Process Development of a Second Generation β-Amyloid-Cleaving Enzyme Inhibitor - Improving the Robustness of a Halogen-Metal Exchange Using Continuous Stirred-Tank Reactors

Article abstract of DOI:10.1021/acs.oprd.1c00126

Process development for the synthesis of a second generation β-amyloid-cleaving enzyme (BACE1) inhibitor (1) is described. The lithiothiazole addition to the isoxazolene (5) under batch conditions was not scalable because of reaction gelling and anion instability. A continuous stirred-tank reactor flow process was developed and successfully executed on the 70 kg scale in multiple runs. In a head-to-head comparison between the continuous and batch processes, the former was clearly superior as it gave a higher yield (80 vs 63%) of the adduct (4) and better reaction control for handling the unstable lithiothiazole as a reaction intermediate. Subsequently, 4 underwent Pd-catalyzed amination with t-butyl carbamate, reductive cleavage of the N-O bond, thioamidine cyclization, and deprotection of the Boc group to provide hydropyranothiazine 2. The synthesis of 1 was completed by amidation with 5-(difluoromethoxy)picolinic acid and the successive deprotection of the benzamide group with either Silicycle-diamine or l-lysine.

Full text of DOI:10.1021/acs.oprd.1c00126

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Article
Process Development of a Second Generation β‑Amyloid-Cleaving
Enzyme InhibitorImproving the Robustness of a Halogen-Metal
Exchange Using Continuous Stirred-Tank Reactors
Bryan Li,* Richard W. Barnhart, Amelie Dion, Steven Guinness, Alan Happe, Cheryl M. Hayward,
Jeffrey Kohrt, Teresa Makowski, Mark Maloney, Jade D. Nelson, Asaad Nematalla,
J. Christopher McWilliams, Zhihui Peng, Jeffrey Raggon, John Sagal, Gerald A. Weisenburger,
Denghui Bao, Miguel Gonzalez, Jiangping Lu, Mark D. McLaws, Jian Tao, and Baolin Wu
Cite This: Org. Process Res. Dev. 2021, 25, 1440−1453
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ABSTRACT: Process development for the synthesis of a second generation β-amyloid-cleaving enzyme (BACE1) inhibitor (1) is
described. The lithiothiazole addition to the isoxazolene (5) under batch conditions was not scalable because of reaction gelling and
anion instability. A continuous stirred-tank reactor flow process was developed and successfully executed on the 70 kg scale in
multiple runs. In a head-to-head comparison between the continuous and batch processes, the former was clearly superior as it gave a
higher yield (80 vs 63%) of the adduct (4) and better reaction control for handling the unstable lithiothiazole as a reaction
intermediate. Subsequently, 4 underwent Pd-catalyzed amination with t-butyl carbamate, reductive cleavage of the N−O bond,
thioamidine cyclization, and deprotection of the Boc group to provide hydropyranothiazine 2. The synthesis of 1 was completed by
amidation with 5-(difluoromethoxy)picolinic acid and the successive deprotection of the benzamide group with either Silicycle-
diamine or L-lysine.
KEYWORDS: BACE1, halogen-metal exchange, CSTR, continuous process, lithiothiazole
In this route3b (Scheme 2), 2,4-dibromothiazole (6) under-
1. INTRODUCTION
went lithium-halogen exchange regioselectively at the C-2
position under cryogenic conditions. The rigid isoxazolene (5)
ring structure allowed a high degree of stereocontrol leading to
thiazole anion addition to the convex face, proving 4 as the
sole product. Nevertheless, there were multiple complications
in scaling this reaction:
β-site amyloid precursor protein-cleaving enzyme 1 (BACE1)
is involved in the production of the β-amyloid (Aβ) peptide, a
neurotoxin which is believed to play a key role in the
development of Alzheimer’s disease.1 Reducing levels of Aβ by
inhibition of BACE1 have been vigorously pursued as a
therapeutic strategy to improve cognitive function in
Alzheimer’s models.2 Among these candidates, thiazinamines
have shown potential as selective BACE1 inhibitors with
improved potency and reduced side effects and are the subject
of ongoing clinical development.3 Recent endeavors from the
Pfizer research team led to the discovery of a potent, selective
BACE1 inhibitor (1, Scheme 1)3b targeting Alzheimer’s
disease. The compound displayed good safety, tolerability,
and pharmacokinetics from phase I clinical trials.4 As BACE1
was an ongoing drug-discovery program, our exploratory active
pharmaceutical ingredient support strategy was to develop
convergent synthesis such that it would accommodate
structural modifications of each of the substructures of the
thiazinamine series (A, B, and C, Scheme 1) from SAR studies
without compromising the delivery timelines.
1 Gelling of the reaction mixture. On gram scale reaction
in the laboratory, the solution of 5 in toluene/THF
(10:1 ratio) was added to the reaction mixture
containing lithiothiazole 7 as fast as possible. Within
minutes of the addition, gelling occurred causing the
stirring to stop. It was found that the reaction gelling
would not occur if the reaction mixture was maintained
at −90 °C during the addition of isoxazolene (5). While
this approach effectively resolved the gelling issue, the
reaction yield was modest at 57% yield after chromato-
graphic isolation. Furthermore, batch size was limited to
Received: April 12, 2021
Published: May 24, 2021
2. RESULTS AND DISCUSSION
2.1. Thiazole Anion Addition to Isoxazolene 5. To
support the continuous development of the thiazinamine series
of clinical candidates, our efforts were focused on enabling a
similar route3b by the Discovery chemistry team for scale up.5
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Scheme 1. Retrosynthesis of 1
Scheme 2. Lithiation of 2,4-Dibromothiazole and Synthesis of 4
∼9 kg in a 500 L reactor due to the stringent
reaction, coupled with the poor solubility of 6 in all
aprotic solvent systems studied, made it a challenge for
scale up. Lithiothiazole 7 was capable of undergoing
further lithiation to give dilithiated species 8 and 9,
which further reacted with 6 to generate monolithiated
species 10 and 11, respectively, giving rise to impurities
requirement for heat exchange.
2 Anion stability issue. During the lithium-bromo
exchange, n-BuLi was slowly added to a suspension of
2,4-dibromothiazole (6) in a solvent under cryogenic
conditions.6 The extremely exothermic Li−Br exchange
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Figure 1. UPLC reaction profile of lithiothiazole addition to isoxazolene 5.
Table 1. Study of the Impact of the Toluene/THF Ratio on
Reaction Fluidity
12 and 13. The formation of 14 was also observed from
the reaction of dilithiothiazole 9 with 5. A des-bromo
impurity 15 was also noted, albeit at a relatively low
level, presumably from the addition of 8 to 5. A typical
chromatogram of the reaction under very careful control
of all operational steps is shown in Figure 1. When the
reaction was carried out above the 10 g scale, the UPLC
reaction profile started to become messy because the
reaction time was prolonged to accommodate both
stages of n-BuLi and isoxazolene addition. Elevating the
reaction temperature above cryogenic conditions was
known to be detrimental giving higher levels of both
isomeric and other impurities.
u1
ratio
reaction time conversion of
entry toluene/THF
(min)
5 (%)
comments
1
2
3
2:1
4:1
10:1
60
60
60
97
97
97
fluid below −75 °C
gelling at −69 °C
gelling at
−55 to −65 °C
that THF also acts as a coordinating solvent to enhance the
reactivity by dissociating n-BuLi hexamers in hexane to form
more reactive tetramers and dimers.7 Conversely, the
significantly increased amount of THF has a deleterious effect
on the reaction kinetics, and this necessitated a super-
stoichiometric amount of BF3·OEt2. Overall, the use of THF
as a cosolvent was a good trade-off by allowing the reaction to
be operable on scale by eliminating the gelling concern.
2.1.2. Methanesulfonic Acid Salt Formation of 4. In
parallel to the reaction development, efforts were directed to
streamline the workup and isolation. As the calculated pKa of 4
is 3.28 (in water), there was a reasonable hope to find a
suitable salt of the product to facilitate product isolation. A salt
screen led to discovery of a highly crystalline methanesulfonic
acid salt of 4 (4·MSA). A solvent screen identified acetonitrile
as being ideal for crystallization, which allowed direct isolation
of 97.7% pure product from the ∼80% pure crude mixture
(Table 2). Further enhancement of the purity of 4 could be
accomplished with a salt break from methanol and aqueous
NaOH. High recovery of the free base 4 could be achieved by
recrystallization from methanol. With this process, laboratory
scale up afforded crystalline product 4 as a free base in 77.4%
isolated yield with 99.2% purity. It is noteworthy that 4-
bromothiazole (16), which would interfere in the downstream
reaction, was completely rejected. In this improved product
isolation protocol, carbon dioxide quenching was no longer
required and a simple aqueous NH4Cl solution quench was
used instead to enable more convenient scale up.
3 Chromatographic isolation of the product 4. While pure
4 was a crystalline solid, direct crystallization of the
product from the reaction mixture was elusive. This was
attributed to a modest crude product purity of ∼80% or
lower, which impeded the crystallization; it could not be
facilitated by seeding. Under the circumstance, silica gel
chromatography was utilized for product isolation. A
major byproduct as shown in Figure 1 was bromothia-
zole 16, generated from excess lithiothiazole 7 after
reaction quench. In early attempts, carbon dioxide was
used to quench the reaction, followed by aqueous
quench, which converted 7 to its corresponding
carboxylic acid 17, providing a purging handle for
aqueous extraction. However, it introduced operational
complexity for further scale up.
As the project quickly moved through the development
pipeline, we were tasked to develop a scalable process under an
expedited timeline. It was clear that the anion addition step
was a bottleneck for the overall synthesis, and we started to
tackle multiple challenges (vide supra) simultaneously.
2.1.1. Selection of Solvents. The solubility of dibromothia-
zole 6 in all solvents was rather poor at cryogenic temperatures.
As the reaction appeared to perform best in a THF/toluene
mixture, we focused on the volumetric ratio of these solvents
for the reaction. A quick survey showed that the gelling issue
was closely related to the volumetric ratio of toluene and THF.
When the toluene/THF ratio was 10:1 and total volume was
∼40 vol, a serious gelling problem was observed. At the same
total solvent volume, gelling was no longer observed at −75 °C
or lower temperature when the solvent ratio of 2:1 (v/v) was
employed (Table 1). While the cause for gelling is not fully
understood, it appears to be related to complexation of
isoxazolene 5 with BF3·OEt2. THF complexes with BF3
competitively, hence breaking up the gelling. It is noteworthy
2.1.3. Adapting the Process to Continuous Flow. The
highly exothermic nature of the Li−Br exchange dictates the
slow combination of n-BuLi and 6 to control the reaction
temperature.10 The adiabatic temperature rise could be as high
as 61.9 °C under a worst-case scenario.8 Since the surface-to-
volume ratio decreases substantially as the reaction is scaled up
in a batch reactor, a dramatic decrease in heat exchange
capacity results. The poor solubility of dibromooxazole 6 in the
reaction solvent(s) further complicated the matter. On an early
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Table 2. Workup with Methanesulfonic Acid Salt Formation for 4
UPLC profile (area %)
16
materials
5
4
yield
product loss
crude product before salt formation
salt formation with MSA in MeCN
2.4%
0.1%
10.7%
0.1%
3.0%
11.6%
0.4%
46.6%
ND
79.1%
97.7%
10.1%
99.2%
49.0%
83.6%
81.8%
MSA salt
mother liquor
product
2.1%
3.1%
a
salt break in MeOH
77.4%
mother liquor
10.2%
a
Overall yield from the reaction, salt formation, and salt break.
Figure 2. PFR schematic diagram for the continuous flow process.
Figure 3. Laboratory CSTR design for the continuous process.
9.0 kg scale batch reaction in a 500 L reactor, the addition time
was 12.7 h. As discussed earlier, prolonged Li−Br exchange
time is detrimental to the reaction due to the formation of
impurities 12, 13, 14, and 15. Since the anion stability and the
long duration of the Li−Br exchange reaction were major
concerns, it became evident that continuous flow technology
could be more optimal for this reaction. Continuous processes
offer many advantages over batch methods, including precise
control of stoichiometry, reaction time and temperature, high
reproducibility, and often better reaction yields.9 The much
higher surface area-to-volume ratio under flow conditions
allows highly efficient heat transfer. When coupled with a
much smaller volume in the reaction train, safety hazards in
handling exothermic reactions are minimized. This is
particularly attractive as the stability of lithiothiazole 7 is a
major concern in this process. Thus, subsequent development
of this critical transformation was directed toward enablement
of a continuous option. Both plug-flow reactor (PFR) and
continuous stirred-tank reactor (CSTR) approaches were
considered. A PFR approach was first tested.10 The solution
of 6 in toluene at RT was pumped into a coil and mixed with
n-BuLi solution in hexane/toluene via a T-mixer. The lithiation
was designed to take place in the first coil and the resulting
anion would react with 5 in a second coil. Both coils were set
in a cooling bath between −60 and −70 °C (Figure 2), and
precooling of each feed stream was carried out to address the
high exothermicity. Unfortunately, tube clogging at the T-
mixer was observed due to precipitation of 6 at this low
temperature. The clogging issue was mainly due to the
significantly decreased solubility of 6,11 and this appeared as an
insurmountable problem under PFR conditions.
CSTR arrangements are more able to handle heterogeneous
mixtures, so we redirected our laboratory assessment (Figure
3). Thus, the solution of 6 in toluene at RT and the solution of
n-BuLi (in hexane, diluted with toluene) were independently
pumped (precooled at −20 to −30 °C before mixing at CSTR)
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a
Table 3. Flow Parameter Study of the CSTR Process at −60 to −70 °C
conditions
residence time
IPC (area %)
b
entry
toluene/THF volume ratio
total volume (vol)
lithiation (min)
anion addition (min)
5
16
4
in situ yield
1
2
3
4
2:1
2:1
2:1
2:1
45
45
30
15
8
11
6
25 × 2
15 × 2
20 × 2
10 × 2
3.0%
2.4%
2.8%
2.4%
12.8%
10.7%
10.9%
11.6%
77.6%
81.4%
80.9%
79.1%
82.4%
85.4%
84.9%
83.6%
30
a
b
20 g of 5 was used for each experiment. Potency adjusted yield by UPLC area %.
Figure 4. CSTR (5 × 13 L) for continuous process scale up.
into and mixed in the first CSTR for 15 min at −60 to −70 °C.
The in situ-generated anion solution overflowed by gravity into
a second CSTR, which contained a solution of 5 premixed with
BF3·OEt2 in THF that was continuously charged. The
combined reaction mixture stayed in the second and third
CSTR at −60 to −70 °C for 25 min each (τ, average residence
time of volume/flow rate) each, before the reaction was
quenched by aqueous NH4Cl solution in the reservoir tank. In
a representative laboratory CSTR run (Figure 3), the collected
reaction mixture indicated ∼97% conversion of 5 to 4 in 82.4%
in situ yield. It was notable that the contents in CSTR1 through
CSTR3 did not appear fully homogeneous but remained highly
fluid.12 The gravity overflow between reactors enabled mass
transfer obviating any need for a mechanical pumping
mechanism. At the designed flow rates, passage time for the
reaction mixture through the well-insulated transfer lines (1/4″
ID) was on the order of seconds, and clogging was not
observed.
impurities, which was attributed to the shorter lithiation time
compared to batch conditions.
2.1.4. CSTR Design for Production. We next moved to scale
up the continuous process and selected 5 × 20 L jacketed
stainless-steel CSTRs installed in series, as shown in Figures 4
and 5. The CSTRs were designed with two exit outlets
allowing adjustment of the maximum residence volume at
either 13 or 20 L. To reduce safety concerns with handling n-
BuLi, the first CSTR was used to prepare and precool (−75 to
−85 °C) a solution of n-BuLi in toluene. Lithiation was
designed to occur in the second CSTR, with the last three
Using the same CSTR setup, the impact of residence time
and solvent volume was investigated (Table 3). The toluene/
THF ratio was kept at 2:1 to maintain fluidity of the reaction
mixture, but the total reaction solvent was reduced from 45 to
30 vol successfully. Residence times for both lithiation (6−15
min) and anion addition (20−50 min) stages were found to
have a good degree of flexibility, which enabled a wide process
window for scale up reactor design. With the CSTR process,
12, 13, 14, and 15 were no longer observed as major
Figure 5. In-series CSTR used in the continuous process.
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Table 4. Comparison of Process Parameters of the Previous and Updated Process
process
previous batch mode
batch mode with improved conditions
24 kg
flow mode with improved conditions
scale
reactor size
(L)
9.0 kg
500 L
70 kg (2×) & 43 kg
13 L × 5 CSTR
1500 L
lithiation
Solvents
Temperature −70 to −77 °C
reaction time 14.8 h
toluene/THF (2:1)
toluene/THF (4.4:1)
−94 to −87 °C
12.7 h
toluene/THF (2:1)
−70 to −77 °C
32 min
anion
addition
Solvents
toluene/THF (2:1)
toluene/THF (4.4:1)
toluene/THF (2:1)
Temperature −58 to −62 °C
−92 to −97 °C
−58 to −62 °C
reaction time 2.1 h
9.7 h
65 min
work
up/isolation
CO2 quench SiO2 chromatography
recrystallization with EtOAc/heptane
aq NH4Cl quench MSA formation
recrystallization with MeOH/NaOH
(aq)
aq NH4Cl quench MSA formation
recrystallization with MeOH/NaOH
(aq)
isolated yield
product
purity
57%
96%
63%
99%
78−80%
99%
a
Scheme 3. Synthesis of Hydropyranothiazine 2
a
Reagents and conditions: (a) Mo(CO)6, CH3CN, H2O, NaBH4, 70 °C, 95%; (b) benzoyl isothiocyanate, DCM, rt, 66%; (c) Ghosez’s reagent,
DCM, rt, 77%; (d) BocNH2, Pd2(dba)3, tBuXPhos and K2CO3, toluene, reflux. 80−85% in situ; (e) BocNH2, Pd2(dba)3, tBuXPhos and K2CO3,
toluene, reflux. 78%; (f) Raney-Ni, H2, 2-PrOH, 25 to 35 °C, 69% overall 2 steps d and f; (g) benzoyl isothiocyanate, THF, 50 °C, not isolated; (h)
48 to 60 °C, not isolated; (i) benzoyl isothiocyanate, THF, 50 °C, not isolated; (j) CDI, THF, 50 °C, not isolated; (k) aq HCl, EtOAc. RT, 82%
overall 4 steps g, j, h and k; (l) concn HCl, then quenched to TEA/EtOAc, 70%.
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CSTRs dedicated to anion addition. Material transfer between
CSTRs was enabled by gravity via insulated stainless-steel
transfer lines (1/4″ ID). Finally, the product stream was
directed to a 5000 L glass-lined reactor containing an aqueous
NH4Cl solution for reverse batch quenching and isolation.
Special attention was paid to the heat exchange requirements
as the Li−Br exchange reaction is a fast and extremely
exothermic reaction (vide supra). Due to limitations of the
available cooling media (−70 °C at its lowest temperature)
and the limited cooling capacity available within the plant, flow
rates were decreased to maintain the continuous lithiation
temperature and subsequent anion addition at −55 to −65 °C.
The residence time for lithiation and anion addition was
increased to 32 and 65 min, respectively, compared to 15 and
50 min employed in laboratory pilots. Fortunately, the reaction
conversion and impurity profile were not affected by the longer
reaction time (Tables 3 vs 4). This may be explicated by the
fact that processing duration of the lithiation in the CSTR
mode was only ∼3% of that in the batch mode (Table 4).
Three batches with 43−70 kg input were completed using this
CSTR process, providing product in 78.1−80.3% isolated yield
with 99% purity (Table 4).
In this campaign, we also ran a head-to-head comparison
between the continuous process and the batch process
incorporating all improvements. Thus, a 24 kg scale was
carried out under batch conditions using a 1500 L stainless-
steel reactor. The isolated yield was ∼15% lower than that of
the flow process. It was noticed that in batch, there was
significantly more tar-like material formed that may be
attributed to the much longer lithiation time (888 min vs 32
min). In addition, the flow process also provided other benefits
such as reduced equipment size (Table 4), reduced safety risk
(much smaller amount of n-BuLi in the reactor train), and
reduced failure risk, that is, allowing reaction control by timely
adjustment of the flow rate or pausing the feed as needed;
therefore, the entire batch of starting materials was not
committed should there be an unforeseen equipment or
reaction failure.
identified. The reaction provided 4a in 80−85% in situ yield,
but chromatography was required to isolate the product. Thus,
we chose to telescope the crude product into the next step for
scalability purpose and avoid yield loss.
2.2.2. N−O Reductive Cleavage. Raney-Ni-catalyzed
hydrogenolysis was found optimal and it offered quantitative
conversion to amino alcohol 21. Isopropanol was found to be a
good solvent for the reaction, while use of methanol gave rise
to an N-methylated impurity. The crude product 4a could be
telescoped directly into the Raney-Ni hydrogenolysis. The
amino alcohol 21, with a calculated pKa of 6.1, offered an
opportunity to remove the organic soluble ligands and
impurities (from the upstream Pd-coupling reaction) with an
acid−base aqueous extractive workup.13 Isolation of 21 was
accomplished with good yield and high purity by crystallization
from toluene. However, we found it more convenient and
efficient to carry the crude product forward as a solution to the
immediate downstream step, where purity upgrade oppor-
tunities were more apparent (vide infra).
2.2.3. Thioamidine Cyclization. While the formation of
thioamidine 22 was straightforward, subsequent ring closure
under acid catalysis (Ghosez’s reagent, methanesulfonic and
trifluoroacetic acids) was complicated by the formation of up
to 20% of oxamidine 25 (Scheme 3). In light of the competing
cyclization driven by the hydroxyl group as a nucleophile, we
envisioned that this pathway could be blocked by forming bis-
benzoyl isothiocyanate adduct 23 via the addition of two
equivalents of BzNCS such that the sulfur-mediated
nucleophilic displacement of the BzNHC(S)O group becomes
the predominant pathway for cyclization. During the reaction,
22 was observed to form initially as an intermediate and then
slowly (6 h at RT) converted to 23. Upon heating the reaction
mixture, 2a was generated as a major product. Using this
method, the oxamidine byproduct 25 was reduced to less than
3% and was fully rejected during crystallization of the product.
This protocol worked sufficiently well for delivery of the first
two campaigns (4.4 and 25 kg, respectively). In the third
campaign, however, we employed an improved process
developed by DeBaillie and co-workers14 for an analogous
transformation, in which 1 equiv of CDI was used to convert
22 to 24, and subsequent heating gave 2a with a cleaner
reaction profile. The use of CDI helped reduce stoichiometry
of odorous BzNCS to 1.0 equiv, although oxamidine 25 was
still present at similar levels in the reaction mixture. While 2a is
a crystalline solid, the product isolation by crystallization
incurred substantial loss in all solvent systems examined. The
crude product was therefore telescoped directly into the next
step.
2.2. Synthesis of Hydropyranothiazine 2. In the
original synthesis,3b the preparation of 2 proceeded with a
reductive cleavage of the N−O bond to give amino alcohol 18,
followed by the formation of thioamidine 19 and ring closure
to give 20 (Scheme 3). To keep the C-3 bromo group on the
thiazole intact, N−O bond cleavage was accomplished using
Mo(CO)6 and NaBH4. The removal of Mo-related byproducts
and the addition of NaBH4 proved challenging when scaled to
tens of grams. The addition of NaBH4 required careful control
as it was accompanied by hydrogen gas evolution and a large
exotherm. Since this was clearly not ideal for scale up, we
reordered the synthetic steps by moving the amination step
upfront (Scheme 3), therefore circumventing the need of
chemoselective reduction of the hydroxylamine in the presence
of the thiazoyl bromide.
2.2.4. Deprotection of the Boc Group to Form 2. When
crude 2a was subjected to the Boc deprotection reaction by
treatment with conc. HCl, the HCl salt of 2a unexpectedly
crystallized from ethyl acetate with the Boc group intact. This
offered a great opportunity for impurity purge and allowed
isolation of 2a·HCl in high purity and yield. Robustness of the
HCl salt formation was ensured as up to 2 equiv of concn HCl
(aq) solution at RT could be tolerated and resulted in no sign
of Boc removal. Deprotection of the Boc group from 2a·HCl
was readily effected with methanolic HCl solution (6 equiv).
Surprisingly, the isolation of 2 as a free base or HCl salt turned
out to be a challenge. Direct quench of the Boc deprotection
reaction by adding a base led to product decomposition that
was not fully understood. Nevertheless, inverse quench by
adding the reaction mixture to an agitated mixture of aqueous
2.2.1. C−N Cross Coupling. The amination of 4 worked
well using dimethoxybenzylamine (DMB-NH2) to give 4b
(Scheme 3). However, deprotection of the DMB group in the
downstream step resulted in moderate purity of the product
and modest yield despite the use of chromatography for
product isolation. Considering the convenience of Boc
deprotection, t-butyl carbamate (BocNH2) was selected as
the coupling partner. After briefly screening both Pd- and Cu-
mediated coupling reactions with BocNH2, a set of conditions
t
employing Pd2(dba)3, BuXPhos, and K2CO3 in toluene were
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a
Scheme 4. Endgame Synthesis of 1
a
Reagents and conditions: (a) T3P, EtOAc, RT; iPrOH, aq HCl, 93%; (b) Silicycle-diamine, DCM/THF, 93%; (c) L-lysine, 1-PrOH, water, 90 °C;
aq NaOH; 86%. (d) aq HCl, MEK, 87%.
base (Na2HPO4 or TEA) and organic solvent (EtOAc)
addressed the problem.
requirements perfectly; the Bz-Lysine byproduct 27 is water-
soluble so it could be expediently rejected in the aqueous
workup under appropriate pH. The reaction was carried out in
nPrOH/water at reflux. Upon reaction completion, water was
added, followed by a heat/cool cycle to help particle size
growth of the crystalline product (1) to facilitate the filtration.
The pH was then adjusted by the addition of aqueous NaOH
to allow for the removal of 27, its residual level was found to be
critical on the overall filtration time of the reaction mixture.
The basic aqueous isopropanol mixture was noted to be quite
viscous at cool temperature (0 to 20 °C), which dramatically
slowed down the filtration. By increasing the temperature of
prefiltered slurry to 40 °C, the filtration proceeded much
speedily. With those improvements, the filtrate rate was
increased by nearly 50-fold; in a 67 kg batch, the filtration was
completed in 2 h, and the product was isolated in 86% yield
with 99.6% purity.
2.3. Endgame Development. 2.3.1. Amidation Reac-
tion. The coupling of hydropyranothiazine 2 with 5-
(difluoromethoxy)picolinic acid (3) was straightforward
(Scheme 4) with the use of propane phosphonic acid
anhydride (T3P solution in ethyl acetate) as the coupling
reagent. The product isolation was streamlined with the
addition of aqueous HCl and isopropanol, which resulted in
direct product crystallization. Upon removal of ethyl acetate by
concentration, the product (26) was isolated in 95% yield. The
process was robust as it offered great opportunities to purge
most incoming impurities from 2a, notably a carried forward
impurity from des-bromo compound (15) that might be
present in the starting material.15 The reaction was found
readily adaptable to a continuous flow process, which was
demonstrated in the laboratory using either T3P or EDC/
HOPO as coupling reagents to give comparable yields and
quality,16 although a batch approach was implemented for
initial manufacturing.
2.3.2. Benzamide Deprotection. Deprotection of the
benzamide group from 26 required differentiation (Scheme
4) between the benzamide and picolinamide. Methoxyamine
was identified initially, and it offered high selectivity, but the
byproduct N-methoxybenzamide could not be easily rejected
as it cocrystallized with the desired product. To circumvent
this issue, the use of SiliaMet Diamine (Si-DIA) was explored;
it was found to be highly chemoselective and gave an extremely
clean reaction profile. The workup was straightforward by
simply removing the silica gel by filtration as the benzoyl group
coming off 26 was solid-phase bound. This protocol was
employed in the first and second campaigns to give 86−88%
yield of 1. With the increased clinical demand, we recognized
that the use of Si-DIA was not a long-term option due to its
relatively high cost. Thus, alternative methods were inves-
tigated. Nucleophilic amines (n-butylamine, pyrrolidine, and
piperidine) were found to offer reasonable chemoselectivity.
However, the corresponding amide byproducts were difficult
to remove in the product isolation. With this in mind, we
envisioned that a water-soluble amine could be the answer.
Thus, L-lysine, an inexpensive commodity chemical, fit the
2.3.3. Final HCl Salt Formation. As the mono-hydro-
chloride salt was selected for clinical development, the final
processing involved an HCl salt formation. HCl (aq) was used
in combination with methyl ethyl ketone; the protocol offered
good recovery of 1·HCl as a crystalline product in excellent
purity.
3. SUMMARY
The development of an efficient synthetic process for
manufacture of thiazolyl hydropyranoisoxazole (4) has been
successfully achieved. The CSTR process established a robust
and reliable platform for scalable synthesis featuring a
continuous Li−Br exchange reaction, followed by the addition
of the lithiothiazole to the isoxazolene (5). By taking advantage
of significant modifications of the first-generation process to
avoid issues with gelling, in conjunction with developing a
methanesulfonic acid salt formation for product isolation, the
overall efficiency of the synthesis and the product quality was
dramatically improved, resulting in 78−80% isolated yield with
99% purity at up to the 70 kg scale. The CSTR continuous
flow process exhibited significant advantages over the batch
process in terms of yield, product quality, and risk manage-
ment. A gravity overflow design obviated the need for multiple
pumping mechanisms for transfer of the heterogeneous
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reaction mixture. It is noteworthy that the cooling capacity of
the jacketed CSTRs was a key factor impacting process
efficiency for these highly exothermic lithiation and anion
addition reactions to prepare the isoxazolene. The subsequent
sequence of the synthesis was successfully developed, most
notably with the novel use of SiliaMet Diamine and L-lysine for
the deprotection of the benzamide group, which allowed
convenient isolation of the product.
chloride solution (175 kg). The mixture was warmed to 15−25
°C and filtered through a celite pad to remove insoluble
materials. The filter cake was rinsed with toluene (78 kg). The
organic phase of the filtrate was separated and washed with aq
12% sodium carbonate solution (50 kg), followed by 10% aq
sodium chloride (132 kg). The organic phase was concentrated
at ≤45 °C under reduced pressure ≤−0.08 MPa until 50−75 L
of the mixture remained. Then, acetonitrile (120 kg) was
added into the mixture. Then, it was sampled for toluene
residual by GC analysis until toluene residual ≤20%. The
mixture was cooled to 0−10 °C; then, methanesulfonic acid
(16.9 kg, 176 mol) was added into the mixture. The mixture
was stirred at 5−10 °C to induce crystallization and held for
2−4 h. The mixture was filtered, and the filter cake was rinsed
with acetonitrile (47.0 kg). The wet filter cake (4-MSA) was
combined with methanol (198 kg) in a reactor and stirred until
the solid dissolved. It was then cooled to 15 °C. 2.3% sodium
hydroxide solution (300 kg) was added, and the pH analysis
was confirmed at >9. The mixture was concentrated at ≤45 °C
under reduced pressure ≤−0.08 MPa until ∼400 L remained.
The mixture was sampled for methanol residual at ≤15%. The
mixture was cooled to 10 °C and stirred for 3 h. The mixture
was filtered and the filter cake was rinsed with purified water
(60.0 kg × 2). The filter cake was vacuum oven-dried at 25−35
°C until KF reached ≤1.0% and methanol residual reached
≤0.5%. 34.0 kg gross weight (33.5 kg corrected for potency,
63.2% yield) product 4 was isolated as a light-yellow solid
(achiral purity 98.4%; chiral purity 99.0%). The spectroscopic
data were identical to those reported in the literature.3b
4.1.1.2. CSTR Continuous Flow Mode. Pump A. Directly
feeding from anhydrous toluene as a solvent line.
4. EXPERIMENTAL SECTION
4.1. General. NMR chemical shifts are reported in parts
per million (ppm) with TMS as an internal standard. Reagents
and solvents were obtained from commercial sources and used
without further purifications. The reactions were monitored
offline by UPLC or HPLC during the laboratory development
and continuous flow production. UPLC method: Agilent SB-
CN column (2.1 × 50 mm) with mobile phases A (0.05% TFA
in water) and B (acetonitrile). Detection wavelength 210 nm;
flow rate 0.65 mL/min, 0−2.9 min, ramp from 5% B to 100%
B; 2.9−3.4 min, stay at 100% B; 3.4−3.5 min, ramp to 5% B;
3.5−4.0 min, 5% B. HPLC method: Zorbax Eclipse Plus C18
column; (4.6 × 100 mm; 3.5 μm, P/N: 959961-902) at 40 °C
with mobile phases A (0.1% H3PO4 in water) and B
(acetonitrile). Detection wavelength 210 nm; flow rate 1.5
mL/min, 0−8 min, ramp from 10% B to 60% B; 8−11 min,
ramp to 95% B; 11−13 min, stay at 95% B; 13−16 min, ramp
to 10% B. Chiral HPLC method: Chiralpak AD-H, 250 × 4.6
mm, 5 μm, column temperature 30 °C. Flow rate 1.0 mL/min;
mobile phase isocratic 60% n-hexane/40% EtOH with 0.05%
diethylamine, run time 19 min. GC method: column
RTX1701, 30 m × 0.32 mm ID × 1.0 μm film, Inlet
temperature is 250 °C; 160.0 kPa pressure; helium gas flow
rate 6.5 mL/min; detector temperature 270 °C; nitrogen as
make up gas H2/air/N2 = 40:450:30 (mL/min); equilibration
time 0.5 min; injection volume 1 mL; run time 17 min.
Temperature gradient: rate 25 °C/min, final temperature 250
°C, final time 4 min.
4.1.1. tert-Butyl (2-((3aR,5S,7aR)-5-Methyltetrahydro-1H-
pyrano[3,4-c]isoxazol-7a(7H)-yl)thiazol-4-yl)carbamate (4).
4.1.1.1. Batch Mode. A clean and dry 1500 L titanium reactor
was evacuated to −0.05 to −0.08 MPa and then filled with
nitrogen to normal pressure. This was repeated three times.
Toluene (210 kg) was charged into the reactor and a sample
was pulled to confirm KF ≤0.03%. The mixture was cooled to
−85 to −75 °C. 2.5 M n-BuLi solution in n-hexane solution
(63.0 kg, 227 mol, 90.9 L) was added at −85 to −75 °C. To
this reactor was added slowly a solution of 2,4-dibromothiazole
(54.0 kg, 222 mol) in a solvent mixture of toluene (210 kg)
and THF (210 kg) that was prepared in a separate reactor and
held at 0−10 °C. During the transfer, the 1500 L titanium
reactor was maintained at −79 to −70 °C, and the addition
time was 7 h 57 min. After the addition was complete, the
reaction mixture was sampled by quenching a sample aliquot
into an ammonium chloride solution and checked by HPLC to
confirm that the Li−Br exchange was complete. To this
reaction mixture was then slowly added a solution mixture of
isoxazolene 5 (24.5 kg, 173 mol) and boron trifluoride etherate
(32.3 kg, 286 mol) in THF (210 kg) that was previously
prepared. During the transfer, the 1500 L titanium reactor was
maintained at −85 to −70 °C. The transfer time was 3 h 23
min. 1 h after the transfer was complete, the reaction was
confirmed to be complete, and the reaction mixture was
warmed to −10 °C and quenched with 20% ammonium
Pump B. Directly feeding from 23 wt % n-BuLi solution in
hexane.
Pump C feed preparation. A dry and clean 500 L glass-lined
reactor was nitrogen purged. Toluene (204 kg) was added to
the reactor and the mixture was sampled for KF analysis until
KF ≤0.03%. At 15−30 °C, 2,4-dibromothiazole (79.0 kg, 325
mol) was added into the mixture and stirred until the solid
dissolves completely by visual check (this feed solution was
made up twice to support one continuous run).
Pump D feed preparation. Anhydrous THF (70.2 kg) was
added into the 3000 L glass-lined reactor and the mixture was
sampled to confirm KF ≤0.05%. Additional THF (615 kg) was
added, and the internal temperature was maintained at 15−30
°C. Isoxazolene 5 (70.0 kg, 496 mol) was added and stirred
until a solution was obtained. Borontrifluoride etherate (94.6
kg, 838 mol) was added into the abovementioned mixture at
15−30 °C. After addition, the mixture was stirred for 0.5−1 h
and held for use.
4.1.1.3. Cooling of the CSTR Prior to Start. The first- and
second-stage CSTR jacket temperature were cooled to −70 to
−90 °C and the third-, fourth-, and fifth-stage CSTR jacket
temperatures were cooled to −60 to −75 °C and then
maintained for at least 0.5−1 h.
4.1.1.4. Setup of Pump Flow Rates. The flow rate of feed
pump A was set at 141.0 g/min, pumps B at 45.0 g/min, pump
C at 181.5 g/min, and pump D at 199.5 g/min. The adjusting
solvent was toluene (59.5 kg). When toluene was pumped into
the fifth-stage CSTR, the mixture was sampled from the lower
port of the fifth-stage CSTR for KF analysis until KF ≤0.03%.
4.1.1.5. Receiving Tank. 0−10 °C ammonium chloride
(70.0 kg) solution in water (2309 kg).
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4.1.1.6. System Startup and Shutdown. The jacket
temperatures of the CSTRs were achieved to the specified
ranges vide supra. Both pumps A and B were started
simultaneously. Feed pump C was timed to be switched on
when CSTR-1 was about to overflow to CSTR-2, which was
∼60 min after the start of pumps A and B. Approximately 20
min after the start of pump C, feed pump D was switched on.
The reaction was monitored by HPLC analysis at CSTR-2 for
the conversion of 6 and at CSTR-5 for both the conversion of
5 and the formation of 4. Upon completion of solution D
feeding, all pumps were stopped, and the CSTRs were stirred
for 0.5−1 h at −55 to −65 °C. The contents of the last three
CSTRs were transferred to the receiving tank, and the first two
CSTRs were diverted to a separate quench container for
disposal. The combined quenched reaction mixture was filtered
through a celite pad (20 kg), and the resulting mixture was
phase-separated. The organic phase was washed with sodium
carbonate (18.0 kg) solution in water (341 kg), followed by
saturated brine solution (385 kg). The resulting organic phase
was fully concentrated under partial vacuum. Acetonitrile (343
kg) was added, and the resulting solution was cooled to 0−10
°C. Methanesulfonic acid (47.7 kg, 496 mol) was added slowly,
and the resulting mixture was stirred for 1−3 h at 5−10 °C to
induce crystallization. Upon further stirring for 1−2 h at the
same temperature, the slurry was filtered, and the filter cake
was rinsed with acetonitrile (4 × 34 kg). The resulting wet
filter cake as a crude 4-MSA salt was combined with methanol
(560 kg) at 5−15 °C. Aqueous sodium chloride (20 kg) in
water (848 kg) was added. The resulting mixture was
concentrated under vacuum until the total volume reached
∼1000 L and then stirred at 0−10 °C for ∼2 h. The resulting
slurry was filtered and rinsed with water (5 × 58 kg). The filter
cake was dried under vacuum at 25−35° for 12 h to give 4 as a
light-brown solid (67.4 kg 78.1%) in HPLC purity 99.4% (KF
0.4%. qNMR potency 97.4%). The spectroscopic data were
identical to those reported in the literature.3b
4.1.2. tert-Butyl (2-((3aR,5S,7aR)-5-Methyltetrahydro-1H-
pyrano[3,4-c]isoxazol-7a(7H)-yl)thiazol-4-yl)carbamate
(4a). A dry and nitrogen-purged 2000 L glass-lined reactor was
charged with toluene (984 kg), and the reactor was evacuated
three times and refilled with nitrogen, followed by cooling to
15−30 °C. 4 (112.5 kg, 368.6 mol), tert-butyl carbamate
(106.1 kg, 905.6 mol), and potassium carbonate (151.3 kg,
1095 mol) were added to the mixture. The mixture was
evacuated to ∼−0.08 MPa and filled with nitrogen. This was
repeated five times. The reactor was sampled for oxygen
content to ensure it was ≤400 ppm. A mixture of
tris(dibenzylideneacetone)dipalladium (29.9 kg, 3.26 mol)
and 2-di-t-butylphosphino-2′,4′,6′-tri-i-propyl-1,1′-biphenyl
(7.80 kg, 18.4 mol) was added to the reaction mixture under
protection of nitrogen. The resulting reaction mixture was
heated at reflux for 5 h upon reaction completion confirmed by
HPLC sampling. The mixture was cooled to 20 °C, and THF
(399 kg) was added. The resulting mixture was filtered and the
filter cake washed with THF (380 kg). The combined filtrate
was concentrated at ≤55 °C under reduced pressure (−0.08
MPa) until ∼250 L of the mixture remained. The mixture was
cooled to 25−35 °C, and THF (191 kg) was added. The total
weight of the final solution was 446 kg (100% yield assumed),
and it was carried over to the next step. This step was repeated
three times on similar scales.
carbamate (21). A dry and nitrogen-purged 3000 L autoclave
reactor was charged with Raney-nickel (64.6 kg) under
nitrogen. The reactor was again evacuated and purged with
nitrogen five times. At 15−25 °C, isopropanol (814 kg) and
the abovementioned crude 4a solution (514 kg of THF
solution 129 kg by potency, 378 mol) were charged to the
reactor with the agitation off. The agitation was then started
and argon was bubbled from the bottom of the reactor for 20
min. While maintaining at 15−35 °C, the reaction mixture was
purged with argon to 0.15−0.2 MPa and then vented to 0.05
MPa; the purge and vent operation was repeated five times.
The reactor was sampled for oxygen content to ensure it was
≤0.1%. At 15−25 °C, the resulting mixture was purged with
hydrogen to 0.3−0.5 MPa and then vented to 0.05 MPa; the
purge and vent operation was repeated five times. The pressure
was then increased to 0.35−0.5 MPa with hydrogen. The
mixture was maintained at 25−35 °C under 0.35−0.5 MPa for
8 h, and HPLC analysis showed reaction completion. The
mixture was purged with argon to 0.15−0.2 MPa and vented to
0.05 MPa. This was repeated five times. The mixture was
filtered over celite (14.6 kg), and the reactor wall and filter
cake were rinsed with isopropanol (410 kg). The combined
filtrate was concentrated at ≤45 °C under reduced pressure
until ∼270 L remained. The mixture was cooled to 20−30 °C,
and dichloromethane (969 kg) was added. At 20−30 °C, the
pH of the mixture was adjusted to 2.5−3.0 with aqueous citric
acid (prepared by mixing 411 kg of citric acid in 2067 kg of
water). The organic phase was extracted with the above-
mentioned pre-prepared citric acid solution (895 kg). The
product-rich, combined aqueous phase was mixed with ethyl
acetate (1154 kg), and the pH of the aqueous phase was
adjusted to ∼8 with a solution of potassium carbonate (527
kg) in purified water (649 kg) at a rate of 10−30 kg/h. The
biphasic mixture was filtered through celite (12.6 kg), and the
filter cake was rinsed with ethyl acetate (465 kg). The phases
were separated, and the organic phase was concentrated at ≤45
°C under reduced pressure until 220−260 L remained. The
resulting solution was solvent-exchanged with THF (3 × 450
kg) at ≤45 °C under reduced pressure until 220−260 L of the
mixture remained. The mixture was sampled for until KF
≤0.1%. The mixture was cooled to 15−25 °C. THF (220 kg)
was added to give a solution of 21 in gross weight 564 kg (89.3
kg product corrected for potency, 260 mol, 68.8%). The
resulting solution was directly carried forward to the next step.
This step was repeated three times on similar scales. The
spectroscopic data were identical to those reported in the
literature.3c
4.1.4. tert-Butyl (2-((4aR,6S,8aR)-2-Benzamido-6-methyl-
4,4a,5,6-tetrahydropyrano[3,4-d][1,3]thiazin-8a(8H)-yl)-
thiazol-4-yl)carbamate Hydrochloride (2a·HCl). A dry and
nitrogen-purged 3000 L glass-lined reactor was charged with
THF (306 kg) and 562 kg of crude 21 solution (84.5 kg
corrected for potency, 246 mol). The mixture was sampled for
KF at ≤0.1%. The mixture was heated to 48−60 °C, benzoyl
isothiocyanate solution which was prepared with benzoyl
isothiocyanate (40.9 kg, 250 mol) and THF (158.0 kg) was
added into the mixture with a peristaltic pump in 3 h. The
mixture was reacted at 48−60 °C for 3 h and then sampled by
HPLC analysis to confirm that the formation of intermediate
22 was complete. While maintaining the temperature at 48 to
60 °C, 1,1′-carbonyldiimidazole (43.4 kg, 268 mol) was added.
The mixture was heated at 48 to 60 °C for 24 h, and the
reaction was deemed complete by HPLC analysis. The mixture
4.1.3. tert-Butyl (2-((3R,4R,6S)-3-Amino-4-(hydroxymeth-
yl)-6-methyltetrahydro-2H-pyran-3-yl)thiazol-4-yl)-
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was cooled to 20 to 35 °C and concentrated at ≤45 °C under
reduced pressure (P ≤ −0.08 MPa) until ∼160 L remained.
The resulting mixture was solvent-exchanged with ethyl acetate
(378 kg) until THF residual reached ≤5%. Ethyl acetate (899
kg) was added. While maintaining at 15−30 °C, citric acid
solution which was prepared with citric acid (89.5 kg) and
purified water (803 kg) was added into the mixture, and the
resulting phases were separated. The organic phase was washed
with 15% sodium chloride solution (480 kg) and then
concentrated at ≤45 °C under reduced pressure (P ≤ −0.08
MPa) until ∼450 L remained. The resulting mixture was
cooled to −10 to 0 °C. While maintaining at this temperature,
aq HCl solution which was prepared with conc. HCl (38.0 kg)
and water (74 kg) was added into the mixture. The resulting
mixture was stirred at −10 to 0 °C for 6 h, and the resulting
slurry was filtered with a centrifuge and the filter cake was
washed with ethyl acetate (3 × 51 kg) and then with water (5
× 85 kg). The filter cake was dried in a rotary conical dryer at
30−60 °C under reduced pressure (P ≤ −0.08 MPa) until KF
was ≤0.5% and ethyl acetate ≤2.0%, respectively. Product 2a-
HCl was isolated as a yellow solid (112.7, 106.6 kg corrected
for potency, 82.2%). This step was repeated two times on
similar scales. LCMS m/z (M + 1): 489.2, 354.3, 175.1, 103.1
1H NMR (DMSO-d6, 400 MHz): δ 10.27 (br 1s),8.15 (d, J =
8.0 Hz, 2H), 7.68−7.62 (m, 1H), 7.58−7.51 (m, 2H), 7.25 (s,
1H), 4.09−3.98 (m, 1H), 3.84−3.60 (m, 2H), 3.12−2.82 (m,
3H), 1.78−1.72 (m, 1H), 1.65−1.36 (m, 10H), 1.26−1.15 (m,
3H).
4.1.5. N-((4aR,6S,8aR)-8a-(4-Aminothiazol-2-yl)-6-methyl-
4,4a,5,6,8,8a-hexahydropyrano[3,4-d][1,3]thiazin-2-yl)-
benzamide (2). A dry and nitrogen-purged 1000 L glass-lined
reactor was charged with methanol (316 kg). The reactor was
cooled to 0−10 °C. While maintaining at this temperature,
acetyl chloride (121.5 kg, 1548 mol) was added slowly. After
addition, the resulting mixture was stirred for 2 h and then
warmed to 25−30 °C. 2a·HCl (136.6, 130.1 kg corrected for
potency, 247.7 mol) was added into the mixture in portions.
The solid addition funnel and reactor wall were rinsed with
methanol (14 kg). The resulting reaction mixture was stirred at
20−30 °C for 4 h, and at this point, the reaction was confirmed
to be complete by HPLC analysis. While maintaining the
temperature at 0−30 °C, the mixture was inversely quenched
into a mixture of ethyl acetate (1794 kg), purified water (2027
kg), and triethylamine (272.2 kg) that was precooled to 0−10
°C. The mixture was warmed to 20−30 °C, and the phases
were separated. The aqueous phase was backextracted with
ethyl acetate (2 × 480 kg) once. The combined organic phase
was washed with water (4 × 1350 kg) and then concentrated at
≤45 °C under reduced pressure ≤−0.08 MPa until ∼220 L
remained. At 30−40 °C, methanol (33.0 kg) and MTBE (1003
kg) were added, and the resulting slurry was stirred at 20−30
°C for 2 h and then cooled to 0−10 °C. The mixture was
filtered with a stainless-steel nutsche filter; the filter cake was
rinsed with MTBE (100 kg). The filter cake was dried in a
rotary conical dryer at 35 to 45 °C under reduced pressure (P
≤ −0.08 MPa) until both ethyl acetate residue and MTBE
residue reached ≤0.5%. Product 2 was isolated as a light-brown
solid (69.0, 67.6 kg corrected, 70.2%) in 100% HPLC purity
(98% potency). This step was repeated two times on similar
scales. The spectroscopic data were identical to those reported
in the literature.3b
5-(difluoromethoxy)picolinamide (26). A dry and nitrogen-
purged reactor with jacket temperature at 15−25 °C was
charged with ethyl acetate (124 kg), followed by 3 (13.0 kg,
67.2 mol). The resulting mixture was stirred for 30 min and
then cooled to 10 °C. Triethylamine (27.5 kg, 272 mol) was
added, followed by the addition of 2 (24.8 kg, 63.6 mol). The
reaction was cooled to 0 °C. 50% T3P solution in ethyl acetate
(102 kg, 160 mol) was slowly added to the reaction while
maintaining the reaction temperature below 5 °C. After the
addition, the reaction was warmed to 25 °C and held for 12 h.
The reaction was then cooled to 10 to 15 °C, and isopropanol
(115 kg) was added, followed by the addition of 0.5 N aq HCl
(99 kg). The resulting mixture was concentrated to ∼350 L
under atmospheric pressure, then cooled to 0 to 10 °C over 3
h, and held at this temperature for 6 h. The resulting slurry was
filtered, and the filter cake was rinsed with a 1:1 volumetric
mixture of isopropanol (39.3 kg)/water (50 kg). The filter cake
was dried at 50 °C under vacuum (P ≤ −0.08 MPa) to give the
desired product as an off-white solid (34.0, 33.2 kg for potency,
59.3 mol, 93.2%) in 99% UPLC purity. The spectroscopic data
were identical to those reported in the literature.3b
4.1.7. N-(2-((4aR,6S,8aR)-2-Amino-6-methyl-4,4a,5,6-
tetrahydropyrano[3,4-d][1,3]thiazin-8a(8H)-yl)thiazol-4-yl)-
5-(difluoromethoxy)picolinamide (1). A nitrogen-purged
reactor with jacket temperature at 25 °C was charged with
30 (32.5 kg, 58.1 mol), 1-propanol (154 kg), and water (65
kg). L-lysine (46.7 kg, 319 mol, 5.5 equiv) was then added. The
resulting mixture was heated at 90 °C for 24 h. When the
reaction was confirmed complete, it was cooled to 55 °C over
30 min, and 3.2 N aq NaOH solution (100 kg) was added
slowly. After the addition was complete, the reaction mixture
was cooled to 25 °C in 2 h. The resulting slurry was heated to
55 °C, then cooled to 20 °C in a 2 h cycle to increase the
particle size, and then concentrated at ∼40 °C under vacuum
(110 mbar) to ∼230 L. Water (130 kg) was added slowly over
1 h at the same temperature, and the agitation was continued
at 40 °C for 1 h and then cooled to 20 °C. The resulting slurry
was filtered, and the filter cake was rinsed with a 7:1 volumetric
ratio of water/1-propanol (2 × 63 kg). The filter cake was
dried at 50 °C under vacuum (P ≤ −0.08 MPa) to give the
desired product which was a white solid (22.9 kg, 50.3 mol,
86.4%) in 99.6% UPLC purity. The spectroscopic data were
identical to those reported in the literature.3b
4.1.8. N-(2-((4aR,6S,8aR)-2-Amino-6-methyl-4,4a,5,6-
tetrahydropyrano[3,4-d][1,3]thiazin-8a(8H)-yl)thiazol-4-yl)-
5-(difluoromethoxy)picolinamide (1-HCl). A nitrogen-purged
reactor with jacket temperature at 25 °C was charged with 2
(22.8 kg, 500 mol) and MEK (238 kg). The resulting mixture
was heated to 45 to 55 °C for ∼1 h, and a complete solution
was obtained. The resulting solution was held at 45 to 55 °C,
filtered through a 0.5 μm in-line cartridge filter, and rinsed with
MEK (18 kg). To the filtrate that was cooled to 30 to 40 °C
was added a solution of aq HCl solution in MEK prepared by
combining 120 kg of 5 N aq HCl (600 mol, 1.2 equiv) and 110
kg of MEK, while maintaining at 30 to 40 °C. After the
addition was complete, the reaction mixture was stirred at 30
to 40 °C for >2 h and then cooled to 0 to 10 °C over 3 h. The
resulting slurry was filtered and rinsed with MEK (55 kg). The
filter cake was dried at 50 °C under vacuum (P ≤ −0.08 MPa)
to give the desired product as a white solid (21.5 kg, 87.2%) in
99.8% UPLC purity. The spectroscopic data were identical to
those reported in the literature.3b
4.1.6. N-(2-((4aR,6S,8aR)-2-Benzamido-6-methyl-4,4a,5,6-
tetrahydropyrano[3,4-d][1,3]thiazin-8a(8H)-yl)thiazol-4-yl)-
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Article
Miguel Gonzalez − Asymchem Life Science Co. Limited,
Tianjin 300457, China; Present Address: Alcon, Fort
Worth, Texas, United States.
AUTHOR INFORMATION
■
Corresponding Author
Jiangping Lu − Asymchem Life Science Co. Limited, Tianjin
Bryan Li − Chemical Research & Development, Worldwide
Research Development & Medical, Pfizer Inc., Groton,
Connecticut 06340, United States; Chemical Research &
Development La Jolla Laboratory, Worldwide Research
Development & Medical, Pfizer Inc., San Diego, California
92121, United States; orcid.org/0000-0002-7972-9966;
Email: bryan.li@pfizer.com
300457, China
Mark D. McLaws − Asymchem Life Science Co. Limited,
Tianjin 300457, China
Jian Tao − Asymchem Life Science Co. Limited, Tianjin
300457, China
Baolin Wu − Asymchem Life Science Co. Limited, Tianjin
300457, China
Authors
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.1c00126
Richard W. Barnhart − Chemical Research & Development,
Worldwide Research Development & Medical, Pfizer Inc.,
Groton, Connecticut 06340, United States; orcid.org/
0000-0002-7666-8942
Amelie Dion − Chemical Research & Development,
Worldwide Research Development & Medical, Pfizer Inc.,
Groton, Connecticut 06340, United States; Present
Address: Merck and Co. Rahway, New Jersey, United
States.
Steven Guinness − Chemical Research & Development,
Worldwide Research Development & Medical, Pfizer Inc.,
Groton, Connecticut 06340, United States
Alan Happe − Chemical Research & Development Worldwide
Research Development & Medical, Pfizer Inc., Sandwich
CT13 9ND, U.K.
Notes
The authors declare no competing financial interest.
¶Retired from Pfizer.
ACKNOWLEDGMENTS
■
We thank Mark Barilla and the staff of Groton Kilo Lab,
Sandwich Product Development Facility, and Asymchem
Production Plant for their support.
DEDICATION
■
⊥Dedicating this paper to Professor Elie Abushanab on the
occasion of his 85th birthday.
Cheryl M. Hayward − Chemical Research & Development,
Worldwide Research Development & Medical, Pfizer Inc.,
Groton, Connecticut 06340, United States
Jeffrey Kohrt − Chemical Research & Development,
Worldwide Research Development & Medical, Pfizer Inc.,
Groton, Connecticut 06340, United States
Teresa Makowski − Chemical Research & Development,
Worldwide Research Development & Medical, Pfizer Inc.,
Groton, Connecticut 06340, United States
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Worldwide Research Development & Medical, Pfizer Inc.,
Groton, Connecticut 06340, United States
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Worldwide Research Development & Medical, Pfizer Inc.,
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Worldwide Research Development & Medical, Pfizer Inc.,
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Pfizer Inc., Groton, Connecticut 06340, United States
Denghui Bao − Asymchem Life Science Co. Limited, Tianjin
300457, China
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