Development of a Large-Scale Cyanation Process Using Continuous Flow Chemistry en Route to the Synthesis of Remdesivir

Article abstract of DOI:10.1021/acs.oprd.0c00172

The implementation of cyanation chemistry at manufacturing scales using batch equipment can be challenging because of the hazardous nature of the reagents employed and the tight control of reaction parameters, including cryogenic temperatures, that help t

Full text of DOI:10.1021/acs.oprd.0c00172

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Full Paper
Development of a Large-Scale Cyanation Process Using Continuous
Flow Chemistry en Route to the Synthesis of Remdesivir
Tiago Vieira, Andrew Stevens, Andrei Chtchemelinine, Detian Gao, Pavel Badalov, and Lars Heumann
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00172 • Publication Date (Web): 21 May 2020
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Development of a Large-Scale Cyanation Process
Using Continuous Flow Chemistry en Route to the
Synthesis of Remdesivir
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Tiago Vieira, †* Andrew C. Stevens,†* Andrei Chtchemelinine,‡ Detian Gao,† Pavel Badalov,†
and Lars Heumann ‡
†Gilead Alberta ULC, 1021 Hayter Road, Edmonton, Alberta T6S 1A1, Canada
‡Gilead Sciences, Inc. 333 Lakeside Drive, Foster City, California 94404, United States
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Table of Contents Graphic
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NH2
N
NH2
N
NH2
N
TFA, TMSOTf,
TMSCN
O
O
N
N
N
N
O
O
O
P
N
N
O
N
BnO
BnO
OH
HN
O
N
DCM, -30 °C
Flow Chemistry
O
BnO
OBn
BnO
OBn
9
HO
OH
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1
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remdesivir
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ABSTRACT: The implementation of cyanation chemistry at manufacturing scales using batch
equipment can be challenging due to the hazardous nature of the reagents employed, and the
tight control of reaction parameters, including cryogenic temperatures, that help to afford
acceptable selectivity and conversion for the desired reaction. Application of continuous flow
chemistry offers a means to mitigate the risk associated with handling large amounts of
hazardous reagents and to better control the reaction parameters. A case study describing the
cyanation of a glycoside using continuous flow chemistry towards the synthesis of the drug
candidate remdesivir is presented.
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KEYWORDS: Continuous flow chemistry, cyanation, remdesivir, COVID-19
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Introduction
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In response to the 2014 outbreak of Ebola virus disease in Libera, Guinea and Sierra Leone,
Gilead Sciences Inc., in collaboration with the Centers for Disease Control and Prevention
(CDC) and the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID),
identified an adenosine nucleotide analogue that demonstrated strong antiviral activity against
multiple strains of the Ebola virus in vitro and in animal models (remdesivir, Scheme 1).1
Remdesivir has also demonstrated broad-spectrum antiviral activity against coronaviruses,
including SARS-CoV and MERS-CoV in primary human cell culture models, and in vivo
models of viral pathogenesis.2 More recently, remdesivir has been found to possess in vitro
inhibition of the novel 2019 coronavirus (SARS-CoV-2) responsible for the outbreak of
COVID-19.3 Based on this observed activity towards coronaviruses, remdesivir has been
applied, on compassionate use grounds, in response to the outbreak of COVID-19 infection4,5
and clinical trials have been initiated.6,7
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The synthesis of remdesivir has been previously reported8 and proceeds through a 6-step
sequence (Scheme 1) which includes the installation of a cyano group through the
stereoselective addition of cyanide to the 1’-position of riboside 1. In the initial process, this
transformation, required cryogenic conditions (-78 °C) and had the potential to liberate
significant quantities of hydrogen cyanide. Pre-clinical and clinical evaluation of remdesivir
would necessitate production of multi-kilogram quantities of compound 2, requiring the
establishment of a safe, robust process for its synthesis. Herein we report the development of
continuous flow conditions for the preparation of 2.
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Scheme 1. Synthetic Route to Remdesivir
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NH2
N
NH2
N
NH2
N
TfOH, TMSOTf,
TMSCN
O
O
N
N
N
N
O
O
O
P
N
N
O
N
BnO
BnO
OH
OBn
HN
O
N
9
DCM, -78 °C
O
BnO
BnO
OBn
HO
OH
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remdesivir
1
2
Results and Discussion
Batch Mode Preparation of Compound 2
Two manufacturing processes for the preparation of compound 2 were developed: i) a batch
cyanation process for pre-clinical and early-stage clinical remdesivir demands; ii) a cyanation
process using continuous flow for advanced phase clinical trials and commercial
manufacturing.
As previously reported,8 the cyanation reaction is performed through combination of
compound 1 (Scheme 1) in dichloromethane (DCM) with trifluoromethanesulfonic acid
(TfOH), trimethylsilyl trifluormethanesulfonate (TMSOTf), and trimethylsilyl cyanide
(TMSCN) while maintaining the reaction temperature at -78 °C. These reaction conditions
afforded a clean reaction profile, with good diastereoselectivity (Table 1, entry 1). The first
challenge to be addressed was the requirement of cryogenic reaction temperatures. When the
reaction was conducted at -15 °C, a rapid and significant decomposition of the starting material
was observed along with reduced diastereoselectivity for the transformation (Table 1, entry 2).
Alternative Brønsted acids were evaluated and trifluoroacetic acid (TFA) was found to be a
promising lead (Table 1, entries 3–7), providing good solution purity of compound 2 and
acceptable diastereoselectivity. Decreasing the reaction temperature to -30 °C was found to
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increase the solution purity as a result of decreased degradation of reactive intermediates,
while providing a modest improvement to the diastereoselectivity (Table 1, entry 5 vs entry
8). The order of addition was also found to impact the diastereoselectivity. For example, adding
the TMSCN prior to the addition of TFA and TMSOTf significantly eroded the
diastereoselectivity of the reaction (Table 1, entry 5 vs entry 9). Reagent equivalents were
investigated and observed to play a role for reaction performance, as increasing the quantities
of both TMSOTf and TMSCN to 6 equivalents increased the selectivity of the reaction,
affording a high diastereoselectivity of 93:7. (Table 1, entries 10–12).
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With the optimized conditions in hand, a larger scale experiment was performed (Table 1,
entry 13). During the scale-up experiment, TMSOTf and TMSCN were each added over
approximately 30 minutes to maintain the reaction at the target temperature of -30 °C. The
30 min addition time resulted in significant decomposition of the reaction mixture and a lower
diastereoselectivity (Table 1, entry 13). This finding was addressed by first combining the
starting material 1 in DCM with TFA at -30 °C, followed by adding a mixture of TMSOTf and
TMSCN in DCM that had been pre-cooled to -30 °C. This new order of addition prevented the
starting material from decomposing while maintaining acceptable diastereoselectivity (Table
1, entry 14). The stoichiometry of TMSOTf was revisited, and it was found that a decrease
would result in compromised diastereoselectivity (Table 1, entry 15).
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Scheme 2. Preparation of Compound 2
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NH2
N
NH2
N
NH2
N
Brønsted Acid
TMSOTf, TMSCN
N
N
N
O
O
O
N
N
N
BnO
BnO
BnO
+
OH
OBn
N
N
DCM, temperature
BnO
BnO
OBn
BnO
OBn
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1
2
3
Undesired Diastereomer
Table 1. Optimization of Cyanation Batch Mode Reaction Conditions
Brønsted
Acid
Acid
(equiv)
TMSOTf TMSCN
(equiv)
Reaction
Solution Purity
of 2 (%)a
In-Process
d.r.
(2:3)
Entry Input of 1
(equiv) Temperature (°C)
Content of 3 (%)a
1
2
3
4
5
6
7
8
9b
100 mg
100 mg
100 mg
100 mg
100 mg
100 mg
100 mg
200 mg
200 mg
TfOH
TfOH
TsOH
MsOH
TFA
2
2
2
2
2
2
2
3
2
2
2
4
4
4
3
3
3
3
3
3
4
4
4
3
3
3
3
-78
-15
-15
-15
-15
-15
-15
-30
-15
90.4
32.3
41.0
16.4
54.1
11.1
2.4
3.6
13.3
5.8
96: 4
71:29
88:14
68:32
85:15
66:34
64:36
90:10
60:40
7.8
9.5
HCO2H
AcOH
TFA
5.7
1.4
75.7
34.8
8.7
TFA
23.0
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200 mg
200 mg
200 mg
66 g
TFA
TFA
TFA
TFA
TFA
TFA
1
1
3
3
3
2
1
3
6
6
6
4
3
3
6
6
6
6
-30
-30
-30
-30
-30
-30
48.6
84.0
88.6
69.2
83.2
78.3
17.4
8.4
74:26
91: 9
93: 7
90:10
85:15
79:21
11
12
6.8
13c
14d
15d
8.0
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200 mg
200 mg
14.7
20.4
Reaction Conditions: Compound 1 in DCM was cooled to the indicated temperature and the indicated Brønsted acid, TMSOTf,
and TMSCN were added sequentially. a Purity was determined by LC analysis. b Reaction Conditions: Compound 1 in DCM was
cooled to the indicated temperature and TMSCN was added, followed by TFA and TMSOTf. c Addition of TMSOTf and TMSCN
were performed over about 30 min each. d Reaction Conditions: TFA was added to a solution of compound 1 in DCM at -30 °C
and a solution of TMSCN and TMSOTf in DCM pre-cooled to -30 °C was subsequently added.
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Having identified viable conditions for the formation of compound 2, attention was directed
towards isolation of the product. On the basis of a number of laboratory trials in solvent
screening (over 40 single solvent or solvent combinations were investigated), it was
determined that compound 2 exhibited favorable solid-state properties and could be selectively
crystallized from either a mixed solvent system (such as ethyl acetate and heptane) or a single
solvent (such as 2-propanol or toluene). Following additional development studies, including
hazards assessments and safety studies, the batch cyanation process was successfully executed
for the early deliveries of remdesivir (Table 2). This enabled acceleration of the program at a
time when there was an urgent need of remdesivir for clinical evaluation due to the escalating
Ebola virus epidemic. For the planned large scale batch size, two key challenges needed to be
addressed: improvement of the safety measures due to the increased operational hazards
associated with the larger quantities of acidic reaction mixtures containing cyanide; and the
increased operational time required to combine large quantities of compound 1/TFA with
TMSOTf/TMSCN which could negatively impact diastereoselectivity and yield.
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Table 2. Batch Mode Preparation of 2
Reaction d.r.
Input
(kg)
Crystallization
Conditions
Product
Product d.r.
(2:3)
Batch
Yield (%)
Purity (%)a
(2:3)
1
EtOAc /
heptane
9.6
84:16
70
100.0
99.9:0.1
2
3
22.0
23.1
86:14
N/Ab
IPA
57
71
99.0
99.2
99.7:0.3
99.1:0.9
Toluene
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Reaction Conditions: TFA (3 equiv) was added to a solution of compound 1 in DCM at -30 °C
and a solution of TMSCN (6 equiv) and TMSOTf (6 equiv) in DCM pre-cooled to -30 °C was
subsequently added. a Purity was determined by LC analysis. b Reaction samples not analyzed
to minimize exposure to hazardous reaction mixtures.
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Feasibility Studies for the Preparation of Compound 2 via Continuous Flow Chemistry
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Based on the above challenges associated with further increasing batch size, alternative
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processing modes were evaluated. Isolated literature reports discuss the use of continuous flow
chemistry to perform C-glycosylations,9 O-glycosylations,10 and the use of cyanide in flow
chemistry directly11 or by using a cyanide precursor.12 This mode of processing offers several
unique advantages to traditional batch processing, including enabling large scale chemistry to
be performed with reduced reaction volumes, and improved temperature control and mixing
efficiency.13-17 Application of these advantages of continuous flow chemistry could, in
principle, address the challenges associated with the batch cyanation process to prepare
compound 2. For instance, smaller quantities of hazardous reaction mixture would be present
during the reaction and could be rapidly quenched once the reaction stream exited the flow
reactor, thereby mitigating a portion of the safety risks associated with the process. The
continuous flow chemistry manufacturing mode would also enable processing facilities to
perform the process at about -30 °C with relatively inexpensive cooling units. An appropriately
designed flow reactor would improve mixing and cooling compared to batch mode chemistry,
both of which could impact the process (Table 1, entries 13 and 14). Lastly, the process was
believed to be viable for continuous flow chemistry as the reaction components were all clear
solutions (once dissolved in DCM) and the reaction mixture was not observed to become
heterogeneous throughout the course of the reaction.
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The development of the continuous flow chemistry process was first investigated on a
VapourtecTM R-series system consisting of four HPLC pumps and 1/16” PFA tubing. The initial
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configuration can be found in Figure 1, where compound 1 is sequentially combined with TFA,
followed by TMSOTf, and then TMSCN. Based on the fast reaction times observed during
development of the batch mode process, the residence times were initially set at 0.5 min, 0.4
min, and 1.8 min for the reaction of compound 1 with TFA, TMSOTf, and TMSCN,
respectively. These initial conditions resulted in high conversion and good diastereoselectivity
(Table 3, entry 1). It appeared that the Brønsted acid (TFA) was not necessary at small scale
for the continuous flow chemistry process (Table 3, entry 2). The results at larger scale
(discussed later) suggested that the presence of TFA was important for process robustness.
Evaluation of the residence times indicated that 0.5 min was optimal for the reaction with
TMSOTf, and that a 2 min residence time was sufficient for the reaction with TMSCN (Table
3, entry 2 vs entry 3–4). Decreasing the equivalents of reagents was found to negatively impact
reaction performance, with a decrease in both conversion and diastereoselectivity under the
flow chemistry conditions, regardless of whether TFA was present or absent (Table 3, entries
9, 10, and 11).
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With acceptable continuous flow conditions established, an in-house model system was
assembled, consisting of three diaphragm metering pumps and 3/16” PFA tubing. The more
powerful diaphragm pumps (compared to the VapourtecTM HPLC pump system) allowed a
significant increase in throughput (ca. 317 g/h consumption of compound 1). Using this system,
the process was tested at 100 g input of compound 1, affording high diastereoselectivity (94:6)
and a solution purity of compound 2 of 93.5% (Table 3, entry 12). Subsequent isolation of the
quenched reaction stream afforded compound 2 in 84% yield and 99.8% purity. This larger
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scale test demonstrated the viability of the continuous flow process and enabled the shift from
batch mode to continuous flow for future preparation of compound 2.
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Figure 1. A Schematic Drawing of Continuous Flow Chemistry Setup
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1
Compound
in DCM
TMSOTf
in DCM
TMSCN
in DCM
Pre-cooling
Loop
Pre-cooling
Pre-cooling
Loop
Lloop
Pre-cooling
Loop
Reaction
Loop 1
Reaction
Loop 2
Reaction
Loop 3
Temperature controlled to -40 °C
TFA
in DCM
KOH(aq) quench
Table 3. Development of Continuous Flow Chemistry Conditions
TFA /
TMSOTf /
TMSCN
(equiv)
Residence Time (min)
In-Process
Solution
In-Process
d.r.
Entry
Content of 1 Purity of 2 Content of 3
(2:3)a
Reaction Reaction Reaction
(%)a
(%)a
(%)a
Loop 1
Loop 2
Loop 3
1
2
3
4
5
6
7
8
9
10
3 / 6 / 6
0 / 6 / 6
0 / 6 / 6
0 / 6 / 6
0 / 6 / 6
0 / 6 / 6
0 / 6 / 6
0 / 6 / 6
1 / 3 / 3
0 / 3 / 3
0.5
0.4
1.8
0.5
0.6
1.3
2.1
1.7
4.0
3.8
0.3
3.7
4.7
89.3
93.4
92.1
91.6
90.5
81.5
87.4
77.8
81.9
69.1
6.3
5.6
6.0
5.8
7.1
9.0
7.8
6.4
13.1
15.3
93:7
94:6
94:6
94:6
93:7
90:10
92:8
92:8
86:14
82:18
—
—
—
—
—
—
—
0.8
—
0.5
0.5
2.5
2.4
0.5
0.4
2.1
0.7
0.7
2.0
3.9
2.0
3.7
2.0
1.8
1.8
2.9
2.9
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2 / 2 / 2
0 / 6 / 6
0.5
0.4
0.5
1.9
2.0
5.7
0.7
74.4
93.5
10.8
5.6
87:13
94:6
12b
—
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Reactions were performed at 1 g input of compound 1 on a VapourtecTM R-Series flow
chemistry system using 1/16” (OD) PFA tubing. Reaction components were combined
according to Figure 1. Residence times were achieved through increasing the volume of the
appropriate reaction loop while maintaining the flow rates constant. Reaction temperatures
were maintained at about -40 °C through immersion of the flow reaction loops in an IPA/dry
ice bath. Only steady state reaction streams were used to analyze in-process reaction profiles.
a Purity was determined by LC analysis of an aliquot of the quenched reaction mixture.
b Experiment was performed at 100 g input of compound 1 on an in-house build system
consisting of 3 diaphragm pumps and 1/4” (OD) PFA tubing. The quenched reaction stream
was isolated via crystallization to afford compound 2 in 84% yield and 99.8% purity by LC.
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Kilo-Lab Scale Preparation of Compound 2 via Continuous Flow Chemistry
Following the successful 100 g demonstration with the in-house model continuous flow
chemistry system (Table 3, entry 12), an improved system was designed for the preparation of
20 kg of compound 2. The following criteria were considered necessary during the design of
this continuous flow reactor: improved safety for larger-scale manufacturing, compatibility of
the reagents and product with the materials of construction of the reactor system,
heating/cooling requirements, mixing requirements, and increased throughput relative to the
lab-scale system. A stainless-steel reactor was designed and fabricated (Figure 2a), which
improved the durability of the reactor and helped the prevention of potential loss of
containment. Stainless-steel (SS) was selected as the material of construction, as it was found
to be compatible with the individual reaction components or mixtures thereof (elemental
impurities due to leaching were not detected in the reaction streams by ICP-MS). With regards
to the cooling requirements for the continuous flow chemistry system, the heat of reaction
was found to be -133 kJ/mol for the reaction of compound 1 with TMSOTf, and -68 kJ/mol for
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the reaction of this stream with TMSCN. These exotherms would give adiabatic temperature
rises of 9 K and 4 K, respectively, which could be easily controlled by bath cooling when using
the SS reactor. Quenching of the reaction mixture was accomplished in a semi-batch mode by
constant delivery of the reaction mixture into a caustic aqueous mixture. A complete system
that was assembled based on the established conditions is shown in Figure 2b.
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Figure 2. Kilo Lab Flow Chemistry Equipment a) Flow Reactor. b) Kilo Lab Flow Reactor
Assembly
a)
b
)
With the SS continuous flow system, an experimental run was performed with 100 g input
of compound 1 (without the use of TFA) to confirm the system performance. The results
obtained were within expectations, although a slight increase in the residual amount of starting
material (2.5%), along with a decrease in diastereoselectivity (91:9) (Table 4, entry 1). At the
time, these lower than expected results were attributed to diffusion at the beginning and end
of the experiment, as a significant amount of non-steady state reaction mixture was collected
for this smaller trial run using the SS reactor system. With the operation of the SS system
confirmed, the process was piloted at 2.75 kg scale (compound 1 input) using the preferred
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conditions identified in Table 3, entry 12 with 0 equiv TFA, however, more pronounced
challenges were encountered. Incomplete conversion of the starting material and a significant
reduction in diastereoselectivity were observed (Table 4, entry 2). Investigation into
equipment setup and execution with the previously used systems (small-scale experiments
with the VapourtecTM system and 100 g experiments with either the in-house 3/16” PFA
reactor system or the SS reactor system) and the 2.75 kg pilot run with the SS reactor system,
when TFA was not added to the reaction mixture (Table 3 entries 11–12 and Table 4 entry 1
vs Table 4 entry 2), suggested that the reactor configuration and materials of construction were
not the cause of the change in reaction performance.
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An alternative explanation for the discrepancy between the small-scale experiments and
2.75 kg pilot run was sought. It was hypothesized that residual water could have generated
trace amounts of TfOH through hydrolysis of TMSOTf. The generated TfOH could then act as
a catalyst, driving both the selectivity and the conversion of the transformation. This effect
would likely have a larger impact on smaller scale reactions (i.e., 1 g or even 100 g) with
decreasing impact as the scale increased (i.e., 2.75 kg). To test this hypothesis, TfOH was
introduced into the TMSOTf/DCM stock solution of a 150 g reaction using the SS reactor
system, and it was found that the conversion and diastereoselectivity were fully restored (Table
4, entry 3). The use of TFA in the reaction mixture was assessed further, and experiments at
150 g scale were conducted with the SS reactor system (Table 4, entries 4–6). It was found that
0.5 mol equiv TFA afforded acceptable conversion and selectivity during the process (Table 4,
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entry 5). To accommodate potential fluctuations in the flow rates of the three-pump system
and increase the robustness of the process, 1.0 equiv TFA was selected for subsequent runs.18
During implementation of this change, the TFA/TMSOTf feed line experienced increased
back-pressure and ultimately plugged. After additional experimentation to evaluate the source
of the plugging, it was discovered that TFA (melting point of -15.4 °C) had limited solubility
in the TMSOTf/DCM mixture at -40 °C and was likely crystallizing during pre-cooling of the
feed line. To circumvent this issue, the TFA/TMSOTf/DCM feed pre-cooling loop was
removed. Additionally, the reaction temperature was adjusted from -40 °C to -30 °C to
decrease the risk of TFA crystallizing from the TFA/TMSOTf/DCM feed solution. These
changes did not have substantial impact on the performance of the reaction, as the solution
purity of compound 2 (and diastereoselectivity of the reaction) was similar despite the increase
in reaction temperature and removal of the TFA/TMSOTf/DCM pre-cooling loop (Table 4,
entries 5 and 6).
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Table 4. Performance of the Stainless Steel Continuous Flow Reactor System
In-Process
Content of 1 Purity of 2 Content of 3
(%)
(%)a (%)a
Solution
In-Process
Reaction
Temp (°C)
TFA / TMSOTf /
TMSCN (equiv)
d.r.
Entry Input of 1
(2:3)
1
100 g
2.75 kg
150 g
150 g
150 g
-40
0 / 6 / 6
2.4
14.1
1.4
3.9
1.2
87.7
8.8
91:9
2
-40
-40
-40
-30
0 / 6 / 6
63.6
86.8
77.3
86.4
18.4
7.7
78:22
92:8
3b
4
0.5 / 3 / 3
0.1 / 6 / 6
0.5 / 6 / 6
13.3
7.1
85:15
92:8
5
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150 g
-30
1 / 6 / 6
0.9
88.7
7.9
92:8
5
6
7
8
Reactions were performed with the SS continuous flow reactor system displayed in Figure 2.
Reactions were performed with residence times of 0.5 min for the reaction of compound 1
with TFA/TMSOTf and 2.0 min for the reaction with TMSCN. Reaction temperatures were
maintained at about -40 °C through immersion of the flow reaction loops in an IPA/dry ice
bath. a Purity was determined by LC analysis of an aliquot of the quenched reaction mixture.
b TfOH was used in place of TFA.
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Under these newly established conditions, the subsequent 5 runs with 2.75 kg input of
compound 1 using the SS reactor system provided high solution purity and diastereoselectivity
(Table 5, entries 2 to 6). The quenched effluents from the multiple runs were combined and
13.0 kg of compound 2 was isolated in 66% yield with 99.7% purity.
Table 5. In-Process Results for the Stainless Steel Continuous Flow Reactor Runs
In-Process
Solution
In-Process
TFA / TMSOTf /
TMSCN (equiv)
d.r.
(2:3)
Content of 1 Purity of 2 Content of 3
Entry
Scale (kg)
(%)a
14.1
3.3
(%)a
63.6
88.3
91.3
93.0
90.8
92.6
(%)a
18.4
5.0
1
2
3
4
5
6
0 / 6 / 6
1 / 6 / 6
1 / 6 / 6
1 / 6 / 6
1 / 6 / 6
1 / 6 / 6
2.75
2.76
2.75
2.75
2.75
2.76
78:22
95:5
95:5
94:6
94:6
94:6
2.4
4.7
0.4
5.6
2.1
5.7
0.7
5.6
Reactions were performed with the SS continuous flow reactor system displayed in Figure 2.
Reactions were performed with residence times of 0.5 min for the reaction of compound 1
with TFA/TMSOTf and 2.0 min for the reaction with TMSCN. Reaction temperatures were
maintained at about -30 °C through immersion of the flow reaction loops in an IPA/dry ice
bath. a Purity was determined by LC analysis of an aliquot of the quenched reaction mixture.
Plant Manufacturing Scale Preparation of Compound 2 via Continuous Flow Chemistry
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The results from the gram scale to kilogram scale runs using the various continuous flow
reactor systems, along with the process parameters applied to each system served as the basis
for design of the plant continuous flow reactor system. The following major factors were used
to define the plant system.
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First, the plant system needed to be well contained to ensure safe handling of the cyanide-
containing reaction mixture. To address this concern, the flow reactor and ancillary equipment
were constructed from stainless-steel and underwent rigorous pressure testing prior to use.
Second, the plant system needs to have the capacity to produce hundreds of kilograms of
compound 2 in reasonable time frame. Table 6 presents a comparison of process parameters for
the different systems. The interaction between residence time, reactor volume and throughput
(consumption of compound 1) can be clearly observed. From this table, it suggests that a
stainless-steel tube reactor with a volume of 2.5 L would achieve a throughput of
approximately 2 kg/h, enabling processing of 200 kg of compound 1 in about 4 days. This
duration was deemed acceptable for the manufacturing requirements. In order to consistently
achieve the desired flow rates of each of the three feeds, three diaphragm pumps constructed
of stainless steel and PTFE, equipped with back pressure regulators, were selected as the feed-
delivery devices and were calibrated prior to use.
Lastly, the plant system needs to have sufficient cooling capacity and efficiency. The cooling
capacity would be required both to adjust the reagent solutions to about -30 °C prior to
reaction, as well as rapidly scavenge heat generated during the reaction to ensure high-quality
product streams are generated. These factors were mitigated through use of the SS construction
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of a tube-in-shell reactor system, enabling rapid heat transfer, along with use of a high capacity
heating/cooling unit.
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Figure 3. A Schematic Drawing of the Plant Manufacturing Scale Continuous Flow Reactor
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TFA / TMSOTf
in DCM
TMSCN
in DCM
Pre-cooling
Loop
1
Compound
in DCM
Pre-cooling
Loop
Reaction
Loop 1
Reaction
Loop 2
Temperature controlled to -30 °C
KOH(aq) quench
Table 6. Key Parameters for Continuous Flow Reactor Systems
Residence Time
Reactor Volume
(min)
System
Description
Material of
Construction
Throughput of
Compound 1
Entry
Reaction Reaction Reaction Reaction
Loop 1
Loop 2
Loop 1
Loop 2
Vapourtec
R-Series
1
2
3
4
PFA
PFA
0.5
2.0
2 mL
10 mL
9 g/h
317 g/h
317 g/h
1.98 kg/h
In-House PFA
Reactor System
0.5
0.5
0.5
2.0
2.0
2.0
59 mL
60 mL
0.5 L
300 mL
300 mL
2.0 L
Kilo-lab SS
Reactor System
Stainless
Steel
Plant Scale
Reactor System
Stainless
Steel
Following the fabrication and installation of the plant scale continuous flow reactor system,
a pilot run was performed at 86 kg input of compound 1 to confirm operability of the plant
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system for manufacturing of compound 2. As observable in Figure 4, after a brief induction
period whereby steady-state operation of the system was obtained, a consistent reaction profile
was observed throughout the duration of the flow chemistry run. Numerical results can be
found in Table 7 (entry 1), demonstrating the plant scale system afforded similar reaction
diastereoselectivity and solution purity of compound 2 when compared to the SS kilo lab
system (Table 7, entry 1 vs Table 5, entries 2–6). The quenched reaction mixture was carried
through an aqueous workup in the same fashion as the kilo lab batch (see experimental details)
to afford a stock solution of compound 2 in DCM. Following solvent exchange into toluene,
the product was crystallized and filtered to obtain 68 kg of compound 2 (Table 7, entry 1). This
delivery enabled preparation of remdesivir to support clinical evaluation of the drug candidate.
Two additional deliveries were subsequently performed using the same plant scale continuous
flow reactor system, at 280 kg and 250 kg input of 1, to provide 204 kg and 198 kg of compound
2, respectively (Table 7, entries 2 and 3).
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Figure 4. 86 kg Flow Chemistry Reaction Profile
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8
Flow Chemistry Reaction Profile
100
90
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20
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0
9
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Compound 1
Compound 2
Compound 3
0
5
10
15
20
25
30
35
Flow Chemistry Run TIme
Reaction profile obtained through sampling an aliquot of the quenched reaction mixture and
LC analysis.
Table 7. In-Process and Final Results for Plant Scale Batches to Generate Compound 2.
Input
Weight Content of
of 1 (kg)
In-Process
Solution
Purity of
2 (%)a
In-Process
Content of
3 (%)a
Output
Weight of
2 (kg)
d.r.
(2:3)
Yield
(%)
Purity
(%)a
Entry
1 (%)a
0.9
1
2
3
86
88.6
89.6
89.8
6.1
5.9
5.9
94:6
94:6
94:6
68
78
72
78
99.6
99.7
99.9
280
0.7
204
198
250
0.5
Reaction Conditions: See experimental details for reaction and isolation conditions. a Purity
was determined by LC analysis.
Conclusion
The development of batch cyanation process to prepare compound 2 and enable
development of the clinical candidate remdesivir was described. Translation of the batch-mode
chemistry into a continuous flow process was accomplished, providing improved control over
the reaction conditions and increased diastereoselectivity. Four unique continuous flow
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chemistry reaction systems were evaluated, including two systems that were utilized for large-
scale manufacture of compound 2. The preparation of about 500 kg of compound 2 through
continuous flow chemistry was reported, enabling preparation and clinical evaluation of
remdesivir for the treatment of Ebola virus and coronavirus diseases.
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Experimental Details
Commercially available solvents and reagents were used as received without further
purification. LC data were collected on Waters Acuity or Waters Acuity H-Class UPLC
instruments with detection by UV. UPLC conditions were as follows: Acuity UPLC BEH C18,
130 Å, 1.7 μm, 2.1 x 100 mm, 75–5% gradient of 20 mM ammonium acetate at pH 9.5 in water
and 100% acetonitrile; flow rate 0.4 mL/min; acquisition time, 12 min; UV at 245 nm. Purity
was calculated through % area normalization.
Preparation of Compound 1
NH2
N
NH2
N
O
N
i) TMSCl, PhMgCl,
i-PrMgCl, THF, -20 °C
O
O
BnO
N
BnO
OH
OBn
N
BnO
OBn
5,
ii) NdCl3, n-Bu4NCl, THF, -20 °C
N
BnO
I
4
1
5
Anhydrous neodymium (III) chloride (1.0 equiv), n-tetrabutylammonium chloride (1.0 equiv),
and tetrahydrofuran (THF, 9 volumes) were combined and the resulting mixture was
concentrated to about 4.5 volumes under ambient pressure at about 66 °C. THF (4.5 volumes)
was charged and the concentration was repeated. The mixture was cooled to about 22 °C and
2,3,5-tri-O-benzyl-D-ribono-1,4-lactone (compound 5, 282 kg, 1.0 equiv) was charged. After
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about 30 min the mixture was cooled to about -20 °C and held. In a separate reaction vessel, 7-
iodopyrrolo[2,1-f][1,2,4]triazin-4-ylamine19 (compound 4, 1.10 equiv) and THF (5 volumes)
were combined and cooled to about 0 °C. Chlorotrimethylsilane (1.10 equiv) was added slowly
and, after about 30 min the mixture was cooled to about -10 °C. Phenylmagnesium chloride (2
M in THF, 2.17 equiv) was added slowly and, after about 30 min, the reaction mixture was
cooled to about -20 °C. iso-Propylmagnesium chloride (2 M in THF, 1.13 equiv) was added
slowly and, after about 2 h, the Grignard reaction mixture was transferred into the
lactone/NdCl3/n-Bu4NCl/THF mixture. The resulting mixture was agitated at about -20 °C and,
after about 8 h, a solution of acetic acid (1 volume) and water (4 volumes) was added. The
resulting mixture was warmed to about 22 °C and iso-propylacetate (i-PrOAc) was added. The
layers were separated and the organic layer was washed sequentially with aqueous potassium
bicarbonate and aqueous sodium chloride. The organic layer was concentrated under vacuum
to about 4.5 volumes and i-PrOAc was charged. The organic layer was washed with water
twice and concentrated to about 4.5 volumes under vacuum. i-PrOAc was charged and the
concentration was repeated. The mixture was filtered and the filtrate was concentrated under
vacuum to about 3 volumes. Methyl tert-butyl ether (MTBE) was charged and the mixture was
adjusted to about 22 °C. Seed crystals of compound 1 were charged, followed by n-heptane,
and the mixture was cooled to about 0 °C. The solids were isolated by filtration and rinsed
forward with an MTBE/n-heptane mixture. The resulting solids were dried under vacuum to
afford compound 1 in 69% yield and 100% purity. Analytical data was consistent with data
previously reported in the literature.8
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Preparation of Compound 2
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Safety Notice: The below chemistry was conducted using TMSCN which may generate HCN
upon exposure to water, protic solvents, or in the presence of acids. HCN is a highly toxic,
colorless, flammable liquid that boils at 25.6 °C. The SDS should be consulted and, in
consultation with appropriate environmental, health and safety personnel, adequate
precautions should be taken. Additional precautions may include items such as the following:
 written documentation outlining handling procedures for reagents, stock solutions,
process streams, waste, and loss of containment or exposure
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 higher level of personal protective equipment use
 cyanide detectors
 trained response personnel with access to cyanide antidote
Batch process to prepare compound 2: To a solution of compound 1 (23 kg, 1.0 equiv) in
DCM (10 volumes) pre-cooled to about -40 °C was charged trifluoroacetic acid (3.0 equiv),
followed by a solution of TMSOTf (6.0 equiv) and TMSCN (6.0 equiv) in DCM (5 volumes),
pre-cooled to about -30 °C, while maintaining the internal temperature below about -25 °C.
The reaction mixture was agitated at below about -30 °C for no less than 10 minutes and
quenched into a pre-cooled (about -10 °C) solution of KOH (35.8 equiv) in water (14 volumes).
The bi-phasic mixture was warmed to ambient temperature. The organic layer was separated
and washed three times with aqueous sodium chloride. The organic phase concentrated under
vacuum to about 4 volumes, diluted with DCM (8 volumes) and concentrated under vacuum
to about 6 volumes. The mixture was filtered and the filtrate was concentrated to about 4
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volumes. The concentrate was diluted with toluene (19 volumes) and concentrated under
vacuum to about 14 volumes at about 50 °C. The mixture was heated to about 90 °C, cooled to
about 55 °C, and seeded with compound 2. The mixture was agitated at about 55 °C for about
one hour and cooled to about 0 °C over about 6 hours. The solids were isolated by filtration
and the filter cake was washed with toluene (3 volumes). The solids were dried under vacuum
at about 50 °C to afford compound 2 in 71% yield and 99.2% purity.
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Continuous flow chemistry process to generate compound 2: Stock solutions of compound 1
(250 kg, 1.0 equiv) in DCM (15.0 volumes) (Feed 1), TMSOTf (6.0 equiv) and TFA (1.0 equiv)
in DCM (4.4 volumes) (Feed 2), and TMSCN (6.0 equiv) in DCM (4.5 volumes) (Feed 3), were
prepared in separate reactors or feed vessels. Feed 1 was pumped at a flow rate of
approximately 504 mL/min through a pre-cooling loop at about -30 °C, and Feed 2 was pumped
at a flow rate of approximately 207 mL/min. Feeds 1 and 2 were combined in Reaction Loop
#1 at about -30 °C for about 30 seconds. The effluent was then combined with Feed 3 (pumping
at approximately 189 mL/min through a pre-cooling loop at about -30 °C) in Reaction Loop #2
at about -30 °C for about 2 minutes. The effluent of the combined feeds was collected directly
into a vessel containing a solution of KOH (19.7 equiv) in water (8 volumes) at about -10 °C.
The mixture was adjusted to about 22 °C, then 2-propanol was added and the layers were
separated. The organic layer was washed with aqueous sodium chloride twice and
concentrated. The resulting solution was filtered. Toluene was charged to the filtrate and the
mixture was concentrated. The mixture was heated to about 55 °C, then cooled to about 0 °C.
The resulting slurry was filtered, rinsed with toluene and dried at about 60 °C to afford
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compound 2 in 78% yield with 99.9% purity. Analytical data were consistent with data
previously reported in the literature.8 Waste streams from the cyanation process (both
laboratory and plant scale) were monitored for cyanide content and were treated with bleach
until cyanide levels were below the limit of detection by cyanide test strips.
AUTHOR INFORMATION
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Corresponding Authors
*E-mail: tiago.vieira@gilead.com, andrew.stevens@gilead.com
ACKNOWLEDGMENT
The authors would like to thank Anna Chiu, Dragos Vizitiu, Jeff Ng, Kevin Allan, Mark
Scott, Richard Yu, and Zhongxin Zhou for thoughtful discussions and feedback during
manuscript preparation.
References
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