Rapid On-Demand Synthesis of Lomustine under Continuous Flow Conditions

Article abstract of DOI:10.1021/acs.oprd.8b00387

Lomustine, an important agent for treatment of brain tumors and Hodgkin's lymphoma, has been synthesized using continuous flow methodology. Desorption electrospray ionization mass spectrometry (DESI-MS) was used to quickly explore a large number of reaction conditions for one of the reaction steps and guide the efficient translation of optimized conditions to continuous lomustine production. Using only four inexpensive commercially available starting materials and a total residence time of 9 min, lomustine was prepared via a linear sequence of two chemical reactions performed separately in two telescoped flow reactors. Sequential offline extraction and filtration resulted in a 63% overall yield of pure lomustine at a production rate of 110 mg/h. The primary advantages of this approach are the rapid manufacture of lomustine with two telescoped steps to avoid isolation and purification of a labile intermediate and the mild conditions used in the nitrosylation step, thereby significantly increasing the purity and yield of this active pharmaceutical ingredient.

Full text of DOI:10.1021/acs.oprd.8b00387

Article
pubs.acs.org/OPRD
Cite This: Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Rapid On-Demand Synthesis of Lomustine under Continuous Flow
Conditions
Zinia Jaman,† Tiago J. P. Sobreira,† Ahmed Mufti,‡ Christina R. Ferreira,† R. Graham Cooks,†
and David H. Thompson*,†
†Department of Chemistry, Purdue University, Bindley Bioscience Center, 1203 West State Street, West Lafayette, Indiana 47907,
United States
‡School of Chemical Engineering, Purdue University, 480 West Stadium Avenue, West Lafayette, Indiana 47907, United States
S
* Supporting Information
ABSTRACT: Lomustine, an important agent for treatment of brain tumors and Hodgkin’s lymphoma, has been synthesized
using continuous flow methodology. Desorption electrospray ionization mass spectrometry (DESI-MS) was used to quickly
explore a large number of reaction conditions for one of the reaction steps and guide the efficient translation of optimized
conditions to continuous lomustine production. Using only four inexpensive commercially available starting materials and a
total residence time of 9 min, lomustine was prepared via a linear sequence of two chemical reactions performed separately in
two telescoped flow reactors. Sequential offline extraction and filtration resulted in a 63% overall yield of pure lomustine at a
production rate of 110 mg/h. The primary advantages of this approach are the rapid manufacture of lomustine with two
telescoped steps to avoid isolation and purification of a labile intermediate and the mild conditions used in the nitrosylation
step, thereby significantly increasing the purity and yield of this active pharmaceutical ingredient.
KEYWORDS: Lomustine, continuous synthesis, reaction telescoping,
desorption electrospray ionization mass spectrometry (DESI-MS), high throughput experimentation
problems, thus motivating our effort to develop a lomustine
synthesis using continuous flow methods.
INTRODUCTION
■
Lomustine, a widely used anticancer agent, is a highly
lipophilic alkylating agent that produces chloroethyl carbenium
ions and carbamylating intermediates in vivo.1,2 These
electrophilic compounds attack the nucleophilic sites on
DNA to form alkylated products.1 Other anticancer agents
such as mitomycin C, streptonigrin, bleomycin, and the
anthracyclines require bioactivation to react with their cellular
targets, whereas lomustine does not require preactivation.3
Unlike alkylating agents that form adducts at the most reactive
N7 position of guanine, chloroethylating compounds like
lomustine form adducts at O6, leading to interstrand DNA
cross-linking. If DNA repair does not occur, this cross-linking
can cause double strand breaks during DNA replication,
eventually leading to cell death via apoptosis.4
Lomustine, 1-(2-chloroethyl)-3-cyclohexyl-l-nitroso-urea
(commercial names: CCNU, CeeNU, Gleostine), is used as
an oral antineoplastic agent that is administered every 6 weeks.
It was first evaluated in clinical trials in the late 1960s5 and
approved by the US FDA in 19766 for primary and metastatic
brain tumors as well as Hodgkin’s lymphoma.4 Bristol-Myers
Squibb originally held the patent for the agent under the brand
name CeeNu. In 2014, Next Source Biotechnology LLC was
approved by the FDA for the rebranding of lomustine under
the trade name Gleostine.6 The average wholesale price for one
dose of rebranded Gleostine has recently experienced a
substantial price increase,5 in part due to the new regulatory
challenges of handling carcinogenic compounds of this type.1,4
Unfortunately, this situation has created patient access
Flow chemistry approaches have been reported as an
efficient methodology and have recently been explored in
both industry and academic laboratories.7−18 Compared to
traditional batch synthesis processes, flow transformations
provide better control over reaction conditions and selectivity
owing to rapid mixing and precise control of reaction
parameters such as temperature, stoichiometry, pressure, and
residence time. The enhanced heat and mass transfer
capabilities also provide safer operational conditions.8,19−25
Generally, these aspects of continuous flow synthesis
contribute to improved chemical reaction efficiency25,26 and
shorter reaction times, enabling process intensification,19 and
more facile scale-up, with improved quality and consistency in
production. Motivated by these factors, continuous flow
synthesis of active pharmaceutical ingredients has recently
become more attractive.23,27−39 Efficient execution of multistep
reactions in a telescoped manner, however, still remains a
challenge due to issues arising from workup conditions,32,40,41
solvent switches,42,43 and flow rate differences.42,44 Moreover,
optimization of continuous flow conditions and analysis
require significant investments in time and material.32,45−50
Recently, our group reported a robust high throughput
experimentation method using desorption electrospray ioniza-
tion mass spectrometry (DESI-MS) to explore reaction
conditions for scalable synthesis in continuous flow reactors
Received: November 19, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.oprd.8b00387
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Organic Process Research & Development
Scheme 1. Synthesis Plan for Lomustine in Continuous Flow, Where 4 = NaNO2/HCO2H and 5 = BuONO
Article
t
at scale.49 Herein, we report the continuous flow synthesis of
lomustine using an optimization protocol that was guided by
DESI-MS. The final method consists of two reactions
telescoped without isolation of the reaction intermediate.
This approach can reduce production costs radically by using a
simple reactor setup and inexpensive starting materials
(Scheme 1). To the best of our knowledge, this is the first
synthesis of lomustine telescoped in continuous flow using an
approach that does not interrupt the flow sequence due to
intermediate workup requirements.
conditions. As shown in Table 1, product yields dropped
sharply in EtOH at longer residence times (entries 1−3) due
Table 1. Reaction Conditions Evaluated for the Synthesis of
3 in Flow Using a Chemtrix S1 Glass System Fitted with a
3225 SOR Reactor Chip
temp
(°C)
residence time
(s)
isolated yield
(%)
entry solvent 2 (equiv)
1
2
3
4
5
6
7
8
EtOH
EtOH
EtOH
ACN
ACN
ACN
toluene
ether
THF
THF
THF
THF
THF
THF
THF
THF
THF
THF
THF
THF
THF
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1.2
1.2
1.2
1.4
1.4
1.4
1.4
50
50
50
50
50
25
50
50
25
25
50
50
65
65
50
50
50
50
50
50
50
10
30
60
10
30
30
10
10
10
30
10
30
10
30
10
30
60
10
30
60
180
42.8
0.00
0.00
56.2
clogged
clogged
clogged
clogged
50.0
56.4
59.5
62.1
47.5
29.1
61.2
64.8
71.3
RESULTS AND DISCUSSION
■
Continuous Synthesis of 1-(2-Chloroethyl)-3-cyclo-
hexylurea, 3. The first step in the synthesis of lomustine
(Scheme 1) is a fast reaction at room temperature. This
transformation was optimized in continuous flow as a function
of temperature, solvent, and stoichiometry to discover the
conditions for maximum product yield. The reactions were
monitored by TLC and MS using a triple quadrupole mass
spectrometer operating in positive ion mode to afford rapid
investigation of full mass spectra and product ion distributions
for each reaction condition.
9
10
11
12
13
14
15
16
17
18
19
20
21
A cascade method was designed to reveal the best conditions
for the first step. Cyclohexylamine, 1, 1-chloro-2-isocyanato-
ethane, 2, and triethylamine (TEA) solutions were pumped
through a Chemtrix 3225 SOR chip with automatic collection
of the products in vials via an autosampler (Figure 1). The
67.1
82.0
91.7
82.8
to ethanolysis of the 1-chloro-2-isocyanato-ethane starting
material. Though the yield of 3 in ACN was significant, its low
solubility in this solvent led to significant chip clogging (entries
4−6). Similar clogging problems were found for toluene and
Et2O, even at low concentrations (entries 7−8). The yield
increased to 50−62% with longer residence times (entries 9−
14) in THF, reaching a maximum when τ = 30 s at 50 °C and
decayed rapidly with increased temperature (entries 13, 14)
due to increased product decomposition at elevated temper-
atures. The yield of 3 was also found to increase with
proportions of 2 ratio (entries 15−21), whereas longer
residence times promoted product decomposition (entry 21).
Consequently, a maximum yield of intermediate 3 (92%) was
achieved under conditions of τ = 60 s, T = 50 °C, and 1.4
equivalents of 1-chloro-2-isocyanatoethane (entry 20).
Synthesis of Lomustine, Guided by DESI-MS High
Throughput Experimentation. We employed DESI-MS to
evaluate the impact of solvent, concentration, and nitrosation
reagent type on the efficiency of lomustine production. DESI-
MS was originally applied to the surface analysis of intact
samples such as biological tissues for cancer diagnosis or
human fingerprints for drug detection,51,52 although, more
recently, the DESI-MS approach has been used for chemical
reaction analysis.49 This approach is thought to involve the
phenomenon of reaction acceleration that occurs within
Figure 1. Microfluidic synthesis of intermediate 3 in a glass reactor
chip (SOR 3225). A = 1 in THF; B = 2 in THF, C = TEA in THF.
individual reaction mixtures were evaporated, and the white
solid was washed with cold Et2O before drying under vacuum
for overnight prior to analysis by TLC, MS, MS/MS, and
NMR (1H and 13C).
Parameters such as residence time (τ), reaction temperature
(T), and 1-chloro-2-isocyanatoethane/cyclohexylamine ratio
were investigated systematically under continuous flow
B
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Organic Process Research & Development
Article
confined volumes such as microdroplets that originate from
spray-based MS ionization processes.51 MS analysis speeds
approaching 104 reaction spots/h can be achieved by this
technique.49
identified as the best solvent for nitrosation using 4. Excess
4 was also used to maximize conversion of 3 to lomustine since
the nitrosation reagents are hygroscopic and readily oxidize to
nitrate.53,54 Overall, the DESI-MS experiments were in
excellent agreement with the outcomes of flow reaction
conditions in terms of stoichiometry, reagents, and solvents.
A series of reactions (Table 2) were performed to maximize
the conversion of 3 to lomustine under continuous synthesis
As shown in Scheme 1 (second step), two nitrosation
methods were investigated. First, the efficiency of sodium
nitrite, 4, in formic acid as the nitrosation reagent toward the
conversion of 3 to lomustine was evaluated. This conversion
was then compared with tert-butyl nitrite (TBN, 5) as the
nitrosation reagent. DESI-MS was used to evaluate the
NaNO2/HCO2H transformation under different reactant
concentrations and solvents. The expected m/z 234 value for
lomustine was not observed. Analysis of a commercial
lomustine sample yielded a very similar MS, suggesting that
fragmentation occurs readily during the ionization process.
Triple quadrupole MS with electrospray ionization, as well as
ion trap mass spectrometry coupled with DESI, revealed the
presence of a stable lomustine fragment ion at m/z 169. This
was further confirmed by the fact that the lomustine fragments
from this reaction matched with commercially available
lomustine (see Supporting Information for details, Figures
S10 and S11). Reactions for all NaNO2 concentrations in
THF/H2O worked better than in the case of ACN/H2O
(Figure 2A). Also, the fragments from 3 were most abundant
Table 2. Isolated Yields of Lomustine under Different
Reaction Conditions Using 4 as the Nitrosation Reagent
a
isolated yield
(%)
(Method 1)
isolated yield
(%)
(Method 2)
isolated yield
(%)
(Method 3)
residence
entry time (min)
1
2
3
4
5
6
0.5
1
3
5
8
26.8
48.8
51.6
79.0
59.0
50.2
−
−
41.2
54.4
−
43.7
50.6
63.1
74.5
68.7
65.3
10
−
a
Temperature = 0 °C, solvent = MeOH/H2O (4:1).
Figure 2. DESI-MS plate maps showing the presence or absence of
expected ions at each spot where the nitrosation reaction conditions
were tested using different stoichiometries and with commercially
available standards. Blue dots indicate the presence of the m/z 169
expected stable fragment for the reaction product (successful
reaction). Red dots indicate that the expected m/z for the reaction
product was not present at the reaction spot (unsuccessful reaction
condition). (A) Concentration screening using the lomustine ion (m/
z 169) intensity and NaNO2 as nitrosation reagent. (B) Solvent
screening using the lomustine ion (m/z 169) intensity and NaNO2 as
nitrosation reagent. (C) Solvent screening using the lomustine ion
Figure 3. Microfluidic synthesis of lomustine using NaNO2/HCO2H
as nitrosation reagent in a Chemtrix 3225 SOR glass reactor chip. A =
3 in THF; B = HCO2H (neat), C = NaNO2 in MeOH/H2O (4:1).
t
(m/z 169) intensity and BuONO as nitrosation reagent.
conditions (Figure 3). Initially, we examined the reaction at 0
°C with a residence time of 30 s using TLC and MS to monitor
the reaction progress. Gratifyingly, lomustine was obtained
through this very short reaction time, albeit in low conversion
(Table 2, entry 1). A systematic evaluation of residence times
led to good lomustine yields and starting material conversions;
however, longer residence times appeared to enhance the
decomposition of lomustine (Table 2, entries 2−6). Con-
sequently, we kept the temperature constant at 0 °C to avoid
NaNO2 decomposition to the diazonium salt that occurs at
higher temperatures.54
in the ACN/H2O reactions compared to reactions in THF/
H2O, suggesting that the conversion of starting material was
comparatively sluggish in ACN/H2O. Next, eight different
solvents (EtOH, MeOH, DCM, ACN, toluene, DMSO, THF,
and EtOAc) were compared to optimize the continuous flow
synthesis. All eight solvents produced similar outcomes using 4
as the nitrosation reagent (Figure 2B). When 5 was used,
lomustine was detected in all solvents except THF and EtOAc
(Figure 2C).
Nitrosation Reaction Optimization in Continuous
Flow. We utilized the reaction conditions emerging from the
DESI-MS high throughput experiments to optimize the flow
synthesis and also check whether unsuccessful reactions
identified by DESI-MS would negatively translate under flow
conditions. From the DESI-MS experiments, THF was
Different purification methods were evaluated to isolate pure
lomustine. Initially, the product was extracted with Et2O (3
times) to exploit the low solubility of 3 in this solvent (Method
1). Unfortunately, TLC analysis revealed the presence of 3 in
the organic layer as well as in the aqueous layer. Therefore, the
combined organic extracts were dried over anhydrous Na2SO4
C
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Article
and concentrated in vacuo to produce a yellowish oil that was
redissolved in Et2O, heated, and cooled in an ice bath to
precipitate 3 from the mixture. NMR analysis of the filtrate
revealed that 22% of 3 remained in the isolated lomustine
product. Next, we purified the product by recrystallization
from ACN (Method 2). Although we obtained very pure
lomustine as determined by NMR analysis, recovery using this
approach was low. Finally, we found that hot filtration from
petroleum ether removed the insoluble 3 impurity (Method 3).
Concentration of the filtrate after drying gave pure lomustine
without a detectable amount of 3 by NMR. Using this method,
we obtained a 74% isolated yield of pure lomustine under the
conditions of 0 °C reaction and a residence time of 5 min using
3 equivalents of 4.
Table 3. Synthesis of Lomustine under at Different Reaction
Conditions Using 5 as the Nitrosation Reagent
solvent
(3.7:1)
temp
residence time
(min)
isolated lomustine
yield (%)
entry
1
(°C)
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
ACN/
EtOH
THF
(100%)
50
50
50
50
50
50
25
25
25
25
25
25
25
0.5
68.3
69.8
60.0
58.8
51.9
49.4
48.8
54.1
66.5
79.3
91.2
89.8
36.5
2
1
3
3
4
5
5
8
6
10
0.5
1
The reaction was also optimized with respect to residence
time, temperature, and solvent using 5 as the nitrosation agent
(Figure 4 and Table 3). This optimization process led to the
7
8
9
3
10
11
12
13
5
8
10
3
Figure 4. Microfluidic synthesis of lomustine using 5 as nitrosation
reagent in a Chemtrix 3223 SOR glass reactor chip. A = 3 in ACN/
EtOH (3.7:1); B = 5 in ACN.
Figure 5. Schematic for the telescoped synthesis of lomustine using
NaNO2/HCO2H (4) as nitrosation reagent.
Table 4. Lomustine Synthesis Yields for Telescoped
Reactions under Different Reagents, Residence Time,
Stoichiometry, and Temperature Conditions
finding that an elevated residence time (8 min), lower
temperature (25 °C), and increased 5 ratio (3 equiv) resulted
in the most efficient conversion to lomustine (91% isolated
yield) after Method 3 purification (Table 3, entry 11).
nitrosation
reagent
TEA
isolated lomustine
yield (%)
entry
1
step 1
step 2
(equiv)
Telescoped Synthesis of Lomustine. The next step was
to adapt the two-step sequence to a continuous flow setup. We
sought to telescope the carbamylation and nitrosation
reactions using the Chemtrix reactor chips. However, an
extraction was required between the steps to remove the TEA
present in the first reaction to avoid competitive consumption
of the nitrosation reagent in the second step. We achieved this
objective by incorporating a commercially available Zaiput
liquid−liquid extractor to remove the base before the
nitrosation step and by reoptimizing the synthesis using a
FEP tubing reactor (Figure 5). In the beginning, the best
reaction conditions included a 1 min residence time at 50 °C
for the first step and a 5 min residence time at 0 °C for the
second step, yielding 43 mg (51.8% overall yield) of pure
lomustine (Table 4, entry 1). Efforts to improve the lomustine
yield by changing the residence times of either the first or
second steps were unsuccessful in the FEP tubing reactor using
4 as the nitrosation reagent (Table 4, entries 1−4).
4
4
4
4
5
5
5
5
5
1 min,
50 °C
5 min,
0 °C
1
51.8
44.2
38.6
24.0
41.5
21.8
24.6
55.5
63.7
2
3
4
5
6
7
8
9
2 min,
5 min,
1
50 °C
0 °C
10 min,
50 °C
10 min,
50 °C
1 min,
50 °C
1 min,
50 °C
1 min,
50 °C
1 min,
50 °C
1 min,
50 °C
5 min,
0 °C
3 min,
0 °C
5 min,
50 °C
5 min,
25 °C
8 min,
25 °C
8 min,
25 °C
8 min,
25 °C
1
1
1
1
1
0.1
0.01
D
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Subsequent telescoping experiments indicated that 5 was a
better nitrosation reagent than 4 for the efficient synthesis of
lomustine in flow. This is also true when the synthesis of
lomustine was performed separately in glass reactor chips
(Table 2 and 3). The final optimized conditions included a 1
min reaction time at 50 °C for the first step and an 8 min
reaction time at 25 °C for the second step (Figure 6). As the
to avoid clogging due to the low solubility of 3. tert-Butyl
nitrite, 5, was found to be a milder and more efficient
nitrosation reagent in this process to enable the isolation of
pure lomustine via simple extraction, filtration, and washing.
This synthesis is a faster and greener process that affords a
significant reduction in reaction time, lower waste production,
and avoidance of any chromatographic steps. Using this
method, 110 mg/h of lomustine can be produced, equivalent
to one dose/2 h for an agent that is administered orally every 6
weeks. Scale up and in-line recrystallization of lomustine are in
progress.
EXPERIMENTAL SECTION
■
All chemicals and reagents were purchased from Sigma-Aldrich
(St Louis, Missouri) and used without any purification.
Intermediate 3 standard was purchased from 1Click Chem-
istry, Inc. (Kendall Park, NJ). Lomustine was purchased from
ApexBio (Houston, TX).
Figure 6. Schematic for the final telescoped synthesis of lomustine
using 5 as a nitrosation reagent.
Liquid Handling Robot. Assay plate setup and sample
preparation steps for DESI-MS were done using a Biomek i7
(Beckman Coulter, Inc., Indianapolis, IN) dual-bridge liquid
handling robot. A 384-tip head was employed to enable
simultaneous transfer of 384 samples under the same
conditions (speed of aspiration and dispensing, height of
pipetting at source and destination positions, pattern of
pipetting, etc.). An 8-channel head was used to provide more
flexibility in terms of volumes transferred, layout of source and
destination places, pipetting height, speed, and reaction
stoichiometry. The high capacity deck accommodated all
labware (robotic tips, plates, reservoirs) needed for assembling
one reaction step. All robotic tips were made of chemically
resistant polypropylene and disposable. Polypropylene multi-
well plates and reservoirs, as well as custom-made Teflon
reservoirs, were used during the experiments for reagent
solutions. Methods were developed and validated using the
Biomek point-and-click programming tool. Standard pipetting
techniques used in this software were modified to optimize
accurate transfer of highly volatile liquids.
Mass Spectrometry. Samples were analyzed using a
Thermo Fisher TSQ Quantum Access MAX mass spectrom-
eter that was connected with a Dionex Ultimate 3000 Series
Pump and WPS-3000 Autosampler (Thermo Fisher Scientific,
Waltham, MA). Electrospray ionization (ESI) analysis in full
scan mode was used to monitor each reaction in both positive
and negative ion modes. These data were recorded using
optimized parameters for the ESI source and MS as follows:
spraying solvent, ACN; spray voltage +3.5 kV (positive mode)
and −4.0 kV (negative mode); capillary temperature, 250 °C;
small amount of extracted TEA from the first step negatively
impacted TBN activity, lowering the amount of TEA used led
to an increased production of lomustine (Table 4, entries 7−9)
such that 1% TEA produced 110 mg (63%) of pure isolated
lomustine via this reaction setup (Table 4, entry 9). TLC,
NMR (1H and 13C), MS, and MS/MS data of lomustine
obtained by this telescoped continuous route were a direct
match with values measured for a commercially available
lomustine sample.
HPLC-MS was used to certify the purity of the synthesized
lomustine and compare the total ion chromatogram (TIC)
values of this material with a commercial lomustine standard.
The TIC profile fully overlapped with the commercial standard
without the appearance of any new byproducts (Figure 7).
CONCLUSION
■
In summary, we have developed a rapid continuous synthesis
of lomustine using DESI-MS to guide the selection of reaction
conditions in the second step of the two-step overall
transformation. A total residence time of 9 min was employed
to produce pure lomustine in 63% overall isolated yield
compared to over 2 h to generate a lower product yield using
batch conditions.2,3,55,56 The two synthetic steps were
optimized separately in glass chips and then translated using
FEP tubing for telescoped scaling of the whole process. Only
one in-line workup step was required for the two-step reaction
sequence. Mixed solvents were used in the telescoped reaction
Figure 7. Total ion chromatogram (TIC) from HPLC-MS/MS and comparison between flow synthesized lomustine (top, red) and commercially
available lomustine (bottom, black).
E
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sheath gas pressure, 10 psi; scan time, 1 s; Q1 peak width
(fwhm), 0.70 Th; micro scans, 3. The autosampler settings
were as follows: MS acquire time, 2 min; sample injection
volume, 10 μL. Thermo Fisher Xcalibur software was used to
process the data from the MS spectrometer.
temp, 325 °C; drying gas flow rate, 8.0 L/min; fragmentor
voltage, 130 V; skimmer, 45 V; and OCT RF V, 750 V.
Carbamylation of Cyclohexylamine in Flow. CAU-
TION: The 1-chloro-2-isocyanatoethane (1) starting material and
cyclohexylurea product (3) are potent alkylating agents.
Appropriate chemical inhalation masks and impermeable gloves
must be worn while handling all reaction operations and product
analyses in a ventilated hood. A solution of cyclohexylamine (1,
500 mmol, 1 equiv) in THF was loaded into a 1 mL Hamilton
gastight glass syringe. Triethylamine (TEA) (500 mmol, 1
equiv) and 1-chloro-2-isocyanatoethane (2, 700 mmol, 1.4
equiv) solutions in THF were individually loaded into another
two 1 mL Hamilton gastight glass syringes. Each solution was
simultaneously dispensed into the SOR 3225 reactor to engage
the reactants. The syringe containing 2 was protected from
light by covering it with aluminum foil. The reactions were run
at 25, 50, and 65 °C, with each temperature condition run at
residence times of 10, 30, 60, and 180 s. The reactions were
monitored by TLC and ESI-MS. Product 3 was collected after
evaporation and washing with cold Et2O. The white solid
product was stored in the dark at 4 °C. The subsequent TLC,
ESI-MS, MS/MS, and NMR (1H and 13C) analyses were
performed after purification (see Supporting Information for
details).
DESI-MS Screening. CAUTION: The cyclohexylurea
starting material (3) and lomustine product are potent alkylating
agents. Appropriate chemical inhalation masks and impermeable
gloves must be worn while handling all reaction operations and
product analyses in a ventilated hood. Stock solutions of 3 and
nitrosation reagents were made at four different concentrations
(50, 100, 150, 200 mmol) in THF and ACN (see Supporting
Information, Figure S2). Each reagent solution was also
prepared (0.1 M) in eight different solvents (EtOAc, THF,
DMSO, toluene, ACN, DCM, EtOH, and MeOH; see
Supporting Information, Figure S3). Initially, 20 μL of 3
solution were dispensed into a 384-well master plate and then
the corresponding nitrosation reagents were added to the plate
in a 1:1 stoichiometry using a Beckman i7 liquid handling
robot, resulting in a final volume of 40 μL in each well.
Moreover, a master plate was made using only commercially
available 3 and lomustine to compare the data (see Supporting
Information, Figure S4). Rhodamine was dissolved in ACN
(0.25 mg/mL) and transferred to a reservoir. A pin tool fitted
with 50 nL pins was used to transfer solutions from the master
plates as well as from the rhodamine reservoir onto the DESI-
MS substrates. The master plate was pinned three times in
separate locations with reaction mixtures and rhodamine once,
resulting in 1536 density on the microtiter plate used as the
DESI-MS substrate (see Supporting Information for details).
Nitrosation of 3 in Flow. CAUTION: The cyclohexylurea
starting material (3) and lomustine product are potent alkylating
agents. Appropriate chemical inhalation masks and impermeable
gloves must be worn while handling all reaction operations and
product analyses in a ventilated hood. A solution of 3 (245
mmol, 1 equiv) in 98% formic acid was loaded into a 1 mL
Hamilton gastight glass syringe. A NaNO2 (735 mmol, 3
equiv) solution in MeOH/H2O (4:1) was separately loaded
into another 1 mL Hamilton gastight glass syringe and
dispensed into the SOR 3225 reactor to engage the reactants.
The reactions were run at 0 °C, using residence times of 30 s,
1, 3, 5, 8, and 10 min. For nitrosation with 5, solutions of 3 in
ACN/EtOH (3.7:1) (200 mmol, 1 equiv) and 5 in ACN (600
mmol, 3 equiv, protected from light by covering the syringe
1
NMR Analysis. H NMR and 13C NMR were acquired
using a Bruker AV-III-500-HD NMR spectrometer (Billerica,
MA, USA). Samples for NMR were prepared by dissolving ∼5
mg of sample in CDCl3. MestReNova 10.0 software was used
1
to analyze the H NMR and 13C NMR.
Optimization in Microfluidic Glass Chips. The first and
second steps of the lomustine synthesis were individually
optimized using a Labtrix S1 (Chemtrix, Ltd., Netherlands)
fitted with a glass microreactor containing staggered oriented
ridges (SOR). Microchips 3223 (three inlets and one outlet,
volume = 10 μL) or 3225 (four inlets and one outlet, volume =
10 μL) having channel widths of 300 μm and channel depth
120 μm were used. The S1 unit is able to independently drive
five syringes connected to the microreactor mounted on a
Peltier heating and cooling stage. All the gastight glass syringes
used were obtained from Hamilton Company (Hamilton,
Reno, NV). FEP tubing and fittings were used to connect the
syringes with the selected connection port on the microreactor.
All operations were controlled using a ChemTrix GUI software
installed on a laptop that was connected to the Labtrix S1 via
an USB cable.
Liquid−Liquid Separator. All the liquid−liquid separa-
tions were performed using a SEP-10 unit (Zaiput Flow
Technologies, Cambridge, MA). The separator uses a porous
hydrophobic PTFE membrane (OB-900) which allows flow of
the organic phase through the membrane. The organic phase
wets the membrane while the aqueous phase does not. A built-
in pressure controller is used to maintain the appropriate
pressure differential that is required of the flow for both sides
of the membrane.
DESI-MS Analysis. The DESI-MS evaluation was done
following the previously published method of Wleklinski et
al.,49 except that the density of reaction spots was 1536 spots/
plate instead of 6144 spots/plate using reagents that were
pipetted into standard polypropylene 384-well plates via the
liquid handling robot. DESI-MS slides were fabricated from
porous PTFE sheets (EMD, Millipore Fluoropore, Saint-
Gobain, France) glued onto a glass support (Foxx Life
Sciences, Salem, NH). The PTFE sheet was cut with scissors
and bonded to the glass slides using spray adhesive (Scotch
Spray mount) (see Supporting Information for details).
HPLC-MS Analysis. HPLC/MS analysis was performed
using an Agilent 6545 UPLC/quadrupole time-of-flight (Q-
TOF) mass spectrometer (Palo Alto, CA), fitted with an
Agilent XDB-C18 column (3.5 μm, 150 mm × 2.1 mm i.d.)
and a 5 μL injection volume. A binary mobile phase, consisting
of solvent systems A and B, was used. Solvent A was 0.1%
formic acid (v/v) in ddH2O, and Solvent B was 0.1% formic
acid (v/v) in ACN. Isocratic elution of A/B at 95:5 was used,
with a column flow rate of 0.3 mL/min. Following separation,
the column effluent was introduced by positive mode
electrospray ionization (ESI) into the mass spectrometer.
High mass accuracy spectra were collected between 70 and
1000 m/z. Mass accuracy was improved by continuously
infusing Agilent Reference Mass Correction Solution (G1969−
85001). The MS detection conditions were as follows: ESI
capillary voltage, 3.5 kV; nebulizer gas pressure, 30 psig; gas
F
DOI: 10.1021/acs.oprd.8b00387
Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
Article
Notes
with aluminum foil) were loaded into two separate 1 mL
Hamilton gastight glass syringes and dispensed into the SOR
3223 reactor. All the reactions were monitored at two different
temperatures (50 and 25 °C) at residence times of 30 s, 1, 3, 5,
8, and 10 min. Reaction progress was monitored by TLC and
ESI-MS. The reaction mixtures were extracted by Et2O,
evaporated, and dried over anhydrous Na2SO4. The crude oily
product was purified by dissolving it in hot petroleum ether,
hot filtering the solution, and evaporating the filtrate to dryness
under reduced pressure to give the yellowish solid lomustine
that was stored at −20 °C. TLC, ESI-MS, MS/MS, NMR (1H
and 13C), and yield analyses were performed after purification
(see Supporting Information for details).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thankfully acknowledge the financial support from the
Department of Defense: Defense Advanced Research Projects
Agency (Award No. W911NF-16-2-0020) and NIH Grant P30
CA023168 of the Purdue University Center for Cancer
Research (NMR, MS, and MPF Shared Resources). The
efforts of Samyukta Sah, Yuta Moriuchi, Deborah M. Aremu,
Ryan Hilger (Amy Facility at Purdue University, Department
of Chemistry, Teflon reservoir fabrication), Larisa Avramova,
and Andy Koswara are also noted with great appreciation.
Telescoped Synthesis of Lomustine in Flow. CAU-
TION: The starting material (2), intermediate (3), and lomustine
product are potent alkylating agents. Appropriate chemical
inhalation masks and impermeable gloves must be worn while
handling all reaction operations and product analyses in a
ventilated hood. A solution of a 1:1 mixture of 1 (0.5 M, 1
equiv) and TEA (0.05 M, 0.01 equiv) in DCM was loaded into
a 5 mL Hamilton gastight glass syringe. A solution of 2 in THF
(0.7 M, 1.2 equiv) was separately loaded into another 5 mL
Hamilton gastight glass syringe. Each solution was simulta-
neously dispensed through a T-connector into a 0.8 mm
diameter chemically resistant fluorinated ethylene propylene
(FEP) tube reactor at 50 °C for 1 min. The output from the
first reactor was connected to a four-way connector where H2O
and DCM were added from two additional syringes. This
mixture was introduced into a Zaiput liquid−liquid separator
(Cambridge, MA), and the organic phase was fed to another
four-way connector where a formic acid and sodium nitrite
(1.5 M, 3 equiv) solution in MeOH/H2O (4:1) was added via
another two 5 mL Hamilton gastight glass syringes. This
mixture was passed through a second FEP tube reactor at 0 °C
for 5 min. In the telescoped reactions using 5, a three-way
connector was connected to the organic phase emerging from
the Zaiput separator using doubled concentrations of all
starting materials (1, 1 M; 2, 1.4 M). An ACN solution of 5 (3
M, 3 equiv) was introduced from a 5 mL Hamilton gastight
syringe. Two separate syringe pumps and three/four syringe
pumps integrated into the Chemtrix system were used to
dispense all solutions and solvents. The reactions were
monitored by TLC and ESI-MS. The purification and analyses
were conducted as described above, except that HPLC-MS
analysis was performed to evaluate the purity of the product
(see Supporting Information for details).
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.oprd.8b00387.
■
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Details of the experimental outline, DESI-MS analysis,
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for the continuous flow synthesis, impurity analysis, and
spectral data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: davethom@purdue.edu.
ORCID
R. Graham Cooks: 0000-0002-9581-9603
David H. Thompson: 0000-0002-0746-1526
■
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