Continuous Process Improvement in the Manufacture of Carfilzomib, Part 1: Process Understanding and Improvements in the Commercial Route to Prepare the Epoxyketone Warhead

Article abstract of DOI:10.1021/acs.oprd.0c00051

Epoxyketone 4 is an isolated intermediate in the manufacturing route to the commercial proteasome inhibitor carfilzomib (Kyprolis). Commercial process development and optimization efforts toward the preparation of epoxyketone 4 highlighted several opportunities for process improvement. In this article, three case studies are presented that demonstrate how a detailed understanding of the reaction mechanism led to improvements that increased the overall robustness of the process. In the first case study, the mechanism of racemization of an α-chiral enone was investigated, resulting in the development of an improved aqueous workup procedure. Next, the stability of a bleach/pyridine mixture used for the step 3 epoxidation reaction was studied, leading to the identification of pyridine as a key raw material and improved reaction conditions and control strategy to meet the conversion target. Finally, oxidized butylated hydroxytoluene (oBHT) was identified as an impurity arising from the use of BHT-stabilized tetrahydrofuran in steps preceding the oxidation. The process understanding obtained from these investigations led to the implementation of process improvements that improved the robustness of the process. The development of a second-generation route to 4 is the subject of part 2 in this series (DOI: 10.1021/acs.oprd.0c00052).

Full text of DOI:10.1021/acs.oprd.0c00051

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Article
Continuous Process Improvement in the Manufacture of
Carfilzomib, Part 1: Process Understanding and Improvements in
the Commercial Route to Prepare the Epoxyketone Warhead
Peter K. Dornan,* Travis Anthoine, Matthew G. Beaver, Guilong Charles Cheng, Dawn E. Cohen,
Sheng Cui, William E. Lake, Neil F. Langille, Susan P. Lucas, Jenil Patel, William Powazinik IV,
Scott W. Roberts, Chris Scardino, John L. Tucker, Simone Spada, Alicia Zeng, and Shawn D. Walker
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ABSTRACT: Epoxyketone 4 is an isolated intermediate in the manufacturing route to the commercial proteasome inhibitor
carfilzomib (Kyprolis). Commercial process development and optimization efforts toward the preparation of epoxyketone 4
highlighted several opportunities for process improvement. In this article, three case studies are presented that demonstrate how a
detailed understanding of the reaction mechanism led to improvements that increased the overall robustness of the process. In the
first case study, the mechanism of racemization of an α-chiral enone was investigated, resulting in the development of an improved
aqueous workup procedure. Next, the stability of a bleach/pyridine mixture used for the step 3 epoxidation reaction was studied,
leading to the identification of pyridine as a key raw material and improved reaction conditions and control strategy to meet the
conversion target. Finally, oxidized butylated hydroxytoluene (oBHT) was identified as an impurity arising from the use of BHT-
stabilized tetrahydrofuran in steps preceding the oxidation. The process understanding obtained from these investigations led to the
implementation of process improvements that improved the robustness of the process. The development of a second-generation
route to 4 is the subject of part 2 in this series (DOI: 10.1021/acs.oprd.0c00052).
KEYWORDS: racemization, α-chiral enone, bleach, epoxidation, carfilzomib
INTRODUCTION
■
Multiple myeloma is a cancer of plasma cells that originates in
the bone marrow.1 Carfilzomib is the active pharmaceutical
ingredient (API) in the marketed proteasome inhibitor
Kyprolis, initially approved by the U.S. Food and Drug
Administration in 2012. Kyprolis is indicated for use in
combination with dexamethasone or with lenalidomide plus
dexamethasone for the treatment of patients with relapsed or
refractory multiple myeloma who have received one to three
lines of therapy and as a single agent for the treatment of
patients with relapsed or refractory multiple myeloma who
have received one or more lines of therapy. Carfilzomib
(Figure 1) is a synthetic epoxyketone-containing tetrapeptide
inspired by the structure of epoxomicin, a natural product with
proteosomal inhibition activity.2 This article presents case
Figure 1. Structures of epoxomicin and carfilzomib.
studies in the continual drive to improve the manufacturing
route to the key epoxyketone intermediate 4 in the synthesis of
identified during familiarization and process development
carfilzomib.
activities include (1) the requirement for silica gel column
The route to prepare epoxyketone 4 involves three steps
from L-Boc-leucine monohydrate: (1) Weinreb amide
formation via the intermediacy of a mixed anhydride, (2)
Received: February 10, 2020
addition of isopropenylmagnesium bromide to generate enone
3, and (3) epoxidation with bleach in pyridine to generate the
isolated intermediate epoxyketone 4 (Scheme 1). Boc
deprotection of 4 and subsequent coupling with intermediate
6 completes the synthesis of carfilzomib. Notable challenges
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Scheme 1. Synthesis of Epoxyketone 4 and Conversion to Carfilzomib
chromatography and resulting stereochemical lability associ-
ated with purification of enone 3, (2) latent exothermic activity
observed for the bleach oxidation, (3) low diastereoselectivity
(ca. 2:1) for the epoxidation to prepare epoxyketone 4, and (4)
the low melting point of the target compound 4 (mp = 41 °C),
which challenges direct isolation from the crude reaction
mixture.
Scheme 2. Synthesis of Enone 3
Since carfilzomib was an in-licensed commercial program, to
address these challenges a two-stage strategy was adopted to
improve the robustness of the current route to epoxyketone 4
and deliver an improved synthesis in the life-cycle management
stage of the program:
phase is then concentrated via distillation and filtered through
a plug of silica gel with ethyl acetate and n-hexane to deliver
enone 3 after concentration of the appropriate fractions.
Control of the stereochemical purity of enone 3 was critical
because of the limited ability to purge stereoisomeric
impurities in the downstream crystallization of epoxyketone
4. However, during development it was noted that varying
levels of racemization were observed (0.1 to >5%) confirming
the lability of the α-enone stereocenter. In order to assess
possible factors affecting racemization, an Ishikawa diagram3
was constructed relating equipment, material, and process
factors to racemization (Figure 2). A detailed sampling plan
was then designed and implemented to identify the origin of
racemization. Table 1 provides an overview of chiral purity
data for four representative batches. Racemization was not
detected in any unit operation prior to distillation of the final
organic phase after aqueous workup (Table 1, entry 1).4
Varying levels of racemization were observed after the
distillation operation, with increased levels noted upon silica
gel column chromatography for batches A and C (Table 1,
entries 2 and 3). These data focused development efforts on
evaluation of the distillation and chromatography unit
operations.
(1) Initiate process understanding workstreams to identify
potential risks to process performance and implement
solutions to mitigate these risks.
(2) Leverage process understanding gained from the first
stage to deliver an improved second-generation process
for the manufacture of epoxyketone 4.
This article presents three case studies from the process
understanding stage: (i) mechanism and mitigation of the
racemization in the step 2 Grignard reaction, (ii) bleach
decomposition in the step 3 epoxidation, and (iii)
identification of an oxidized butylated hydroxytoluene
(oBHT) impurity. In all cases, process improvements were
implemented on the basis of a fundamental understanding of
identified failure modes to improve the robustness of the
process. The development of a second-generation process for
the manufacture of epoxyketone 4 is the subject of part 2 of
this series (DOI: 10.1021/acs.oprd.0c00052).
RACEMIZATION OF ENONE 3
■
Step 2 of the manufacturing route involves the preparation of
enone 3 by addition of Weinreb amide 2 to a solution of
isopropenylmagnesium bromide in tetrahydrofuran (THF)
(Scheme 2). Aqueous ammonium chloride is charged upon
confirmation of reaction completion, followed by acidification
with 5 M HCl. After separation of the phases, the organic layer
is washed with aqueous NaHCO3 and brine. The final organic
An in-depth investigation of in-process samples for batch C
highlighted the presence of white crystalline solids in
distillation samples prior to the chromatography operation.
The following observations were made with regard to these
solids:
B
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Figure 2. Ishikawa diagram to summarize potential root causes for racemization of enone 3.
these solids, presumably because of efficient extraction in the
upstream HCl wash.
Table 1. Levels of ent-3 for Four Representative Batches A−
D
The presence of process salts, combined with the high levels
of racemization observed for batch C, led to the hypothesis
that these salts caused the epimerization; however, exposure of
enone 3 to these individual species did not result in the
expected racemization (Figure 3). Silica gel alone also did not
result in racemization of enone 3. Notably, exposing enone 3
to a representative mixture of silica gel and the process salts
resulted in significant levels of racemization. This unexpected
cooperative effect could be reproduced by exposing enone 3 to
silica in combination with either NaHCO3 or Na2CO3. The
magnitude of the effect is consistent with the observation that
the pH of an aqueous solution of the isolated process salts is
between that of NaHCO3 and Na2CO3. These data suggest
that a synergistic effect is operative where the interaction of
silica with either NaHCO3 or Na2CO3 produces a more potent
racemizing medium than any of the individual components
alone. We hypothesize that upon adsorption of the enone to
silica gel, the acidity of the α-proton is enhanced either by the
Lewis acidic nature of silica or through hydrogen bonding with
silica,5 facilitating a base-catalyzed deprotonation.6 It should be
noted that the carbonyl of the Boc group may also serve as a
point of interaction with silica, increasing the extent of
adsorption.
entry
1
sampling point
A
B
C
D
prior to distillation
(step iv)
after distillation (step
iv)
after chromatography
(step v)
<0.05%
<0.05%
<0.05%
<0.05%
2
3
0.19%
0.41%
0.15%
0.14%
0.66%
3.39%
0.05%
<0.05%
• They were soluble in water (>100 mg/mL).
•
1H NMR spectra obtained in CDCl3 or D2O did not
reveal organic impurities.
• ICP-MS analysis showed 30.5 wt % Na and 75 ppm Mg.
All other metals (Si, V, Cr, Ni, Cu, As, Mo, Ru, Rh, Pd,
Cd, Ir, Pt, Hg, Bi) were below the limit of detection.
• Capillary electrophoresis showed 8.2 wt % chloride and
the presence of carbonate/bicarbonate (not quantified).
• The pH of a 6.7 mg/mL aqueous solution was 10.0 (cf.
measured values of 8.4 for NaHCO3 and 11.3 for
Na2CO3).
• Addition of 1 M HCl to a 60 mg/mL aqueous solution
produced vigorous bubbling.
Consistent with these observations, the white solids (“process
salts”) were shown to be a mixture of NaHCO3, Na2CO3, and
NaCl derived from precipitation during the aqueous wash and
distillation sequence. Mg was found only in trace amounts in
The observation that the recovered process salts were more
basic than NaHCO3 (used in the wash steps) warranted
C
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increase from 8.4 to 9.0, confirming that removal of CO2 can
drive the bicarbonate−carbonate equilibrium to higher pH.
Additionally, under relevant process distillation conditions, the
azeotropic removal of water would also be expected to drive
the equilibrium from bicarbonate to carbonate.
With this understanding of the mechanism of racemization,
the aqueous workup and purification process was redesigned to
mitigate the risk of racemization of enone 3. An inverse quench
of the reaction stream into a mixture of 30 wt % aqueous citric
acid and hexane resulted in a clean phase cut and the absence
of precipitates. The organic layer was then washed sequentially
with water, 5 wt % aqueous NaHCO3, and water prior to a
polish filtration operation to ensure complete purging of
process salts from the organic layer or reactor walls. Finally,
evaluation of the organic stream resulting from the optimized
wash sequence demonstrated that the silica plug could be
removed without any impact on the quality of epoxyketone 4.
These process changes, driven by a detailed understanding of
the factors influencing racemization, resulted in an improved
procedure for manufacturing that removed the risk of
bicarbonate/carbonate-induced racemization.
Figure 3. Racemization of enone 3 in the presence of additives at 35
°C.
EPOXIDATION TO PREPARE EPOXYKETONE 4
■
The manufacture of epoxyketone 4 proceeds via bleach-
promoted epoxidation of enone 3 in pyridine.8 In a typical
process, a solution of enone 3 in pyridine is charged to a
precooled (−15 °C) solution of commercial 10−15 wt %
aqueous NaOCl and pyridine over 60−90 min. The crude
reaction mixture is warmed to 0 °C, at which point an
exotherm to 30 °C is observed at production scale. A lab-scale
reaction was performed at −10 °C with slow addition (1 h) of
the substrate solution to replicate the production-scale
additional consideration. We hypothesized that the distillation
conditions employed in the production of enone 3 could
impact the equilibrium described in Figure 4a through the
continuous removal of CO2 and H2O. To probe the effect of
CO2 removal, the pH of an aqueous solution of NaHCO3 was
monitored under vacuum and constant-volume conditions
(Figure 4b).7 The pH of the aqueous solution was found to
Figure 4. (a) Equilibrium between bicarbonate, carbonate, and carbonic acid species. (b) pH increase of an aqueous NaHCO3 solution under
representative distillation conditions.
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Figure 5. Reaction progress kinetics via in situ infrared spectroscopy10 and corresponding pH and temperature profiles for the bleach epoxidation of
enone 3.
conditions but without purposefully warming to 0 °C.
Evaluation of the reaction progress kinetics (as determined
by in situ infrared spectroscopy) determined that this latent
exothermic activity (at t = 5 h) was not associated with the
productive reaction to generate epoxyketone 4 (Figure 5).9
Product formation was observed during and after the addition
of substrate, and a gradual decrease in pH was observed,
corresponding to the consumption of bleach. At t = 5 h, a
sudden decrease of pH was observed that coincided with the
latent exotherm, at which point the desired reaction was nearly
complete. The pH continued to decrease after the exothermic
event, reaching a second inflection after an additional 5 h of
aging.
of enone 3 was introduced as a reactivity probe, since the
reaction is known to retain the stereochemical integrity of the
α-chiral center. At reaction completion, analysis of the chiral
purity of crude epoxyketone 4 can provide a quantitative
measure of bleach activity at the time of addition of (R)-3.
As shown in Table 2, 10% (R,S)-4 was formed when (R)-3
was added after the first pH drop (pH < 12.5), indicating that
the bleach/pyridine mixture retains some oxidizing ability at
this time point. Conversely, no (R,S)-4 was observed upon
addition of (R)-3 at the second pH drop (pH < 10), consistent
with the complete decomposition of bleach despite the excess
(2.0 equiv) bleach charge. This study also indicates that the
pyridine system may not have sufficient buffer capacity to
maintain high pH throughout the reaction. As a preventative
action, the addition time of the enone substrate must not be
extended longer than necessary (typically <2 h) to ensure that
the bleach is still active by the end of the substrate addition.
Continued familiarization and development activities
revealed an unexpected dependence of the stability of the
bleach solution on the source of pyridine. Bleach/pyridine
mixtures prepared from a variety of pyridine lots under
conditions representative of the process were titrated for
hypochlorite content. As shown in Table 3, pyridine from
vendor 1 (entry 1) retained a high titer of hypochlorite after
the bleach was charged to pyridine. Conversely, pyridine from
vendor 2 (entry 2) resulted in nearly complete decomposition
of bleach as assayed after the bleach charge. Of note, a
diagnostic brown color was observed during the bleach
addition (Figure 6) that was associated with low bleach
activity in the final mixture. A second lot from vendor 2
showed similar behavior (entry 3), demonstrating that the root
cause of bleach decomposition was not limited to contami-
nation of a single lot but instead linked to the manufacturing or
storage method. Of note, the rate of bleach addition also
impacted the titer; for example, the hypochlorite titer remained
high when the bleach addition was performed quickly (10 min
versus 100 min), as shown in entry 4.
Bleach stability is known to decrease at lower pH,11 and
therefore, we hypothesized that the spontaneous pH shift
could be associated with a loss of hypochlorite. Because of the
potential for incomplete reaction conversion upon bleach
decomposition, an experiment was designed to assess the
oxidative potential of residual bleach in the reaction mixture at
various time points. As depicted in Table 2, the R enantiomer
Table 2. Addition of (R)-3 as a Labeled Substrate to Assess
Bleach Potency at Various Time Points
expt
pH at which (R)-3 is charged
(S,R)-4:(R,S)-4
A
B
12.5
10
90:10
100:0
E
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pretreating the pyridine from vendor 2 with K2CO3 and then
filtering led to retention of the hypochlorite titer. Furthermore,
addition of 20 ppm FeCl3 to the vendor 1 pyridine indeed led
to loss of bleach activity. The binary response demonstrated in
all of these studies (either full retention or absence of
hypochlorite) suggests a catalytic and irreversible decom-
position pathway. Given the effect of basic and acidic additives
on the stability of bleach titer shown in Table 3, we next
monitored the pH of the mixture throughout the course of the
bleach addition. As shown in Figure 7, the pH measured for
bleach addition to pyridine from vendor 1 showed a gradual
increase from 12 to 14, consistent with slow charge of a basic
species. When bleach was added to pyridine from vendor 2, the
pH initially increased to a pH of 12 but then decreased over
the remaining course of addition. These data suggest that
below a critical pH, the bleach decomposed as it was being
charged, leading to a drop in pH and continued decom-
position. This can also explain the effect of the addition rate: a
high pH is achieved immediately upon rapid bleach addition,
resulting in a stable solution. This scenario is also consistent
with the effect of added acid or base, which can trigger or
prevent the decomposition (see Table 3, entries 5 and 6).
Furthermore, the observed pH sensitivity of the bleach/
pyridine mixture also explains the bleach decomposition
behavior observed upon extended aging of the reaction
mixture (Figure 5).
Table 3. Effect of the Pyridine Input on the Bleach Titer
after Charging Bleach to Pyridine
bleach/
pyridine
a
entry
pyridine source
vendor 1
vendor 2 (lot 1) none
vendor 2 (lot 2) none
modification
activity
color
b
1
2
3
4
none
99%
2.6%
2.3%
94%
pale yellow
brown
brown
vendor 2 (lot 1) addition over
10 min
pale yellow
5
vendor 2 (lot 1) pyridine pretreated
with K2CO3, then
96%
pale yellow
filtered
6
vendor 1
20 ppm FeCl3
1.9%
brown
a
Relative to the theoretical amount of hypochlorite based on titration
of bleach input. See the Experimental Section for the bleach titration
b
method modified for use with pyridine. Addition over 100 min.
Although rapid (<10 min) addition of bleach in lab-scale
experiments was shown to generate active bleach/pyridine
mixtures with all lots of pyridine tested, this strategy is not
practical on production scale because of the exotherm
associated with the addition of bleach to pyridine caused by
the heat of mixing and precipitation of solids. Therefore,
several controls were used to ensure process robustness. First,
the pyridine used in manufacturing was use-tested as a raw
material under stressed conditions to ensure solution stability.
Second, a use test of the bleach/pyridine mixture as an in-
process control, which was already part of the procedure, was
shown to be capable of detecting bleach decomposition, thus
securing the batch.12
Figure 6. Visual appearance of the bleach/pyridine mixture with
vendor 1 pyridine (left) and vendor 2 pyridine (right).
In summary, detailed understanding of the stability of
bleach/pyridine mixtures before and after epoxidation
facilitated the characterization of acceptable operating
Given the pH dependence of the stability of the bleach, we
hypothesized that the difference in pyridine lots is related to
trace acidic or basic impurities. To probe this possibility,
Figure 7. Variation of the pH of the mixture as bleach was added to pyridine from vendor 1 (blue) or vendor 2 (red) over 200 min.
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Scheme 3. Progression of BHT and oBHT through the Three-Step Synthesis of Epoxyketone 4
parameters and led to improvements that increase the process
robustness.
of bleach to pyridine were critical factors affecting the stability
of bleach in pyridine. Furthermore, exothermic activity after
reaction completion was found to be related to decomposition
of residual bleach. Optimized processing conditions, testing of
the pyridine raw material, and an in-process control to confirm
the oxidizing power of the bleach/pyridine mixture resulted in
a robust manufacturing process. Finally, oBHT was identified
as an impurity resulting from the use of BHT in THF upstream
of the oxidation step. Fate-and-purge studies of this impurity
confirmed an effective control strategy. These three case
studies demonstrate that detailed process understanding is
critical to ensure robust commercial manufacturing. Part 2 in
this series will present the development of a second-generation
route to epoxyketone 4.
IDENTIFICATION OF THE oBHT IMPURITY
■
BHT is an antioxidant and radical scavenging additive
commonly found in many commercial ethereal solvents,
commercial reagents supplied as solutions in these solvents,13
various reagents that are prone to oxidative degradation, and
certain commercial polymers. Impurity analysis performed in
lab-scale studies identified oBHT (8) (Scheme 3) as an
impurity formed during the bleach-mediated oxidation
protocol (vide supra). In both of the preceding synthetic
steps, Weinreb amide formation and Grignard addition, the
reaction solvent is THF containing BHT as a stabilizer. The
overall telescoped process results in accumulation of 3 wt %
BHT (relative to enone 3), which is converted to oBHT in the
step 3 epoxidation reaction (Scheme 3).14 By HPLC analysis,
the liquid chromatography area percent (LCAP) of this
impurity is amplified by the relative response factor of 4.3
relative to epoxyketone 4. Crystallization was identified as the
primary control point for purging of this impurity, as
chromatographic separation was inefficient because of
coelution of oBHT with epoxyketone 4. An authentic sample
of oBHT15 was synthesized independently using PhI(OAc)2 in
order to perform fate-and-purge studies, which demonstrated
that typical levels could be purged under the standard
crystallization conditions. In view of the widespread use of
ethereal solvents stabilized with BHT in manufacturing, this
example highlights the importance of tracking related
impurities, such as oBHT.
EXPERIMENTAL SECTION
■
Preparation of tert-Butyl (S)-(2,6-Dimethyl-3-oxo-
hept-1-en-4-yl)carbamate (3). Isopropenylmagnesium bro-
mide (target 0.5 M in THF) was titrated prior to use, and
charges were adjusted on the basis of the solution potency.
Isopropenylmagnesium bromide (30.0 kg, 0.41 M, 13.3 mol)
was charged to an inerted reactor, and the contents were
cooled to an internal temperature of −10 °C. A solution of
Weinreb amide 2 (1.46 kg, 5.30 mol) in THF (3.8 L, 2.6 L/kg)
was then added to the reactor over 60 min while the internal
temperature was maintained below −5 °C. The reaction
mixture was warmed to an internal temperature of 25 °C and
aged for 19 h. To a separate reactor, 30 wt % citric acid (8.7 L,
6.0 L/kg) and n-hexane (8.7 L, 6.0 L/kg) were charged, and
the internal temperature was adjusted to 0 °C. The crude
reaction mixture was charged to the biphasic citric acid/hexane
mixture over 75 min while the internal temperature was
maintained below 10 °C, and then the internal temperature
was raised to 20 °C. The phases were allowed to separate, and
the bottom aqueous phase was removed. The organic layer was
washed sequentially with water (8.7 L, 6.0 L/kg), 8 wt %
NaHCO3 (8.7 L, 6.0 L/kg), and water (8.7 L, 6.0 L/kg). The
resulting organic phase was concentrated to a volume of 2 L/
kg under vacuum at a jacket temperature of 35 °C. n-Hexane
(2.0 L, 1.4 L/kg) was charged, and the solution was polish-
filtered. The solution was then concentrated to a volume of 2
L/kg under vacuum at a jacket temperature of 35 °C to afford
a hexane solution of 2 (1.09 kg potency-corrected, 80%
solution assay yield, 93.2 LCAP) with spectra identical to that
previously reported in the literature.16
CONCLUSION
■
Detailed mechanistic understanding of the workup and
isolation of an α-chiral enone, a bleach/pyridine epoxidation,
and an impurity arising from the THF stabilizer BHT were
performed that led to the identification of several opportunities
for process improvements. Silica and carbonate/bicarbonate
were found to have a synergistic effect leading to enhanced
racemization of enone 3, and bicarbonate was found to be
partially converted to carbonate in the distillation step,
exacerbating the effect. An improved workup procedure that
prevents the entrainment of salts after workup, and removal of
the silica plug, have been implemented. An investigation into
the stability of bleach/pyridine mixtures for epoxidation
showed that the quality of pyridine and the time for addition
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Titration of Bleach/Pyridine Mixtures. Bleach concen-
tration can be routinely measured by iodometric titration. This
is typically accomplished by reducing hypochlorite using an
excess of potassium iodide under acidic conditions according
to
followed by a rinse of the addition vessel with MeCN (70 mL,
1.6 mL/g). A modest exotherm was observed with a maximum
internal temperature of 26 °C. The reaction mixture was aged
at 20 °C for 18 h, diluted with EtOAc (340 mL, 8 mL/g), and
washed twice with a saturated aqueous solution of NaCl (170
mL, 4 mL/g). The final organic phase was concentrated in
vacuo and purified by column chromatography (5−15%
EtOAc/heptane) to afford oBHT as a white crystalline solid
(17.7 g, 38% yield, 97.1 wt %) with spectra identical to that
previously reported in the literature.18
ClO− + 2I− + 2H+ → I2 + Cl− + H2O
The I2 species is then titrated with thiosulfate according to
2−
I2 + 2S2O3 → 2I− + S4O62−
AUTHOR INFORMATION
However, pyridine has been observed to interfere with this
method. To titrate bleach/pyridine mixtures, standard bleach
titration protocols were adapted by adding additional HCl. In a
typical procedure, a 0.2 M solution of sodium thiosulfate was
standardized by titration of a solution of KIO3. A bleach/
pyridine mixture (prepared from addition of 11 wt % sodium
hypochlorite (4.8 mL) to pyridine (12.9 mL) was diluted with
water in a 100 mL volumetric flask. A 25 mL aliquot of this
solution was then transferred to a conical flask and further
diluted with ∼100 mL of water. Potassium iodide (2 g) was
then added, and the flask was agitated until complete
dissolution was observed. HCl (1 M, 200 mL) was then
added. The solution was titrated with the standardized
thiosulfate solution until a straw-yellow color was observed.
A starch indicator solution (1 mL) was then added, forming a
dark-blue solution. The titration was then continued until a
clear end point was observed.
■
Corresponding Author
Peter K. Dornan − Process Development, Amgen Inc.,
Cambridge, Massachusetts 02142, United States;
Email: pdornan@amgen.com
Authors
Travis Anthoine − Process Development, Amgen Inc.,
Cambridge, Massachusetts 02142, United States
Matthew G. Beaver − Process Development, Amgen Inc.,
Cambridge, Massachusetts 02142, United States; orcid.org/
0000-0003-3816-4601
Guilong Charles Cheng − Process Development, Amgen Inc.,
Cambridge, Massachusetts 02142, United States
Dawn E. Cohen − Process Development, Amgen Inc.,
Cambridge, Massachusetts 02142, United States
Sheng Cui − Process Development, Amgen Inc., Cambridge,
Massachusetts 02142, United States
Preparation of tert-Butyl ((S)-4-Methyl-1-((R)-2-meth-
yloxiran-2-yl)-1-oxopentan-2-yl)carbamate (4). Aqueous
sodium hypochlorite (10 wt %, 3.4 L, 5.5 mol) was added over
90 min to a precooled (−10 °C) solution of pyridine (9.0 L, 13
L/kg) and water (0.84 L, 1.2 L/kg) at such a rate that the
internal temperature remained below −5 °C. A sample of the
bleach/pyridine suspension was pulled, and a use test was
performed to ensure activity. A solution of enone 3 (700 g,
2.74 mol) in pyridine (0.78 L, 1.1 L/kg) was then charged over
45 min while the internal temperature was maintained below
−10 °C. The batch was aged for an additional 4 h at −15 °C
and then diluted with precooled (−10 °C) n-hexane (21 L, 30
L/kg) and water (5.6 L, 8.0 L/kg) at such a rate to maintain
the internal temperature below −10 °C. The temperature of
the biphasic mixture was adjusted to 0 °C, and the phases were
allowed to separate. The lower aqueous layer was separated
and back-extracted with n-hexane (14 L, 20 L/kg). The
combined organic extracts were then washed sequentially with
water (16 L/kg, 11.2 L), 10 wt % NaHSO4 (10 kg/kg, 7 kg)
two times, water (10 L/kg, 7.0 L), 30 wt % Na2S2O3 (8 kg/kg,
5.6 kg), and water (10 L/kg, 7.0 L) two times. The resulting
solution was distilled to an oil to provide crude epoxyketone 4
(284 g, 42% solution assay yield, 32.9 LCAP) with spectra
identical to that previously reported in the literature.17
William E. Lake − Process Development, Amgen Inc.,
Cambridge, Massachusetts 02142, United States
Neil F. Langille − Process Development, Amgen Inc., Cambridge,
Massachusetts 02142, United States
Susan P. Lucas − Process Development, Amgen Inc., Cambridge,
Massachusetts 02142, United States
Jenil Patel − Process Development, Amgen Inc., Cambridge,
Massachusetts 02142, United States
William Powazinik IV − Process Development, Amgen Inc.,
Cambridge, Massachusetts 02142, United States
Scott W. Roberts − Process Development, Amgen Inc.,
Thousand Oaks, California 91320, United States;
orcid.org/0000-0001-8963-3282
Chris Scardino − Process Development, Amgen Inc., Thousand
Oaks, California 91320, United States
John L. Tucker − Process Development, Amgen Inc., Thousand
Oaks, California 91320, United States
Simone Spada − Process Development, Amgen Inc., Cambridge,
Massachusetts 02142, United States
Alicia Zeng − Process Development, Amgen Inc., Cambridge,
Massachusetts 02142, United States
Shawn D. Walker − Process Development, Amgen Inc.,
Cambridge, Massachusetts 02142, United States
General Procedure for Racemization Studies. To a 2
dram vial were added enone 3 (25 mg), n-hexane (0.25 mL),
and mesitylene as an internal standard. The appropriate solid
(12.5 mg) was then added to the vials, and the mixtures were
aged with stirring at the prescribed temperature. Samples were
pulled for analysis via chiral HPLC.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.0c00051
Notes
The authors declare no competing financial interest.
2,6-Di-tert-butyl-4-hydroxy-4-methylcyclohexa-2,5-
dien-1-one (oBHT). To a suspension of iodobenzene
diacetate (93.2 g, 289 mmol) in MeCN (100 mL, 2.4 mL/g)
and water (106 mL, 2.5 mL/g) at 20 °C was added a solution
of BHT (42.5 g, 193 mmol) in MeCN (150 mL, 3.5 mL/g),
ACKNOWLEDGMENTS
■
We thank Tsang-Lin Hwang for NMR characterization and
Jessica Mix for support in the kilo lab for demonstration
purposes.
H
https://dx.doi.org/10.1021/acs.oprd.0c00051
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
pubs.acs.org/OPRD
Article
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https://dx.doi.org/10.1021/acs.oprd.0c00051
Org. Process Res. Dev. XXXX, XXX, XXX−XXX