Why Did Compound Three Require Chromatography for Purification Discussion Questions

Description

7. Look at Scheme 1 and the synthesis of compound 9. Why is the synthesis of compound 9 not ideal in the context of industrial chemistry? (2 marks) 
8. Why did compound 3 require chromatography for purification? (1 mark) Why is chromatography disfavored in industrial processes? (2 marks) 
9. Why is the presence of a chromophore helpful during chemical synthesis? (2 marks) 
10. Look at Figure 3 and compound 17. Was compound 17 a desired product? (1 mark) How was compound 17 made? (2 marks) Why was the formation of compound 17 problematic? (2 marks) 
11. Figure 6 shows 5 impurities that were identified during the synthesis of lactam 25. A lactam is a cyclic amide. Circle the amide functional group in lactam 25. (1 mark) Speculate how each of the by-products might be formed. (10 marks – 2 per impurity) 
12. Scheme 7 shows the route to compound 8. How many stereocenters are present in compound 8 (2 marks) Identify the stereocenters as R or S. (4 marks – show your working!) 
13. Why was compound 8 preferred as an intermediate in the synthesis of Lufotrelvir. (2 marks) 
14. What is MEK? (1 mark) Draw the structure (1 mark). Why is MEK used so much in the synthesis of Lufotrelvir? (2 marks) 
15. What is an anti-solvent? (2 marks) 
16. What was used as an anti-solvent in the synthesis of compound 2? (1 mark) 
17. Why did the authors spend so much effort getting to compound 2? What was special about compound 2? (4 marks) 
18. During the development of Lufotrelvir, various synthetic routes were developed to access the target material. Provide 4 reasons, as stated in the manuscript, why different synthetic routes were required.This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use
and analyses in any form or by any means with acknowledgement of the original source.
These permissions are granted for the duration of the World Health Organization (WHO)
declaration of COVID-19 as a global pandemic.
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Article
Early Clinical Development of Lufotrelvir as a Potential Therapy for
COVID-19
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Christophe Allais, David Bernhardson, Adam R. Brown, Gary M. Chinigo, Jean-Nicolas Desrosiers,
Kenneth J. DiRico, Ian Hotham, Brian P. Jones, Samir A. Kulkarni, Chad A. Lewis, Ricardo Lira,
Richard P. Loach, Peter D. Morse, James J. Mousseau, Matthew A. Perry, Zhihui Peng, David W. Place,
Anil M. Rane, Lacey Samp, Robert A. Singer,* Zheng Wang, Gerald A. Weisenburger, Hatice G. Yayla,
and Joseph M. Zanghi
Cite This: https://doi.org/10.1021/acs.oprd.2c00375
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ABSTRACT: Lufotrelvir was designed as a first in class 3CL protease inhibitor to treat COVID-19. Development of lufotrelvir was
challenged by its relatively poor stability due to its propensity to epimerize and degrade. Key elements of process development
included improvement of the supply routes to the indole and lactam fragments, a Claisen addition to homologate the lactam, and a
subsequent phosphorylation reaction to prepare the prodrug as well as identification of a DMSO solvated form of lufotrelvir to
enable long-term storage. As a new approach to preparing the indole fragment, a Cu-catalyzed C−O coupling using oxalamide
ligands was demonstrated. The control of process-related impurities was essential to accommodate the parenteral formulation.
Isolation of an MEK solvate followed by the DMSO solvate ensured that all impurities were controlled appropriately.
KEYWORDS: lufotrelvir, PF-07304814, COVID-19, Claisen addition, amidation, phosphate prodrug

INTRODUCTION
few crystalline intermediates as isolation points and about a 5%
overall yield from lactam 3. In particular, the reactive
chloromethyl ketone moiety was introduced early in the
sequence and carried across nearly all of the steps from lactam
4 to ultimately prepare the highly elaborated compound 11.
The homologation of 3 to furnish 4 was one of the most
problematic steps with highly variable yields which were
generally poor. Lactam 3 was prepared by the route described
in the literature but required chromatography for purification
due to the relatively poor crystallinity of 3.9 The route to the
indole fragment (9) was extremely efficient, but not deemed
suitable for larger scale manufacture due to the high energy
azide intermediates.10 Despite this, smaller batches (1−3 kg
scale) of 9 were prepared using flow methods to minimize the
hazardous handling of the azides.
Degradation under alkaline and acidic conditions plagued 2
and the closely related precursors in the process (Figure 2).
The phosphate moiety of the prodrug (2) would hydrolyze to
yield 1. Even though 1 is the active metabolite, the level of 1 as
an impurity in 2 needed to be minimized, otherwise 1 would
precipitate during iv formulation. In addition, 1 or 2 was
susceptible to epimerization at the stereocenter alpha to the
ketone moiety at a pH above 5. As the pH was increased into
In 2019, a novel coronavirus disease emerged that was caused
by SARS-CoV-2, a closely related virus to SARS-CoV-1 that
caused severe acute respiratory syndrome (SARS) in 2002.1
Because of the rapid spread of SARS-CoV-2, a global pandemic
emerged with health agencies seeking therapeutic options.2 A
number of approaches to treating patients infected with the
SARS-CoV-2 have been in clinical development (Figure 1),
including remdesivir and molnupiravir.3 One treatment
strategy is to target the 3CL protease to selectively inhibit
replication of the coronavirus. Toward this approach, PF00835231 (1) was designed as a potent and first in class 3CL
protease inhibitor which would be administered by intravenous
infusion.4 Unfortunately, the low aqueous solubility of 1 (200 mg/mL).5
Lufotrelvir (2) is metabolized in vivo by phosphatases to
liberate the active metabolite 1. After clearing preclinical
toxicology and ADME safety, 2 began clinical evaluation. Not
long after clinical evaluation of lufotrelvir was underway, a
closely related analog, nirmatrelvir, entered clinical development as a potent and orally bioavailable SARS-Cov-2 3CL
protease inhibitor, the development of which is the subject of a
separate manuscript.6,7 Herein is reported the rapid process
development of lufotrelvir (2) to enable clinical studies and to
develop a potential commercial route.
The original route used by the medicinal chemistry team is
shown below (Scheme 1).8 The overall route was linear with
© XXXX American Chemical Society
Received: December 1, 2022
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Figure 1. Potential therapeutic agents for treatment of SARS-CoV-2.
Scheme 1. Original Route to Lufotrelvir, PF-07304814 (2)
the alkaline range, the epimerization of 2 became more facile.
Finally, under acidic conditions, 1 or 2 degraded via hydrolysis
to an indole-leucine fragment (13). Managing the limited
stability of 2 and its related precursors in the process route was
a key challenge for development.
indole fragment (9) was unsafe, new routes needed to be
identified that would be amenable to large scale manufacture.
The current homologation of the lactam (3) to introduce the
chloromethyl ketone was highly unreliable on scale and
therefore required a more robust process or a new approach.
Finally, the route to lactam 3 required identifying a means of
purification via crystallization to avoid chromatography.
With these research priorities established, one of the primary
concepts adopted was to build 2 starting from the coupling of
the indole fragment (9) with leucine and later introduction of
the lactam fragment (3). In this way, not only would all
intermediates possess a chromophore, but also would possess
more crystallinity (imparted by the indole moiety) to improve
the control strategy. This approach was reliant on demonstrat-

EARLY CLINICAL DEVELOPMENT
As development began, a number of key priorities were
established. One of the highest priorities was to consider the
order of the steps to minimize intermediates with stability
issues while introducing more viable control points with
crystalline intermediates. For the endgame of the process, the
route would need to address the limited stability of 2 and its
precursors to maximize yield. Because the current route to the
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Figure 2. Degradation pathways for 2.
Scheme 2. Preparation of Indole 9, Leucine Amidation, and Hydrolysis
degradation. At 55 °C and higher, it was observed that the
methyl ether of the indole would undergo cleavage to yield the
4-hydroxyindole. To facilitate driving the hydrolysis toward
completion, it was essential to distill the methanol and methyl
acetate byproducts under vacuum (which operated more easily
with H2SO4 to avoid loss of HCl). Upon completion of the
reaction, dilution with water (7 vol) crystallized 13 in 80−90%
yield.
As scale up of 9 commenced toward the preparation of 13,
the vendors depleted the available supply of 9A (2-methyl-3nitroanisole) and its phenol precursor. As a result, some of the
vendors needed to identify alternative alpha raw materials. As
this problem emerged, another route was identified that
utilized a known Cu-catalyzed C−N coupling to form the
indole.12 The new sequence (Scheme 2B) featured a Cucatalyzed C−N ring closure that relies only on the substrate
and triethylamine as a ligand13 as well as a Cu-catalyzed C−O
coupling to install the methoxy group.14 During the development of this latter Cu-catalyzed C−O coupling, oxalamide
ligands were found to be quite effective. A few new oxalamide
ligands were designed with better water solubility to facilitate
removal of the Cu complex. The ligands were prepared by
combining one equivalent of dimethyl oxalate and at least two
equivalents of amine in methanol or other solvents depending
ing early amidation of indole 9 with leucine followed by an
amidation with the lactam fragment (3).
Preparation of this new indole-leucine fragment, 13, was
straightforward to achieve, and the supply of indole 9 was
enabled by another known route based on the Reissert indole
synthesis (Scheme 2A).11 With access to indole 9, amidation
between methyl leucine (14) and 9 was demonstrated with
T3P in high yield followed by hydrolysis of the methyl ester.
For the amidation, it was crucial to avoid alkaline conditions
which would otherwise lead to epimerization. With triethylamine, this was more difficult to achieve and required careful
matching of the stoichiometry of the base to the amidation
reagent; however, when using N-methylimidazole, pyridine or
a related mild base, epimerization was not observed even when
used in excess. Optimal conditions for the amidation utilized
T3P (1.25 equiv) and N-methylimidazole (3 equiv) in
acetonitrile (7 vol) at ambient conditions. Upon completion
of the amidation, the solvent was partially removed to
crystallize 15 after addition of water (10 vol). For the
hydrolysis step, alkaline conditions were avoided again to
minimize epimerization. The methyl ester was efficiently
hydrolyzed by heating concentrated hydrochloric acid or
sulfuric acid (1.5 equiv) in acetic acid (5 vol) as solvent. Mild
heating (35−45 °C) under the acidic conditions avoided
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Scheme 3. Homologation of Lactam 3
Figure 3. Formation of 17, a problematic impurity in lufotrelvir (2).
Scheme 4. Endgame for Toxicology Lot
on the physical properties of the ligand and filtering the
resulting symmetrical ligands as solids. Through these efforts,
the most effective ligands found were DPEO15 and other
closely related oxalamide ligands including DMAPO (Scheme
2B). While these two ligands performed similarly for this
coupling, an analog of DMAPO derived from 1,2-diaminoethane (rather than 1,3-diaminopropane) was found to be
ineffective most likely due to the highly favorable chelation
leading to a stable and inactive complex.
monolithium salt of chloroacetic acid which then failed to
undergo enolization to the more soluble dilithium salt. Starting
from a more soluble acetate salt and forming the enolate would
help resolve this issue if the route had been further developed
using flow chemistry; however, ultimately an alternative route
was selected though still utilizing the Claisen addition for a
different substrate (see discussion below of commercial route
development).
Using this flow protocol, the supply of compound 5 became
surmountable after a deprotection with either HCl, MsOH or
TsOH. Though the HCl or MsOH salt of 5 was an oil, the
TsOH salt was highly crystalline which could be a potential
control point. At this stage, 5 was typically formed in situ and
used immediately in the downstream amidation.
When studying the amidation of 13 and 5, it was quickly
realized that this combination of substrates was more prone to
epimerization via formation of an oxazolone intermediate of 13
during activation of the carboxylic acid (shown in Figure 3).
For preparation of the first toxicology lot, epimerization was
limited by combining both reactants in DMF with HATU and
slowly adding diisopropylethylamine at −20 °C. With this
protocol on 10−20 g scale, the dr typically ranged from 90:10
to 96:4 and 8 was isolated in 55−60% yield. When scaling this
protocol to ∼500 g, it was found to be challenging due to the
sensitivity of pH connected with addition rate of base and
HATU, which resulted in poorer purity (∼90−94%) and
typically lower dr (80:20 to 85:15), and consequently required
chromatography to upgrade the purity to >99% in 57% yield.
As an alternative to this protocol, it was recognized during
screening of Lewis acids that Zn salts could help suppress
epimerization of 13. Using stoichiometric amounts of Zn(OTf)2 with EDCI (rather than HATU) in acetonitrile
typically delivered a dr of 85:15 to 92:8 in situ on ∼600 g
scale, which after crystallization of 8 improved the dr to >95:5

HOMOLOGATION OF THE LACTAM AND
ENDGAME
From this point, the most convergent route to 2 would be to
carry out an amidation of 13 with lactam 5 to intercept 8, an
intermediate in the original route (Scheme 1). To pursue this
route, supply of 5 required homologation of 3 which was a
highly problematic (Scheme 3). The first challenge was that
purification of 3 originally required chromatography which was
an acceptable option for small scale but would be an issue
long-term. Even with purified 3, the first-generation homologation protocol was highly variable in yield (∼15−50% yield)
and generally did not scale well using LHMDS and/or LDA
with chloroiodomethane.16 When examining the literature for
alternative protocols, the use of chloroacetic acid or
chloroacetate derivatives seemed to be a potential alternative.17
The chloroacetatic acid lithium or magnesium enolate
performed much more reliably, providing 40−50% yield of 4
in a batch mode at −10 °C. When switching over to a plug
flow reactor, the isolated yield improved to about 60% with
90% conversion. LHMDS in THF was used out of
convenience, but the magnesium enolate delivered comparable
results. It was later realized that the lithium enolate had issues
with solids forming and clogging the flow reactor. The likely
cause of the insoluble material was the formation of the
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Scheme 5. Endgame for First Clinical Batch of 2
Figure 4. Typical levels of impurities in isolated lufotrelvir (2) hydrate and purge factors.
(10 equiv) in iPrOH (15 vol) at 30−50 °C for deprotection
and directly crystallized 2 as a nonstoichiometric hydrate. With
this protocol, the removal of residual iPrOH from 2 required
extensive heating under vacuum in the tray dryer, but this
approach was sufficient for the toxicology lot and provided
some insight into solid form challenges of 2 which favored
forming hydrates and solvates.
Almost immediately after delivery of the toxicology batch,
the first clinical batch was manufactured (Scheme 5). The
chemistry remained largely unchanged with the exception of a
few adjustments. The reaction solvent for the phosphate
alkylation was changed from acetonitrile to MEK (15 vol),
which also was used during the deprotection to 2. For the
deprotection protocol in MEK, HCl was substituted for TFA
(20 equiv) which allowed lowering the reaction temperature to
the range of 20−30 °C. Upon reaction completion, 2
crystallized as the MEK solvate in 61% yield (for 2 steps)
and 97.9% purity. The isolation of 2 as the MEK solvate was
crucial for efficient control of impurities but was undesirable as
in 43% yield. A remaining concern was that this step resulted
in modest yields regardless of the protocol used and so
alternative disconnections warranted examination (see further
discussion below).
Rather than using the original approach of converting the
chloromethyl ketone to the hydroxyl derivative and subsequently converting to the phosphate through an additional 3
step sequence (Scheme 1), directly alkylating 8 with a
protected phosphate was examined (Scheme 4). The alkylation
proceeded smoothly with di-tert-butylpotassium phosphate
(DTBKPhos) in aprotic solvents. For the phosphorylation
which was carried out at 25−35 °C with DTBKPhos (2.0
equiv), viable solvents included acetonitrile, acetone, or methyl
ethyl ketone (15 vol) in combination with a catalytic iodide
source for an in situ Finkelstein reaction, which in this case was
tetrabutylammonium iodide (0.2 equiv). The reaction was
typically worked up to remove salts, and 16 was carried
forward as a solution to the deprotection of the t-butyl groups
to furnish 2. The first-generation route used concentrated HCl
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Scheme 6. Optimized Endgame to 2 for Clinical Supply
a final form for parenteral formulation due to the residual
organic solvent present. For the formulation of 2, the
nonstoichiometric hydrated form of 2 had been selected for
clinical development and could be accessed most readily by
crystallization in protic media. Generally, protic solvents
favored conversion to the desired hydrate crystal form, even
in the absence of water. For this crystal form, the extent of
hydration depended upon the level of water during the
polymorph transition as well as the relative humidity of the
storage environment but would often equilibrate toward the
monohydrate under standard relative humidity. Consequently,
the MEK solvate of 2 was converted to the desired hydrate via
a reslurry in ethanol (15 vol) at 40−45 °C over 2−4 h. After
cooling and filtration of 2, removal of residual ethanol by tray
drying was effective for lowering the level below 0.5%. From
the polymorph conversion, 844 g of 2 was isolated for this first
clinical batch in 98.6% purity and 96% yield after correcting for
the potency based on 3.5% water content.
This initial clinical batch of 2 was acceptable for early
studies, but there were concerns of highly insoluble impurities
leading to precipitation during parenteral formulation. The
insoluble impurities of concern were mainly introduced during
the phosphorylation step to afford 16. A number of side
products resulted from competing side reactions between 8
and reactive contaminating salts present in the DTBKPhos
(such as acetate, hydroxide or mono-tert-butylphosphate) or
from other side reactions including hydrolysis of 8 to 1. In
general, 8, 16, and 2 (as well as other related intermediates)
degraded to 13 via the oxazolone intermediate (shown in
Figure 3) which was also highlighted in Figure 2 (degradation
pathways). For this reason, 13 was often observed as a lowlevel impurity in the concluding steps of the process and as a
degradant for 2. One interesting impurity was 17 that arose
from degradation of 8 to 13 and subsequent reaction between
13 and unreacted 8 (Figure 3). Impurity 17 required strict
control limits in the process due to its high insolubility which
would lead to precipitation during liquid formulation. After the
acid deprotection of 16 to yield 2 as the MEK solvated form,
most impurities were well-controlled (see purge factors in
Figure 4). The subsequent conversion of 2 to the desired
hydrate had modest control of impurities.
the solvent, the stoichiometry of the sodium iodide catalyst,
and stoichiometry of the DTBKPhos. Using too little sodium
iodide would result in more opportunity for 8 to degrade to 13
rather than undergoing the desired alkylation with DTBKPhos.
If the concentration of DTBKPhos is higher, this can
preferentially outcompete 13 for alkylation of 8 (or its iodo
analog), thereby reducing the level of 17 formed. The more
interesting finding was that the solvent had a profound impact
on limiting the formation of 17. By blending a polar aprotic
solvent such as DMSO or DMF with acetone or MEK, the
alkylation proceeded more cleanly and resulted in less
degradation to 13 and consequently reduced levels of 17. In
MEK alone as solvent, the level of 17 could be as high as 5%,
which after isolation of crude 2 as the MEK solvate, 17 was
reduced to a level of 0.5%. This level of 17 was considered
unacceptable for formulation which required levels ideally
below 0.10%. Comparing DMSO and DMF, DMSO
consistently provided lower levels of 17. Blending MEK with
DMSO tended to provide overall higher assay levels of 16. It
was desirable to limit the total volumes of DMSO in the
process to facilitate the workup of 16. As a compromise, an
85:15 blend of MEK to DMSO was implemented which
typically formed about 1% of 17 in situ, resulting in a level of
about 0.1% in crude 2 when isolated as the MEK solvate. The
optimized phosphorylation protocol involved reacting 8 with
DTBKPhos (1.5 equiv) with NaI (0.2 equiv) in DMSO (1.5
vol) and MEK (8.5 vol) at 25−35 °C over about 24 h (Scheme
6).
Another concern with the initial process was the use of TFA
in the deprotection which could lead to highly variable purity
results depending on the reaction time and temperature due to
the propensity for epimerization, and degradation. After
examining a number of potential acids for the deprotection,
oxalic acid emerged as a leading option. The tert-butyl groups
of 16 were smoothly cleaved with oxalic acid (5−6 equiv) at
50−60 °C over 6−12 h in MEK (∼10 vol) which maintained
the same primary solvent across the two steps. The milder acid
limited most side reactions though typically epimer formed at a
level of 2−5% in situ which was readily purged to a level below
0.2% in crude 2 and is further lowered to below 0.1% during
isolation of 2 as the hydrate. Limiting the level of water to
NMT 0.5% in the deprotection to 2 was important to avoid
higher levels of epimerization and additional degradation to
give 1. The water level was routinely monitored and lowered as
necessary through distillation of MEK to remove residual
water. The deprotection of 16 slowly liberated isobutylene
over 6−7 h which then reacted with oxalic acid, MEK, and
residual water. The lengthy reaction time and heating led to
less volatile fate products of isobutylene.
With the optimal protocols for the phosphate alkylation and
deprotection, the MEK solvate of 2 was isolated in 64−72%
yield and in 99% purity for the two telescoped steps. These
adjustments delivered a more robust process which demonstrated comparable results in several different manufacturing

REVISION OF ENDGAME FOR MANUFACTURE
Following the delivery of the first clinical batch, the
formulation team raised additional concerns of problematic
insoluble impurities in the process for 2. Of the greatest
concern was the pseudodimer impurity 17. The isolation of
crude 2 as the MEK solvate had a purge factor of about 90%
for 17; however, this could still result in measurable levels of
17 that could precipitate during parenteral formulation. Studies
were conducted relating to the conversion of 8 to 16 to
improve process understanding of all impurities and especially
to limit the formation of 17. The process parameters with the
greatest influence on the formation of 17 were the choice of
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Figure 5. Route to 25 via lactam 3.
Figure 6. Main Impurities Tracked in Lactam Synthesis.
quality of 8 was generally poor (variable levels of impurities,
total impurities at a level of 3−6%) via the amidation of 5 and
13 as a result of poor control of the quality of 5 and the
capricious nature of the amidation. Relating to this supply of 8
was the general need for more robust preparation of the lactam
fragment.
facilities at varying scales of operation. All impurities were
controlled to a level of NMT 0.20% during isolation of the
MEK solvate and further reduced to a level below 0.1% during
isolation of the hydrate with the exception of the des-chloro
impurity 8A and methyl ester 31 (derived from lactam 3
undergoing amidation with 13). As discussed earlier, the purge
factors were generally quite high during isolation of the MEK
solvate, but were much more modest during isolation of the
hydrate (Figure 4).
For the polymorph conversion step, the drying of the
hydrate was still challenging on scale when using ethanol as the
primary solvent. In addition, this transformation offered
minimal control of impurities due to the poor solubility and
reslurry nature of the process. As a compromise to this, the
combination of MEK and MeOH was found to be a viable
alternative. By starting the process in MEK (4.5 vol), the crude
2 remains as the MEK solvate which upon addition of MeOH
(3 vol) can then undergo the transition to the desired hydrate.
The solubility of 2 in MeOH is significantly higher than in
EtOH which greatly facilitates the form conversion. With the
improved solubility not only comes a more reliable form
conversion but also a slight improvement for the control of
impurities (mainly the epimer and a couple of other impurities
that may persist). Upon completion of the polymorph
transition, the slurry is further diluted with MEK (2.5 vol) to
improve the recovery of 2 while still remaining within the
boundary conditions favoring the hydrate (so that the solvent
composition is NLT 20% v/v MeOH). As with the original
process, drying to remove residual solvents is difficult but
easier than drying EtOH. Drying the crystals was greatly
facilitated with a high nitrogen bleed and heating to 50−60 °C.
Higher temperatures were avoided for drying the solids due to
the propensity of 2 to degrade and epimerize. Even at 50−60
°C, epimerization was possible. When drying at 55−60 °C over
35 h, the level of epimer increased from nondetectable to
0.72%.
With the updated route to 2 as described above in Scheme 6,
clinical supply was enabled, but, from a commercial
manufacture perspective, a few challenges remained. The
stability of hydrated 2 was limited due to degradation at
ambient conditions to 1. Ideally, a more stable solvate or new
solid form could resolve the stability concerns of 2. The purity
of 2 was improved with the new clinical route, but the
formulation team preferred further improvements to ensure all
potentially insoluble impurities were under even tighter control
for future commercial production. Another point was that the

DEVELOPMENT OF THE ROUTE TO THE LACTAM
3
The route to lactam 3 is well documented in the literature18
and is available commercially; however, the purification of 3
has been limited to chromatographic methods for manufacture
(Figure 5). For early deliveries of 3, chromatography was
consistently used, though crystallization from diisopropyl ether
was viable once 3 was prepared in high purity. As a result, there
was not an ideal means of reliably purifying 3 and much of the
chemistry possessed low yielding steps which required further
process understanding.
The first step of the process required enolization of 22 at
−78 °C followed by diastereoselective trap of the enolate with
bromoacetonitrile to yield 23 as an oil in about 75% assay yield
and typically >99:1 dr.19 Both the enolization and quench of
the enolate required careful cryogenic control as originally
conceived by the Hanessian group, otherwise the diastereoselectivity suffered greatly. In fact, the charge of
bromoacetonitrile was so sensitive to temperature control
that precooling of the bromoacetonitrile as a THF solution and
performing a subsurface charge was typically implemented in
batch mode. The quench of the reaction was equally important
and required a sequential charge of methanol followed by a
nonaqueous acid (acetic acid or citric acid work well) charged
as a solution in THF. Because of the challenges with scaling
this process in batch mode, the process was often adapted to a
flow chemistry protocol to enable better temperature control
during enolization, enolate alkylation, and eventual quench.
The solution of 23, which was obtained following workup,
was carried forward in methanol for reduction of the nitrile to
the amine to allow cyclization to the lactam. The reduction had
been demonstrated by a number of methods in the literature
including NaBH4/NiCl2,20 Pd/C,21 PtO2,22 and NaBH4/
CoCl2.23 For conventional hydrogenation, it was observed
that PtO2 outperformed Pd/C, but required acidic conditions.
As a result, a subsequent step was required for neutralization of
the amine to allow cyclization to the lactam. For this approach,
a screen was performed which showed that triethylamine,
TMEDA, Hunig’s base, and sodium carbonate all gave some
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Figure 7. Tosylate salt of 5 attempted amidation and impurities from side reactions.
level of epimerization for the cyclization to the lactam, while
DBU and alkoxide bases did not have any epimerization. To
avoid adjustment of pH, Raney Ni hydrogenation could be
carried out under alkaline or neutral conditions which allowed
for in situ cyclization to the lactam.
Using Raney Ni 2400 seemed optimal for the process in
methanol at ambient conditions with 5 bar hydrogen as a
starting point for optimization. A dimer lactam impurity (26,
Figure 6) was observed to form under these standard
conditions which could be limited by dilution (25−30 mL/g
solvent) or with the addition of ammonia or other amines.
However, epimerization was more competitive if ammonia,
triethylamine, or other alkaline additives were used, and so as a
compromise, NMT 1 equiv of ammonia was used as an
additive. Cooling to 5 °C was explored as an option to
minimize epimerization which seemed to help; however,
oxidation of the lactam to a pyrrole (29) became more
competitive under these conditions or if using higher loadings
of ammonia (2−10 equiv). The pyrrole impurity (29) could
not be purged easily and therefore required limits on the extent
to which it was formed. From these studies, the revised
conditions were 100 wt % Raney Ni 2400, 1 equiv ammonia at
ambient conditions with 5 bar hydrogen in methanol (30 mL/
g). The optimal conditions with Raney Ni 2400 provided the
most economical approach, but were identified too late for
most of the early manufacture of lactam 3.
As an alternative to hydrogenation, a process was developed
using sodium borohydride and cobalt chloride. The process
closely mirrors protocols described in the literature.18
Typically, sodium borohydride (4−6 equiv) is charged as a
solution in EtOH (or slurry in THF) or solid to a methanol
solution of 23 and CoCl2 (0.5−1.0 equiv) at 0 °C. Often
cobalt chloride hexahydrate was used, but switching to the
anhydrous form generally gave less lactam dimer (26)
formation. As with the hydrogenation protocol, more dilute
conditions helped limit dimer (26) formation. If less polar
cosolvents (including ethanol, THF or ethyl acetate) were
used with methanol, this also helped reduce the level of lactam
dimer impurities (26). The reduction proceeded well at 0−5
°C, but the lactam cyclization was relatively slow under these
conditions and typically required warming to 10−25 °C to
reach completion. While the use of ethanol for dissolution of
sodium borohydride may appear attractive in that it is safer
(minimal reactivity even at ambient conditions), transesterification was encountered using this protocol which
required minimizing the reaction time at ambient conditions
while still alkaline (prior to acidic quench). A more practical
alternative was to use THF with methanol as a blend. It was
deemed unsafe to prepare a solution of sodium borohydride in
MeOH due to the reactivity between the two components, and
so manufacture was conducted with a portionwise slurry
charge of sodium borohydride in THF or as a solid charge into
cooled MeOH with a solution of 23 and CoCl2. Early on, the
process involving sodium borohydride and CoCl2 was used for
manufacture, but it was later recommended to use the process
with Raney Ni 2400 for improved safety and yield metrics.
Regardless of which means of reduction was used, crude
lactam 3 did not crystallize readily. A number of approaches
were explored to purify and crystallize the lactam including
enzymatic transformations and coformers. While examining
coformers and potential salt forms, it was observed that
deprotecting the Boc group with acid could provide a salt form
of 25 that would crystallize more readily and enable
purification. The tosylate salt of 25 was found to be optimal
in this respect and crystallized readily from methanol or
isopropanol. This process was implemented with either
reduction protocol (Raney Ni or NaBH4/CoCl2) such that
after workup, 3 was azeotropically dried by distillation of
iPrOH to limit water levels to avoid any saponification of the
ester. To the solution of 3 in iPrOH was charged anhydrous
tosic acid (azeotropically dehydrated with iPrOH as well). The
solution was typically heated to 45−55 °C to enable Boc
cleavage and direct crystallization of 25 as the tosylate salt.24
To improve the yield, MTBE, EtOAc, or iPrOAc could be
added as cosolvents. The overall yield for reduction,
cyclization, deprotection, and salt formation of 25 was 35−
45% using the NaBH4/CoCl2 protocol, while the yield for the
Raney Ni protocol was generally 50−60%.
The isolation of 25 as the tosylate salt provided remarkable
control of impurities. Typically, the lactam dimer impurity
(26) was formed at levels of 5−25% depending on processing
conditions, but the high purge factor during isolation of 25
limited levels to NMT 2% (Figure 6). The various other
impurities (epimers, unreacted intermediates (i.e., 27), ethyl
ester (28), ester hydrolysis (30)) were generally wellcontrolled at levels of NMT 0.25%, the only exception being
the oxidized impurity (29) which needed to be controlled to
levels of NMT 0.25% during its formation due to a lack of its
purge during isolation of 25.

DEVELOPMENT OF COMMERCIAL ROUTE TO
LUFOTRELVIR
Resolving the quality of 8 was a more pressing matter due to
the volumes increasing rapidly as clinical demand escalated. As
mentioned earlier, the tosylate salt of 5 was crystalline and
could serve as a control point prior to the amidation with 13 to
furnish 8. Unfortunately, when using the tosylate salt of 5 in
the amidation with 13 (Figure 7), the reaction did not perform
well probably due to the lack of solubility which resulted in
more competitive oxazolone formation. In addition, 5 is prone
to competitive dimer formation via alkylation and condensation pathways which did not bode well for this strategy,
especially for scaling a heterogeneous process that would be
highly sensitive to equipment configuration. It was noticed that
25 as an impurity in 5 would readily undergo amidation with
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Scheme 7. New Route to 8
13 to yield 31, suggesting that this might be an ideal substrate
for an amidation reaction (Scheme 7).
Because the tosylate salt of 25 was identified as an ideal
control point in the preparation of the lactam, it was examined
as an alternative substrate to 5 in the amidation with 13
(Scheme 7). When comparing the amidations, of 5 and 25, it
was immediately noticed that 25 cleanly reacted with 13 to
give 31. The solvent (MEK or acetone) and amidation
conditions (EDCI and HOPO) were critical for minimizing
epimerization. Using EDCI (1.25 equiv) in combination with
HOPO (0.25 equiv) and diisopropylethylamine (2.5 equiv) in
MEK consistently delivered 96:4 dr and >99% conversion over
6 h at rt. After workup, 31 could be crystallized from MTBE or
2-MeTHF as a solvated form in 80−85% yield. As another
alternative, crude 31 could be telescoped directly into the next
step because it was formed so cleanly.
To complete the formal synthesis by intercepting the key
intermediate 8, 31 required homologation with chloroacetic
acid analogous to the Claisen reaction previously carried out
with lactam 3. Several different protocols were evaluated for
the homologation reaction including use of a sulfoxonium
ylide,25 malonate additions and additions with chloroacetic
acid or esters,17 though none of which performed well26 except
for the chloroacetic acid additions as the magnesium
enolate.17a,27 Because 31 has four easily exchangeable protons
in addition to requiring deprotonation and enolization of
chloroacetic acid, a high loading of t-BuMgCl was required
(originally 20 equiv) which was optimized down to 12−14
equiv. To minimize the extent of dehalogenation, it was
essential to use a tertiary amine as a ligand for the magnesium.
Triethylamine gave acceptable results (7−10% dehalogenation), but much less dehalogenation (97% conversion with minimal
impurities other than ∼3% dehalogenation. After workup, 8
was isolated in 60−75% yield and 95−98% purity on 100−250
kg scale.
Earlier on in development, 8 had been isolated as an ethyl
acetate, MTBE or 2-MeTHF solvate with poor physical
properties. With all of these solvated forms, the crystallization
was not very effective at controlling impurities, but
diastereomers could be well-controlled with these solvates as
long as the starting dr was at least ∼85:15. With this new
protocol for 8, a new, highly desirable 2-MeTHF solvated form
was observed that possessed superior stability (less prone to
desolvation) and improved physical properties (higher bulk
density) for handling. With lower purity of 8 (90−94%), only a
less desirable 2-MeTHF solvate of 8 was readily accessible that
had a low bulk density and was not as easily handled.28 To
access the desired 2-MeTHF solvate, the crystallization needed
to be carried out from a blend of 2-MeTHF with either iPrOH
or water. It was not ideal to use the blend with water because
there was a very narrow range for which sufficient water would
enable access to the desired form but too much water led to
dissolution of the crystals. The combination of 2-MeTHF and
iPrOH was preferred (at least 25% v/v iPrOH in the solvent
composition), allowing for easier and more robust access to the
desired form. As mentioned earlier, chemical purity was one
key aspect for allowing access to the desired form. If the purity
was insufficient, then the purity would need to be first
upgraded by recrystallization from methanol or isopropanol
and readily converted to the desired 2-MeTHF solvate in a
blend of 2-MeTHF and iPrOH.
Proceeding with the more consistent quality of 8 into the
downstream phosphorylation and deprotection resulted in
more reliable performance for preparing 2. Despite this, it was
desired to further improve the quality of 2 and identify a
superior solvate for storage of 2. As the nonstoichiometric
hydrate, 2 is prone to hydrolysis to afford 1 via the residual
water present. At ambient conditions, this degradation can be
shelf life limiting over just a month, but with cold storage may
provide 1−2 years of shelf life. Ideally, a solid form that would
provide multiyear shelf life was desired. In addition to the
challenges of storing the hydrate of 2, the MEK solvate was
found to be a poor candidate for long-term storage due to the
potential for desolvation.
While studying various approaches to dissolution and
recrystallization of 2, it was noticed that dissolution in
DMSO and crystallization with MEK or iPrOH led to new
solid forms. While it was expected that the use of MEK as
antisolvent would provide the MEK solvate, closer inspection
of the crystal form showed that a DMSO solvate had been
formed. This was also true when using iPrOH as the
antisolvent with DMSO and when using a mixture of the 3
solvents. More remarkable is that the DMSO solvate was found
to be relatively stable to storage even at ambient conditions.
For comparison, the hydrate of 2 would degrade at ambient
conditions over 12 weeks so that the level of 1 was in the range
of 0.3% to 0.4%, while the level of 1 as an impurity in the
DMSO solvate of 2 would remain at NMT 0.05% over this
same time period. With cold storage the shelf life of the DMSO
solvate of 2 was projected to be at least 3−4 years. The
degradation of 2 was also problematic for the parenteral
formulation, and so this finding of a more stable solvate
ultimately benefited the overall supply and storage strategy.
This DMSO solvated form of 2 had a number of desirable
physical and chemical properties beyond the improved
chemical stability. Among them was the excellent control of
nearly all process-related impurities and degradants, high
stability of the solvate (other solid forms desolvate more
readily), and excellent bulk density and flow properties. The
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Scheme 8. Endgame for Proposed Commercial Route to Lufotrelvir
Scheme 9. Final Manufacturing Route to Lufotrelvir

bulk density of the hydrated form of 2 was generally about 0.2
g/mL with a needle morphology, while the more favorable
DMSO solvated form of 2 was 0.3−0.4 g/mL with a plateshaped or rod-shaped morphology.
With the realization that the MEK solvated form of 2 could
potentially desolvate during storage at ambient conditions over
several weeks, it became critical to incorporate the isolation of
the DMSO solvate in the process. It was attempted to directly
prepare the DMSO solvate of 2 during the deprotection of 16
in MEK with oxalic acid; however, the synergistic solubility of
2 with blends of MEK and DMSO resulted in a low-yielding
process. Instead, the DMSO solvate isolation was integrated
into the route as an independent step by redissolving the MEK
solvated form of 2 in MEK (∼2 vol) and DMSO (∼1 vol) at
ambient conditions and crystallizing 2 as the DMSO solvate
with the addition of iPrOH (∼15 vol) as antisolvent. The
process delivered the DMSO solvated form of 2 in about 83−
90% yield for the individual step (Scheme 8). With the
inclusion of this step, there was now a viable recrystallization of
2 to ensure that all impurities and endotoxins were wellcontrolled, in addition to allowing for longer term storage.
CONCLUSIONS
Identification of a reliable supply route to the tosylate salt of
lactam 25, which was subsequently converted to 8 in high
yielding transformations, and excellent control of processrelated impurities paved the way for large scale production of 2
(Scheme 9). Facile incorporation of the phosphate moiety via
alkylation and the subsequent deprotection further enabled
robust manufacture. Finally, discovery of the DMSO solvate of
2 was essential to providing a stable intermediate for longer
term storage with excellent physical properties while also
providing another important isolation for control of processrelated impurities. By passing through the MEK solvated
intermediate and DMSO solvate of 2, all process-related
impurities were well-controlled below 0.1% to enable routine
parenteral formulation. With the concluding polymorph
conversion to deliver the desired hydrate, the overall yield
from 8 is 54%, producing 2 in 99.5−99.8% purity on 30−40 kg
scale.
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Article
was controlled to a temperature of 15−30 °C. Charged 15
(404.6 kg, (335 kg assay corrected)) to the mixture; heated the
reaction mixture to NMT 50 °C and held for at least 6 h
(typically 16−24 h) until NMT 0.5% of 15 remaining by
reaction assay; and charged purified water (2345 kg) at 35−50
°C and held for 1−2 h. The reaction mixture was cooled to
15−25 °C, at a rate of 15−30 °C/h. The slurry was held at
15−25 °C for at least 2 h. The slurry was filtered with a
stainless-steel centrifuge, and the filtercake was washed twice
with purified water (2 × 836 kg). The solid was dried at 45−55
°C for at least 8 h until KF NMT 0.3%. 13 was isolated as offwhite to dark yellow crystals (271 and 271 kg assay corrected,
84% yield) in 99.2% purity and 100% assay. mp 174−175 °C;
1
H NMR (400 MHz, DMSO-d6) δ 12.56 (s, 1H), 11.56 (s,
1H), 8.50 (d, J = 8.0 Hz, 1H), 7.35 (s, 1H), 7.10 (t, J = 7.9 Hz,
1H), 7.01 (d, J = 8.2 Hz, 1H), 6.51 (d, J = 7.6 Hz, 1H), 4.49−
4.41 (m, 1H), 3.88 (s, 3H), 1.81−1.66 (m, 2H), 1.64−1.53
(m, 1H), 0.93 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H);
13
C NMR (100 MHz, methanol-d4) δ 176.30, 164.13, 155.65,
139.78, 130.35, 126.24, 120.12, 106.17, 102.82, 100.28, 55.69,
52.17, 41.50, 26.24, 23.43, 21.80.
EXPERIMENTAL SECTION
General. All reactions were carried out in dry reaction
vessels under an atmosphere of dry nitrogen unless otherwise
specified. All reagents and solvents were used as received
without further purification unless otherwise specified. For
most steps, reactions were monitored, and purity was assessed
by HPLC.
Methyl (4-Methoxy-1H-indole-2-carbonyl)-L-leucinate (15). Acetonitrile (1106.8 kg) was charged into the
8000 L glass-lined reactor 1 at 15−30 °C. Indole 9 (208.0 kg,
(191.2 kg assay corrected)) was charged at 15−30 °C. NMethylimidazole (301.0 kg) was charged at a rate of 100−200
kg/h at 15−30 °C. L-Leucine methyl ester hydrochloride
(209.2 kg) was charged to the mixture at 15−30 °C and held
for 0.5−1 h; charged 50% T3P in acetonitrile (831.0 kg) at a
rate of 200−300 kg/h at 15−30 °C. The reaction mixture was
held at 15−30 °C until the UPLC reaction assay indicated
NMT 1% of indole 9 remaining. The mixture was filtered with
a stainless steel nutsche filter which was preloaded with Celite
(3−5 cm depth) (25.0 kg) and transferred to a 1250 L glasslined reactor 2, and the transfer was washed with acetonitrile
(159 kg). The filtrate of 12500 L glass-lined reactor 2 was
concentrated at NMT 45 °C under reduced pressure (NMT
−0.07 MPa) until 1400−1600 L (6.7−7.7 L/kg) remained.
The batch temperature was adjusted to 15−30 °C; charged
purified water (300.0 kg) at 15−30 °C at a rate of 100−200
kg/h and held for 0.5−1 h; and charged purified water (1600.0
kg) at 15−30 °C at a rate of 100−200 kg/h and held for at
least 6 h (this completed the crystallization). The mixture was
filtered with a stainless-steel centrifuge. Each portion of the
filter cake was rinsed with purified water (200.0 kg × 4). After
drying the solids with vacuum at NMT 50 °C, 15 was isolated
as off-white to light yellow crystals (367 kg, (290 kg assay
corrected), 84% yield) in 96.5% purity and 79% assay.
Mp160−163 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.57
(s, 1H), 8.62 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 1.7 Hz, 1H),
7.10 (t, J = 7.9 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.51 (d, J =
7.6 Hz, 1H), 4.58−4.44 (m, 1H), 3.89 (s, 3H), 3.65 (s, 3H),
1.85−1.62 (m, 2H), 1.64−1.53 (m, 1H), 0.93 (d, J = 6.4 Hz,
3H), 0.89 (d, J = 6.4 Hz, 3H); 13C NMR (101 MHz, DMSOd6) δ 173.14, 161.20, 153.65, 137.88, 129.54, 124.57, 118.05,
105.41, 101.16, 99.26, 55.09, 51.91, 50.51, 39.38, 24.44, 22.85,
21.16.
(4-Methoxy-1H-indole-2-carbonyl)-L-leucine (13). To
a 5000 L glass-lined reactor was charged acetic acid (1794 kg),
purified water (266 kg), and sulfuric acid (27 kg). The batch
Tosylate Salt of Methyl (S)-2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate (25). The preparation of 23 via a
batch process has been described previously.18,19
Preparation of 23 via flow chemistry. Preparation of process
streams: To a 12500 L glass-lined reactor was charged THF
(5695 kg) at 20 °C and 22 (790 kg). In a separate 2000 L
glass-lined vessel was charged bromoacetonitrile (206 kg) at 20
°C. In another vessel was charged THF (680 kg) and MeOH
(53 kg) for the quench. In a 3000 L glass-lined vessel was
charged THF (1025 kg) and acetic acid (344 kg) at 20 °C
which was also intended for the quench. In a 5000 L glass-lined
vessel was charged purified water (3740 kg) followed by
sodium chloride (515 kg) at 20 °C.
Flow reaction: The solution of 22 in THF was precooled in
a coil to −70 °C and was pumped at a rate of 55 kg/h into
PFR1 at −70 °C, which was reacted with 1.0 M LHMDS in
THF (5309 kg) and was pumped at a rate of 46 kg/h at −70
°C. The stream leaving from PFR1 was maintained under
cooling and was pumped into PFR2 where it was blended with
a precooled stream of bromoacetonitrile in THF (pumped at a
rate of 34 kg/h) at −75 °C. The stream exiting PFR2 was
maintained under cooling and pumped into PFR3 where it was
blended at −75 °C with a precooled stream of MeOH in THF
(pumped at a rate of 33 kg/h). The resulting stream from
PFR3 was sent to CSTR1 at −65 °C where it was blended with
a precooled solution of acetic acid in THF (pumped at a rate
of 24 kg/h). The stream exiting CSTR1 was maintained at −70
°C and was sent to CSTR2 which was at −10 °C and had a
solution of aqueous sodium chloride (pumped in at a rate of 37
kg/h). The stream exiting CSTR2 was sent to a liquid−liquid
separator, and the organic phase was concentrated with a thin
film evaporator. The feed rate to the evaporator was 150−300
kg/h. The resulting solution was concentrated under vacuum
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in 96.5% purity and 92.5% assay. Residual solvent analysis
indicated 0.4% iPrOH, 0.1% MeOH, and 0.1% MTBE. Mp
175−177 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 8.53 (br
s, 3 H), 8.00 (s, 1 H), 7.48 (d, J = 8.20 Hz, 2 H), 7.12 (d, J =
7.80 Hz, 2 H), 4.22 (br s, 1 H), 3.76 (s, 3 H), 3.15−3.26 (m, 2
H), 2.53−2.62 (m, 1 H), 2.30 (s, 3H), 2.23−2.32 (m, 1 H),
1.87−2.09 (m, 2 H), 1.62−1.73 (m, 1 H); 13C NMR (100
MHz, DMSO-d6) δ 178.8, 170.2, 146.1, 138.2, 128.6, 126.0,
53.4, 51.8, 40.4, 38.9, 32.2, 28.2, 21.3.
to about 2−3 L/kg in a 8000 L glass-lined reactor. To the
solution was charged with toluene (2734 kg). The solution was
concentrated under vacuum to about 2−3 L/kg. To the
solution was charged toluene (2367 kg). Any insoluble
material remaining was removed by filtration. The filtrate
was concentrated under vacuum until about 1.5−2 L/kg
remained. To the residue was charged MeOH (3130 kg) which
was concentrated under vacuum until 3−4 L/kg remained. The
resulting solution in MeOH was assayed (typically 31% assay
and 60% yield) to yield 23 as a red to brown solution (∼1735
kg, (545 kg assay corrected). This solution in MeOH was
carried forward directly into the reduction and subsequent salt
formation.
To a 8000 L glass-lined reactor was charged MeOH (1991
kg) and cobalt(II) chloride hexahydrate (275 kg) at 20 °C.
The solution was cooled to −5 °C. A solution of 23 in MeOH
(1574 kg, (360 kg assay corrected) was charged while keeping
the temperature in the range of −15 to 0 °C. Sodium
borohydride (173 kg) was added to the reaction mixture in
portions. After charging about 50% of the sodium borohydride,
it was held for 1−3 h, monitored progress (confirming NMT
50% 23 remaining), and resumed charge. The reaction mixture
was held at −10 °C for about 6−10 h and monitored for
progress until NMT 2% of 23 remained (charged more sodium
borohydride (25 kg portion) to reach completion as needed).
Upon meeting reaction completion criteria, the reaction
mixture was warmed to 10−15 °C and held for at least 12 h.
The reaction mixture was sampled every 4−6 h until
conversion to 3 from 24 indicated NMT 2% of 24 remained.
The reaction mixture was quenched at 0−10 °C with a
solution prepared from purified water (1936 kg) and citric acid
monohydrate (650 kg). After the addition, the resulting
mixture was allowed to warm to 25 °C and was held for about
1 h. The reaction mixture was concentrated under vacuum
until about 5−6 L/kg and checked for residual MeOH (criteria
NMT 20% w/w). The reaction mixture was extracted with
DCM (2862 kg) at 20 °C by stirring for 30−60 min and
settling and discarding the aqueous layer. The lower organic
layer was concentrated under vacuum until 2−3 L/kg
remained. To the solution was charged iPrOH (1434 kg),
and the mixture was concentrated under vacuum at NMT 60
°C until 3−4 L/kg remained (confirmed that residual DCM is
NMT 5% and water by KF is NMT 1%). To the solution was
charged MTBE (801 kg) which was held at 20 °C for 30−60
min. The solution with MTBE was concentrated under
atmospheric conditions until ∼2 L/kg remained. In a separate
3000 L glass-lined reactor was charged toluenesulfonic acid
monohydrate (286 kg), iPrOH (569 kg), and trimethoxymethane (128 kg). The solution of tosic acid and trimethoxymethane was heated to 45 °C for 2−4 h until KF was NMT
0.5%. The iPrOH solution of tosic acid was concentrated
under vacuum until ∼1.5 L/kg remained. To the tosic acid
solution was charged iPrOH (569 kg) which was concentrated
under vacuum until 1.5−2 L/kg remained and residual MeOH
was NMT 3% w/w. The solution of tosic acid was charged to
the solution of 3 at 45−55 °C. The resulting reaction mixture
was held at 45−55 °C for 8−12 h and then sampled for
reaction assay (NMT 2% 3 remaining relative to 25). The
slurry was cooled to 5 °C and held for at least 2 h. The crystals
were filtered, and the transfer was washed twice with iPrOH
(284 kg) cooled to 5 °C. The filtercake was dried under
vacuum at 50−60 °C for 16 h. Isolated 25 as an off-white
crystalline solid (170 kg, (157 kg assay corrected), 38% yield)
Methyl (S)-2-((S)-2-(4-Methoxy-1H-indole-2-carboxamido)-4-methylpentanamido)-3-((S)-2-oxopyrrolidin-3yl)propanoate (31). To a 5000 L glass-lined reactor was
charged 13 (125 kg), 25 (155 kg), 2-hydroxypyridine N-oxide
(11.4 kg), and methyl ethyl ketone (515 kg). The reaction
mixture was cooled to 0 °C, and N,N-diisopropylethylamine
(119 kg) was charged. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (94.5 kg) was charged in a single
portion, and the mixture stirred for 20 min, then warmed to 20
°C, and stirred for at least 8 h or until NMT 1% 13 remained
by reaction assay. A 23.5 wt % aqueous solution of sodium
chloride (438 L) was added to the reaction mixture, followed
by a solution of orthophosphoric acid (71.4 kg) in water (375
L) and MTBE (625 L). The resulting biphasic mixture was
stirred for 15 min, then the layers were separated. The organic
layer was washed with 23.5 wt % aqueous saturated brine (438
L). The organic layer was concentrated under reduced pressure
(250 mbar, 50 °C) to a volume of ∼625 L (5 L/kg), charged
with MTBE (625 L), and repeated the distillation. Additional
MTBE (625 L) was charged, and the mixture cooled to 35 °C
over 15 min. Then, another portion of methyl tert-butyl ether
(313 L) was charged slowly, resulting in precipitation. The
slurry was granulated for 30 min before a final portion of
MTBE (313 L) was charged to achieve a final solvent ratio of
approximately 4:1 methyl tert-butyl ether:methyl ethyl ketone.
The final slurry was granulated for 30 min, then cooled to 10
°C at 0.25 °C/min, and held for 4 h. The final slurry was
filtered, rinsing with methyl tert-butyl ether (313 L) and dried
on the filter, then in a vacuum oven at 25 °C. Isolated 31 as
off-white crystals (233 kg, (163 kg assay corrected), 82% yield)
and was isolated as a MTBE solvate (13% w/w MTBE by
HSGC residual solvent assay) in 70% assay and 95.5% purity.
Mp 143−147 °C; 1H NMR (600 MHz, DMSO-d6): δ 11.56
(d, J = 2.1 Hz, 1H), 8.53 (d, J = 7.9 Hz, 1H), 8.37 (d, J = 8.1
Hz, 1H), 7.65 (s, 1H), 7.35 (dd, J = 2.1, 0.6 Hz, 1H), 7.09 (t, J
= 7.8 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.50 (d, J = 7.7 Hz,
1H), 4.52 (ddd, J = 10.2, 8.3, 5.0 Hz, 1H), 4.37 (ddd, J = 11.2,
7.7, 4.3 Hz, 1H), 3.88 (s, 3H), 3.62 (s, 3H), 3.11 (m, 2H),
3.08 (s, 3H), 2.36 (m, 1H), 2.14−2.06 (m, 2H), 1.76−1.65
(m, 2H), 1.65−1.59 (m, 2H), 1.59−1.52 (m, 1H), 1.11 (s,
9H), 0.94 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H); 13C
NMR (150 MHz, DMSO-d6): δ178.0, 172.6, 172.4, 160.9,
153.6, 137.8, 129.9, 124.4, 118.1, 105.4, 101.1, 99.2, 72.1, 55.1,
L
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136−139 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 11.59
(d, J = 1.95 Hz, 1 H), 8.62 (d, J = 7.81 H, 1 H), 8.45 (d, J =
7.41 Hz, 1 H), 7.65 (s, 1 H), 7.38 (d, J = 1.56 Hz, 1 H), 7.10
(t, J = 8.00 Hz, 1 H), 7.02 (d, J = 8.20 Hz, 1 H), 6.51 (d, J =
7.80 Hz, 1 H), 4.60 (d, J = 2.34 Hz, 2 H), 4.47 (ddd, J = 11.22,
7.71, 3.71 Hz, 2 H), 3.84−3.92 (m, 2H), 3.54 (m, 1H), 3.33 (s,
1 H), 3.06−3.18 (m, 2 H), 2.25−2.34 (m, 1 H), 2.07−2.15 (m,
1 H), 1.95−2.03 (m, 1 H), 1.53−1.91 (m, 9 H), 1.12 (s 3 H),
0.89- 0.97 (m, 6 H); 13C NMR (100 MHz, DMSO-d6) δ ppm
201.1, 178.8, 173.5, 161.8, 154.1, 138.3, 130.3, 124.9, 118.5,
105.9, 101.7, 99.7, 74.8, 67.2, 55.6, 55.2, 52.1, 48.2, 39.9, 37.9,
31.3, 27.7, 25.9, 24.9, 23.5, 21.8, 21.4.
51.9, 51.3, 50.1, 48.7, 40.3, 39.4, 37.6, 32.4, 27.2, 26.8, 24.3,
23.1, 21.5.
2-Methyl Tetrahydrofuran Solvate of N-((S)-1-(((S)-4Chloro-3-oxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-4-methoxy-1H-indole-2-carboxamide (8). To a 3000 L glass-lined reactor
under nitrogen at 20−25 °C is charged THF (609 kg), charged
31 (329 kg, (228.7 kg assay corrected)), and rinsed charge
with more THF (179 kg). Concentrated under vacuum
targeting a batch volume of 687−916 L (3−4 L/kg) to
remove any residual water and MEK before proceeding to the
Claisen reaction, charged THF (1618 kg) followed by
chloroacetic acid (82.4 kg, 1.8 equiv), and held for 1−2 h at
20−25 °C. To a separate 12500 L glass-lined reactor was
charged 1.7 M THF solution of tert-butyl magnesium chloride
in tetrahydrofuran (2753 kg, 12 equiv) and either triethylamine (490 kg, 10 equiv) or preferably N-methylpiperidine
(485 kg, 10 equiv). The reaction mixture in the 12500 L vessel
was cooled to 10 °C. The mixture of chloroacetic acid and 31
in THF was charged slowly to the solution of Grignard and Nmethylpiperidine at 5−20 °C (target of 10 °C) at a rate of
180−250 kg/h. The resulting reaction mixture was held at 5−
20 °C for at least 6 h (typically 12−32 h) and then was
sampled for reaction assay until NMT 5% 31 remained
(typically 2% 31 remained). In a 20000 L glass-lined reactor
was charged purified water (3000 kg) and citric acid
monohydrate (1225 kg) which was cooled to 5 °C. To this
cooled quench solution of aqueous citric acid was charged the
reaction mixture while keeping the batch temperature below 15
°C. Once the quench was complete, the resulting reaction
mixture was allowed to warm to 20 °C. The mixture was
extracted with MTBE (851.6 kg) by holding for at least 30 min
at 20 °C with agitation, settling for 30 min, and discarding the
aqueous phase. The upper, organic layer was cooled to 10 °C
and stirred for 10−15 min with a cold solution prepared from
sodium bicarbonate (91 kg) and purified water (1840 kg) at 10
°C. The mixture was settled, and the aqueous phase was
discarded. The organic layer was stirred for 30 min with a
solution prepared from sodium chloride (287 kg) and purified
water (2067 kg) at 20 °C. The mixture was settled, and the
aqueous layer discarded. The organic phase was concentrated
in vacuo to a target volume of 2290−3435 L (10−15 L/kg).
The reaction mixture was filtered to remove any particulates
and then concentrated to a volume of 687−916 L (3−4 L/kg).
Isopropanol was charged (530 kg, ∼ 2 L/kg) and concentrated
to 3−4 L/kg. This was repeated twice more to prepare the
iPrOH solvate in situ (could isolate this if desired with
addition of heptane to iPrOH) and confirmed by residual
solvent analysis that THF and MTBE were NMT 3% w/w.
The suspension of crude 8 in iPrOH (∼4 L/kg volume) was
heated to 45−55 °C, charged 2-MeTHF (3 L/kg) and
concentrated to target of 3−4 L/kg (residual water should be
low ∼2−3% by KF), and charged 2-MeTHF (3 L/kg),
concentrated to target of 3−4 L/kg again, and held at ∼50 °C
for about 2 h to enable form conversion to desired solvate (rod
morphology by PLM). Then charged heptane (2 L/kg) slowly
over 30−60 min, held for 1 h, cooled to 10−20 °C over at least
1 h, and held for at least 1 h. The crystals were filtered and
washed the transfer with 2-MeTHF cooled to 10 °C. The
crystals were dried under vacuum at NMT 45 °C for about 8−
12 h. Isolated 8 2-MeTHF solvate as off-white crystals (197 kg,
(166 kg assay corrected), 70% yield, 84% assay, 98% purity).
Residual solvent analysis indicated 11.3% w/w 2-MeTHF. mp
MEK Solvate of (S)-3-((S)-2-(4-Methoxy-1H-indole-2carboxamido)-4-methylpentanamido)-2-oxo-4-((S)-2oxopyrrolidin-3-yl)butyl Dihydrogen Phosphate (2). To
a glass-lined reactor under nitrogen was charged MEK (515
kg) and DMSO (124 kg) at ambient conditions. Then charged
potassium di-tert-butylphosphate (48.4 kg) and sodium iodide
(3.9 kg), charged 8 (75.0 kg, (63 kg assay corrected)), heated
the mixture to 30 °C, and held for 24 h. Assayed the reaction
mixture for completion (criteria is NMT 2% of the sum of 8
and iodo intermediate remaining). After confirming completion, charged water (600 kg) and MTBE (555 kg), held the
mixture for about 15 min with agitation, settled for about 30
min, and discarded the lower aqueous layer. At 30 °C, charged
water (600 kg), agitated for about 15 min, settled for 30 min,
and discarded the aqueous layer. At 30 °C, charged water (600
kg), agitated for about 15 min, settled for 30 min, discarded
the aqueous layer, cooled to 20−25 °C, and concentrated in
vacuo at 20−25 °C until the volume was about 750 L. Then
charged MEK (605 kg) and concentrated in vacuo at 20−25
°C until the volume was about 750 L a second time. Charged
MEK (605 kg) and concentrated in vacuo at 20−25 °C a third
time until the volume was about 900 L. Sampled the reaction
mixture for KF to confirm water was NMT 0.5%. After criteria
for water level was met, resumed concentration to reach a
volume of 750 L. To the slurry was charged oxalic acid (67.9
kg) which dissolved the substrate to give a solution, heated to
60 °C, and held for 12 h. Assayed the reaction mixture for
completion (criteria NMT 1% SM and mono-tert-butyl
intermediate remaining). After confirming completion, cooled
the reaction mixture to 10 °C over at least 1 h and held for at
least 5 h. Filtered the crystals and washed the transfer twice
with MEK (180 kg). Dried the crystals with a nitrogen stream
for several hours and dried with vacuum with mild heating at
NMT 35 °C for 6 h. Isolated MEK solvate of 2 as tan crystals
(53.44 kg, 67% yield). Mp 135−138 °C; 1H NMR (400 MHz,
DMSO-d6): δ 11.57 (d, J = 1.8 Hz, 1H), 8.52 (d, J = 8.0 Hz,
M
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Article
heated to 40 °C and held for at least 1 h to ensure the
polymorph conversion is complete. After confirming the
polymorph conversion was complete by PXRD, the slurry
was further diluted with MEK (180 kg) at 40 °C and held for
an additional 1 h. The slurry was cooled to 10 °C over about 2
h and held at 10 °C for 2 h. The cold slurry was filtered and
the transfer washed with a blend of cold MEK (116 kg) and
MeOH (28 kg). The solids (∼41 kg) were resuspended in a
blend of MEK (66 kg) and MeOH (16 kg). The slurry was
held at rt with agitation for 2−3 h. The slurry was filtered and
blown dry with nitrogen for several hours at rt. The solids were
dried under vacuum at 45−56 °C for 44 h and isolated the
hydrate of 2 as off-white crystals (34.3 kg, 93% yield) in 99.5%
purity. Residual solvent analysis indicated 0.16% MeOH,
0.41% MEK and NMT 0.05% DMSO. Mp 155−157 °C;
1
HNMR (400 MHz, DMSO-d6): δ 11.57 (d, J = 1.8 Hz, 1H),
8.52 (d, J = 8.0 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.63 (s,
1H), 7.36 (d, J = 1.6 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 7.00 (d,
J = 8.2 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 4.68 (dd, J = 17.6,
8.1 Hz, 1H), 4.57 (dd, J = 17.6, 7.2 Hz, 1H), 4.53−4.43 (m,
2H), 3.88 (s, 3H), 3.15−3.03 (m, 2H), 2.42 (q, J = 7.3 Hz,
2H), 2.32 (m, 1H), 2.06 (s and m, 4H), 1.95 (m, 1H), 1.77−
1.51 (m, 5H), 0.94 (d, J = 6.2 Hz, 3H), 0.90 (t, J = 7.1 Hz, 3
H), 0.89 (d, J = 6.2 Hz, 3H); 13CNMR (100 MHz, DMSOd6): δ 203.9 (d, J = 7.4 Hz), 178.8, 173.3, 161.6, 154.1, 138.3,
130.4, 124.9, 118.5, 105.9, 101.6, 99.7, 68.2 (d, J = 4.4 Hz),
55.5, 54.0, 51.9, 39.9, 37.8, 31.2, 27.6, 24.9, 23.5, 21.9.
1H), 8.42 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H), 7.36 (d, J = 1.6
Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H),
6.50 (d, J = 7.6 Hz, 1H), 4.68 (dd, J = 17.6, 8.1 Hz, 1H), 4.57
(dd, J = 17.6, 7.2 Hz, 1H), 4.53−4.43 (m, 2H), 3.88 (s, 3H),
3.15−3.03 (m, 2H), 2.42 (q, J = 7.3 Hz, 2H), 2.32 (m, 1H),
2.06 (s and m, 4H), 1.95 (m, 1H), 1.77−1.51 (m, 5H), 0.94
(d, J = 6.2 Hz, 3H), 0.90 (t, J = 7.1 Hz, 3 H), 0.89 (d, J = 6.2
Hz, 3H); 13CNMR (100 MHz, DMSO-d6): δ 203.9 (d, J = 7.4
Hz), 178.8, 173.3, 161.6, 154.1, 138.3, 130.4, 124.9, 118.5,
105.9, 101.6, 99.7, 68.2 (d, J = 4.8 Hz), 55.5, 54.0, 51.9, 39.9,
37.8, 36.3, 31.2, 29.8, 27.6, 24.9, 23.5, 21.9, 8.2.
DMSO Solvate of (S)-3-((S)-2-(4-Methoxy-1H-indole2-carboxamido)-4-methylpentanamido)-2-oxo-4-((S)-2oxopyrrolidin-3-yl)butyl dihydrogen phosphate (2). To
a glass-lined reactor under nitrogen was charged DMSO (85
kg) and MEK (111 kg). To the vessel at rt was charged MEK
solvate of 2 (55 kg). The reaction mixture was held for at least
1 h to ensure complete dissolution was achieved. The solution
was passed through a micron filter and rinsed the transfer with
a blend of MEK (44 kg) and DMSO (6 kg). The solution was
heated to 30 °C. To the solution was charged iPrOH (216 kg)
at 30 °C. The reaction mixture was held at 30 °C for 3 h
during which time crystals formed. To the slurry was charged
iPrOH (432 kg) over at least 20 min at 30 °C. The slurry was
held at 30 °C for at least 2 h. Cooled the slurry to 5 °C over at
least 2 h. Held at 5 °C for 11 h. Filtered the crystals and
washed the transfer twice with iPrOH (90 kg). Dried the
crystals with nitrogen stream at rt for several hours. Dried
crystals under vacuum at 40−50 °C for 7 h. Isolated DMSO
solvate of 2 as off-white crystals (46.7 kg, 89% yield) in 99.9%
purity. Analysis of residual solvents indicated 0.47% iPrOH,
0.10% MEK, 11.3% DMSO. Mp 166−168 °C; 1H NMR (400
MHz, DMSO-d6) δ ppm 11.58 (d, J = 1.95 Hz, 1 H), 8.52 (d, J
= 7.81 Hz, 1 H), 8.43 (d, J = 7.80 Hz, 1 H), 7.64 (s, 1 H), 7.36
(d, J = 1.56 Hz, 1 H), 7.10 (t, J = 8.00 Hz, 1 H), 7.01 (d, J =
8.20 Hz, 1 H), 6.51 (d, J = 7.80 Hz, 1 H), 4.46−4.72 (m, 4 H),
4.07 (br s, 1 H), 3.61−3.85 (m, 1 H), 3.41−3.60 (m, 1 H),
3.23−3.39 (m, 4 H), 2.99−3.21 (m, 3 H), 2.70 (s, 6 H), 2.28−
2.46 (m, 1 H), 2.15−2.21 (m, 4 H), 1.87−2.11 (m, 6 H),
1.52−1.78 (m, 5 H), 1.04 (d, J = 5.85 Hz, 1 H), 0.93 (br d, J =
12.49 Hz, 3 H), 0.87−0.98 (m, 3 H); 13CNMR (100 MHz,
DMSO-d6): δ 203.9 (d, J = 7.4 Hz),178.8, 173.4, 161.6, 154.1,
138.3, 130.4, 124.9, 118.5, 105.9, 101.6, 99.7, 68.2 (d, J = 4.4
Hz), 55.6, 54.0, 52.0, 40.9, 39.9, 37.8, 31.2, 27.6, 24.9, 23.5,
21.9.

ASSOCIATED CONTENT
sı Supporting Information
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.2c00375.
Includes 1H NMR and 13C NMR spectra for all of the
isolated compounds. Also includes X-ray powder
diffraction data for compounds 13, 15, 31, 8, MEK
solvate of 2, DMSO solvate of 2, and hydrate of 2
(PDF)

AUTHOR INFORMATION
Corresponding Author
Robert A. Singer − Chemical Research and Development,
Pfizer Inc., Groton, Connecticut 06340, United States;
orcid.org/0000-0001-9730-1261;
Email: robert.a.singer@pfizer.com
Authors
Christophe Allais − Chemical Research and Development,
Pfizer Inc., Groton, Connecticut 06340, United States;
orcid.org/0000-0002-3443-9391
David Bernhardson − Chemical Research and Development,
Pfizer Inc., Groton, Connecticut 06340, United States
Adam R. Brown − Chemical Research and Development,
Pfizer Inc., Groton, Connecticut 06340, United States
Gary M. Chinigo − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
Jean-Nicolas Desrosiers − Chemical Research and
Development, Pfizer Inc., Groton, Connecticut 06340, United
States
Kenneth J. DiRico − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
Ian Hotham − Chemical Research and Development, Pfizer
Inc., Groton, Connecticut 06340, United States
Hydrate of (S)-3-((S)-2-(4-Methoxy-1H-indole-2-carboxamido)-4-methylpentanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butyl dihydrogen phosphate (2). To a
glass-lined reactor under nitrogen at 25 °C was charged MEK
(235 kg) followed by DMSO solvate of 2 (45 kg). To the
slurry was charged MeOH (125 kg). The resulting slurry was
N
https://doi.org/10.1021/acs.oprd.2c00375
Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
pubs.acs.org/OPRD
Brian P. Jones − Chemical Research and Development, Pfizer
Inc., Groton, Connecticut 06340, United States
Samir A. Kulkarni − Chemical Research and Development,
Pfizer Inc., Groton, Connecticut 06340, United States
Chad A. Lewis − Chemical Research and Development, Pfizer
Inc., Groton, Connecticut 06340, United States;
orcid.org/0000-0002-2000-6456
Ricardo Lira − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
Richard P. Loach − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
Peter D. Morse − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
James J. Mousseau − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States; orcid.org/0000-00025712-9222
Matthew A. Perry − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
Zhihui Peng − Chemical Research and Development, Pfizer
Inc., Groton, Connecticut 06340, United States
David W. Place − Chemical Research and Development, Pfizer
Inc., Groton, Connecticut 06340, United States;
orcid.org/0000-0001-6174-0721
Anil M. Rane − Chemical Research and Development, Pfizer
Inc., Groton, Connecticut 06340, United States
Lacey Samp − Chemical Research and Development, Pfizer
Inc., Groton, Connecticut 06340, United States
Zheng Wang − Chemical Research and Development, Pfizer
Inc., Groton, Connecticut 06340, United States
Gerald A. Weisenburger − Chemical Research and
Development, Pfizer Inc., Groton, Connecticut 06340, United
States
Hatice G. Yayla − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
Joseph M. Zanghi − Chemical Research and Development,
Pfizer Inc., Groton, Connecticut 06340, United States
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Complete contact information is available at:
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Notes
The authors declare the following competing financial
interest(s): The authors were all employees of Pfizer Inc. at
the time of this work.

ACKNOWLEDGMENTS
Our team is indebted to Cheryl Hayward for organizing
support on the project with one of the largest development
teams ever assembled. We thank John Weaver, Catherine
Buckley, and Remzi Duzguner for supporting the safety testing
on this program. This project could not have moved quickly
without the analytical support assistance of Doug Farrand,
Beth Greenberg, Kerri Bradshaw, Jorge Lopez, Morgan Duffy,
Andy Davidson, and Mark Olivier. The scale up of the various
routes within Pfizer was supervised by Steve Hoagland, Tim
Houck, Andy Fowler, Kudzai Saunyama, and Kevin Meldrum.
We thank Cuong Lu, Hud Risley, and Emma McInturff for
communication with vendors who ultimately provided
advanced intermediates and API.

Article
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13104−13110. (b) Tian, Q.; Nayyar, N. K.; Babu, S.; Chen, L.; Tao,
J.; Lee, S.; Tibbetts, A.; Moran, T.; Liou, J.; Guo, M.; et al. An
efficient synthesis of a key intermediate for the preparation of the
rhinovirus protease inhibitor AG7088 via asymmetric dianionic
cyanomethylation of N-Boc-L-(+)-glutamic acid dimethyl ester.
Tetrahedron Lett. 2001, 42, 6807−6809.
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Nonno, R.; Pannacci, M.; Lucini, V.; Fraschini, F.; Stankov, B. M. 2[N-Acylamino(C1−C3)alkyl]indoles as MT1Melatonin Receptor
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(13) Preparation of methyl 4-bromo-1H-indole-2-carboxylate:
Combined 2,6-dibromobenzaldehyde, hippuric acid (1.2 equiv),
acetic anhydride (3.3 equiv) and potassium acetate (1.2 equiv) in
THF. Heated mildly (40−50 °C) to enable aldol condensation and
dehydration to form the oxazolone product which crystallized from
solution. Upon completion, diluted the reaction with isopropanol (6
mL/g) and water (4 mL/g). The suspension was cooled to rt and
filtered and dried. The oxazolone product, 4-(2,6-dibromobenzylidene)-2-phenyloxazol-5(4H)-one, was isolated as a yellow, crystalline
solid in 86−89% yield. Heated 4-(2,6-dibromobenzylidene)-2-phenyloxazol-5(4H)-one to 50 °C in methanol (1.5 mL/g) and toluene (5
mL/g) with triethylamine (1.0 equiv) which rapidly converted the
oxazolone to the methyl ester. Charged potassium carbonate (3
equiv) and copper (I) iodide (0.1 equiv) and increased heating to
reflux. Upon completion, worked up with EtOAc and aq ammonium
hydroxide followed by a wash with citric acid. Crystallized the methyl
4-bromo-1H-indole-2-carboxylate from 10:1 MeOH/water in 85%
yield and ∼70% yield overall.
(14) Preparation of 4-methoxy-1H-indole-2-carboxylic acid: Combined 2 M potassium tert-butoxide in 2-MeTHF (3eq), methanol (3.5
equiv), copper (I) iodide (0.05 equiv), and ligand (either DPEO or
DMAPO, 0.05 equiv). To the resulting blue mixture was added
methyl 4-bromo-1H-indole-2-carboxylate, and the reaction was
heated to 80 °C. Upon completion, the reaction mixture was cooled
to 20 °C, and 1 mL/g of water was added. The reaction was warmed
to 60 °C and held for hydrolysis to the carboxylic acid. After
hydrolysis was complete, the reaction mixture was filtered through
Celite, washed with 0.5 mL/g MeTHF and 0.5 mL/g water. The
resulting filtrate was acidified with phosphoric acid (4 equiv) and the
resulting biphasic mixture was split. To the resulting organic layer was
added 10 mL/g ethyl acetate, and the solution was distilled to a
Article
volume of roughly 7 mL/g. After distillation, added 3.5 mL/g
methylcyclohexane and crystallized. Filtering the resulting slurry
provided an 85% yield of 4-methoxy-1H-indole-2-carboxylic acid.
Preparation of N1,N2-bis(3-(dimethylamino)propyl)oxalamide
(DMAPO): To a 150 mL reactor with overhead stirring was charged
diethyl oxalate (68.4 mmol, 9.29 mL, 10.0 g, 1.00 equiv), and
methylcyclohexane (100 mL, 10 mL/g). Heated to 30 °C. Charged
N,N-dimethyl-1,3-propanediamine (205 mmol, 26 mL, 21.0 g, 3.00
Equiv) dropwise while controlling the temperature between 35 and 50
°C. Held at 35−40 °C for 2 h. Cooled to 25 °C to allow crystals to
form. Held for 1 h. Cooled to 10−20 °C and held for 1 h. Filtered the
crystals. Isolated PF-01041726 (58.4 mmol, 15.1 g, 85.4% Yield, 0.854
Equiv) as white crystals. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.78
(br t, J = 5.85 Hz, 1 H), 3.15 (q, J = 6.89 Hz, 2 H), 2.19 (t, J = 6.83
Hz, 2 H), 2.11 (s, 6 H), 1.58 (quin, J = 7.02 Hz, 2 H); 13CNMR (100
MHz, DMSO-d6): δ 160.4, 57.4, 45.6, 38.0, 27.1.
(15) Chen, Z.; Jiang, Y.; Zhang, L.; Guo, Y.; Ma, D. Oxalic Diamides
and tert-Butoxide: Two Types of Ligands Enabling Practical Access to
Alkyl Aryl Ethers via Cu-Catalyzed Coupling Reaction. J. Am. Chem.
Soc. 2019, 141, 3541−3549.
(16) (a) Rajkumar, S.; He, S.; Yang, X. Kinetic Resolution of
Tertiary 2-Alkoxycarboxamido-Substituted Allylic Alcohols by Chiral
Phosphoric Acid Catalyzed Intramolecular Transesterification. Angew.
Chem., Int. Ed. 2019, 58, 10315−10319. (b) Parisi, G.; Colella, M.;
Monticelli, S.; Romanazzi, G.; Holzer, W.; Langer, T.; Degennaro, L.;
Pace, V.; Luisi, R. Exploiting a “Beast” in Carbenoid Chemistry:
Development of a Straightforward Direct Nucleophilic Fluoromethylation Strategy. J. Am. Chem. Soc. 2017, 139, 13648−13651.
(17) (a) Fujieda, H.; Maeda, K.; Kato, N. Efficient and Scalable
Synthesis of Glucokinase Activator with a Chiral ThiophenylPyrrolidine Scaffold. Org. Process Res. Dev. 2019, 23, 69−77.
(b) Chen, P.; Cheng, P. T. W.; Spergel, S. H.; Zahler, R.; Wang,
X.; Thottathil, J.; Barrish, J. C.; Polniaszek, R. P. A Practical Method
for the Preparation of α-Chloroketones of N-Carbamate Protected-αAminoacids. Tetrahedron Lett. 1997, 38, 3175−3178. (c) Ganiek, M.
A.; Ivanova, M. V.; Martin, B.; Knochel, P. Mild Homologation of
Esters through Continuous Flow Chloroacetate Claisen Reactions.
Angew. Chem., Int. Ed. 2018, 57, 17249−17253.
(18) (a) Tian, Q.; Nayyar, N. K.; Babu, S.; Chen, L.; Tao, J.; Lee, S.;
Tibbetts, A.; Moran, T.; Liou, J.; Guo, M.; Kennedy, T. P. An
Efficient Synthesis of a Key Intermediate for the Preparation of the
Rhinovirus Protease Inhibitor AG7088 via Asymmetric Dianionic
Cyanomethylation of N-Boc-L-(+)-Glutamic Acid Dimethyl Ester.
Tetrahedron Lett. 2001, 42, 6807−6809. (b) Vuong, W.; Vederas, J. C.
Improved Synthesis of a cyclic Glutamine Analogue Used in Antiviral
Agents Targeting 3C and 3CL Proteases Including SARS-CoV-2
Mpro. J. Org. Chem. 2021, 86 (18), 13104−13110.
(19) Hanessian, S.; Margarita, R. 1,3-Asymmetric Induction in
Dianionic Allylation Reactions of Amino Acid Derivatives-Synthesis
of Functionally Useful Enantiopure Glutamates, Pipecolates and
Pyroglutamates. Tetrahedron Lett. 1998, 39, 5887−5890.
(20) Kong, D.; Li, M.; Wang, R.; Zi, G.; Hou, G. Highly efficient
asymmetric hydrogenation of cyano-substituted acrylate esters for
synthesis of chiral γ-lactams and amino acids. Organic & Biomolecular
Chemistry 2016, 14, 1216−1220.
(21) Yang, S.; Chen, S.-J.; Hsu, M.-F.; Wu, J.-D.; Tseng, C.-T. K.;
Liu, Y.-F.; Chen, H.-C.; Kuo, C.-W.; Wu, C.-S.; Chang, L.-W.; et al.
Synthesis, Crystal Structure, Structure-Activity Relationships, and
Antiviral Activity of a Potent SARS Coronavirus 3CL Protease
Inhibitor. J. Med. Chem. 2006, 49, 4971−4980.
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K. X.; Liu, H. Improved Synthesis of Rupintrivir. Science China:
Chemistry 2012, 55, 1101−1107. (b) Ghosh, A. K.; Xi, K.; Ratia, K.;
Santarsiero, B. D.; Fu, W.; Harcourt, B. H.; Rota, P. A.; Baker, S. C.;
Johnson, M. E.; Mesecar, A. D. Design and Synthesis of
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A.; Vatansever, E. C.; Drelich, A. K.; Sankaran, B.; Geng, Z. Z.;
Blankenship, L. R.; Ward, H. E.; et al. A Quick Route to Multiple
Highly Potent SARS-CoV-2 main protease inhibitors. ChemMedChem.
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J.; Su, H.; Chang, D.; Wang, J.; Peng, J.; Zhu, L.; Nian, Y.; Hilgenfeld,
R.; Jiang, H.; Chen, K.; Zhang, L.; Xu, Y.; Neyts, J.; Liu, H. Design,
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(24) An alternative approach was to carry out the deprotection in
MeOH (3−5 mL/g) at 20−35 °C over 24−48 h and dilute with
EtOAc or MTBE (10−15 mL/g) to improve the yield.
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Shevlin, M. A Concise Synthesis of a β-Lactamase Inhibitor. Org. Lett.
2011, 13, 5480−5483. (b) Chung, J. Y. L.; Meng, D.; Shevlin, M.;
Gudipati, V.; Chen, Q.; Liu, Y.; Lam, Y.-H.; Dumas, A.; Scott, J.; Tu,
Q.; Xu, F. Diastereoselective FeCl3 •6H2O/NaBH4 Reduction of
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(26) The sulfoxonium ylide had been previously examined with 3
and had shown to proceed well through the addition but was slow to
hydrolyze to the desired methyl chloro ketone. For this substrate, 31,
the reaction did not even perform very well with the sulfoxonium
ylide. Malonates also failed to progress well with 31.
(27) While chloroacetates performed well in the Claisen addition,
alpha-alkoxy acetates did not, even with the protected phosphate on
the acetate. This would have been a more convergent means of
introducing the protected phosphate moiety and avoids the need for
the alkylation step with the phosphate but could not be realized.
(28) Many factors can influence crystallization and polymorph
transitions such as temperature, solvent and purity of starting material.
For one example, see the discussion relating to axitinib and the
references therein: Chekal, B. P.; Campeta, A. M.; Abramov, Y. A.;
Feeder, N.; Glynn, P. P.; McLaughlin, R. W.; Meenan, P. A.; Singer,
R. A. The Challenges of Developing an API Crystallization Process for
a Complex Polymorphic and Highly Solvating System. Part I. Org.
Process Res. & Dev. 2009, 13, 1327−1337.
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