Online Inductive Electrospray Ionization Mass Spectrometry as a Process Analytical Technology Tool to Monitor the Synthetic Route to Anagliptin

Article abstract of DOI:10.1021/acs.oprd.6b00039

Inductive electrospray ionization (iESI) is an ambient ionization method that is particularly well-suited to online reaction monitoring. It allows the potential of electrospray mass spectrometry (MS) to be realized as a routine process analytical technology (PAT) tool to monitor practical synthetic reactions in real time. In this study, a synthetic route to Anagliptin (target API) was successfully monitored using online iESI-MS. Starting materials not seen by traditional reaction monitoring tools (HPLC-UV/Vis and GC-FID) were observed, as well as water-sensitive reagents and intermediates which cannot easily be followed by other methods. Online tandem mass spectrometry (MS/MS) was used to characterize chemical species in the reaction mixture. Impurities and byproducts were identified, and information on the progress of byproduct formation enabled implementation of strategies to eliminate these byproducts in the course of the reaction. This work demonstrates how iESI-MS can be employed to obtain comprehensive information and solutions to some practical problems that occur in small-molecule synthetic reaction monitoring.

Full text of DOI:10.1021/acs.oprd.6b00039

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Full Paper
On-line Inductive Electrospray Ionization Mass Spectrometry as a Process
Analytical Technology Tool to Monitor the Synthetic Route to Anagliptin
Xin Yan, Ryan M Bain, Yafeng Li, Ran Qiu, Tawnya G. Flick, and R. Graham Cooks
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00039 • Publication Date (Web): 12 Apr 2016
Downloaded from http://pubs.acs.org on April 15, 2016
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On-line Inductive Electrospray Ionization Mass
Spectrometry as a Process Analytical Technology
Tool to Monitor the Synthetic Route to Anagliptin
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Xin Yan†, Ryan M. Bain†, Yafeng Li†, Ran Qiu†, Tawnya G. Flick*‡, R. Graham Cooks*†
† Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907,
United States
‡ Department of Analytical Research & Development, Amgen Inc., 1 Amgen Center Dr,
Thousand Oaks, CA 91320, United States
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ABSTRACT: Inductive electrospray ionization (iESI) is an ambient ionization method that is
particularly well suited to online reaction monitoring. It allows the potential of electrospray mass
spectrometry (MS) to be realized as a routine process analytical technology (PAT) tool to
monitor practical synthetic reactions in real time. In this study, a synthetic route to Anagliptin
(target API) was successfully monitored using on-line iESI-MS. Starting materials not seen by
traditional reaction monitoring tools (HPLC-UV/Vis and GC-FID) were observed as well as
water sensitive reagents and intermediates which cannot easily be followed by other methods.
On-line tandem mass spectrometry (MS/MS) was used to characterize chemical species in the
reaction mixture. Impurities and byproduct were identified and information on the progress of
byproduct formation enabled implementation of strategies to eliminate these byproducts in the
course of the reaction. This work demonstrates how iESI-MS can be employed to obtain
comprehensive information and solutions to some practical problems that occur in small
molecule synthetic reaction monitoring.
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Key words: Ambient Ionization • Spray-based Ionization • Reaction Mechanism • Chemical
Synthesis • Bulk Reaction
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INTRODUCTION
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Process analytical technology (PAT) methodology, as endorsed by the U.S. Food and Drug
Administration (FDA)1,2, is a system of analytical techniques for real-time process
characterization in the pharmaceutical industry which is applied increasingly in pharmaceutical
development, scale-up, and manufacture.3-7 The PAT concept is embraced in the FDA’s Quality-
by-Design (QbD) framework8-10 which aims at process understanding and control so that product
quality is built into the manufacturing process.11 Such processes can produce consistent quality
products, making product quality control less dependent on analytical testing at the endpoint, i.e.
the final active pharmaceutical ingredient (API).12-14 This initiative has encouraged the
pharmaceutical industry to increase research and use of new analytical technologies to perform
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timely measurements on critical quality attributes of raw materials, intermediates and products.15-
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Successful implementation of PAT requires the appropriate selection of process analytical
methods, the particulars of which depend on the application and molecule. In the FDA’s PAT
definition1,11 (“a system for designing, analyzing, and controlling manufacturing through timely
measurements (i.e., during processing) of critical quality and performance attributes of raw and
in-process materials and processes, with the goal of ensuring final product quality”), “analyzing”
equates to the use of in situ analytical tools. A variety of analytical methods have been
incorporated into the PAT toolbox for on-line process monitoring, including focused beam
reflectance measurement (FBRM)18-20, particle video microscopy (PVM)18,19, infrared (IR)
spectroscopy11,21-28, UV/Vis spectroscopy20, Raman spectroscopy22,29,30, nuclear magnetic
resonance (NMR) spectroscopy21,22,31 and high-performance liquid chromatography (HPLC)31.
However, little information is provided on chemical structures by many of these measurements
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which only give signals for characteristic functional groups, and this remains one of the barriers
to the application of spectroscopy in PAT applications. On the other hand, just as there is no
single off-line analytical tool that meets all needs for process development, understanding or
control strategy for any particular product, there is no single in situ analytical tool that will work
for all applications. New analytical technologies need to be developed and added to the PAT
toolbox. Because of its inherent sensitivity, speed, and molecular selectivity32-37, mass
spectrometry (MS) has seen some use in process analysis, including in-process monitoring of
exhaust gases of fermentation processes38, real-time deuterium abundance measurements in
water vapor39, analysis of trace gases in food products and environmental monitoring.40,41
However, MS has not been used as a routine PAT tool to monitor reactions in real time.42-44
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Recent studies have shown that a particular version of electrospray mass spectrometry, namely
inductive electrospray ionization mass spectrometry (iESI-MS)45,46, can be used to continuously
monitor reacting systems in a preparation-free on-line process. This analysis tool applies to
organic chemical reactions and allows the study of reaction progress.47 This method has the
notable advantage of being applicable to concentrated solutions of the type encountered in
pharmaceutical manufacturing. An early version of such a system (Figure S1) was used to
automatically sample reaction mixtures in-situ and deliver them to the MS inlet, thereby enabling
the continuous reaction mixture monitoring and providing virtually real-time structural
information on the intermediates and products in the mixture. Three important reactions in
pharmaceutical synthesis of different types including reductive amination, Negishi cross-
coupling (an air and moisture-sensitive reaction) and Pd/C-catalyzed hydrogenolysis (a
heterogeneous reaction) were successfully monitored.47 This system enabled short-lived
intermediates to be observed, and tracked through the synthetic reaction. The development of
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iESI-MS gave opportunities for on-line MS as a PAT tool in reaction monitoring. In the course
of developing the system further to allow process analysis, the Anagliptin synthetic route (target
API 1, Scheme 1)48 was monitored using on-line iESI-MS. Solutions to practical problems which
occurred in this realistic application were provided mainly by using additional capabilities of the
mass spectrometer. Note that neither of the starting materials 2 or 3 can be detected by traditional
reaction monitoring tools such as HPLC-UV/Vis and GC-FID. Also water sensitive reagents and
intermediates are involved which cannot readily be followed by regular ESI-MS.
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Scheme 1. Synthetic route to API Anagliptin
O
CN
O
O
O
NaI, K2CO3,
acetone
CN
H
Cl
N
NH2
N
O
N
O
N
N
H
H
4
2
3
C
16H28N4O3
C7H9ClN2O
Mol. wt. 172.61
C9H20N2O2
Mol. wt. 188.27
Mol. wt. 324.43
O
CN
H
4N HCl / dioxane
N
H2N
N
5
C11H20N4O
O
O
O
CN
H
Mol. wt. 224.31
N
Et3N, THF
HN
N
N
H
N
N
N
O
O
N
N
N
7, CDI
N
N
N
Anagliptin (1)
C7H6N4O
N
OH
N
Mol. wt. 162.15
C19H25N7O2
N
N
N
Mol. wt. 383.46
N
9
THF, rt
N
8
6
C11H9N5O
Mol. wt. 227.23
HN
C8H7N3O2
Mol. wt. 177.16
N
9
C3H4N2
Mol. wt. 68.08
RESULTS AND DISCUSSION
Monitoring of Amidation of (S)-1-(2-Chloroacetyl)pyrrolidine-2-carbonitrile 2, with (2-
Amino-2-methyl-propyl)-carbamic acid tert-butyl ester 3. The first step in the synthesis of
Anagliptin, amidation of (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile 2, with (2-amino-2-
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methyl-propyl)-carbamic acid tert-butyl ester 3 (Scheme 1), leading to the intermediate product 4
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was monitored by on-line iESI-MS.
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The reagent 3 was first mixed with sodium iodide and potassium carbonate in acetone. The
reagent 2 was then added dropwise to an ice-cooled acetone suspension and the reaction mixture
was stirred at room temperature for 10 h. Time-resolved mass spectra (Figure 1) were recorded
to show the progress of the reaction with designation of the detected ions, each time point
requiring 1.8 seconds (average 9 scans). Figure 1a was collected before the addition of reagent 2.
Sodium adducts of the reagent 3 and its dimer were detected at m/z 211 and 399. Ions at m/z 311
and 499 are assigned as the sodium adduct of dicarbamate 10 (Scheme 2) and the cluster ion of
[M3+ M10+Na]+. Dicarbamate 10 arises from impurities in reagent 3 as shown by MS/MS
(Figure S2). After the addition of reagent 2, the abundance of the ions at m/z 211 increased
(Figure 1b), due to a contribution by the isobaric potassium adduct of reagent 2; that is, the ions
of sodiated 3 overlap in nominal mass with potassiated 2 at m/z 211. This mass overlap was
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13
confirmed by the change of their C isotopic distributions (Figure S3). The proportion of each
species was calculated from the experimentally observed isotopic pattern. With time, the signal
due to product ion 4, observed at m/z 363 [M4+K]+ increased and it became dominant after 3 h
(Figure 1d). The formation of dimers of product 4 at m/z 671 [2M4+Na]+ and m/z 687 [2M4+K]+
was also observed. The spectra then showed no significant changes out to 8 h (Figure 1f).
Halogen exchange proceeded readily as was indicated by the formation of sodiated and
potassiated ions of activated iodide intermediate 11 at m/z 287 and m/z 303 (Figure 1b,c,S6). The
progression of the reaction is shown in Figure 2, which displays the changing abundance
(normalized) of the ions corresponding to the reagent 3, intermediate 11 and the product 4. The
product 4 was isolated and purified by column chromatography. The mass spectrum of 4 after
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purification is shown in Figure S4a and its identity was evident from its MS/MS product ion
spectrum (Figure S4b,c,d; tandem MS of 4 and its dimer in solution were shown in Figure S12).
This reaction was also monitored by traditional off-line nanoelectrospray ionization (nESI) MS.
The time resolved mass spectra (Figure S5) show the formation of product 4 and impurity 12
(discuss later), but the ions of reagent 2 and 3 were suppressed by the strong signal from the
protonated acetone dimer at m/z 117 and thus could not be followed. The fact that more species
were ionized well in online-iESI makes this monitoring system an effective tool to follow the
reactions.
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Figure 1. Time-resolved mass spectra of amidation of (R)-1-(2-chloroacetyl)pyrrolidine-2-
carbonitrile 2 with (2-amino-2-methyl-propyl)-carbamic acid tert-butyl ester 3 by iESI-MS.
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Scheme 2. Structures and molecular masses of impurities 10, active intermediate 11 and
byproduct 12, 13 in the synthetic route to Anagliptin 1
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NC
N
O
O
O
O
O
CN
H
H
O
O
CN
CN
N
10
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N
O
I
N
N
O
N
N
N
N
O
N
H
H
N
O
H
N
10
14H28N2O4
Mol. wt.288.39
11
C7H9IN2O
Mol. wt. 264.07
13
C15H22N6O2
Mol. Wt.318.38
12
C23H36N6O4
Mol. wt. 460.58
C
Figure 2. Reaction progress of the amidation of (R)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile
2 with (2-amino-2-methyl-propyl)-carbamic acid tert-butyl ester 3. Average total ion intensity is
around 8E5.
Ions at m/z 499 were observed before the addition of reagent 2 and the ions are assigned from
the MS/MS data (Figure 3a,b) as corresponding to the sodium binding adduct of 10 and 3. The
dicarbamate 10 also reacted with excess reagent 2 to form the product 4, resulting in the
decreasing abundance of the ions at m/z 499 with full disappearance occurring within 2 h.
However, the signal intensity at m/z 499 started to increase again at 5 h and reached 7% of the
base peak at 8 h. This latter species was identified as the product 12 of a second amidation of 4
with reagent 2 and its identity was confirmed by the different tandem mass spectrum from that of
the earlier species (Figure 3c,d). The MS spectrum of the latter species 12 after purification was
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in agreement with this assignment (Figure S7). These results demonstrate the advantage of using
MS as a PAT tool to monitor low-level impurities that are formed and tracked through the
synthetic reaction mixture.
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Figure 3. Tandem MS spectra of ions at (a) m/z 499 and (b) ions formed before the addition of
reagent 2; (c)and (d) show MS3 spectra of ions formed after 5 h.
Another phenomenon worthy of note is the presence of both sodium adducts and potassium
adducts of the same species. Both sodiated or potassiated ions are readily formed in the presence
of sodium iodide and potassium carbonate in the reaction mixture. They include m/z 211
[M3+Na]+, 227 [M3+K]+; 287 [M11+Na]+, 303 [M11+K]+; 311 [M12+Na]+, 327 [M12+K]+ and 347
[M4+Na]+, 363 [M4+K]+ (Figure 1, S11), and their formation is competitive. The concentration
of K+ is twice than that of Na+, however, all the ions formed are sodium adducts (Figure 1a,b) at
the beginning of the reaction. This shows that potassium competes less well than sodium in
adduct formation with this suite of compounds. As the reaction went on, the disappearance of
sodiated ions and formation of potassiated ions for all these three species in the reaction mixture
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was observed. This is consistent with the preferential removal of sodium ions as an insoluble (in
acetone) precipitate of sodium chloride.49
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Monitoring the Deprotection of Compound 4. In step 2 (Scheme 1) of the synthetic route to
Anagliptin, the reaction of 4 with 4 N hydrochloric acid in 1,4-dioxane and dichloromethane,
deprotects the Boc group. The reaction mixture was analyzed after dilution with the reaction
solvent, 1,4-dioxane and dichloromethane (1:1, v:v). The time-resolved mass spectra (Figure 4)
show the progress of the reaction. Figure 4a was recorded 1 min after the addition of 4 N
hydrochloric acid in 1,4-dioxane. Protonated 4 and its dimer were detected at m/z 325 and 649,
respectively. Meanwhile, a new ionic species at m/z 223 had started to be observed. In
subsequent reaction monitoring MS spectra (representative spectra shown at 10 min, 20 min and
50 min, Figure 4b-d), showed ions of protonated 4 and its dimers decreasing in abundance, while
ions at m/z 223 continually increased and eventually dominated the spectra after 50 min. This
latter signal represents the major product of this reaction.
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Figure 4. Time-resolved mass spectra of reaction of 4 with 4 N hydrochloric acid in 1,4-dioxane
and dichloromethane by iESI-MS.
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The observed cations at m/z 223 are designated as [M5 - H]+, loss of hydride from the neutral
molecules with its MS/MS shown in Figure S8b. The key step in ESI is normally the generation
of the protonated form of the molecule: M + AH+ ꢀ [M+H]+ + A. However, hydride abstraction
is a well-known route to positive ion formation in chemical ionization (CI) MS. Factors such as
the composition of the reaction mixture are known44 to affect the outcome of the competition
between a proton donotion and hydride loss. In order to confirm the species at m/z 223, methanol
was used as dilution solvent before analysis of the reaction mixture. Ions at m/z 223 moved to
m/z 225 while other major ions in the solution remained unaffected (Figure S8a, c). The ions at
m/z 225 were assigned to [M5+H]+ based on their fragmentation (Figure S8d).
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Monitoring of Formation of Anagliptin 1. The third step in the synthetic route to Anagliptin
includes two reactions which occur sequentially without separation of intermediates: activation
of 2-methylpyrazolo[1,5-a]pyrimidine-6-carboxylic acid 6 with N, N’-carbonyldiimidazole (CDI)
7 to yield the corresponding acylimidazole 8, followed by amination with (S)-1-((1-amino-2-
methylpropan-2-yl)glycyl)pyrrolidine-2-carbonitrile 5, leading to the final product, Anagliptin 1
(Scheme 1).
CDI 7 was first added to a solution of 6 in tetrahydrofuran (THF), and the mixture was stirred
at room temperature. Figures S9a,b show spectra recorded before the addition of CDI 7. Reagent
6 is poorly protonated in the positive ion mode (Figure S9a), while it is easily deprotonated in the
negative ion mode due to the carboxyl group (Figure S9b). Considering that other major ionic
species involved in this reaction step will likely be observed as the protonated molecules, the
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positive ion mode was used as the ionization mode of choice when monitoring the reaction, but
the negative ion mode offered complementary information. After adding CDI 7 to the reaction
solution, protonated imidazole and its dimer were rapidly destroyed due to the sensitivity of CDI
7 to water. Protected by the use of a N2 sheath gas in online-iESI, the protonated CDI 7 at m/z
163 could still be seen with low intensity in the spectra (Figure 5a). The activated carboxylic
acid product 8 is also water sensitive, while protonated 8 was detected at m/z 288 and confirmed
by MS/MS (Figure 5d). The comparison experiment was done using traditional nESI-MS with
CDI 7 in anhydrous THF (Figure 5b) and then reaction with 6 (Figure 5c). Neither protonated
CDI 7 nor product 8 could be detected in the traditional nESI-MS. The current iESI reaction
monitoring system is well suited to follow moisture sensitive reactions which are commonly
applied in the pharmaceutical industry.
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Figure 5. MS spectra of activation of 2-methylpyrazolo[1,5-a]pyrimidine-6-carboxylic acid 6
with N, N’-carbonyldiimidazole (CDI) 7 in THF analyzed by (a) online iESI-MS (b,c) traditional
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nESI. Note, (b) is the mass spectrum of pure CDI 7 in THF. (c,d) tandem MS of acylimidazole
(8).
6
7
8
9
The reaction mixture was added dropwise to 5 in an ice-cooled solution of triethylamine in
THF in order to form the product 1. The mixture was warmed to room temperature and stirred.
The spectrum shown in Figure 6a was recorded before adding the reaction mixture. Protonated 5,
its dimer, water cluster and triethylamine cluster were detected separately at m/z 225, 449, 243
and 326, respectively. New ionic species at m/z 543 and 319 were seen right after the reaction
mixture containing the acylimidazole was added dropwise to the solution (Figure 6b,c). Ions at
m/z 319 are assigned to protonated 13 (Scheme 2) based on MS/MS data (Figure S10c,d). The
major byproduct 13, originates from the side reaction of amine 5 with CDI 7, and the ions at m/z
543 are ionic clusters of 5 with protonated 13 (Figure S10a). The purity of commercial CDI 7
may be variable due to its water sensitivity, therefore it is common to employ excess in the
activation step to ensure complete conversion of the carboxylic acid to the acylimidazole. The
unreacted CDI 7 also reacted with the amine 5. From the reaction monitoring data, the amine 5
was shown to react faster with residual CDI 7 than with the acylimidazole 8 (Figure 6b, c). Once
the byproduct formed, its signal remained relatively constant. This suggests the byproduct
cannot be avoided by stopping the reaction early, instead reducing the amount of CDI 7 in the
previous step can decrease the byproduct formation. After the reaction was allowed to occur for
30 mins, the product ions, protonated Anagliptin 1 at m/z 384, started to form and reached its
maximum at the end of the reaction period (Figure 6d,e,f). The structure of the product 1 was
confirmed by MS/MS (Figure S10b). The other product, the imidazole 9, was observed at m/z 69.
The reported yield45 of this step is only 33% without corrections for impurities formed. The
information on impurities generated in this reaction was provided from the above experiment.
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Figure 6. Time-resolved mass spectra of amidation of (R)-1-(2-chloroacetyl)pyrrolidine-2-
carbonitrile 6 with (2-amino-2-methyl-propyl)-carbamic acid tert-butyl ester 5 by iESI-MS.
CONCLUSION
The iESI-MS was investigated as a useful PAT tool on monitoring the synthetic route of
Anagliptin in real time. The chemical species involved in the reactions were identified using
MS/MS data. Active intermediates, impurities and byproducts were identified in the reaction
mixture. Two pairs of ions with the same m/z were differentiated based on isotopic distribution
and tandem MS and their structures were assigned. Moreover, the current iESI-MS reaction
monitoring system shows advantages in better ionization of species in the reaction mixture
compared with the analysis using traditional nESI-MS. It also shows the capability to monitor
water sensitive reagents and intermediates. The formation of byproducts due to the excess water-
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sensitive reagent were followed, and its reaction rate was found to be much faster than that of the
target reaction. The work provides a comprehensive demonstration of process monitoring by
mass spectrometry including solutions to some practical problems that occur in small molecule
synthetic reaction monitoring. The probe used for this analysis could also be used to monitor
reactions at different scales.
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EXPERIMENTAL SECTION
Instrumentation. A linear ion trap mass spectrometer (LTQ, Thermo Fisher Scientific, San
Jose, CA, USA) was used to record positive ion mode full scan mass and MSn spectra. Typical
MS parameters included averaging of 3 microscans, 100 ms maximum injection time, 15 V
capillary voltage, 150°C capillary temperature, and 65 V tube lens voltage. Data were acquired
and processed using Xcalibur 2.0 software (Thermo Fisher Scientific). The identification of
analyte ions was confirmed by tandem mass spectrometry (MS/MS) using collision-induced
dissociation (CID). An isolation window of 1.5 Th (mass/charge units) and normalized collision
energy of 30 % – 40 % (manufacturer’s unit) were selected for the CID experiments.
Chemicals and Reagents. All reagents and solvents were used directly without any further
purification. (2-Amino-2-methyl-propyl)-carbamic acid tert-butyl ester 3 was purchased from
J&W Pharmlab, LLC; (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile 2 was purchased from
Alchem Pharmtech. 2-Methylpyrazolo[1,5-a]pyrimidine-6-carboxylic acid 6 was purchased from
Advanced ChemBlocks Inc; N, N’-carbonyldiimidazole 7, sodium iodide, potassium carbonate,
acetone were purchased from Sigma-Aldrich (St. Louis, MO, USA). OmniSolv HPLC grade
methanol was purchased from EMD (Bedford, MA, USA). Water was purified and deionized
using a Milli-Q system (Millipore, Bedford, MA, USA).
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Synthesis
of
(S)-t-butyl
(2-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)-2-
5
6
7
8
methylpropyl)carbamate (4). A solution of (S)-1-(2-chloroacetyl) pyrrolidine-2-carbonitrile 2
(3 mmol, 673.4 mg) in acetone (6 ml) was added to an ice-cooled stirred suspension of (2-amino-
2-methyl-propyl)-carbamic acid tert-butyl ester (3 mmol, 565 mg) 3, NaI (3.9 mmol, 584.8 mg),
and K2CO3 (3.9 mmol, 539.2 mg) in acetone (14 ml). The reaction mixture was stirred at room
temperature for 10 h. The resulting mixture was filtered to remove insoluble materials, and
concentrated under reduced pressure. The residue was purified by column chromatography on
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silica gel
methylpropyl)carbamate (4).
Synthesis of (2-amino-2-methyl-propyl)-carbamic acid tert-butyl ester (5). (2-((2-(2-
Cyanopyrrolidin-1-yl)-2-oxoethyl)amino)-2-methylpropyl)carbamate (210.3 mg) in
to
give (S)-t-butyl
(2-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)-2-
4
dichloromethane (40 ml) was added to 4 N hydrochloric acid/1,4-dioxane (40 ml) and the
mixture was stirred at room temperature until reaction was complete in 1 hour. The product was
concentrated under reduced pressure to give 5.
Synthesis of (S)-N-(2-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)-2-methylpropyl)-2-
methylpyrazolo[1,5-a]pyrimidine-6-carboxamide (1). Carbonyl diimidazole 7 (17.8 mg, 0.11
mmol) was added to a solution of 6 (17.7 mg, 0.1 mmol) in tetrahydrofuran (5.7 mL), and the
mixture was stirred at room temperature for 4 h. The reaction mixture was slowly added to 5
(29.7 mg, 0.1 mmol) in an ice-cooled solution of triethylamine (69 uL) in tetrahydrofuran (5.7
mL). The mixture was warmed to room temperature and stirred until reaction completion. The
reaction mixture was concentrated under reduced pressure, and then dichloromethane was added
to the residue. Insoluble materials were removed by filtration and the filtrate was concentrated
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under reduced pressure. The residue was subjected to column chromatography (eluting solvent;
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dichloromethane/methanol, 50:1) to yield pure 1 (30%).
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ASSOCIATED CONTENT
Supporting information
Additional information as noted in text.
AUTHOR INFORMATION
Corresponding Author
*Flick G. Tawnya
E-mail: tflick@amgen.com
*R. Graham Cooks
E-mail: cooks@purdue.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Authors acknowledge funding support from Amgen Inc. and the National Science Foundation
(CHE-1307264) as well as contributions of Dr. Laura Blue and Dr. Yuan-Qing Fang.
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