Post on 20-Jan-2016
Gedeon Richter Ltd
Spectroscopic division
Structure of
and quality control
with NMR spectroscopy
Vinblastine
WHAT WAS MY PROJECT?
Zsófia Sólyom
Vinblastine Vinblastine (VLB)(VLB)
Vinca RoseaMadagascar
NH
N
H
OH
CH3
MeOOC
N
N
MeO
CH3
HCOOMe
OH
OAc
CH3
H
bis-indole alkaloid
Vinblastine Vinblastine (VLB)(VLB)
One of most One of most powerfulpowerful drugs in drugs in the the treatment of cancertreatment of cancer
VLB treatment
<<<< <<
pure(!)pure(!)
VLBVLB
pure(!)pure(!)
VLBVLB
ExtractioExtraction n
More than 90 similar
alkaloids!!! VLB
VLBvincristine
des-acethyl-VLB
leurosydine
etc…
It is impossible to produce 100% pure VLB!
regulatory regulatory demands are demands are
constantlconstantly y increasing increasing on all on all
three frontsthree fronts !!
Impurity profiling (QC):Impurity profiling (QC):1. 1. detectdetect 2. structurally ID2. structurally ID3. quantify3. quantify
ChromatograChromatogramm
AU
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0,70
Minutes
0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 45,00 50,00 55,00 60,00
VLBVLB
AU
-0,002
0,000
0,002
0,004
0,006
0,008
0,010
0,012
0,014
0,016
0,018
0,020
Minutes
0,00 10,00 20,00 30,00 40,00 50,00 60,00
VLBVLB
leurosineleurosine
?? ?? ?? ??
NH
N
H
O
CH3
MeOOC
N
N
MeO
CH3
HCOOMe
OH
OAc
CH3
H
NH
N
H
OH
CH3
MeOOC
NH
N
MeOH
COOMe
OH
OAc
CH3
H
des-acethyl-VLBdes-acethyl-VLB
NH
N
H
OH
CH3
MeOOC
N
N
MeO
CH3
HCOOMe
OH
OH
CH3
H
? = unknowns
similar structure (origin of plant,
mutation, technology, etc…)
des-methyl-VLBdes-methyl-VLB
NH
N
H
OH
CH3
MeOOC
N
N
MeO
CH3
HCOOMe
OH
OAc
CH3
H
= stereogenic center= stereogenic center
210= 1024 possible
stereoisomers
210= 1024 possible
stereoisomers
The challenge is enormous!The challenge is enormous!
Huge stakesHuge stakes
The product can not be sold until all the
impurities have been identified!
PRECIZE structure
quickly
and
from the smallest possible amount of sample
Task (in this case):
NMR!NMR!
What does NMR
mean?
NNuclearuclear M Magneticagnetic R Resonanceesonance
How does it work?
(extremely simplefied approach)
miniature spinning compass needles
H
nuclei
S
NE
qu
ilib
riu
m s
tate
Tra
nsi
tio
n s
tate
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!!!
The oscillation frequency depends on the atom type as well!
13C
nuclei
RESULT: THE SPECTRUM!
1H 13C
VLBNot informative enough in this case
series of pulses applied withdifferent direction, strength,
length, frequency…
pulse sequences:
What’s the purpose??Neighbours “feel” each other!
HSQC
One possible result from such spin manipulation:
Only one of the possibilities
Only one of the possibilities
HSQC
2D measurements2D measurements
Information on: constitution stereochemistry
molecular dynamics
Information on: constitution stereochemistry
molecular dynamics
sample and time-consuming
sample and time-consuming
Assignment With thorough interpretation of the
spectra we can decide unambiguously
„Who is Who”
Difficulties: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
But:
There is paucity of impurities
?
New techniques!New techniques!
CRYO PROBE
(cooled electronics)
CRYO PROBE
(cooled electronics)
2005: first cryogenic probe in Hungary
Jel/zaj viszonyJel/zaj viszonyKétféleképpen növelhetőKétféleképpen növelhető
cooled electronicscooled electronics
more sensitivitymore sensitivity More information More information
normal electronicsnormal electronics
How much sample is required to identify an unknown impurity with
the cryo probe?
How much sample is required to identify an unknown impurity with
the cryo probe?
My projectMy project
How can the structure of a VLB impurity be assigned and what measurements are essential for
this?
How can the structure of a VLB impurity be assigned and what measurements are essential for
this?
LeurosineLeurosine
NH
N
H
O
CH3
MeOOC
N
N
MeO
CH3
HCOOMe
OH
OAc
CH3
H
Model compound
NH
N
H
OH
CH3
MeOOC
N
N
MeO
CH3
HCOOMe
OH
OAc
CH3
H
VLB
Typical impurity
Two-dimensional
measurements
Two-dimensional
measurements
Two-dimensional
measurements
Two-dimensional
measurements
500 µg500 µg
HSQC spectraHSQC spectra
Measurement time: 11 h
Almost every signal is visible,
leurosine can be identified
Normal electronics
Cooled electronics
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 mg necessary
AcknowledgementsAcknowledgements
Dr. Csaba Szántay
Dr. Zoltán Béni
Gedeon Richter Ltd
Spectroscopic Division
Hungarian Association for Innovation
Thank You for your attention!
NMRmagnet