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

• One of most powerful drugs in the treatment of
  cancer

VLB treatment
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pure(!) VLB
lt
lt

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Extraction
VLB
vincristine
des-acethyl-VLB
leurosydine
etc
More than 90 similar alkaloids!!!
VLB
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It is impossible to produce 100 pure VLB!
regulatory demands are constantly increasing on
all three fronts !
Impurity profiling (QC)1. detect 2.
structurally ID3. quantify
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Chromatogram
VLB
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des-acethyl-VLB
des-methyl-VLB
leurosine
VLB
? unknowns similar structure (origin of plant,
mutation, technology, etc)
?
?
?
?
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The challenge is enormous!
stereogenic center
210 1024 possible stereoisomers
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Huge stakes
The product can not be sold until all the
impurities have been identified!
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STRUCTURE RESEARCH
Task (in this case)
PRECIZE structure quickly and from the smallest
possible amount of sample
NMR!
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What does NMR mean?
Nuclear Magnetic Resonance
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How does it work?
(extremely simplefied approach)
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miniature spinning compass needles
H nuclei
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N
Equilibrium state
S
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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
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13C nuclei
The oscillation frequency depends on the atom
type as well!
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RESULT THE SPECTRUM!
Not informative enough in this case
VLB
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pulse sequences
series of pulses applied with different
direction, strength, length, frequency
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Whats the purpose?? Neighbours feel each
other!
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HSQC
One possible result from such spin manipulation
Only one of the possibilities
HSQC
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2D measurements
Information on constitution
stereochemistry molecular dynamics
sample and time-consuming
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Assignment
With thorough interpretation of the spectra we
can decide unambiguously Who is Who
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• VLB - relatively large, such as the impurities
  and derivatives
• Conformational dynamics (9-membered ring,
  rotation)

Difficulties
Even more amount of sample is needed
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But
There is paucity of impurities
?
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New techniques!
CRYO PROBE (cooled electronics)
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2005 first cryogenic probe in Hungary
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Jel/zaj viszony
normal electronics
Kétféleképpen növelheto
more sensitivity
More information
cooled electronics
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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?
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Model compound
Leurosine
VLB
Typical impurity
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Two-dimensional measurements
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Two-dimensional measurements
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Normal electronics
HSQC spectra
Almost every signal is visible, leurosine can be
identified
Cooled electronics
500 µg
Measurement time 11 h
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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


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Conclusions

• 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
• 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.
• I have learnt a lot, not only about NMR, but also
  about pharmaceutical research in general.

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Acknowledgements
Dr. Csaba Szántay
Dr. Zoltán Béni
Gedeon Richter Ltd Spectroscopic Division
Hungarian Association for Innovation
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NMR magnet
Thank You for your attention!