All schistosomiasis projects currently in process should be created as child pages to this page. See the "add child" link at the bottom if you want to initiate a new open research project for schisto.
Project A: Development of a low-cost enantioselective synthesis of PZQ. It's an open project, like everything on TSL.
Meaning?: contributors can change anything they wish on these pages.
If you wish to contribute: please don't leave comments here. Instead, edit the pages below directly or leave comments.
Other ways to interact: The Friendfeed page and the Lab Book Blog (for the Pictet-Spengler approach)
How about email? No. Please avoid emails if at all possible, since they're closed, and people can't be part of the process.
What's needed? Right now we need your help with these things:
1) CATALYSTS: A key reaction in the Pictet-Spengler approach to PZQ will be the reaction creating the stereogenic center. If you lab is involved in this area of research, and you have catalysts you are willing to share, please let us know (on this website). We would like to screen catalysts for this reaction. We can send starting material, or you can send us samples of catalysts. Primary data for this part of the project are now posted here.
2) CLASSICAL RESOLUTION: The specific problem we need help with is here. In a nutshell: we've had spectacular success with the cleavage of the amide in PZQ to give an intermediate called praziquanamine. Praziquanamine can also be synthesised from scratch. Syncom have discovered how to resolve this molecule. We now need to optimise this process, and could use more help from process chemists. If you work in this field, or know people who do, please pass on the link. Development Chemicals have discovered another resolution, using a different intermediate in PZQ synthesis. While this intermediate is not commercially-available (in small quantities), there is work needed here too - to replicate the resolution, increase its efficiency and to demonstrate that the inactive enantiomer of the intermediate can be recycled.
3) HYDROGENATION CATALYSTS/CATALYST SCREENING: A powerful approach would be the asymmetric hydrogenation of an oxidised form of PZQ, described here. The asymmetric reduction in question is a challenge. We need people with experience in this area to screen catalysts. We can mail out starting material. DSM have already started to help us with this - the more, the faster here.
4) LITERATURE: To help with aim 1 above, we need a good catalyst for the catalytic, asymmetric Pictet-Spengler reaction. Could someone help us assemble a comprehensive list here? This is a way you can really help us if you don't have access to a lab. Another thing we need help with is finding out the synthetic route employed by the main suppliers of PZQ. There's a lack of clarity there - more on this soon.
1) HPLC advice: We previously required advice on enantioselective HPLC: The promising intermediate in the resolution work mentioned above is PZQamine. To assay its e.e. by HPLC we need to be able to separate the enantiomers on a column. Chiralcel-OD was not working for us but the appropriate conditions were rapidly found by Syncom here].
1) PRICES: We needed help obtaining prices for bulk starting materials for racemic PZQ syntheses. This required input from industrial chemists who know of suppliers of these materials for low prices. This problem is now less important since we are focussing on the use of an enantioselective Pictet-Spengler reaction, which employs the existing large-scale synthetic route precursor, making discussions of starting material costs redundant.
This thread starteded as a place to locate anything related to the final API form(s) in which PZQ might be isolated (ie Drug Substance), or the formulation of these into Drug Product.
A hub for alternative drugs to the currently used PZQ
To assemble a list below of naturally-occurring alternatives to PZQ for the treatment of schistosomiasis.
Synthetic compounds that are alternatives to PZQ - anyone can assemble a list below.
Page dedicated to possible PZQ analogs. Either add below, or create child pages.
The analog containing benzoyl in place of cyclohexanoyl was described in one of the early PZQ patents. Interestingly the patent says this (column 2, top): "[The enantiopure compound] exhibits its althelmintic activity on [organism] in a dose which is up to five times smaller than that of the racemic [compound]. This effect could not be expected, since one would anticipate a doubling of the efficiency at most, if only one isomer were active."
Project to determine if it is feasible to automate / accelerate the catalyst screening project through automated software systems.
Currently the project is in requirements initiation. We are defining top level requirements of what an automated tool will look like. Please feel free to contribute
Following link will be where requirements are documented. (This should support anonymous access).
When more tasks are required, issues can be edited and viewed using the following issues management system. Unfortunately the issues tool doesn't support anonymous entry of issues so you will need to make an account for yourself.
A start has finally been made on determining the phase diagram for PZQAmine from 0 to 100% ee, based upon melting point determination of known % w/w mixtures of racemicand single enantiomer PZQAmine. Samples of the latter were provided by Michael early this year.
The "version 1" phase diagram is attached, along with a second file containing the raw data. Note that the data has been generated from mixtures of racemic and (R)- PZQAmine, and the data simply "mirrored" into the (S)- rich region of the diagram. The ee of the supplied (R)- amine has been taken as stated (97% ee) and not rechecked.
Explanation: Whilst the "pure" racemate and (R)- enantiomer show sharp melting point behaviour typical of pure materials, mixtures of the two exhibit melting point depression. When this reaches a minimum the mixture is known as the eutectic. By using DSC (differential scanning calorimetry) to observe the melting event in detail, two melting endotherms can be observed for most of the mixtures. The onset of melting is determined graphically from the DSC, and is more informative than the temperature of the endotherm peaks. These data can be plotted to map out the phase diagram, which is of relevance because melting point is a surrogate for solubility, and hence the behaviour of these materials during recrystallisation.
I will next be attempting to improve the resolution of the DSC method by adjusting the scan rate, so as to better define the location of the eutectic point. For the present it is clear that crystallisation of PZQAmine mixtures with below 50% ee will deliver racemic crystals, whereas mixtures of above 95% ee can be crystallised up to enantio-purity.
It is therefore important at present not to convert the diastereomeric salt to free amine until confident that the salt is of at least 95% ee.
Best regards, Nick
The attached file contains X-ray powder diffraction patterns (XRPDs) for racemic and (R)- PZQAmine, as well as a mixture of the two. These confirm that (as is often the case) the racemic compound and the single enantiomer have different crystal structures. This is consistant with the phase diagram for PZQ Amine gathered from the melting point of mixtures of the two.
Thanks to Jana Galbraith (Almac Sciences Physical Sciences Dept) for recording these patterns.
Praziquantel (PZQ, 1) is the drug of choice for the treatment of schistosomiasis.1 It is synthesized as a racemate, where one enantiomer is active, and the other has no effect on the parasite.2 Half the currently-administered pill is hence unnecessary. The World Health Organisation and the international schisto community recommend that a way be found to produce PZQ tablets containing only the active enantiomer.3 This would have the following pharmacological advantages:
i) Optically pure PZQ is twice as active as the racemate;4 but with a reduced ‘drug burden.'
ii) An increased dose would be possible. It has been argued convincingly that the current dose is subcurative, favouring the progressive selection of partially resistant survivors.5
iii) The size of tablets could be reduced, thereby helping children take the drug.
The (S)-(+)-enantiomer is inactive and harmless [NB It is said to contribute significantly to the bitter taste of the racemate; see "Resolutions of PZQ"], and the (R)-(-)-enantiomer is active.6 The control of the single stereogenic centre is at the centre of the project.
In general it is easier to synthesise racemic molecules than it is to synthesise one enantiomer of a racemate. The current very low price of racemic PZQ reflects this. PZQ is being distributed in mass chemotherapy programmes in six African countries as part of the Schistosomiasis Control Initiative (SCI), funded by the Gates Foundation.7 (Other African countries are attempting to follow suit with their own national control programmes.) The drug has been purchased by SCI from the South Korean company Shin Poong for US¢7 per 600 mg tablet. The target efficiency for a competitive synthesis of optically pure PZQ is hence US¢23 per gram.
Many syntheses of PZQ have been reported. The original synthesis was in 1977.8 The more recent report based on peptide acetal chemistry may reflect the current generics route.9 Our group has also published new radical- and solid phase-based methods.10, 11
The original patent literature detailed a method for the separation of PZQ enantiomers, based on co-crystallisation and recycling of the unwanted enantiomer.12 It is likely that such a method is expensive (it has never been adopted). [See also a 2009 paper in "Resolutions of PZQ"]. It is likely an efficient enantioselective synthesis would be less expensive.
The first enantioselective synthesis of PZQ was reported in 2004 using a chiral auxiliary-mediated Pictet Spengler reaction.13 The method is not suitable for scale-up, not least because chiral auxiliary-mediated processes are inherently less efficient than catalytic ones.
The aim of the project is to design and execute a catalytic, asymmetric synthesis of the active enantiomer of PZQ. The synthesis must be highly efficient and low cost. It is not enough that the synthesis works because the route needs to be scaled up for multi-ton production. The object of this open source chemistry project is therefore to share synthetic strategies and results towards this target.
There are two ways in which organic chemists can help. One is to design synthetic routes or share experience on those being discussed. The other is to attempt one or two steps and share results, so that an optimized route may be arrived at.
As an example, our group at Sydney has devised a new synthesis of PZQ and are currently exemplifying it in the lab. We would value advice on this route, and, once we report it, practical improvements on any given step (in other words beat the yield or enantiomeric excess!) On the other hand, people are encouraged to devise alternative routes and post these as new entries on Synaptic Leap.
1. A. Fenwick, L. Savioli, D. Engels, R. Bergquist and M. H. Todd, Trends in Parasitology, 2003, 19, 509-515.
2. P. Andrews, H. Thomas, R. Pohlke and J. Seubert, Med. Res. Rev. 1983, 3, 147-200.
3. Second meeting of the second EU Concerted Action on Praziquantel, Yaound&eactute;, Cameroon, March 2004.
4. M. H. Wu et al., Am. J. Trop. Med. Hyg. 1991, 45, 345-349.
5. M. J. Doenhoff, Parasitol. Today, 1998, 14, 434-435.
6. C. A. Redman et al. Parasitol. Today 1996, 12, 14-20.
8. J. Seubert, R. Pohlke and F. Loebich, Experientia 1977, 33, 1036-1037.
9. J. H. Kim, Y. S. Lee, H. Park and C. S. Kim, Tetrahedron 1998, 54, 7395-7400.
10. M. H. Todd, C. O. Ndubaku and P. A. Bartlett, J. Org. Chem. 2002, 67, 3985-3988.
11. S. El-Fayoummy, W. Mansour, and M. H. Todd, Tetrahedron Lett. 2006, 47, 1287-1290.
12. J. Seubert, German Patent Application 2,418,111.
13. C. Ma, Q.-F. Zhang, Y.-B. Tan and L. Wang, J. Chem. Res. 2004, 186-187.
I was looking at all the difficulty that is occuring with finding a cheap source of chiral catalyst for the Pictet-Splenger and mused about using Phenylglycine, as this avoids the issue due to it being part of the chiral pool.
Anyway here is my attempt at getting the key ring system together for the system. Don't think anything I used is particularly expensive. Sorry about the conditions being vague, was just a quick thought that I came up with.
We have designed a new synthesis of PZQ based on a catalytic, asymmetric aza-Henry reaction (Scheme 1). The key step is the generation of the new stereogenic centre in 4. From here, the reduction to 5 should be facile with e.g. samarium iodide.1 From 5, the two steps to PZQ are known from the original report.2
A final issue is that the dihydroisoquinoline 3 is not commercially available. There are many simple routes to this compound, such as a potassium permanganate-mediated oxidation from tetrahydroisoquinoline. However, the synthesis of 3 also needs to be efficient and inexpensive if the whole route is not to be compromised.
Recrystallisation of PZQ may be achieved with diethyl ether in hexane.8
What the Community can do
With regards our first proposed route:
a) Advice from groups experience in this chemistry would be very useful.
b) Donations of catalysts for screening of the aza-Henry reaction would also be very useful.
c) Students in such groups can attempt one or more of the reactions in our proposed route and try to maximize the route's efficiency. This applies to all steps, but is particularly relevant for the asymmetric step.
With regards scale-up, advice from process/industrial chemists on any given step with regards to scale-up issues would be very valuable. This applies to both synthesis and purification.
For discussions of routes, a regular blog-style of discussion is probably fine. For posting chemical structures, I would recommend using e.g. Chemdraw and posting gifs. To do this you 'creat content' and submit gifs to the image gallery. There is an option when doing this of attaching Chemdraw .cdx files, which may be useful for us. When inserting images please use thumbnails - image sizes can be a little erratic.
For reporting of experimental data, it would be most useful if quality scientific conventions were maintained, and that if a result is claimed, it is properly reported. The open source community relies on honesty and repeatability rather than peer-review. Certainly the route we arrive it will need to work well in the real world!
1. K. Yamada, S. J. Harwood, H. Gröger and M. Shibasaki, Angew. Chem. Int. Ed. 1999, 38, 3504-3506.
2. J. Seubert, R. Pohlke and F. Loebich, Experientia 1977, 33, 1036-1037.
3. J. Seayad and B. List, Org. Biomol. Chem. 2005, 3, 719-724.
4. A. P. Venkov and S. M. Statkova-Abeghe, Tetrahedron 1996, 52, 1451-1460.
5. J. H. Kim, Y. S. Lee, H. Park and C. S. Kim, Tetrahedron 1998, 54, 7395-7400.
We have been working for a little while on the aza-Henry route to PZQ. We're going to submit a paper to an open access journal on some of this work, but I thought we should post on something we're looking at now, since we've come up against an unexpectedly difficult step and need some help.
We've been trying to reduce an aliphatic nitro group (picture is below). The compound is a model case for PZQ that we've been looking at. This reduction looks to be a very simple reaction, and we did not expect problems.
Aliphatic nitro reduction
Shibasaki has reported on the reduction of aza-Henry beta-nitroamines like this.1 The literature contains few reliable procedures for aliphatic nitro reduction2 – aromatic nitro groups are no problem. We've tried regular hydrogenation, Raney Nickel, SmI2 and LiAlH4. Jim Anderson at Nottingham recently published a very nice paper resurrecting Al-Hg amalgam as a reagent for this transformation, and we are having luck with it.3 This post is a very overdue appeal to the community at large:
a) Does anyone know of any other good reaction conditions for this reaction?
b) Does anyone have any advice on good ways to isolate the resulting diamines?
1. K. Yamada, S. J. Harwood, H. Groger and M. Shibasaki, Angew. Chem. Int. Ed. 1999, 38, 3504-3506.
2. S. L. Ioffe, V. A. Tartakovskii and S. S. Novikov, Russ. Chem. Rev. 1966, 35, 19-32.
3. J. C. Anderson and H. A. Chapman, Synthesis 2006, 3309-3315.
This may come too late, but you could look into using conditions published by AGMBarrett for aliphatic nitro group reductions, by transfer hydrogenation. Its in Tet Lett but there's an open-access link here:
The Pictet-Spengler reaction is used in the current industrial synthesis of rac-PZQ, and these reactions have been looked at on the Synaptic Leap here.
The PS has been used in the synthesis of PZQ, and we used it as the final step in our solid phase synthesis of PZQ, but both these syntheses were racemic.
General aim is this:
We have now started an Electronic Lab Notebook for the raw data for this specific aspect of the project here. Comments can be left here or there.
Essentially we need to start with literature catalysts for the catalytic, asymmetric Pictet-Spengler reaction. We therefore need to assemble a list of these catalysts here (literature help needed from anyone please!):
2010 Wu et al.
Need more candidate catalysts!
We are looking for people to help screen known catalysts for the relevant reaction. If your lab has any such catalysts we can send you starting material to screen. Substantial contributors to the project will be invited to be authors on the resulting papers.
Log of labs contacted for help with screening: (Important note: declining a request to participate will never be criticised - people may have very valid reasons for not wanting to participate, or may just be too busy. This log is here purely for record, to stimulate readers to suggest alternative/additional collaborating labs, and to avoid duplication of effort. If you know of a lab working on the asymmetric Pictet-Spengler reaction please feel free to contact them and leave a note of the request here.)
Wayne Best of Epichem has suggested an alternative approach based on a PS reaction of a chiral phenylethanolamine. This is an interesting idea, since 1 to 2 (below) might work well with asymmetric induction, and the products resemble PZQ. The use of a chiral starting material, in the generation of some analogue of PZQ that contains an extra stereocentre, could be an interesting project, but it's not clear these analogs would be active. Anyone have a suggestion for a suitable commercially-available chiral starting material? Anyone want to try this?
A chiral auxiliary-mediated PS was reported by Ma et al. (J. Chem. Res. 2004, 2004, 186-187 - paper doesn't seem to have a DOI?) where the key asymmetric step is shown below.
Other examples: Comins 1991
The use of an auxiliary is of course atom-inefficient, however, and likely to be expensive.
We started catalyst screenings for the asymmetric Pictet-Spengler reaction. So far, we have tested several achiral Brønsted-acidic catalysts to determine acid strength which is required to cyclize the open-chain praziquantel intermediate 1 to the tetrahydroisoquinoline heterocycle 2.
(+/-)-BINOL-N-triflyl phosphoramide 3 and N,N'-bis[3,5-bis(trifluoromethyl)phenyl]thiourea 4
As previous experiments showed only strong Brønstedt-acids (e.g. conc. sulphuric acid, methane sulfonic acid) are able to convert the inactivated arene compound 1 to the Pictet-Spengler product 2. Therefore we are looking for strong chiral proton donors to catalyse this specific reaction.
We are using the racamate/achiral equivalents 3 and 4 of chiral catalysts in order to prove the activity of these Brønsted-acids for the acid-catalysed PS reaction. However, the conversion of the dimethoxyacetal 1 hasn't been successful yet as a result of the too low acidity of the catalysts.
To increase the acidity of the catalyst we have started the synthesis of 1,1-Binaphthyl-2,2-disulfonate 5 and 1,1-Binaphthyl-2,2-disulfonimide 6.
The recent results and the raw data of these experiments can be viewed at our Electronic Lab Notebook here.
This is a post of random synthesis ideas for praziquantel that have been sitting in my inbox for too long. Both sources are industrial. If anyone has any comments, please post. If anyone has any further ideas, please post directly here – you can see how long it can take things to emerge if you use email...
Back in June I gave a talk at Stanford to a group called SPARK hosted by Kevin Grimes. This was a very interesting collection of people - highly interdisciplinary. Students and staff give talks, and the idea of the meetings is to brainstorm ideas on the projects underway, many of which were relevant to tropical diseases. These were like normal science group meetings, but with a much broader audience who were not shy of asking tough questions on detail and overall project direction. I really liked the way that a) students were actively involved along with faculty in meetings, and b) much of the stuff under discussion was in progress and incomplete, allowing real input from the audience.
My talk about the stuff The Synaptic Leap's project with praziquantel was interesting because of the diverse nature of the questions I got. This is one of the benefits of talking to a mixed audience of smart people. One of the most vocal skeptics of the approach was Steve Schow from Telik. His questions were excellent because they were grounded in economics. Essentially the arguments centered around where the money would come from for open compound development (or even drug discovery). I won't rehash the discussion here, because that's for a separate post that's building, but after the meeting Steve (who is an organic chemist by training) sent in a bunch of suggestions for other syntheses of enantioenriched PZQ. After checking with him, I am posting them here - his schemes posted directly. I'm posting them for completeness, in case the ideas chime with anyone or anyone wants to chip in. I want to thank Steve for contributing them.
This first idea is essentially the approach that's already been described. DSM are currently conducting catalyst screens for this reaction on the enamide shown and Development Chemicals had a hit with a related benzoyl substrate.
Steve's appeal to literature was the following:
Most of these examples have coordinating groups such as carbonyls that are well-placed to deliver the metal (and H) from a defined side. This has already been discussed here. There are a couple of examples above that do not require this. Perhaps the relevant ligands would be of interest - if anyone has the literature references for these? Steve's second suggestion involves an asymmetric hydrogenation at an earlier stage.
With the following precedents:
We have found working with dihydroisoquinolines to be rather annoying, given how prone they are to oxidation, but certainly the chances of a successful asymmetric hydrogenation here look to be better. Anyone have any comments on this approach, or literature sources for a comparison? Steve's final suggestions were as follows:
We'd thought of the first two, but have not done much/any work on them. It's a challenge because the enamide is not terribly reactive - it takes a lot to protonate the molecule. In the middle case, the tautomer shown is less stable than that where the double bond is conjugated with the ring. In the final case, the asymmetric reduction approach is no use since introduction of acid is required for the cyclization and this produces the acyliminium ion which scrambles stereochemistry. The introduction of the chiral acid is essentially the problem we're having with the Pictet-Spengler approach, in that the Ar ring needs methoxy groups to be active in this reaction. Thus far we have drawn blanks.
More recently, the guys at Creative Chemistry, who were looking into a route to enantioenriched PZQ, and developed a resolution approach, also contributed a few ideas they had been playing around with, but had not pursued. Again, I want to thank them for sending these along, which is in the spirit of this open project. Apologies for taking too long to post these. The following is essentially as received from DC. Idea 1: Reaction of tetrahydroisoquinoline with peroxide/tungstate/cyanide gives the unsaturated nitrile (TL, 1987, 6469). Yields when this was attempted were lower than described and the product was obtained by distillation. It may be possible to reduce this material enantioselectively, but the centre adjacent to the nitrile will be very acidic. This could also offer an easy entry to the diamine for resolution.
Idea 2: The asymmetric synthesis of praziquantel via a Bischler-Napieralski reaction was attempted as described by Czarnocki. Unfortunately, on reading the experimental, it became apparent that the 96% yield of the B-N reaction was based on recovered starting material and that the yield of product was only 15%. In our hands, the reaction gave several products and very little remaining starting material. Using the simpler chloromethyl amide the reaction went very cleanly. Unfortunately, the material is possibly too unstable for any further reaction unless kept under acidic conditions.
Idea 3: The preparation and hydrogenation of the tartaric acid derivative has been documented (Can J Chem, 1992, 70, 1555 (MHT - annoying journal website - the paper is meant to be here); Acta Chim Acad Sci Hung, 1976, 89, 161 (seemingly not online)). It may be possible to perform this reaction with the unsubstituted analogue shown. If so the amine could be reacted with glycine followed by cleavage and reductive amination (or cleavage/reduction to create a chirally stable centre). A similar reaction using R-(+)-glyceraldehyde has been documented and, after hydrogenation, gives product of the desired stereochemistry (Can J Chem, 1986, 2205).
Idea 4: Speculatively, a suitable B-N product could be converted to the amide and the resulting product hydrogenated using, for example, a Binap type catalyst. This type of reaction has been documented.
There are two general ways to generate enantioenriched PZQ starting from the racemate - either by destroying the stereocentre, or not.
1. Destroying the stereocentre, then reinstating it (a "stereoablative" approach). Craig Williams suggested this interesting possibility: take rac-PZQ and oxidize to the enediamide 2. This allows a catalytic, asymmetric hydrogenation to give (R)-PZQ. This is attractive because asymmetric hydrogenations are efficient, and both enantiomers of rac-PZQ can be taken through to the enantiopure material.
This is a great suggestion. According to the original review of PZQ by Peter Andrews et al., a similar idea is contained in the original patent literature on PZQ. The reference is "J. Seubert, German Pat. Appl. 2,418,111". I tracked down this patent, and it's unsurprisingly in German. While I can order a beer in German and ask the way to the cinema, I can't translate this. Does anyone with some German have some time to extract relevant information from this patent and give the conditions that were used for the generation of the enediamide, and whether this was done on PZQ or an analog? I'll post it below. [update - translation now shown below]
We have started to look at the oxidation reaction to the enediamide, but need help with this reaction. [update - this approach has now been given its own page here]. The review has no experimental details - but the reaction has been published in...[need reference]
According to the same review above, intermediates in the synthesis of PZQ (3 and 4 below) could be resolved. (PZQ itself presumably cannot, unless anyone has any bright ideas). Obviously this is a less attractive approach than a catalytic, asymmetric synthesis of such intermediates. However, the unwanted enantiomer, after conversion to (S)-PZQ, can be transformed to the enediamide 2 and hydrogenated to rac-PZQ. This therefore converts the inactive enantiomer of PZQ to rac-PZQ, giving another 25% yield of the desired enantiomer.
The review describes this process without specifiying what R is (where for PZQ it's cyclohexanoyl).
For the resolution via 4 to be effective, this molecule either needs to be synthesised from scratch, or it can be made from PZQ. For an industrial approach, it is probably best to simply make this molecule from scratch, as in the current industrial synthesis, but either way can easily be used to generate quantities of 4. What is needed now is a robust method for the resolution of amine 4. This idea now has its own page here.
A possible solution to the large-scale preparation of enantiopure PZQ is the approach shown below, originally suggested by Craig Williams.
We have a decent approach to this involving heating in a sulfur melt. This needs improvement, but we can generate gram quantities of the intermediate PZQ-enamide easily.
This is the key. A number of different catalysts for this reaction have been tried [data coming soon, sorry] but no conversion has been observed. Reduction with rhodium on carbon quantitatively generates rac PZQ, so the enamide is reactive. The geometry of the enamide here is awkward in that the exocyclic carbonyl is not able to direct a metal-based asymmetric catalyst to the double bond, as in the classical model. It's a challenging reduction.
This second part of the synthetic route needs people who have expertise in screening hydrogenation catalysts, or people who have in their posession unusual catalysts for asymmetric hydrogenation that might be appropriate for this kind of reaction. The Todd group can mail people samples of the PZQ-enamide for this screening (plus samples of PZQ for the assay). Most any column will separate PZQ enantiomers. If you are interested in helping with this part of the project, please reply below.
Relevant groups/papers to consider (please fee free to suggest others here by editing the page):
August 2010: We received our first offer of help with the asymmetric hydrogenation. Laurent Lefort from DSM in the Netherlands, has offered to include our substrate in future catalyst screening that they are undertaking. This is a great offer. Laurent has a great deal of relevant experience in this area. Aug 31st: The Todd lab has mailed a sample of the enamide plus a reference sample of PZQ to DSM. October 20: Second set of DSM results posted here. January 25 2011: Third, larger screen from DSM posted in the same place as the others.
The PZQ-enamide is shown below. This is an intermediate in the stereoablative route to enantiopure PZQ.
HPLC trace for PZQ-enamide (using ChiralcelOD-H, solvents: Hex:IPA:TEA 60:40:0.1, Flow Rate: 0.7 mL/min) gives retention time: 15.772 mins. Original HPLC trace attached below.
(Note comparison HPLC trace for PZQ itself is here.)
Other data: RF =0.35(1:1 Hexane: EtOAc); m.p. 131-134 ºC; 1H NMR (300 MHz, CDCl3): δ 1.15-1.85 (10H, m, cy), 2.35-2.65 (1H, m, cy ), 2.84 (t, 2H, J 5.2, H7), 3.82 (t, 2H, J 5.5, H6), 4.35 (1H, s, H4), 6.70 (1H, s, H2), 7.10-7.51 (4H, m, Ar); (Proton NMR spectrum attached below) 13C NMR (75 MHz, CDCl3): δ 26.1, 29.4, 38.8, 41.4, 45.9, 48.7, 106.0, 106.0, 123.1, 123.6, 126.9, 127.7, 128.2, 128.4, 128.6, 128.8, 129.0, 134.5, 164.3, 174.5; IR (CHCl3): 2952-2870 cm-1, 1652 cm-1, 1463 cm-1; MS (ESI) m/z: 311.1 [(MH)+, 100%], 333.3 [(MNa)+, 55%] HRMS (ESI) Calcd. for C19H22N2NaO2 (MNa+): 333.15735. Found: 333.15765.
This page will contain all examples of failed reactions in the attempted asymmetric hydrogenation of the PZQ enamide.
We are very grateful to Sigma-Aldrich for an initial donation of the catalysts employed below. Please note that we do not hold a library of other hydrogenation catalysts. Any suggestions for alternative catalysts are welcome, but in the interests of speed, the best suggestions here are catalysts + who could run the reactions (other than the Todd group) and to whom we could send PZQ enamide. This will accelerate the research.
In the Todd lab, we used a bespoke hydrogenator vessel of 15 mL capacity able to take pressures of 2000 psi and where the addition of reagents can be performed under Ar/H2. To verify that this set-up was able to reproduce literature results, we carried out the following asymmetric hydrogenation:
See a review of phospholane ligands for asymmetric catalysis for more, as well as the original paper [need ref] that the above reaction is taken from. While the results don't exactly match, the experimental set-up is clearly adequate for carrying out asymmetric hydrogenation reactions.
Under the same conditions as above, using the PZQ enamide, no reaction was observed, even after heating at 50 oC for 24 hours or increasing the pressure to 600 psi. Starting material is quantitatively recovered in each case.
Other conditions tried (no conversion in any case. Unless otherwise stated, the reactions were carried out in 5 mL solvent on 100 mg PZQ enamide) using the same conditions as for the test reaction above.
2 mol% (R)-RuCl[(p-cymene)(BINAP)]Cl.
(Formed in situ from 5 mol% Ru2(C6H6)2Cl4 and 10 mol% ligand)
PipPHOS (2 mol%) and Rh(COD)2BF4 (4 mol%)
Let's assume that catalysts like those above do actually function by delivery of the metal centre (and bound H) to the olefin by intramolecular coordination of e.g. a neighbouring carbonyl:
(taken from this PNAS paper)
...then the geometry of the PZQ enamide prevents such delivery. This is the basic problem here.
Update September 20th - First results from DSM (results will periodically be posted as screens are carried out):
Two conclusions from this:
1) The method of preparation of the enamide (using a sulfur melt) does not in this case lead to contamination with sulfur that deactivates catalyst.
2) MonoPhos does not give conversion under these conditions.
Update October 20th 2010 - Second update from DSM
Conditions were: cat = 0.01mmol, substrate PZQ = 0.2mmol, 5mL solvent, 25 bar H2, 16h
Catalyst structures are:
Update Jan 25th 2011 (data from DSM received December 21st 2010)
New attempts with Rh. Preparation of the 48 catalysts: The catalyst is preformed by stirring Rh(COD)2BF4 with 1.1 eq ligand for 1 h in DCM at rt. [Rh] = 0.042 M. See table below for the list of the 48 ligands used.
Preparation of the 48 reaction mixtures: The catalyst Rh/L (0.0042 mmol Rh in 0.5 mL DCM) in DCM solution is transferred to hydrogenation vials with the liquid handling robot. The substrate in MeOH (0.035 mmol in 2.2 mL MeOH) is added. S/C = 8.
Hydrogenation conditions: 25 bar H2, 60°C, 18 h, stirring = 300 rpm
No product was obtained for almost all catalysts - except for traces (a few %) of product for vials 29, 40, 42 corresponding to ligands CTH-PhanePhos (E4), JosiPhos-2-1 (H5), JosiPhos-212-1 (B6). These results are rather consistent. PhanePhos was already identified as a good substrate for a related substrate. The 2 JosiPhos present similar structural features: aryl-aliphatic phosphines with in both cases t-Bu groups.
See attached file “DSM 48 vial screen” for raw data for this screen, including the structures of the ligands employed.
Update July 3 2012 (using results obtained from DSM 22/09/2011)
Some promising results - the best to date. In the first run, below, there is full conversion for the first two entries, with some enantiomeric excess. The solvent change to TFE was probably important.
Conditions: cat = 0.01mmol, substrate PZQ = 0.2mmol, 5mL solvent, 25 bar H2, 16h. SL-M004-1 is the MandyPhos ligand of Solvias - structure may be seen in their catalog.
The second run used TFE. There is a lot of variation depending on ligand, but again some promising results here, with JosiPhos and PipPhos both doing well but wth low conversion.
Note added July 3 2012 - all results to date have been collated in a Google Spreadsheet for easier viewing.
PZQ can't be resolved as-is (unless anyone has any bright ideas how to resolve amides). One of the most promising strategies to prepare enantiopure praziquantel (using a strategy that starts from the racemate) is a classical resolution of praziquanamine (1, "PZQamine"). This molecule can either be made from scratch (it's an intermediate in the current PZQ synthesis) or can be obtained in high yield from PZQ itself.
PZQamine a secondary amine. We need a resolution that is inexpensive, and will work on a large scale.
One of the original patents in this area (US 3,993,760, 1976) details a way to do this resolution, shown below. This patent actually concerns a benzoyl analog of PZQ, but the effect is the same since PZQamine is the amine resolved.
Quinic acid is employed. The initially-formed crystals are the salt formed from the undesired enantiomer of PZQamine, so the mother liquor is concentrated to yield new crystals of the desired enantiomer. The exact procedure as reported is (page 5, column 7, "Example 1" lines 32-50):
"[(-)-PZQamine] may be obtained as follows: 24.3 g. of [(rac)-PZQamine] (m.p. 118-119 oC; obtainable by treating [(rac)-benzoyl analog of PZQ] with methanolic hydrochloric acid and subsequent heating to 180 oC at 15 mm Hg) are dissolved in 100 mL methanol and added to a warm solution of 30 g. quinic acid in 500 mL methanol. The mixture is refluxed for 15 minutes and then cooled to 20 oC. The crystals obtained are separated by filtration, the mother-liquor is evaporated to 100 mL and the crystals precipitating now are separated. The quinic acid salt of [(-)-PZQamine] is obtained; m.p. = 196 oC. The salt is dissolved in water, the solution is made alkaline and extracted with chloroform. After drying and evaporating the organic solvent the laevorotatory free base is obtained; m.p. = 120 oC, [a]D 20 = -306 o C (sic)"
The most obvious thing about this procedure is that the mass recovery of the desired enantiomer is not given. It is also doubtful that enantiopure material is produced without a final recrystallisation, but this is not specified. Nor is the solvent/concentration employed in the measurement of optical activity. Having to obtain the desired material from the mother liquor is also sub-optimal. This is a problem with using quinic acid as a resolving agent, since the unnatural enantiomer is not available in quantity.
Update August 27th 2010: Syncom BV rapidly identified a simple resolution method for PZQamine below. This method employs either quinic acid (which was also mentioned in the original Merck patent on PZQ) or dianisoyltartaric acid. (-)-PZQ is the active enantiomer desired in this project. Resolution with naturally-occurring D-(-)-quinic acid gives (+)-PZQamine. If we make the assumption that (-)-PZQamine leads to (-)-PZQ, and if we assume that we want to isolate PZQ from the solid component of the resolution rather than the mother liquor, then we would need L-(+)-quinic acid for this PZQ resolution which is not available. However (-)-dianisoyl-L-tartaric acid also gives (+)-PZQamine, implying (+)-dianisoyl-D-tartaric acid will give the desired (-)-PZQamine and hence (-)-PZQ. Both enantiomers of dianisoyltartaric acid are available commercially (for the same price), e.g. from TCI. The procedure and spectra are shown below, and will now be repeated in our lab here in Sydney. We'd encourage others to verify/check this also.
Syncom have done a fantastic job here. If there are remaining questions, they are these:
1) Which other chiral acids are good candidates for this resolution?
2) Which other solvents should we examined?
3) Which conditions should we optimize to keep the eventual cost of the large-scale process low?
4) Are there any good guidelines for procedures to maximize the yield of crystalline salt, and the yield of the subsequent re-isolation of the free amine?
5) Does anyone know of papers where other amines resembling praziquanamine (with a stereocentre a couple of carbons away from the NH) have been resolved?
6) What can we do with the undesired (S)-enantiomer of praziquanamine? Throw it away? Our default plan is to dehydrogenate and re-hydrogenate (via 2, above) to re-form (rac)-praziquanamine, but this is not elegant.
Relevant publications and groups (please just edit the page and add below using DOI to link):
Screen for resolution conditions using composition of mother liquor (Eli Lilly)
Please feel free to post any other suggestions below. Even better – plan to have a go in your own lab and post data here.
Anyone have an estimate of the genetic similarity between S. haematobium and S. mansoni? I have not been able to locate a figure.
Let's think long-term: How best to scale up production of PZQ?
This is likely most economically done in India or China (possibly Brazil or South Africa), all of which have well-established pharmaceutical manufacturing sectors. However, building manufacturing capacity in poorer disease-endemic countries stands to have enormous benefits for economic development and potential to eventually better meet local and regional general pharmaceutical demand!
Best places to start? Perhaps Uganda (starting with Quality Chemicals Industries, which opened a facility in Kampala recently pre-qualified by WHO to manufacture antiretroviral HIV drugs), other relatively disease-endemic countries (e.g. Rwanda) or other regional economic powers (e.g. South Africa).
Some of the African science and drug development networks could be helpful in identifying partners and determining how best to proceed: ANDI, SSI, ISHReCA, or the Emory South Africa Drug Discovery Program.
A major challenge may lie in weak regulatory capacity (complex and difficult to navigate) in many African countries.
Based on past experience with "Chiral Switch" projects in a commercial environment, I have drafted the attached document as a summary of my understanding of the aims of the PZQ project in the context of what would be needed (technically) to get (R)-PZQ to market in the western world. This does not examine manufacture per se, but rather the many necessary steps to obtain permission to market. I am sure there are some which I may have missed, but the point is that there would (in my past experience) be far more going on than focussing as at present on "manufacture" and "cost of goods", however important these will be in the long-run.
It is necessary to prove both safety & effectiveness. Now this may already be in place, but it is not yet evident on this site, and the expected standards change over time. This is why I suggest a critical review of the existing data, to identify any gaps in 21st-Century terms. If these are found to exist, then they can only presently be addressed through re-supply of (R)-PZQ by chromatographic means, which is a project in itself. Commercially, this would be pursued in parallel with the efforts to identify a viable manufacturing route to the single enantiomer.
This is intended as a discussion document, and I am receptive to amendments or additions. Some of the points made by "guest" under the PZQ Manufacturing Capacity thread are quite relevant.
Possible molecular targets for schisto research should be child pages off this one.
A recent paper suggests that thioredoxin glutathione reductase from S. mansoni is an interesting drug target for schisto, particularly given that PZQ was inactive against this target. From a quick look at the paper it appears as though no crystal structure is available for the enzyme, but screening is required. I wonder: is there anything the community can do here as a research project in virtual screening, given the data in the paper on compounds already tested? Mat
This is a parent page for improving the current methods for the purification of PZQ.
Methods for the purification of PZQ can be collated here.
1. This paper describes the purification of enantiopure PZQ using "chloroform/methanol 0–0.3% MeOH as a solvent system."
2. This paper uses 1:1 EtOAc/petroleum ether ramping to pure EtOAc.
3. This paper uses "thick layer preparative chromatography [prep TLC] benzene/ethyl acetate 1/1, silica gel."
Proton NMR assignment for PZQ has been carried out twice in the literature. Once in a review article: Analytical Profiles of Drug Substances and Excipients, 1998, 25, 463, and once in Arch. Pharm. (Weinheim), 1989, 322, 795-799. Of interest are the large differences in chemical shift between the diastereotopic protons on positions 1 and 6. Need image capture from the pdfs posted below...
Ryan has posted NMR data in a separate post.
This page details attempts to optimise the recrystallization of praziquantel. Anybody can add to/edit this page.
1. This patent (US patent 4,523,013) recrystallizes PZQ from "a mixture of petroleum ether and acetone," obtaining a 95% yield on the reaction.
2. This paper recrystallizes from ethyl acetate and hexanes to obtain a 70% yield on the reaction.
The best system the Todd lab has observed for the recrystallization of racemic praziquantel, as of Oct. 26, 2009, is the following:
Praziquantel is dissolved in a minimal amount of 50 ± 2ºC ethanol (solubility ca. 190 mg/mL; some results as high as 240 mg/mL; comfortably, 200 mg PZQ/mL 50ºC EtOH). This solution is allowed to cool to rt, and is left at 5ºC overnight before filtration, rinsing the crystals minimally with 5ºC EtOH. After drying under high vacuum, purity can be determined by melting temperature (138-139ºC, ref. RyanPakula blog, supporting NMR spectra coming soon) and proton NMR spectroscopy (CDCl3 works well).
This page details the various approaches to rac-PZQ
A new patent application using a multistep reaction (Ugi reaction) for the synthesis of Praziquantel
This recent patent application describes a new method for the synthesis of rac-Praziquantel. The key step in this procedure is a multi-step reaction, also known as the Ugi reaction, which enables the formation of the intermediate 5 up to 89%. [2,3] Under Broensted-acidic conditions a Pictet-Spegler-cyclization yields 6 or Praziquantel 7, depending on the various substituents R2.
 “Novel Synthesis of Praziquantel“, A. Dömling, Patent Application 2009, WO 2009/11533(A1), Language: German.
 “Multicomponent Reactions with Isocyanides” A. Dömling, I. Ugi, Angew. Chem.2000, 39, 18, 3168 – 3210.
 "Recent Developments in Isocyanide Based Multicomponent Reactions in Applied Chemistry", A. Dömling, Chem. Rev. 2006, 106 ,1, 17–89; DOI: 10.1021/cr0505728.
We just tried this exact reaction 2 weeks ago :) http://usefulchem.wikispaces.com/Exp258 [JCB]
The synthesis of rac-PZQ via the Pictet-Spengler route was developed by the Korean Shin Poong Pharmaceutical Company and obtains very low production costs of US¢7 per 600 mg tablet of the drug.
First step (amide bond formation to give 3)
Second step (attachment of acetal to give 4) - there's also a one-pot procedure to combine the first 2 steps
Third step (cyclization to praziquanamine to give 5)
Combination of steps 1-3 to give the dimethoxy analog of PZQ
 Formation of pyrazinoisoquinoline ring system by the tandem amidoalkylation and N-acyliminium ion cyclization: An efficient synthesis of Praziquantel, J. H. Kim, Y. S. Lee, H. Park and C. S. Kim, Tetrahedron 1998, 54, 7395-4000.
 Synthesis of Praziquantel via N-Acyliminium Ion Cyclization of Amido Acetals through Several Synthetic, J. H. Kim, Y. S Lee and C. S. Kim, Heterocycles 1998, 48, 11, 2279-2285.
 History of Praziquantel: http://www.stanford.edu/group/parasites/ParaSites2006/Praziquantel/history.html
The most obvious, and known, approach to rac-PZQ is using Reissert chemistry.
This approach has been used in the following publications:
 Organic Sonochemistry. Synthesis and Use of Reissert compounds under PTC-Ultrasound, J. Ezquerra and J. Alvarez-Builla, J. Heterocyclic Chem. 1988, 25, 917-925.
 Alkylation of isoquinolines via 2-benzoyl-1,2-dihydroisoquinaldonitriles: 1-Benzylisoquinoline, B. C. Uff, J. R. Kershaw and J. L. Neumeyer, Org. Synth. 1988, 6, 115; Org. Synth. 1977, 56, 19.
Child pages from this one detail attempts at this synthesis, and can host comment about it.
For an inexpensive route to rac-PZQ (and potentially the enantioenriched material), the relevant starting materials (below) are required. While prices may be obtained for these from commercial catalogues, we require realistic prices of these materials on a large (ton) scale to assess whether this approach to PZQ is economically viable. The starting materials are:
Cyclohexane carboxylic acid
Example price from Sigma-Aldrich = US$59 for 500 g = 12 cents per gram. Bulk?
Cyanide sources: KCN (InChI=1/CN.K/c1-2;/q-1;+1)
or NaCN (InChI=1/CN.Na/c1-2;/q-1;+1)
US$ 96 for 1 L (Sigma-Aldrich)
US$ 532 for 500 mL (Sigma-Aldrich)
An early step in the Reissert approach to PZQ is the synthesis of cyclohexanoyl chloride (InChI=1/C7H11ClO/c8-7(9)6-4-2-1-3-5-6/h6H,1-5H2) from cyclohexane carboxylic acid, (InChI=1/C7H12O2/c8-7(9)6-4-2-1-3-5-6/h6H,1-5H2,(H,8,9)). This step is needed if it is found that the acid chloride is prohibitively more expensive than the acid.
Cyclohexanoyl chloride was prepared by refluxing cyclohexanecarboxylic acid (390 mmol, 50.0 g) and SOCl2 (770 mmol, 56.0 mL, 1.95 eq.) for 4 h with exclusion of moisture. The mixture was allowed to cool. Gas bubbles continued to evolve from the solution and hence the mixture was allowed to stir overnight at rt.
The residue was purified by fractional distillation (1 atm, bp 186°C) to give the acid chloride as a light yellow liquid (47.0 g, 321 mmol, 82%).
1H NMR Cyclohexanoyl chloride
I'd like to suggest that this approach deserves its own thread - at present it is mentioned within the stereoselective synthesis project. I think it is more important than that - if you agree, there is existing content that should be migrated across to here.
Now that both racemic and single enantiomer PZQAmine are available, it is possible to determine phase diagrams - either binary, based on MPt, or ternary based upon solubility. But why do this ?
The essence of the matter is that options for resolving a racemic mixture or or purifying a partially resolved mixture are defined in the phase diagram, which is a represention of the significance of the solid crystalline form(s) which may exist. Pasteur's original resolution of tartaric acid was achieved by hand-picking apart individual crystals of the two enantiomers, but this was only possible because it naturally crystallises as what is called a conglomerate (ie it spontaneously resolves into separate crystals)(and that these were recognisably different, and physically large enough to handle). This resolution self-evidently happens because this is a "lower energy option" than if the molecules of (+) and (-) were to try to arrange themselves into the same crystal. If any of the crystals are characterised they will be found to be identical (xrpd, IR, MPt, DSC etc), with the exception of the sense of their chirality.
More often, a mixture of enantiomers will be happier to crystallise as a "racemic compound" in which (typically) pairs of opposite enantiomers first associate (think shaking hands), and then the pairs stack to form the larger crystal lattice. In such cases, if the single enantiomer can be isolated or made, it will typically crystallise in quite a different way, since it is unable to pair with itself in the same way it can with the opposite form. Thus crystals of the racemate and the single enantiomer will have different physical properties (xrpd, DSC, IR, MPt etc).
If you then consider a mixture of single enantiomer and racemate crystals, the two types of crystals behave effectively as impurities to each other, and an intimate mixture will display a depressed melting point relative to either "pure" crystal type. Somewhere on the scale from 100% racemate to 100% single enantiomer the melting point of the mixture will exhibit a minimum, called the eutectic composition. This is highly significant because if by some means you happen to obtain a euctic mixture it will be impossible to change its composition by crystallisation, either from the melt or from a solution. [caveat: - unless you can change the crystal form, but that's another story]
If you obtain a composition between that of the eutectic and the single enantiomer, then it will in theory be possible to crystallise your mixture up to enantiopurity. It is even possible to predict the ultimate possible yield/efficency given some basic data. However, if you have a composition between that of the eutectic and the racemate, you will never be able to obtain a pure enantiomer by crystallisation - instead it will be the racemate that crystallises first, and then once the liquor composition has reached the eutectic composition, it will be the eutectic that crystallises.
NB There is no way I know to predict where the eutectic will lie - it has to be determined experimentally.
So, when performing a diastereomeric salt resolution, it remains important to understand the crystallisation properties of the "free amine", as well as to know the behaviour of your salt. Without this, you cannot tell whether you need to recrystallise your salt up to enantiopurity before you crack it back to the amine, or whether you can rely upon crystallisation of the amine to play its part. Obviously it would be desirable to get 100% de salt from your resolution - but this too is subject to the energetics of that system, as expressed in its phase diagram. The latter are (even) more complicated than what I have described above, and beyond my scope to adequately explain, though there is plenty to be found in textbooks.
I have attached a file with a picture of a typical binary phase diagram, and some explanations...
Note - the raw data from this trial (HPLC traces for all the different columns) may be downloaded with this link. (large PDF file)
Here is a procedure from a German Patent Application from 1975 which uses quinic acid for the racemic resolution of praziquanamine. The corresponding US patent (US 3,993,760, 1976) provides the translated procedure (page 5, column 7, "Example 1" lines 32-50) here. However, this procedure gives less information about the yield obtained.
The result of the first trial conducted under the conditions described in the patent procedure was disappointing (see Procedure 1 and Patent Procedure). Methanolic solutions of Praziquanamine (1 eq.) and quinic acid (1.3 eq.) were combined and heated at reflux for 15 min. Upon cooling to room temperature, the precipitate which formed was filtered off. The filtrate was concentrated to a fifth of the volume and a second precipitate was collected. The amorphous precipitates contained a different enantiomeric excess of each enantiomers:
Precipitate 1: yield 72% (ee 11% (+)-PZA)*
Precipitate 2: yield 13% (ee 45% (-)-PZA)*
*) ee determined by optical rotation of the liberated amine from the diastereomeric salt (so far, all attempts to determinate ee by HPLC have been unsuccessful)
With small variations in the conditions the ee and yield of the praziquanamine resolution were improved (see Procedure 2). The methanolic solutions of amine and acid were combined in a sealed tube, heated to reflux and cooled to room temperature. The first precipitate was filtered off, the liquor was concentrated untill a second solid started to precipitate and then the mixture was stored for 10 h in the fridge at 6°C.
Precipitate 1: yield 59% (ee 48% (+)-PZA)*
Precipitate 2: yield 23% (ee 85% (-)-PZA)*
The current issues I am working on are finding a suitable method to determine the ee by using chromatography (HPLC, GC) and optimizing the resolution conditions.
Syncom BV found a resolution method for PZQamine by screening of a number of chiral acids and identified (-)-dianisoyl-L-tartaric as an excellent resolving agent for the (+)-enantiomere.
Praziquanamine (202 mg, 1.00 mmol) was dissolved in methanol (0.8 mL) and added to a warm solution of quinic acid (1.30 mmol, 250 mg) in methanol (4.2 mL). After a few seconds an amorphous precipitate formed. The stirred mixture was heated at reflux for 15 min and allowed to cool to room temperature. The amorphous precipitate was filtered off, the remaining solution was concentrated to about 0.8 mL and the resulting second amorphous precipitate was also filtered off. Both precipitates and the remaining filtrate were adjusted to pH 12 by adding a 2 N NaOH solution, extracted with DCM and dried over sodium sulfate. The concentrated residues were analyzed by optical rotation (and chiral HPLC).
*Calculation of the enantiomeric excess by optical rotation:
ee = ([α]obs/[α]max) x 100
[α]D20 (-)-praziquanamine = -306° 
Praziquanamine (202 mg, 1.00 mmol) was dissolved in methanol (0.8 mL) and added to a warm solution of quinic acid (1.30 mmol, 250 mg) in methanol (4.2 mL) in a sealed tube. The stirred mixture was heated to reflux and cooled to room temperature. The amorphous precipitate was filtered off, the remaining solution was concentrated to about 1.5 mL when a solid started to precipitate. The mixture was stored in the fridge at 6°C for 10 h and then the second amorphous precipitate was filtered off. Both precipitates and the filtrate were adjusted to pH 12 by adding a 2 N NaOH solution, extracted with DCM and dried over sodium sulfate. The concentrated residues were analyzed by optical rotation (and chiral HPLC).
Patent Procedure (Translated from the original patent application, language: German, page 11-12): 
24.3 g (+/-)-4-oxo-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isochinoline (m.p. 118-119°C […]) is dissolved in 100 mL methanol and added to a warm solution of 30 g quinic acid in 500 mL methanol. It is heated to reflux for 15 min and then cooled to 20°C. The obtained crystals are filtered off, the mother liquor is reduced to 100 mL and the precipitated crystals are separated. The quinic acidic salt of (-)-4-oxo-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isochinoline is obtained, m.p 196°C. The salt is dissolved in water, the solution is made basic and extracted with chloroform. After drying and evaporation the left rotating free base is obtained, m.p. 120°C, [α]D20 = -306°.
There are many ways to analyse PZQ with enantioselective chromatography. The approach used in-house at Sydney. We used ChiralcelOD-H column, Solvents: Hex:IPA:TEA 60:40:0.1, Flow Rate: 0.7 mL/min. The retention times were 12.362 & 14.932 mins. Sample HPLC trace is posted below.
Paper detailing the preparative separation of PZQ enantiomers by Intervet:
The preparative scale chromatography was performed on microcrystalline cellulose triacetate using methanol as the mobile phase, conditions under which the enantiomer having the negative optical rotation emerged first from the column . After crystallisation from methanol/water, (−)-PZQ was obtained in enantiomeric excess >99%, as determined by HPLC (column used Chiralcel OD-H). No residual other enantiomer (+)-PZQ was detected in this sample. X-ray structural analysis, using Cu-Kα radiation, of a monoclinic crystal in hemi-hydrate form obtained from said fraction by crystallization from methanol/water unequivocally proved the R-configuration. Full paper is here.
(Edits by Mat Todd on this page)
This page is intended as a place where people can discuss the student-led optimization of PZQ resolution. Relevant ELN is here, containing background here. Description of how students can help out is here (PDF). Paper will be assembled here. Blog post describing the project is here.
If you have questions/comments/suggestions, feel free to comment below. What we need at the outset are: 1) volunteers to carry out lab experiments, and 2) suggestions of ways to improve the existing process.
One of the enduring research problems associated with Praziquantel is that its mechanism of action is unknown, which is to say the biological target is unknown. If we were to find this target, we could design drugs rationally. There is evidence in the literature that the parasite's calcium ion channel is involved in praziquantel's mode of action. Robert Greenberg reviewed this area in 2005. Conor Caffrey has also summarised this research in a recent article on schistosomiasis chemotherapy. In general, the chain of events between PZQ contact, calcium entry and parasite death is still unknown. The hypothesis of direct binding between PZQ and ion channel proteins is unsatisfactory because: a) Resistant strains of the parasite show no changes in this protein; Immature worms are almost completely refractory to PZQ, although they express the same calcium channel beta-subunit proteins; the functional aspects of PZQ effects on calcium channels have been documented only indirectly by current changes in oocytes expressing single components of the schisto channel; no indication exists as to whether calcium channels would interact directly with PZQ or through other intermediates. Paper b) The existence of voltage gated calcium channels at the worm surface has not been demonstrated. c) Calcium influx and muscle contraction are not always followed by worm death. Indeed, it is still an assumption that calcium influx is the cause of schistosome death. Paper Rather than me sifting through the literature, I'd like to ask you all for help in collating links to relevant papers here, so suggestions for and against calcium channels? Possible other target proteins will form separate project pages of their own linking off this one. Mat Todd
Does praziquantel bind actin? Tallima and El Ridi recently proposed actin as the molecular target of PZQ, based on affinity chromatography studies. Todd and Cioli performed similar experiments (with a different support-bound PZQ) and found actin bound to PZQ-free support, implying that actin is a false positive, identified due to its cellular abundance. Other evidence? Required projects?
Another interesting PZQ paper has come out of the cell biology group at the CNR in Rome, concerning the mode of action of PZQ. Using radioactive calcium, the group have demonstrated that calcium influx in itself is not enough to kill parasites. Pre-incubation with cytochalasin D promoted calcium influx but protected the worms from PZQ's schistosomicidal effects.
There are three obvious methods to identify PZQ's in vivo target: 1) Radiolabelled PZQ (tritiated probably best). It's likely that the interaction between PZQ and its target is not long-lived, which means purely radiolabelled PZQ is not the best strategy. 2) Solid-supported PZQ. It may be possible to attach PZQ to a solid support and pass the parasite proteome down a column of this support to identify retarded proteins. For preliminary results in this direction see the actin project page. 3) A strong approach is the attachment of a photoaffinity probe to PZQ, e.g. an azide. Incubation of photoaffinity-PZQ ("hv-PZQ") with schistosome extract followed by irradiation with UV light should cross-link the PZQ with the target protein. Naturally such proteins need to be identified, implying a radioactive photoaffinity probe is needed, which is more difficult and expensive to make. For approaches 2 and 3, a suitable place to attach things to PZQ must be found, which links to the search for effective analogs of PZQ where variation in the structure of the molecule is tolerated.
There may be members of this community who do not yet have direct experience of developing a new medicine from concept to pharmacy shelf. Be assured, there is a lot more to it than "just" the synthesis of the API. Why not take advantage of a Free ACS Webcast on May 6, 2010 Thurs 2:00-3:00 PM ET
From a Beaker to a Bottle: Overview of the Drug Discovery and Development Process for Small Molecule Therapeutics
A free webcast from the American Chemical Society as part of their Professional Growth and Development Series. Learn about the drug development process and find out how long it takes, how much it costs, and the odds of getting a new drug approved.
I am sure this should make an excellent introduction, so I urge you to sign up at:
Information at http://www.SeventhStreetDev.com/SignUp.aspx
We could use this page afterwards to discuss matters arising, especially wrt PZQ
Best Regards, Nick