Continuous Synthesis and Purification by Coupling a Multistep Flow Reaction with Centrifugal Partition Chromatography

Article abstract of DOI:10.1002/anie.201703852

Continuous-flow multistep synthesis is combined with quasi-continuous final-product purification to produce pure products from crude reaction mixtures. In the nucleophilic aromatic substitution of 2,4-difluoronitrobenzene with morpholine followed by a het

Full text of DOI:10.1002/anie.201703852

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Accepted Article
Title: Continuous Synthesis and Purification by Coupling a Multistep
Flow Reaction with Centrifugal Partition Chromatography
Authors: Róbert Örkényi, János Éles, Ferenc Faigl, Péter Vincze, Anita
Prechl, Zoltán Szakács, János Kóti, and István Greiner
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201703852
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10.1002/anie.201703852
Angewandte Chemie International Edition
COMMUNICATION
Continuous Synthesis and Purification by Coupling a Multistep
Flow Reaction with Centrifugal Partition Chromatography
Róbert Örkényi,* János Éles, Ferenc Faigl, Péter Vincze, Anita Prechl, Zoltán Szakács, János Kóti and
István Greiner*
The continuous-flow synthesis of active pharmaceutical
ingredients (APIs) and their intermediates[1–9] is actively
encouraged by regulatory agencies,[10] not to mention its
advantages compared to batch processing.[11,12] However, the
continuous manufacturing of the final dosage form of drugs by
coupling the synthesis with formulation demands highly pure
APIs. Consequently, continuous-flow purification is inevitable in
most cases. Nonetheless, continuous synthesis is usually
followed by ‘discontinuous’ purification because of the fact that
the number of available options for continuous purification is
limited[1,13]. The existing methods[6,14] can be classified as in-line
work-up and final-product purification depending on their primary
place of application within a multistep sequence[1]. In-line work-
up can remove co-products while it cannot eliminate by-products
which are structurally related to the desired product. High purity
can be achieved by final-product purification using multicolumn
by inversing the stationary and mobile phase with each other
multiple times and re-injecting the sample solution in between.
Choosing the biphasic liquid system (BLS) is like choosing the
column and the eluent in high-performance liquid
chromatography (HPLC). As a rule of thumb, the partition
coefficients should be around 0.5 – 1.5 for the compound of
interest, and the settling time of the phases should not be more
than 20 s[38]
.
chromatography,[15]
simulated
moving
bed
(SMB)
[16–19]
chromatography,
catch and release chromatography,[20–23]
crystallization[15,19,24–26] or recrystallization,[2,27] although these
methods have their drawbacks: crystallization usually requires
semi-batch processing, and catch and release chromatography
can only be categorized as a truly continuous purification
method when automated switching between multiple columns is
employed.[28] The operation of SMB chromatography is
technically complex; furthermore, it utilizes expensive solid
adsorbent, and challenging separations may require additional
crystallization.[15,18]
Centrifugal partition chromatography (CPC) is a counter-current
separation technique[29–33] widely used for the purification of
natural products, small molecules and biologics. CPC does not
require a solid stationary phase, two non-miscible phases are
applied instead; one of them is used as the mobile phase and
the other as the stationary phase which is maintained inside the
rotating column by the centrifugal force. In ascending mode
(AM) the upper (lighter) phase is the mobile phase and the lower
(denser) phase is the stationary phase, while in descending
mode (DM) it is just the opposite (Figure 1.). The batch-wise
separations can be converted into semi-continuous purification
by using multiple dual-mode (MDM)[30,34–37], which means that
the liquid nature of the stationary phase is used to regenerate it
Figure 1. Scheme of the working principle of CPC device in AM and DM.
CPC has many advantages as compared to HPLC, such
as higher resolution, no need for column replacement or silica to
recycle, and it is absolutely free of irreversible adsorption.[38]
In order to address the purification issues currently faced
in flow synthesis, we decided to develop a new continuous final-
product purification method by using CPC. Herein we report the
first successful coupling of a multistep flow synthesis and MDM
CPC to accomplish quasi-continuous purification and production
of the pure product.
[*]
R. Örkényi, Prof. Dr. F. Faigl
Our aim was to develop a novel final-product purification
method by coupling a multistep continuous-flow synthesis with
MDM CPC. This paper is organized into five sections as follows:
flow reaction, finding the proper BLS, CPC method development,
automation, and coupling the reaction with the purification.
The target molecule, 4-fluoro-2-(morpholin-4-yl)aniline (5a),
which is a key intermediate in the synthesis of bioactive
Department of Organic Chemistry and Technology,
Budapest University of Technology and Economics,
Budapest, H-1521, Hungary
E-mail: orkenyi.robert@gmail.com
Dr. J. Éles, P. Vincze, A. Prechl, Dr. Z. Szakács, J. Kóti,
Dr. I. Greiner
Gedeon Richter Plc.,
Gyömrői út 19-21., H-1103 Budapest, Hungary
E-mail: i.greiner@richter.hu
carbazoles,[39] was synthesized in
substitution (SNAr) reaction[16] of 2,4-difluoronitrobenzene (1)
a nucleophilic aromatic
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201703852
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COMMUNICATION
with morpholine (2) followed by heterogeneous catalytic
hydrogenation (Figure 2.).
of 5 mLmin-1 and a rotation speed of 2000 rpm. Owing to the
distinctively higher partition coefficient of the desired product
(5a) as compared to the by-products (5b,c), the product eluted
first in AM (the upper phase is the mobile phase) and it eluted
last in DM (the lower phase is the mobile phase), which is ideal
for our purification purposes.
Using these optimized conditions without modification, an
MDM method was developed to achieve quasi-continuous
purification. After the column had been equilibrated and the first
sample injection took place, the by-products (5b, c) were simply
washed out from the column in DM. Next, the sample solution
was injected into the column again, and finally the product from
both injections was eluted and collected in AM. This process
could be repeatedly reversed several times, without post-
washing and equilibration of the column between cycles. The
efficiency and recovery were not affected as compared to the
single AM and DM separation. In this way a stable, uninterrupted
MDM CPC separation could be conducted for more than 5 h.
The purity of the product was more than 99.9% (GCMS) and the
recovery of 5a was 91%.
Figure 2. Continuous-flow SNAr reaction of 2,4-difluoronitrobenzene (1) with
morpholine (2) followed by a heterogeneous continuous hydrogenation of the
nitro compounds (3a-c) to the corresponding anilines (5a-c), using a loop
reactor and the H-Cube Pro™ device.
In order to connect the reaction stream with the purification
unit it was essential to automate the sample intake, which was
enabled by the SpotPrepII device’s magnetic valves that can be
programmed. Due to the increased dead volume before the
column, a prolonged elution time was necessary in AM to
achieve the same recovery.
The first reaction step was performed in ethanol (EtOH) at
100°C with 10 min of residence time in a loop reactor connected
to a Zaiput® back pressure regulator adjusted to 10 bar. The
resulting crude reaction mixture of compounds 3a-c and the
morpholine hydrofluoride salt (4) were introduced into the
H-Cube Pro™ reactor containing a 10% Pd/C cartridge at 50°C
and atmospheric pressure. The product (5a) and all of the
intermediates (3a-c) and by-products (5b,c) were isolated and
characterized (see the Supporting Information). The average 5a
content of the reaction mixture was about 81% along with 5% of
5b and 12% of 5c.
First, an adequate BLS was developed through extensive
experimentation to differentiate between regioisomers (5a,b)
that are similar in every physicochemical property, including pKa
(Table 1.). The mixture of n-hexane (n-Hex) / methyl tert-butyl
ether (MTBE) / EtOH / water system in a volumetric ratio of
1/1/1/1 gave ideal partition coefficients (KU/L) for anilines 5a-c
(Table 1.) and exhibited a short settling time of 16 s (see the
Supporting Information).
In order to match the composition of the sample intake of
the CPC separation with the output of the continuous-flow
reactor, the EtOH solution of the product was mixed with the
other components of the chosen BLS. After phase separation
using a separatory funnel, the upper and lower phase of the
resulting biphasic mixture was separately introduced according
to the program (see Supporting Information) into the CPC device
(Figure 3A). The phase separation unit served as a buffer flask
and also allowed the escape of the excess hydrogen from the
reduction step (which would otherwise be forcing out the liquid
from the CPC column). In order to reach an overall continuous
operation, the total inlet throughput of the reaction stream and
the other components of the sample solution into the buffer flask
must be equal or greater than the throughput of the outlet in a
certain period of time. For this purpose the elution times (both in
AM and DM), the time and flow rate of the sample intakes, the
flow rates of the reaction stream and the other components of
the sample solution, and their volume contraction factor were
considered.
Table 1. Measured physicochemical parameters of anilines 5a-c.
Entry
Parameter
5a
5b
5c
[a]
1
2
KU/L
1.86
0.49
0.24
The whole system (two-step reaction and purification)
could be continuously operated, the isolated yield, purity and
productivity values were satisfactory (Table 2: Entry 1).
[b]
pKa
4.08±0.015
4.06±0.029
4.76±0.023
[a] The partition coefficients (KU/L) were determined by GCMS measurements
in the biphasic solvent system of n-Hex/MTBE/EtOH/H2O=1/1/1/1 v/v ratio.
(KU/L = peak area of the compound in the upper phase divided by the peak
area of the compound in the lower phase). [b] pKa values were determined by
UV-spectrophotometric titrations (See Supporting Information).
In order to increase the productivity by increasing the
sample solution concentration and its throughput, a one-phase
sample intake method was developed (Figure. 3B). The sample
solution was prepared as a single-phase mixture of the reaction
stream in EtOH combined with MTBE and water using two
additional pumps (195/25/150 µL min-1 of flow rates of
Employing the chosen BLS in the initial batch-wise CPC
experiments performed on a 100 mL capacity column (Armen
SCPC-100+1000-B apparatus with SpotPrepII system) showed
practically baseline separation for the product both in AM and
DM. The operating conditions on the equilibrated column were
the following: 5-10 mL sample injection, mobile phase flow rate
H2O/MTBE/EtOH, respectively), in
a
composition that
corresponds to the lower phase of the BLS (ratios were
determined using GC-FID or 1H NMR for the organic compounds
and Karl-Fischer titration for the water content, see Supporting
Information). The more concentrated sample solution and the
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10.1002/anie.201703852
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higher throughput of the reaction stream gave a productivity that
was 60% higher (Table 2: Entry 2) than that of the two-phase
sample intake method.
In summary, we have developed a system for the multistep
continuous-flow synthesis and purification of a complex reaction
mixture, utilizing quasi-continuous multiple dual-mode centrifugal
partition chromatography that can be operated in a truly
continuous manner by using buffer flasks and a few pumps (See
Supporting Information) and by synchronizing the flow reaction
with the purification. The productivity increased significantly by
the one-phase intake of the sample solution.
Table 2. Results obtained in the coupled system of the two-step synthesis
with purification.
Yield[a]
(%)
Purity[b]
(%)
Productivity[c]
(g h-1L-1)
Entry
Sample intake method
Throughput could be easily elevated by scaling up the
column capacity[40–43] or by converting to a true moving bed
system[44–47] via introducing the sample solution continuously into
the intermediate point of the column (e.g. between two columns).
This system is the very first continuous-flow adsorbent-free
final-product purification technique, and should prove to have a
wide applicability in the synthesis of APIs or its intermediates.
1
2
Two-phase[d]
One-phase[e]
57
59
> 99.9
> 99.9
1.44
2.27
[a] Isolated yield for the overall two synthetic steps followed by the quasi-
continuous purification. [b] Measured by GCMS. [c] Mass of the pure product
divided by the time of the process and the volume of the column. [d] Scheme
of the process shown in Figure 3A. [e] Scheme of the process shown in Figure
3B.
Figure 3. Flow chart of the two-step synthesis followed by a quasi-continuous MDM CPC purification, using: A, two-phase sample intake or B, one-phase sample
intake. U: Upper phase of the chosen BLS, L: Lower phase of the chosen BLS; S(L): Sample solution in lower phase, S(u): Sample solution in upper phase.
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
COMMUNICATION
Róbert Örkényi*, János Éles, Ferenc
Faigl, Péter Vincze, Anita Prechl, Zoltán
Szakács, János Kóti and István Greiner*
Page No. – Page No.
Continuous Synthesis and
Purification by Coupling a Multistep
Flow Reaction with Centrifugal
Partition Chromatography
Text for Table of Contents
Continuous-flow multistep synthesis meets quasi-continuous final-product purification to produce pure products from crude reaction mixtures.
In the nucleophilic aromatic substitution of 2,4-difluoronitrobenzene with morpholine followed by a heterogeneous catalytic hydrogenation, the
desired monosubstituted product can be continuously separated from the by-products in a purity of over 99ꢀ% by coupling a flow reactor
sequence to a multiple dual-mode (MDM) centrifugal partition chromatography (CPC) device. It has many advantages as compared to HPLC,
such as higher resolution, no need for column replacement or silica to recycle, and it is absolutely free of irreversible adsorption.
This article is protected by copyright. All rights reserved.