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
The reports for the larger scale synthesise from phenylacteonitrile (Compound 1) through to Daraprim via Steps A,B and C are itemized below. The isolation of pure samples of the phenylketonitrile (Compound 2) and the enol ethers (Compounds 3 and 4) has provided NMR spectra for compounds that are not usually isolated and characterised in commercial literature.
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.
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.