Process Development for the Synthesis of a Monobactam Antibiotic-LYS228

Article abstract of DOI:10.1021/acs.oprd.9b00330

A scalable process for the novel monobactam antibiotic LYS228 has been developed. This paper covers a novel scalable process for producing β-lactam 10 in the multiple kilogram scale. An alternative approach to compound 7 without protecting-group exchange

Full text of DOI:10.1021/acs.oprd.9b00330

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Full Paper
Process Development for the Synthesis of a Monobactam Antibiotic - LYS228
Zhongbo Fei, Quanbing Wu, Wanben Gong, Peng Fu, Cheng Li, Xianwen Wang,
Yufeng Han, Bin Li, Lei Li, Bin Wu, Yi Zhao, Jinjing Li, Wenya Zhu, Wenlong Qiu,
Jing Guo, Jianguang Zhou, Yuanqiang Li, Marco Villa, and Chi Ming CHEUNG
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00330 • Publication Date (Web): 07 Feb 2020
Downloaded from pubs.acs.org on February 7, 2020
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Process Development for the Synthesis of a Monobactam Antibiotic -
LYS228
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Zhongbo Fei, Quanbing Wu, Wanben Gong, Peng Fu, Cheng Li, Xianwen Wang, Yufeng Han, Bin Li, Lei
Li, Bin Wu, Yi Zhao, Jinjing Li, Wenya Zhu, Wenlong Qiu, Jing Guo, Jianguang Zhou, Yuanqiang Li a, Marco
Villa b and Chi Ming Cheung*
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Chemical and Analytical Development, Suzhou Novartis Pharma Technology Co. Ltd, Changshu, Jiangsu,
China 215537
a Zhejiang Jiuzhou Pharmaceutical Technology Co., Ltd., Waisha Road 99, Jiaojiang, Taizhou, Zhejiang,
China 318000
b Euticals SpA, Rozzano, Quinto de Stampi, Milan. Italy
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ABSTRACT: A scalable process for a novel monobactam antibiotic LYS228 has been developed. This
paper covers a novel scalable process for producing β-lactam 10 in multiple kilograms scale. An alternative
approach to compound 7 without protecting group exchange has been demonstrated to enable a shortened
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synthesis. A scalable process for the side chain 20 which used an alternative reagent to mitigate a safety
risk from the initial med-chem approach was performed. The final assembling to LYS228 to a stable isolated
product was identified and executed in multiple kilogram scale.
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KEYWORDS: monobactam, safety risk, tri-hydrate form, LYS228
INTRODUCTION
β-Lactams are a major class of antibiotic drugs, but their utility against Gram-negative organisms has been
eroded due to drug resistance development.1 As members of this class of antibiotics, carbapenems are
regarded as the treatment of the last resort. However, an increase of clinical strains being able to degrade
carbapenems was observed via expressing carbapenemases. One solution is to develop a monocyclic β-
lactam antibiotic such as monobactams.2,3 Aztreonam is currently the only monbactam drug which is stable
to a part of carbapenemases. Novartis has discovered a novel monobactam LYS228, which is stable to all
carbapenemases while exhibiting potent activity against carbapenem-resistant Enterobacteriaceae.4 To
support the drug development of LYS228 we have developed a scalable process for LYS228. Herein we
report our results.
OH
O
OH
O
O
O
N
N
N
N
H2N
H2N
O
O
O
S
S
HN
O
HN
O
O
N
N
N
SO3H
SO3H
LYS228
Aztreonam
Figure 1. Structures of Aztreonam and LYS228
RESULTS AND DISCUSSION
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The overall synthesis of LYS228 is outlined in Scheme 1. The key synthetic intermediates 10, 14 and 20
7
8
are used, and their synthesis will be discussed as well as the final assembling to LYS228.
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Scheme 1. The overall synthesis of LYS228
O
Ph
O
Ph
O
O
N
OH
N
OH
O
BocHN
H2N
O
S
O
20
O
N
O
OCH3
CbzHN
O
OH
O
CbzHN
O
N
N
H2N
O
+
O
O
N
S
NH
14
HN
O
O
DMB
N
OCH3
10
1
2
N
SO3H
LYS228
1. Synthesis of β-lactam 10. The reported asymmetric synthesis of 9 was a key intermediate for process
development.5,6 Thus optically pure (S)-glyceraldehyde acetonide 1 (8~10%wt in water) was condensed with
2,4-dimethoxybenzylamine 2 in DCM to provide aldimine 3 (Scheme 2). After phase separation and drying,
the solution of 3 in DCM was subject to a Staudinger synthesis with 4 to produce β-lactam 5 in the presence
of Et3N. The stability of 3 in DCM was sufficient for isolation and telescoping to the next step. To prevent
hydrolysis of acyl chloride 4 to the carboxylic acid due to presence of trace amount of water, we found
charging 0.3 equivalent of SOCl2 to the reaction was reliable to keep the performance of the reaction
consistent. Due to the instability of the phthalimido group in the following steps, we exchanged the
protecting group to N-Cbz as reported.5 The crude 5 was deprotected and further telescoped to 7. Isolation
and purification was carried out at this stage of the synthesis by crystallization in methanol, providing 7 with
99.8% ee and in 30.7% overall yield from 1. Acetonide 7 was telescoped to 10 through deprotection of the
acetonide, oxidative cleavage of the resulting diol 8 and reduction of aldehyde 9. β-Lactam 10 was isolated
and recrystallized in ethyl acetate to give a 75.1% overall isolated yield from 7 with 99.9% ee.
Scheme 2. Synthesis of β-lactam 10
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O
O
Cl
4
5
6
7
N
O
H2N
O
O
O
4
O
O
O
H
O
O
H2N
O
O
O
OCH3
DCM
20 o
H2NNH2
EtOH
H
SOCl2 (0.3 eq)
Et3N, DCM
O
CbzCl
CbzHN
O
H
N
N
O
+
C
N
O
O
aq. NaHCO3, EtOAc,
N
DMB
OCH3
N
DMB
OCH3
DMB
1
30.7% from
8
6
7
1
2
3
5
9
OCH3
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OH OH
CbzHN
O
OH
CbzHN
H
O
CbzHN
O
TsOH-H2O
THF, H2O
NaBH4
DCM, MeOH
NaIO4
EtOAc
N
N
N
DMB
O
DMB
DMB
7
75.1% from
8
10
9
To avoid protecting-group exchange from N-phthalyl to N-Cbz we explored using N-Cbz-protected glycine
4’ in the Staudinger synthesis (Scheme 3) to reduce steps in the plant. The costs in dedicated manufacturing
facilities for β-lactam steps can be higher than normal facilities. The N-Cbz -protected glycine 4’ was
activated as a mixed anhydride by treating with isopropyl chloroformate in the presence of Et3N followed
by addition of the solution of 3 in DCM. The reaction occurred at room temperature to directly provide 7 in
50.4% isolated yield.
This route was not developed further as we envisioned a different synthetic strategy to incorporate the β-
lactam ring at a later stage in the synthesis (this synthesis would be reported in due course). The reason
to avoid the formation of β-lactam in the early stages of the synthesis would enable more flexibility for
manufacturing of the non β-lactam steps and hence only manufacture the β-lactam compound steps in
designated β-lactam facilities.
Scheme 3. Alternative synthesis of β-lactam 7
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O
CbzHN
OH
O
4'
6
7
Cl
O
Et3N
8
9
O
O
10
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O
CbzHN
O
O
O
O
O
H2N
H
CbzHN
O
O
DCM
20 o
OCH3
N
O
+
N
C
OCH3
O
DMB
DCM
50.4% yield from
1
OCH3
7
3
1
2
OCH3
2. Conversion of 10 to β-lactam 14. The hydroxyl group of 10 was mesylated to 11 by employing a slight
excess MsCl to obtain a full conversion (Scheme 4). The product was isolated in 87% yield. From 11, we
initially attempted to install the oxazolidinone moiety via a direct substitution with 2-oxazolidinone. However,
it was proven to be unproductive after several attempts.
Scheme 4. Conversion of 10 to 14
O
O
O
O
H2N
CbzHN
O
OH
CbzHN
O
NH
CbzHN
O
OMs
CbzHN
O
N
CbzHN
O
N
K2S2O8, K2HPO4
OH
CDI
MsCl (1.2 eq)
OH
N
N
N
NH
14
N
ACN, H2O
51.0%
DCM
DMB
70 o
C
DMB
TEA, DCM
87%
DMB
DMB
61.9% over
two steps
11
13
12
10
O
O
CbzHN
O
N
CbzHN
O
O
NH2
NH
N
HO
DMB
14'
12'
The more nucleophilic 2-amino ethanol was thus attempted in the substitution. The reaction proceeded with only 2%
conversion over 20 h at room temperature by using 10 equivalents of 2-amino ethanol and 3 equivalents of DIPEA as
base in ACN. Warming the reaction to 50°C for 20 h afforded 58.3% product 12 (by HPLC area) with 13.2%
mesylate 11 remaining, however with concomitant formation of 7.0% regioisomeric impurity 12’. More
forcing conditions were employed to perform the reaction in neat 2-aminoethanol at 70°C, which resulted in
a full conversion in 2 h to afford 66.9% product 12 and 4.9% impurity 12’. Prolonging the reaction time or
increasing the reaction temperature led to gradual decomposition of the product likely resulting to the ring
opening of the β-lactam. Impurity 12’ was carried over to the next step, in which crude 12 was treated with
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CDI to give product 13, 12’ did not cyclize and was removed in the workup. Product 13 was slurried in ethyl
acetate to provide the product in 61.9% isolated yield over two steps with 98.9% purity by HPLC area. The
DMB protecting group was then removed. The initial reductive conditions under Pd on carbon only led to
cleavage of N-Cbz with the DMB protecting group intact, while other conditions including TFA-Et3SiH and
acidic conditions of MsOH and BBr3 gave complex mixtures without detection of the desired product. The
most effective oxidative conditions were using K2HPO4-buffered K2S2O8 in a mixed solvent system of ACN
and water. These conditions also produced 5~8% oxidized impurity 14’ as determined by LC-MS and H-
NMR analysis. Isolation and crystallization of 14 lowered the impurity to 3.0%, which was purged in the
downstream process (vide infra).
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3. Synthesis of side chain 20. The synthesis of side chain 204a started from the known 1-hydroxy-1-
cyclopropanecarboxylic acid 16 (Scheme 5), whose conversion to 17 is reported using (diazomethylene)dibenzene.7
Concerning the safety risk with the reagent using (diazomethylene)dibenzene, we chose to use
(bromomethylene)dibenzene for the alkylation. Upon warming in toluene in the presence of DBU, the alkylation
provided 17 in 79.4% isolated yield along with 5.0% bis-alkylation impurity 17’ which was removed by
recrystallization. By portion-wise charging NaOtBu at a rate maintaining a consistent reaction temperature, the
amination of 17 with O-(diphenylphosphinyl)hydroxylamine proceeded smoothly to provide 18 in 78% isolated yield.
Condensation of 18 with α-keto acid 19 at room temperature provided the desired Z-oxime 20 with a favorable
selectivity of 96.4/3.6. The undesired E-oxime was lowered to 0.6% by recrystallization in ACN-H2O.
Scheme 5. Synthesis of side chain 20
O
O
Ph
O
OH
N
Ph
BocHN
P
NH2
O
Ph
O
Ph
Ph
O
O
O
Ph
S
19
Ph
Br
Ph
O
O
O
O
Ph
N
O
Ph
O
NaOtBu, THF
78%
OH
OH
N
OH
H2N
THF, EA
73%
DBU, Toluene
79%
OH
16
BocHN
17
18
S
20
O
Ph
O
Ph
Ph
O
Ph
17'
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4. Final assembling to LYS228. The final assembling to LYS228 is shown in Scheme 6. N-Cbz cleavage of
14 by Pd-catalyzed hydrogenation was feasible in MeOH but on isolation the loss was high. Therefore,
DMF was chosen as solvent for the hydrogenation with the idea of telescoping the product solution. The
hydrogenation in DMF was a clean reaction which provided 15 in 95.5% assay yield with a catalyst loading
of 5 wt% palladium on carbon. Since the next step is sensitive to water, using low catalyst loading is critical
to avoid introducing water into the system from the wet catalyst. Therefore, 5 wt% catalyst loading provided
a solution of 15 in DMF containing ca. 0.88% water which was acceptable for the next step without the need
for drying. Vacuum was applied to the solution in order to remove a small amount of volatile dimethylamine
which could participate in the coupling reaction to form the dimethyl amide impurity. Alternatively, co-
evaporation of this solution with DCM removed dimethylamine as well as simultaneously lowering the water
content further to 0.33%. Coupling of 15 with 20 was completed employing CDMT as condensation reagent.
Thus the optimized process was activation of acid 20 with two equivalents of CDMT in the presence of
NMM. The completion was monitored by derivatizing a sample of the reaction mixture with aniline to the
corresponding anilide, followed by charging 1.4 equivalents of 15 solution in DMF. The potential adduct of
15 to CMDT was not detected in the reaction. Upon completion of the reaction, inverse addition of reaction
mixture to water was carried out to precipitate the product. This suspension was stirred for 12 h to
decompose the dimer impurity 21a, presumably to the monomer via hydrolysis, which would otherwise
remain in the filter cake and be difficult to remove in the downstream process. Filtration of the resulting
suspension provided the wet product 21, drying by heating caused decomposition and washing with water-
miscible ethanol caused unacceptable product loss. Since it is critical to have dry 21 for the following water-
sensitive sulfonylation reaction a second precipitation using all organic solvents was implemented by adding
the solution of 21 in THF to n-heptane. The precipitate containing volatile solvents collected by filtration and
was easily dried to provide product 21 with water content below 1% and in 92% isolated yield. The product
purity was 95.7% by HPLC area with ca. 2% impurity 21b based on LC-MS analysis, resulting from coupling
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of the carboxylic acid 20 with the oxidized impurity from N-Cbz cleavage of 14’ from the previous
5
6
hydrogenation step.
7
8
Scheme 6. Final assembling to LYS228
9
O
Ph
Ph
Ph
10
11
12
13
14
15
16
17
18
19
20
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Ph
Ph
O
Ph
O
O
O
O
N
O
OH
O
N
O
O
BocHN
N
N
O
SO3.DMF
DMF
N
O
N
S
BocHN
BocHN
O
O
5 wt% Pd-C
H2
O
O
O
20
O
H2N
O
N
S
S
CbzHN
O
N
HN
O
O
HN
O
O
N
N
aq. NaHCO3
DMF
NH
15
CDMT, NMM, DMF
NH
NH
N
SO3Na
21
14
22
Ph
Ph
O
MeO
N
O
N
OMe
O
N
N
N
N
O
N
N
BocHN
OMe
OMe
O
O
21a
The dimer impurity
S
HN
O
N
NH
HO
O
21b
OH
OH
O
OH
O
OH
O
O
O
O
N
O
O
N
N
N
N
N
N
S
H3N
N
BocHN
H2N
H2N
TFA, anisole
DCM, H2O
O
O
N
O
O
O
O
O
O
S
S
S
HN
O
O
HN
O
HN
O
O
O
N
HN
O
N
N
N
N
O
NH
HN
3H2O
SO3
SO3H
OH
24
SO3H
LYS228
25
23
With dried 21, the sulfonylation step was attempted using DMF-SO3 complex, particular attention paid to
the purity control. Portion wise charging four equivalents of DMF-SO3 secured the complete conversion of
21 in DMF. The sulfonate-activated β-lactam product became less stable, so the reaction solution was
quenched by inverse addition using a stirred biphasic solvent system of ethyl acetate and aqueous NaHCO3
which allowed immediate partition of the product to the organic phase and prevented decomposition from
occurring in the aqueous phase. The excess DMF-SO3 was removed by quenching and washing with
aqueous NaHCO3. This quenching converted the sulfonic acid product to the sodium salt 22,
recrystallization of which in MTBE-heptane provided the product in 92% isolated yield with 95% HPLC purity.
The oxidized impurity was removed in this step. It is worth mentioning, the sodium salt was also obtained
when neutral brine was used instead of aqueous NaHCO3 for the quenching and washing process, which
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indicates the obtained sulfonic acid is a stronger acid than HCl, thus the sulfonic acid proton exchanges
with NaCl to form the sodium salt which partitions to the organic phase and HCl to aqueous phase.
Further optimization with TFA led us to develop a two-step deprotection procedure; the first step was to
deprotect the ester group to form 23 by introducing 20 equivalent of TFA and anisole in DCM, in which
anisole served as a scavenger for the formed diphenylmethyl cation. The second step was the removal of
the Boc group by introducing water to the reaction, for which the excess TFA was needed to achieve full
conversion but was more conveniently charged at the first step. Without introduction of water for the second
deprotection, the reaction would result in significant decomposition before complete conversion of the
mono-deprotected intermediate 23, presumably due to the strong acidic environment in the absence of
water. We fortunately found the product solution in TFA-water, obtained by phase separation of the reaction
solution, could be directly used for product precipitation upon inverse addition with acetone. Filtration of the
precipitants obtained in this manner required a nitrogen atmosphere until complete removal of solvents in
order to prevent the filter cake from becoming gummy in the course of filtration. The solids were not
hygroscopic after complete removal of solvents and stable to air for material transfer. Isolation in this way
provided the product in 80.6% assay yield with 90.5% HPLC purity. The impurity profile from this process
was consistent from batch to batch with impurities greater than 1.0% being mono-deprotected intermediate
23 (1.02%), ring-opened impurity 24 (2.90%) and desulfonylated impurity 25 (1.45%) as determined by LC-
MS analysis. This crude product also contained 2000 ppm residual TFA. These impurities as well as
residual TFA were efficiently removed by a single recrystallization in THF-water to provide the product as
the desired trihydrate form4c with 97.8% HPLC purity and in a 70.7% yield The product containing both a
strong acidic group and a basic amino group forms a zwitter ion, thus TFA did not form a salt with the
product and removal of TFA did not require base treatment.
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CONCLUSION
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We have developed a manufacturing process for LYS228 to support drug development activities. In this
work, we have developed a scalable process for LYS228 as a new improved synthesis without protecting
group exchange for β-lactam 10. In addition, optimising conditions of 10 to 14 by overcoming a challenging
alkylation and a scalable process for the side chain 20 featuring a selection of an appropriate amination
reagent. We have also developed the final assembling process to LYS228 by tackling the challenges of
identifying optimum reaction conditions, isolating and purifying the labile β-lactam compound. The
chemistry and isolation knowledge accumulated in this study and described herein should be useful for
developing other monobactam antibiotic drugs.
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EXPERIMENTAL SECTION
NMR spectra were run on Bruker AVANCE 400MHz spectrometers using ICON-NMR, under TopSpin
program control. Spectra were measured at 298K, unless indicated otherwise, and were referenced relative
to the solvent resonance or tetramethylsilane resonance. HPLC analysis was performed using Agilent 1200,
and the results were described as area %. LC-MS analysis was performed using Agilent 1200 and 6130
Quadrupole system.
Benzyl
((2S,3S)-1-(2,4-dimethoxybenzyl)-2-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-4-oxoazetidin-3-
yl)carbamate (7)12 To a solution of (S)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde 1 (9 wt% in water, 82.4
kg, 633.2 mol, 1.0 eq) in DCM (1100 kg) was added 2,4-dimethoxybenzylamine 2 (106.3 kg, 635.8 mol, 1.0
eq) dropwise at 10~15°C over 20 min. The resulting solution was stirred at 10~25°C for 2 h, and the organic
layer was separated. The aqueous layer was extracted with DCM (440 kg), and the combined organic layers
were washed with 5% NaCl aqueous solution (440 kg), dried with sodium sulfate (88.2 kg), filtered, and
rinsed with DCM (220 kg). To the filtrate was added triethylamine (128.6 kg, 1270.8 mol, 2.0 eq). The
solution was cooled to 0°C and SOCl2 (19.6 kg, 164.7 mol, 0.3 eq) was added, followed by N-phthalylglycyl
chloride 4 (137.0 kg, 612.7 mol, 1.0 eq) over a period of 20 min. The reaction mixture was stirred at 20~25°C
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for 3 h, washed with water (440 kg) two times. The aqueous solution was extracted with DCM (440 kg). The
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combined organic layer was concentrated to give the crude product 5 as an oil.
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To the above residue was added absolute EtOH (1330 kg) and hydrazine hydrate (158.7 kg, 3135 mol) at
20~25°C. The resulting mixture was heated under reflux for 3 h. The reaction mixture was cooled to
20~25°C, filtered and rinsed with EtOH (123 kg). A solvent exchange was performed at 40~45°C under
vacuum EtOAc (1522 kg). The insoluble solids were filtered. The filter cake was rinsed with EtOAc (190
kg). The filtrate was washed with 5% NaCl aqueous solution (760 kg). The aqueous layer was extracted
with EtOAc (380 kg). The combined organic layers, containing 6, was used directly in the next step.
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To the above solution of 6 in EtOAc was added saturated sodium bicarbonate solution (1408 kg) at 10~15°C
followed by dropwise addition of benzyl chloroformate (140.8 kg, 825.3 mol, 1.3 eq). The reaction mixture
was stirred at 10~25°C for 2 h. The organic layer was separated, and the aqueous layer was extracted with
EtOAc (111 kg). The combined organic layers were washed with 10% NaCl aqueous solution (760 kg).
The aqueous layer was extracted with EtOAc (140.8 kg). The combined organic layers were dried over
Na2SO4 (132 kg) for 1~1.5 h and filtered. The filtrate was concentrated to give the crude product as a pale
yellow oil. To the crude product was added MeOH (792 kg) with stirring, followed by cooling to 0-5°C over 3
h and stirring for 5 h to obtain more solids. The solids were filtered and rinsed with cold MeOH (~5°C, 79.7
kg) to give the desired product 7 (91.9 kg, 98.3 % HPLC purity, 99.8% ee, 30.7% overall yield from 1 as a
white solid.
Alternative synthetic route.
To a solution of (S)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde 1 (2.6 g, 20.0 mmol, 1.0 eq) in DCM (40
mL) was added 2,4-dimethoxybenzylamine 2 (3.4 g, 20.0 mmol, 1.0 eq). The reaction solution was stirred
at 20°C for 2 h. Na2SO4 (8 g) was added to the reaction, filtered and rinsed with DCM (10 mL) to give a
solution of 3 in DCM to be used for the next step. The solution of 2-benzyloxycarbonyl-amino acetic acid 4’
(5.5 g, 26.3 mmol, 1.3 eq) and triethylamine (7.9 g, 78.2 mmol, 3.9 eq) in DCM (60 mL) was cooled to -
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40°C, to which was added isopropyl carbonochloridate (1.0 M solution in DCM, 78.0 ml, 78.0 mmol, 3.9 eq)
over 5 min, and the resulting reaction mixture was stirred for 5 min at -40 °C. The above freshly prepared
imine 3 solution was added to the reaction. The reaction was warmed up to room temperature and stirred
for 10 h. The reaction was washed with 3M HCl (40 mL), saturated NaHCO3 (40 mL), brine (40 mL), dried
over Na2SO4 (8 g) and filtered. The filter cake was rinsed with DCM (15 mL), and the filtrate was
concentrated to give the crude product 7 as a yellow solid. The crude product was suspended in MeOH (35
mL) and stirred for 30 min at 0°C. The suspension was filtered and the filter cake was washed with cold
MeOH (5°C, 10 mL) to give 7 (4.74 g, 97.0% purity, 50.4 % yield) as a white solid.
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Benzyl ((2S,3S)-1-(2,4-dimethoxybenzyl)-2-(hydroxymethyl)-4-oxoazetidin-3-yl)carbamate (10)13 To a
solution of 7 (72.0 kg, 153.0 mol, 1.0 eq) in THF (649 kg) was added a solution of p-toluenesulfonic acid
monohydrate (21.6 kg, 113.6 mol, 0.7 eq) in water (180 kg). The reaction mixture was stirred at 65°C for
3~4 h. The reaction mixture was cooled to 20~30°C and neutralized to pH = 7 by sat. NaHCO3 solution
(~110 kg solution). The reaction solution was concentrated to remove excess THF. To the residue had
water (79.5 kg) added and the mixture was stirred at 20~25°C for 1~1.5 h. The solids were filtered and
rinsed with water (108 kg) to give the desired wet product 8 as an off-white solid which was used in the next
step without further purification.
To a solution of wet 8 in EtOAc (712 kg) was added a solution of sodium periodate (36.5kg, 170.7 mol, 1.1
eq) in water (216 kg). The reaction mixture was stirred at 50°C for 2 h. The reaction mixture was cooled to
20~25°C and the layers were separated. The aqueous layer was extracted with EtOAc (216 kg). The
combined organic layers were washed with 5% NaCl aqueous solution (360 kg) and concentrated to give
product 9 as an off-white solid which was used in the next step without further purification.
To a solution of 9 in DCM (935 kg) was added sodium borohydride (11.6 kg, 306.9 mol, 2.0 eq) and lithium
chloride (3.4 kg, 80.2 mol, 0.5 eq) and stirred under nitrogen. To the reaction was added dropwise MeOH
(97 kg) over 0.5 h at 0~5°C. The resulting reaction mixture was stirred at 0~5°C for 2 h and the reaction
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was completed. The reaction was quenched with cold water (~5°C, 360 kg), stirred at 0°C for 2 h and the
layers were separated. The organic layer was washed with water (360 kg). The aqueous layer was
extracted with DCM (66 kg). The combined organic layers were concentrated to give the crude product. To
the crude product was added EtOAc (252 kg) and heated to 60~70 °C until obtaining a clear solution then
slowly cooled to 0~5°C and stirred for 1~2 h. The solids were filtered and dried at 40-50°C under vacuum
for 12 h to give the desired product 10 (46 kg, 99.4% purity, 99.9% ee, 75.1% yield from 7).
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Benzyl ((3S,4R)-1-(2,4-dimethoxybenzyl)-2-oxo-4-((2-oxooxazolidin-3-yl)methyl)azetidin-3-yl)carbamate
(13)14 To a stirred solution of 10 (50.0 kg, 124.9 mol, 1 eq) in DCM (665 kg) was added triethylamine (28.5
kg, 281.6 mol, 2.3 eq) at 0°C. Then, MsCl (17.2 kg, 149.9 mol, 1.2 eq) was slowly added over 0.5 h in
maintaining the temperature below 5°C. The resulting light yellow solution was stirred at 0~5°C for 2 h. The
reaction mixture was quenched with water (355 kg) at 0°C, and the layers were separated. The aqueous
layer was extracted twice with DCM (75 kg). The combined organic layers were concentrated until ca. 5~6
V, and followed by adding hexane (385 kg). The suspension was stirred for 1 h, filtered and dried to give
product 11 (52.0 kg, 87%) as an off-white solid.
A mixture of 11 (52.0 kg, 108.8 mol, 1.0 eq) in 2-aminoethanol (405 kg) was heated to 70°C for 2.5 h. The
reaction mixture was cooled to 20~30°C, and water (1215 kg) was slowly added over 0.5 h. The mixture
was stirred for 1 h filtered and washed with water (226 kg) to provide wet 12.
The wet 12 was dissolved in DCM (428.5 kg) and washed with 5% NaCl aqueous solution (50 kg). The
solution was dried with Na2SO4 (70 kg), filtered and rinsed with DCM (70 kg). To the filtrate was added CDI
(26.6 kg, 164.0 mol, 1.7 eq) and stirred at 20~25°C for 2 h. The reaction mixture was cooled to -5~5°C,
adjusted to pH = 3~4 with 1N HCl, and stirred for 0.5 h. The layers were separated and the organic layer
was washed with water (261 kg). The aqueous layer was extracted with DCM (48 kg). The combined organic
layers were concentrated followed by addition of EtOAc (177 kg). The suspension was stirred for 2 h at
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5~10°C and filtered. The filter cake was washed with EtOAc (23 kg), and dried to give product 13 (31.6 kg,
98.9% purity, 100% ee, 61.9% yield from 11).
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Benzyl ((3S,4R)-2-oxo-4-((2-oxooxazolidin-3-yl)methyl)azetidin-3-yl)carbamate (14)14 To a suspension of
13 (5.8 kg, 12.4 mol, 1 eq) in ACN (23 kg) and water (58 kg) was added K2S2O8 (6.7 kg, 24.8 mol, 2.0 eq)
and K2HPO4-3H2O (7.3 kg, 32.0 mol, 2.6 eq). The reaction mixture was heated to reflux at internal
temperature ~80°C and the mixture turned to a clear pink solution. The reaction was stirred at reflux for 3
h. HPLC analysis showed the reaction was completed. The reaction mixture was cooled to 20~25°C and
added EtOAc (26 kg). The reaction mixture was stirred for 0.5 h and filtered to remove undissolved inorganic
salts and washed with EtOAc (3 kg). The filtrate was phase-separated and the aqueous phase was
extracted with EtOAc (26 kg x 3). The combined organic layers were concentrated until 3 volume of 13 to
which was added hexane (26 kg). The suspension was filtered, and washed with a mixed solvent of EtOAc
and Hexane (V/V = 1/1, 5 kg) to give the crude product as a yellow solid. The crude product was suspended
in a mixed solvent of MeOH and EtOAc (V/V = 1/1, 52 kg), and heated to 55~65°C to give a clear solution.
The clear solution was slowly cooled to 0~3°C and stirred for 2 h. The suspension was filtered, washed with
a mixed solvent of MeOH and EtOAc (V/V=1/1, 6 kg), and dried to afford 14 (5.8 kg, 96.5% HPLC purity,
2.9 % the over oxidized impurity 14’, 100% ee, 51%) as a pale yellow solid. A sample was chromatographed
to isolate 14’ for structure determination. 1H NMR (400 MHz, DMSO-d6) δ 8.37 (s, 1H), 8.23~8.33 (m, 1H),
8.18 (d, J = 9.2 Hz, 1H), 7.31~7.40 (m, 4H), 6.51 (t, J = 7.2 Hz, 1H), 5.21~5.30 (m, 1H), 5.17 (br t, J = 5.9
Hz, 1H), 4.89~5.10 (m, 3H), 4.16~4.37 (m, 1H), 3.86~4.01 (m, 2H), 3.42~3.51 (m, 1H), 3.27~3.36 (m, 1H),
3.17 (dd, J = 14.1, 4.1 Hz, 1H). MS: m/z = 336.2 ([M + H]+), 358.1 ([M + Na]+).
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Benzhydryl
1-((((Z)-1-(2-((tert-butoxycarbonyl)amino)thiazol-4-yl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2-
oxooxazolidin-3-yl)methyl)azetidin-3-yl)amino)ethylidene)amino)oxy)cyclopropane-1-carboxylate (21)14 To
a solution of 14 (5.7 kg, 17.8 mol, 1.0 eq) in DMF (200 L) was charged 5 wt% Pd-C (900 g). The system
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was degassed (3 times) and exposed to hydrogen atmosphere (1.5 bar). The reaction was stirred at 20°C
for 2 h. The reaction mixture was filtered and washed with DMF (12 L). The filtrate was put under vacuum
for 1 h at 25°C to remove dimethyl amine impurity, and resulting solution containing 15 (3.14 kg in 212 L
solution, 0.88% water content, assay yield 95.5%) was used for the next step.
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4a
To a solution of carboxylic acid 20 (8.64 kg, 16.0 mol, 1.0 eq) in DMF (36.4 L) followed by dropwise
adding NMM (2.43 kg, 24.0 mol, 1.5 eq) was cooled to 0°C and CDMT (4.21 kg, 24.0 mol, 1.5 eq) was
added with internal temperature controlled below 10°C. The reaction solution was stirred for 1 h at 0~5°C.
HPLC analysis of a derivatized sample (obtained by quenching a sample of the reaction solution with
excess aniline) showed full conversion of the carboxylic acid to the activated intermediate. To the reaction
was added a solution of 15 in DMF (3.14 kg in 212 L solution, 0.88% water content, assay yield 95.5%).
The reaction was stirred 0~5°C for 2 h and HPLC analysis showed complete conversion. EtOAc (375 L)
was added to the reaction and a 20% brine solution (3 x 125 L) was added to the reaction and The brine
solutions were back extracted with EtOAc (250 L) and the organics were combined, followed by washing
with 0.1N HCl solution (2 x 125 L) and the organics were washed with a solution of 5% NaHCO3 (125 L)
and separated. The organics were concentrated under vacuum at 30°C to a volume of 56L and transferred
to a solution of ipropyl ether (225 L) and cooled and stirred 0~5°C for 1 h. The suspension was filtered and
washed with ipropyl ether (2 x 20 L) and dried under vacuum at 24 h at 30°C to give product 21 (9.02 kg,
93.3 wt%, 95.7% purity, containing 2.0% 21b, 92.1 % yield) as a solid.
Sodium
(3S,4R)-3-((Z)-2-((1-((benzhydryloxy)carbonyl)cyclopropoxy)imino)-2-(2-((tert-
butoxycarbonyl)amino)thiazol-4-yl)acetamido)-2-oxo-4-((2-oxooxazolidin-3-yl)methyl)azetidine-1-sulfonate
(22)14 A solution of 21 (11.1 kg, 15.76 mol, 1 eq) in DMF (49.2 L) was cooled to 0~5°C for 10 min was
added DMF-SO3 (9.17 kg, 59.88 mmol, 3.8 eq). The reaction was stirred at 0~5°C for 3 h and HPLC analysis
showed completion of the reaction and the reaction was further diluted with EtOAc (2 x 98 L). The reaction
solution was slowly added to a stirred biphasic solution of the aqueous solution containing NaHCO3 (9.05
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kg) and NaCl (19.68 kg) in water (196.8 L) keeping the temperature below 10°C, and the pH of aqueous
phase was ~6.3. The phases were separated. The organic phase was washed with 10% NaCl aqueous
solution (2 x 65 L) and separated. The organics were concentrated at 40-50°C to 86 L and MTBE (2 x 197
L) was added and the organics concentrated to 86 L. Heptanes (197 L) was added to the solution dropwise
and the suspension was left to stir for 1 h at 20°C and filtered and the cake was washed with heptanes (3
x 20 L) and dried at 40°C for 24 h to give product 22 (8.03 kg, 90.2 wt%, 95.0% purity with no over-oxidized
impurity detected, 93.8% yield).
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(3S,4R)-3-((Z)-2-(2-ammoniothiazol-4-yl)-2-((1-carboxycyclopropoxy)imino)acetamido)-2-oxo-4-((2-
oxooxazolidin-3-yl)methyl)azetidine-1-sulfonate (LYS228)14 To a solution of 22 (12.9 kg, 85 wt% assay,
13.61 mol, 1.0 eq) in DCM (129 L) had anisole (2.94 kg, 27.2 mol, 2.0 eq) added and cooled to 0~5°C. TFA
(31 kg, 272.2 mol) was added dropwise maintaining the internal temperature below 10°C. The reaction
mixture was stirred at 0~5°C for 1 h and HPLC analysis showed the complete conversion of 22 to the acid
intermediate 23. Water (6.12 L) was added and stirred at 25°C for 24 h and HPLC analysis showed the
complete conversion. The stirring was stopped and the layers separated. The lower aqueous/TFA phase
was added dropwise to a cooled solution of acetone (211 L) at 0~5°C over 0.5 h with vigorous stirring and
the suspension was stirred for 2 h at 0~5°C. The product was filtered under vacuum with protection with
nitrogen atmosphere. The filter cake was washed with acetone (2 x 32 L) under nitrogen atmosphere and
thoroughly dried under a nitrogen flow to provide crude LYS228 (8.0 kg, assay 70 wt% (5.6 kg), 90.5%
purity, containing 1.0% impurity 23, 2.9% impurity 24 and 1.5% impurity 25, 80.6% yield).
Crude LYS228 (8.0 kg, assay 70 wt% (5.6 kg), 10.88 mol) was dissolved in a mixed solvent of water (11 L)
and THF (22 L) to give a clear yellow solution. The solution was filtered through a cartridge and was added
dropwise to a solution of THF (78 L) over 1 h. The suspension was stirred at 25°C for 2 h and filtered under
vacuum protected from the light. The filter cake was washed with cold water (2 x 11 L) and dried under
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nitrogen (with 30-40% water humidity) at 25°C for 4 h to give LYS228 as a trihydrate (4 kg, 97.8% purity by
HPLC area, 70.7% yield).
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AUTHOR INFORMATION
Corresponding Author
* (C.M.C) Tel: +86 512 5225 6538. E-mail: chiming.cheung@novartis.com
ORCID
Chi Ming Cheung: 0000-0002-2470-1712
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We would like to thank our partners on this project Zhejiang Jiuzhou Pharmaceutical Technology Co., Ltd. and
Euticals SpA (and all the supporting teams for both companies especially Palma Fedeli at Euticals) for their
successful execution of the process on kilogram scale.
Finally, we would like to thank Boston Pharmaceuticals for allowing Novartis to publish this research.
ABBREVIATIONS
ACN, acetonitrile; BBr3, boron tribromide; Boc, tert-Butyloxycarbonyl; CBZ, benzyloxycarbony; CDI,
carbonyldiimidazole; CDMT, 2-chloro-4,6-dimethoxy-1,3,5-triazine; DCM, dichloromethane; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DIPEA, diisopropylethylamine; DMB, 2,4-dimethoxylbenzyl; DMF,
dimethylformamide; DMF-SO3, sulfur trioxide N,N-dimethylformide complex; EtOAc; ethyl acetate; EtOH,
ethanol; Et3SiH, triethylsilane; H-NMR, proton nuclear magnetic resonance; HPLC, high performance liquid
chromatography; K2HPO4, dipotassium phosphate; K2S2O8, potassium persulfate; LC-MS,
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Liquid Chromatography-Mass Spectrometry; MeOH; methanol; MsCl, methanesulfonyl chloride; MsOH,
methanesulfonic acid; MTBE, methyl tert-butyl ether; NaCl, sodium Chloride; NaBH4, sodium borohydride;
NaHCO3, sodium hydrogen carbonate; NaIO4, sodium periodate; NaOtBu, sodium tert-butoxide; NEt3,
triethylamine; NMM, N-methyl morpholine; Pd, palladium; SOCl2, thionyl chloride; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; TsOH.H2O, Toluene-4-sulfonic acid monohydrate;
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REFERENCES
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New β-Lactamases from Gram-Negative Bacteria, Annu. Rev. Microbiol. 2011, 65, 455.
(2) Felici, A.; Amicosante, G.; Oratore, A.; Strom, R; Ledent, P.; Joris, B.; Fanuel, L.; Frere, J. M. An
overview of the kinetic parameters of class B β-lactamases, Biochem. J. 1993, 291, 151.
(3) Felici, A.; Amicosante, G. Kinetic Analysis of Extension of Substrate Specificity with Xanthomonas
maltophilia, Aeromonas hydrophila, and Bacillus cereus Metallo-β-Lactamases, Antimicrob Agents
Chemother. 1995, 39, 192
(4) (a) Reck, F.; Bermingham, A.; Blais, J.; Capka, V.; Cariaga, T.; Casarez, A.; Colvin, R.; Dean, C.
R.; Fekete, A.; Gong, W.; Growcott, E.; Guo, H.; Jones, A. K.; Li, C.; Li, F.; Lin, X.;; Lindvall, M.;
Lopez, S.; McKenney, D.; Metzger, L.; Moser, H. E.; Prathapam, R.; Rasper, D.; Rudewicz, P.;
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Sethuraman, V.; Simmons, R. L. SALTS AND SOLID FORMS OF MONOBACTAM ANTIBIOTIC,
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(12)The lab-scale synthesis is reported in references (4b), (5) and (6); herein reported are a scale-up
procedure and an alternative synthesis of 7.
(13)The lab-scale synthesis is reported in reference (4b). Here reported is a scale-up procedure.
(14)The lab-scale synthesis is reported in reference (4c). Here reported is an improved procedure for
scale-up.
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