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Gedeon Richter Ltd Spectroscopic division

Structure of. Vinblastine. and. quality control. with NMR spectroscopy. Zsófia Sólyom. Gedeon Richter Ltd Spectroscopic division. WHAT WAS MY PROJECT?. Vinblastine (VLB). bis -indole alkaloid. Vinca Rosea Madagascar. Vinblastine (VLB).

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Gedeon Richter Ltd Spectroscopic division

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

  2. Vinblastine (VLB) bis-indole alkaloid Vinca RoseaMadagascar

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

  4. pure(!) VLB < < $

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

  6. It is impossible to produce 100% pure VLB! regulatory demands are constantly increasing on all three fronts! Impurity profiling (QC):1. detect2. structurally ID3. quantify

  7. Chromatogram VLB

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

  9. The challenge is enormous! = stereogenic center 210= 1024 possible stereoisomers

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

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

  12. What does NMR mean? Nuclear Magnetic Resonance

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

  14. miniature spinning compass needles H nuclei

  15. N Equilibrium state S

  16. 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!!! Transition state

  17. 13C nuclei The oscillation frequency depends on the atom type as well!

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

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

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

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

  22. 2D measurements Information on: constitution stereochemistry molecular dynamics sample and time-consuming

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

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

  25. But: There is paucity of impurities ?

  26. New techniques! CRYO PROBE (cooled electronics)

  27. 2005: first cryogenic probe in Hungary

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

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

  30. Model compound VLB Leurosine Typical impurity

  31. Two-dimensional measurements

  32. Two-dimensional measurements

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

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

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

  36. NMR magnet Thank You for your attention!

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