Key words P218 - pyrimidine - antimalarial drugs - process development
Malaria is a communicable disease caused by Plasmodium parasites and can be life-threatening, especially in tropical and subtropical countries such
as Africa and Asia, including Thailand.[1 ]
[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.[5 ] Severe malaria is more likely to develop in children under 5 years old and individuals
with immune failures. Plasmodium falciparum (P. falciparum ) is the most dangerous species to human life.[5 ]
[6 ]
[7 ]
[8 ] Despite the availability of several antimalarial drugs, the emergence of drug resistance
poses a significant threat to human existence.[9 ]
[10 ] Moreover, affordable treatment and low production cost are necessary in view of
the economic status of the most affected people.[11 ] Therefore, 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]propoxy}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
wild-type and resistant P. falciparum.
[12 ] Recently, 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 ]
[15 ] P218 and its hydroxylated metabolite P218-OH (Figure [1 ]), together with their glucuronide forms were identified.[15 ]
[16 ] Consequently, 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.
Figure 1 Structure of P218 and P218-OH as hydrochloride salt
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 (POCl3 ), 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.[21 ] The 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.
Scheme 1 Retrosynthetic analysis of P218 derivatives and previous synthetic route of 2,4-diamino-6-ethyl-5-hydroxypyrimidine
(5 )
To avoid the aforementioned challenges during scale-up, 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.[21 ] The synthetic procedure allowed us to produce P218 and its metabolite P218-OH in
ten and twelve steps, respectively. Moreover, our development of these synthetic procedures
will be a foundation for further derivatization and optimization of antimalarial drugs
and other antifolate agents.[22 ]
[23 ]
[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-2-enenitrile (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-hydroxypyrimidine
(5 ) in excellent yields (96%). In summary, hydroxypyrimidine (5 ) was prepared in five steps in 18% yield without chromatographic purification (Scheme
[2 ]).
Scheme 2 Synthesis of 2,4-diamino-6-ethyl-5-hydroxypyrimidine (5 )
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 ]).
Scheme 3 Synthesis of 3-(2-{3-[(2,4-diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}phenyl)propanoic
acid (P218)
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. Notably, 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 (12 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 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 ]).
Scheme 4 Synthesis of 3-(2-{3-[(2,4-diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}-5-hydroxyphenyl)propanoic
acid (P218-OH)
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 melting-point apparatus.
3-Oxopentanenitrile (1)
Anh MeCN (72 mL, 1.37 mol) was added dropwise to a suspension of NaH (24.5 g, 1.02
mol, 60% dispersion in mineral oil) in anh THF (300 mL) at –78 °C for 1 h under a
N2 atmosphere. A solution of methyl propionate (60.0 g, 0.68 mol) in anh THF (100 mL)
was added to the reaction mixture, which was then heated to 70 °C overnight. After
completion of the reaction, the mixture was acidified to pH ~2–3 with 3 M aq HCl.
The solution was then extracted with DCM (2×), dried over Na2 SO4 , filtered, and evaporated to dryness under reduced pressure; this gave crude product
1 as a yellow-brown oil (66.37 g) (the yields were determined by weighing the product
obtained after removal of the mineral oil). Crude product 1 was directly used on the same day for the next step without further purification.
1 H NMR (500 MHz, CDCl3 ): δ = 3.45 (s, 2 H), 2.65 (q, J = 7.2 Hz, 2 H), 1.13 (t, J = 7.2 Hz, 3 H).
13 C NMR (100 MHz, CDCl3 ): δ = 197.9, 113.6, 35.3, 31.3, 7.0.
ESI-HRMS: m /z [M]+ calcd for C5 H7 NO: 97.0528; found: 97.0545.
3-Methoxypent-2-enenitrile (2)
3-Methoxypent-2-enenitrile (2)
Conc. H2 SO4 (8 mL, 0.18 mol) was added slowly to a solution of 1 (66.37 g, 0.69 mol) and trimethyl orthoformate (264 mL, 2.4 mol) in anh MeOH (180
mL); the mixture was stirred at 70 °C overnight. The reaction mixture was basified
to pH ~8–9 with K2 CO3 . MeOH was then removed by evaporation under reduced pressure. The mixture was diluted
with H2 O and extracted with EtOAc (2×). The organic layer was washed with sat. aq NaCl, dried
over Na2 SO4 , filtered, and evaporated to dryness under reduced pressure; this gave crude product
2 as a brown oil (80.0 g). The crude mixture of product 2 was directly used for the next step without purification.
1 H NMR (500 MHz, CDCl3 ): δ = 4.29 (s, 1 H), 3.63 (s, 3 H), 3.21 (s, 6 H), 2.65 (s, 2 H), 2.48 (q, J = 7.6 Hz, 2 H), 1.83 (q, J = 7.6 Hz, 2 H), 1.14 (t, J = 7.6 Hz, 3 H), 0.93 (t, J = 7.6 Hz, 2 H).
13 C NMR (100 MHz, CDCl3 ): δ = 179.9, 118.3, 116.5, 101.1, 68.8, 56.2, 48.5, 27.2, 26.8, 22.9, 11.4, 7.9.
ESI-HRMS: m /z [M + Na]+ calcd for C6 H9 NO: 134.0582; found: 134.0580.
ESI-HRMS: m /z [M + Na]+ calcd for C7 H13 NO2 : 166.0844; found 166.0834.
6-Ethylpyrimidine-2,4-diamine (3)
6-Ethylpyrimidine-2,4-diamine (3)
A mixture of guanidine hydrochloride (207.1 g, 2.16 mol) and NaOMe (116.6 g, 2.16
mol) in anh MeOH (300 mL) was left stirring at r.t. for 15 min under a N2 atmosphere. The reaction mixture was filtered under vacuum and the filtrate was added
to a solution of 2 (80 g, 0.72 mol) in anh DMSO (480 mL). The mixture was heated at 110 °C overnight.
MeOH was then removed by evaporation in vacuo. The mixture was subsequently extracted
with EtOAc (2×). The organic layer was washed with sat. aq NaCl, dried over Na2 SO4 , filtered, and evaporated to dryness under reduced pressure; this gave the crude
product as a brown oil. DMSO was removed after distillation under reduced pressure.
The crude product was crystallized with Et2 O; this gave 3 as a white solid; yield: 20.8 g (22%); mp 161.1–164.8 °C.
1 H NMR (500 MHz, DMSO-d
6 ): δ = 6.08 (s, 2 H), 5.69 (s, 2 H), 5.56 (s, 1 H), 2.26 (q, J = 7.6 Hz, 2 H), 1.08 (t, J = 7.6 Hz, 3 H).
13 C NMR (100 MHz, DMSO-d
6 ): δ = 169.5, 164.7, 163.2, 91.9, 29.8, 12.7.
ESI-HRMS: m /z [M + H]+ calcd for C6 H10 N4 : 139.0984; found: 139.0987.
2,4-Diamino-6-ethylpyrimidin-5-yl Hydrogen Sulfate (4)
2,4-Diamino-6-ethylpyrimidin-5-yl Hydrogen Sulfate (4)
A suspension of 3 (20.8 g, 0.15 mol) and 5 N aq NaOH solution (226 mL, 1.13 mol) was stirred at r.t.
for 30 min and then cooled to 0 °C. A solution of ammonium persulfate (69 g, 0.3 mol)
in H2 O (93 mL) was added and the mixture was stirred at 0 °C for 1 h. The mixture was left
at r.t. for 1 h and heated at 95 °C overnight. The mixture was cooled to 0 °C and
acidified to pH 4.5 with 3 M aq HCl. The precipitate obtained was collected by filtration
and washed with H2 O; this gave 4 as a yellow solid; yield: 29.7 g (84%); mp 220 °C (decomp).
1 H NMR (400 MHz, DMSO-d
6 ): δ = 11.75 (br s, 1 H), 8.33 (br s, 1 H), 7.26 (br s, 3 H), 2.61 (q, J = 7.4 Hz, 2 H), 1.14 (t, J = 7.5 Hz, 3 H).
13 C NMR (100 MHz, DMSO-d
6 ): δ = 161.6, 153.1, 149.0, 123.2, 20.7, 11.7.
ESI-HRMS: m /z [M + Na]+ calcd for C6 H10 N4 O4 SNa: 257.0320; found: 257.0316.
2,4-Diamino-6-ethyl-5-hydroxypyrimidine (5)
2,4-Diamino-6-ethyl-5-hydroxypyrimidine (5)
A solution of conc. H2 SO4 (6.72 mL, 0.127 mol) in H2 O (10 mL) was heated to 120 °C for 30 min. Compound 4 (29.68 g, 0.127 mol) was then added to the solution, which was then stirred for 20
min. After completion of the reaction, the mixture was cooled to 0 °C, and then basified
to pH 8 with K2 CO3 . A pale brown solid precipitated; it was washed with H2 O; this gave 5 ; yield: 18.8 g (96%); mp 220 °C (decomp).
1 H NMR (500 MHz, DMSO-d
6 ): δ = 7.36 (br s, 1 H) 5.96 (br s, 2 H), 5.35 (br s, 2 H), 2.42 (q, J = 7.6 Hz, 2 H), 1.08 (t, J = 7.6 Hz, 3 H).
1 H NMR (400 MHz, MeOD): δ = 2.64 (q, J = 7.6 Hz, 2 H), 1.24 (t, J = 7.6 Hz, 3 H).
13 C NMR (100 MHz, DMSO-d
6 ): δ = 159.2, 154.9, 147.9, 125.2, 22.0, 12.5.
ESI-HRMS: m /z [M + H]+ calcd for C6 H11 N4 O: 155.0933; found: 155.0936.
Methyl 3-(2-Hydroxyphenyl)propanoate (6)
Methyl 3-(2-Hydroxyphenyl)propanoate (6)
3,4-Dihydrocoumarin (20 mL, 157.8 mmol) was dissolved in anh MeOH (300 mL); then conc.
H2 SO4 (2 mL, 37.3 mmol) was added. The reaction mixture was heated to 45 °C overnight under
a N2 atmosphere. MeOH was removed under vacuum, and then the mixture was neutralized with
K2 CO3 . The mixture was diluted with H2 O and extracted with DCM (2×). The organic layer was collected and washed with sat.
aq NaCl and dried over Na2 SO4 . The solution was filtered and concentrated under reduced pressure to dryness. The
crude product was further purified by precipitation with cooled hexane; this gave
6 as a white solid; yield: 25.9 g (90%); mp 39.4–41.1 °C.
1 H NMR (500 MHz, DMSO-d
6 ): δ = 9.35 (s, 1 H), 7.03 (d, J = 6.3 Hz, 1 H), 7.03–6.99 (m, 1 H), 6.77 (d, J = 7.9 Hz, 1 H), 6.69 (t, J = 7.4 Hz, 1 H), 3.57 (s, 3 H), 2.76 (t, J = 7.7 Hz, 2 H), 2.55 (t, J = 7.7 Hz, 2 H).
13 C NMR (125 MHz, DMSO-d
6 ): δ = 172.9, 155.1, 129.6, 127.2, 126.4, 118.8, 114.8, 51.2, 33.3, 25.4.
ESI-HRMS: m /z [M + Na]+ calcd for C10 H12 O3 : 203.0684; found: 203.0684.
Methyl 3-[2-(3-Bromopropoxy)phenyl]propanoate (7)
Methyl 3-[2-(3-Bromopropoxy)phenyl]propanoate (7)
A solution of 6 (12.0 g, 66.6 mmol) in acetone (200 mL) was stirred at r.t. and then 1,3-dibromopropane
(40.5 mL, 398.7 mmol) and K2 CO3 (18.2 g, 131.7 mmol) were added. The reaction mixture was heated to 65 °C under reflux
for 2 h. After completion of the reaction, the mixture was subjected to solvent removal
under vacuum. The reaction mixture was then quenched with H2 O and extracted with EtOAc (2×). The combined organic layers were dried over Na2 SO4 , filtered, and concentrated under reduced pressure to give the crude product which
was further purified by column chromatography (silica gel, 10% EtOAc/hexane); this
gave 7 as a pale yellow oil; yield: 17.2 g (86%).
1 H NMR (400 MHz, CDCl3 ): δ = 7.13–7.19 (m, 2 H), 6.86 (m, 2 H), 4.12 (t, J = 6.1 Hz, 2 H), 3.68 (s, 3 H), 3.64 (t, J = 6.1 Hz, 2 H), 2.96 (t, J = 7.8 Hz, 2 H), 2.62 (t, J = 7.8 Hz, 2 H), 2.35 (qt, 2 H).
13 C NMR (100 MHz, CDCl3 ): δ = 173.7, 156.4, 129.9, 128.9, 127.6, 120.7, 111.1, 65.0, 51.5, 34.1, 32.3, 30.1,
26.1.
ESI-HRMS: m /z [M + Na]+ calcd for C13 H17 BrO3 : 323.0259; found: 323.0277.
Methyl 3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}phenyl)propanoate (8)
Methyl 3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}phenyl)propanoate (8)
The pale brown solid of 5 (3.4 g, 18.7 mmol) and LiOH·H2 O (2.1 g, 50.0 mmol) were dissolved in anh DMF (25 mL) at r.t. under a N2 atmosphere. After the mixture had stirred for 1 h, 7 (3.75 g, 12.5 mmol) and KI (3.1 g, 18.7 mmol) were added, and the mixture was then
stirred overnight. After completion of the reaction, DMF was removed under reduced
pressure. The residue was diluted with H2 O, followed by extraction with EtOAc (2×). The organic layer was collected and dried
over Na2 SO4 , filtered, and concentrated under reduced pressure; this gave the crude product.
The crude product was purified by column chromatography (silica gel, 2% MeOH/DCM);
this gave 8 as a pale yellow oil; yield: 2.4 g (52%).
1 H NMR (500 MHz, MeOD-d
4 ): δ = 7.20–7.16 (m, 1 H), 7.14 (dd, J = 7.1, 1.6 Hz, 1 H), 6.97 (brd, J = 7.4 Hz, 1 H), 6.87–6.84 (m, 1 H), 4.23 (t, J = 6.0 Hz, 2 H), 3.95 (t, J = 6.0 Hz, 2 H), 3.61 (s, 3 H), 2.92 (t, J = 7.7 Hz, 2 H), 2.59 (t, J = 7.7 Hz, 2 H), 2.45 (q, J = 7.6 Hz, 2 H), 2.29–2.24 (m, 2 H), 1.10 (t, J = 7.6 Hz, 3 H).
13 C NMR (125 MHz, MeOD-d
4 ): δ = 175.4, 160.5, 157.9, 130.9, 130.3, 129.9, 128.8, 121.7, 112.3, 70.9, 65.3,
52.0, 35.1, 31.0, 27.2, 24.9, 13.3.
ESI-HRMS: m /z [M + H]+ calcd for C19 H27 N4 O4 : 375.2032; found: 375.1960.
3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}phenyl)propanoic Acid Hydrochloride
(P218·HCl)
3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}phenyl)propanoic Acid Hydrochloride
(P218·HCl)
Ester intermediate 8 was then hydrolyzed by 10% NaOH in EtOH and heated to 90 °C. After 2 h, EtOH was
removed under reduced pressure; this gave the crude product which was then acidified
to pH 2–3 with conc. HCl, affording a salt form of P218 without the need for column
chromatography; this gave P218·HCl as a white solid; yield: 2.2 g (92%); mp 195.4–197.6
°C. The NMR data are in accordance with the literature,[12 ] with the signal of the quaternary carbon on the pyrimidine ring missing.
1 H NMR (500 MHz, DMSO-d
6 ): δ = 12.45 (br s, 1 H), 12.09 (br s, 1 H), 8.31 (br s, 1 H), 7.84 (br s, 1 H), 7.44
(br s, 2 H), 7.19–7.14 (m, 2 H), 6.98 (d, J = 8.0 Hz, 1 H), 6.85 (t, J = 7.3 Hz, 1 H), 4.15 (t, J = 5.9 Hz, 2 H), 3.90 (t, J = 6.3 Hz, 2 H), 2.78 (t, J = 7.7 Hz, 2 H), 2.48–2.45 (m, 2 H), 2.24–2.19 (m, 2 H), 1.12 (t, J = 7.6 Hz, 3 H).
13 C NMR (125 MHz, DMSO-d
6 ): δ = 173.9, 161.1, 156.2, 153.3, 147.3, 129.5, 128.6, 127.5, 120.3, 111.3, 70.7,
63.9, 33.7, 29.1, 25.3, 19.8, 11.9.
ESI-HRMS: m /z [M + H]+ calcd for C18 H24 N4 O4 : 361.1847; found: 361.1876.
5-(Benzyloxy)-2-hydroxybenzaldehyde (9)
5-(Benzyloxy)-2-hydroxybenzaldehyde (9)
A round-bottomed flask was charged with 4-(benzyloxy)phenol (10.0 g, 50.0 mmol), anh
MgCl2 (15.5 g, 175.0 mmol), and paraformaldehyde (6.0 g, 200 mmol); the addition of anh
MeCN followed (0.5 M) at 0 °C. The resulting mixture was stirred at r.t. for 30 min,
before triethylamine (26.3 mL, 175 mmol) was added. The reaction mixture was then
heated to reflux for 18 h. After completion of the reaction, the resulting mixture
was cooled to r.t. and treated with 1 M aq HCl. The two phases were separated and
the aqueous layer was extracted with EtOAc (2×). The combined organic layers were
dried over Na2 SO4 , filtered, and concentrated under reduced pressure to give the crude product, which
was further purified by column chromatography (silica gel, 5% EtOAc/hexane); this
gave 9 as a white solid; yield: 8.2 g (72%); mp 95.5–97.6 °C.
1 H NMR (500 MHz, CDCl3 ): δ = 10.67 (s, 1 H), 9.83 (s, 1 H), 7.45–7.38 (m, 4 H), 7.37–7.32 (m, 1 H), 7.22
(dd, J = 9.0, 3.1 Hz, 1 H), 7.08 (d, J = 3.1 Hz, 1 H), 6.94 (d, J = 9.0 Hz, 1 H), 5.07 (s, 2 H).
13 C NMR (125 MHz, CDCl3 ): δ = 196.1, 156.2, 151.8, 136.5, 128.7 (2C), 128.2, 127.5 (2C), 126.1, 120.0, 118.7,
116.8, 71.0.
ESI-HRMS: m /z [M – H]+ calcd for C14 H12 O3 : 227.0714; found: 227.0709.
Ethyl (E )-3-[5-(Benzyloxy)-2-(3-bromopropoxy)phenyl]acrylate (11)
Ethyl (E )-3-[5-(Benzyloxy)-2-(3-bromopropoxy)phenyl]acrylate (11)
To a stirred solution of 9 (10.7 g, 46.8 mmol) in DCM (0.2 M) at r.t. was added ethyl (triphenylphosphoranylidene)acetate
(19.6 g, 56.2 mmol) and the reaction mixture was stirred for 1 h. After completion
of the reaction, the mixture was quenched with water and the two phases were separated.
The aqueous layer was extracted with DCM (2×). The combined organic layers were dried
over Na2 SO4 , filtered, and concentrated under reduced pressure to afford the crude product, which
was used in next step without further purification. To the resulting mixture of 9 in acetone (0.1 M) was added 1,3-dibromopropane (280.8 mmol, 28.5 mL) and K2 CO3 (12.9 g, 93.6 mmol) and the mixture was stirred at r.t. The reaction mixture was
then heated to reflux, at which it was stirred for 2 h. Then the acetone was removed
under reduced pressure. The reaction residue was then added to water and extracted
with EtOAc (2×). The combined organic layers were dried over Na2 SO4 , filtered, and concentrated under reduced pressure to give the crude product, which
was further purified by column chromatography (silica gel, 5% EtOAc/hexane); this
gave 11 as a white solid; yield: 15.5 g (79% over two steps); mp 57.1–59.9 °C.
1 H NMR (500 MHz, CDCl3 ): δ = 7.97 (d, J = 16.2 Hz, 1 H), 7.45–7.37 (m, 4 H), 7.36–7.31 (m, 1 H), 7.15 (d, J = 3.0 Hz, 1 H), 6.97 (dd, J = 9.0, 3.0 Hz, 1 H), 6.87 (d, J = 9.0 Hz, 1 H), 6.44 (d, J = 16.1 Hz, 1 H), 5.04 (s, 2 H), 4.26 (q, J = 7.1 Hz, 2 H), 4.12 (t, J = 5.8 Hz, 2 H), 3.63 (t, J = 6.3 Hz, 2 H), 2.36 (p, J = 6.1 Hz, 2 H), 1.34 (t, J = 7.1 Hz, 3 H).
13 C NMR (125 MHz, CDCl3 ): δ = 167.2, 152.9, 151.8, 139.3, 136.8, 128.6 (2C), 128.0, 127.4 (2C), 124.4, 118.9,
118.1, 114.1, 113.8, 70.7, 66.6, 60.4, 32.3, 30.0, 14.3.
ESI-HRMS: m /z [M + Na]+ calcd for C21 H23 BrNaO4 : 441.0672; found: 441.0666.
Ethyl (E )-3-(5-(Benzyloxy)-2-{3-[(2,4-diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}phenyl)acrylate
(12)
Ethyl (E )-3-(5-(Benzyloxy)-2-{3-[(2,4-diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}phenyl)acrylate
(12)
To a stirred solution of 11 (7.1 g, 16.9 mmol) in DMF (0.5 M) at r.t. was added 2,6-diamino-4-ethyl-5-hydroxypyrimidin-1-ium
chloride (5 ·HCl; 4.8 g, 25.4 mmol), KI (4.2 g, 25.4 mmol), and LiOH·H2 O (1.1 g, 25.4 mmol); then the mixture was stirred for 18 h. After completion of the
reaction, EtOAc and water were added and the two phases were separated. The aqueous
layer was extracted with EtOAc (2×). The combined organic layers were extracted many
times with water to remove the DMF and dried over Na2 SO4 , filtered, and concentrated under reduced pressure, to give the crude product, which
was further purified by column chromatography (silica gel, 2% MeOH/DCM); this gave
12 as a white foamy solid; yield: 4.2 g (51%).
1 H NMR (500 MHz, DMSO-d
6 ): δ = 7.88 (d, J = 16.1 Hz, 1 H), 7.49–7.42 (m, 2 H), 7.41–7.36 (m, 3 H), 7.34–7.28 (m, 1 H), 7.07
(s, 2 H), 6.66 (d, J = 16.1 Hz, 1 H), 6.11 (s, 2 H), 5.56 (s, 2 H), 5.10 (s, 2 H), 4.19 (t, J = 5.9 Hz, 2 H), 4.16 (q, J = 7.1 Hz, 2 H), 3.78 (t, J = 6.1 Hz, 2 H), 2.30 (q, J = 7.6 Hz, 2 H), 2.18 (t, J = 6.1 Hz, 2 H), 1.22 (t, J = 7.1 Hz, 3 H), 0.97 (t, J = 7.6 Hz, 3 H).
13 C NMR (125 MHz, DMSO-d
6 ): δ = 166.5, 159.7, 159.0, 158.2, 152.3, 151.7, 138.9, 137.2, 128.4 (2C), 128.2,
127.8, 127.8 (2C), 123.1, 119.0, 118.6, 114.0, 113.8, 69.8, 68.9, 65.4, 60.0, 29.4,
23.5, 14.2, 12.6.
ESI-HRMS: m /z [M + H]+ calcd for C27 H33 N4 O5 : 493.2445; found: 493.2449.
Ethyl 3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}-5-hydroxyphenyl)propanoate
(13)
Ethyl 3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}-5-hydroxyphenyl)propanoate
(13)
Anh and degassed MeOH (0.1 M) was added to a round-bottomed flask charged with 12 (3.5 g, 7.1 mmol) and 10% Pd/C (10% w/w, 636.3 mg) at r.t. The reaction mixture was
allowed to stir under a H2 atmosphere. After completion of the reaction, the mixture was filtered through Celite
in a Buchner funnel. The solution was concentrated under reduced pressure, to give
the crude product, which was further purified by column chromatography (silica gel,
4% MeOH/DCM); this gave 13 as a white foamy solid; yield: 2.0 g (71%).
1 H NMR (500 MHz, MeOD-d
4 ): δ = 6.80 (d, J = 8.3 Hz, 1 H), 6.69–6.52 (m, 2 H), 4.14 (t, J = 5.8 Hz, 2 H), 4.07 (q, J = 7.1 Hz, 2 H), 3.94 (t, J = 6.2 Hz, 2 H), 2.85 (t, J = 7.7 Hz, 2 H), 2.56 (dd, J = 8.2, 7.1 Hz, 2 H), 2.47 (q, J = 7.6 Hz, 2 H), 2.22 (p, J = 6.0 Hz, 2 H), 1.19 (t, J = 7.1 Hz, 3 H), 1.12 (t, J = 7.6 Hz, 3 H).
13 C NMR (125 MHz, MeOD-d
4 ): δ = 175.0, 161.3, 160.6, 160.1, 152.1, 151.3, 131.1, 130.3, 118.0, 114.5, 113.8,
71.0, 66.0, 61.5, 35.4, 31.1, 27.2, 24.9, 14.5, 13.3.
ESI-HRMS: m /z [M + H]+ calcd for C20 H29 N4 O5 : 405.2132; found: 405.2137.
3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}-5-hydroxyphenyl)propanoic
Acid Hydrochloride (P218-OH·HCl)
3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy}-5-hydroxyphenyl)propanoic
Acid Hydrochloride (P218-OH·HCl)
To afford the final product (P218-OH), the intermediate 13 (180.9 mg, 0.45 mmol) was then hydrolyzed by 10% NaOH (4 mL). After 10 min, EtOH
was removed from the mixture under reduced pressure, to give the crude product, which
was then acidified to pH 2–3 with concentrated HCl, affording a salt form of P218-OH,
with no need for column chromatography; this gave a white solid; yield: 142.2 mg (72%);
mp 208.3–212.8 °C.
1 H NMR (400 MHz, MeOD): δ = 6.81 (d, J = 8.6 Hz, 1 H), 6.67–6.57 (m, 2 H), 4.14 (t, J = 5.7 Hz, 2 H), 4.04 (t, J = 6.2 Hz, 2 H), 2.84 (dd, J = 8.5, 7.1 Hz, 2 H), 2.65–2.50 (m, 4 H), 2.27 (p, J = 6.0 Hz, 2 H), 1.18 (t, J = 7.6 Hz, 3 H); .
13 C NMR (100 MHz, MeOD): δ = 177.0, 163.5, 155.0, 152.2, 151.2, 149.1, 131.3, 129.7,
117.9, 114.4, 113.7, 72.3, 65.7, 35.3, 30.8, 27.2, 21.5, 12.5.
ESI-HRMS: m /z [M + H]+ calcd for C18 H24 O4 N5 : 377.1825; found: 377.1817.
