© pfizer limited 2008 1 cap and case: a step forward towards automatic structure elucidation anna...

© Pfizer Limited 2008 1

Cap and CASE: a step forward towards automatic structure elucidation

Anna Codina, Ruth Boetzel, Ingrid Hamernig and Don Richards

Structure Elucidation, ARD, PGRD


Good Results Micro + Training


1. Introduction

2. Applications/Examples:

• Identification of Impurities

• Challenging molecules: Arylpyrazoles

• Regulatory documents generation/checking

3. Conclusions


Structure Elucidation

LC - NMR (1H)

Structure

~> 0.5%

Isolation

2D NMR + MS

Report / Database

LC-MS

• Low level impurities identification

• Proof of Structure / Structure confirmation for regulatory documentation


NMR: How can we work faster?

acquisition

H1_2D.esp

processing

analysis

results/report/LNB

database

7.25 7.00 6.75 6.50F2 Chemical Shift (ppm)

120

125

130

135

140

145

150

155

160

F1

Che

mic

al S

hift

(pp

m)

(24, 22)

(21, 19)

(23, 21)

(21, 23)

(23, 19)

(24, 20)

(24, 19)

22

23

21

24

20

19

26

OH27

OH25

28

OH29

30NH31 32

CH333

CH334

CH335

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5Chemical Shift (ppm)

1.0

1.0

2.0

1.0

0.9

0.9

0.9

26

21 24

2328

30<''>

30<'>

22

23

21

24

20

19

26

OH27

OH25

28

OH29

30NH31 32

CH333

CH334

CH335


Acquisition - Sensitivity of NMR spectroscopy

• MS low nanogram

• UV low nanogram

• NMR microgram


NMR signal to noise

Coil Sensitivity

Magnetic Field

Sample Massin CoilS/N

Scoil B02 m sample

Vnoise

Noise RcoilNoise RcoilRcoil


Improving sensitivity

High magnetic fields

Performance enhancement ofRF probe and electronics

• < V detection coil (microcoils)

• Cold probes and preamplifiers

• Coil built from low-loss materials (superconductors)

Energy levels & population distribution

Hyperpolarisation(DNP)


Microcoils

5 mm Probe CapNMRTM Microcoil


Microcoils and microflow NMR

Flow cell


Automation - OMNMR


Analysis: Computer Assisted Structure Elucidator

NMR: pp 2D MS: MF

Generate MCD

Generate StructuresFrom MCD/s

Check MCD

Find the Best Structure !

Check data/add information

Accurate Mass Fragments

Formulae Fragment Formulae

Accurate Mass Fragments

Formulae Fragment Formulae

www.bdal.de/uploads/media/LCGC_March_2008_Appl._Books.pdf


1. Introduction





3. Conclusions


Structure elucidation of low level impurities

D:\Xcalibur\data\R0129\R0129_132 03-Nov-05 12:42:43 E010002700

RT: 0.00 - 65.00

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

300000

320000

340000

360000

380000

400000

420000

440000

460000

480000

500000

uAU

16.03

9.46

48.2813.09 63.091.07

55.8726.63 56.4237.2918.18 53.81 61.5950.6938.23 44.8139.6625.091.57 30.1719.51 35.0633.823.35 23.025.02

NL:2.11E6

Channel A UV R0129_132

?

LC - NMR (1H)

Structure

~> 0.5%

Isolation

2D NMR + MS

Report / Data base

LC-MS


CapNMR data for 1 of the impurities

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10Chemical Shift (ppm)

7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6F2 Chemical Shift (ppm)

115

120

125

130

135

140

145

F1

Che

mic

al S

hift

(ppm

)

9 mg/60ul injection 20750ug in the active volume13C 27 hHMBC 4 h


4 full data sets to analyse!


Computer Assisted Structure Elucidation (CASE)

CH315.35(fb)

CH264.76(ob)

CH240.50

CH72.50

CH83.04(ob)

CH115.06

CH117.52

CH121.32

CH122.30

CH127.83

CH127.83

CH128.35

CH128.62 CH

128.62

C138.98

C148.12

C149.68

R

N

OO

O

HH

Molecular ConnectivitiesDiagram (MCD)

GENERATION of STRUCTURESfrom MCD


Database: G enerated M oleculesd_{N}(^{13}C) (ListA) (285 pts)d_{N}(^{1}H) (ListA) (285 pts)

25020015010050# in ListA

1

2

3

4

5

6

285/97953 – 6h 52 m

CH315.35

CH240.50

64.76

72.50CH83.04

115.06

CH117.52

CH121.32

CH122.30

CH127.83

CH127.83

CH128.35

CH128.62

CH128.62

R

C138.98

C148.12

C149.68

NH

O

OOH

THE BEST STRUCTURE

d_{N}(^{13}C) 0.662d_{N}(^{1}H) 0.091d_{A}(^{13}C+^{1}H) 1.570


First 2 structures

0

50

100

150

200

0 50 100 150 200

Experimental C13 CS (ppm)

Ca

lcu

late

d C

13

CS

(p

pm

)

First Stucture

2nd Structure

R R R


1. Introduction





3. Conclusions


Arylpyrazoles

NH2 N

N

F

F

F

F

F

FF

F

Cl Cl

R


CASE of Example 1 - 12 mg

C15H8O1N4F8Cl2 1H, 13C, 1H-13C HSQC, 1H-13C/N- HMBC


dA (1H ): 0.029 (v.9.02)

d (13C ): 1.082 (v.9.02)

1 (ID =611)

The B est S tructure19.62

126.53

126.53

137.15135.24

135.24

132.68

27.4186.72

110.77

122.78

124.43

146.97

150.78

162.64

NH252.49

NH2102.44

N181.74

N

OF

F

FF

F

F

F

F

Cl

Cl


No Filter: 3325/3409 – 2 min Filter: C CS and internal bad list3/3409 – 2 min


CASE of Example 2 – 0.5 mg

C15H7O2N3F8Cl2 1H, 13C, 1H-13C HSQC, 1H-13C/15N- HMBC, 1H19F-HSQC


From MCD 26: 1476 – 11 sACD + Arylpyrazole fragments


Base degradant Acid degradant Oxidation

Structure

Molecular Formula C15H8O1N4F8Cl2 C15H7O2N3F8Cl2 C15H6O3N4F8Cl2 Total amount 12 mg 0.530 mg < 0.5 mg Experimental data H-C HSQC H-C/N HMBC H-C HSQC/BC H-C HSQC/BC

User data H-N HSQC H-N HSQC/BC H-F HSQC

H-F HSQC

SE protocol

N properties ACD fragments Filter: good list and 5

member ring

C ring info added 26 MCDs ACD and Arylpy-

razole fragments No lists

2 drawn connectivities

Good list: 3 fragments

NN

NH2

ClCl

CF3

FF

CF3

O

NH2

NN

NH2

ClCl

CF3

FF

CF3

O

OH

NN

ClCl

CF3

FF

CF3

O

NH2

NO2

CASE of Arylpyrazoles – challenging molecules

Less informationMore Human intervention


1. Introduction





3. Conclusions


Cap and CASE in regulatory

• Regulatory documentation is required for drugs to go through clinical trials

• Structure confirmation or full prove of structure

• Not sample limited

• Cap is ideal: ‘all in one’ (13C) and automation

• CASE:

– Used as a checking tool for H and C assignments: data are peak picked and contain 13C direct

– Can be used as a automatic structure verification and assignment tool and maybe for proof of structure documents


MW NM Fragments Comments 114 C8H4O1N2 symmetry 162 C7H11O2Cl1 symmetry 173 C9H19NO2 ½ h 199 C13H13O1N1 CS degeneracy 200 C11H20O3 ½ h 201 C11H23NO2 ½ h 241 C13H23NO3 ½ h 248 C12H12O4N2 Pfizer db 266 C14H9O2F3 Pfizer db 356 C20H15F3N2O 15N HSQC 364 C18H21ClN2O4 444 C28H32N2O3 Pfizer db 466 C26H21O3N2F3 Pfizer db

CASE and regulatory data


Conclusions

• CASE can be used to elucidate the structure of unknowns, such as

impurities. •Training systems are a powerful tool, especially for molecules with low

number of connectivities.• Good data required and limited overlapping.• Not a easy tool to use. • Generation of structures from an unbiased position, avoiding wrong

assumptions.• The combination of capillary microcoil NMR and CASE significantly

decreases the level of human intervention in SE.


THE FUTURE?


N

NH

O

S

O

NO

O

N

N

N


Acknowledgements

•SE team: Ingrid Hamernig

•Protasis: Dean Olson, Bob Albrecht, Tim Peck and David Strand

•ACD labs: Ryan Sasaki

© pfizer limited 2008 1 cap and case: a step forward towards automatic structure elucidation anna...

Documents

pfizer limited

h slide

arylpyrazoles slide

mcd slide

limited cap

limited overlapping

training slide

pgrd slide