Development of a Practical Synthetic Method for Clinical Candidate 3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy} phenyl)propanoic acid (P218) and Its Hydroxylated Metabolites

3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]prop-oxy}phenyl)propanoic acid, known as P218, has demonstrated great potency and safety in preclinical and human studies. However, the previous synthetic methods for P218 gave low yields and required hazardous reagents and challenging procedures. In this study, we have successfully developed a decagram-scale synthetic route for P218 with practical and scalable methods for large-scale production. Furthermore, this is also a first report of a novel synthetic approach for P218-OH, a hydroxylated metabolite of P218, by modification of our discovery route. Our synthetic procedures for P218 and P218-OH are a significant advancement in drug development processes, including manufacturing processes and drug metabolism studies.

2][3][4] In 2020, during the COVID-19 pandemic, the World Health Organization (WHO) reported approximately 241 million cases of malaria and more than 627,000 deaths worldwide, with an 11% increase in the mortality rate compared to the previous year -two thirds of which were due to the COVID-19 disruption. 5Severe ma-laria is more likely to develop in children under 5 years old and individuals with immune failures.Plasmodium falciparum (P.6][7][8] Despite the availability of several antimalarial drugs, the emergence of drug resistance poses a significant threat to human existence. 9,10Moreover, affordable treatment and low production cost are necessary in view of the economic status of the most affected people. 11Therefore, the development of new antimalarial candidates and a practical process to produce them are urgently needed. 3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]prop-oxy}phenyl)propanoic acid (P218) (Figure 1) was discovered by Yuthavong et al. 12,13 P218, prepared in hydrochloride salt form, has demonstrated high potency against wildtype and resistant P. falciparum. 12Recently, first-in-human and sporozoites challenge clinical studies have shown favorable safety and pharmacokinetic profiles of P218, as well as its chemoprotective antimalarial activity against P. falciparum. 14,15P218 and its hydroxylated metabolite P218-OH (Figure 1), together with their glucuronide forms were identified. 15,16Consequently, standard P218 and metabolites are needed for subsequent clinical and related studies, and the development of a practical synthetic route for P218 and its metabolites is required.

PSP Synthesis
Retrosynthetic analysis of P218 envisioned 2,4-diamino-6-ethyl-5-hydroxypyrimidine (5) and the bromo-substituted derivative 7 as the common core (Scheme 1).However, the synthesis of 5 has encountered challenges, [17][18][19][20] including poor overall yields, poor reproducibility, the use of hazardous reagents such as phosphorus oxychloride (POCl 3 ), and the need for extensive purification processes (Scheme 1).Recently, an alternative synthetic route for P218 was proposed through C-6 late-stage modification starting from commercially available 2,4-dichloro-5-methoxypyrimidine (Scheme 1).However, this synthetic route also poses significant challenges due to the use of complex, expensive chemical reagents and extensive purification methods. 21The disadvantages of both synthetic routes have raised concerns regarding the high cost of production.Therefore, a simple and robust synthetic route is required for producing P218 to serve as a medicine at low cost.
To avoid the aforementioned challenges during scaleup, we report here an alternative and more practical method for the synthesis of P218 and its derivatives, with significant improvements in the synthetic processes and overall yields of the products.We synthesized the key intermediate 5 in parallel, followed by conjugation of two bromo-substituted derivatives.In particular, a chromatography-free synthetic method for 2,4-diamino-6-ethyl-5-hydroxypyrimidine (5), as a key intermediate for P218 and P218-OH, has been developed.This method is scalable, up to multigram scale, allowing access to key intermediate 5, which has been a bottleneck in previous methods. 21The synthetic procedure allowed us to produce P218 and its metabolite P218-OH in ten and twelve steps, respectively.[24] The synthesis of 2,4-diamino-6-ethyl-5-hydroxypyrimidine (5) began with a low-cost commercially available material, methyl propionate, using a chromatography-free synthetic method on a 60-gram scale, as depicted in Scheme 2. Methyl propionate underwent nucleophilic attack by acetonitrile in a presence of sodium hydride as a base in anhydrous tetrahydrofuran.The starting material was initially prepared at a low temperature of -78 °C and then refluxed at 70 °C, resulting in a crude of 3-oxopentanenitrile (1).Trimethoxybenzene of known purity was chosen as internal standard for 1 H NMR quantitative analysis (qHNMR).The purity of compound 1 was obtained by 1 H NMR (73.18%,STD = 0.02).To establish a more viable synthetic procedure, the temperature for the preparation step was optimized by varying it from -78 to 0 °C.The reaction was also optimized at -10 °C, followed by reflux at 70 °C, and it still showed a similar yield.Subsequently, nitrile 1 was transformed into a crude mixture of 3-methoxypent-2enenitrile (2a) and 3,3-dimethoxypentanenitrile (2b) in the presence of trimethyl orthoformate under acidic conditions.The crude containing impurities was quantified by using trimethylbenzene as internal standard.The yields of 2a and 2b (53:47) in the crude mixture were obtained by 1 H qNMR (86.64%,STD = 0.04).The enol ether 2a and acetal 2b were subjected to guanidine, affording 6-ethylpyrimidine-2,4-diamine (3) in low yield (22% over 3 steps).Pyrimidine 3 underwent a Boyland-Sims oxidation reaction in the presence of ammonium persulfate under basic condition, leading to the formation of 2,4-diamino-6-ethylpyrimidin-5-yl hydrogen sulfate (4) in high yields (84%).The resulting sulfate ester 4 was further hydrolyzed under concentrated acidic conditions to provide 2,4-diamino-6-ethyl-5-hy-
On the other hand, the key bromo-substituted intermediate 7 was prepared (Scheme 3).The process began with the acidic hydrolysis of 3,4-dihydrocoumarin, producing ring-opened methyl 3-(2-hydroxyphenyl)propanoate (6) in excellent yield (90%) without column chromatography.To reduce the costs of chemical reagents used in the Mitsunobu reaction as described in the previous study, 12 e.g., 3-bromopropan-1-ol, triphenylphosphine, and diisopropyl azodicarboxylate, a modified process was developed.A simple O-alkylation of the phenol group of 6 with an excess of 1,3-dibromopropane under basic condition was performed, resulting in methyl propanoate 7 in good yield (86%) without the observation of di-O-alkylated side product (Scheme 3).
The bromo-substituted intermediate 7 was then subjected to nucleophilic attack by the hydroxyl group of the prepared pyrimidine 5 under basic conditions (Scheme 3).This led to the formation of the desired ester intermediate 8 in moderate yield (50%).The observed preferential formation of ester 8 indicates that O-alkylation was favored.No-tably, the N-alkylated side product was not observed.This might be due to the low reactivity of the amine moiety of the pyrimidine ring and the use of LiOH as a base.Consequent hydrolysis of the methyl ester under basic conditions, followed by precipitation in a hydrochloric acid solution, resulted in the formation of the final product P218 as a hydrochloride salt in excellent yield (92%) (Scheme 3).
To prepare the hydroxylated metabolite P218-OH, we unsuccessfully attempted to perform late-stage modification of P218 under various conditions (unpublished data).Therefore, P218-OH was totally synthesized from intermediate 5 and a new counterpart (Scheme 4).4-(Benzyloxy)phenol, commercially available, was used as the starting material to produce P218-OH in a multistep reaction steps), including 5 steps to form intermediate 5. First, 5-(benzyloxy)-2-hydroxybenzaldehyde (9) was obtained in good yield (72%) by formylation by using paraformaldehyde and magnesium chloride (Scheme 4).The Wittig coupling reaction of aldehyde 9 with ethyl (triphenylphosphoranylidene)acetate was then carried out.Subsequently, the two subsequent alkylation reactions were performed as previously described for P218, resulting in ethyl acrylate 12 in satisfactory yield (40% over 3 steps).Then, alkene 12 was

PSP Synthesis
hydrogenated and the benzyl protecting group was removed, resulting in the ethyl propanoate intermediate 13 in good yield (71%).Finally, the desired final product, P218-OH, was obtained through hydrolysis under basic conditions and subsequent precipitation with concentrated hydrochloric acid; this led to the formation of P218-OH as a hydrochloride salt in good yield (72%) (Scheme 4).
In summary, this study presents an efficient synthetic procedure for the crucial dihydrofolate reductase-targeting moiety, 2,4-diamino-6-ethyl-5-hydroxypyrimidine (5), to overcome the limitations of previous methods.The optimized procedures resulted in high yields of P218 and its metabolite, P218-OH, and offer a practical approach for further large-scale studies and manufacturing.This could have significant implications for the development of pyrimidine derivatives for combating malaria as a major global health issue.
All chemicals used for the synthesis were purchased from commercial suppliers and used without further purification.Reactions were monitored by TLC.The products were collected by either precipitation or column chromatography as indicated in the procedures. 1 H and 13 C NMR spectra were obtained on Bruker DRX400 or AV500D spectrometers (100 or 125 MHz for 13 C NMR).Quantitative 1 H NMR spectroscopy (qNMR) was applied for purity assessment.Mass spectra were obtained on an Agilent 6540 UHD Q-TOF LC/MS spectrometer.Melting points were recorded using Electrothermal IA9100 digital meltingpoint apparatus.

Figure 1
Figure 1 Structure of P218 and P218-OH as hydrochloride salt