<title>Introduction</title> <p>Oxazolo[5,4-<italic>d</italic>]pyrimidines have been reported to possess a variety of biological activities including kinase inhibition [##UREF##0##1##–##REF##15050645##2##], adenosine receptor antagonism [##REF##16289482##3##] and tumor growth inhibition [##UREF##1##4##]. In particular, 5-aminooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones [9-oxa-guanines] and related heterocycles have been shown to inhibit the ability of ricin to inactivate ribosomes [##REF##11754581##5##]. Given these reports, and the expectation of further applications based upon similarity to naturally occurring nucleic acid bases, this heterocyclic ring system continues to generate interest.</p> <p>Approaches to the oxazolo[5,4-<italic>d</italic>]pyrimidine ring system generally involve either cyclodehydration of an 5-(acylamino)-4-hydroxypyrimidine [##UREF##2##6##–##UREF##6##10##] or elaboration of a 4-cyano- or 4-(alkoxycarbonyl)-5-aminooxazole [##UREF##7##11##–##UREF##11##15##] (##FIG##0##Figure 1##), with only isolated reports of alternative routes [##UREF##12##16##–##UREF##13##17##]. However, few publications have described the preparation of 5-aminooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones [##REF##11754581##5##,##UREF##4##8##,##UREF##10##14##] or 5-mercaptooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones [9-oxa-2-thio-xanthines] [##UREF##3##7##,##UREF##11##15##], and in both cases, the conditions do not appear to be amenable to the preparation of oxazolo[5,4-<italic>d</italic>]pyrimidines with variations at the 2-position, particularly those with additional functional groups or increased steric demand.</p> <p>In pursuing 8-methyl-9-oxa-guanine [2-methyloxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-one] as an initial lead in the design of inhibitors of the ribosome-inactivating protein ricin [##REF##11754581##5##], we set out to prepare a series of 2-substituted 5-aminooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones and related 5-thio-oxazolo[5,4-<italic>d</italic>]pyrimidines. Here we report these studies, which have led to the preparation of analogs suitable for further elaboration, for example by “click” chemistry employing copper-catalyzed Huisgen 1,3-dipolar cycloadditions [##UREF##14##18##].</p>
<title>Results and Discussion</title> <p>Our previous route [##REF##11754581##5##] to 8-methyl-9-oxa-guanine (<bold>2a</bold>) involved the thermal cyclodehydration of 5-(acetylamino)-2-amino-4,6-dihydroxypyrimidine (<bold>1a</bold>) [##UREF##15##19##] (##FIG##1##Figure 2##). Unfortunately, for other 5-acylamino analogs of <bold>1a</bold>, this route failed to afford the required oxazolo[5,4-<italic>d</italic>]pyrimidines, presumably due to decomposition of the product at the temperatures required for cyclodehydration. Other cyclodehydration conditions (POCl<sub>3</sub>, PPA) were also unsuccessful.</p> <p>An alternative route was sought in which the oxazole ring was formed first, followed by elaboration of the pyrimidine ring. Our previous work had demonstrated that in the case of ethyl 5-amino-2-methyloxazole-4-carboxylate <bold>3a</bold> [##UREF##16##20##], direct annulation with chloroformamidine in DMSO at 120 °C or with 2-methylisothiourea sulfate neat or in ethylene glycol at 170 °C did not afford the oxazolo[5,4-<italic>d</italic>]pyrimidine <bold>2</bold> (##FIG##1##Figure 2##). Thus, a stepwise annulation [##UREF##11##15##] strategy was explored for the preparation of analogs of <bold>2</bold> bearing different 2-positions substituents. The required 5-aminooxazoles <bold>3b,c</bold> were prepared from the (acylamino)cyanoacetates <bold>5b,c</bold>, which in turn were derived from ethyl cyanoglyoxylate oxime (<bold>4</bold>) by reduction followed by acylation by isobutyric anhydride or azidoacetyl chloride (##FIG##2##Figure 3##). Reaction of the aminooxazoles <bold>3b,c</bold> with benzoylisothiocyanate afforded the thioureas <bold>6b,c</bold>. Direct cyclization of <bold>6b,c</bold> to oxazolo[5,4-<italic>d</italic>]pyrimidines <bold>2b,c</bold> by treatment with ammonia in methanol was unsuccessful. However, thioureas <bold>6b,c</bold> were converted to the desired 2-substituted the oxazolo[5,4-<italic>d</italic>]pyrimidines <bold>2b,c</bold> by stepwise methylation followed by cyclization in the presence of ammonia in methanol. Although the intermediate products after methylation were not purified, in the case of <bold>6c</bold> the <sup>1</sup>H NMR spectrum of the crude product following methylation retains the signals for the ethoxy group of <bold>6c</bold> and displays a new resonance for the <italic>S</italic>-methyl group at 2.0 ppm (see ##SUPPL##1##Supporting Information File 1##), indicating that the intermediate is the <italic>S</italic>-methylisothiourea <bold>A</bold> (##FIG##2##Figure 3##). The mechanism involved in the conversion of intermediates such as <bold>A</bold> to <bold>2</bold> is not clear. It is unlikely that the 5-(methylthio)oxazolo[5,4-<italic>d</italic>]pyrimidine is an intermediate because treatment of 5-(methylthio)oxazolo[5,4-<italic>d</italic>]pyrimidine <bold>8a</bold> (see below) with ammonia in methanol fails to afford <bold>2a</bold>.</p> <p>The thiourea <bold>6b</bold> and its analog <bold>6a</bold>, which was prepared from aminooxazole <bold>3a</bold> in 51% yield, were also subjected to cyclization in the presence of ethanolic KOH to afford the 5-mercaptooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones <bold>7a,b</bold> (##FIG##3##Figure 4##). Further elaboration of <bold>7a</bold> by alkylation with methyl iodide, benzyl bromide, propargyl bromide, or methyl 6-<italic>O</italic>-(tolylsulfonyl)-α-D-glucopyranoside [##REF##17564440##21##] afforded the thioethers <bold>8a</bold>–<bold>d</bold>, respectively. The structural assignment of <bold>8a</bold>–<bold>d</bold> as thioethers rest principally on their <sup>1</sup>H and <sup>13</sup>C NMR spectra, which contain resonances more commensurate with groups attached to sulfur (e.g., for <bold>8a</bold>: 2.2 ppm in <sup>1</sup>H and 13 ppm in <sup>13</sup>C NMR, respectively) than to nitrogen, as would be the case for 4- or 3-alkylated 5-mercaptooxazolo[5,4-<italic>d</italic>]pyrimidine-7(6<italic>H</italic>)-ones.</p> <p>The thioether <bold>8c</bold> bearing a propargyl substituent was subjected to “click” chemistry coupling with benzyl azide or 2-morpholinoethyl azide in the presence of catalytic CuSO<sub>4</sub> and sodium ascorbate to afford the triazoles <bold>9</bold> and <bold>10</bold>, respectively, in good yield (##FIG##4##Figure 5##). The copper-catalyzed Huisgen cycloaddition of terminal alkynes and alkyl azides favors formation of the 1,4-triazole regioisomers [##UREF##17##22##], and in the case of triazole <bold>9</bold>, this regiochemistry was confirmed by <sup>1</sup>H NOE spectra (see ##SUPPL##1##Supporting Information File 1##).</p>
<title>Results and Discussion</title> <p>Our previous route [##REF##11754581##5##] to 8-methyl-9-oxa-guanine (<bold>2a</bold>) involved the thermal cyclodehydration of 5-(acetylamino)-2-amino-4,6-dihydroxypyrimidine (<bold>1a</bold>) [##UREF##15##19##] (##FIG##1##Figure 2##). Unfortunately, for other 5-acylamino analogs of <bold>1a</bold>, this route failed to afford the required oxazolo[5,4-<italic>d</italic>]pyrimidines, presumably due to decomposition of the product at the temperatures required for cyclodehydration. Other cyclodehydration conditions (POCl<sub>3</sub>, PPA) were also unsuccessful.</p> <p>An alternative route was sought in which the oxazole ring was formed first, followed by elaboration of the pyrimidine ring. Our previous work had demonstrated that in the case of ethyl 5-amino-2-methyloxazole-4-carboxylate <bold>3a</bold> [##UREF##16##20##], direct annulation with chloroformamidine in DMSO at 120 °C or with 2-methylisothiourea sulfate neat or in ethylene glycol at 170 °C did not afford the oxazolo[5,4-<italic>d</italic>]pyrimidine <bold>2</bold> (##FIG##1##Figure 2##). Thus, a stepwise annulation [##UREF##11##15##] strategy was explored for the preparation of analogs of <bold>2</bold> bearing different 2-positions substituents. The required 5-aminooxazoles <bold>3b,c</bold> were prepared from the (acylamino)cyanoacetates <bold>5b,c</bold>, which in turn were derived from ethyl cyanoglyoxylate oxime (<bold>4</bold>) by reduction followed by acylation by isobutyric anhydride or azidoacetyl chloride (##FIG##2##Figure 3##). Reaction of the aminooxazoles <bold>3b,c</bold> with benzoylisothiocyanate afforded the thioureas <bold>6b,c</bold>. Direct cyclization of <bold>6b,c</bold> to oxazolo[5,4-<italic>d</italic>]pyrimidines <bold>2b,c</bold> by treatment with ammonia in methanol was unsuccessful. However, thioureas <bold>6b,c</bold> were converted to the desired 2-substituted the oxazolo[5,4-<italic>d</italic>]pyrimidines <bold>2b,c</bold> by stepwise methylation followed by cyclization in the presence of ammonia in methanol. Although the intermediate products after methylation were not purified, in the case of <bold>6c</bold> the <sup>1</sup>H NMR spectrum of the crude product following methylation retains the signals for the ethoxy group of <bold>6c</bold> and displays a new resonance for the <italic>S</italic>-methyl group at 2.0 ppm (see ##SUPPL##1##Supporting Information File 1##), indicating that the intermediate is the <italic>S</italic>-methylisothiourea <bold>A</bold> (##FIG##2##Figure 3##). The mechanism involved in the conversion of intermediates such as <bold>A</bold> to <bold>2</bold> is not clear. It is unlikely that the 5-(methylthio)oxazolo[5,4-<italic>d</italic>]pyrimidine is an intermediate because treatment of 5-(methylthio)oxazolo[5,4-<italic>d</italic>]pyrimidine <bold>8a</bold> (see below) with ammonia in methanol fails to afford <bold>2a</bold>.</p> <p>The thiourea <bold>6b</bold> and its analog <bold>6a</bold>, which was prepared from aminooxazole <bold>3a</bold> in 51% yield, were also subjected to cyclization in the presence of ethanolic KOH to afford the 5-mercaptooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones <bold>7a,b</bold> (##FIG##3##Figure 4##). Further elaboration of <bold>7a</bold> by alkylation with methyl iodide, benzyl bromide, propargyl bromide, or methyl 6-<italic>O</italic>-(tolylsulfonyl)-α-D-glucopyranoside [##REF##17564440##21##] afforded the thioethers <bold>8a</bold>–<bold>d</bold>, respectively. The structural assignment of <bold>8a</bold>–<bold>d</bold> as thioethers rest principally on their <sup>1</sup>H and <sup>13</sup>C NMR spectra, which contain resonances more commensurate with groups attached to sulfur (e.g., for <bold>8a</bold>: 2.2 ppm in <sup>1</sup>H and 13 ppm in <sup>13</sup>C NMR, respectively) than to nitrogen, as would be the case for 4- or 3-alkylated 5-mercaptooxazolo[5,4-<italic>d</italic>]pyrimidine-7(6<italic>H</italic>)-ones.</p> <p>The thioether <bold>8c</bold> bearing a propargyl substituent was subjected to “click” chemistry coupling with benzyl azide or 2-morpholinoethyl azide in the presence of catalytic CuSO<sub>4</sub> and sodium ascorbate to afford the triazoles <bold>9</bold> and <bold>10</bold>, respectively, in good yield (##FIG##4##Figure 5##). The copper-catalyzed Huisgen cycloaddition of terminal alkynes and alkyl azides favors formation of the 1,4-triazole regioisomers [##UREF##17##22##], and in the case of triazole <bold>9</bold>, this regiochemistry was confirmed by <sup>1</sup>H NOE spectra (see ##SUPPL##1##Supporting Information File 1##).</p>
<title>Conclusion</title> <p>In conclusion, routes to functionalized oxazolo[5,4-<italic>d</italic>]pyrimidines from 2-substituted 5-aminooxazole-4-carboxylic acid ethyl esters were developed, the key to which is the relatively mild conditions employed in the step-wise elaboration of the pyrimidine ring. Compounds <bold>2b,c</bold>, <bold>7a,b</bold>, <bold>8a</bold>–<bold>d</bold>, <bold>9</bold>, and <bold>10</bold> were evaluated for their ability to inhibit recombinant, catalytically active ricin A-chain (RTA) employing a modification of the previously reported assay [##REF##11754581##5##]. Briefly, the synthesis of protein from endogenous globin mRNA by rabbit reticulocyte lysate was determined in the absence of RTA, in the presence of sufficient RTA to inhibit 90% of protein synthesis (<italic>ca.</italic> 10 pM), and in the presence of both RTA and increasing concentrations of the oxazolo[5,4-<italic>d</italic>]pyrimidines. The ability of these compounds to inhibit RTA and thereby rescue protein synthesis was determined. Compounds <bold>2b</bold> (IC<sub>50</sub> = 2.8 mM) and <bold>9</bold> (IC<sub>50</sub> = 1.6 mM), displayed some activity; whereas, none of the other compounds examined showed any significant RTA inhibitory activity.</p>
<p>Oxazolo[5,4-<italic>d</italic>]pyrimidines can be considered as 9-oxa-purine analogs of naturally occurring nucleic acid bases. Interest in this ring system has increased due to recent reports of biologically active derivatives. In particular, 5-aminooxazolo[5,4-<italic>d</italic>]pyrimidine-7(6<italic>H</italic>)-ones (9-oxa-guanines) have been shown to inhibit ricin. The preparation of a series of 2-substituted 5-aminooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones and related 5-thio-oxazolo[5,4-<italic>d</italic>]pyrimidines is described, including analogs suitable for further elaboration employing “click” chemistry utilizing copper-catalyzed Huisgen 1,3-dipolar cycloadditions. Two of the compounds prepared were found to inhibit ricin with IC<sub>50</sub>
<italic>ca.</italic> 1–3 mM.</p>
<title>Supporting Information</title>
<p>Support from DOD/USAMRC DAMD17-03-C-0088 to JDR is gratefully acknowledged.</p>
<fig id="F1" position="float"><label>Figure 1</label><caption><p>The two general synthetic approaches to oxazolo[5,4-<italic>d</italic>]pyrimidines.</p></caption></fig> <fig id="F2" position="float"><label>Figure 2</label><caption><p>Thermal cyclodehydration route to 9-oxo-guanine.</p></caption></fig> <fig id="F3" position="float"><label>Figure 3</label><caption><p>Preparation of 2-substituted 5-aminooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones.</p></caption></fig> <fig id="F4" position="float"><label>Figure 4</label><caption><p>Preparation of 2-substituted 5-aminooxazolo[5,4-<italic>d</italic>]pyrimidin-7(6<italic>H</italic>)-ones and related thioethers.</p></caption></fig> <fig id="F5" position="float"><label>Figure 5</label><caption><p>Click chemistry elaboration of a 5-(propargylthio)oxazolo[5,4-<italic>d</italic>]pyrimidine.</p></caption></fig>
<supplementary-material content-type="local-data" id="SD2"><label>File 1</label><caption><p>This file includes full experimental details for all new compounds.</p></caption></supplementary-material> <supplementary-material content-type="local-data" id="SD1"><label>File 2</label><caption><p>Copies of <sup>1</sup>H and <sup>13</sup>C NMR spectra of compounds <bold>2b</bold>,<bold>c</bold>; <bold>3a</bold>–<bold>c</bold>; <bold>5b</bold>,<bold>c</bold>; <bold>6a</bold>–<bold>c</bold>; <bold>A</bold>; <bold>7a</bold>,<bold>b</bold>; <bold>8a</bold>–<bold>d</bold>; <bold>9</bold>; and <bold>10</bold>.</p></caption></supplementary-material>
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Beilstein J Org Chem. 2008 Jul 25; 4:26