Multistep Flow Synthesis of Diazepam Guided by Droplet-Accelerated Reaction Screening with Mechanistic Insights from Rapid Mass Spectrometry Analysis

Article abstract of DOI:10.1021/acs.oprd.7b00218

Electrospray and Leidenfrost droplet accelerated reactions were used as a predictive tool for estimating the outcome of microfluidic synthesis as demonstrated by Wleklinski et al. Rapid analysis by electrospray-mass spectrometry (ESI-MS) also provided immediate feedback on reaction outcomes in flow reactions. Significant reaction acceleration was observed in electrospray relative to the corresponding bulk reaction. This rapid reaction screening and analysis method has allowed for the detection of previously unreported outcomes in the reaction between 5-chloro-2-(methylamino)benzophenone and haloacetyl chloride (halo = Cl or Br) in the continuous synthesis of diazepam. In our current study, a more detailed extension of the previous work, we report acceleration factors that are solvent dependent; additional byproducts that were observed on the microfluidic scale that were absent in the droplet reactions. Gaining insight from this combined droplet and microfluidic screening/rapid ESI-MS analysis approach, we have helped guide the synthesis of diazepam and showcased the potential of this method as a reaction optimization and discovery tool. Informed by these new insights, diazepam was synthesized in a high-yield two-step continuous flow process.

Full text of DOI:10.1021/acs.oprd.7b00218

Article
pubs.acs.org/OPRD
Multistep Flow Synthesis of Diazepam Guided by Droplet-
Accelerated Reaction Screening with Mechanistic Insights from
Rapid Mass Spectrometry Analysis
†
†
H. Samuel Ewan, Kiran Iyer, Seok-Hee Hyun, Michael Wleklinski, R. Graham Cooks,*
and David H. Thompson*
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States
*
S Supporting Information
ABSTRACT: Electrospray and Leidenfrost droplet accelerated reactions were used as a predictive tool for estimating the
outcome of microfluidic synthesis as demonstrated by Wleklinski et al. Rapid analysis by electrospray-mass spectrometry (ESI-
MS) also provided immediate feedback on reaction outcomes in flow reactions. Significant reaction acceleration was observed in
electrospray relative to the corresponding bulk reaction. This rapid reaction screening and analysis method has allowed for the
detection of previously unreported outcomes in the reaction between 5-chloro-2-(methylamino)benzophenone and haloacetyl
chloride (halo = Cl or Br) in the continuous synthesis of diazepam. In our current study, a more detailed extension of the
previous work, we report acceleration factors that are solvent dependent; additional byproducts that were observed on the
microfluidic scale that were absent in the droplet reactions. Gaining insight from this combined droplet and microfluidic
screening/rapid ESI-MS analysis approach, we have helped guide the synthesis of diazepam and showcased the potential of this
method as a reaction optimization and discovery tool. Informed by these new insights, diazepam was synthesized in a high-yield
two-step continuous flow process.
1
. INTRODUCTION
In the present study, we use the synthesis of diazepam
Scheme 1) as a model system to showcase how the droplet
(
The potential for the efficient synthesis of active pharmaceutical
ingredients (APIs) through continuous flow chemistry
continues to draw interest from a broad range of disciplines
Scheme 1. Proposed Pathway for the Continuous Synthesis
of Diazepam
1
−5
throughout academia and industry.
Mass spectrometry
(MS) has proven to be a useful tool in reaction monitoring
as it allows monitoring of the kinetics and outcome of a
6
reaction. MS also helps in identifying reactive intermediates
and hence in understanding the mechanistic details of a
7
chemical reaction. Additionally, MS can be used to study
8
chemical reactions in droplets. Previous studies have shown
1
3
screening demonstrated by Wleklinski et al. guided the
continuous synthesis of diazepam. Since a key step in this
pathway involves N-acylation, these observations may have a
more general bearing on N-acylation reactions in the synthesis
that most chemical reactions are accelerated in microdroplets
formed by electrospray ionization (ESI) relative to the
9
,10
corresponding bulk reactions.
This acceleration is due, in
part, to solvent evaporation and its effect on reagent
14
11
of common bioactive molecules. Although microdroplet
concentration at the interface. This rapid method of reaction
screening can be useful in guiding microfluidic reactions and in
scale up. An alternative way of generating microdroplets for
studying chemical reactions is by employing the Leidenfrost
effect. This effect occurs when a solution is dropped onto a
heated surface such as a glass Petri dish which is held at a
temperature higher than the boiling point of the solvent used.
As the droplet approaches the heated surface, the solvent
begins to evaporate, and a layer of insulating vapor is formed
around the droplet. This prevents rapid evaporation of the
solvent and causes the droplet to levitate. Although the
droplets that are formed by this technique are larger than ESI
droplets, they have some of the same properties; therefore,
reactions in these droplets can also be a useful tool in guiding
microfluidic transformations.
conditions do not always directly translate into microfluidic
scale conditions, this method can serve as a rapid yes/no
prediction tool for the likelihood of a productive microfluidic
reaction. Flow chemistry systems, coupled with online
monitoring by ESI-MS, enable rapid screening of reaction
conditions with real-time feedback. Not only does this allow for
facile and efficient synthesis of APIs, but it also brings an
opportunity for new insights into reaction pathways and
byproduct formation. Diazepam may be obtained in two
synthetic steps, starting from the N-acylation reaction of 5-
chloro-2-(methylamino)benzophenone 1 with 2-haloacetyl
chloride 2 giving amide 3. Subsequent treatment with ammonia
12
Received: June 22, 2017
©
XXXX American Chemical Society
A
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Figure 1. N-Acylation screen. Synthesis of 3 in using bromoacetyl chloridecomparison of spray and flow in toluene and acetonitrile.
then results in cyclization, giving diazepam 4 (Scheme 1). For
each reaction screened in droplets or examined in flow reaction
systems, the outcome was immediately ascertained by ESI-MS
analysis.
The screening of flow reaction conditions for the first step
also began with a solvent screen. NMP and DMF performed
poorly, giving limited conversion and significant impurities. N-
Acylation occurred rapidly and with good conversion in
toluene, whereas in ACN, the formation of side products (5,
7
) arising from the S 2 reaction pathway was observed (Figure
2
. RESULTS AND DISCUSSION
.1. N-Acylation Reaction Screen. Reaction screening
N
13
1
). In addition to solvents, temperatures and residence times
2
were varied (Table S3). With increasing temperature, in most
cases, conversion was poorer due to increasingly prevalent side
reactions. By exchanging chloroacetyl chloride for bromoacetyl
chloride, we obtained evidence of an alternative reaction
pathway (previously reported by Wleklinski et al.). The
molecular weight of the expected product of this N-acylation
reaction using bromoacetyl bromide is 367; however, we saw an
additional peak at m/z 322. This corresponds to a loss of
bromine rather than chlorine, as expected from N-acylation. A
began with examination of the N-acylation step in ESI droplets,
Leidenfrost droplets, bulk, and flow reaction systems. Initial ESI
spray (offline) reaction screening across several solvents
13
[
dimethylacetamide (DMA), tetrahydrofuran (THF), dime-
thylformamide (DMF), acetonitrile (ACN), N-methylpyrroli-
done (NMP), and toluene] revealed significant acceleration in
ACN (∼35×) and toluene (∼100×) relative to a 30 min screen
of the reaction in bulk at the same initial concentrations (Figure
1, Figure S1, Table 1). Although the spray experiment was
mixture of the S 2 and N-acylation products (from the flow
N
performed in different solvents, the final extraction before
analysis was done in ACN for all experiments.
experiment) was isolated by column chromatography, and
though they could not readily be separated from one another,
NMR analysis showed two distinct sets of peaks at 3.7 and 3.9
ppm, corresponding to the methylene protons of the N-
Table 1. Accelerations Factors for the N-Acylation Reaction
in the Synthesis of Diazepam
acylation and S 2 products, respectively (Figure 2, Figure S2).
N
a
Guided by these observations regarding the mechanism of the
reaction where one solvent favored the more desired pathway
and products, we determined that toluene was the optimal
solvent for the continuous synthesis of diazepam.
acceleration factor
solvent
starting material
ESI spray
Leidenfrost
acetonitrile
chloroacetyl chloride
bromoacetyl chloride
chloroacetyl chloride
bromoacetyl chloride
35
29
97
38
25
24
14
34
In addition to this S 2 product, we also observed a peak at
N
toluene
m/z 288 in our flow experiments, consistent with ring closure
to produce a seven-membered lactone. We propose that this m/
z 288 compound (mol. wt. 287) is a result of an initial SN2
a
Acceleration factors reported in Table 1 are apparent acceleration
reaction, followed by nucleophilic attack by the carbonyl on the
factors given by the ratio of the mass spectrometry signals for the
products relative to the starting materials (i.e., the conversion ratio) in
the micro droplets relative to the bulk. This is not the same as the ratio
of the rate constants for the two media.
1
acyl chloride to form the seven-membered ring. H and
13
CNMR analysis confirmed this proposed structure (Figures
S3 and S4, respectively). This result suggests that the selection
B
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microfluidic screen. A microfluidic screen of the second step
from the N-acylation product 3A, prepared previously, was
carried out prior to attempting both steps in continuous flow.
The solvent screen was limited by the low solubility of the
chloro version of the intermediate (previously prepared in
batch). For each solvent, varying temperatures and residence
times were screened. The material was insoluble at a target
concentration of 250 μM in ACN and toluene but dissolved
well in NMP at this concentration. Fortunately, a good
conversion to diazepam was observed in NMP (Table S3).
Another interesting observation when studying this step was
the appearance of a peak at m/z 303. We anticipated that this
might represent a substitution of nitrogen at the methyl
position. Due to the appearance of this peak in the MS at lower
temperatures and residence times, and disappearance at higher
temperatures, we also believed that it might be an intermediate
in the diazepam synthesis. LC-MS analysis revealed that,
despite the previous MS observation, the quantity of this m/z
1
Figure 2. Methylene proton H NMR signals of the S 2 and N-
acylation product mixture.
N
3
03 material remained relatively constant throughout the
temperature and residence time screen, even as the quantity
of diazepam steadily increased. This could be in part due to
high ionization efficiency of the m/z 303 material drowning out
the signal from other compounds present in the reaction
mixture. This m/z 303 material (6) is believed to be a
of reaction solvent and halide can be used to exert control over
the reaction outcome in a microfluidic system. It is noteworthy
that these alternative outcomes were observed in the
microfluidic experiment but were absent in the corresponding
droplet experiments. We believe that this is due to the high
temperature and pressure conditions accessible in microfluidic
reactions, but absent in droplet reactions, making alternative
reaction pathways with higher energy requirements more likely.
15
previously reported hydrolysis product of diazepam.
2.3. Continuous Diazepam Synthesis. On the basis of
these observations, we were able to develop a more complete
understanding of the possible outcomes of each synthetic step
of our synthetic route to diazepam. These possible reaction
pathways are summarized in Scheme 2. Guided by this
knowledge from our screening, we next attempted to optimize
the synthesis of diazepam in two continuous steps. We used a
two-chip reactor system to allow for finer control of
2
.2. Cyclization Reaction Screen. Previous screening of
the cyclization reaction space revealed that a greater
concentration of ammonia was required to form diazepam in
Leidenfrost droplets (Figure 3). Reaction acceleration was
observed in these droplet reactions, and they corroborated the
Figure 3. Cyclization reaction screen. Synthesis of diazepam, comparing ACN (left) and toluene (right) solvents in spray, Leidenfrost, and flow
reactions using bromoacetyl chloride.
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Article
Scheme 2. Reaction Pathway to Diazepam and Its
Byproducts Formed during Continuous Flow Reaction
nuances observed in flow reactions were not observed in
droplet experiments, these experiments still consistently
provided a yes/no indication of the viability of a reaction.
Further, we have demonstrated the continuous synthesis of
diazepam in two steps in a microfluidic flow reactor. Our
synthesis features the use of a mixed solvent system, as well as
two microfluidic chips in sequence, allowing for optimized
temperature control at each step. Additionally, we have
identified previously unknown reaction pathways. These results
showcase the possibility for microfluidic synthesis coupled with
rapid ESI-MS analysis to identify previously unknown reaction
pathways and optimize continuous synthesis of APIs.
temperature and residence time in each chip. The first chip
combined 5-chloro-2-(methylamino)benzophenone and haloa-
cetyl chloride in a 1:2 ratio, respectively (Figure 4, R1 and R2),
4. EXPERIMENTAL SECTION
4
.1. Reagents. Reagents were purchased from Sigma-
Aldrich and used without further purification.
.2. NMR Analysis. NMR samples were prepared by
4
microscale SiO2 column chromatography. Samples were
analyzed using a Bruker AV-III-500-HD NMR spectrometer.
4
.3. Mass Spectrometry. A mass spectral analysis of
reaction products was performed using an LTQ ion trap mass
spectrometer (Thermo Fisher Scientific, San Jose, CA) with
nanoESI (nESI) ionization. All product samples (spray,
Leidenfrost, or flow reactions) were diluted 1:100 into ACN
before analysis, unless otherwise noted. The distance between
the tip of the spray emitter and ion transfer capillary to the MS
was kept constant at ca. 1 mm. Experiments were performed
using borosilicate glass pulled to a ca. 1−3 μm aperture. A spray
voltage of either positive or negative 2.0 kV was used for all
analyses. Positive-ion mode was used for all chemical analyses,
unless otherwise noted. Product ion (MS/MS) spectra were
recorded using collision-induced dissociation (CID) with a
normalized collision energy of 25 (manufacturer’s unit).
Figure 4. Reactor schematic for continuous diazepam synthesis.
before dilution of the resultant N-acylation product mixture
(
R3) with methanol and subsequent addition of ammonia/
methanol (R4) in a 7-fold excess in the second chip.
Solvent screening was again limited by solubility, particularly
upon addition of ammonia/methanol in the second step. To
alleviate this problem, a dilution step after the first reaction step
was incorporated to improve solubility. Good solubility was
achieved using toluene for the first step and diluting 1:4 with
methanol or NMP. The use of ACN in the first step and for
dilution was also effective. As in the previous flow reaction
screens, we varied temperature and residence time, as well as
the selection of bromo- vs chloroacetyl chloride (Table S4).
The results of this screen seemed to corroborate our previous
observations, with bromoacetyl chloride resulting in some SN2
product, particularly in ACN. Furthermore, the overall
conversion from the intermediate to diazepam was higher
when bromoacetyl chloride was used.
Yields for each reaction were determined using a quantitative
ESI-MS/MS method. The optimum result was achieved using
bromoacetyl chloride in the toluene/methanol solvent system,
which yielded diazepam in 100% yield, based on our ESI-MS
quantitation method. This was also the optimal solvent system
in Leidenfrost and spray microdroplets (Table S1).
4
.4. Quantitative MS Analysis. An MS based calibration
−7
−6
−6
was made from mixtures of 0, 1 × 10 , 1 × 10 , 2.5 × 10 ,
and 8 × 10− M diazepam with 3.88 × 10 M diazepam-D3.
Each point was measured with nESI in triplicate, and the
calibration is based on the diazepam to diazepam-D3 ratio.
Crude reaction samples were quantified by first diluting an
appropriate amount (×10 000 typically) and then adding the
same amount of internal standard. Each crude sample was
diluted in duplicate and analyzed by nESI.
6
−6
4.5. ESI Experiments. These experiments were performed
by spraying the reaction mixture directly onto glass wool and
then extracting the sprayed residue with ACN. The extract was
diluted 1:100 then analyzed by MS. A home-built electrospray
ionization source was used. Reagents 1 and 2 were premixed at
concentrations of 100 mM and 200 mM, respectively, and
loaded into a syringe. Offline spray was carried out at a flow
rate of 10 μL/min, 100 psi N sheath gas, and 5 kV voltage. The
2
total spray time was 10 min. After MS analysis, the washed
material was drawn back into the syringe and mixed with
ammonia in methanol and then electrosprayed to synthesize
diazepam.
3
. CONCLUSIONS
4
.6. Leidenfrost Droplet Experiments. This experiment
This study, using the diazepam synthesis as a model reaction,
demonstrates the ability of MS analysis and droplet reactions to
guide microfluidic synthesis. MS can be used not only as an
analytical tool but can also serve as a quick way to predict
reactivity and guide microscale synthesis. The use of spray and
Leidenfrost droplet reactions as a screening step to guide the
larger scale microfluidic screening proved a useful tool in
predicting the overall outcome of a reaction. While some
was carried out on a hot plate with a heat setting of 540 °C
(although the droplet temperature was much lower). Reagents
1 and 2 were premixed and loaded into a Pasteur pipet. The
reaction mixture was then dropped onto a glass Petri dish that
was placed on the hot plate. The reaction mixture was added in
aliquots over a time period of about 2 min. After 2 min, the
mixture was collected from the surface using a Pasteur pipet
and then analyzed by MS after diluting 1:2.
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Organic Process Research & Development
.7. Microfluidic Experiments. All microfluidic reactions
were carried out using a Chemtrix Labtrix S1 system, equipped
with 3223 or 3224 reactor chips.
Article
4
product was isolated via SiO column chromatography in 68%
yield. The structure was confirmed by H NMR (Figure S5).
2
1
ASSOCIATED CONTENT
Supporting Information
4
.8. N-(2-Benzoyl-4-chlorophenyl)-2-halo-N-methyla-
cetamide (3). Solutions (100 mM) of 5-halo-2-
methylamino)benzophenone (1 equiv) and of haloacetyl
■
*
S
(
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.oprd.7b00218.
chloride (1 equiv) in toluene, ACN, DMF, or NMP were
prepared. In the DMF reaction screen, 500 mM solutions were
used; in the NMP reaction screens, 250 mM solutions of
benzophenone and 500 mM (2 equiv) chloroacetyl chloride
were used. A syringe was loaded with each of these two
solutions and positioned on the first two inlets of a 10 μL
Labtrix 3223 chip. A third syringe was loaded with toluene and
positioned on the third port of the same chip as a diluent. The
reaction was flowed with 30, 60, and 180 s residence times at
temperatures of 50, 100, and 150 °C. Samples were collected
and immediately analyzed by ESI-MS (1 μL of each sample was
diluted with 99 μL of ACN, then loaded into a glass
electrospray tip for analysis). Samples were saved and stored
at −20 °C.
Structure index, Figures S1−S5, Tables S1−S4 (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*
E-mail: davethom@purdue.edu.
E-mail: cooks@purdue.edu.
*
ORCID
R. Graham Cooks: 0000-0002-9581-9603
David H. Thompson: 0000-0002-0746-1526
Author Contributions
†
H.S.E. and K.I. contributed equally.
Notes
4
.9. Diazepam (4) from N-(2-Benzoyl-4-chlorophenyl)-
The authors declare no competing financial interest.
2
-chloro-N-methylacetamide (3A). A 250 mM solution of
N-(2-benzoyl-4-chlorophenyl)-2-chloro-N-methylacetamide (1
equiv) was prepared. Syringes were loaded with the prepared
solution and with 7 N ammonia in methanol (7 equiv) and
positioned on the first two inlets of a 10 μL Labtrix 3223 chip.
A third syringe was loaded with NMP and positioned on the
third port of the same chip as a diluent. The reaction was
flowed with 30, 60, and 180 s residence times at temperatures
of 50, 100, and 150 °C. Samples were collected and
immediately analyzed by ESI-MS (1 μL of each sample was
diluted with 99 μL of ACN, then loaded into a glass
electrospray tip for analysis). Samples were saved and stored
at −20 °C.
ACKNOWLEDGMENTS
■
This work was supported by the Defense Advanced Projects
Agency of the United States of America and the National
Science Foundation (CHE-1307264).
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4.11. Batch Synthesis of N-(2-Benzoyl-4-chlorophen-
yl)-2-chloro-N-methylacetamide (3A). Chloroacetyl chlor-
ide (0.39 mL, 4.9 mmol, 1 EQ) was added to a solution of 5-
chloro-2-(methylamino)benzophenone (1.2 g, 4.9 mmol) in
1
4
00 mL NMP (50 mM). The reaction was heated to 90 °C for
0 min. The solution was then concentrated in vacuo, and the
E
DOI: 10.1021/acs.oprd.7b00218
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