gedeon richter ltd spectroscopic division structure of and quality control with nmr spectroscopy...

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