Use of Lipase Catalytic Resolution in the Preparation of Ethyl (2 S,5 R)-5-((Benzyloxy)amino)piperidine-2-carboxylate, a Key Intermediate of the β-Lactamase Inhibitor Avibactam

Article abstract of DOI:10.1021/acs.oprd.8b00173

Here we describe an efficient and cost-effective chemoenzymatic synthesis of the β-lactamase inhibitor avibactam starting from commercially available ethyl 5-hydroxypicolinate hydrochloride. Avibactam was synthesized in 10 steps with an overall yield of 23.9%. The synthetic route features a novel lipase-catalyzed resolution step during the preparation of (2S,5S)-ethyl 5-hydroxypiperidine-2-carboxylate, a valuable precursor of the key intermediate ethyl (2S,5R)-5-((benzyloxy)amino)piperidine-2-carboxylate. Our synthetic route was used to produce 400 g of avibactam sodium salt.

Full text of DOI:10.1021/acs.oprd.8b00173

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Full Paper
Use of Lipase Catalytic Resolution in the Preparation of
ethyl (2S,5R)-5-((benzyloxy)amino) piperidine-2-carboxylate,
a Key Intermediate of the #-lactamase Inhibitor Avibactam
tao wang, Liang-dong Du, Ding-jian Wan, Xiang Li, Xinzhi Chen, and Guofeng Wu
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00173 • Publication Date (Web): 05 Nov 2018
Downloaded from http://pubs.acs.org on November 6, 2018
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Use of Lipase Catalytic Resolution in the Preparation of
ethyl (2S,5R)-5-((benzyloxy)amino) piperidine-2-carboxylate,
a Key Intermediate of the -lactamase Inhibitor Avibactam
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Tao Wanga,c, Liang-Dong Du b, Ding-jian Wan b, Xiang Li b, Xin-Zhi Chen c*,
Guo-Feng Wu a*
a Research &Development Center, Zhejiang Medicine CO., Ltd, 59 East Huangcheng Road,
Xinchang, Zhejiang, 312500, P. R. China
b Shanghai Laiyi Center for Biopharmaceuticals R&D, 5B, Building 8 200 Niudun Road
Pudong District, Shanghai, 201203, P. R. China
c Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang
University, 38 Zhejiang University Road, Xihu District, Hangzhou, 310007, P. R. China
*E-mail:xcwuyuanzhang@163.com
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For Table of Contents Only
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HO
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N
CO2Et
HO
H
(Boc)2O, TEA
THF
1) Rh/C , H2
HCl
single enantiomer
dr > 99:1
N
CO2Et
2) Lipozyme CALB
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+
lipase catalytic resolution
HO
HO
N
CO2H
H
O
N
CO2Et
(S)
Boc
OBn
HN
H2N
(R)
N
+
N
OSO3Na
O
N
CO2Et
H
(R)
H
N
HO
Avibactam
The total yield : 23.9%
(R)
CO2
N
Boc
water-soluble
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ABSTRACT
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Here we describe an efficient and cost-effective chemoenzymatic synthesis of the
-lactamase inhibitor avibactam starting from commercially available
ethyl-5-hydroxypicolinatehydrochloride. Avibactam was synthesized in 10 steps with
an overall yield of 23.9%. The synthetic route features a novel lipase-catalyzed
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resolution
step
during
the
preparation
of
(2S,5S)-ethyl
5-hydroxypiperidine-2-carboxylate, a valuable precursor of the key intermediateethyl
(2S,5R)-5-((benzyloxy)amino) piperidine-2-carboxylate. Oursynthetic route was used
to produce 400 grams of avibactam sodium salt.
KEYWORDS
avibactam,lipozyme CALB,lipase-catalyzed resolution, chiral isomers
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INTRODUCTION
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The production of β-lactamases by pathogenic bacteria is a common mechanism of
intrinsic and acquired immunity to β-lactam antibiotics. β-Lactamase inhibitors can
bind to β-lactamases and inactivate them, which can significantly improve the
efficacy of β-lactam antibiotics in the treatment of penicillin- and penicillin
derivative-resistant bacterial infections.
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Avibactam (1, Figure 1), sodium (2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo
[3.2.1]octan-6-yl sulfonate, is a novel non-β-lactam β-lactamase inhibitor that
contains a diazabicyclo[3.2.1]octane (DBO) heterocyclic core structure 1–4. The
clinical use of avibactam in combination with ceftazidime, a cephalosporin antibiotic,
was approved by the European Medicines Agency (EMA) in 2015 (sold as Zavicefta)
and the Food and Drug Administration (FDA) in 2016 (sold as Avycaz).
Ceftazidime/avibactam has been used for the successful treatment of multidrug
Gram-negative bacterial infections including complicated intra-abdominal infections
(CIAI), complicated urinary tract infections (CUTI), and hospital acquired pneumonia
(HAP) 5,6. Furthermore, compared with the other three currently available β-lactamase
inhibitors, clavulanic acid, sulbactam, and tazobactam, avibactam is more potent and
has a broader spectrum of activity. Avibactam is active against class Aβ-lactamases
including Klebsiella pneumoniaecarbapenemases (KPCs) and extended-spectrum
β-lactamases(ESBLs),
class
C
β-lactamases,
and
some
class
D
β-lactamases1,7,8.Additionally, avibactam acts as a reversible covalent inhibitor, which
is a unique mechanism of inhibition among β-lactamase inhibitors 2,9
.
Figure 1 Structure of Avibactam (Sodium Salt) (1).
O
S
H2N
N
R
N
O
O
O
O- Na+
S
O
1
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In recent years, considerable research has focused on the synthesis of
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avibactam10–22(Scheme 1);and in particular, on the preparation of a key intermediate,
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10–22
ethyl (2S,5R)-5-((benzyloxy)amino) piperidine-2-carboxylate
and its
9
derivatives(Scheme 1). In the early stages of β-lactamase inhibitor research,
Sanofi-Aventis developed route A for the synthesis of avibactam 13, 15. In this process,
piperidine derivatives with two chiral centers were used as starting materials to
synthesizeavibactam via inversion of configuration. Miller and colleagues also
prepared avibactam analogue using an approach based on route A19.However, the raw
materials for these synthetic routes are expensive and the processes require the use of
several environmentally undesirable reagents and solvents.
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Later, route B was developed to synthesizeavibactam from L-glutamate acid or
L-pyroglutamic acid 14. In this route, ethyl (2S,5R)-5-((benzyloxy)amino)
piperidine-2-carboxylate was constructed via multiple synthetic steps including
ring-opening, ring-closing,reduction, and deprotection. Recently, AstraZeneca and
Forest Laboratories optimized this process using commercially available Boc-benzyl
glutamate as a starting material (achieving a 55 %overall yield) 22.
The final established route to synthesize the ethyl (2S,5R)-5-((benzyloxy)amino)
piperidine-2-carboxylateanalogue is based on the olefin metathesis reaction, which is
20
promoted by transition metal-based catalysts
(Route C).However, there are
disadvantages to this route, including substrate instability that leads to a lower yield,
the requirement for chiral ligand participation, and lower stereoselectivity. The
limitations of the current routes result in difficulties implementing them on an
industrial scale.
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Scheme 1. Reported Synthetic Routes to the Avibactam Key Intermediate
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6
7
8
Route A
OBn
R
OH
R
NH
OH
R
R
R
S
O
TFA anhydride
O
O
N
N
N
H
H
O
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O
O
O
CF3
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(2R,5S)-vinyl 5-((benzyloxy)amino)
piperidine-2-carboxylate
Route B
O
S
COOH
NH2
S
COOEt
COOEt
N2C
N
HOOC
HN
Boc
O
H
OBn
OBn
N
R
HN
S
S
N
COOEt
COOEt
N
H
H
(2S,5R)-ethyl 5-((benzyloxy)amino)
piperidine-2-carboxylate
Route C
S
TBSO
TBSO
Boc
Hoveyda-Grubbs Catalyst
N
O
N
Boc
O
O
S
TBSO
R
HN
NH
OBn
O-benzyl-N-((3R,6S)-6-(((tert-butyldimethylsilyl)oxy)
methyl)piperidin-3-yl)hydroxylamine
Here, we report the use oflipase catalyticresolution as a central step to construct ethyl
(2S,5R)-5-((benzyloxy)amino) piperidine-2-carboxylate, the key intermediate of
avibactam (Scheme 2).
Scheme 2.Use of Lipase Catalytic Resolution to Construct the Avibactam Key
Intermediate
HO
N
CO2Et
H
HO
1) Rh/C , H2
HCl
CO2Et 2) lipase catalytic resolution
4
N
+
2
HO
N
CO2H
H
4'
RESULTS AND DISCUSSION
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In this study, we developed an optimized approach to synthesize avibactam based on
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12-16
previously published protocols
were made: first, the
(Scheme 3, route D). Two fundamental changes
commercially available and affordable
9
ethyl-5-hydroxypicolinatehydrochloridewas used as the starting material, which
avoids the Complex construction process of piperidine ringused in previously reported
routes; second, a novel lipase-catalyzed resolution step was used to synthesize
(2S,5S)-ethyl 5-hydroxypiperidine-2-carboxylate (4), the valuable precursor of the key
intermediate ethyl (2S,5R)-5-((benzyloxy)amino) piperidine-2-carboxylate (7).
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Scheme 3. Route D: Our Approach to the Synthesis of Avibactam
Route D
HO
HO
HO
Lipozyme CALB
Rh/C H2
EtOH
HCl
HCl
Phosphate solution
N
CO2Et
N
H
CO2Et
N
CO2Et
H
2
3
4
O
O
OBn
HN
H2N
NH3,MeOH
Toluene
H2N
N
HN
NH
N
N
CO2Et
OSO3Na
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O
OBn
H
1
7
To start, ethyl 5-hydroxypicolinatehydrochloride (2) was converted into ethyl
5-hydroxypiperidine-2-carboxylatehydrochloride (3;cis/trans 97:3) via
=
a
hydrogenation reaction in ethanol. Trans isomers can be easily removed by
reslurrying in ethyl acetate at 35℃ and isolation by filtration.Next, we screened a
series of lipases for one suitable
to catalyze the resolution of (2S,5S)-ethyl
5-hydroxypiperidine-2-carboxylate(4) from compound 3 (Table 1). When porcine
pancreas lipase, porcine liver esterase, lipase AS “Amano” lipase AK “Amano”, and
lipase PS “Amano” SD were separately used to catalyze this reaction, we were unable
to detect any formation of the target product in each case. (entry 1–entry 5). However,
when Chiralzyme IM-100 lipase and Lipozyme CALB were screened as potential
catalysts, we detected formation of 4 (entries 6 and 7). Further investigation revealed
that crude 3 underwent Chiralzyme IM-100 lipase and Lipozyme CALB17-catalyzed
resolution to yield enantiomerically pure compound 4 with a diastereomeric ratio (dr)
of ≥99:1. Although Chiralzyme IM-100 lipase and Lipozyme CALB gave equivalent
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products and yields, we selected Lipozyme CALB as the catalyst, because it was
difficult to remove the Chiralzyme IM-100 lipase from the system following the
completion of the reaction.
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Table 1 Screening of a Series of Lipases for the Lipase-Catalyzed Resolution
Step
HO
(S)
(R)
HO
HO
lipase
HCl
(S)
CO2Et
(R)
CO2H
+
N
CO2Et
N
N
H
H
H
3
4
4'
entry
lipase
solventa
Time
24h
Yieldb
none
1
Porcine pancreas
lipase
isopropyl ether solution
2
Porcine liver
esterase
isopropyl ether solution
24h
none
3
4
5
Lipase AS “Amano” isopropyl ether solution
Lipase AK “Amano” isopropyl ether solution
24h
24h
24h
none
none
none
Lipase PS “Amano”
SD
isopropyl ether solution
isopropyl ether solution
isopropyl ether solution
6
7
Chiralzyme IM-100
lipase
24h
24h
30%
34%
Lypozyme CALB
a
isopropyl ether solution: isopropyl ether:water (99.5:0.5, v/v). bYield after purification with silica gel
chromatography.
To optimize the Lipozyme CALB-catalyzed resolution step, we then investigated the
effect of a range of pH values on the reaction (Table 2)17. We found that compound 3
was almost completely hydrolyzed at pH 8 or pH 10 (entry 1, 2). However, we were
able to detect product 4 at lower pH values (pH 7.5, pH 7, and pH 6.8) following 12h
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incubation (entries 3, 4, and 5, respectively), with the highest yield of 4 obtained at
pH 6.8. When the reaction buffer was below pH 6.8, the reaction progressed very
slowly, and a longer incubation time (up to 30 h) was required to obtain a satisfactory
product yield. Additionally, in buffer solutions below pH 7,Lipozyme CALB is
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efficient
to
identify
its
substrate:
we
obtained
(2S,5S)-ethyl
5-hydroxypiperidine-2-carboxylate(4) with nearly 100% chiral purity as the R,R
configuration was almost completely hydrolyzed.
Table 2 Optimization of pH Conditions for theLipozyme CALB-catalyzed
Resolution Step
(S)
(R)
HO
HO
HO
Lypozyme CALB
KH2PO4 Buffer
HCl
(S)
CO2Et
(R)
CO2H
+
N
N
H
CO2Et
N
H
H
3
4
4'
a
entry
PH
10
8
7.5
7
6.8
time
12 h
12 h
12 h
12 h
12 h
Yiel
1
2
3
4
5
Trace
Trace
12%
34%
48%
a Yield after purification with silica gel chromatography.
Synthesis of the Key Intermediate 7 and Avibactam
An efficient and cost-effective synthesis of intermediate 7 was crucial for the viability
of our proposed synthetic route (route D). Starting from commercially available
ethyl-5-hydroxypicolinatehydrochloride (2) (Scheme 4),compound 3, with an
isomeric ratio of 97:3 (cis/trans), was synthesized via a hydrogenation reaction.Trans
isomers can be easily removed by reslurrying in ethyl acetate at 35℃ and isolation
by filtration23. the pure product 3 underwent lipase (Lipozyme CALB17)-catalyzed
resolution to yield compound 4 as a single enantiomer with dr ≥99:1, together with
the side product 4' 11b. Subsequently, a mixture of di-tert-butyl carbonate (Boc2O) and
triethylamine was added dropwise to the aqueous solution of 4 and 4', and the mixture
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4
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6
was
stirred
at
room
temperature
for
18h
to
and
form
the
(2S,5S)-1-tert-butyl-2-ethyl-5-hydroxy-piperidine-1,2-dicarboxylate(5)
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8
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byproduct 5'11b. As compound 5' is water-soluble, it was easy to yield the pure
product 5 by extraction with EtOAc. Overall, compound 5 was obtained using relatively
environment-friendly methods and the average total yield for the three-step process
was 42.9%.
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Next, compound 5 was dissolved in acetonitrile and stirred at −30°C for 30 min,
followed by the successive dropwise addition of 2,6-dimethylpyridine and
trifluoromethanesulfonic anhydride. A solution of O-benzylhydroxylamine in
2,6-dimethylpyridine was subsequently added and the mixture was stirred at room
temperature for 12h to yield compound 6. The key intermediate 7 was prepared by
treating compound 6 with trifluoroacetic acid to remove the tert-butyloxycarbonyl
(Boc) protecting group. The total yield for the final two steps was 75.2%.
Scheme 4. Synthesis of the Key Intermediate 7
(S)
(R)
HO
HO
HO
HO
Rh/C , H2
Lipozyme CALB
HCl
HCl
(S)
CO2Et
(R)
CO2H
+
reslurrying in EA
Phosphate solution
N
N
N
H
CO2Et
N
CO2Et
H
H
4
4'
3
2
(a single enantiomer,
dr >99 :1)
OBn
(R)
OBn
(S)
HN
(R)
HO
HN
TFA
(S)
1) Tf2O, 2,6-Dimethylpyridine
2) NH2OBn, CH3CN
(S)
CO2Et
(S)
N
CO2Et
N
DCM
N
H
CO2Et
Boc
Boc
7
6
5
total yield for the three steps: 42.9%
the yield for the last two
steps : 75.2%
(Boc)2O, TEA
THF
+
(R)
H
N
HO
(R)
N
CO2
Boc
5'
Route D
water soluble
Finally, we use compound 7 as the key intermediate to complete the synthesis of
avibactam. Because subsequent reactions are relatively simple and do not involve the
construction of complex chiral centers. So we synthesized avibactam on the basis of
previously published routes12, 14–16, 19, 21 and refered to the detailed experiments
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reported by Michael Golden et al22(Scheme 5). That is, Amminolysis of esters
(compound 7) with NH3 in MeOH leads to amide (compound 8) ,which upon
protection with Fmoc-Cl in the presence of DIEA in chlorobenzene, followed by
cyclization of the piperidine with CDI and subsequent deprotection with Et2NH
affords bicyclic compound 9. simultaneous debenzylation and sulfonylation of
compound 9 with H2 over Pd/C and SO3-Me3N in the presence of Et3N in
i-PrOH/H2O. Treatment of sulfonic acid (compund10) with Bu4NOAc, gives rise to
the corresponding quaternary ammonium salts (compound 11) which upon sodium
exchange reaction with BuCH(Et)COONa in EtOH/H2Ofurnishes the target avibactam
sodium. Thus,An efficient synthesis of pure, crystalline avibactam was completed in
10 steps with a 23.9% overall yield from 5-hydroxypicolinatehydrochloride (2). This
route was efficient, cost-effective, used environment-friendly reagents, and we were
able to successfully perform the synthesis on a 400-gram scale.
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Scheme 5. Conversion of 7 to Avibactam (1)
O
O
O
(S)
(S)
OBn
(R)
H2N
H2N
89.0% yield
HN
H2,Pd/C, SO3.NMe3
aq IPA,NEt3
(R)
96.5% yield
(R)
N
HN
(S)
NH
1) FMOC-Cl,DIPEA,PhCl
2) CDI
3) Diethylamine
4) HCl (aq)
NH3, MeOH
Toluene
N
N
CO2Et
OBn
OBn
H
8
9
7
O
O
(S)
O
O
O
(S)
(S)
H2N
H2N
(R)
H2N
N
1)nBu4NOAc
2) Swap to MIBK
Sodium 2-ethylhexanoate
H2O/EtOH
N
(R)
(R)
N
N
N
O
N
O
O
O
O
O
S
O
1
S
O
O
Na+
O-
S
11
O
10
OH
yield 96.2%
O
Bu4N+
yield 89.6%
CONCLUSION
In summary, we have developed an efficient and cost-effective process for the
synthesis of avibactam (1) from commercially available and affordable ethyl
5-hydroxypicolinatehydrochloride (2). A lipase-catalyzed (Lipozyme CALB) step to
achieve kinetic resolution of (2S,5S)-5-hydroxypiperidine-2-carboxylate acid (4), the
valuable precursor of the key intermediate ethyl (2S,5R)-5-((benzyloxy)amino)
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piperidine-2-carboxylate (7) was successfully accomplished, and this reaction
achieved excellent stereoselectivity. Finally, we finished the synthesis of avibactam
from 7 based on previously published protocols. This enzyme resolution method not
only provides us a rapid ,mild, and efficient synthesis of avibactam , but also provides
a potential tool for rapidly building a family of chiral piperidine-2-carboxylate with
other substituting groups.
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EXPERIMENTAL SECTION
General. Unless otherwise mentioned, all commercial reagents and solvents were
used directly as purchased. Flash chromatography was performed using silica gel with
petroleum ether/ethyl acetate or dichloromethane/methanol as the eluent. Melting
points were uncorrected. Optical rotations were measured with a sodium lamp. NMR
spectra were recorded on a spectrometer at 500 MHz (1H NMR), 101 MHz or 126
MHz (13C NMR). Mass spectrometry data were recorded on a high-resolution mass
spectrometer in the electrospray ionization (ESI) mode. Chemical shifts (δ) are
reported in parts per million and referenced to the residual solvent peak, and J values
are given in hertz (Hz).
Preparation of ethyl 5-hydroxypiperidine-2-carboxylatehydrochloride (3)
Rh/C
(25.0
g,
10%)
was
added
to
a
mixture
of
ethyl-5-hydroxypicolinatehydrochloride(2) (250.0 g, 1.496 mol) and ethanol(1.5 L) in
a vessel. The reaction mixture was stirred under the atmosphere of hydrogen (200.0
psi) at room temperaturefor 12h until completion of the reaction (by TLC analysis).
Then, the mixture was filtered and the solvent was removed under reduced pressure to
obtain the crude product, The reaction mass was treated with ethyl acetate (1.00 L) at
35℃ which leads to precipitation of the solid. The precipitated solid was filtered,
washed
with
the
ethyl
acetate
and
dried
in
oven
to
give
ethyl5-hydroxypiperidine-2-carboxylate hydrochloride (3) (253.0 g,94%).1H NMR
(500 MHz, DMSO) δ 5.37 (d, J = 2.5 Hz, 1H), 4.22 (dq, J = 7.0,1.0 Hz, 2H), 4.10 (dd,
J = 9.5, 4.5 Hz, 1H), 3.94 (s, 1H), 3.11– 3.08 (m, 2H), 2.00 – 1.94 (m, 2H), 1.76 –
1.67 (m, 2H), 1.25 (t, J = 7.0 Hz, 1H),. 13C NMR (126 MHz, DMSO) δ 168.05 (s),
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61.72(s), 60.60 (s), 54.59 (s), 48.04 (s),28.32 (s), 20.08 (s), 13.89 (s).IR (cm−1): 3397,
2959,1747, 1415, 1374, 1169, 1135, 1077, 1031. MS (ESI) m/z: 174.1 [M + H]+
Preparation of (2S, 5S)-ethyl 5-hydroxypiperidine-2-carboxylate (4)
Ethyl 5-hydroxypiperidine-2-carboxylate hydrochloride3 (253.0 g, 1.460 mol) was
dissolved in a potassium phosphate buffer solution(8.53 L, 0.1 M, pH = 8). The pH of
the solution was adjusted to 6.8 with dipotassium hydrogen phosphate followed by the
addition ofLipozyme CALB (253.0 g, 5000 LU/g ). The reaction mixturewas stirred at
room temperature for 12 h until completion of the reaction (chiral HPLC,detected by
BOC derivatization of 4 ). The aqueous solution of 4 (8.53 L) was obtained by filtration
and directly used for the next step.
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Preparation of (2S, 5S)-1-tert-butyl-2-ethyl-5-hydroxypiperidine-1,2-dicarboxy-
late (5)
Triethylamine(407 mL, 2.92mol) wasadded to a mixture of the aqueous solution of
4 (8.53 L) and THF(8.00 L) at room temperature. Then (Boc)2O(101.0 g,1.752 mol)
was added dropwise to the mixture at 0oC. The reaction mixture was stirred at room
temperature for 18 h until completion of the reaction (by TLC analysis). Then, the
reaction mixture was extracted three times with ethyl acetate (3.00 L × 3). The
by-product 5' is water-soluble and remained in the aqueous solution. The combined
organic layers were dried over anhydrous MgSO4. Evaporation of the solvent under
vacuum, followed by flash column chromatography on silica gel(petroleum
ether/ethyl acetate = 4/1), gave the corresponding product 5, as a pale yellow liquid
(74.73g, the two step total yields :42.9%), 20= −101.59 (c = 0.62, CHCl3);1H NMR
[α]D
(500 MHz, CDCl3) δ 4.73 (d, J = 87.9 Hz, 1H), 4.21 (s, 2H), 4.10 (d, J = 9.0 Hz, 1H),
3.64 (s, 1H), 2.72 (dt, J = 50.3, 11.5 Hz, 1H), 2.29 (s, 1H), 1.98 (d, J = 10.3 Hz, 1H),
1.81 – 1.67 (m, 1H), 1.64 (s, 1H), 1.60 (s, 1H), 1.46 (d, J = 17.5 Hz, 9H), 1.28 (t, J =
6.9 Hz, 3H).13C NMR (101 MHz, CDCl3) δ 171.47 (s), 155.55 (s), 80.55 (s), 66.57 (d,
J = 22.1 Hz), 61.27 (s), 53.35 (d, J = 119.3 Hz), 48.00 (d, J = 84.9 Hz), 30.12 (d, J =
58.6 Hz), 28.29 (s), 24.91 (d, J = 22.5 Hz), 14.22 (s).IR (cm−1): 2979, 1741, 1699,
1403, 1370, 1149, 1073, 1024.MS (ESI) m/z: 296.1[M+Na]+
Preparation of (2S, 5R)-1-tert-butyl-2-ethyl 5-((benzyloxy)amino) piperidine-1, 2-
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dicarboxylate (6)
5
6
2, 6-dimethylpyridine (95.0 mL, 805.0 mmol) and Tf2O (131 mL, 778 mmol)
7
8
wereadded successively, dropwise, to a mixture of 5 (202.0 g, 741.0 mmol) in CH3CN
9
o
(2.00L) at -30 C, and the reaction mixture was stirred at this temperature for 15
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min.NH2OBn and 2, 6-dimethylpyridine (95 mL , 805.0 mmol) was slowly added to
o
the reaction solution at -30 C. Then, the reaction mixture was stirred at 0oC for 30
min and at room temperature for 12 h. The reaction mixture was extracted with ethyl
acetate (600 mL × 3), and the combined organic layers were dried over anhydrous
MgSO4. Evaporation of the solvent under vacuum gave the corresponding crude
product 6 (280.2 g).The crude product 6 was directly used to next step without further
[α]2D0
purification.The pure product 6 is a white solid, m.p. 122.3−125.2 °C;
=
−81.59 (c = 0.63, CHCl3);1H NMR (500 MHz, CDCl3) δ 7.39 – 7.32 (m, 4H), 7.32 –
7.27 (m, 1H), 5.47 (s, 1H), 4.72 (dd, J = 25.9, 11.5 Hz, 3H), 4.19 (dd, J = 14.0, 7.0 Hz,
3H), 3.14 (t, J = 36.2 Hz, 2H), 1.96 (s, 2H), 1.69 (d, J = 13.9 Hz, 1H), 1.46 (s, 10H),
1.29 – 1.24 (m, 3H).13C NMR (126 MHz, CDCl3) δ 137.92 (s), 128.53 (s), 128.37 (s),
127.80 (s),80.19 (s), 76.66 (s), 61.07 (s), 53.68 (s),53.26 (s), 42.76 (s), 28.37 (s),
22.98 (s), 21.26 (s), 14.28 (s). IR (cm−1): 3242, 2947, 1738, 1674, 1427, 1152, 1195,
1030,701.MS (ESI) m/z: 379.2 [M+H]+
Preperation of ethyl (2S, 5R)-5-((benzyloxy) amino) piperidine-2-carboxylate (7)
Trifluoroacetate acid (124.0 mL, 1.665 mol) was added dropwise to the solution of the
crude product 6 (280.2 g) in CH2Cl2 (1.50 L) at 0 oC. The mixture was stirred at r.t.for
15h. The pH of reaction solution was adjusted to 10 with saturated sodium
bicarbonate solution. Then, the reaction mixture was extracted with DCM (600 mL ×
3), and the combined organic layers were dried over anhydrous MgSO4. Evaporation
of the solvent under vacuum, followed by flash column chromatography on silica
gel(DCM/MeOH = 20/1)), gave the corresponding crude product 7,as a pale yellow
20
liquid(155.4g, the two step total yields :75.2% ),
= -10.40 (c = 0.92, CHCl3);1H
[α]D
NMR (500 MHz, CDCl3) δ 7.41 – 7.32 (m, 4H), 7.33 – 7.21 (m, 1H), 4.89 – 4.42 (m,
2H), 4.18 (q, J = 7.1 Hz, 2H), 3.38 (ddd, J = 11.9, 4.0, 1.8 Hz, 1H), 3.27 (dd, J = 11.0,
3.1 Hz, 1H), 2.99 (tt, J = 10.5, 4.0 Hz, 1H), 2.44 (dd, J = 11.9, 9.8 Hz, 1H), 2.07 (ddd,
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J = 17.0, 8.5, 4.6 Hz, 1H), 2.01 – 1.89 (m, 1H), 1.53 (tdd, J = 13.0, 11.1, 3.8 Hz, 1H),
1.27 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ173.07 (s), 137.78 (s), 128.37
(s), 127.84 (s), 76.78 (s), 60.84 (s),58.41 (s), 57.10 (s), 49.44 (s), 28.10 (s), 28.01 (s),
14.20 (s).IR (cm−1): 3270, 2930, 1736, 1453, 1191, 1033, 746, 695. MS (ESI) m/z:
279.0 [M + H]+.
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Preperation of (2S,5R)-5-((benzyloxy)amino)piperidine-2-carboxamide (8)
Ethyl(2S, 5R)-5-((benzyloxy) amino) piperidine-2-carboxylate7(593.0g, 2.130 mol)
was mixed with a solution of 7M ammonia in methanol (2.00 L).The reaction mixture
was stirred at r.t for 8 h until completion of the reaction (by TLC analysis). The solid
was removed by filtration andthe obtained cake was washed withmethanol (2 x
500mL). The filtrateswerecombined and concentrated under vacuum to 1.50 L
(Cautiouly, warming from 0oC) followed by the addition of toluene (3.00 L). This
o
operation is repeated twice in succession. The obtained solution was stirred at 80 C
for 0.5 h.Thenit was cooled to 0oC. The crude product 9precipitated and was obtained
by filtration and washed with methyl tert-butyl ether(1.00 L× 3) and dried in vacuum
[α]2D0
to give the pure 8as a white solid (510.1 g, 96.5%),m.p. 148.2−150.9 °C;
=
−19.20. (c = 0.36, CHCl3);1H NMR (400 MHz, DMSO) δ 7.58 – 7.17 (m, 5H), 7.10 (s,
1H), 6.92 (s, 1H), 6.48 (d, J = 6.0 Hz, 1H), 4.57 (s, 2H), 3.13 (dd, J = 11.9, 2.4 Hz,
1H), 2.87 (dd, J = 11.0, 2.7 Hz, 1H), 2.81 – 2.68 (m, 1H), 2.21 (dd, J = 11.8, 10.2 Hz,
1H), 1.88 – 1.76 (m, 2H), 1.34 – 1.20 (m, 1H), 1.11 (ddd, J = 17.5, 12.9, 4.0 Hz,
1H).13C NMR (101 MHz, CDCl3) δ 176.31 (s), 137.69 (s), 128.45 (s), 128.43 (s),
127.94 (s), 76.90 (s), 59.74 (s), 57.01 (s), 49.25 (s), 28.17 (s), 27.76 (s).IR (cm−1):
3378, 2946, 1624, 1438, 1323, 1122, 824, 698. MS (ESI) m/z: 250.2 [M + H]+.
Preperation of (2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-
carboxamide (9)
(2S,5R)-5-[(benzyloxy)amino]piperidine-2-carboxamide 8 (510.1 g, 2.045 mol) was
mixed with di-isopropylethylamine (381 mL, 2.188mol) and chlorobenzene (3.00 L)
at 20 °C. The solution of 9-Fluorenylmethyl chloroformate (540.2 g, 2.086mol) in
chlorobenzene (3.10 L) was added to the reaction mixture.The mixture was stirred at
30 °C until completion of the reaction. Then carbonyl diimidazole (331.3 g, 2.658
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mol,dropwisely) was added and agitation was continued at 15 °Cfor 11 h until
completion of the reation. Diethylamine (529 mL , 5.112 mol) was added and
agitation was continued at r.t. for 2 h Aqueous 3 M hydrochloric acid (3.20L, 9.600
mol) was added (to achieve a pH of 2 to 6) and the mixture was cooled to 0 °C. The
solid was isolated by filtration, washed with water (1.50 L × 2.0) and 1-chlorobutane
(1.50 L × 2.0), and dried to give the title compound as a white crystalline solid (495.0
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[α]2D0
g, 89%), m.p. 154.2−156.1 °C;
= −23.57 (c = 0.65, CHCl3);1H NMR (400
MHz, DMSO) δ 7.45 (dd, J = 7.8, 1.6 Hz, 2H), 7.43 – 7.33 (m, 4H), 7.30 (s, 1H), 4.93
(q, J = 11.3 Hz, 2H), 3.69 (d, J = 6.9 Hz, 1H), 3.62 (s, 1H), 2.90 (s, 2H), 2.22 – 1.94
(m, 1H), 1.93 – 1.76 (m, 1H), 1.75 – 1.50 (m, 2H). 13C NMR (101 MHz, CDCl3) δ
172.27 (s), 167.70 (s), 135.60 (s), 129.24 (s), 128.84 (s), 128.59 (s), 78.30 (s), 59.18
(d, J = 253.0 Hz), 57.92 (s), 47.80 (s), 20.86 (s), 17.31 (s). IR (cm−1): 3406, 2991,
1761, 1663, 1455, 1386, 1022, 755. MS (ESI) m/z: 298.1[M + Na]+.
Preparation of Tetrabutylammonium [(2S, 5R)-2-Carbamoyl-7-oxo-1, 6-diazabi-
cyclo[3.2.1]octan-6-yl] Sulfate (11)
(2S,5R)-6-(Benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxamide9(495.0 g,
1.792mol) was mixed with sulfur trioxide trimethylamine complex (301.0 g, 2.150
mol), triethylamine (65 mL, 890 mmol), 10%w/w Pd/C (40 g, 0.025 wt), isopropanol
(2.50L), and water (2.50 L). This mixture was then held in a hydrogenation vessel and
flushed with nitrogen at ambient pressure. Hydrogen was then fed into the vessel at
0.4 mol equiv per hour until the debenzylation reaction was completed. The catalyst
was removed by filtration and washed with water. The combined filtrates were
washed with n-butyl acetate (2.00 L). A solution of tetrabutylammonium acetate
(812.0 g, 2.698 mol), acetic acid (11 g, 180 mmol) in water (1.00 L) was prepared. 72%
of the tetrabutylammonium acetate solution was added to the reaction mixture, which
was then extracted with DCM (2.00 L). The remaining 28% of the
tetrabutylammonium acetate solution was added to the reaction mixture which was
then extracted with DCM (1.50 L). The organic extracts were combined and
concentrated to 1.50 L followed by the addition of 4-methyl-2-pentanone (1.25 L).
The reaction mixture was cooled to 0 °C to precipitate the desired product
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13.Compound 13 was collected by filtration and washed with 4-methyl-2-pentanone
5
6
(1.00 L), and was dried to yield a white crystalline solid 11 (814.0 g,
7
8
[α]2D0
89.6%),m.p.180.3−182.4 °C (decomposition);
= −23.20 (c = 0.57, CHCl3);1H
9
NMR (500 MHz, DMSO) δ 7.36 (d, J = 78.9 Hz, 1H), 3.98 (s, 1H), 3.68 (d, J = 6.6
Hz, 1H), 3.33 (s, 1H), 3.27 – 3.12 (m, 4H), 3.01 (d, J = 11.6 Hz, 1H), 2.92 (d, J = 11.7
Hz, 1H), 2.14 – 1.97 (m, 1H), 1.84 (d, J = 6.0 Hz, 1H), 1.66 – 1.47 (m, 5H), 1.44 –
1.18 (m, 4H), 0.94 (t, J = 7.3 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 172.29 (s),
166.04 (s), 60.52 (s), 58.69 (s), 57.86 (s), 48.13 (s), 23.95 (s), 20.78 (s), 19.71 (s),
17.24 (s), 13.68 (s).IR (cm−1): 3474, 2964, 1761, 1694, 1488, 1272, 1007, 612.MS
(ESI) m/z: 288.0[M + Na]+.
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Preparation of Avibactam Sodium Salt (1)
A solution of sodium 2-ethyl hexanoate (475.0 g, 2.850 mol) in ethanol (2.00 L) was
added
to
a
solution
of
tetrabutylammonium
[(2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo-[3.2.1]octan-6-yl] sulfate 11 (723.0 g)
in ethanol (2.50L) and water (50 mL ) over approximately 1 h and the temperature
was maintained at r.t. The reaction mixture was held for 2 h. The product was
filtered, washed with ethanol (2 × 2.00L), and dried to yield a white crystalline solid
[α]2D0
1(395.0 g, 96.2%),m.p.259.1−262.4 °C(decomposition);
= −46.40 (c = 0.79,
MeOH/H2O = 1/1); 1H NMR (500 MHz, D2O) δ 4.15 (dd, J = 5.8, 2.8 Hz, 1H), 4.01
(d, J = 7.5 Hz, 1H), 3.28 (d, J = 12.2 Hz, 1H), 3.06 (d, J = 12.2 Hz, 1H), 2.23 – 2.09
(m, 1H), 2.06 – 1.96 (m, 1H), 1.94 – 1.82 (m, 1H), 1.81 – 1.69 (m, 1H). 13C NMR
(126 MHz, D2O) δ 174.72 (s), 169.53 (s), 60.43 (s), 59.93 (s), 47.33 (s), 20.03 (s),
18.31 (s).IR (cm−1): 3459, 1749, 1675, 1361, 1270, 1013, 857, 768. MS (ESI) m/z:
279.0 [M + H]+.
AUTHOR INFORMATION
Corresponding Athours
*Guo-fengWu E-mail:xcwuyuanzhang@163.com
*Xin-zhi Chen E-mail:xzchen@zju.edu.cn
ACKNOWLEDGMENT
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The authors would like to thank the Research ＆ Development Center, Zhejiang
Medicine CO. and Zhejiang University. The research was supported in part by the
Postdoctoral Advanced Programs of Zhejiang Province(No. BSH201673)
Supporting Information Available.The Supporting Information is available free of
charge on the ACS Publications website.
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Chiral HPLC Report of Compound 5(PDF)
Chiral HPLC Report of Compound 7 (PDF)
Copies of the1H and 13C NMR spectra of compounds (PDF)
REFERENCES
1) Bonnefoy, A.; Dupuis-Hamelin, C.; Steier, V.; Delachaume, C.; Seys, C.;
Stachyra, T.; Fairley, M.; Guitton, M.; Lampilas, M. In vitro activity of
AVE1330A, an innovative broad-spectrum non--lactam -lactamase inhibitor,
Journal of Antimicrobial Chemistry, 2004, 54 (2), 410−417.
2) Aszodi, J.; Fromentin, C.; Lampilas, M.; Rowland, D. Novel heterocyclic
compounds which are active as inhibitors of beta-lactamases. PCT Int. Appl.
WO/2003063864 A2, Aug 7, 2003.
3) Ehmann, D. E.; Jahić, H.; Ross, P. L.; Gu, R. F.; Hu, J.; Kern, G.; Fisher, S. L.
Avibactam is covalent, reversible, non--lactam -lactamase inhibitor, Proc. Natl.
Acad. Sci. 2012, 109(29), 11663-11668.
4) Zhanel, G. G.; Lawson, C. D.; Adam, H.; Schweizer, F.; Zelenitsky, S.;
Lagacé-Wiens, P. R.; Denisuik,A.; Rubinstein, E.; Gin, A. S.; Hoban, D. J.; Lynch,
J. P. Imipenem-relebactam and meropenem-vaborbactam: two novel
carbapenem--lactamase inhibitor combinations, Drugs. 2013, 73(2), 159-177.
5) Stachyra, T.; Péchereau, M. C.; Bruneau, J. M.; Claudon, M.; Frère, J. M.;
Miossec, C.; Coleman . K; Black, M. T. Mechanistic studies of the inactivation of
TEM-1 and P99 by NXL 104, a novel non--lactam -lactamase inhibitor,
Antimicrob. Agents. Chemoth. 2010, 54(12), 5132-5138.
6) Liscio, J. L.; Mahoney, M. V.; Hirsch, E. B. Ceftolozane/tazobactam and
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ceftazidime/avibactam: two novel -lactam/-lactamase inhibitor combination
agents for the treatm ent of resistant Gram-negative bacterial infections, Int.
J.Antimicrob. Ag. 2015, 46(3), 266-271.
9
7) Lahiri, S. D.; Mangani, S; Durand-Reville,Benvenuti, M.; De Luca, F.; Sanyal, G.;
Docquier, J. D. Structural insight into potent broad-spectrum inhibition with
reversible recyclization mechanism: avibactam in complex with CTX-M-15 and
pseudomonas aeruginosa AmpC -lactamases, Antimicrob. Agents. Chemoth.
2013, 57(6), 2496-2505.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8) US FDA. FDA approves new antibacterial drug Avycaz [ EB/OL ] [ 2014-02-25 ].
9) Coleman, K. Diazabicyclooctanes (DBOs): a potent new class of non-β-lactam
β-lactamase inhibitors, Curr. Opin. Microbiol. 2011, 14(5), 550-555.
10) Dubreuil, L. J.; Mahieux, S.; Neut, C.;Miossec, C.; Pace, J. Anti-anaeobic activity
of a new -lactamase inhibitor NXL 104 in combination with -lactams and
metronidazole, Int. J.Antimicrob. Ag. 2012, 39(6), 500-504.
11) (a)Mangion, I. K.; Ruck, R. T.; Rivera, N.; Huffman, M. A.; Shevlin, M. A
concise synthesis of a -lactamase inhibitor, Org.Lett. 2011, 13(20), 5480–5483
(b) Krishnamurthy, S.; Venkataprasad, J.; Vagvala, T. C.; Moriguchi, T.; Tsuge, A.
α-Chymotrypsin andꢀL-acylase aided synthesis of 5-hydroxypipecolic acidꢀvia
Jacobsen's hydrolytic kinetic resolution of epoxy amino acids, RSC.
Advances. 5(64), 52154-52160.
12) (a)Lampilas, M.; Aszoid, J.; Rowlands, D. A.; Fromentin, C. Azabicyclic
compounds, preparation thereof and use as medicines, in particular as antibacterial
agents. PCT Int. Appl. WO/2002010172 A1, Feb 7, 2002. (b) Boyd, J.A.;
Cherryman, J. H.; Golden, M.; Kalyan, Y. B.; Lawton, G. R.; Milne, D.; Phillips,
A. J.; Racha, S.; Ronsheim, M. S.; Telford, A.; Zhou, S. H.; Dedhiya, M. Process
for
preparing
heterocyclic
compounds
including
trans-7-oxo-6-
(sulphooxy)-1,6-diazabicyclo [3,2,1]octane-2-caroxamide and salts thereog. PCT
Int. Appl. WO/2012172368 A1, Dec 20, 2012. (c) Blizzard, T. A.; Chen, H.; Gude,
C.; Hermes, J. D.; Imbriglio, J. E.; Kim, S.; Wu, J. Y.; Ha, S.; Mortko, C. J.;
Mangion, I.; Rivera, N.; Ruck, R. T.; Shevlin, M. Beta-lactamase inhibitors. PCT
ACS Paragon Plus Environment

Organic Process Research & Development
Page 20 of 22
1
2
3
4
5
6
7
8
Int. Appl. WO2009/091856 A2, Jul 23, 2009. (d) Abe, T.; Okue, M.; Sakamaki, Y.
Optically-active diazabicyclooctane derivative and method for manufacturing
same. PCT Int. Appl. WO2012/086241 A1, Jun 28, 2012.
9
13) (a)Aszodi, J.; Lampilas, M.; Fromentin, C.; Rowlands, D. A. Nouveaux composes
heterocycliques, actifs comme inhibiteurs de beta-lactamases. FR2835186A1, Aug
1, 2003. (c) Lampilas, M.; Rowlands, D. A.; Kebsi, A.; Ledoussal, B.; Pierres, C.
Nitrogenous heterocyclic compounds, preparation thereof and use thereof as
antibacterial medicaments. PCT Int. Appl. WO/2008142285 A1, Nov 27, 2008.
14) (a) Patil,V. J.; Tadiparthi, R.; Birajdar, S.; Bhagwat, S. Nitrogen containing
compounds and their use. U.S. Patent Application 8969334 B2, Mar 3, 2015. (b)
Hecker, E. A.; Baldwin, A. B-Lactamase Inhibitor Picoline Salt. U.S. Patent
Application 9120796 B2, Sep 1, 2015. (c) Aszodi, J.; Fromentin, C.; Lampilas, M.;
Rowlands, D. A. Heterocyclic compounds as inhibitors of beta-lactamases. U.S.
Patent Application 7612087 B2, Nov 3, 2009. (d)Gu, Y. G.; He,Y.; Yin, N.;
Alexander, D. Isoxazole beta-lactamase inhibitors. PCT Int. Appl.
WO2013/149136 A1, Oct 3, 2013. (e) Hwang, Y. S.; Gu, J. Q.; Jain, A.; Garad, S.;
Jacob, P. S. Crystalline form of a beta-lactamase inhibitor. U.S. Patent
Application 2014/0275001 A1, Sep 18, 2014.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15) Baldwin, J. E.; Adlington, R. M.; Godfrey, C. R.; Gollins, D. W.; Smith, M. L.;
Russel, A. T. A new approach to the synthesis of γ-keto-α-amino acids: synthesis
of optically pure 5-hydroxy-4-oxo-L-norvaline, L-HON, Synlett. 1993, 1993(01),
51-53.
16) Abe, T.; Okue, M.; Sakamaki, Y. Optically active diazabicyclooctane derivatives
and process for preparing the same. U.S. Patent Application 8772490 B2, Jul 8,
2014.
17) (a)Pietruszk, J.; Simon, R. C.;Kruska, F.; Braun, M. Dynamic enzymatic kinetic
resolution of methyl 2,3-dihydro-1H-indene-1-carboxylate, Eur. J. Org. Chem.
2009, 2009(35), 6217-6224. (b) BenjaminA. Anderson.; Marvin M. Hansen.;
Allen R. Harkness.; Cynthia L. Henry.; Jeffrey T. Vicenzi.; Milton J. Zmijewski.
Application of a practical biocatalytic reduction to an enantioselective synthesis of
ACS Paragon Plus Environment

Page 21 of 22
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
the 5H-2,3-benzodiazepine LY 300164, J. Am. Chem. Soc. 1995, 117(49),
12358-12359. c) Yoshihiko, Hirose.; Kinya, Kariya.; Ikuharu, Sasaki.; Yoshiaki,
Kurono.; Hirosato, Ebiikea.; Kazuo, Achiwaa. Drastic solvent effect on lipase-
catalyzed enantioselective hydrolysis of prochiral 1,4-dihydropyidines,
Tetrahedron. Lett. 1992, 33(47), 7157-7160.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18) (a)Mangion, I. K.; Ruck, R. T.; Rivera, N.; Huffman, M. A.; Shevlin, M. A
concise synthesis of a β-lactamase inhibitor, Org. Lett. 2011, 13(20), 5480-5483.
(b)Chavan, V. P.; Wankhede, K. S.; Deshmukh, V. V.; Bhavsar, S.; Patil, K. R.;
Yeole, R. D.; Patel, M. V. Process for preparation of sodium
(2S,5R)-6-(Benzyloxy)-7-Oxo-1,6-Diazabicyclo[3.2.1]Octane-2-Carboxylate. U.S.
Patent Application 9790223 B2, Oct 17, 2015.
19) Miller, S. P.; Zhong,Y. L.; Liu, Z.;Simeone, M.; Yasuda, N.; Limanto, J.; Chen, Z.;
Lynch, J.; Capodanno, V. Practical and cost-effective manufacturing route for the
synthesis of a β-lactamase inhibitor, Org. Lett. 2014, 16(1), 174-177.
20) Xiong, H.; Chen, B.; Durand-Réville, T. F.; Joubran, C.; Alelyunas, Y. W.; Wu,
D.;
Huynh, H. Enantioselective synthesis and profiling of two novel
diazabicyclo- octanone β-lactamase inhibitors, ACS. Med. Chem. Lett. 2014, 5(10),
1143-1147.
21) Priour, A.; Bonnet, A.; Oddon, G.; Mazurie, A. Method for preparing
disubstituted piperidine and intermediate. PCT Int. Appl. WO/2009090320 A1,
Jul 23, 2009. (b) Lampilas, M.; Aszodi, J.; Rowlands, D. A.; Fromentin, C.
Azabicyclic compounds, preparation thereof and use as medicines, in particular as
antibacterial agents. PCT Int. Appl. WO/2002010172 A1, Feb 7, 2002. (c)
Dedhiya, M. G.; Bhattacharya, S.; Ducandas, V.; Giuliani, A.; Ravaux, V.; Bonnet,
A.;
Priour,
A.;
Spargo,
P.
Crystalline
forms
of
trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3,2,1]octane-2- carboxamide sodium
salt. U.S. Patent Application 8471025, Jun 25, 2013.
22) Ball, M.; Boyd, A.; Ensor, G. J.; Evans, M.; Golden, M.;Linke, S. R.;Milne, D.;
Murphy, R.; Telford, A.; Kalyan, Y.; Lawton, G. R.; Racha, S.; Zhou, S. H.
Development of a manufacturing route to avibactam, a β-lactamase inhibitor, Org.
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23)In the process of amplification, we found that under the conditions of enzyme
resolution, the crude product containing 3% trans-isomer had no effect on our
resolution and no trans-isomer was detected in the resolved product. For the reason,
we suspect that the first possibility is that the lipase is unrecognizable to the trans
isomers and completely hydrolyzed in buffer solution. Another possibility is that the
enzyme can also recognize the trans-isomer. Half of this was hydrolyzed, leaving
about 1.5% of the isomers, which, due to their low content, gradually hydrolyzes in
our buffer solution as the reaction time increases. This is consistent with the reduction
in yield of our main product if it is not treated for a long time during the resolution
process.
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