Todd:PZQ Student Optimization

=Student-led Optimization of the Resolution of Praziquantel=

New authors - add yourselves here in alphabetical order:

Charles T. Cox, Department of Chemistry, Stanford University, Stanford, CA 94305, USA Bojan Milic, Departments of Chemistry and Biology, Stanford University, Stanford, CA 94305, USA Piero Olliaro, Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization, Avenue Appia 20, 1211 Geneva 27, Switzerland Matthew H. Todd, School of Chemistry, The University of Sydney, NSW 2006, Australia Michael Woelfle, School of Chemistry, The University of Sydney, NSW 2006, Australia

Authors have contributed to the open electronic lab notebook where the original research was first disclosed. Full, original reports from students who have contributed my be found here: PZQ Student Optimization Reports.

Abstract
A preparation of enantiopure praziquantel was recently discovered that involves the classical resolution of a hydrolysis product, praziquanamine. The protocol is experimentally simple, and involves no chromatography. For the procedure to be of the greatest impact, the price must be kept very low, meaning that the process must be highly optimized. This paper describes the results obtained by a distributed group of students working on this problem in an open source manner.

Introduction
Description of schisto, PZQ and the need for (R)-PZQ: Schistosomiasis is: Praziquantel is: It is desirable to deploy the drug enantiopure because:

Description of the existing process

Identification of current shortcomings:

Brief outline of how the project was intended to operate:

Optimization of Hydrolysis of (rac)-PZQ
The starting point (published procedure) was the use of 1:4 EtOH:1 N HCl at reflux for 26 hours (750 mL total for 20 g PZQ). The cyclohexanecarboxylic acid byproduct was removed by washing with ethyl acetate, and the aqueous portion was basified with 5 N NaOH and PZQamine was extracted with dichloromethane. The crude solid thus obtained could be recrystallised from toluene, giving PZQamine in 92% yield.

Attempts: Original optimization results. BM 1-1: 1 g scale, at 0.5, 1 and 2 N HCl, 37.5 mL, basified with 6 N NaOH. But >100% yields obtained since samples contained solvent. TLC indicated incomplete reaction for reaction employing 0.5 MN HCl. BMSPDYL 1-2: 2 g scale, 75 mL, 22/23 h reflux, use of 10 N NaOH for basification, 79% yield. BMSPDYL 1-2: 2 g scale, 75 mL, 22 h reflux, use of 10 N NaOH for basification, 64% yield. MW 2-14:

Formation of PZQamine salt
Milic: While resolution of PZQamine had previously been achieved through the implementation of a number of L-tartaric acid derivatives, the two particular resolving agents applied in this experiment, namely (−)-O,O′-Di-pivaloyl-L-tartaric acid (3) and (−)-O,O′-Di-p-toluoyl-L-tartaric acid (4), had not been utilized or tested in the isolation of a single enantiomer of PZQamine.1 For reasons which remain unclear, (3) did not result in the formation of a salt/precipitate when subjected racemic PZQamine in the presence of isopropanol and water, despite its structural similarity to successfully implemented resolving agents (Figure 3).1 Further experimentation notwithstanding, the experimental results strongly, if not conclusively, indicate that (3) unfortunately cannot be utilized as a resolving agent in the resolution of PZQamine. Based on spectral data collected, (4) was successful in the resolution of PZQamine to yield R-(–)-PZQamine (5) (0.1110 g; 44%; 79% ee). Given that the resolution of PZQamine does not yield a product which has different IR or 1H NMR spectra from the starting material, the IR and 1H NMR results only serve to confirm that the product is the desired PZQamine. Most importantly, the IR spectrum (Thin Film) of the resolved PZQamine reveals, among others, a peak at 3367.36 cm-1, consistent with the presence of N–H stretching, as well as a peak at 1636.16 cm-1 corresponding to a C=O amide stretch. Indeed, the IR spectrum of the resolved PZQamine is wholly consistent with that of racemic PZQamine, as expected. Furthermore, the 1H NMR spectrum of the resolved intermediate is in complete agreement with NMR results reported in literature, with the exception of peaks at 4.0 ppm and 1.2 ppm, respectively.1 The two incompatible peaks mentioned are consistent with peaks for isopropanol. As such, the isolated compound is not 100% pure. It follows from such a conclusion that the calculated 44% yield is in fact higher than the actual yield. Nevertheless, the yield value of 44% is consistent with literature values available for similar resolving agents implemented in PZQamine resolution, and is not far away from the optimal figure of 50%.1 It should be noted that, although performing the experiment a second time resulted in a yield of 118% (0.8275 g), such a yield was obtained due to inadequate solvent evaporation. As such, the figure of 44% used in the above discussion is far more realistic and representative of the reaction. Repeating the experiment and diligently drying the isolated substance would provide valuable information regarding the yield of resolved PZQamine achieved by (4). Furthermore, given that 1H NMR data strongly suggests that isopropanol is the only impurity present in a detectable quantity, it is not unreasonable to conclude that non-trivial quantities of side products were not synthesized. The 1H NMR spectrum of the product with a shift reagent, in this case Eu(hfc)3, provides the information necessary to determine the enantiomeric excess of the PZQamine, and thereby judge the effectiveness of (4) as a resolving agent. Using the peaks at 4.33 ppm and 4.7 ppm, which correspond to the chiral hydrogen of PZQamine, the enantiomeric excess achieved was determined to by 79%. As such, the resolving agent (4) contributed to the fairly successful enantioselective isolation of the desired R-(–)-PZQamine. Indeed, the enantiomeric excess achieved is comparable to that attained by other previously implemented resolving agents, such as (–)-dibenzoyl-L-tartaric acid.1 Overall, the resolution of PZQamine using (4) to yield R-(–)-PZQamine (5) (0.1110 g; 44%; 79% ee) can be declared to have been highly successful. Future endeavors aimed at the resolution of PZQamine should perhaps center upon the exploration and development of new resolving agents, both those derived from L-tartaric acid and those from perhaps less conventional resolving agents. Furthermore, subsequent undertakings of this synthesis should likely include further purification steps, most likely by recrystallization, in the interest of increasing product purity.

Synthesis of (R)-PZQ from PZQamine
Interestingly, given that it has been established above that the resolved PZQamine (5) contains the comparatively more nucleophilic isopropanol as a substantial impurity, a side product ester, isopropyl cyclohexanecarboxylate, would result from the reaction of the residual isopropanol and cyclohexanoyl chloride, as shown below (Figure 7): The spectral data accumulated convincingly indicates that the desired synthesis of the target R-(–)-PZQ (6) (0.3846 g; 83%; 90% ee) did indeed occur. Among the many peaks produced, the IR (Thin Film) spectrum of the isolated powder reveals a significantly smaller peak corresponding to N–H stretching (3490.75 cm-1) than was observed in the IR spectrum of (5). It is reasonable to conclude that, while still present in the final product, the PZQamine starting material (5) was consumed by the reaction to a considerable extent. Nevertheless, the presence of the N–H stretch peak convincingly implies that the final step of the synthesis did not go to completion. The proposed explanation for such an occurrence, namely the reaction of residual isopropanol with the cyclohexanoyl chloride to form an ester side product, is itself given credence by the presence of a peak at 1722.49 cm-1, which is consistent with the C=O stretching of esters. Therefore, the IR spectrum provides strong evidence that the desired reaction occurred to a significant extent, in addition verifying the presence of the postulated side-product ester. As such, the IR spectrum lends significant credibility to the mechanism of the synthesis of (6) proposed above. Furthermore, as the IR spectrum identifies both unreacted PZQamine (5) and the side product ester as impurities present in the product powder, it follows that the true yield of (6) is less than the calculated 83%. Nevertheless, the product yield of the final step of 83% is largely consistent with the yield values reported in literature (90%).1 The 1H NMR spectrum of the product, while not optimal due to excessive dilution of the product leading to an inadequate and unclear spectrum, is in agreement with the expected NMR of PZQ. In essence, while the NMR spectrum does not contradict the above interpretation of the IR spectrum, it is not suitable for any detailed analysis or confirmation of potential side products. It would therefore be of interest to produce a more adequate NMR spectrum of the isolated product in order to potentially pursue a more rigorous product analysis. Moreover, the 1H NMR spectrum of the product with the shift reagent Eu(hfc)3 unfortunately does not lend itself at all to any analysis of the enantiomeric excess achieved. Perhaps most importantly, the polarimetry measurement of the final product (λ = 589 nm; c = 1.039 g/(100 mL); DCM) of  –127° corresponds to a calculated enantiomeric excess of 90% based on polarimetry values of enantiopure R-(–)-PZQ available in literature.1 As such, despite the inadequacy of the shift NMR in determining enantiomeric excess, the polarimetry data is a clear and unambiguous indicator that the synthesis undertaken was indeed extensively enantioselective. Interestingly, the calculated enantiomeric excess value of 90% exceeds the measured enantiomeric excess of the precursor substance (79%). Given that resolution had occurred in the synthesis of the precursor (5), it is not possible that the enantiomeric excess of the subsequent product is greater. Interestingly, the fact that the measured enantiomeric excess value of the final product (6) is unrealistic can be accounted for by the presence of unreacted resolved PZQamine (5) in the product powder. As the specific rotation of enantiopure R-(–)-PZQamine is more negative (–152°) than that of R-(–)-PZQ (–140°) based on values available in literature, the presence of R-(–)-PZQamine as an impurity in the final product would have resulted in a more negative polarimetry measurement, which in turn translates to a greater calculated enantiomeric excess value.1 It must be noted that, while the desired final product (6) and precursor (5) were enantioselectively synthesized, neither are enantiopure. As such, while the synthesis was successful in the isolation of enantioenriched R-(–)-PZQ, the evidence discussed above indicates that the goal of synthesizing enantiopure R-(–)-PZQ was not achieved.

Conclusion
The hydrolysis product of racemic praziquantel (PZQ), namely racemic praziquanamine (PZQamine) (1.7883 g; 69%; under 1M HCl) was successfully resolved using (−)-O,O′-Di-p-toluoyl-L-tartaric acid to enantioselectively isolate R-(–)-PZQamine (0.1110 g; 44%; 79% ee), which was itself then implemented in the successful enantioselective synthesis of R-(–)-PZQ (0.3846 g; 83%; 90% ee). Although the synthesis undertaken achieved the isolation of enantioenriched R-(–)-PZQ, evidence suggests that the goal of synthesizing enantiopure R-(–)-PZQ was not attained. Overall, this work illustrates the feasibility of implementing simple and comparatively inexpensive laboratory techniques and reactions towards the enantioselective synthesis of select medicinally-relevant compounds.

Methods and Materials
The following section contains the best procedures identified to date for each step in the resolution.

Hydrolysis of PZQ
Procedure for the 1-Gram Scale Hydrolysis of Racemic Praziquantel (1) to Racemic Praziquanamine (2) Under Varying HCl Concentrations Three separate mixtures of HCl, anhydrous ethanol, and PZQ were prepared, as follows: (a) To a mixture of 30 mL 0.5 M HCl and 7.5 mL anhydrous ethanol was added PZQ (1.0102 g). (b) To a mixture of 30 mL 1M HCl and 7.5 mL anhydrous ethanol was added PZQ (1.0069 g). (c) To a mixture of 30 mL 2M HCl and 7.5 mL anhydrous ethanol was added PZQ (1.0012 g). Each mixture was stirred and heated to reflux for 23 hours. The reactions were then cooled to room temperature, upon which each was washed with 10 mL of EtOAc. Upon cooling in an ice bath, the each solution was treated with 6N NaOH until the pH of the solution was determined to be 12 (using pH paper). The solutions were then each extracted twice with 15 mL of methylene dichloride and the organic layers were subsequently washed with brine (pH 12). The organic layers were each then dried with anhydrous sodium sulfate, upon which the solvent was evaporated off using a rotary evaporator.

Under 0.5 M HCl Oil Yield: 0.9716 g; 149% TLC (Silica; 1:1 Hexanes : EtOAc; CAM Visualization): 2 spots; Rf1 = 0.31, Rf2 = 0.69 Appearance: clear, colorless oil. The oil solidified after approximately 45 minutes to yield a solid, yellowish-white powder IR (KBr pellet): 3319.93 cm-1 (N–H stretch), 3052.72 cm-1 (sp2 C–H stretch), 2966.19 cm-1 (sp3 C–H stretch), 2896.01 cm-1 (sp3 C–H stretch), 1635.34 cm-1 (C=O amide stretch), ~1600 cm-1 (C=C aromatic stretch), 1440.69 cm-1 (C=C aromatic stretch), 1297.29 cm-1 (C–N stretch) 1H NMR (300 MHz, CDCl3): (δ 7.2 ppm, m), (δ 5.3 ppm, s, DCM), (δ 4.9 ppm, m), (δ 4.1 ppm, m), (δ 3.7 ppm, m), (δ 3.6 ppm, d), (δ 2.9 ppm, m), (δ 2.0 ppm, s), (δ 1.7 ppm, s), (δ 1.1 ppm, m), (δ 0.0 ppm, s, TMS)

Under 1M HCl Oil Yield: 0.7940 g; 122% TLC (Silica; 1:1 Hexanes : EtOAc; CAM Visualization): 1 spot; Rf = 0.70 Appearance: clear, colorless oil. The oil solidified after approximately 45 minutes to yield a solid, yellowish-white powder IR (KBr pellet): 3314.18 cm-1 (N–H stretch), 3052.74 cm-1 (sp2 C–H stretch), 2966.34 cm-1 (sp3 C–H stretch), 2896.12 cm-1 (sp3 C–H stretch), 1719.67 cm-1 (C=O stretch), 1635.24 cm-1 (C=O amide stretch), ~1600 cm-1 (C=C aromatic stretch), 1440.31 cm-1 (C=C aromatic stretch), 1297.36 cm-1 (C–N stretch) 1H NMR (300 MHz, CDCl3): (δ 7.2 ppm, m), (δ 5.3 ppm, s, DCM), (δ 4.8 ppm, m), (δ 4.1 ppm, m), (δ 3.7 ppm, m), (δ 3.5 ppm, d), (δ 2.9 ppm, m), (δ 2.0 ppm, s), (δ 1.8 ppm, s), (δ 1.1 ppm, m), (δ 0.0 ppm, s, TMS)

Under 2M HCl Oil Yield: 0.6761 g; 104% TLC (Silica; 1:1 Hexanes : EtOAc; CAM Visualization): 1 spot; Rf = 0.74 Appearance: clear, colorless oil. The oil solidified after approximately 45 minutes to yield a solid, yellowish-white powder IR (Thin Film): 3310.70 cm-1 (N–H stretch), 3049.68 cm-1 (sp2 C–H stretch), 2966.85 cm-1 (sp3 C–H stretch), 2907.21 cm-1 (sp3 C–H stretch), 1735.49 cm-1(C=O stretch), 1638.21 cm-1 (C=O amide stretch), ~1600 cm-1 (C=C aromatic stretch), 1459.31 cm-1 (C=C aromatic stretch), 1296.61 cm-1 (C–N stretch) 1H NMR (300 MHz, CDCl3): (δ 7.2 ppm, m), (δ 5.3 ppm, s, DCM), (δ 4.8 ppm, m), (δ 4.1 ppm, m), (δ 3.7 ppm, m), (δ 3.6 ppm, d), (δ 2.9 ppm, m), (δ 2.0 ppm, s), (δ 1.7 ppm, s), (δ 1.2 ppm, m), (δ 0.0 ppm, s, TMS)

Procedure for the 4-Gram Scale Hydrolysis of Racemic Praziquantel (1) to Racemic Praziquanamine (2) Under 1M HCl To a mixture of 120mL 1 M HCl and 30 mL anhydrous ethanol was added PZQ (4.0823g). The mixture was then stirred and heated to reflux for 23 hours. The reaction was allowed to cool to room temperature, upon which it was washed twice with 10 mL of EtOAc. Upon cooling in an ice bath, the solution was treated with 10M NaOH until the pH of the solution was determined to be 12 (using pH paper). The solution was then extracted once with 12 mL of methylene dichloride and twice with 10 mL of methylene dichloride. The combined organic layers were subsequently washed with brine (pH 12). The organic layers were then dried with anhydrous sodium sulfate, upon which the solvent was evaporated off using a rotary evaporator.

Yield: 1.7883 g; 69% Appearance: clear, colorless oil. The oil solidified after approximately 45 minutes to yield a solid, yellowish-white powder IR (KBr pellet): 3314.39 cm-1 (N–H stretch), 3052.88 cm-1 (sp2 C–H stretch), 2966.44 cm-1 (sp3 C–H stretch), 2896.65 cm-1 (sp3 C–H stretch), 1636.04 cm-1 (C=O amide stretch), ~1600 cm-1 (C=C aromatic stretch), 1440.69 cm-1 (C=C aromatic stretch), 1297.36 cm-1 (C–N stretch) 1H NMR (300 MHz, CDCl3): (δ 7.2 ppm, m), (δ 4.8 ppm, m), (δ 4.1 ppm, m), (δ 3.7 ppm, m), (δ 3.5 ppm, d), (δ 2.9 ppm, m), (δ 1.8 ppm, s), (δ 1.2 ppm, m), (δ 0.0 ppm, s, TMS)

The Resolution of PZQamine
Procedure for the 0.25-Gram Scale Resolution of Racemic Praziquanamine (2) Using L-Tartaric Acid Derivatives (4,5) Two separate mixtures of PZQamine, an L-tartaric acid derivative, isopropanol, and deionized water were prepared, as follows: (a) Racemic PZQamine (0.2514 g) and (−)-O,O′-Di-pivaloyl-L-tartaric acid (0.4011 g, 1 eq.) were dissolved in 11 mL isopropanol and 2.2 mL water by heating and stirring. (b) Racemic PZQamine (0.2432 g) and (−)-O,O′-Di-p-toluoyl-L-tartaric acid (0.4863 g, 1 eq.) were dissolved in 11 mL isopropanol and 2.2 mL water by heating and stirring. The solutions were cooled to room temperature and stored for 22 hours at 5°C. Unlike solution (b), which produced a white precipitate, solution (a) did not yield a salt/precipitate and was therefore not subjected to the subsequent steps. The salt of (b) was vacuum filtered and subsequently suspended in 5 mL of deionized water. NaOH (2M) was added to the suspension until the pH of the solution was 11 and the salt was allowed to dissolve. The solution was extracted three times with 7 mL methylene dichloride, upon which the organic layer was dried with NaSO4 and the solvent evaporated off with a rotary evaporator.

Using (−)-O,O′-Di-pivaloyl-L-Tartaric Acid as Resolving Agent (4) Yield: 0 g; 0%

Using (−)-O,O′-Di-p-toluoyl-L-tartaric acid as Resolving Agent (5) Yield: 0.1110 g; 44% Appearance: clear, transparent, colorless solid/solidified oil 1H NMR (300 MHz, CDCl3): (δ 7.2 ppm, m), (δ 5.3 ppm, s, DCM), (δ 4.8 ppm, m), (δ 4.0 ppm, m), (δ 3.7 ppm, m), (δ 3.5 ppm, d), (δ 2.9 ppm, m), (δ 2.0 ppm, s), (δ 1.2 ppm, m), (δ 0.0 ppm, s, TMS)

Procedure for the 0.70-Gram Scale Resolution of Racemic Praziquanamine (2) Using the Resolving Agent (−)-O,O′-Di-p-toluoyl-L-tartaric acid (5) Racemic PZQamine (0.7015 g) and (−)-O,O′-Di-p-toluoyl-L-tartaric acid (1.3382 g, 1 eq.) were dissolved in 31.5 mL isopropanol and 6.5 mL water by heating and stirring. The solutions were cooled to room temperature and stored for 22 hours at 5°C. The precipitate was vacuum filtered and subsequently suspended in 9 mL of deionized water. NaOH (3M) was added dropwise to the suspension until the pH of the solution was 11 and the salt was allowed to dissolve. The solution was extracted four times with 5 mL methylene dichloride, upon which the organic layer was dried with NaSO4 and the solvent evaporated off with a rotary evaporator.

Yield: 0.8275 g; 118% Appearance: unclear, white-ish oil IR (KBr pellet): 3367.36 cm-1 (N–H stretch), 2971.27 cm-1 (sp2 C–H stretch), 2931.70 cm-1 (sp3 C–H stretch), 1766.15 cm-1 (C=O stretch), 1636.16 cm-1 (C=O amide stretch), ~1600 cm-1 (C=C aromatic stretch), 1466.77 cm-1 (C=C aromatic stretch), 1301.20 cm-1 (C–N stretch) 1H NMR (300 MHz, CDCl3): (δ 7.3 ppm, m), (δ 5.8 ppm, m), (δ 5.2 ppm, m), (δ 4.8 ppm, m), (δ 4.0 ppm, m), (δ 3.7 ppm, m), (δ 3.5 ppm, d), (δ 2.9 ppm, m), (δ 2.5 ppm, m), (δ 1.6 ppm, s), (δ 1.4 ppm, s), (δ 1.2 ppm, m), (δ 0.0 ppm, s, TMS) 1H NMR (300 MHz, CDCl3, Eu(hfc)3): (δ 7.4 ppm, s), (δ 7.25 ppm, s), (δ 4.7 ppm, s, peak of interest, 0.12H), (δ 4.33 ppm, s, peak of interest, 1.00H), (δ 2.6 ppm, s), (δ 2.4 ppm, s), (δ 1.5 ppm), (δ 1.35 ppm), (δ 0.0 ppm, s, TMS) Enantiomeric Excess: 79%

Conversion of PZQamine to PZQ
Procedure for the 0.30-Gram Scale Synthesis of Enantiopure R-(–)-Praziquantel (6) from Enantiopure Praziquanamine (3) To resolved PZQamine (0.30 g) was added triethylamine (0.2302 g, 1.5 eq.) and methylene dichloride (8 mL). Cyclohexanoyl chloride (0.2402 g, 1.1 eq.) was added dropwise to the mixture at 0°C and the mixture was stirred at room temperature for 45 hours. The reaction was then quenched with 1 mL of deionized water and stirred for a further 30 minutes. The reaction mixture was separated and the organic layer washed, in this order, with 5 mL of 10% sodium carbonate, 5 mL of 0.5N HCl, and 5 mL brine. After drying with NaSO4, the solvent was evaporated off using a rotary evaporator and the product stored at 5°C for 24 hours.

Yield: 0.3846 g; 83% TLC (Silica; 1:1 Hexanes : EtOAc; CAM Visualization): 2 spots; Rf1 = 0.34, Rf2 = 0.91 Appearance: white solid/powder Melting Point: 113°C – 123°C IR (Thin Film): 3490.75 cm-1 (N–H stretch), 3052.22 cm-1 (sp2 C–H stretch), 2928.21 cm-1 (sp2 C–H stretch), 2855.01 cm-1 (sp3 C–H stretch), 1722.49 cm-1 (C=O stretch), 1647.45 cm-1 (C=O amide stretch), ~1600 cm-1 (C=C aromatic stretch), 1450.07 cm-1 (C=C aromatic stretch), 1297.62 cm-1 (C–N stretch) 1H NMR (300 MHz, CDCl3): (δ 7.3 ppm, m), (δ 5.3 ppm, s, DCM), (δ 5.2 ppm, m), (δ 5.0 ppm, m), (δ 4.8 ppm, m), (δ 4.5 ppm, m), (δ 4.1 ppm, d), (δ 3.9 ppm, d), (δ 2.9 ppm, m), (δ 2.5 ppm, m), (δ 2.2 ppm, s), (δ 1.9 ppm, m), (δ 1.3 ppm, m), (δ 0.0 ppm, s, TMS) 1H NMR (300 MHz, CDCl3, Eu(hfc)3): (δ 7.3 ppm, m), (δ 5.3 ppm, s, DCM), (δ 5.1 ppm, m), (δ 4.6 ppm, s), (δ 3.0 ppm, m), (δ 2.3 ppm, m), (δ 2.2 ppm, s), (δ 1.8 ppm, m), (δ 1.3 ppm, m), (δ 0.4 ppm, d), (δ 0.0 ppm, s, TMS) Polarimetry: [α]589nm = –127°  (c = 1.039, DCM) Enantiomeric Excess: 90%