ethoxy-1-ﬂuoro-2-oxoethoxy)-7-oxo-1,6-

diazabicyclo[3.2.1]octane-2-carboxylic acid (25). To a solution of 47

(17.3 g, 49.9 mmol) in MeNO₂(86 mL) was added 2 M solution of

TiCl₄in CH₂Cl₂(49.9 mL, 100 mmol, 2.00 equiv) at −30 °C. After

being stirred for 20 min at −30 °C, the mixture was poured into iced

water, and the aqueous layer was extracted with EtOAc. The organic

CDCl₃): δ 7.35−7.30 (10H, m), 6.95 (1H, s), 5.98 (1H, d,³J_H−F= 53.2

Hz), 4.50−4.43 (2H, m), 4.20 (1H, d, J = 14.2 Hz), 4.00−3.97 (1H, br

m), 3.14 (1H, d, J = 12.4 Hz), 2.92 (1H, d, J = 28.9 Hz), 2.57 (3H, s),

2.35−2.29 (1H, m), 2.24−2.15 (2H, m), 1.97−1.90 (1H, m).

Benzhydryl (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-(2-hydroxyethyl)-

sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (40).

To a solution of 34 (2.93 g, 4.96 mmol) were added 3A molecular

sieves, 1 M tetra-n-butylammonium ﬂuoride (TBAF) in tetrahydrofur-

an (THF) (5.95 mL, 5.95 mmol, 1.20 equiv) and AcOH (0.340 mL,

5.95 mmol, 1.20 equiv) at 0 °C. After being stirred for 4 h at room

temperature, the mixture was ﬁltered, and the solvent was removed

under reduced pressure. The crude product was puriﬁed by ﬂash

column chromatography (0−10%, MeOH/EtOAc) to give 40 (246 mg,

1

10%) as a white amorphous solid. H NMR (400 MHz, CDCl₃): δ

7.41−7.26 (10H, m), 6.95 (1H, s), 5.98 (1H, d,³J_H−F= 53.3 Hz), 4.36−

4.33 (1H, m), 4.21−4.17 (2H, m), 3.98−3.96 (1H, br m), 3.21−3.14

(2H, m), 3.01−2.95 (1H, m), 2.67 (1H, d, J = 11.9 Hz), 2.38−2.31

(1H, m), 2.24−2.11 (2H, m), 2.09 (1H, s), 1.97−1.91 (1H, m). MS-

ESI (m/z): 477 [M + H]⁺.

Benzhydryl (2R)-2-(((2R,5R)-2-((R)-(2-(carbamoyloxy)ethyl)-

sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroace-

tate (41). To a solution of 40 (232 mg, 0.487 mmol) in THF (4 mL)

was added CSI (0.076 mL, 0.876 mmol, 1.80 equiv) at −30 °C. After

the mixture was stirred for 30 min at −30 °C, 8% aq NaHCO₃was

added, and the mixture was stirred for 1 h at room temperature. Brine

was added, and the aqueous layer was extracted with EtOAc twice. After

the combined organic layers were dried over Na₂SO₄, the solvent was

removed under reduced pressure. The crude product was puriﬁed by

ﬂash column chromatography (0−10%, MeOH/EtOAc) to give 41

1

(212 mg, 84%) as a white amorphous solid. H NMR (400 MHz,

CDCl₃): δ 7.40−7.29 (10H, m), 6.96 (1H, s), 5.98 (1H, d, J = 53.1 Hz),

4.59−4.54 (2H, m), 4.45−4.42 (1H, m), 3.99−3.97 (1H, br m), 3.24−

3.17 (1H, m), 3.12 (1H, d, J = 12.1 Hz), 3.07−3.01 (1H, m), 2.71 (1H,

d, J = 12.1 Hz), 2.38−2.31 (1H, m), 2.22−2.10 (2H, m), 1.97−1.90

(1H, m). MS-ESI (m/z): 520 [M + H]⁺.

Ethyl (2R)-2-(((2R,5R)-2-((R)-((S)-2,3-dihydroxypropyl)sulﬁnyl)-7-

oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (42).

To a solution of 35 (340 mg, 0.832 mmol) in CH₂Cl₂(3.4 mL) were

added anisole (0.546 mL, 4.99 mmol, 6.00 equiv) and 2 M solution of

AlCl₃in MeNO₂(2.50 mL, 4.99 mmol, 6.00 equiv) at −30 °C. After

being stirred for 20 min at −30 °C, the mixture was poured into brine.

The aqueous layer was extracted with THF four times, and the solvent

was removed under reduced pressure. The crude was puriﬁed by ﬂash

column chromatography (0−10%, MeOH/CHCl₃) to give 42 (240 mg,

78%) as a pale-yellow gum. ¹H NMR (400 MHz, CDCl₃): δ 5.87 (1H,

d,³J_H−F= 52.3 Hz), 4.40−4.28 (4H, m), 4.09−4.06 (1H, br m), 3.77

(1H, dd, J = 11.4, 3.7 Hz), 3.63 (1H, dd, J = 11.4, 5.6 Hz), 3.33 (1H, d, J

= 12.2 Hz), 3.26−3.20 (2H, m), 2.87 (1H, dd, J = 13.6, 2.8 Hz), 2.43−

2.36 (1H, m), 2.30−2.16 (2H, m), 2.04−1.97 (1H, m), 1.33 (3H, t, J =

7.2 Hz). MS-ESI (m/z): 369 [M + H]⁺.

Benzhydryl (2R)-2-(((2R,5R)-2-((R)-(3-amino-2-(((4-

methoxybenzyl)oxy)imino)-3-oxopropyl)sulﬁnyl)-7-oxo-1,6-

diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (43). To a sol-

ution of 36 (929 mg, 1.23 mmol) in DMF (10 mL) was added TASF

(407 mg, 1.48 mmol, 1.20 equiv). After being stirred for 1 h, the mixture

was poured into 10% aq citric acid, and the aqueous layer was extracted

with EtOAc twice. The combined organic layers were washed with

water and dried over Na₂SO₄. The solvent was removed under reduced

pressure to give a crude product. To a solution of the crude carboxylic

acid in CH₂Cl₂(10 mL) were added Et₃N (0.256 mL, 1.85 mmol, 1.50

equiv) and isobutyl chloroformate (0.178 mL, 1.36 mmol, 1.10 equiv)

at 0 °C. After being stirred for 15 min at 0 °C, 28% aq. ammonia (0.286

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layer was washed with water and brine before being dried over MgSO₄.

The solvent was removed under reduced pressure to give 25 (12.9 g,

89%). ¹H NMR (400 MHz, DMSO-d₆): δ 6.29 (1H, d,³J_H−F= 55.1 Hz),

4.30−4.17 (2H, m), 3.98−3.95 (2H, m), 3.07−3.00 (2H, m), 2.04−

1.77 (4H, m), 1.22 (3H, t, J = 7.1 Hz).

(d,³J_H−F= 58.6 Hz). HPLC: 99.4% (method-A). Anal. Calcd for

C₁₀H₁₃FN₃O₆SNa(H₂O)_1.7: C, 31.95; H, 4.40; F, 5.05; N, 11.18; S,

8.53; Na, 6.12. found: C, 32.03; H, 4.27; F, 4.92; N, 11.30; S, 8.08; Na,

6.49.

Sodium (2R)-2-(((2R,5R)-2-((R)-(cyanomethyl)sulﬁnyl)-7-oxo-1,6-

diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (8). Compound

8 (115 mg, 66%) was prepared from 27d in a similar manner to 2. ¹H

NMR (400 MHz, D₂O): δ 5.82 (1H, d,³J_H−F= 58.5 Hz), 4.59 (1H, t, J =

6.7 Hz), 4.28−4.26 (1H, br m), 3.44−3.32 (2H, m), 2.43−2.20 (3H,

m), 2.11−2.04 (1H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ 171.8

(d,³J_C−F= 27.1 Hz), 170.6, 115.3, 110.6 (d,²J_C−F= 241.4 Hz), 77.4,

Ethyl (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-(3-hydroxypropyl)sulﬁnyl)-

7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (48). To a

solution of 27f (240 mg, 0.514 mmol) in CH₂Cl₂(2 mL) was added

2 M solution of AlCl₃in MeNO₂(0.771 mL, 1.54 mmol, 3.00 equiv) at

−30 °C. After being stirred for 30 min at −30 °C, the mixture was

poured into brine, and the aqueous layer was extracted with EtOAc

three times. The combined organic layers were dried over Na₂SO₄, and

the solvent was removed under reduced pressure. The crude was

puriﬁed by ﬂash column chromatography (0−10%, MeOH/EtOAc) to

give 48 (131 mg, 72%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃):

δ 5.88 (1H, d,³J_H−F= 52.6 Hz), 4.33−4.25 (3H, m), 4.08−4.05 (1H, br

m), 3.80−3.77 (2H, br m), 3.36 (1H, d, J = 11.8 Hz), 3.22−3.11 (2H,

m), 2.93−2.86 (1H, m), 2.50 (1H, br s), 2.42−2.37 (1H, m), 2.27−1.97

(5H, m), 1.34 (3H, t, J = 7.0 Hz). MS-ESI (m/z): 353 [M + H]⁺.

Ethyl (2R)-2-(((2R,5R)-2-((R)-((1H-imidazole-4-yl)methyl)sulﬁnyl)-

7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (49).

To a solution of 27k (90.0 mg, 0.190 mmol) in CH₂Cl₂(1.4 mL)

was added TFA (1.4 mL) at 0 °C. After the mixture was stirred for 1 h at

room temperature, the solvent was removed under reduced pressure to

give 49 which was used for the next step without any puriﬁcation.

Methyl (2R)-2-(((2R,5R)-2-((R)-((2-aminothiazol-5-yl)methyl)-

sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroace-

tate (50). Compound 50 (41.4 mg, 66%) was prepared from 27l in a

similar manner to 48. The compound was used for the next step without

any puriﬁcation. MS-ESI (m/z): 393 [M + H]⁺.

Sodium (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-methylsulﬁnyl)-7-oxo-

1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (2). To a solution of

32 (50.0 g, 112 mmol) in THF/H₂O (v/v = 2/1, 915 mL) was added 1

N aq NaOH (112 mL, 112 mmol, 1.00 equiv) at 0 °C. After being

stirred for 20 min at 0 °C, the reaction was quenched by dry ice. The

organic solvent was removed under reduced pressure, and the resulting

solution was washed with EtOAc. The aqueous solution was applied

onto HP20SS resin and subjected to ODS column chromatography

(0−10%, MeCN/H₂O) to give 2 (26.5 g, 78%) as a white amorphous

solid. ¹H NMR (400 MHz, D₂O): δ 5.81 (1H, d, J = 58.5 Hz), 4.32 (1H,

t, J = 6.3 Hz), 4.24−4.22 (1H, br m), 3.42 (1H, d, J = 12.0 Hz), 3.30

(1H, d, J = 12.0 Hz), 2.77 (3H, s), 2.38−2.29 (1H, m), 2.23−2.12 (2H,

m), 2.07−1.99 (1H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ 171.9

(d,³J_C−F= 27.1 Hz), 171.4, 110.7 (d,²J_C−F= 241.4 Hz), 78.3, 62.6, 48.1,

37.7, 21.4, 18.5. ¹⁹F NMR (377 MHz, D₂O): δ −130.2 (d,³J_H−F= 59.9

Hz). HPLC: >99.9% (method-A). Anal. Calcd for C₉H₁₂FN₂O₅SNa-

(H₂O)_2.1: C, 31.79; H, 4.80; F, 5.59; N, 8.24; S, 9.43; Na, 6.76. found:

C, 31.81; H, 4.83; F, 5.40; N, 8.45; S, 9.28; Na, 6.64.

62.4, 48.1, 21.1, 19.5. ¹⁹F NMR (377 MHz, D₂O): δ −130.2 (d,³J_H−F

=

58.6 Hz). HPLC: 99.5% (method-A). Anal. Calcd for

C₁₀H₁₁FN₃O₅SNa(H₂O)_0.9: C, 34.97; H, 3.76; F, 5.53; N, 12.23; S,

9.33; Na, 6.69. found: C, 34.96; H, 3.71; F, 5.40; N, 12.32; S, 9.31; Na,

6.60.

Sodium (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-(2-hydroxyethyl)sulﬁnyl)-

7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (9). To a sol-

ution of 34 (518 mg, 0.877 mmol) were added anisole (0.383 mL, 3.51

mmol, 4.00 equiv) and 2 M solution of AlCl₃in MeNO₂(1.75 mL, 3.51

mmol, 4.00 equiv) at −30 °C. After being stirred for 30 min at −30 °C,

the mixture was poured into aq NaHCO₃, and the aqueous layer was

washed with EtOAc. The aqueous solution was applied onto HP20SS

resin and subjected to ODS column chromatography (0−10%, MeCN/

H₂O) to give 9 (221 mg, 76%) as a white amorphous solid. ¹H NMR

(400 MHz, D₂O): δ 5.82 (1H, d,³J_H−F= 58.6 Hz), 4.49 (1H, t, J = 6.1

Hz), 4.27−4.24 (1H, br m), 4.12−4.00 (2H, m), 3.47 (1H, d, J = 12.4

Hz), 3.37−3.30 (2H, m), 3.06 (1H, dt, J = 13.9, 4.2 Hz), 2.40−2.31

(1H, m), 2.26−2.15 (2H, m), 2.07−1.99 (1H, m). ¹³C{¹H} NMR (100

MHz, D₂O): δ 171.8 (d,³J_C−F= 27.1 Hz), 171.2, 110.6 (d,²J_C−F= 240.6

Hz), 77.1, 62.5, 57.4, 55.0, 48.1, 21.4, 18.8. ¹⁹F NMR (377 MHz, D₂O):

δ −130.2 (d,³J_H−F= 58.6 Hz). HPLC: 96.3% (method-C). Anal. Calcd

for C₁₀H₁₄FN₂O₆SNa(H₂O)_2.3: C, 32.14; H, 5.02; F, 5.08; N, 7.50; S,

8.58; Na, 6.15. found: C, 32.01; H, 4.77; F, 4.99; N, 7.69; S, 8.50; Na,

6.52

Sodium (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-(3-hydroxypropyl)-

sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (10).

Compound 10 (81.4 mg, 64%) was prepared from 48 in a similar

manner to 2. ¹H NMR (400 MHz, D₂O): δ 5.81 (1H, d,³J_H−F= 58.7

Hz), 4.42 (1H, t, J = 6.1 Hz), 4.26−4.23 (1H, br m), 3.75 (2H, t, J = 6.3

Hz), 3.46 (1H, d, J = 12.3 Hz), 3.32 (1H, d, J = 12.0 Hz), 3.19−3.12

(1H, m), 2.99−2.91 (1H, m), 2.38−2.29 (1H, m), 2.25−2.13 (2H, m),

2.06−1.99 (3H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ 171.8 (d,³J_C−F

= 27.1 Hz), 171.2, 110.7 (d,²J_C−F= 241.4 Hz), 76.8, 62.9, 62.5, 48.6,

48.1, 27.4, 21.4, 18.8. ¹⁹F NMR (377 MHz, D₂O): δ −130.2 (d, J = 59.9

Hz). HPLC: 98.3% (method-A). Anal. Calcd for C₁₁H₁₆FN₂O₆SNa-

(H₂O)_1.7: C, 35.05; H, 5.19; F, 5.04; N, 7.43; S, 8.51; Na, 6.10. found:

C, 35.29; H, 5.14; F, 4.69; N, 7.60; S, 8.08; Na, 5.83.

Sodium (2R)-2-(((2R,5R)-2-((R)-(2-(carbamoyloxy)ethyl)sulﬁnyl)-

7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (11).

Compound 11 (121 mg, 79%) was prepared from 41 in a similar

manner to 9. ¹H NMR (400 MHz, D₂O): δ 5.82 (1H, d,³J_H−F= 58.5

Hz), 4.58−4.46 (3H, m), 4.27−4.24 (1H, br m), 3.51−3.43 (2H, m),

3.33 (1H, d, J = 10.8 Hz), 3.20 (1H, ddd, J = 14.4, 6.0, 3.7 Hz), 2.40−

2.30 (1H, m), 2.26−2.15 (2H, m), 2.09−1.99 (1H, m). ¹³C{¹H} NMR

(100 MHz, D₂O): δ 171.8 (d,³J_C−F= 27.1 Hz), 171.1, 161.5, 110.6

(d,²J_C−F= 241.4 Hz), 77.0, 62.5, 60.8, 51.5, 48.0, 21.3, 18.9. ¹⁹F NMR

(377 MHz, D₂O): δ −130.2 (d,³J_H−F= 58.6 Hz). HPLC: >99.9%

(Method-A). Anal. Calcd for C₁₁H₁₅FN₃O₇SNa(H₂O)_2.0: C, 32.12; H,

4.66; F, 4.62; N, 10.22; S, 7.79; Na, 5.59. found: C, 32.07; H, 4.73; F,

4.52; N, 10.48; S, 7.67; Na, 5.62.

Sodium (2R)-2-ﬂuoro-2-(((2R,5R)-2-((S)-methylsulﬁnyl)-7-oxo-

1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (6). Compound 6

1

(261 mg, 73%) was prepared from 27b in a similar manner to 2. H

NMR (400 MHz, D₂O): δ 5.82 (1H, d,³J_H−F= 58.0 Hz), 5.70 (1H, dd, J

= 47.9, 9.8 Hz), 5.64 (1H, dd, J = 46.6, 9.8 Hz), 4.28−4.26 (1H, br m),

3.45 (1H, d, J = 12.3 Hz), 3.34 (1H, d, J = 12.3 Hz), 2.37−2.17 (3H, m),

2.10−2.04 (1H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ 171.8 (d,³J_C−F

= 27.1 Hz), 170.8, 110.7 (d,²J_C−F= 241.4 Hz), 94.6 (d,²J_C−F= 220.8

Hz), 72.6 (d,⁴J_C−F= 8.1 Hz), 62.4, 48.2, 21.3, 19.5. ¹⁹F NMR (377

MHz, D₂O): δ −130.2 (d,³J_H−F= 58.2 Hz), −228.4 (t,³J_H−F= 47.2 Hz).

HPLC: >99.9% (method-A). Anal. Calcd for C₉H₁₁F₂N₂O₅SNa-

(H₂O)_1.3: C, 31.46; H, 3.99; F, 11.06; N, 8.15; S, 9.33; Na, 6.69.

found: C, 31.76; H, 4.11; F, 10.88; N, 8.26; S, 8.99; Na, 6.46.

Sodium (2R)-2-(((2R,5R)-2-((R)-(2-amino-2-oxoethyl)sulﬁnyl)-7-

oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (7).

Compound 7 (208 mg, 64%) was prepared from 33 in a similar

manner to 2. ¹H NMR (400 MHz, D₂O): δ 5.82 (1H, d,³J_H−F= 58.9

Hz), 4.58 (1H, t, J = 6.6 Hz), 4.27−4.25 (1H, br m), 3.42 (1H, d, J =

12.4 Hz), 3.33 (1H, d, J = 11.6 Hz), 2.41−2.32 (1H, m), 2.26−2.16

(2H, m), 2.09−2.03 (1H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ

171.9 (d,³J_C−F= 27.1 Hz), 171.6, 171.0, 110.6 (d,²J_C−F= 241.4 Hz),

76.9, 62.5, 48.1, 21.2, 19.1. ¹⁹F NMR (377 MHz, D₂O): δ −130.2

Sodium (2R)-2-(((2R,5R)-2-((R)-((S)-2,3-dihydroxypropyl)sulﬁnyl)-

7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (12).

Compound 12 (27.9 mg, 43%) was prepared from 42 in a similar

manner to 2. ¹H NMR (400 MHz, D₂O): δ 5.81 (1H, d,³J_H−F= 58.7

Hz), 4.45 (1H, t, J = 6.1 Hz), 4.26−4.23 (1H, br m), 4.19−4.16 (1H,

m), 3.72 (1H, dd, J = 11.8, 4.0 Hz), 3.62 (1H, dd, J = 11.9, 5.9 Hz), 3.46

(1H, d, J = 12.0 Hz), 3.33 (1H, d, J = 11.8 Hz), 3.19 (1H, dd, J = 13.6,

10.8 Hz), 3.02 (1H, dd, J = 13.6, 2.3 Hz), 2.36−2.30 (1H, m), 2.25−

2.14 (2H, m), 2.07−2.02 (1H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ

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171.9 (d,³J_C−F= 27.1 Hz), 171.2, 110.7 (d,²J_C−F= 241.4 Hz), 77.3, 68.5,

67.7, 62.6, 56.2, 48.2, 21.4, 18.8. ¹⁹F NMR (377 MHz, D₂O): δ −130.1

(d,³J_H−F= 59.9 Hz). HPLC: >99.9% (method-A).

(1H, d, J = 14.1 Hz), 3.46 (1H, d, J = 12.3 Hz), 3.34 (1H, d, J = 12.0

Hz), 2.31−2.12 (3H, m), 2.08−1.99 (1H, m). ¹³C{¹H} NMR (100

MHz, D₂O): δ 173.7, 171.9 (d,³J_C−F= 27.1 Hz), 171.0, 140.7, 113.1,

110.7 (d,²J_C−F= 240.6 Hz), 76.3, 62.5, 52.6, 48.4, 21.5, 18.8. ¹⁹F NMR

(377 MHz, D₂O): δ −130.1 (d,³J_H−F= 58.6 Hz). HPLC: 97.4%

(method-A).

Sodium (2R)-2-(((2R,5R)-2-((R)-(3-amino-2-(hydroxyimino)-3-

oxopropyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-

ﬂuoroacetate (13). Compound 13 (121 mg, 79%) was prepared from

43 in a similar manner to 9. The oxime was obtained as a single

Sodium 2,2-diﬂuoro-2-(((2R,5R)-2-((R)-methylsulﬁnyl)-7-oxo-1,6-

diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (1). Compound 1 (173

mg, 68%) was prepared from 70³³in a similar manner to 2. ¹H NMR

(400 MHz, D₂O): δ 4.38 (1H, t, J = 6.0 Hz), 4.24−4.21 (1H, br m),

3.48 (1H, d, J = 12.3 Hz), 3.39 (1H, d, J = 12.0 Hz), 2.78 (3H, s), 2.38−

2.31 (1H, m), 2.24−2.14 (2H, m), 2.08−2.05 (1H, m). ¹³C{1H} NMR

1

geometrical isomer, but the geometry (E/Z) is unknown. H NMR

(400 MHz, D₂O): δ 5.82 (1H, d, J = 58.6 Hz), 4.53 (1H, t, J = 6.3 Hz),

4.31 (2H, s), 4.26−4.23 (1H, br m), 3.42 (1H, d, J = 12.4 Hz), 3.33−

3.30 (1H, m), 2.39−2.32 (1H, m), 2.26−2.21 (2H, m), 2.05−2.01 (1H,

m). ¹³C{¹H} NMR (100 MHz, D₂O): δ 171.9 (d,³J_C−F= 27.1 Hz),

170.9, 169.2, 146.9, 110.7 (d,²J_C−F= 240.6 Hz), 78.2, 62.4, 48.3, 46.0,

21.4, 18.9. ¹⁹F NMR (377 MHz, D₂O): δ −130.2 (d,³J_H−F= 58.6 Hz).

HPLC: 96.1% (method-D).

(2R)-2-(((2R,5R)-2-((R)-(3-Aminopropyl)sulﬁnyl)-7-oxo-1,6-

diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetic acid-2,2,2-tri-

ﬂuoroacetic acid (1/1) (14). To a solution of 37 (286 mg, 0.497

mmol) in CH₂Cl₂(3 mL) was added TFA (3 mL) at 0 °C. After the

mixture was stirred for 15 min at 0 °C, the solvent was removed under

reduced pressure. The crude was triturated with ⁱPrOAc to give 14 (107

mg, 51%). ¹H NMR (400 MHz, D₂O): δ 5.81 (1H, d, J = 58.4 Hz), 4.52

(1H, t, J = 6.7 Hz), 4.26−4.24 (1H, br m), 3.59−3.47 (3H, m), 3.41

(1H, d, J = 12.1 Hz), 3.34−3.25 (2H, m), 2.36−2.30 (1H, m), 2.24−

2.19 (2H, m), 2.07−2.01 (1H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ

171.3, 170.9 (d,³J_C−F= 24.9 Hz), 165.9 (q,³J_C−F= 35.5 Hz) (TFA),

119.3 (q,²J_C−F= 291.7 Hz) (TFA), 110.0 (d,²J_C−F= 242.1 Hz), 77.3,

63.0, 48.5, 47.9, 36.8, 21.2, 19.3. ¹⁹F NMR (377 MHz, D₂O): δ −75.6

(s) (TFA), −132.5 (d,³J_H−F= 55.9 Hz). HPLC: 96.7% (method-A)

Anal. Calcd for C₁₀H₁₆FN₃O₅S(TFA)_0.7(ⁱPrOAc)_0.1(H₂O)_0.6: C, 34.85;

H, 4.64; F, 14.36; N, 10.25; S, 7.82. found: C, 35.04; H, 4.58; F, 13.90;

N, 10.29; S, 7.67.

(100 MHz, D₂O): δ 172.0, 166.1 (t,³J_C−F= 31.9 Hz), 120.0 (t,²J_C−F

=

281.0 Hz), 78.4, 63.5, 47.7, 37.6, 21.3, 18.4. ¹⁹F NMR (377 MHz,

D₂O): δ −83.1 (d,³J_F−F= 136.2 Hz), −83.7 (d,³J_F−F= 134.9 Hz).

HPLC: 88.1% (method-A). Anal. Calcd for C₉H₁₁F₂N₂O₅SNa-

(H₂O)_1.3: C, 31.46; H, 3.99; F, 11.06; N, 8.15; S, 9.33; Na, 6.69.

found: C, 31.69; H, 4.04; F, 11.20; N, 8.33; S, 9.03; Na, 6.71.

Sodium 2,2-diﬂuoro-2-(((2R,5R)-7-oxo-2-sulfamoyl-1,6-

diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (3). Compound 3 (706

mg, 88%) was prepared from 87³³in a similar manner to 2. ¹H NMR

(400 MHz, D₂O): δ 4.65 (1H, t, J = 8.3 Hz), 4.25−4.22 (1H, br m),

3.71 (1H, d, J = 12.4 Hz), 3.40 (1H, dt, J = 12.4, 1.4 Hz), 2.33−2.24

(2H, m), 2.16−2.02 (2H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ

172.7, 166.2 (t,³J_C−F= 31.9 Hz), 120.0 (t,²J_C−F= 280.6 Hz), 77.5, 64.0,

46.0, 20.7, 20.5. ¹⁹F NMR (377 MHz, D₂O): δ −83.1 (d,³J_F−F= 136.2

Hz), −83.8 (d,³J_F−F= 134.9 Hz). HPLC: >99.9% (method-A). Anal.

Calcd for C₈H₁₀F₂N₃O₆SNa(H₂O)_1.3: C, 26.64; H, 3.52; F, 10.54; N,

11.65; S, 8.89; Na, 6.37. found: C, 26.92; H, 3.27; F, 10.28; N, 11.88; S,

8.90; Na, 6.63.

Sodium (2R)-2-ﬂuoro-2-(((2R,5R)-7-oxo-2-sulfamoyl-1,6-

diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (4). Compound 4 (121

mg, 82%) was prepared from 89³³in a similar manner to 2. ¹H NMR

(400 MHz, D₂O): δ 5.80 (1H, d,³J_H−F= 58.5 Hz), 4.60 (1H, t, J = 8.2

Hz), 4.25 (1H, t, J = 3.8 Hz), 3.67 (1H, d, J = 12.3 Hz), 3.33 (1H, dt, J =

12.3, 1.5 Hz), 2.34−2.19 (2H, m), 2.17−2.01 (2H, m). ¹³C{¹H} NMR

Sodium (2R)-2-(((2R,5R)-2-((R)-(2-acetamidoethyl)sulﬁnyl)-7-

oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroacetate (15).

Compound 15 (96.4 mg, 66%) was prepared from 44 in a similar

manner to 2. ¹H NMR (400 MHz, D₂O): δ 5.81 (1H, d,³J_H−F= 58.6

Hz), 4.45 (1H, t, J = 6.3 Hz), 4.26−4.23 (1H, br m), 3.68 (2H, t, J = 6.2

Hz), 3.43 (1H, d, J = 12.3 Hz), 3.35−3.28 (2H, m), 3.05 (1H, dt, J =

13.7, 5.5 Hz), 2.35−2.28 (1H, m), 2.24−2.12 (2H, m), 2.06−1.97 (4H,

m). ¹³C{¹H} NMR (100 MHz, D₂O): δ 177.3, 171.8 (d,³J_C−F= 27.1

Hz), 171.1, 110.7 (d,²J_C−F= 241.4 Hz), 77.1, 62.5, 52.1, 48.0, 36.1, 24.7,

21.3, 18.9. ¹⁹F NMR (377 MHz, D₂O): δ −130.0 (d,³J_H−F= 58.6 Hz).

HPLC: 99.8% (method-A). Anal. Calcd for C₁₂H₁₇FN₃O₆SNa-

(H₂O)_1.8: C, 35.52; H, 5.12; F, 4.68; N, 10.36; S, 7.90; Na, 5.67.

found: C, 35.49; H, 5.12; F, 4.77; N, 10.51; S, 7.69; Na, 5.39.

Sodium (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-((5-methyl-1,3,4-oxadia-

zol-2-yl)methyl)sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)-

oxy)acetate (16). Compound 16 (21.5 mg, 72%) was prepared from 39

(100 MHz, D₂O): δ 172.1, 171.9 (d,³J_C−F= 27.1 Hz), 110.6 (d,²J_C−F

=

240.6 Hz), 77.2, 63.1, 46.2, 20.6, 20.5. ¹⁹F NMR (377 MHz, D₂O): δ

−130.1 (d,³J_H−F= 58.6 Hz). HPLC: >99.9% (method-A). Anal. Calcd

for C₈H₁₁FN₃O₆SNa(H₂O)_1.0: C, 28.49; H, 3.89; F, 5.63; N, 12.46; S,

9.51; Na, 6.82. found: C, 28.58; H, 4.12; F, 5.69; N, 12.52; S, 9.53; Na,

6.41.

Sodium (2R)-2-ﬂuoro-2-(((2R,5R)-2-((S)-methylsulﬁnyl)-7-oxo-

1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (5). Compound 5

(63.4 mg, 64%) was prepared from 71³³in a similar manner to 2. ¹H

NMR (400 MHz, D₂O): δ 5.80 (1H, d,³J_H−F= 59.0 Hz), 4.27−4.23

(2H, m), 3.53 (1H, d, J = 12.3 Hz), 3.37 (1H, dd, J = 12.0, 2.5 Hz), 2.75

(3H, s), 2.22−2.12 (3H, m), 2.07−1.98 (1H, m). ¹³C{¹H} NMR (100

MHz, D₂O): δ 173.0, 172.0 (d,³J_C−F= 27.1 Hz), 110.7 (d,²J_C−F= 240.6

Hz), 77.7, 63.1, 47.4, 36.3, 21.3, 21.2. ¹⁹F NMR (377 MHz, D₂O): δ

−130.1 (d,³J_H−F= 58.6 Hz). HPLC: >99.9% (method-A). Anal. Calcd

for C₉H₁₂FN₂O₅SNa(H₂O)_2.1: C, 31.79; H, 4.80; F, 5.59; N, 8.24; S,

9.43; Na, 6.76. found: C, 32.03; H, 4.86; F, 5.53; N, 8.51; S, 8.84; Na,

6.27.

1

in a similar manner to 2. H NMR (400 MHz, D₂O): δ 5.82 (1H,

d,³J_H−F= 58.6 Hz), 4.63−4.61 (2H, m), 4.28−4.25 (1H, br m), 3.43

(1H, d, J = 12.3 Hz), 3.33 (1H, d, J = 11.8 Hz), 2.59 (3H, s), 2.38−2.16

(3H, m), 2.08−2.05 (1H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ

171.8 (d,³J_C−F= 26.4 Hz), 170.7, 170.2, 162.0, 110.7 (d,²J_C−F= 240.6

Hz), 76.8, 62.3, 48.2, 21.1, 19.2, 13.0. ¹⁹F NMR (377 MHz, D₂O): δ

−130.0 (d,³J_H−F= 58.6 Hz). HPLC: 99.1% (method-A).

Ethyl (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-methylsulﬁnyl)-7-oxo-1,6-

diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (19). To a solution of 32

(5.00 g, 11.2 mmol) in EtOH/CH₂Cl₂(v/v = 1/2, 150 mL) was added

K₂CO₃(1.55g, 11.2 mmol, 1.00 equiv) at 0 °C. After the mixture was

stirred for 3 h at 0 °C, 10% aq citric acid and brine were added, and the

layers were separated. The aqueous layer was extracted with EtOAc

twice, and the combined organic layers were dried over Na₂SO₄. After

the solvent was removed under reduced pressure, the crude was puriﬁed

by ﬂash column chromatography (0−10%, EtOH/EtOAc) to give 19

Sodium (2R)-2-(((2R,5R)-2-((R)-((1H-imidazole-4-yl)methyl)-

sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroace-

tate (17). Compound 17 (39.0 mg, 50%) was prepared from 49 in a

similar manner to 2. ¹H NMR (400 MHz, D₂O): δ 7.91 (1H, s), 7.33

(1H, s), 5.83 (1H, d,³J_H−F= 58.7 Hz), 4.44−4.37 (2H, m), 4.26−4.23

(2H, m), 3.46 (1H, d, J = 12.3 Hz), 3.35 (1H, d, J = 12.0 Hz), 2.29−2.09

(3H, m), 2.06−1.99 (1H, m). ¹³C{¹H} NMR (100 MHz, D₂O): δ

171.9 (d,³J_C−F= 26.4 Hz), 171.0, 139.7, 128.7, 122.4, 110.7 (d,²J_C−F

=

241.4 Hz), 75.7, 62.5, 49.1, 48.4, 21.5, 18.8. ¹⁹F NMR (377 MHz,

D₂O): δ −130.1 (d,³J_H−F= 58.6 Hz). HPLC: 94.7% (method-A).

Sodium (2R)-2-(((2R,5R)-2-((R)-((2-aminothiazol-5-yl)methyl)-

sulﬁnyl)-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)-2-ﬂuoroace-

tate (18). Compound 18 (27.5 mg, 65%) was prepared from 50 in a

similar manner to 2. Compound 18 contains a stereoisomer of sulfoxide

1

(2.67 g, 77%) as a white solid. H NMR (400 MHz, CDCl₃): δ 5.88

(1H, d,³J_H−F= 52.2 Hz), 4.38−4.26 (2H, m), 4.10 (1H, t, J = 6.1 Hz),

4.07−4.05 (1H, br m), 3.36 (1H, d, J = 12.0 Hz), 3.20 (1H, d, J = 12.3

Hz), 2.68 (3H, s), 2.48−2.39 (1H, m), 2.29−2.11 (2H, m), 2.03−1.94

(1H, m), 1.34 (3H, t, J = 7.2 Hz). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ

167.1, 162.8 (d,³J_C−F= 35.9 Hz), 105.2 (d,²J_C−F= 239.2 Hz), 76.8, 62.9,

60.5, 45.1, 37.1, 19.3, 15.9, 14.0. ¹⁹F NMR (377 MHz, CDCl₃): δ

1

(dr = 3/1). H NMR (400 MHz, D₂O): δ 6.74 (1H, s), 5.82 (1H,

d,³J_H−F= 58.7 Hz), 4.48 (1H, t, J = 5.9 Hz), 4.31−4.25 (2H, m), 4.08

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−136.7 (d,³J_H−F= 51.8 Hz). HPLC: 99.9% (method-B). HRMS (ESI)

m/z: [M + H]⁺calcd for C₁₁H₁₈O₅N₂FS, 309.0915; found, 309.0911.

Isopropyl (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-methylsulﬁnyl)-7-oxo-

1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (20). Compound 20

(159 mg, 67%) was prepared from 68³³and (R)-isopropyl

plasma samples. Plasma samples were stored at −20 °C until LC/MS/

MS analyses.

LC/MS/MS Determination of Test Compounds. Aliquots (5−

40 μL) of each samples were transferred to a 96-well plate, mixed with

250 or 500 μL of methanol, and centrifuged at 1580g for 10 min at 4 °C.

A portion of supernatant was injected to a Shimadzu Nexera HPLC

system interfaced with an API-5000 mass spectrometer (AB Sciex,

Framingham, MA). Test compound plasma concentrations were

determined by LC/MS/MS with electrospray ionization in the positive

ion mode using an external standard method.

Pharmacokinetic Analysis. The elimination half-life (T_1/2) was

estimated as ln 2/k_el, where k_elis the elimination rate constant derived

from the slope of the log concentration versus time proﬁle. The area

under the concentration−time curve from 0 h to the last time point

(AUC) was calculated by linear trapezoidal approximation. Total

clearance (CL_tot) was estimated as dose/AUC.

Serum Stability. Each experiment was performed in duplicate.

Pooled human, rat, mouse, dog, and monkey serum were incubated

with test compound for 30 min at 37 °C, respectively. The

concentration of test compounds was 5 μmol/L. After incubation, an

aliquot (20 μL) of each sample was transferred to a 96-well plate, mixed

with 120 μL of methanol, and centrifuged at 1580g for 10 min at 4 °C.

The supernatant was analyzed by LC/MS/MS, and the plasma stability

(%) of test compounds was calculated.

Serum Protein Binding. Serum PB was evaluated by the

ultraﬁltration method. Test compounds were spiked in the serum

collected from fasted mice, rats, dogs, and monkeys at 10 μg/mL. After

incubation for 5 min at 37 °C, serum samples were transferred to

Centrifree (Merck KGaA, Darmstadt, Germany) and centrifuged.

Initial serum samples and ﬁltrates were analyzed by LC/MS/MS.

In Vivo Eﬃcacy Studies. In vivo eﬃcacy of CTB and prodrug 21

was evaluated in the murine urinary tract infection model against K.

pneumoniae strains SR08667. Brieﬂy, 5 week aged ICR female mice (N

= 5 per arm, CLEA Japan, Inc.) were used in this study. This animal

study was approved by the Institutional Animal Care and Use

Committee of Shionogi & Co., Ltd. K. pneumoniae SR08667 suspension

was diluted with MHB to be 1.2 × 10⁷cfu/mL. A 0.1 mL of the diluted

bacterial suspension was transurethrally inoculated (1.2 × 10⁶CFU/

mouse), and the urethra was tightened for 4 h for the infection. The

CTB and 21 were orally administered four times at the time points of 4,

8, 28, and 32 h after the infection. The dose of the agents was 30 mg/kg

of CTB alone, 10 mg/kg of CTB combined with 3, 10, or 30 mg/kg of

21, or 30 mg/kg of CTB combined with 30 mg/kg of 21. Both of CTB

and 21 were formulated in 0.5% methylcellulose. At 4- or 48-h post-

infection, animals were euthanized, and kidneys (two samples/mouse)

were collected and homogenized in 2.7 mL of MHB. The tissue

homogenates were serially diluted, and the viable cells in the samples

were counted using modiﬁed Drigalski agar.

bromoﬂuoroacetate 90³³in a similar manner to 32. H NMR (400

1

MHz, CDCl₃): δ 5.85 (1H, d,³J_H−F= 52.5 Hz), 5.19−5.10 (1H, sep, J =

6.3 Hz), 4.12−4.07 (2H, m), 3.37 (1H, d, J = 12.0 Hz), 3.19 (1H, d, J =

12.0 Hz), 2.68 (3H, s), 2.48−2.39 (1H, m), 2.28−2.11 (2H, m), 2.02−

1.94 (1H, m), 1.33 (3H, d, J = 6.3 Hz), 1.30 (3H, d, J = 6.3 Hz).

¹³C{¹H} NMR (100 MHz, CDCl₃): δ 167.0, 162.3 (d,³J_C−F= 35.9 Hz),

105.3 (d,²J_C−F= 237.7 Hz), 76.8, 71.2, 60.6, 45.1, 37.1, 21.6, 21.4, 19.3,

15.8. ¹⁹F NMR (377 MHz, CDCl₃): δ −136.5 (d,³J_H−F= 51.8 Hz).

HPLC: >99.9% (method-B). HRMS (ESI) m/z: [M + H]⁺calcd for

C₁₂H₂₀O₅N₂FS, 323.1071; found, 323.1067.

Cyclohexyl (2R)-2-ﬂuoro-2-(((2R,5R)-2-((R)-methylsulﬁnyl)-7-oxo-

1,6-diazabicyclo[3.2.1]octan-6-yl)oxy)acetate (21). Compound 21

(23.5 g, 83%) was prepared from 68³³and (R)-cyclohexyl

bromoﬂuoroacetate 91³³in a similar manner to 32. H NMR (400

1

MHz, CDCl₃): δ 5.87 (1H, d,³J_H−F= 52.2 Hz), 4.93−4.87 (1H, m),

4.12−4.07 (2H, m), 3.36 (1H, d, J = 12.0 Hz), 3.19 (1H, d, J = 12.3 Hz),

2.68 (3H, s), 2.47−2.39 (1H, m), 2.27−2.11 (2H, m), 2.02−1.89 (3H,

m), 1.78−1.21 (8H, m). ¹³C{¹H} NMR (100 MHz, CDCl₃): δ 167.0,

162.3 (d,³J_C−F= 35.9 Hz), 105.3 (d,²J_C−F= 238.4 Hz), 76.8, 76.1, 60.6,

45.1, 37.1, 31.4, 31.2, 25.1, 23.7, 23.7, 19.3, 15.8. ¹⁹F NMR (377 MHz,

CDCl₃): δ −136.2 (d,³J_H−F= 53.1 Hz). HPLC: >99.9% (method-B).

HRMS (ESI) m/z: [M + H]⁺calcd for C₁₅H₂₄O₅N₂FS, 363.1384;

found, 363.1379.

β-Lactamase Inhibition Assay. The inhibition of β-lactamases

was determined by the hydrolysis of nitroceﬁn. The hydrolysis of

nitroceﬁn was recorded as absorbance at 492 nm using Envision2013

(PerkinElmer). The concentration of β-lactamase enzyme (KPC-2,

CTX-M-15, CMY-2, and OXA-48) used was determined by the

measurement of hydrolysis of nitroceﬁn (50 μg/mL), where the change

of the absorbance was approximately 0.2 from the negative control (no

enzyme). The enzyme was added to the sample solution including

nitroceﬁn (ﬁnal concentration, 50 μg/mL) and an inhibitor at a volume

ratio of 1:3 (ﬁnal, 80 μL). The absorbance was measured at 35 °C after

20 min incubation. The concentration of the inhibitor, needed to

reduce the hydrolysis of the substrate by 50% (IC₅₀), was calculated

using XLﬁt (IDBS, UK).

Antibiotic Susceptibility Testing. Strains with a “SR” designation

are from previous surveillance which are kept in the Shionogi collection

of clinical isolates and have been characterized by multiplex PCR and

sequencing analysis including whole genome sequencing. All strains

with an “ATCC” designation are purchased from American Type

Culture Collection (ATCC). The MICs were determined using the

broth microdilution method in accordance with Clinical Laboratory

Standards Institute (CLSI) guidelines.³⁴Brieﬂy, twofold serial dilution

of test compounds was prepared and transferred to a 96-well plate

including cation-adjusted Mueller Hinton broth (CAMHB). The

bacterial suspension for inoculum were prepared based on the optical

density of 625 nm to be approximately 5 × 10⁴cfu/well as the ﬁnal

inoculum size. The 96-well plates were incubated at 35 °C for 16−20 h.

The MIC endpoint was deﬁned as the lowest concentration of the

compound that inhibited bacterial growth as detected by the unaided

eye. The MPC values were used to deﬁne BLI. The MPC₁was deﬁned

as the concentration of BLI that was required to reduce the CTB MIC

to ≤1 μg/mL. The MPC₁values were determined using the broth

microdilution method. Brieﬂy, twofold serial dilution of test BLIs was

prepared and transferred to a 96-well plate including CAMHB with 1

μg/mL of CTB. The bacterial suspension for inoculum was prepared

based on the optical density of 625 nm to be approximately 5 × 10⁴cfu/

well as the ﬁnal inoculum size. The 96-well plates were incubated at 35

°C for 16 to 20 h. The MPC₁endpoint was deﬁned as the lowest

concentration of the compound that inhibited bacterial growth as

detected by the unaided eye.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00799.

Molecular formula strings and microbiological data

(CSV)

Typical reaction setup for light-promoted reaction;

procedure for synthesis of all compounds; supercritical

ﬂuid chromatography condition and chromatogram for

isolation of sulfoxide epimer 71; comparison of ¹H NMR

1

of sulfoxide 27a with its diastereomer 92; H and ¹³C

Pharmacokinetic Studies. PK studies were performed in mice,

rats, dogs, and monkeys. Blood samples were collected after intravenous

and oral administration at designated time and centrifuged to obtain the

NMR spectra of 2 and 19−21; and HPLC charts of 2 and

19−21 (PDF)

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droxysuccinimide; DABCO, 1,4-diazabicyclo[2.2.2]octane;

DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMF, N,N-dime-

thylformamide; TBAF, tetra-n-butylammonium ﬂuoride; TFA,

triﬂuoroacetic acid; THF, tetrahydrofuran; DMAP, 4-dimethy-

laminopyridine; PBP, penicillin-binding protein

AUTHOR INFORMATION

Corresponding Author

Motohiro Fujiu − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan; orcid.org/0000-

0002-5976-7041; Email: motohiro.fujiu@shionogi.co.jp

■

Authors

REFERENCES

■

Katsuki Yokoo − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Jun Sato − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan; orcid.org/0000-

0002-0225-9476

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25Feb-ET_NM_WHO.pdf?ua=1 (accessed Oct 16, 2020).

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(2) González-Bello, C.; Rodríguez, D.; Pernas, M.; Rodríguez, A.;

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Toyonaka-shi, Osaka 561-0825, Japan

Kazuo Komano − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Hiroki Kusano − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Soichiro Sato − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Toshiaki Aoki − Shionogi CMC Research Innovation Center,

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Ryosuke Watari − Shionogi Pharmaceutical Research Center,

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Tomoyuki Kawachi − Shionogi Pharmaceutical Research

Center, Toyonaka-shi, Osaka 561-0825, Japan

Yuya Hirakawa − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Daiki Nagamatsu − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Emi Kashiwagi − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Hideki Maki − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Kenji Yamawaki − Shionogi Pharmaceutical Research Center,

Toyonaka-shi, Osaka 561-0825, Japan

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00799

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We appreciate the management for this research by Dr. Shuji

Yonezawa. The evaluations of compounds were supported by

Shuhei Matsumoto. We thank Yumiko Takagi for the element

analysis data.

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ABBREVIATIONS USED

■

ESBL, extended-spectrum β-lactamase; DBO, diazabicyclooc-

tane; CTB, ceftibuten; CRE, carbapenem-resistant Enter-

obacteriaceae; BLI, β-lactamase inhibitor; AVI, avibactam;

CAZ, ceftazidime; MIC, minimum inhibitory concentration;

MPC, minimum potentiating concentration; NOAEL, no

observed adverse eﬀect level; dr, diastereomeric ratio; CSI,

chlorosulfonyl isocyanate; TASF, tris(dimethylamino)-

sulfonium diﬂuorotrimethylsilicate; Bzh, benzhydryl; EDC, 1-

ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOSu, N-hy-

(15) Durand-Réville, T. F.; Comita-Prevoir, J.; Zhang, J.; Wu, X.; May-

Dracka, T. L.; Romero, J. A. C.; Wu, F.; Chen, A.; Shapiro, A. B.; Carter,

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J. Med. Chem. 2021, 64, 9496−9512

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J. Med. Chem. 2021, 64, 9496−9512

Article Doi

Discovery of 2-Sulfinyl-Diazabicyclooctane Derivatives, Potential Oral β-Lactamase Inhibitors for Infections Caused by Serine β-Lactamase-Producing Enterobacterales

DOI: 10.1021/acs.jmedchem.1c00799

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