Daraprim Synthesis

 

 

Overview

The synthesis of Daraprim by Sydney Grammar School students, with the assistance of their teachers, Dr Malcolm Binns and Dr Erin Sheridan, was completed in three steps as shown in the diagram above.

Significant investigation was undertaken to

• elucidate the optimal conditions to produce the keto-nitrile Compound 2 in Step A
• determine why Step D and related reactions one stepreactions would not work when Compound 2 was the starting material 
• methylate Compound 2 to form Compound 4

Discussion of Grammar syntheses

2-(4-chlorophenyl)-3-oxopentanenitrile. (Compound 2). Keto, enol and enolate forms.

At the onset of this project it was understood that the phenylketonitrile , Compound 2, might exist in both keto and enol forms. It was also suggested by Thomas McDonald that the red colour of the reaction mix, which disappeared on acidification may be due the production of the corresponding enolate (enoxide). Our investigation of the NMR spectra of Compound 2 in CDCl₃ indicated that it was 100% keto-form displaying the characteristic δ4.66 shift for the benzylic proton, whilst in d⁶DMSO Compound 2 was 100% enol-form displaying evidence for E and Z isomerism and no signal at δ4.66.  The spectrum submitted by Thomas McDonald for the Compound 2 had been also been run in d⁶DMSO and displayed the characteristics of the enol-form.

We found that Compound 2 was surprizingly acidic, presumeably as a result of the benzylic carbanion being stabilized by phenyl, nitrile and carbonyl groups. Addition of excess triethylamine to a solution of Compound 2 in ethanol immediately resulted in the generation of the enolate, as evidenced by the immediate appearance of a very polar spot in the TLC.  Intermediate levels of triethylamine resulted in a smear on the TLC running between protonated and deprotonated species. The enolate was isolated as its triethylammonium salt by direct reaction between Compound 2 and triethylamine and a confirmatory NMR spectrum in d⁶DMSO was obtained.

A very polar by-product was formed alongside Compound 2. It has tenaciously been identified as 4-chlorophenylbenzoate until further analysis becomes available. It is surmised that it is generated by the addition of molecular oxygen to the phenylacetonitrile anion to form the peroxide anion which then undergoes further reaction. If this is the case the Sydney Grammar decision to work with larger quantities (~20g) of material would have mitigated the problem of oxygen contamination somewhat.  It is noted that the Vasiliki Theologia Chioti group were working on the synthesis of Compound 2 using much smaller (part gram) quantities of materials and were not able to indentify Compound 2 in any of their eraction fractions after purification, perhaps a result of complete reaction of their cabanion with oxygen. 

Another advantage of using larger quanties of materials in the synthesis was that the exotherm of the reaction took the temperature quickly from room temperature to 40°C or so, accelerating the desired process and reducing the solubility of oxygen in the reaction mix (stirring of the reaction mix was stopped as soon as the potassium tert butoxide had dissolved). To make the reaction more school friendly and foolproof, reaction quantities of all reagents were based on the consumption of a whole bottle of potassium tert butoxide.  This enabled the rapid addition of the potassium butoxide directly from the bottle (safe and efficient handling) and did not leave any residue in the bottle to "expire" during storage.

Finally, in the earlier reaction workups by Thomas Mcdonald and Sydney Grammar, the highly basic reaction mix was quenched with water, extracted with a solvent, acidified and extracted again. Later recognition of the acidic nature of compound two led to the more efficient immediate quenching of the reaction mix with an excess of hydrochloric acid.

 

2-(4-chlorophenyl)-3-methoxy-2-pentenenitrile. Compound 4

In spite of indications that Compound 4 could be synthesised from Compound 2 and trimethyl orthoformate in an acidic aprotic environment, this was found not to be the case in our hands. Neither milder alkylating conditions using acidic silica nor more aggressive conditions using concentrated sulfuric acid were found to be effective.  

The alternative reaction system, Step E, pictured above with methanol as the alkoxylating agaent and trimethyl orthoformate acting as a dehydrating agent worked to some extent but the reaction yield was never more than about 50% according to TLC analysis. TLC analysis of the reaction mix indicated that two new products were present. Isolation and susequential NMR analysis of these products suggested they were the E and Z isomers of the desired Compound 4. One of the isomers tended to crystalise out of the Compound 4 oil on standing.

As the much of the reaction solvent (methanol) was lost during the overnight reaction in the hot water bath (presumably due to competing reactions between the methanol and the triethyl orthoformate) the reaction scheme was considered inconvenient for larger scale reactions and an alternative enol ether was sought as an intermediate in the synthetic pathway.

 

2-(4-chlorophenyl)-3-(2-methylpropoxy)-2-pentenenitrile. Compound 3

This reaction started well with water formed in the reaction effectively removed from the reaction by condensing in the condenser of the Dean Stark apparatus and dropping into the reservoir. As the reaction proceeded, the water still condensed in the condenser but did not drop into the reservoir, rather it reincorporated back into the refluxing solvent and was returned to the reaction mix.  As a result the reaction never went to completion.  Perhaps varying the tolulene and isobutanol content would further improve the yield.

Removal of unreacted Compound 2 was effected by adding triethylamine to the products of the reaction and then stirring in some silica to remove the very polar triethylammonium salt of Compound 2.

Seperate E and Z isomers were apparent in the TLC analysis and the NMR spectra.

 

Daraprim

The use of DMSO as the reaction solvent in Step C has many advantages. The guanidine hydrochloride, sodium methoxide and resultant sodium chloride (and guanidine) were all soluble in the DMSO. Alternative syntheses in ethanol typically filtered out the sodiumchloride precipitate before proceeding. Additionally, the DMSO is supposed to accelerate the isomeration and aromatization phase of the reaction after the intial addition of the guanidine to Compound 3, a significant rate limiting step in ethanol based reactions.

After standing overnight at room temperature, much of the Daraprim had crytallised from solution even though there was still starting material present according to TLC analysis. The yield of the reaction could be improved by holding the reaction mix at an elevated temperature overnight to push the reaction towards completion.  The isolation of the Daraprim could be improved by filtering the solution containing Daraprim crystals under reduced pressure. The School chemistry laboratory did not have sufficient vacuum available to filter the majority of the highly viscous mixture. A laborious and messy workup was required.

Guanidine carbonate is not as soluble in DMSO as guanidine chloride, however may have been suitable as an alternative.  It may be that guanidine carbonate would react with Compound 3 without the need for the sodiummethoxide base, but there was insufficient time to trial this system.

Step D,a one step reaction from Compound 2 to Daraprim, was discontinued in part due to the acidic nature of Compound 2 creating previouslydiscussed unwanted side reactions when triethylamine/guanidine hydrochloride systems were investigated and in part to the relative insolubility of the guanidine carbonate system making it difficult to identify whether the relatively insoluble Daraprim had formed. It well may be that a review of the previously dismissed guanidine carbonate reaction products alongside an authentic Daraprim sample may conclude that the attempted one-step reactions were more successful than initially thought.

 

Summary prepared by M.R.Binns on behalf of the Sydney Grammar School team.

2-(4-chlorophenyl)-3-oxopentanenitrile (SGS 10-4, 20.3g, 0.095 mol) was dissolved in a mixture of toluene (200mL) and 2-methylpropan-1-ol (20mL). 18M H₂SO₄ (2.5 mL) was added and the mixture was refluxed for 6 hours in a Dean Stark apparatus. A TLC (isopropanol) of the reaction mixture indicated that some starting material was still present.  The main products were two close spots corresponding to the E and Z isomers of the enol ether. The organic phase was worked up with a solution of saturated sodium hydrogen carbonate and dried over anhydrous sodium sulfate. The organic phase was still clouldy and was passed through a short silica column to remove the cloudiness.  Dichloromethane was passed through the short column until TLC analysis indicated that the reaction material had been removed from the short silica column.

Addition of 10mL of triethylamine to the reaction mix converted the unreacted starting material to its very polar triethylammonium enolate salt (SGS 14-1). Chromatography silica (100g) was added to the organic phase, which was made up to 400mL with dichloromethane and stirred for two hours.  TLC of the organic phase indicated that the polar baseline material had disappeared from solution and absorbed on to the silica (see photo below).  The organic phase was decanted, rinsed twice with dichloromethane. The organic phase was rinsed with 1M HCL (2 x 50 mL) and deionised water (50 mL) to remove all traces of triethylamine. The solvent was then removed from the organic phase to yield 2-(4-chlorophenyl)-3-(2-methylpropoxy)-pent-2-enenitrile (15.1g, 0.057 mol, 60%) as a red oil.  This product was used in the large scale reaction to synthesis Daraprim.

1M HCl (100mL) was added to the silica containing the absorbed enolate and dichloromethane was added to produce a freely flowing, stirrable mixture.  After stirring for an hour, the dichloromethane solution was decanted and the silica extracted with a further 100mL of dichloromethane. The two dichloromethane solutions were combined and then the solvent removed to yield a red oil (5.2 g) that was indicated to be a 50:50 mixture of starting material and  2-(4-chlorophenyl)-3-(2-methylpropoxy)-pent-2-enenitrile. This material was kept for recycling in any further reactions.

2-(chlorophenyl)-3-(2-methylpropoxy)-pent-2-enenitrile (SGS 15-3, 14.0 g, 53.2 mmol) was dissolved in DMSO (140 mL). Guanidine hydrochloride (10.0 g, 105 mmol) was stirred in to the solution followed by sodium methoxide powder (5.0 g, 93 mmol). The solution became dark red in colour on addition of the sodium methoxide, which dissolved into the solution within an hour. No precipitation of sodium chloride was observed. The solution was allowed to stand at room temperature for 18 hours. After this time, some material (Daraprim) had crystallized from the reaction mix and stirring was recommenced to encourage crystallization of the Daraprim from solution and advance the reaction. 

     

After 24 hours of reaction time, 0.2 g of Daraprim was isolated from the reaction mix by filtration, however the high viscosity of the reaction mix made the process too laborious and so the reaction was worked up by pouring it into water (500 mL).  The resultant brown precipitate was separated by filtration and dissolved in DCM (500mL). On washing the DCM extract with water, large amounts of white solid (Daraprim) precipitated at the DCM/water interface. This Daraprim was collected and dried.

     

The DCM extract was reduced in volume to 150 mL and the resulting crystals of Daraprim were filtered off and washed sparingly with ethanol and allowed to dry overnight.  The dried Daraprim samples were combined (3.67 g, 16.7 mmol, 31.4%).  TLC analysis (isopropanol) indicated that the remaining DCM extract was largely comprised of unreacted starting material and a small amount of Daraprim.

        

Comment: The reaction was worked up without further modification after 24 hours due to time pressure to produce a gram quantity sample of Daraprim even though TLC of the reaction mixture indicated that starting material was still present. The reaction yield should be improved by running the reaction at a higher temperature, say 40 or 50 deg C. Vacuum filtration of the reaction mixture is desirable as most of the Daraprim present in the reaction mixture has already crystallized out and is easily rinsed once isolated, obviating the need for copious and wasteful amounts of solvent to intially dissolve and then isolate the Daraprim.

2-(4-chlorophenyl)-3-(2-methylpropoxy)-pent-2-enenitrile (1.0 g, 3.8 mmol) was dissolved in DMSO (10 mL). Guanidine hydrochloride (1.0 g, 10.5 mmol) was stirred in to the solution followed by sodium methoxide powder (0.5 g, 9.3 mmol). All of the reagents dissolved and the solution was allowed to stand at room temperature for 18 hours. After this time, some material had crystallized from the reaction mix. 

The reaction mixture was added to water (100 mL) and the mixture took on a brown curdled appearance (see photo below). The reaction workup was extracted with dichloromethane (3 x 100mL). The solid material dissolved into the DCM. The DCM solution was washed with water (100ml). TLC analysis indicated that the DCM solution was comprised of Daraprim and unreacted 2-(chlorophenyl)-3-(2-methylpropoxy)-pent-2-enenitrile. Evaporation of the DCM solvent yeilded 0.8g of crude product. Due to time restraints, the crude Daraprim was not purified further; a larger scale reaction was carried out instead.

   

4-chlorophenylacetonitrile (17.5 g, 0.109 mol), ethyl propionate (11.4 g, 0.111 mol) and potassium tert-butoxide (25.0 g, 0.227 mol) were combined in THF (170 mL) with stirring. The colourless solution turned red and the temperature of the solution rapidly rose to 40 deg.C.  The reaction flask was covered in aluminium foil in order to retain the heat.  All of the potassium tert-butoxide had dissolved after 40 minutes, leaving a homogeneous solution, and stirrer was turned off.  

The reaction mixture was worked up by pouring it into 250 mL of 1M HCl solution.  The oily mixture was seperated from the aqueous phase (which was retained) and then washed with 100 mL water containing one drop of 1M HCL.  The three combined aqueous washes were extracted with 100 mL of dichloromethane. The original oily mixture and dichloromethane solution were combined and dried (sodium sulfate), after which the solvent was removed to yield 2-(4-chlorophenyl)-3-oxopentanenitrile (SGS 10-4, 20.3g, 0.098 mol, 90%) a as a redish oil.redish oil.  TLC indicated, unlike the previous reactions which sat for 17 hours, that very little polar baseline product was present in the SGS 10-4 crude product.

The crude SGS 10-4 was used without purification in the second step of the Daraprim synthesis.

Comment: This is the second large scale synthesis of 2-(4-chlorophenyl)-3-oxopentanenitrile, en route to Daraprim. Even though potassium butoxide is required, its handling by the schoolboys is minimised as the reaction is scaled to use an entire 25g bottle of potassium butoxide. No weighing is required, there is minimal contact with moist air and there is no potassium butoxide left over to degrade with time and cause uncertainties in future reactions.  The TLC of the crude product suggested that a small amount of starting material was present.  Holding the reaction at 40 deg C for 6-8 hours should completely consume the starting material.