Todd:PZQ Resolution

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The Resolution of Praziquantel

Michael Woelfle,¹ Jean-Paul Seerden,² J. de Gooijer,² K. Pouwer,² Piero Olliaro³ and Matthew H. Todd¹*
1. School of Chemistry, The University of Sydney, NSW 2006, Australia
2. Syncom B.V., Kadijk 3, 9747 AT Groningen, The Netherlands
3. Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization, Avenue Appia 20, 1211 Geneva 27, Switzerland

• To whom correspondence should be addressed: Tel. +61 2 9351 2180, matthew.todd@sydney.edu.au

Authors are members of The Synaptic Leap, where the original research was coordinated and first disclosed.

Abstract

The preparation of enantiopure praziquantel is described through the classical resolution of a hydrolysis product, praziquanamine. The protocol is experimentally simple, and involves no chromatography. The method was found and validated by an unusual research mechanism - open science - where the details of the collaboration (involving academic and industrial partners) and all research data were available on the web as they were acquired, and anyone could take part.

Introduction

Schistosomiasis (Bilharzia) is termed a "neglected" tropical disease owing to the continuing low level of investment in treatments, prevention and research, yet the disease accounts for an extraordinarily high level of suffering around the world.(Ross 2002, Chitsulo 2000) While it is true that hundreds of millions of people are infected or at risk of becoming infected,(Steinmann 2006) typical methods of measuring the impact of chronic infection caused by such diseases can underestimate their impact.(King 2008) Schistosomiasis has been called a "silent pandemic".(King 2005)

Over the past decades several compounds have been used for the treatment of schistosomiasis,(Caffrey 2007; Caffrey 2009; Fenwick 2003) but today there is only one drug of choice, praziquantel (PZQ).(Andrews 1983, Doenhoff 2008) Mass treatment programs, such as that coordinated by the Schistosomiasis Control Initiative, are now reaching and treating millions of people, though there is expected to be a large growth in the demand for PZQ in the coming years.(Hotez 2010)

With increasing use comes an increase in the risk of the development of resistance or tolerance by the parasite. Such resistance was famously developed via an artificial selection experiment in the laboratory,(Fallon 1994) and examples of resistance have already been noted in the field.(Liang 2010, Melman 2009, Doenhoff 2002, Liang 2001) Reliance on a single drug for intensive mass treatment is dangerous. While there have been attempts to find bioactive PZQ analogs,(Dong 2010, Laurent 2008, Ronketti 2007) as well as the discovery of new compounds for the treatment of schistosomiasis based on different modes of action,(ref) in the short term it is sensible to continue to use PZQ in a way that maximises its life as a useful drug.

PZQ is synthesised and administered as a racemate. The (-)-enantiomer is the eutomer(Xiao 1989, Wu 1991) and has the (R) configuration.(Andrews 1983, citing "R. Grosser, Bayer AG, internal report (1982)"; Meyer 2009) Administration of the pure eutomer resulted in fewer side effects than the racemate.(Wu 1991) The inactive (+)-enantiomer is associated with side effects and is also primarily responsible for the extremely bitter taste of the pill;(Meyer 2009) compliance in taking PZQ in the field is already a challenge.(Parker 2008) (ref to bitterness impacting this?) The typical dose per treatment (600 mg)(correct?) is large. To postpone the development of resistance it would be advantageous to increase the dose of the drug administered, while decreasing the pill size, reducing side effects and removing the bitter taste. All this could be accomplished were the drug to be made available as a single enantiomer. For these reasons investigations into the viability of a process-scale route to enantiopure PZQ were included in the WHO/TDR business plan for the coming years.[TDR 2007] Availability of the separate enantiomers would also be a valuable tool for the elucidation of the mechanism of action of the drug, still unknown after more than 30 years of use.

There are typically four methods available for the conversion of a racemic synthesis to one that generates the same molecule as a single enantiomer: 1) Enantioselective synthesis, 2) Chromatographic separation, 3) Stereoablation and 4) Resolution. To date there have been no reports of any such strategies that have promise for the large-scale generation of enantiopure PZQ.[Ref to others] The approaches are all being investigated by the scientific community on an online collaborative open science portal known as The Synaptic Leap. This article describes the recent solution to the resolution of PZQ identified using this open science mechanism. The protocol developed permits the convenient generation of PZQ enantiomers on a gram scale.

Scheme 1. Strategies Towards the Preparation of Enantiopure (R)-(-)-Praziquantel ((R)-1)

An attractive approach to the resolution of PZQ is to employ the commercially-available racemic material which is already available on a large scale for a low price. PZQ contains only amide functional groups that are not amenable to resolution, so the drug needs to be converted to a species that is amenable to cocrystallization, and the obvious solution is to cleave the exocyclic amide bond to give a secondary amine (2, PZQamine). A similar strategy was reported in one of the original patents in this area,(Pohlke 1976) albeit on an aromatic (benzoyl rather than cyclohexanoyl) analog of PZQ; the hydrolysis of this same analog was also reported more recently in a paper concerning the synthesis of other analogs of PZQ. It was found that the resolution of PZQamine could be effected with quinic acid. However, no information was given concerning the yield of material isolated, nor its enantiopurity. Further, use of naturally-occurring D-(-)-quinic acid gave crystals of the quinic acid salt containing the undesired enantiomer of PZQamine, necessitating the isolation of the desired enantiomer of PZQamine from the mother liquor, typically an undesirable approach. This is not a trivial problem to rectify since L-(+)-quinic acid is not available in quantity. The hydrolysis of PZQ itself has been reported for purposes not related to the resolution of PZQ.(Mitsui 2001, Dong 2010) A recent report on the attempted resolution of PZQamine (synthesized de novo, not from the hydrolysis of PZQ) reported that common chiral acids (tartaric acid, camphorsulfonic acid, mandelenic acid and Mosher's acid) under a variety of conditions failed to result in resolution.(Laurent 2008) Ultimately PZQamine enantiomers were obtained by the covalent attachment of Mosher's acid, chromatographic separation, and finally cleavage of the exocyclic amide bond under harsh conditions.

Patents to check (cited in Andrews review): Resolution of intermediates:

• J. Seubert, H. Thomas, and P. Andrews, German Pat. Appl. 2,362,539
• J. Seubert, German Pat. Appl. 2,504,250
• R. Pohlke, F. Loebich, J. Seubert, H. Thomas, and P. Andrews, German Pat. Appl. 2,331,713

Recycling unwanted enantiomer through oxidation and hydrog:

• J. Seubert, German Pat. Appl. 2,418,111

Results

The hydrolysis of PZQ to give PZQamine has been reported to occur in low yield (1 N HCl, reflux, 3 hours, 27% yield)(Mitsui 2001) using conditions which gave efficient cleavage of a PZQ analog where cyclohexanoyl was replaced with benzoyl. (patent, Frehel) More recently the hydrolysis was reported to occur in 2 N HCl solution under reflux for 20 hours in 80% yield.(Dong 2010) In our hands cleavage in a mixture of 1 N HCl solution in ethanol (4:1) under reflux for 26 hours gave PZQamine in 92% yield. The amine could conveniently be isolated by recrystallization from toluene on a multi-gram scale.

To assay any resolution method for this amine, its enantiomers needed to be separable using enantioselective HPLC. Use of a standard stationary phase for this analysis (Chiralcel OD-H) did not permit this separation. A screen of other stationary phases revealed that several were suitable: Chiralpak X Y Z.(ref TSL page and raw data)

With a suitable analytical method in hand a number of different resolution agents and conditions were screened.

Quinic acid

Tartaric

Repeat

Given that many people may not have access to the column necessary for the determination of the enantiomeric excess of PZQamine, we confirmed that, in ethanol at a convenient concentration, the optical rotation and enantiomeric composition of PZQamine are linearly related (Figure X). Thus purification of PZQamine to constant melting point and measurement of the optical rotation under these conditions will give an adequate measure of the enantiomeric excess achieved in the resolution without the need for chromatographic analysis. In fact a linear relationship is also seen for PZQ itself under related conditions, as well as a linear relationship between concentration and observed optical rotation up to 1 g per mL (correct concentration?) ([Figure X]).

The conversion of enantiopure PZQamine to PZQ may be achieved by X. There is no loss of optical purity in this step.

Discussion

Comments on Chemistry: The resolution of PZQamine was reported in one of the original PZQ patents, but there was little experimental detail and the method had a number of shortcomings (expand on this). Since then there have been no further reports of the successful preparation of enantiopure PZQ using this approach, despite attempts along these lines. Resolution is attractive as a means to prepare enantiopure PZQ since costs can be kept low, and operationally the methods are typically simple, requiring crystallisations rather than elaborate chromatographic separations or stereocontrolled syntheses. It is thus the hope that the present method will find use outside chemical laboratories.

Draft comments on open science: Several difficult problems were overcome during this work. A set of conditions were found for the high-yielding hydrolysis of PZQ - a reaction that had previously only been reported in low yield under very harsh conditions.(Mitsui 2001) The identification of an effective resolving agent for PZQamine, however, was a significant undertaking and initially attempts by our academic laboratory failed. Rather than continuing to make and screen potential resolving agents in-house, and appeal was made to the general chemical community on several websites. Since all research data was being made openly available during the project, it was hoped that others with experience in resolving secondary amines would have ideas as to what to try. In the event a private company, Syncom, elected to screen a number of conditions in-house both for the analytical separation of PZQamine enantiomers and the resolution of the amine. Since Syncom are specialists in this field, the identification of an effective resolving agent was rapid. There are several points of interest here. The first is that since the project was taking place in the open, the relevant experts identified themselves. This is a key advantage of open science over closed lab science where collaborators must typically be found and targetted, and where the utilization of experts is necessarily limited by the professional acquaintances of those taking part. Second is the involvement of a private company in an open source undertaking, where there is no promise of IP reward in return for the commitment of resources. The question arises as to what, then, is the motivation for the involvement of industrial partners in open projects. In other fields such as computer science/software development it is well understood that there are advantages in this kind of approach. (examples coming)

Experimental Section

(rac)-PZQ was a gift from WHO/TDR; the sample was originally synthesized by Merck. Analytical samples of (R)- and (S)-PZQ were a gift from Intervet GmbH and were prepared according to the literature method.(X)

Procedures

Analytical data for rac-PZQ Enantiomeric composition of praziquantel can be assayed by a number of enantioselective HPLC columns. For example: Chiralcel OD-H column, Hexane/Isopropanol/triethylamine solvent system (60:40:0.1), flow rate 0.7 mL per minute. Retention times 12.4 and 14.9 minutes. Sample HPLC trace for racemic PZQ: Media:Rac-PZQ_HPLC.JPG

Analytical method for praziquanamine (see http://www.thesynapticleap.org/node/315) Enantioselective HPLC columns suitable for baseline separation of the enantiomers of praziquanamine include Chiralcel OJ-H, Chiralpak IA and AS-H; eluent solvent system: heptane/EtOH/Et₂NH (60:40:0.2) at 0.5 ml/min flow rate. Columns found to be unsuitable include Chiralcel OD (Laurent 2008), OD-H, Chiralpak IB and AD-H. The raw traces may be downloaded from here. Sample trace (smaller file): Media:PZQamine_trace.pdf

(rac)-Praziquanamine, 2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (X)

(rac)-PZQ (20.0 g, 64.0 mmol) was dissolved in a mixture of EtOH (150 mL) and 1 N HCl (600 mL) and heated at reflux for 26 h. The ice-cooled solution was treated with 5 N NaOH until a pH of 12 was reached and extracted three times with DCM (30 mL). The combined organic layers were washed with basified brine solution (pH ~12), dried over sodium sulfate and concentrated under reduced pressure. The residual yellow solid (12.4 g) was recrystallized from toluene and a further batch of analytically identical crystals were obtained from the mother liquor after concentration and recrystallization. PZQamine was thus obtained as a pale yellow solid (11.9 g, 58.8 mmol, 92% yield).

m.p. 117-118^oC (lit. 117-119^oC (Kim et al. Tetrahedron 1998)); 118-119^oC (Merck patent); 116-118^oC (Laurent 2008)). ¹H NMR (CDCl3, 200 MHz): δ 1.76 (bs, 1H), 2.64-3.02 (m, 4H), 3.49 (d, J=17.6, 1H), 3.61 (d, J=17.4, 1H), 3.67 (ddd, J=13.2, 4.7, 1.2, 1H), 4.69-4.85 (m, 2 H), 7.04-7.20 (m, 4H). ¹³C NMR (CDCl3, 50.3 MHz): d = 29.4, 39.4, 50.4, 50,7, 57.5, 125.3, 127.2, 127.6, 130.0, 134.8, 135.6, 167.9. C12H14N2O (202.3). Spectroscopic data match those reported in the literature.(Kim et al. Tetrahedron 1998, Laurent 2008))

Synthesis of Resolving Agent

Procedure: (see: http://www.ourexperiment.org/racres_pzq/665)

(+)-Di-p-anisoly-D-tartaric acid (X)

m.p. 186-188.5°C (lit: 186°C (P. Rabe, Justus Liebigs Annalen der Chemie)), ¹H NMR (DMSO-d6, 200 MHz) : δ = 3.50 (bs, 2H), 3.86 (s, 6H), 5.80 (s, 2H), 7.13 (d, J=8 Hz, 4H), 7.97 (d, J=8 Hz, 4H). ¹³C NMR (DMSO-d6, 50.3 MHz) : δ = 55.7 (2C), 71.2 (2C), 114.4 (4C), 120.7 (2C),131.6 (4C), 163.8 (2C), 164.3 (2C),167.4 (2C). IR (neat): nu = 2943 cm-1, 1720, 1662, 1601, 1243, 1170, 1103, 1012, 847, 762, 691. MS (ESI (+)) m/z (%): 875 (35) [2M+K]⁺, 859 (85) [2M+Na]⁺, 441 (53) [M+Na]⁺, 435 (85), 329 (100). HRMS (ESI (+)) Calcd. for [C20H18O10+Na]⁺: 441.07922, found: 441.0790. [alpha]D²⁰ = +163° (c=1, EtOH), (lit: [alpha]D20 = +167° (c=1, MeOH) (P. Rabe, Justus Liebigs Annalen der Chemie); 163° (c=1, EtOH) (TCI America, Chemical Supplier for (+)-Di-p-anisoyl-D-tartaric Acid)). C20H18O10 (418.4).

Resolution of PZQamine

Merck patent gives m.p. of quinic acid salt as 196 oC. Gives (-)-PZQamine m.p. 120 oC and alpha of -306 o. No indication of enantiopurity, nor solvent/concentration employed for the measurement of alpha. (Laurent 2008) gives Specific rotations of (R)-PZQamine and (S)-PZQamine as [α]D = –152 (c = 4.10–3, CH2Cl2) and [α]D = +146 (c = 5.10–3, CH2Cl2) for enantiomeric excesses higher than 95 and 91%, respectively. Include method for determining ee of PZQamine without HPLC.

Procedure: (see: http://www.ourexperiment.org/racres_pzq/1309)

rac-praziquanamine (10.0 g, 49.5 mmol) and (-)-dibenzoyl-L-tartaric acid * 2 i-PrOH (23.7 g, 49.5 mmol) were dissolved in a mixture of i-PrOH (450 mL) and water (90 mL) by heating the stirred mixture. The solution was allowed to cool to room temperature. After 2 h kept at room temperature the colorless crystals were filtered off and washed with a minimum amount of cold i-PrOH/water (5:1) afforded 12.1 g (21.6 mmol, 44%) of pale yellow crystals (m.p.=143-145°C). The diastereomeric salt was recrystallized from a mixture of i-PrOH (180 mL) and water (90 mL) and the crystalline precipitate was filtered off after the mixture was kept for 2 h at room temperature yielded in 9.54 g (17.0 mmol, 34%) colorless spicular crystals (m.p.=143.5-145.5°C).

Synthesis of enantiopure PZQ from enantiopure PZQamine

Procedure: (see: http://www.ourexperiment.org/racres_pzq/1468)

To an ice-cooled solution of R-(-)Praziquanamine (3.27 g, 16.2 mmol) and triethylamine (2.45 g, 3.38 mL, 24.3 mmol, 1.5 eq.) in dichloromethane (80 mL) was added dropwise cyclohexanoyl chloride (2.62 g, 2.39 mL, 17.8 mmol, 1.1 eq.) at 0°C and stirred for 14 h at room temperature. The solution was quenched with water (10 mL) and stirred for further 30 min. The layers were seperated and the organic layer was washed with sodium carbonate solution, 0.5 N HCl solution and brine, dried over magnesium sulfate and concentrated under reduced pressure. The remaining yellow oil became solid after drying under high vacuum and storing in the fridge at 5°C. The pale yellow solid was recrystallized from acetone/hexane (35 mL, 1:1 mixture) and two further batches of analytically identical crystals were obtained from the mother liquor after concentration and recrystallization. R-(-)PZQ was obtained as colorless crystals (4.56 g, 14.6 mmol, 90%).

m.p. 113.5 - 114.5°C. [α]D20 = -136° (c=1, EtOH). HPLC: Chiracel OD-H column, Eluent: hexane/iso-propanol/triethylamine 60:40:0.1, Flow rate 0.7 mL per minute. RT: R-(-)-PZQ: 11.1 min, S-(-)-PZQ 13.6 min.

Mechanism of Collaboration

The initial request to the community for help with the resolution of PZQ was posted on The Synaptic Leap on X. Initial experiments towards the resolution of PZQ began in X with the report of the cleavage of the cyclohexanoyl moiety. Early attempts to resolve ZAamine were posted in X. A request for assistance was posted on another website (LinkedIn) on X, and a response from JPS was received in X. PZQamine was synthesised in the School of Chemistry, Sydney on X and mailed to Syncom. The samples were received on X, and experiments in the separation of the enantiomers by chiral HPLC were performed on X. Experiments

Acknowledgements

We thank Denis Daumerie (WHO/TDR) for a generous gift of racemic praziquantel and Harald Sekljic (Intervet Innovation GmbH) for a gift of analytically pure samples of (R)- and (S)-PZQ.

References

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