Chiral HPLC method development PZQAmine at Syncom B.V.

Published by Jean-Paul Seerden on 24 May 2010 - 10:17pm

There is good news regarding the separation of the racemic praziquanamine with chiral HPLC.

  • Syncom B.V., The Netherlands, a CRO specialized in all aspects of organic synthesis, with a 20-year track record in resolution of chiral compounds, screened five different chiral HPLC columns, varied the eluent and used UV detection (220 and 260 nm) and a PDR-Chiral Advanced Laser Polarimeter.


  • The S(+)- and R(-)-enantiomers of praziquanamine could be separated well with good baseline resolution using the Chiralcel OJ-H, as well as the Chiralpak IA, and the Chiralpak AS-H column with heptane/EtOH/Et2NH (60/40/0.2) as eluent (0.5 ml/min flow rate).

  • The Chiralpak OD-H or the Chiralpak IB column was not suitable for the separation of these enantiomers, as was also noticed in Eur.J.Org.Chem. 2008, 895.


The diastereomeric salt resolution screening of praziquanamine with some chiral acids is in progress.



Note - the raw data from this trial (HPLC traces for all the different columns) may be downloaded with this link. (large PDF file)


MatTodd's picture

This is great Jean-Paul. We now have a robust analytical method. Thanks also for including the raw data, above, so everyone can see the results. We're really looking forward to the results of the screening with the chiral acids.

The analytical method for the separation of the enantiomers could probably be easily scaled for use in simulated moving bed (SMB) chromatography. This would then provide a preparative method for the desired enantiomer.

A good chromatographer should be able to provide estimates on throughput and cost based on the analytical conditions.

MatTodd's picture

Good suggestion, and this has already been done at Intervet in Germany, see They're being kind enough to send us some enantiopure material generated with this method. I'd be interested in your thoughts (and others') on the costs associated with this approach on a ton scale, and whether there are any drugs available for low prices that have been produced using this technology.

ndt228's picture

Dear Matt,
My previous employer (UCB) have used chiral SMB at industrial scale to resolve an API (Keppra) as part of their strategy to meet high demand.  They worked this in parallel with classical resolution and a chiral hydrogenation - all three processes were registered for supply of marketed API.  You can read about some other large-scale SMB separations here:   UCB piloted at 12 tonnes per annum, and the production unit uses 1m diameter columns.
Clearly SMB is competitive as an alternative to (some) chiral syntheses, but this is in the general context of patented APIs being made for profit.   Where this project differs is in the combination of extreme cost-constraints, and the absence of a developing IP position. 
cheers, Nick

MatTodd's picture

Very interesting, Nick. I guess the other feature of any chromatographic solution is that one loses 50% of the material without some racemisation/recycling strategy, but this all adds to the cost. I agree without such a restriction the efficiencies of modern SMB would make this an attractive option.

ndt228's picture

SMB "resolution" remains of very high value in reliably delivering early tox/clinical supplies when you may only have a "racemic" synthesis - you can actually get 2 chiral products to test from one synthesis.  It can be used to "buy time" whilst synthetic options are investigated.  The chiral hydrog idea newly described elsewhere is a classic example of this, and just what was done for Keppra, whilst classical and SMB resolution met the initial market needs.    In normal commercial API terms a feedstock like rac-PZQ Amine would probably be regarded as cheap enough not to bother with the off-isomer recycle in the short term (ie before getting very serious about commercialisation, based on evidence of efficacy, advantage relative to racemate, etc).  There can be many difficulties to be faced in a recycle stream with build-up of impurities etc.
In our case we need to think of off-isomer recycle from the start - but didn't someone suggest using a "reverse-PS" reaction to re-racemise it ?  Either from existing stocks of resolved PZQ (either enantiomer), or from some of the fresh supply from Intervet, you can hydrolyse back to the amine, and are then ready to try for racemisation.  If/when you succeed in this you'll have significantly improved the chances of SMB being  a viable means of delivering chiral API.  
It is also possible that if specifically chiral syntheses do not deliver product of sufficiently high ee, then SMB could be a cost-effective means to "polish" the product up to purity.  Your only other alternative will be to achieve this by crystallisation, and again, supplies of resolved PZQ can be utilised now to investigate the properties of the single enantiomer wrt solvation, polymorphism, eutectic points etc etc.  Such data will always be relevant to the project, no matter what route is used to get to the single enantiomer.
I did encounter a paper describing use of SMB in an chemical engineering dept at a university in (?) Brazil - I'll try to find it again - they might be prepared to collaborate if the needs for material go beyond what Intervet will supply.
Cheers, Nick

  • Screening of a number of chiral acids at Syncom B.V. revealed that both quinic acid (as was reported in a Merck patent US3993760A1 example 1;  1976) and (-)-dianisoyl-L-tartaric acid displayed excellent resolving abilities for racemic praziquanamine, kindly provided by Matthew Todd. One recrystallization afforded the salt with a de of 95%, enriched in the (+)-enantiomer. The exact yield has not been determined but with some optimization it should be possible to obtain a yield of at least 30% (60% of the theory).
  • Non-optimized Syncom protocol for resolution of praziquanamine with (-)-dianisoyl-L-tartaric acid
    A mixture of rac-praziquanamine (101 mg, 0.5 mmol) and (-)-dianisoyl-L-tartaric acid (214 mg, 0.5 mmol) was dissolved in a mixture of isopropanol (2 mL) and water (0.4 mL) by the application of heat. The obtained clear solution was allowed to cool to room temperature overnight and the resulting crystals were isolated by filtration. The free base was liberated with aqueous base, extracted and analyzed by chiral HPLC using UV detection (220 and 260 nm) and a PDR chiral advanced laser detector. Various chiral HPLC methods were developed at Syncom B.V. and were posted on the website An ee of 65-67% was found for the (+)-isomer, using a chiral advanced laser detector at 675 nm. The filtrate (mother liquor) was enriched in the desired opposite (-)-enantiomer (ca. 60% ee). One recrystallization of the crystals from a mixture of isopropanol and water (2 and 1 mL) increased the ee to 95%. (Dr. Kees Pouwer, Syncom B.V., The Netherlands, May 26, 2010).
  • The diastereomeric salt resolution of praziquanamine with diaroyl tartaric acids is simple and can easily be upscaled to produce kilogram quantities or more. For the preparation of (-)-praziquanamine the resolution with (+)-dianisoyl-D-tartaric acid is preferred. When using the (-)-dianisoyl-L-tartaric acid the (-)-praziquanamine enantiomer should be isolated from the mother liquor.


(NB this information is also posted here)