jm141-84 07172013 10:00am - waters corporation · 2014-01-31 · crt_rxn4_scale-up_t1-1 (2) pda ch1...

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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


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


0.47

0.210.34

0.83

0.68 0.73


0.43

0.190.30

0.86

0.69


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


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


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


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


Range: 5.239e-2

1.04

0.190.21

1.08


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

jm141-84 07172013 10:00am - waters corporation · 2014-01-31 · crt_rxn4_scale-up_t1-1 (2) pda ch1...

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