1. Gedeon Richter Ltd Spectroscopic division Structure of and quality control with NMR spectroscopy Vinblastine WHAT WAS MY PROJECT? Zsófia Sólyom

2. Vinblastine (VLB) Vinca Rosea Madagascar bis-indole alkaloid

3. Vinblastine (VLB) One of most powerful drugs in the treatment of cancer VLB treatment

4. << < < pure(!)VLBpure(!)VLB

5. Extraction More than 90 similar alkaloids!!! VLB vincristine des-acethyl-VLB leurosydine etc…

6. It is impossible to produce 100% pure VLB! regulatory demands are constantly increasing on all three fronts ! Impurity profiling (QC): 1. detect 2. structurally ID 3. quantify

7. Chromatogram VLB

8. leurosine ? ? ? ? ? ? ? ? des-acethyl- VLB ? = unknowns similar structure (origin of plant, mutation, technology, etc…) des-methyl- VLB

9. = stereogenic center 2 10 = 1024 possible stereoisomers The challenge is enormous!

10. Huge stakes The product can not be sold until all the impurities have been identified!

11. PRECIZE structure quickly and from the smallest possible amount of sample Task (in this case): NMR!

12. What does NMR mean? Nuclear Magnetic Resonance

13. How does it work? (extremely simplefied approach)

15. miniature spinning compass needles H nuclei

16. S N Equilibrium state

17. Transition state Oscillation frequency (MHz): A bit different for each H nucleus! Depends on the molecular environment of the nucleus, whereby it is informative of the STRUCTURE!!!

18. The oscillation frequency depends on the atom type as well! 13 C nuclei

19. RESULT: THE SPECTRUM! 1H1H 13 C VLB Not informative enough in this case

20. series of pulses applied with different direction, strength, length, frequency … pulse sequences:

21. What’s the purpose?? Neighbours “feel” each other!

23. HSQC One possible result from such spin manipulation: Only one of the possibilities HSQC

24. 2D measurements Information on: constitution stereochemistry molecular dynamics sample and time- consuming

25. Assignment With thorough interpretation of the spectra we can decide unambiguously „Who is Who”

26. Difficulties: VLB - relatively large, such as the impurities and derivatives Conformational dynamics (9- membered ring, rotation) VLB - relatively large, such as the impurities and derivatives Conformational dynamics (9- membered ring, rotation) Even more amount of sample is needed

27. But: There is paucity of impurities ?

28. New techniques! CRYO PROBE (cooled electronics) CRYO PROBE (cooled electronics)

29. 2005: first cryogenic probe in Hungary

30. Jel/zaj viszony Kétféleképpen növelhető cooled electronics more sensitivity More information normal electronics

31. How much sample is required to identify an unknown impurity with the cryo probe? My project How can the structure of a VLB impurity be assigned and what measurements are essential for this?

32. Leurosine Model compound VLB Typical impurity

33. Two- dimensional measurements

36. 500 µ g HSQC spectra Measurement time: 11 h Almost every signal is visible, leurosine can be identified Normal electronics Cooled electronics

37. Normal electronics: around 100- 150 mg necessary = Collection of sample and purification 100-150 mg few weeks 500 µg 3-4 days ~ Two orders of magnitude!!! Cooled electronics: around 0.500 m g necessary

38. Conclusions Structure determination of technological impurities is a key factor in drug production Structure determination of technological impurities is a key factor in drug production The most powerful tool for the identification of an impurity in solution is NMR spectroscopy The most powerful tool for the identification of an impurity in solution is NMR spectroscopy By using 1D and 2D NMR experiments I have shown that leurozin can be distinguished from VLB. By using 1D and 2D NMR experiments I have shown that leurozin can be distinguished from VLB. Cooled NMR probes allow the measurement of two orders of magnitude less sample, saving much time and money. Cooled NMR probes allow the measurement of two orders of magnitude less sample, saving much time and money. I have learnt a lot, not only about NMR, but also about pharmaceutical research in general. I have learnt a lot, not only about NMR, but also about pharmaceutical research in general.

39. Acknowledgements Dr. Csaba Szántay Dr. Zoltán Béni Gedeon Richter Ltd Spectroscopic Division Hungarian Association for Innovation

40. Thank You for your attention! NMR magnet