1. © 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

2. © Pfizer Limited 2008 2

3. 3  Good ResultsMicro + Training

4. © Pfizer Limited 2008 4 1.Introduction 2.Applications/Examples: Identification of Impurities Challenging molecules: Arylpyrazoles Regulatory documents generation/checking 3.Conclusions

5. © Pfizer Limited 2008 5 Structure Elucidation LC - NMR ( 1 H) Structure ~> 0.5% Isolation 2D NMR + MS Report / Database LC-MS Low level impurities identification Proof of Structure / Structure confirmation for regulatory documentation

6. © Pfizer Limited 2008 6 NMR: How can we work faster? acquisition processing analysis results/report/LNB database

7. © Pfizer Limited 2008 7 Acquisition - Sensitivity of NMR spectroscopy MSlow nanogram UVlow nanogram NMR microgram

8. © Pfizer Limited 2008 8 NMR signal to noise

9. © Pfizer Limited 2008 9 Improving sensitivity High magnetic fields Performance enhancement of RF probe and electronics < V detection coil (microcoils) Cold probes and preamplifiers Coil built from low- loss materials (superconductors) Energy levels & population distribution Hyperpolarisation (DNP)

10. © Pfizer Limited 2008 10 Microcoils 5 mm Probe CapNMR TM Microcoil

11. © Pfizer Limited 2008 11 Microcoils and microflow NMR Flow cell

12. © Pfizer Limited 2008 12 Automation - OMNMR

13. © Pfizer Limited 2008 13 Analysis: Computer Assisted Structure Elucidator NMR: pp 2D MS: MF Generate MCD Generate Structures From MCD/s Check MCD Find the Best Structure ! Check data/ add information www.bdal.de/uploads/media/LCGC_March_2008_Appl._Books.pdf

14. © Pfizer Limited 2008 14 1.Introduction 2.Applications/Examples: Identification of Impurities Challenging molecules: Arylpyrazoles Regulatory documents generation/checking 3.Conclusions

15. © Pfizer Limited 2008 15 Structure elucidation of low level impurities ? LC - NMR ( 1 H) Structure ~> 0.5% Isolation 2D NMR + MS Report / Data base LC-MS

16. © Pfizer Limited 2008 16 CapNMR data for 1 of the impurities 9 mg/60ul injection 20 750ug in the active volume 13 C 27 h HMBC 4 h

17. © Pfizer Limited 2008 17 4 full data sets to analyse!

18. © Pfizer Limited 2008 18 Computer Assisted Structure Elucidation (CASE) Molecular Connectivities Diagram (MCD) GENERATION of STRUCTURES from MCD

19. © Pfizer Limited 2008 19 285/97953 – 6h 52 m THE BEST STRUCTURE d_{N}(^{13}C) 0.662 d_{N}(^{1}H) 0.091 d_{A}(^{13}C+^{1}H) 1.570

20. © Pfizer Limited 2008 20 R R R

21. © Pfizer Limited 2008 21

22. © Pfizer Limited 2008 22 1.Introduction 2.Applications/Examples: Identification of Impurities Challenging molecules: Arylpyrazoles Regulatory documents generation/checking 3.Conclusions

23. © Pfizer Limited 2008 23 Arylpyrazoles

24. © Pfizer Limited 2008 24 CASE of Example 1 - 12 mg C 15 H 8 O 1 N 4 F 8 Cl 2 1 H, 13 C, 1 H- 13 C HSQC, 1 H- 13 C/N- HMBC

25. © Pfizer Limited 2008 25

26. © Pfizer Limited 2008 26 No Filter: 3325/3409 – 2 minFilter: C CS and internal bad list 3/3409 – 2 min

27. © Pfizer Limited 2008 27 CASE of Example 2 – 0.5 mg C 15 H 7 O 2 N 3 F 8 Cl 2 1 H, 13 C, 1 H- 13 C HSQC, 1 H- 13 C/ 15 N- HMBC, 1 H 19 F-HSQC

28. © Pfizer Limited 2008 28 From MCD 26: 1476 – 11 s ACD + Arylpyrazole fragments

29. © Pfizer Limited 2008 29 CASE of Arylpyrazoles – challenging molecules Less information More Human intervention

30. © Pfizer Limited 2008 30 1.Introduction 2.Applications/Examples: Identification of Impurities Challenging molecules: Arylpyrazoles Regulatory documents generation/checking 3.Conclusions

31. © Pfizer Limited 2008 31 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’ ( 13 C) and automation CASE: –Used as a checking tool for H and C assignments: data are peak picked and contain 13 C direct –Can be used as a automatic structure verification and assignment tool and maybe for proof of structure documents

32. © Pfizer Limited 2008 32 CASE and regulatory data

33. © Pfizer Limited 2008 33 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.

34. © Pfizer Limited 2008 34 THE FUTURE?

35. © Pfizer Limited 2008 35

36. © Pfizer Limited 2008 36 Acknowledgements SE team: Ingrid Hamernig Protasis: Dean Olson, Bob Albrecht, Tim Peck and David Strand ACD labs: Ryan Sasaki

37. © Pfizer Limited 2008 37