Practical Considerations and Examples in Adapting Amidations to Continuous Flow Processing in Early Development

Article abstract of DOI:10.1021/acs.oprd.0c00112

Amidation is among the most frequently executed reactions in pharmaceutical research and development. We have explored the feasibility of adapting amidations to plug flow reactor (PFR) process conditions for the preparation of early development compounds. Among coupling reagents possessing good thermal stability, carbodiimides and T3P have been selected, as they are readily soluble, require no preactivation, offer excellent reaction kinetics, and enable convenient product isolation. A carbodiimide/2-hydroxylpyridine oxide (HOPO) protocol was demonstrated in four case studies with homogeneous feed and reaction streams that were readily adaptable to a PFR design. In a head-to-head comparison, T3P was also found to be readily adaptable to a PFR flow process and gave comparable yields. The EDC/HOPO method works well for amidations that do not involve substrates that are highly sensitive to racemization; its water compatibility makes it the reagent of choice when the amine reactant is in a salt form, since water can be added as a cosolvent to aid solubility. For substrates that are extremely sensitive to racemization, we have shown one successful example of peptide coupling using TBTU or COMU under PFR continuous flow conditions.

Full text of DOI:10.1021/acs.oprd.0c00112

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Communication
Practical Considerations and Examples in Adapting Amidations
to Continuous Flow Processing in the Early Development
Bryan Li, Gerald A. Weisenburger, and James Christopher McWilliams
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00112 • Publication Date (Web): 29 Apr 2020
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Practical Considerations and Examples in Adapting
Amidations to Continuous Flow Processing in the
Early Development
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Bryan Li, Gerald A Weisenburger and J. Christopher McWilliams
Chemical Research & Development, Pharmaceutical Science Small Molecules
Division, Worldwide Research and Development, Pfizer Inc., Eastern Point Road,
Groton, Connecticut 06340, United States.
bryan.li@pfizer.com
Table of Contents Graphic
(pharmaceutical intermediates or products)
RNH2 + R'CO2H
R'CONHR
Coupling reagents:
EDC/HOPO
T3P
TBTU/COMU 1 example
4 examples
1 example
Homogeneous
feed stream A
Amide product
receiver bottle
BPR
Homogeneuous
feed stream B
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Abstract: Amidation is among the most frequently executed reactions in pharmaceutical
research and development. We have explored the feasibility of adapting amidations
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to plug flow reactor (PFR) process conditions for preparation of early
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development compounds. Among coupling reagents possessing good thermal
stability, carbodiimides and T3P have been selected, as they are readily soluble,
require no pre-activation, offer excellent reaction kinetics and enable convenient
product isolation. A carbodiimide/2-hydroxylpyridine oxide (HOPO) protocol was
demonstrated in four case studies with homogeneous feed and reaction streams
readily adaptable to a PFR design. In a head to head comparison, T3P was
also found readily adaptable to a PFR flow process and gave comparable yields.
The EDC/HOPO method works well for amidations that do not involve
substrates highly sensitive to racemization; its water compatibility makes it the
reagent of choice when the amine reactant is in a salt form, since water can be
added as a co-solvent to aid solubility. For substrates extremely sensitive to
racemization, we have shown one successful example of peptide coupling using
TBTU or COMU under PFR continuous flow conditions.
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Keywords: Flow amidation, continuous process, peptide coupling, early
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development.
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1. Introduction
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In the recent decade, continuous processing has attracted considerable interest within
the pharmaceutical industry. Continuous flow technology offers many advantages over
batch methods including precise control of stoichiometry, reaction time and temperature, high
reproducibility, and often better yields thanks to improved reaction control.1 Continuous
processing has been applied in the handling of hazardous reactions, unstable reaction
intermediates, high temperature/pressure and the expansion of chemical design
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spaces in the pharma industry. In addition, there has always been a focus on process
intensification to gain production efficiency, reduce manufacturing facility footprints,
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shorten development timelines, and improve quality control. Amidation, as a common,
well-studied chemical transformation, is traditionally carried out under batch conditions, and in
general does not present scale up concerns. Numerous coupling reagents have been developed
to address special needs of reaction rates, epimerization, and efficiency. Recently, a Pfizer
process safety lab published a detailed thermal stability assessment of the most commonly
used peptide coupling reagents.4 As amidation is among the most frequently executed
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reactions5 in the pharma industry, we were interested in understanding the generality of
adapting these reactions to flow processes, and looked for opportunities to apply the
technology in the early phase of development. While solid-phase supported peptide
synthesis via continuous processing has been well documented in the literature,6
solution phase flow amidations have been reported more sporadically. Polster and
colleagues demonstrated a Schotten-Baumann reaction by coupling an acid chloride
with an amine using a plug flow reactor (PFR).7 A continuous stirred-tank reactor (CSTR)
process for the scale-up of a highly potent drug candidate was described by White and
coworkers from the same Eli Lilly process chemistry group.8 In the latter example,
application of continuous processes approaches showed clear benefits in reducing
potential worker exposure by reducing unit operations and allowing commercial-scale
API production in laboratory fume hoods.
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2. Practical considerations in flow amidations
In this study, our primary objective was to explore the feasibility of applying continuous
processing technologies to coupling carboxylic acids with amines to form the
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corresponding amides in early development programs. With emphasis on simplicity,
convenience and speed, we considered the following:
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(1) Pre-activation of the carboxylic acid. Though a pre-activation step can be carried
out in a continuous process and telescoped to the amidation, in general this
introduces operational complexity as the stoichiometry of the coupling
reagent(s) is often critical. Complete conversion of the carboxylic acid and
consumption of the activation reagent are necessary to avoid impurity formation
from reaction of the amine with the coupling reagent. When an amidation
reaction requires a pre-activation of the carboxylic acid, either the acid or the
amine is used in slight excess to factor in reagent quality and the presence of
adventitious water. Commonly used pre-activated acid derivatives include:
a) Acid chloride. As exemplified in both afore-mentioned Lilly publications, acid
chlorides are often isolated before proceeding to the amidation step.
Commonly used chlorinating reagents are oxalyl chloride, thionyl chloride,
cyanuric chloride, phosphorus trichloride or phosphorous pentachloride. As
the chlorinating agent is generally used in excess, and it reacts with the
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amine reactant, it’s difficult to directly telescope to the amidation reaction
under continuous flow.
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b) Mixed anhydride. They are routinely used in solution phase amidations under
batch conditions. One of the most commonly used activating agents is
pivaloyl chloride.9 Cole and coworker preformed the pivaloyl mixed anhydride
under batch conditions, and subsequently used isolated intermediate in a
CSTR process for the manufacture of an active pharmaceutical ingredient
(API).10 Nevertheless pivaloyl chloride and its byproduct pivalic acid carry a
pungent odor detectable even at very low levels, which makes its use
undesirable for the late steps in API manufacture. Furthermore, pivaloyl
chloride is a highly acutely toxic (HAT) reagent.11 Isobutyl chloroformate and
the related family of reagents are also deemed generally unsafe for use on
large scale.4 Phosphoric12 or sulfuric mixed13 anhydrides are also known, but
less commonly utilized. In recent years, the use of propylphosphonic anhydride
(T3P) has gained a great deal of popularity;14 the corresponding phosphonic
acid mixed anhydride is formed in situ, and the reaction is mostly frequently
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carried out in the same vessel without the need to pre-form the mixed
anhydride.
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c) Acyl imidazole. This is a popular method of activation and can be readily
prepared by reacting the carboxylic acid with carbonyl diimidazole (CDI).15
The pre-activation requires precise control of stoichiometry and process
analytical technology (PAT) control to monitor the conversion. While an
under-charge of CDI leads to incomplete reaction, an over-charge of the
reagent often leads to urea byproduct formation. Another major hurdle in
application of CDI in continuous processes is its poor solubility in commonly
used reaction solvents (vide infra).
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(2) Solubility/solvent selection. A homogeneous reaction is highly desirable in adaptation
to a flow process. Green chemistry principles are applied in solvent selection16 with
ease of work up and product isolation also taken into account. In a continuous flow
design, coupling reagents should be readily soluble at ambient temperature in
typical processing solvent(s). In this aspect, CDI, with low to moderate
solubility17 in common aprotic solvents, is limited to reactions carried out in an
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aprotic polar solvent, e.g. DMF, DMSO, and sulfolane when adapting to a PFR
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flow process. The use of a large amount of solvent generally impedes product
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isolation, and is therefore avoided if more efficient alternatives are available.
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(3) Thermal safety. Per the recent reagent selection guideline for amide coupling endorsed
by the Pfizer process safety lab,4 our initial focus was on the “preferred” reagent
category, largely determined by a thermal safety assessment. If it were necessary to
employ a non-preferred reagent, a comprehensive process safety evaluation is completed
prior to scale up.
(4) Flow equipment. With simplicity and convenience in mind, it is desirable to have fewer
feed streams/pumps and PAT requirements for reasons of lower risk of equipment
failure. Therefore, a flow system with two feed streams is targeted whenever feasible.18
A PFR design is preferred since it is more readily adaptable from various laboratory
scales to production scale, particularly in the early developmental phases. Reactions that
generate gases (CO2 from CDI activation, CO and HCl from oxalyl chloride) are more
demanding of reactor design, and preference is given to alternatives that are friendly to
flow adaptation. Furthermore, reagents such as acid chlorides that are corrosive to pump
heads/seals are disfavored.
(5) Product isolation. The ease of workup/product isolation needs to be considered in any
process development program. Therefore, N-Ethoxycarbonyl-2-ethoxy-1,2-
dihydroquinoline (EEDQ) and the associated family of coupling reagents are not
deemed favorable as byproducts of the coupling reagent can be difficult to remove.
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Among the Pfizer process safety lab preferred coupling reagent list, methanesulfonyl chloride
toluenesulfonyl choride, , phosphorus oxychloride (POCl3) oxalyl chloride, carbonyl
diimidazole (CDI), pivaloyl chloride (PivCl), cyanuric chloride (TCT), 2-chloro-4,6-dimethoxy-
1,3,5-triazine (CDMT), diphenyl phosphoryl chloride (DPC), diphenylphosphinyl chloride
(DPPCl), diethyl phosphorocyanidate (DEPC), diphenyl phosphite (DPP), and
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pentafluorophenyl diphenylphosphinate (FDPP) require pre-activation. The dihydroquinone
family (EEDQ, 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline or IIDQ, and 1-tert-
butoxy-2-butoxycarbonyl-1,2-dihydroisoquinoline or BBDI) can lead to impurity formation,19
and generates the quinoline byproduct that adds complexity in the product isolation. The
imidazolium reagents (bromo-tris-pyrrolidino-phosphonium hexafluorophosphate or PyBrOP,
chlorotripyrrolidinophosphonium hexafluorophosphate or PyClOP, 1-(chloro-1-
pyrrolidinylmethylene)pyrrolidinium hexafluorophosphate or PyCIU, 2-chloro-1,3-
dimethylimidazolinium Hexafluorophosphate or CIP) are not widely popular among process
chemists as pyrrolidide derivatives were often observed as byproducts, which is attributed to the
presence of pyrrolidine as a contaminant in commercial sources, and the reagents typically
require crystallized before use.20 It has been reported that PyBroP activation of Boc-amino
acids can give moderate yields due to formation of the N-carboxyanhydride (NCA).21 The use
of tetramethylfluoroformamidinium hexafluorophosphate (TFFH) in a scale up facility will
necessitate the use of Hastelloy reactors.22 Tetrachloro-N-hydroxyphthalimide
tetramethyluronium hexafluorophosphate (CITU) was not considered during the study since it
was introduced after this work was completed; it does not appear widely available on bulk scale.
With these realities in mind, the number of reagents to consider dramatically decreases to the
carbodiimides and T3P. In the carbodiimide family, N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC) is particularly attractive due to fast reaction kinetics
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and the ease of product isolation; the urea byproduct is water soluble.23 The combination of
carbodimides with 2-hydroxylpyridine oxide (HOPO) is known to be a powerful amidation
protocol.24 HOPO is regularly used as an additive to increase reaction kinetics and suppress
epimerization.25 General considerations of EDC/HOPO26 and T3P in their adaptability to flow
processes are summarized in Table 1.
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Table 1. EDC/HOPO and T3P in Flow Adaptability Considerations
Reagent
EDC/HOPO
T3P
Attributes
Cost per mol
EDC $48
HOPO $16
$255
(based on SciFinderTM
)
Yes; a big advantage as amines are often
in organic salt forms and water is
generally needed as a co-solvent for
solubility.
Water compatibility
No
No; in general excess of the reagent does not present risk to
reaction performance. If the reaction is not fully converted,
additional reagent can be added.
Critical stoichiometry
Homogeneous feed
streams
Yes; water can be used as co-solvent if
needed.
Yes; in many
solvents.
Risk of racemization
(for substrate of
concern)
Thermal hazard
Ease of workup/
product isolation
Flow adaptation
feasibility
Low/Medium
Low/Medium
Low26
Low
Good; often direct drop isolation
Good
High
High
3. Results and discussion
Several Pfizer development portfolio examples were chosen to measure flow
adaptability potential based on the above analysis. In this study, the workflow involved:
(1) Identification of stable and homogenous feed and effluent streams, as they are
critical for the development of a continuous flow processes in a PFR.7 Solubility
screening of the reactants, product and reagents. Different combinations of
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reactants/reagent can lead to different solubility behavior. Chemical and thermal
stability of the feed streams is also essential in devising the feed streams.
(2) Optimization of the reaction conditions and studying batch reaction kinetics
under various temperatures with an aim to keep reaction/residence time within
60 min.27 ChemGlass pressure relief vials28 were used if the reaction were kept
under pressure.
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(3) Isolation of the product without chromatography, ideally using direct drop
isolation.
(4) Demonstration of the reactions by flow on multi-gram scales.
It is gratifying to find out that for all reactions investigated, a fully homogeneous
reaction system was attainable. In each example, reaction screening was performed
under batch conditions for optimal stoichiometry, solvent selections, choice of reagents
and residence time. Much effort was directed to assuring a homogeneous reaction
mixture and high reaction conversion in each case. In Entry 1 (Table 2), exactly 1.0
equiv each of the reactants (1 and 2) were used with 1.2 equiv of EDC. When
triethylamine was used as a base, a carbamimidate impurity29 was formed as a major
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impurity at as much as ~11% (by UPLC area). However, when the base was switched
to sodium bicarbonate, a clean reaction was observed. (Table 2). After evaporation of
most THF and acetone, the product (3) was isolated in 90.5% yield in a 10 g flow
demonstration run. In both entries 1 and 2 (Table 1), the amine reactant was a
hydrochloride salt, and had limited solubility in most aprotic organic solvents except the
very polar DMSO or sulfolane. The compatibility of EDC with water30 was a huge
advantage by allowing water as a co-solvent in the reaction, which afforded a
homogeneous reaction system, a critical feature in adapting the reaction to PFR flow
processes. In Entry 2 (Table 2) reaction, chemoselectivity was important for both the
acid and amine reactants, as competing functionalities were present (the hydroxyl
group of glycolic acid and the heteroaryl-NH2 of amine 5). To suppress side reactions
from the self-condensation from glycolic acid, we chose an aqueous biphasic solvent
system. As EDC is water compatible (Table 1), the EDC/HOPO aqueous coupling
conditions were considered optimal for these coupling partners; most other conditions
screened gave impurities from competing side-reactions.31 The reaction was complete
in 5 min at 20 °C to give the product in 99.0% purity by UPLC in the reaction mixture
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(as glycolic acid is a very cheap commodity chemical and readily removed in the
aqueous phase, 3.0 equiv was used to speed up the reaction). The product isolation
was easily accomplished by removal of THF and filtration to give a 92% yield of
compound 6 in a 5 g flow run (Figure 1). The reaction in Entry 3a (Table 2) worked
well using either T3P or EDC as the coupling reagent. The flow adaptation for T3P
facilitated amidation was a simple modification from the batch conditions. The reaction
solvent was changed from EtOAc to DCM to obtain a homogeneous reaction mixture,
this was necessary due to the limited solubility of nicotinic acid 7 in EtOAc. T3P in
acetonitrile and DMF worked reasonably well for the reaction, but was not optimal for
product isolation. The workup and isolation involved the removal of DCM by
evaporation and granulation in aqueous iPrOH; 93% isolated yield was obtained in a
10 g flow run as illustrated in Figure 1. The reaction could also be readily adapted
using the EDC/HOPO method, although the reaction kinetics were slower than using
T3P; a THF/water/acetone solvent mixture provided a homogeneity. In a 10 gram flow
run, the reaction was carried out at 90 °C for a 45 min residence time to give 91.6%
isolated yield (Entry 3b, Table 2). The amidation in entry 4 (Table 2) had been carried
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out in multi-kilogram scale under batch conditions. T3P, used as the coupling reagent,
was the major cost contributor in this reaction as both reactants are cheap commodity
chemicals. This reaction could be simply adapted to the EDC/HOPO method, but the
EDC byproduct, ethyl-(N’,N’-dimethylamino)propyl urea, had similar solubility versus the
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product at the desirable pH range, complicating product isolation. Use of N, N′-
Diisopropylcarbodiimide (DIC) as the coupling reagent circumvented the issue successfully.
The reaction remained fully homogeneous in a solvent mixture of THF/water (10 mL/g each).
At 75 °C in 30 min, the reaction was converted cleanly. The physical and chemical properties of
the product necessitated a more tedious product isolation. Thus, the DIC byproduct (diisopropyl
urea) was removed by filtration after evaporation of THF, the resulting aqueous filtrate was
adjusted to pH 11, and was then back-extracted with nBuOH to enable the final isolation. After
concentration of the extract and granulation in acetonitrile, 70.3% yield was isolated in a 10 g
flow demo run. Higher yield could potentially be achieved with some optimization as in situ
yield was excellent.
While the use of EDC/HOPO or T3P proved to be appropriate for routine amidations,
this could lead to racemization for some sensitive substrates (e.g. entry 5, Table 2).
This calls for the use of coupling reagents that are not preferred as judged solely by
thermal stability. In the scenario where both coupling partners are expensive (e.g.
cyclic peptide (14) and tripeptide (13)), and limited quantities are available for process
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development, a flow process offers the benefit of mitigating scale up risk; i.e. the entire
amount of materials are not committed up front. Reaction performance may be
determined in the very beginning of the run with either online or offline analysis. The
reaction can be fine-tuned if necessary, to maximize yield and minimize formation of
impurities. It was demonstrated that both 1-cyano-2-ethoxy-2-oxoethylidenaminooxy)-
dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) and 2-(1H-
Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) worked remarkably
well for this peptide coupling (entry 5, Table 2). The diastereomeric impurity was
detected at less than 0.2% under both flow and batch conditions. With less than 10
grams of Polymixin cyclic peptide 14, we were able to develop a process that was
ready to be scaled up.32 This was particularly important, as both availability of starting
materials and time for process development are important constraints in early drug
development phases.
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Table 2. Amidation Reaction Conditions Adaptable to Flow33
Acid (Compound No.
and equiv. used)
Amine (Compound No. and
equiv. used)
Product
(Isolated
Yield)
Feed streams and Reaction
Conversions
Entry
10
11
12
13
14
Feed stream A: 1, 2 and NaHCO3 (2.0 eq)
in THF/water (8/2 vol34).
O
OH
O
1
2
Ar2
Feed stream B: EDC (1.2 eq), HOPO (1.0
eq) in THF/water/acetone (2/3/3 vol).
3
Ar1
1
15
(90.5%)
1.0
equiv
1.0
equiv
Ar
N
OMe
16
17
18
19
20
21
22
23
24
25
N
•HCl
H
Conditions: 40 °C, 2 min 22%; 55 °C 15
min, 83%; 85 °C, 45 min, 100%.
Feed stream A: 5 in THF/water (5/0.5
vol).
H
• HCl
N
Feed stream B: 4, EDC (2.2 eq),
HOPO (1.0 eq) in THF/water (5/4.5
vol).
4
5
HO
HO
O
6
N
26
27
2
N
H2N
3.0
equiv
1.0
equiv
linker
(92.0%)
28
29
30
31
32
33
34
35
36
37
38
39
N
Conditions: 20 °C, 1 min, 77%; 20 °C,
5 min. 100%.
Feed stream A: 7, Et3N (4.2 eq) in
THF/water (8/1 vol).
Feed stream B: 8, 3.0 eq EDC, HOPO
(1.0 eq) in THF/water/aceteone (2/4/6
vol).
935
3a
40
(91.6%)
41
42
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48
49
50
H
OH
S
7
O
8
O
N
BzHN
N
Conditions: 75 °C 35 min, 97%;
90 °C 30 min, 99%
1.05
equiv
S
1.0
equiv
N
OCHF2
H2N
Feed stream A: 7, 8 in Et3N (4.2 eq) in
DCM (10 vol).
9
3b
51
Feed stream B: 50 wt% T3P (2.5 eq)
in EtOAc.
(93.0%)
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Conditions: 22 °C, 2 min, 94%; 5 min,
98%; 20 min, 99%
5
6
7
8
9
Feed stream A: 10, 11 and HOPO (1.0 eq)
10
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12
13
14
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16
17
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25
26
27
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60
in THF/water (10/10 vol).
N
OH
10
11
1236
O
Feed stream B: DIC (2.0 eq).
4
5
1.0
equiv
1.0
equiv
(70.3%)
N
H
NH2
Conditions: 20 °C, 2 h, 50%; 75 °C, 30
min, 100%.
OH
O
NHBoc
Boc NH
NH2
O
Ph
O
Feed stream A: 13, 14 and DIPEA (2.4 eq)
in DCM (6.2 vol)/ DMF (3.3 vol).
HN
O
H
N
O
N
H
13
14
HO
NH
NH
Feed stream B: COMU (1.3 eq) in DMF
(3.3 vol).
O
HN
O
1537
OH
1.0
equiv
1.05
equiv
NHBoc
O
O
HN
(88.4%)
NH
NH Boc
NH Boc
HN
O
O
Conditions: 22 °C, 1 min, 90%; 4 min,
100%.
N
H
N
Ph
Figure 1. Examples of Laboratory PFR Flow Diagrams
5
amine (1.0 eq),
THF (5 vol)
water (0.5 vol)
0.20 mL/min
0.354 M (concentration)
Amide product
receiver bottle
BPR
4
acid (3.0 eq)
EDC (2.2 eq),
HOPO (1.0 eq) in
92%
(isolated yield)
22°C
 9.6 min
0.32 mL/min
THF (5 vol)/water (0.5 vol)
total run time 88 min
3
0.665 M (concentration of
)
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Acid (1.0 eq),
0.25 mL/min
8
amine (1.05 eq),
Et3N (4.25 eq)
in DCM (8 vol) solution
5
0.234 M (concentration of
)
6
Amide product
receiver bottle
7
8
9
BPR
91.6%
(isolated yield)
50 wt% T3P solution
in EtOAc (2.5 eq)
DCM (2 vol)
22°C
 25 min
0.136 mL/min
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total run time 440 min
1.07 M (concentration)
13
14
Acid
amine
(1.0 eq),
(1.05 eq),
0.262 mL/min
DIPEA (2.4 eq)
in DCM (6.2 vol)/DMF
(3.3 vol) solution
a
Amide product
receiver bottle
10
0.081M (concentration of
)
BPR
88.4%
(isolated yield)
22°C
COMU (1.3 eq)
in DMF (3.3 vol)
 4.6 min
0.082 mL/min
total run time 75 min
0.328 M (concentration)
a
10
The low molar concentration was attributed to the high molecular weight of
.
4. Conclusion
We have made a brief assessment to develop a general method to adapt amidations to
PFR flow processes. Among coupling reagents exhibiting acceptable thermal stability,
we have shortlisted carbodiimides and T3P as reagents of choice considering minimal
pre-activation requirements, reasonable solubility, adaptability to a range of flow
equipment and straightforward product isolation. EDC/HOPO coupling reagents were
demonstrated to be effective in PFR flow processes, and in the four case studies,
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homogeneous feed and reactions streams could be achieved. In a head to head
comparison, T3P was also found readily adaptable flow, providing comparable yields,
although halogenated (less green) DCM reaction solvent was required to assure
solubility. With its low cost and coupling efficiency, EDC/HOPO is deemed an attractive
choice for amidations that do not involve substrates that are highly sensitive to
racemization; its water compatibility offers the additional advantage of allowing water to
be used as a co-solvent to dissolve amine reactants in salt forms. The workup and
isolation typically involve evaporation of organic solvent, and the product can often
crystallize38directly from the resultant aqueous mixture. For substrates prone to
epimerization, we have shown one successful example of peptide coupling using TBTU
or COMU39 via PFR continuous flow.
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Experimental Section
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9
General Methods. Flow experiments were performed using a Vapourtec R4 reactor
equipped with a T-mixer and the desired reactor volume and a 100 psi back pressure
regulator. The Vapourtec R4 reactor was initially equilibrated with exactly the same
makeup of the solvent or solvent mixture for each feed line under the reaction
temperature. The feed streams were then switched to the reactant/reagent lines with the
intended flow rates as shown in the diagrams. The reaction was monitored offline by
UPLC during the continuous flow. At the end of the reactant/reagent feed completion,
the feed was continued with exactly the same makeup of the solvent or solvent mixture
for at least 10 mL. The effluent collected was then worked up and isolated as described.
An Agilent SB-CN column (2.1 x 50 mm) with mobile phases A (0.05% TFA in water)
and B (acetonitrile). Flow rate 0.65 mL/min, 0 - 2.9 min, ramp from 5% B to 100% B; 2.9
– 3.4 min, stay at 100% B; 3.4 - 3.5 min, ramp to 5% B; 3.5 – 4.0 min, 5% B.
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N-(2-((4aR,6S,8aR)-2-benzamido-6-methyl-4,4a,5,6-tetrahydropyrano[3,4-d][1,3]thiazin-
8a(8H)-yl)thiazol-4-yl)-5-(difluoromethoxy)picolinamide (9): Two feed streams were
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prepared as follows: Stream A – amine 835 (10.0 g, 25.7 mmol) was dissolved in a
solvent mixture of THF (80 mL) and water (10 mL). Triethylamine (11.1 g, 109 mmol)
was added. A homogenous solution was obtained after stirring for 5 min. Stream B –
acid 735 (5.22 g, 27.0 mmol) was dissolved in THF (20 mL). Water (40 mL) and 2-
Hydroxypyridine N-Oxide (2.86 g, 25.7 mmol) were added, followed by acetone (60 mL).
EDC (15.1 g, 77.2 mmol) was subsequently added. The resulting mixture was stirred for
10 min to give a homogeneous solution. Vapourtec reactor R4 with a 10 mL stainless steel
coil equipped with a 100 psi back pressure regulator was equilibrated with the exactly the
same makeup of the solvent mixture for the respective feed lines. Upon reaching
steady temperature and pressure, the reaction system feeds were switched over from
solvent lines to reagents lines, and the flow rates were set at 0.148 mL/min (A) and 0.183
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mL/min (B) with a target residence time of 30 min. At the end of the run, the feeds were
switched over to solvents again until at least 10 mL were pushed out. The effluent was
concentrated by rotovap under reduced pressure (75 mmHg, 30 °C), the product crystallized out
directly. The slurry was filtered and rinsed with water, then dried in a vacuum oven to give 9 as a
white solid (13.2 g, 91.6%). The spectroscopic data is identical to those reported.35
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(4-aminophenyl)(4-(dimethylamino)piperidin-1-yl)methanone (12): 4-Aminobenzoic
Acid (10.0 g, 72.9 mmol), 2-Hydroxypyridine N-oxide (8.20 g, 72.9 mmol) and 4-
(Dimethylamino)piperidine (9.84 g, 72.9 mmol) were dissolved in a mixture of THF (100
mL) and water (100 mL). 1,3-Diisopropylcarbodiimide (18.8g, 146 mmol, 18.8 g) was
added (Note: In batch reaction kinetics study, there was no meaningful reaction at
ambient temperature, therefore it was decided to feed the reaction a single stream in
the flow demonstration run). The reaction mixture was a homogenous solution at RT.
After the reactor coils were equilibrated the solvent makeup of the reaction under the
reaction temperature. The reaction solution was fed at 1 mL/min to a 20 mL coil (with
static mixer inserts) followed by a 10 mL standard TFE coil both at 75°C with targeted
residence time of 30 min (no BPR was attached). After the run is complete, the run was
continued with the solvents for at least 45 mL. The collected effluent was rotorvapped to
remove THF under reduced pressure (60°C, 20 mmHg). The resulting slurry was
cooled to RT, and filtered to remove the diisopropyl urea as white solid. The aqueous
filtrate was pH adjusted to ~11 with 1N NaOH solution, then extracted with BuOH (1 x
10 vol and 1 x 5 vol). The combined BuOH solution was washed with brine solution (1 x
10
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5 vol). The final BuOH phase was concentrated to remove most BuOH, 50 mL of MeCN
was added. After stirring for 5 min, a slurry was obtained. It was granulated for 30 min at
RT and filtered. The filtered solid was dried in a vacuum oven to give the desired
product (12.7 g, 70%). The spectroscopic data were consistent with those of an
authentic sample.36
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Polymixin Analog (15): Two feed streams were prepared as follows: Stream A -
tripeptide 1337 (1.01 g, 1.47 mmol) and PMBH-Boc337 (1.60 g, 1.54 mmol) were
dissolved in DCM (10 mL) and DMF (5.3 mL) in the presence of iPr2NEt (463.8 mg, 2.45
equiv). Stream B - COMU (816 mg, 1.30 equiv) was dissolved in DMF (5.3 mL).
Vapourtec R4 was used for the flow run with 1.6 mL glass chip reactor (Uniqsis
UQ5102) at RT equipped with 100 psi BPR. The flow rates were set at 0.262 mL/min
(A) and 0.082 mL/min (B) with a targeted residence time of 4.6 min. The reaction
workup and isolation were the same as reported earlier with 88% yield isolated.37
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Acknowledgement: The authors thank Drs. Stéphane Caron, Jade Nelson, and
Nicholas Thomson for their support and suggestions, also thank Mr. Remzi Duzguner
for process safety support.
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References and Notes:
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Continuous Flow Nitration of Benzaldehyde. Org. Proc. Res. Dev. 2009, 13, 999-1002. (c)
Porta, R.; Benaglia, M.; Puglisi, A. Recent Developments in the Synthesis of Pharmaceutical
Products. Org. Process Res. Dev., 2016, 20, 2–25. (d) Movsisyan, M.; Delbeke, E. I.P.;
Berton, J. K. E. T.; Battilocchio, C.; Ley, S. V.; C. Stevens, V.Taming hazardous chemistry by
continuous flow technology. Chem. Soc. Rev. 2016, 45, 4892-4928. (e) Li, B.; Guinness, S.
M.; Hoagland, S.; Fichtner, M.; Kim, H.; Li, S.; Maguire, R J.; McWilliams, J. C.; Mustakis,
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methods using continuous-flow microreactors. Tetrahedron 2009, 65, 6611-6625. (b)
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Modular Flow Reactors: Bestmann–Ohira Reagent for the Formation of Alkynes and
Triazoles. Angew. Chem., Int. Ed. 2009, 48, 4017-4021. (c) Li, B.; Widlicka, D.; Boucher, S.;
Hayward, C. M.; Lucas, J.; Murray, J.; O'Neil, B.; Pfisterer, D.; Samp, S.; Van Alsten, J.;
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R.; Vaid, R. K.; Yu, H. Rapid Development and Scale-Up of a 1H-4-Substituted Imidazole
Intermediate Enabled by Chemistry in Continuous Plug Flow Reactors. Org Process Res.
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Microfluidic synthesis of α-ketoesters via oxidative coupling of acetophenones with alcohols
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Active Pharmaceutical Ingredients. Angew. Chem. Int. Ed. 2015, 54, 6688–6728.
4. Sperry, J. B.; Minteer, C. J.; Tao, J.; Johnson, R.; Duzguner, R.; Hawksworth, M.; Oke,
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6. (a) Spare, L. K.; Laude, V.; Harman, D. G.; Aldrich-Wright, J. R.; Gordon, C. P., An
optimised approach for continuous-flow solid-phase peptide synthesis utilising a rudimentary
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continuous-flow peptide synthesis – an abridged account of solid and solution-based
approaches. Org. Biomol. Chem. 2018, 16 (2), 180-196. (c) Frank, R.; Gausepohl, H. In
Continuous flow peptide synthesis, de Gruyter: 1988; pp 41-60. (d) Andrews, R. P.;
Summers, C., Automated continuous flow peptide synthesis. Am. Biotechnol. Lab. 1986, 4
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17. CDI solubility screen in commonly used solvents.
Solubility in volumes
(mL per mg CDI)
Comments
Solvent
Acetone, THF, MeCN
20
40
This data was collected
using serial dilutions at 5,
10, 20, and 40 volumes.
MEK, EtOAc, PrOAc, 2-MeTHF, anisole
MIBK, iPrOAc, BuOAc, iBuOAc, MTBE, t-
AmOMe, toluene, heptane
>40
10
DCM
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