Schistosomiasis Research Community
Welcome, I'm Mat Todd, the community leader for schistosomiasis research.
Why Schistosomiasis is Important
Schistosomiasis is one of the most burdensome of the neglected diseases, with 200 million people infected and 400 million people at risk. Infection is widespread with a relatively low mortality rate, but a high morbidity rate, causing severe debilitating illness in millions of people. The disease is often associated with water resource development projects, such as dams and irrigation schemes, where the snail intermediate hosts of the parasite breed. The drug of choice for the treatment of schistosomiasis is praziquantel (PZQ).
Join Us and Login
As a guest on The Synaptic Leap, you can browse and look all you want. You must login and create a user profile for yourself if you want to participate. Once you are logged in you may begin collaborating with other scientists world-wide and become a part of the collective intelligence trying to help improve the drugs available for schisto.
Get Involved with an Open Research Project
See current projects for a list of our active open research projects. You may learn more about these projects and learn how you can participate. Or, you may "add a child page" (see the link at the bottom) to initiate and describe your own open research project for schistosomiasis.
If you're still in the brainstorming phase of starting a project, write a blog article to discuss your ideas with other scientists around the world also studying schistosomiasis. Working together, we can direct the research towards the most promising ideas.
You can also help shape and direct other schisto research ideas by reading and commenting on other community posts.
Keep Up To Date with The Latest Schisto Information
To assist you with your schisto research we have pulled together a research tools page for schisto as well as an RSS news feeds for schisto. If you know of a useful tool that we don't have on the list, login and add a comment for others to see.
Current Projects for Schistosomiasis
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.
A Summary of What is Needed Right Now
The development of a low-cost enantioselective synthesis of PZQ is an open source project, meaning contributors can change anything they wish on these pages. If you wish to contribute, please don't leave comments here. Instead, go to the pages described below and either edit the pages to insert information or leave comments. You can also ask general questions on the Friendfeed page. Please avoid emails if at all possible.
Current help is needed from you (the community) with the following in schisto projects.
1) Help obtaining prices for bulk starting materials for racemic PZQ syntheses. This really needs input from industrial chemists who know of suppliers of these materials for low prices.
2) Advice on how to increase the yield of, or otherwise improve the procedure employed for, the synthesis of acid chlorides - in our case the synthesis of cyclohexanoyl chloride.
3) As a result of Michael's spectacular success with the cleavage of the amide in PZQ to give praziquanamine, we need suggestions on how to resolve praziquanamine - i.e. which chiral acid should we look at, and what sorts of reaction conditions?
Enantioselective Synthesis of Praziquantel
Aims:
- To develop an efficient enantioselective synthesis of the bioactive enantiomer of Praziquantel
- To optimize such a synthesis so it is suitable for large-scale manufacture.
Background
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, 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). 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.
Summary of the Project
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.
References
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.
7. http://www.schisto.org/
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.
Aza-Henry Route to PZQ
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.
Technical Issues:
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.
Reduction of Aliphatic Nitro Groups
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?
Cheers,
Mat
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.
Updates to Aza-Henry Catalysts
Just a quick update compiled by one of my students, Wing Yan, on some aza-Henry catalysts not included in the first post.
Cheers,
Mat
Pictet-Spengler Route to (R)-PZQ
Wayne Best of Epichem has suggested an alternative route to (R)-PZQ, based on a Pictet-Spengler (PS) reaction of a chiral phenylethanolamine. This is an attractive route, since 1 to 2 should work well (good asymmetric induction) and the products resemble PZQ.
Pictet-Spengler route to PZQ?
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. 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.
Literature PS Approach to PZQ
The use of an auxiliary is of course atom-inefficient. The use of a chiral starting material, in the generation of some analogue of PZQ, could be an interesting project. Anyone have a suggestion for a suitable commercially-available chiral starting material? Anyone want to have a go?
Mat
Starting from the Racemate
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.
Craig's Suggestion
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. The review has no experimental details - but the reaction has been published in...[need reference]
2. Resolution
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.
Resolvable intermediates
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. However, to evaluate the resolution of 4 in the lab, an efficient method for cleavage of the cyclohexanoyl from PZQ is needed. We have started to look at this here. [links]
Mat
Promising Targets for Schistosomiasis
Possible molecular targets for schisto research should be child pages off this one.
Thioredoxin Glutathione Reductase
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
Purification of Praziquantel
This is a parent page for improving the current methods for the purification of PZQ.
Chromatography of PZQ
This page details attempts to optimise the purification of praziquantel by chromatography, with an emphasis on preparative, rather than analytical, work. Anybody can add to/edit this page.
Literature
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."
NMR Spectroscopy of PZQ
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.
Recrystallization of PZQ
This page details attempts to optimise the recrystallization of praziquantel. Anybody can add to/edit this page.
Literature
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).
Racemic Syntheses of PZQ
This page details the various approaches to rac-PZQ
Pictet-Spengler route to rac-PZQ
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.
[1] 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.
[2] 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.
[3] History of Praziquantel: http://www.stanford.edu/group/parasites/ParaSites2006/Praziquantel/history.html
Reissert Approach to rac-PZQ
The most obvious, and known, approach to rac-PZQ is using Reissert chemistry.
This approach has been used in the following publications:
[1] Organic Sonochemistry. Synthesis and Use of Reissert compounds under PTC-Ultrasound, J. Ezquerra and J. Alvarez-Builla, J. Heterocyclic Chem. 1988, 25, 917-925.
[2] 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.
Starting Materials for the Synthesis of rac-PZQ
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:
Reissert route chemicals:
Isoquinoline
(InChI=1/C9H7N/c1-2-4-9-7-10-6-5-8(9)3-1/h1-7H)
Cyclohexanoyl chloride
(InChI=1/C7H11ClO/c8-7(9)6-4-2-1-3-5-6/h6H,1-5H2)
Cyclohexane carboxylic acid
(InChI=1/C7H12O2/c8-7(9)6-4-2-1-3-5-6/h6H,1-5H2,(H,8,9))
Example price from Sigma-Aldrich = US$59 for 500 g = 12 cents per gram. Bulk?
Thionyl Chloride
(InChI=1/Cl2OS/c1-4(2)3)
Chloroacetyl chloride
(InChI=1/C2H2Cl2O/c3-1-2(4)5/h1H2)
Cyanide sources: KCN (InChI=1/CN.K/c1-2;/q-1;+1)
or NaCN (InChI=1/CN.Na/c1-2;/q-1;+1)
Pictet-Spengler route chemicals:
2-Phenylethanamine
(InChI=1/C8H11N/c9-7-6-8-4-2-1-3-5-8/h1-5H,6-7,9H2)
US$ 96 for 1 L (Sigma-Aldrich)
2,2-Dimethoxyethanamine
(InChI=1/C4H11NO2/c1-6-4(3-5)7-2/h4H,3,5H2,1-2H3 )
US$ 532 for 500 mL (Sigma-Aldrich)
Synthesis of Cyclohexanoyl Chloride from Cyclohexane Carboxylic Acid
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.
Experimental:
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
Target of Praziquantel
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
Actin
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?
Role of Calcium Flux
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.
Strategies to Identify the Target of Praziquantel
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.