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JM141-75_06252013_11:00am
Time-0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00
AU
0.0
5.0e-2
1.0e-1
1.5e-1
2.0e-1
2.5e-1
3.0e-1
-0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00
AU
0.0
2.5e-2
5.0e-2
7.5e-2
1.0e-1
1.25e-1
1.5e-1
1.75e-1
2.0e-1
2.25e-1
2.5e-1
2.75e-1
-0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00
AU
0.0
1.0e-2
2.0e-2
3.0e-2
4.0e-2
5.0e-2
6.0e-2
7.0e-2
CRT_RXN8_Path2_T0-1-2 3: Diode Array 270
Range: 8.268e-2
0.53
0.01
1.77
CRT_RXN8_path2_UPLC_T0-2 Diode Array 270
Range: 3.052e-1
1.04
0.92
0.19 1.26
CRT_RXN8_path2_UPLC_T0-1 Diode Array 270
Range: 3.273e-1
1.05
1.17
SM1 = m/z 352 Da
SM2 = m/z 535 DaSM2
SM1
SM2
SM2
INVESTIGATING THE IMPLEMENTATION OF CONVERGENCE CHROMATOGRAPHY FOR MEDICINAL CHEMISTRY AND PROCESS DEVELOPMENT LABORATORIES
Michael D. Jones1, Sean M. McCarthy1, James McKearin2, Jon Kremsky2, Andy Aubin1, Paula Hong1, and Margaret Maziarz1
1. Waters Corporation, 34 Maple Street, Milford, MA 01757; 2. Prime Organics, 25 Olympia Ave, Woburn, MA
INTRODUCTION
Synthetic chemists work in a wide range of industries generating compounds for both final products as well as key intermediates. A thorough understanding of
the optimal synthetic route allows chemists to make knowledgeable decisions related to increasing purity and yield. In pharmaceuticals, medicinal chemistry departments also require high throughput and robust instrumentation. However, the workflow is highly dependent on the instrumentation, which ideally can allow a pharmaceutical organization to compartmentalize new entities into compound libraries. This can further increase the chances of bringing a target through to the lead optimization process.
In this presentation, we will describe the use of Ultra Performance Convergence Chromatography for monitoring several synthetic processes as performed in a medicinal or synthetic chemistry laboratory. The benefits of this new technology in expanding the current analytical capabilities of the synthetic chemist will be illustrated. Specific examples will illustrate the benefits of implementing CC in simplifying workflows, analyzing structurally similar compounds and determining requirements for an orthogonal approach, all in order to aid better decisions to drive compounds to market.
METHODS
Instruments
UPC2
Instrument: ACQUITY UPC2 with 6 column capacity
Detectors: PDA and SQD2
Mobile Phase A: CO2 (tank, medical grade)
Modifier B: 15 mM ammonium formate/1% formic acid
in MeOH
Column: See figure captions
Injection Vol.: 0.5 µL
Column Temp.: Achiral columns: 50 °C
Chiral columns: 35 °C
Flow Rate: 2.0 ml/min
Gradient: See Figure Captions
ABPR pressure: 1885 psi
Wavelength: 254 nm
CDS: MassLynx with OpenLynx and OA Login
Make up Flow: 0.1 % NH4OH in MeOH
SQD2
Capillary (kV) 2.00 kV Cone (V) 20.00 V
Extractor (V) 3.00 V Source Temp. 150 °C
Desolvation Temp. 500 °C Cone Gas Flow 50 L/Hr
Enhancing ‘Intra-Technique’ Selectivity Justifying an Orthogonal Approach to Reversed-Phase LC (RPLC)
Notable Key Instrument Capabilities
Stationary Phase Agnostic
A variety of columns such as RPLC, NPLC, HILIC, and chiral columns can be used without changing mobile phase,
diluents, or instrumentation
Independent Temperature Control Advantageous for achiral/chiral screening. Achiral
experiments may be run at higher temperatures than the
chiral experiments. The column manager allows for independent control of each column compartment
maximizing the life of chiral columns which typically have upper temperature limitations of 40-450C not in use during
storage in the column manager.
Direct Injection of Organic solvents
All of the synthetic processes monitored consisted of organic aliquots of the reaction mixtures. With this technique,
conversion to ‘RPLC friendly’ diluents was not required.
The fourth reaction step in the synthetic route for rosuvastatin (right) was
monitored by UPC2/MS. There is a selectivity difference observed for this mixture when screening columns as evidence by UV and MS detection (below).
With MS, we tracked the elution order of m/z=304 Da as seen with the blue arrow. We also identified two isobaric species (m/z = 362 Da) which were
resolved on the 2-EP and BEH columns (which were not detected by UV) as depicted by the blue rectangle. By utilizing UV/MS, we can see another
isobaric species m/z = 265 Da which splits into 2 peaks when using a UPC2 BEH 2-EP column. The separation of these isobaric species are not observed
on the other columns as depicted by the green parallelogram.
UPC2 BEH 2-EP
UPC2 BEH
CSH Fluoro-
Phenyl
HSS C18 SB
CONCLUSIONS
Mass spectrometry was clearly needed to identify non
-chromophore containing analytes which are typical at
early stages of synthetic reactions
The ‘ease of use’ column management allowed for the
expanded selectivity exploration with a stationary
phase agnostic capability. This can only be provided by the convergence chromatographic approach if
performed on a single system
Chiral Screening is a necessity throughout each step
of the synthesis that contains enantiomeric analytes
Having an Orthogonal Approach to RPLC can aid the
monitoring and quantification of yield and purity as
well as facilitate the purification decision process
JM141-55 Time 2:05PM 2/22/2013
Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
AU
0.0
2.5e-2
5.0e-2
7.5e-2
1.0e-1
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
AU
0.0
2.5e-2
5.0e-2
7.5e-2
1.0e-1
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
AU
2.5e-2
5.0e-2
7.5e-2
1.0e-1
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
AU
0.0
2.5e-2
5.0e-2
7.5e-2
CRT_RXN4_scale-up_T1-1 (2) PDA Ch1 [email protected] -Compens.Range: 1e-10.58
0.570.28
0.150.19
0.30
0.67
1.00
CRT_RXN4_scale-up_T1-2 (2) PDA Ch1 [email protected] -Compens.Range: 1e-10.59
0.47
0.210.34
0.83
0.68 0.73
CRT_RXN4_scale-up_T1-3 (2) PDA Ch1 [email protected] -Compens.Range: 1e-10.61
0.43
0.190.30
0.86
0.69
CRT_RXN4_scale-up_T1-4 (2) PDA Ch1 [email protected] -Compens.Range: 1e-10.53
0.44
0.22 0.36
0.76
0.66
0.630.73
UV
?
JM141-55 Time 2:05PM 2/22/2013
Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
%
0
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
%
0
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
%
0
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40
%
0
CRT_RXN4_scale-up_T1-1 1: Scan ES+ BPI
4.27e7
0.60367
0.50362
0.45440
0.29265
0.00118
0.31265
0.51362 0.58
304
0.68351
1.01214
0.86214
0.74295
1.17200
1.28200
1.19200
CRT_RXN4_scale-up_T1-2 1: Scan ES+ BPI
4.34e7
0.60367
0.56362
0.48265
0.00118
0.53305
0.84351
0.70304
0.68321 0.81
3840.78384
1.17356
0.92323
1.01305
CRT_RXN4_scale-up_T1-3 1: Scan ES+ BPI
4.09e7
0.633670.50
362
0.45265
0.00118
0.51362
0.55362 0.58
305
0.873510.71
304
0.68384
0.73295 0.79
406
0.973050.95;323 1.01
279
CRT_RXN4_scale-up_T1-4 1: Scan ES+ BPI
4.13e7
0.553670.50
362
0.45265
0.00118
0.51362
0.78351
0.67304
0.57367 0.65
321 0.71384
0.923560.85
3230.88305
MS
m-CPBA
DCM
O
O
O
O
S
F
N
NO
O
S
F
N
N
Importance of Screening Chiral Columns
BrCl
N+ NH
S
NaHCO3
MeOH, Reflux 12hrs
Cl
N
S
N
* *
The second reaction step for the synthesis of clopidogrel
utilizes a chiral starting material which is reacted with 4,5,6,7-tetrahydrothieno[3,2-c]pyridine to yield a chiral intermediate
product (below). The chiral screening results for the chiral starting material indicated a Chiralpak ID column provided the
best separation of the two enantiomers. It was hypothesized that the same chiral column would provide separation of the
chiral product as well. Interestingly, the Chiralpak ID did not provide a separation of the enantiomers; however, the worst
column choice from the initial screening results of the chiral starting material (Chiralpak IB) provided the best separation
for the chiral intermediate generated from the second step of the clopidogrel synthetic route. Since the UPC2 was coupled to
MS, the peaks were easily mass confirmed (spectra not shown).
with nitrile cmp_141-36_2:50min
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60
AU
0.0
2.0
4.0
6.0
8.0
1.0e+1
1.2e+1
1.4e+1
1.6e+1
1.8e+1
2.0e+1
2.2e+1
2.4e+1
2.6e+1
2.8e+1
3.0e+1
3.2e+1
3.4e+1
3.6e+1
3.8e+1
4.0e+1
4.2e+1
4.4e+1
4.6e+1
4.8e+1
5.0e+1
5.2e+1
5.4e+1
5.6e+1
5.8e+1
6.0e+1
6.2e+1
6.4e+1
6.6e+1
6.8e+1
7.0e+1
PLV_RXN3_Tr3_T3-6 3: Diode Array Range: 7.233e+11.03
211
0.58210
0.19210
0.84210
0.74210
0.64210
0.68210
1.68210
N
Br
Cl
*
*
N
Cl
N
S
NH
S
with nitrile cmp_141-36_2:50min
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60
AU
0.0
2.0
4.0
6.0
8.0
1.0e+1
1.2e+1
1.4e+1
1.6e+1
1.8e+1
2.0e+1
2.2e+1
2.4e+1
2.6e+1
2.8e+1
3.0e+1
3.2e+1
3.4e+1
3.6e+1
3.8e+1
4.0e+1
4.2e+1
4.4e+1
4.6e+1
4.8e+1
5.0e+1
5.2e+1
5.4e+1
5.6e+1
5.8e+1
6.0e+1
6.2e+1
6.4e+1
PLV_RXN3_Tr3_T3-4 3: Diode Array Range: 6.668e+11.04
215
0.38210
0.19210
0.99210
0.65210
0.61210
2.11210
N
Br
Cl
*
*
N
Cl
N
S
NH
S
Chiralpak ID Results Chiralpak IB Results
Importance of Mass Spectrometry
JM141-84_07172013_10:00am
Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50
%
0
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50
AU
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2
3.0e-2
3.5e-2
4.0e-2
4.5e-2
5.0e-2
5.5e-2
CRT_RXN7_path2_T1-1-2 3: Diode Array 284
Range: 6.362e-2
0.52210
0.112100.10
210
0.01368
0.47210
0.88210
CRT_RXN7_path2_T1-1-2 1: Scan ES+ TIC
3.78e7
1.86118
0.893540.53
352
0.00118
0.67295
0.95102
1.41368
1.88118
1.89118
SM
Product
SMProduct
OHCH3
O
N+
CH3
O
N
In some cases, the starting material, the degradants of a
starting material or other byproduct of the reaction may not contain a chromophore. These non-chromophore containing
constituents, when not detected, can lead to a lack of understanding of the synthetic process. This lack of
understanding can hinder the troubleshooting of poor yield and impure final products.
In the example below, the seventh reaction step in the rosuvastatin synthesis was monitored. The MS data clearly
detects reduction impurities of one of the starting materials. The presence of these impurities may inhibit yield of further
reactions downstream.
N
OH
F
N
N
O
OS
+
O-
N+
O
+ TPAPDCM
molecular sieves
N
O
F
N
N
O
OS
SM Product
Reaction Step #7
The eighth reaction step in the synthesis route for
rosuvastatin involved the use of a Wittig reagent to aid the production of the desired enantiomer of the final product for
rosuvastatin without use of a chiral resolving agent. The details of the reaction are described (right).
It is common for a workflow to screen high and low pH
mobile phases in an effort to promote selectivity for a given separation. The initial time point of this reaction was
monitored with UPLC and UPC2 (above, left). Our results show better separation of the starting materials when using
UPC2 when compared to the RPLC approach. Elution of SM2 is not easily resolved from SM1 for RPLC, regardless of pH.
Additionally, UPC2 provided better peak shape for SM2. Each of these improvements realized by UPC2 aided in monitoring
the reaction and quantitation of the yield and purity.
SM1 SM2
Low pH
UPLC
High pH UPLC
UPC2 JM141-75_06252013_5:00pm
Time-0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00
AU
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2
3.0e-2
3.5e-2
4.0e-2
4.5e-2
-0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00
AU
-5.0e-3
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2
3.0e-2
3.5e-2
4.0e-2
-0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00
AU
-5.0e-3
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2
3.0e-2
3.5e-2
4.0e-2
4.5e-2
5.0e-2
CRT_RXN8_Path2_T5-1-2 3: Diode Array 298
Range: 5.798e-2
0.69
0.53
0.01
1.76
CRT_RXN8_path2_UPLC_T5-2 Diode Array 298
Range: 5.239e-2
1.04
0.190.21
1.08
CRT_RXN8_path2_UPLC_T5-1 Diode Array 298
Range: 4.845e-2
1.05
0.950.20
1.07
1.25
SM1SM2
PDT
PDT + SM1Co-elutionSM2
Not observed to be present in RPLC!!
Low pH
UPLC
High pH UPLC
UPC2
As the reaction progressed, aliquots were taken every hour.
Shown (above, right) are the results for the UPC2 and high/low pH LC chromatograms of an aliquot taken after 6 hours.
The RPLC approach indicates SM2 has been fully consumed; however UPC2 clearly shows its presence. More importantly,
the separation of the product from SM1 was not optimal in either of the RPLC pH results and the product peak was quite
broad. UPC2 provided optimal resolution of all the constituents in the mixture. Because of this added
resolution, purification of this material would be easier .
Time Point 0: Before reaction occurs Time Point 5: 6 hours
OHO
O
OHN
F
N
N
O OS
Si
N
O
F
N
N
O
OS
+
ACN, reflux
Argon
OHO
O
OHN
F
N
N
O OS
Si
PPh3
OCh3
O O O
Si
Wittig reagent
CH3
OO S
CH3
O
CH3
CH3
N
F
N
N
PPh3
OCh3
O O O
Si
Wittig reagent