Synthesis of Nucleo Aminooxy Acid Derivatives

Abstract

Nucleobase-functionalized peptides have attracted increasing interest because of their well-ordered secondary structures and stability toward enzymatic degradations. We have designed and synthesized nucleo aminooxy acids as novel building blocks for nucleopeptides. Four nucleo aminooxy acid derivatives with cytosine or thymine in the side chain linked by an amide or a triazole moiety have been synthesized from l-serine.

Key words

nucleo aminooxy acids - nucleopeptides - N-oxy nucleopeptides - cytosine - thymine - amides - triazoles - l-serine - eliminations

Nucleo amino acids are synthetic amino acids bearing nucleobases covalently linked to their side chains. Various peptides containing nucleo α- and β-amino acids have been reported as being able to form rigid and helical structures as well as well-defined double strands with complementary sequences.[1] [2] [3] [4] [5] [6] [7] Moreover, nucleopeptides have recently emerged as a promising alternative to peptide nucleic acids,[ ^8,9 ] able to penetrate into a cell nucleus without cytotoxic effects.[ ¹⁰ ]

Aminooxy acids are analogues of amino acids bearing an oxyamine function (O–NH₂) in the place of amine. Peptides of aminooxy acids have an ease of forming well-defined­ structures like α-, β- and γ-turns or helices thanks to intramolecular hydrogen-bond formation.[11] [12] It would therefore be interesting to synthesize nucleo aminooxy acids­ containing nucleobases on the side chain of aminooxy acids in order to study the secondary structure and DNA/RNA binding properties of the corresponding N-oxy nucleopeptides, since both the N-oxy peptide and the nucleobases could contribute to structure organization. As part of a continuing program on the synthesis of sugar- and nucleoside-derived aminooxy acids,[13] [14] [15] [16] [17] we report herein the synthesis of nucleo aminooxy acid derivatives with thymine or cytosine connected to the side chain of a β-aminooxy acid through either an amide or a triazole linkage (Figure [1]). To the best of our knowledge, nucleo aminooxy acids have not been previously reported in the literature.

Figure 1 Structures of the target nucleo aminooxy acid derivatives

The target nucleo aminooxy acid derivatives are accessible from the β-phthalimidooxy ester 8 (Scheme [1]). This compound has been previously prepared from l-serine by Burke and co-workers.[ ¹⁸ ] We have synthesized the phthalimidooxy ester 7 from l-Ser–OMe (5) by N-tritylation and Mitsunobu reactions, and purified compounds 6 and 7 by simple precipitation, without column chromatography. Treatment of 7 with 36% hydrochloric acid in dichloromethane as reported led, however, to a mixture of compounds. Removal of the trityl group was then achieved with acetyl chloride in methanol, leading to the amine salt 8 in 65% yield. Coupling of 8 with N ⁴-Cbz-protected cytosin-1-ylacetic acid 9 [ ¹⁹ ] using BOP reagent furnished the cytosin-1-yl-substituted aminooxy ester 2a in 53% yield; however, reaction of 8 with thymin-1-ylacetic acid (14)[ ²⁰ ] using EDC/HOAt led to the corresponding polar thymin-1-yl-substituted aminooxy ester which proved to be difficult to purify.

We then decided to replace the phthaloyl protecting group of the oxyamine in 8 by Boc, through Cbz protection of the amine function, hydrazinolysis and treatment with Boc₂O;[ ¹⁸ ] however, saponification of 10 led to a mixture of the desired carboxylic acid 11 [ ¹⁸ ] and the elimination product 12 [ ²¹ ] in a 1:1 ratio (Scheme [1]), showing that the N-Boc-protected aminooxy ester 10 is sensitive to basic conditions. Deprotection of the Cbz group under hydrogenolysis conditions was also troublesome. In fact, prolonged reaction induced homolytic cleavage of the N–O bond.[ ¹⁵ ]

Scheme 1 Synthesis of nucleo aminooxy acid derivatives 1 and 2

Nevertheless, it was possible to obtain the amine 13 in acceptable yield when the reaction time did not exceed 10 minutes. Coupling of amine 13 with thymin-1-ylacetic acid (14) promoted by EDC/HOAt furnished the thymin-1-yl-substituted aminooxy ester 1a in 43% yield (two steps), along with a small quantity of over-acylation product of the HN–O nitrogen.[ ²² ] Boc protection of the HN–O nitrogen in 10 was then realized with Boc₂O in the presence of 4-(dimethylamino)pyridine to afford a 70% yield of 15 and a 15% yield of the tris-Boc derivative 16. Fast hydrogenolysis of 15 followed by coupling with 14 or 9 gave the corresponding nucleo aminooxy esters 1b and 2b in 55% and 65% yield, respectively. To prepare the fully deprotected nucleo aminooxy acids 1c and 2c, it is preferable to remove the Boc groups before saponification in order to avoid the elimination reaction of 1b and 2b under basic conditions. Compounds 1c and 2c were obtained in quantitative yield (Scheme [1]).

Triazole-linked nucleo aminooxy acid derivatives 3 and 4 could be prepared by click reaction between the azido intermediate 19 and the alkyne derivatives 22 and 21 [ ²³ ] (Scheme [2]). Compound 8 was firstly acylated with chloroacetyl chloride to give compound 17; however, subsequent substitution with sodium azide at 0 °C quantitatively afforded the elimination product 18. We then decided to prepare 19 by condensation of amine salt 8 with azidoacetic acid[ ²⁴ ] promoted by EDC/HOBt in the presence of 1 equivalent of triethylamine. Once again, the elimination reaction mainly occurred: the desired compound 19 was isolated in only 24% yield. To avoid this side reaction, hindered 2,6-di-tert-butyl-4-methylpyridine was chosen to neutralize the amine salt 8, successfully leading to compound 19 in 95% yield. Click reaction of 19 with 1-propargylthymine (22) catalyzed by copper(II) sulfate and sodium ascorbate accomplished the cycloaddition reaction, followed, however, by elimination of the phthalimidooxy moiety to give compound 23 in 68% yield. Fortunately, the use of ascorbic acid[ ²⁵ ] avoided the elimination reaction and afforded the desired nucleo aminooxy acid derivatives 3 and 4 in 87% and 77% yield, respectively.

Scheme 2 Synthesis of nucleo aminooxy esters 3 and 4; DTBMP = 2,6-di(tert-butyl)-4-methylpyridine

In summary, two series of nucleo aminooxy acid derivatives have been synthesized by linking thymine or cytosine to the side chain of an α-amino-β-aminooxy acid prepared from l-serine methyl ester. A more convenient procedure for the synthesis of phthaloyl-protected β-aminooxy ester 8 has been developed. The high polarity of phthaloyl-protected amide-linked nucleo β-aminooxy esters led us to prepare Boc-protected derivatives 1a, 1b and 2b which were fully deprotected to the free nucleo β-aminooxy acids 1c and 2c. Triazole-linked nucleo aminooxy esters 3 and 4 have also been successfully synthesized via click chemistry. During our synthesis, we also observed the instability of phthaloyl- or Boc-protected β-aminooxy esters under basic conditions, leading to the corresponding acrylate elimination products. This side reaction could be avoided by the use of a hindered base or nonbasic conditions. These newly synthesized nucleo aminooxy acid derivatives might constitute useful building blocks for the synthesis of N-oxy nucleopeptides for investigation of their secondary structure and DNA/RNA binding properties.

Commercially available solvents and reagents were used without further purification, except DMF which was distilled over CaH₂. Melting points were measured on a Kofler bench. Optical rotations were measured using a JASCO P-2000 polarimeter. Column chromatography was performed on Carlo Erba silica gel 60A (40–63 μm). Analytical thin-layer chromatography was performed on E. Merck aluminum precoated plates of silica gel 60F-254 with detection by UV light and by spraying with 10% H₂SO₄ in EtOH or ninhydrin soln (3 g·L^–1) and heating for about 20 seconds at 400 °C. ¹H and ¹³C NMR spectra were recorded on a Jeol ECS-400 spectrometer. ESI-HRMS data were recorded on a Bruker micrOTOF-Q II or a Bruker maXis spectrometer using standard conditions.

N-Trityl-l-serine Methyl Ester (6)[ ¹⁸ ]

To a soln of l-Ser–OMe·HCl (5; 9.29 g, 59.9 mmol) in CH₂Cl₂ (250 mL) were added Et₃N (23 mL, 163.7 mmol) and TrCl (19.98 g, 71.9 mmol). The resulting mixture was stirred for 16 h at r.t. and then concentrated under reduced pressure. The crude material was triturated in EtOAc and compound 6 was isolated by precipitation as a white solid; yield: 18.64 g (84%); mp 138 °C.

R_f  = 0.5 (EtOAc–PE, 1:1).

O-Phthalimido-N-trityl-l-serine Methyl Ester (7)[ ¹⁸ ]

To a soln of 6 (10.02 g, 27.76 mmol) in toluene (150 mL) were added N-hydroxyphthalimide (6.33 g, 38.86 mmol) and Ph₃P (10.18 g, 38.86 mmol). At 0 °C, DIAD (7.65 mL, 41.64 mmol) was then added dropwise. The resulting mixture was stirred for 18 h at r.t. and then washed with 1 N NaOH (2 × 50 mL) and brine (50 mL), dried over MgSO₄ and concentrated under reduced pressure. To the orange crude material dissolved in Et₂O (100 mL), ice (100 g) was added. After 2 h of vigorous stirring, the precipitate was collected by filtration to obtain 7 as a white solid; yield: 8.23 g (58.5%); mp 113 °C.

R_f  = 0.74 (EtOAc–PE, 1:1).

O-Phthalimido-l-serine Methyl Ester Hydrochloride (8)

To a soln of 7 (11.12 g, 21.98 mmol) in MeOH (300 mL) was added AcCl (1.73 mL, 24.18 mmol) at 0 °C. After 1 h of stirring, MeOH was evaporated under reduced pressure to give a white crude material which precipitated in CH₂Cl₂ to give 8 as a white solid; yield: 4.28 g (65%); mp 154 °C.

[α]_D ²⁷ +2.6 (c 0.5, MeOH).

¹H NMR (CD₃OD): δ = 7.89–7.86 (m, 4 H, H-Phth), 4.65–4.63 (m, 2 H, CH₂), 4.61–4.58 (m, 1 H, CH), 3.88 (s, 3 H, OCH₃).

¹³C NMR (CD₃OD): δ = 170.6, 167.2 (CO), 138.9 (CH-Phth), 132.7 (Cq), 127.3 (CH-Phth), 78.3 (CH₂), 56.5 (OCH₃), 55.3 (CH).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₂H₁₃N₂O₅: 265.0824; found: 265.0820.

Methyl (S)-2-[2-(4-{[(Benzyloxy)carbonyl]amino}-2-oxopyrimidin-1(2H)-yl)acetamido]-3-[(1,3-dioxoisoindolin-2-yl)oxy]propanoate (2a)

To a soln of {N ⁴-[(benzyloxy)carbonyl]cytosin-1-yl}acetic acid (9; 1.31 g, 4.33 mmol) in DMF (20 mL) were added DIPEA (1.1 mL, 6.66 mmol) and BOP reagent (1.91 g, 4.33 mmol) at r.t. After 10 min, compound 8 (1.00 g, 3.33 mmol) was added. The reaction mixture was stirred for 18 h, then concentrated and dissolved in EtOAc (100 mL). The organic layer was washed with sat. aq NH₄Cl (1 × 30 mL), sat. aq NaHCO₃ (2 × 30 mL) and brine (1 × 30 mL), then dried over MgSO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography (CH₂Cl₂ to CH₂Cl₂–MeOH, 96:4) to give 2a as white crystals; yield: 863 mg (53%); mp 118 °C.

[α]_D ²⁷ +13.3 (c 0.5, CHCl₃); R_f  = 0.40 (CH₂Cl₂–MeOH, 95:5).

¹H NMR (CDCl₃): δ = 8.01 (d, J = 7.8 Hz, 1 H, NH), 7.85–7.79 (m, 2 H, H-Phth), 7.75–7.72 (m, 2 H, H-Phth), 7.69 (d, J = 7.3 Hz, 1 H, H-Ar), 7.37 (m, 5 H, CH), 7.26 (d, J = 7.3 Hz, 1 H, CH), 5.20 (s, 2 H, OCH₂), 4.88–4.86 (m, 1 H, CH), 4.85 (dd, J = 3.2, 7.3 Hz, 1 H, OCH₂), 4.73 (s, 2 H, NCH₂), 4.38 (dd, J = 3.2, 7.3 Hz, 1 H, OCH₂), 3.68 (s, 3 H, OCH₃).

¹³C NMR (CDCl₃): δ = 169.1, 166.8, 163.5, 163.0, 156.0, 149.3 (Cq), 149.5, 135.2 (CH), 134.9 (Cq), 128.8, 128.7, 128.6 (CH), 128.4 (Cq), 123.9, 95.8 (CH), 77.7, 68.0 (OCH₂), 53.6 (NCH₂), 53.1 (OCH₃), 52.2 (CH).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₆H₂₃N₅NaO₉: 572.1393; found: 572.1387.

Methyl (S)-3-{[(tert-Butoxycarbonyl)amino]oxy}-2-{2-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]acetamido}propanoate (1a)

To a soln of methyl (S)-2-{[(benzyloxy)carbonyl]amino}-3-{[(tert-butoxycarbonyl)amino]oxy}propanoate[ ¹⁸ ] (10; 204 mg, 0.55 mmol) in MeOH (10 mL) was added 10% Pd/C (30 mg). H₂ was bubbled into the mixture for 10 min. The mixture was filtered and the filtrate was concentrated under reduced pressure. The resultant oily residue in CH₂Cl₂ (4 mL) was added to a mixture of 2-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]acetic acid (14; 131 mg, 0.72 mmol), EDC (136 mg, 0.72 mmol) and HOAt (97 mg, 071 mmol) in CH₂Cl₂ (26 mL) at r.t. After 18 h of stirring, the mixture was washed with sat. aq NaHCO₃ (2 × 10 mL) and brine (1 × 10 mL). The organic layer was dried over MgSO₄ and concentrated. The residue was purified by column chromatography (CH₂Cl₂–MeOH, 96:4) to obtain 1a as a white foam; yield: 94 mg (43%); mp 110 °C.

[α]_D ²⁷ +2.0 (c 0.5, CHCl₃); R_f  = 0.40 (CH₂Cl₂–MeOH, 9:1).

¹H NMR (CDCl₃): δ = 9.57 (s, 1 H, NH), 8.07 (d, J = 8.2 Hz, 1 H, NH), 7.85 (s, 1 H, NH), 7.11 (s, 1 H, CH), 4.81–4.78 (m, 1 H, CH), 4.50 (s, 2 H, NCH₂), 4.30 (dd, J = 3.7, 11.0 Hz, 1 H, OCH₂), 4.02 (dd, J = 3.7, 11.0 Hz, 1 H, OCH₂), 3.71 (s, 3 H, OCH₃), 1.91 (s, 3 H, CH₃), 1.45 (s, 9 H, t-Bu).

¹³C NMR (CDCl₃): δ = 170.3, 167.2, 164.5, 157.5, 151.5 (CO), 141.0 (CH-Ar), 111.2 (Cq), 82.6 (Cq-t-Bu), 75.6 (OCH₂), 53.0 (OCH₃), 51.7 (CH), 50.2 (NCH₂), 28.2 (t-Bu), 12.5 (CH₃).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₁₆H₂₄N₄NaO₈: 423.1492; found: 423.1488.

Methyl (S)-2-{[(Benzyloxy)carbonyl]amino}-3-{[bis(tert-butoxycarbonyl)amino]oxy}propanoate (15)

To a soln of 10 (106 mg, 0.29 mmol) in THF (3 mL) were added a soln of Boc₂O (75 mg, 0.35 mmol) in THF (3 mL) and DMAP (70 mg, 0.58 mmol) at r.t. After 17 h of stirring, the reaction mixture was concentrated. The crude product was purified by column chromatography (EtOAc–PE, 1:9 to 2:8) to give compound 15 as a colorless­ oil [yield: 94 mg (70%)] and methyl (S)-2-{[(benzyl­oxy)carbonyl](tert-butoxycarbonyl)amino}-3-{[bis(tert-butoxycarb­onyl)amino]oxy}propanoate (16) as a colorless oil [yield: 25 mg (15%)].

Compound 15

[α]_D ²⁷ –2.0 (c 0.5, CHCl₃); R_f  = 0.61 (EtOAc–PE, 3:7).

¹H NMR (CDCl₃): δ = 7.36–7.33 (m, 5 H, H-Ph), 6.16 (d, J = 8.2 Hz, 1 H, NH), 5.14 (s, 2 H, OCH₂), 4.57–4.54 (m, 1 H, OCH₂), 4.57–4.54 (m, 1 H, CH), 4.02 (dd, J = 3.2, 9.2 Hz, 1 H, OCH₂), 3.78 (s, 3 H, OCH₃), 1.52 (s, 18 H, 2 × t-Bu).

¹³C NMR (CDCl₃): δ = 169.9, 156.2, 149.8 (CO), 136.4 (Cq-Ph), 128.5, 128.2, 128.1 (CH-Ph), 84.6 (Cq-t-Bu), 75.6, 67.1 (CH₂), 53.3 (CH), 52.8 (OCH₃), 28.1 (t-Bu).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₂H₃₂N₂NaO₉: 491.2006; found: 491.2015.

Compound 16

[α]_D ²⁷ –16.4 (c 0.5, CHCl₃); R_f  = 0.68 (EtOAc–PE, 3:7).

¹H NMR (CDCl₃): δ = 7.35–7.30 (m, 5 H, H-Ph), 5.37–5.34 (m, 1 H, CH), 5.21 (s, 2 H, OCH₂), 4.62–4.59 (m, 1 H, OCH₂), 4.25–4.23 (m, 1 H, OCH₂), 3.64 (s, 3 H, OCH₃), 1.46 (s, 18 H, 2 × t-Bu), 1.41 (s, 9 H, t-Bu).

¹³C NMR (CDCl₃): δ = 168.5 (CO), 153.5, 151.1, 149.9, 135.2 (Cq), 128.6, 128.5, 128.4 (CH-Ph), 84.1, 83.8 (Cq-t-Bu), 75.1, 69.0 (OCH₂), 57.5 (CH), 52.6 (OCH₃), 28.2, 28.1 (t-Bu).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₇H₄₀N₂NaO₁₁: 591.2530; found: 591.2520.

Methyl (S)-3-{[Bis(tert-butoxycarbonyl)amino]oxy}-2-{2-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]acetamido}propanoate (1b)

To a soln of 15 (100 mg, 0.21 mmol) in MeOH (2 mL) was added 10% Pd/C (40 mg). H₂ was bubbled into the mixture for 10 min. The mixture was filtered and the filtrate was concentrated under reduced pressure. The resultant oily residue in CH₂Cl₂ (2 mL) was added to a mixture of 14 (55 mg, 0.29 mmol), EDC (57 mg, 0.29 mmol) and HOBt (41 mg, 0.29 mmol) in CH₂Cl₂ (8 mL) at r.t. After 18 h of stirring, the mixture was washed with sat. aq NaHCO₃ (2 × 10 mL) and brine (1 × 10 mL). The organic layer was dried over MgSO₄ and concentrated. The residue was purified by column chromatography (EtOAc–PE, 4:6 to 6:4) to obtain 1b as a white foam; yield: 59 mg (55%); mp 100 °C.

[α]_D ²⁷ +11.0 (c 0.5, CHCl₃); R_f  = 0.43 (CH₂Cl₂–MeOH, 9:1).

¹H NMR (CDCl₃): δ = 9.00 (s, 1 H, NH), 7.86 (d, J = 7.8 Hz, 1 H, NH), 7.05 (s, 1 H, CH), 4.70–4.67 (m, 1 H, CH), 4.61 (dd, J = 2.8, 9.6 Hz, 1 H, OCH₂), 4.48 (s, 2 H, NCH₂), 3.97 (dd, J = 3.7, 10.1 Hz, 1 H, OCH₂), 3.73 (s, 3 H, OCH₃), 1.88 (d, J = 0.9 Hz, 3 H, CH₃), 1.49 (s, 18 H, 2 × t-Bu).

¹³C NMR (CDCl₃): δ = 169.2, 166.8, 164.2, 151.0, 150.4 (Cq), 140.6 (CH-Ar), 111.2, 85.1 (Cq), 75.6 (OCH₂), 53.0 (OCH₃), 51.9 (CH), 49.8 (NCH₂), 28.1 (t-Bu), 12.4 (CH₃).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₁H₃₂N₄NaO₁₀: 523.2016; found: 523.2007.

Methyl (S)-2-{2-[4-{[(Benzyloxy)carbonyl]amino}-2-oxopyrimidin-1(2H)-yl]acetamido}-3-{[bis(tert-butoxycarbonyl)amino]oxy}propanoate (2b)

Compound 15 (676 mg, 1.44 mmol) was hydrogenolyzed in the presence of 10% Pd/C (270 mg). The resultant oily residue in CH₂Cl₂ (10 mL) was added to a mixture of 9 (623 mg, 2.06 mmol), EDC (600 mg, 2.02 mmol) and HOBt (278 mg, 2.06 mmol) in CH₂Cl₂ (70 mL) at r.t. After 18 h of stirring, the mixture was washed with sat. aq NaHCO₃ (2 × 50 mL) and brine (1 × 50 mL). The organic layer was dried over MgSO₄ and concentrated. The residue was purified by column chromatography (EtOAc–PE, 4:6 to 6:4) to obtain 2b as a white solid; yield: 587 mg (65%); mp 97 °C.

[α]_D ²⁷ +4.1 (c 0.5, CHCl₃); R_f  = 0.33 (EtOAc).

¹H NMR (CDCl₃): δ = 8.16 (s, 1 H, NH), 7.94 (d, J = 7.3 Hz, 1 H, NH), 7.67 (d, J = 7.4 Hz, 1 H, CH), 7.32–7.28 (m, 5 H, H-Ph), 7.20 (d, J = 7.4 Hz, 1 H, CH), 5.14 (s, 2 H, OCH₂), 4.68–4.65 (m, 1 H, CH), 4.63 (s, 2 H, NCH₂), 4.52 (dd, J = 3.2, 10.1 Hz, 1 H, OCH₂), 3.98 (dd, J = 3.7, 10.1 Hz, 1 H, OCH₂), 3.62 (s, 3 H, OCH₃), 1.40 (s, 18 H, 2 × t-Bu).

¹³C NMR (CDCl₃): δ = 169.3, 168.0, 166.8, 162.9, 155.9, 152.4, 150.2 (Cq), 149.6 (CH), 135.2 (Cq), 128.7, 128.6, 128.3, 95.3 (CH), 84.8 (Cq-t-Bu), 75.4, 67.9 (OCH₂), 52.9 (OCH₃), 52.0 (CH), 50.8 (NCH₂), 28.0 (t-Bu).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₈H₃₈N₅O₁₁: 620.2568; found: 620.2561.

(S)-3-(Aminooxy)-2-{2-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]acetamido}propanoic Acid (1c)

To a soln of 1b (46 mg, 0.092 mmol) in MeOH (2 mL) was added AcCl (200 μL, 2.88 mmol) at 0 °C. After 4 h of stirring, the mixture was concentrated under reduced pressure. The residue was dissolved in THF (1.5 mL) and H₂O (1.5 mL). LiOH (6.6 mg, 0.28 mmol) was added to the solution at r.t. After an overnight stirring, the mixture was neutralized with H⁺ resin (Dowex) and concentrated under reduced pressure to give 1c as a white solid; yield: 26 mg (100%); mp 124 °C.

[α]_D ²⁷ +3.4 (c 0.5, MeOH).

IR (KBr): 3481.6, 3329.1, 2976.3, 1724.2, 1670.0, 1625.5, 1618.3, 1557.3 cm^–1.

¹H NMR (DMSO-d ₆): δ = 11.29 (s, 1 H, OH), 8.80 (d, J = 7.3 Hz, 1 H, NH), 7.43 (s, 1 H, CH), 4.77–4.70 (m, 1 H, CH), 4.51 (m, 1 H, OCH₂), 4.38–4.28 (m, 2 H, NCH₂), 3.91–3.86 (m, 1 H, OCH₂), 1.73 (s, 3 H, CH₃).

¹³C NMR (DMSO-d ₆): δ = 168.0, 165.0, 151.5 (Cq), 142.8 (CH), 108.5 (Cq), 73.0 (OCH₂), 51.8 (CH), 49.6 (NCH₂), 12.5 (CH₃).

HRMS (ESI): m/z [M – H₂O + Na]⁺ calcd for C₁₀H₁₂N₄NaO₅: 291.0705; found: 291.0701.

(S)-3-(Aminooxy)-2-{2-[4-{[(benzyloxy)carbonyl]amino}-2-oxopyrimidin-1(2H)-yl]acetamido}propanoic Acid (2c)

To a soln of 2b (49 mg, 0.079 mmol) in MeOH (2 mL) was added AcCl (200 μL, 2.88 mmol) at 0 °C. After 4 h of stirring, the mixture was concentrated under reduced pressure. The residue was dissolved in THF (1.5 mL) and H₂O (1.5 mL). LiOH (6.6 mg, 0.28 mmol) was added to the solution at r.t. After an overnight stirring, the mixture was neutralized with H⁺ resin (Dowex) and concentrated under reduced pressure to give 2c as a white solid; yield: 31 mg (97%); mp 113 °C.

[α]_D ²⁷ –6.8 (c 0.5, DMSO).

IR (KBr): 3403.3, 3319.1, 1734.4, 1709.2, 1666.0, 1644.7, 1606.9 cm^–1.

¹H NMR (DMSO-d ₆): δ = 11.28 (s, 1 H, OH), 9.82 (s, 1 H, NH), 8.88 (d, J = 7.8 Hz, 1 H, NH), 7.96 (d, J = 7.8 Hz, 1 H, CH), 7.38–7.30 (m, 5 H, H-Ph), 6.95 (d, J = 7.8 Hz, 1 H, CH), 5.14 (s, 2 H, OCH₂), 4.74–4.68 (m, 1 H, CH), 4.55–4.44 (m, 3 H, NCH₂ + OCH₂), 3.90–3.86 (m, 1 H, OCH₂).

¹³C NMR (DMSO-d ₆): δ = 167.8, 163.7, 160.5, 155.5, 153.7 (Cq), 151.5 (CH), 136.5 (Cq), 129.0, 128.7, 128.5, 92.3 (CH), 73.0, 67.0 (OCH₂), 51.9 (CH), 51.6 (NCH₂).

HRMS (ESI): m/z [M – H₂O + H]⁺ calcd for C₁₇H₁₈N₅O₆: 388.1257; found: 388.1250.

Methyl (S)-2-(2-Chloroacetamido)-3-[(1,3-dioxoisoindolin-2-yl)oxy]propanoate (17)

To a soln of 8 (209 mg, 0.70 mmol) in THF (10 mL) were added Et₃N (196 μL, 1.40 mmol) and chloroacetyl chloride (83 μL, 1.05 mmol) at 0 °C. After 17 h of stirring at r.t., the reaction mixture was washed with brine (2 × 10 mL). The aqueous layer was extracted with EtOAc (2 × 10 mL). The combined organic layers were dried over MgSO₄ and concentrated. The crude product was purified by column chromatography (EtOAc–PE, 2:8 to 6:4) to give 17 as a white solid; yield: 126 mg (53%); mp 138 °C.

[α]_D ²⁷ +62.5 (c 0.5, CHCl₃); R_f  = 0.37 (EtOAc–PE, 1:1).

¹H NMR (CDCl₃): δ = 7.98 (d, J = 5.0 Hz, 1 H, NH), 7.88–7.83 (m, 2 H, H-Phth), 7.80–7.77 (m, 2 H, H-Phth), 4.89–4.87 (m, 1 H, CH), 4.89–4.87 (m, 1 H, OCH₂), 4.42 (dd, J = 4.6, 11.9 Hz, 1 H, OCH₂), 4.18 (s, 2 H, CH₂Cl), 3.74 (s, 3 H, OCH₃).

¹³C NMR (CDCl₃): δ = 168.9, 166.6, 163.4 (CO), 135.0 (CH-Phth), 128.7 (Cq), 123.9 (CH-Phth), 77.1 (OCH₂), 53.2 (OCH₃), 52.0 (CH), 42.5 (CH₂Cl).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₄H₁₄ClN₂O₆: 341.0540; found: 341.0535.

Methyl 2-(2-Azidoacetamido)acrylate (18)

To a soln of 17 (211 mg, 0.62 mmol) in DMF (3 mL) was added NaN₃ (61 mg, 0.93 mmol) at 0 °C. After 150 min at 0 °C, EtOAc (20 mL) was added to the mixture and the resultant solution was washed with brine (2 × 15 mL). The aqueous layer was extracted with EtOAc­ (1 × 20 mL). The combined organic layers were dried over MgSO₄ and concentrated. The residue was purified by column chromatography (EtOAc–PE, 2:8 to 3:7) to give 18 as a colorless oil; yield: 155 mg (100%).

R_f  = 0.47 (EtOAc–PE, 3:7).

¹H NMR (CDCl₃): δ = 8.47 (s, 1 H, NH), 6.54 (s, 1 H, =CH), 5.87 (s, 1 H, =CH), 4.04 (s, 2 H, CH₂N₃), 3.78 (s, 3 H, OCH₃).

¹³C NMR (CDCl₃): δ = 165.5, 164.1 (CO), 130.5 (Cq), 109.8 (=CH₂), 53.1 (OCH₃), 52.9 (CH₂N₃).

Methyl (S)-2-(2-Azidoacetamido)-3-[(1,3-dioxoisoindolin-2-yl)oxy]propanoate (19)

To a soln of 2-azidoacetic acid (111 mg, 1.0 mmol) in CH₂Cl₂ (5 mL) were added EDC (83 mg, 0.43 mmol), HOBt (58 mg, 0.43 mmol) and 2,6-di-tert-butyl-4-methylpyridine (68 mg, 0.33 mmol) at r.t. After 10 min, compound 8 (100 mg, 0.33 mmol) was added. After 17 h of stirring, the reaction mixture was washed with sat. aq NaHCO₃ (1 × 10 mL) and brine (1 × 10 mL). The organic layer was dried over MgSO₄ and concentrated. The resulting residue was purified by column chromatography (EtOAc–PE, 5:5 to 10:0) to give 19 as a white solid; yield: 110 mg (95%); mp 158 °C.

[α]_D ²⁷ +45.9 (c 0.5, CHCl₃); R_f  = 0.38 (EtOAc–PE, 1:1).

¹H NMR (CDCl₃): δ = 7.85–7.83 (m, 2 H, H-Phth), 7.79–7.75 (m, 2 H, H-Phth), 4.90–4.88 (m, 2 H, OCH₂), 4.39–4.36 (m, 1 H, CH), 4.10 (s, 2 H, CH₂N₃), 3.73 (s, 3 H, OCH₃).

¹³C NMR (CDCl₃): δ = 169.0, 167.3, 163.4 (CO), 135.0 (CH-Phth), 128.6 (Cq), 123.9 (CH-Phth), 77.3 (OCH₂), 53.1 (OCH₃), 52.7 (CH₂N₃), 51.6 (CH).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₄H₁₄N₅O₆: 348.0944; found: 348.0939.

Benzyl [2-Oxo-1-(prop-2-yn-1-yl)-1,2-dihydropyrimidin-4-yl]carbamate (21)

To a soln of benzyl (2-oxo-1,2-dihydropyrimidin-4-yl)carbamate[ ²⁶ ] (20; 556 mg, 2.27 mmol) in MeCN (25 mL) was added 60% NaH (108 mg, 4.53 mmol) at 0 °C. Propargyl bromide (80% in toluene; 401 μL, 2.72 mmol) was then added. After 18 h of stirring at reflux, the reaction mixture was washed with brine (2 × 15 mL). The organic layer was dried over MgSO₄ and concentrated. The residue was purified by column chromatography (EtOAc–PE, 5:5 to 8:2) to give 21 as a pale yellow solid; yield: 192 mg (30%); mp 154 °C.

R_f  = 0.60 (EtOAc).

¹H NMR (CD₃OD): δ = 8.10 (d, J = 7.3 Hz, 1 H, =CH), 7.38–7.30 (m, 6 H, H-Ar), 5.19 (s, 2 H, OCH₂), 4.67 (s, 2 H, NCH₂), 2.96 (s, 1 H, ≡CH).

¹³C NMR (CD₃OD): δ = 163.9, 156.4, 153.2 (Cq), 147.9 (CH), 135.8 (Cq), 128.3, 128.1, 128.0, 95.9 (CH), 75.1 (≡CH), 70.3 (≡Cq), 67.2 (OCH₂), 38.6 (NCH₂).

Methyl 2-[2-(4-{[5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]methyl}-1H-1,2,3-triazol-1-yl)acetamido]acrylate (23)

To a soln of 19 (230 mg, 0.66 mmol) in DMF (3 mL) were added 5-methyl-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione (22; 109 mg, 0.66 mmol), sodium ascorbate (66 mg, 0.33 mmol) and CuSO₄·5H₂O (41 mg, 0.17 mmol) at r.t. After an overnight stirring, EtOAc (50 mL) was added to the mixture. The solution was washed with sat. aq NaHCO₃ (1 × 30 mL) and brine (2 × 30 mL). The organic layer was dried over MgSO₄ and concentrated under reduced pressure to give 23 as a white solid; yield: 157 mg (68%); mp 150 °C.

R_f  = 0.66 (CH₂Cl₂–MeOH, 9:1).

¹H NMR (DMSO-d ₆): δ = 11.29 (s, 1 H, NH), 9.88 (s, 1 H, NH), 8.01 (s, 1 H, H-triazole), 7.60 (s, 1 H, =CH), 6.22 (s, 1 H, =CH), 5.75 (s, 1 H, =CH), 5.31 (s, 2 H, CH₂N), 4.87 (s, 2 H, CH₂N), 3.73 (s, 3 H, OCH₃), 1.71 (s, 3 H, CH₃).

¹³C NMR (DMSO-d ₆): δ = 165.9, 164.8, 164.0, 151.2, 142.9 (Cq), 141.7 (CH-Ar), 132.6 (Cq), 125.8, 110.9 (CH), 109.4 (Cq), 53.3 (OCH₃), 52.5, 42.7 (NCH₂), 12.5 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₄H₁₇N₆O₅: 349.1260; found: 349.1255.

Methyl (S)-3-[(1,3-Dioxoisoindolin-2-yl)oxy]-2-[2-(4-{[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]methyl}-1H-1,2,3-triazol-1-yl)acetamido]propanoate (3)

To a soln of 19 (200 mg, 0.58 mmol) in DMF (4 mL) were added 22 (95 mg, 0.58 mmol), ascorbic acid (51 mg, 0.29 mmol) and CuSO₄·5H₂O (36 mg, 0.14 mmol) at r.t. After 18 h of stirring, EtOAc­ (40 mL) was added to the solution. The precipitate formed was filtered and the filtrate was washed with brine (2 × 20 mL). The organic layer was dried over MgSO₄ and concentrated under reduced pressure to give 3 as a white solid; yield: 255 mg (87%); mp 156 °C.

[α]_D ²⁷ +3.9 (c 0.5, DMSO); R_f  = 0.14 (EtOAc).

¹H NMR (DMSO-d ₆): δ = 11.31 (s, 1 H, NH), 9.08 (d, J = 7.8 Hz, 1 H, NH), 8.03 (s, 1 H, H-triazole), 7.85–7.79 (m, 4 H, H-Phth), 7.60 (d, J = 1.4 Hz, 1 H, CH), 5.23 (s, 2 H, CH₂N), 4.91 (s, 2 H, CH₂N), 4.79–4.77 (m, 1 H, CH), 4.51–4.42 (m, 2 H, OCH₂), 3.68 (s, 3 H, OCH₃), 1.74 (s, 3 H, CH₃).

¹³C NMR (DMSO-d ₆): δ = 169.6, 166.4, 164.8, 163.4, 162.8, 151.3, 142.8 (Cq), 141.7, 135.4 (CH), 129.1 (Cq), 125.6, 123.9 (CH), 109.4 (Cq), 76.8 (OCH₂), 53.0 (OCH₃), 52.2 (CH), 51.9, 42.6 (NCH₂), 12.5 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₂N₇O₈: 512.1530; found: 512.1522.

Methyl (S)-2-(2-{4-[(4-{[(Benzyloxy)carbonyl]amino}-2-oxopyrimidin-1(2H)-yl)methyl]-1H-1,2,3-triazol-1-yl}acetamido)-3-[(1,3-dioxoisoindolin-2-yl)oxy]propanoate (4)

To a soln of 19 (50 mg, 0.14 mmol) in DMF (1 mL) were added 21 (41 mg, 0.14 mmol), ascorbic acid (13 mg, 0.07 mmol) and CuSO₄·5H₂O (9 mg, 0.035 mmol) at r.t. After 18 h of stirring, EtOAc­ (15 mL) was added to the solution. The precipitate formed was filtered and the filtrate was washed with brine (2 × 10 mL). The organic layer was dried over MgSO₄ and concentrated under reduced pressure to give 4 as a white solid; yield: 70 mg (77%); mp 126 °C.

[α]_D ²⁷ +0.9 (c 0.5, CHCl₃); R_f  = 0.15 (EtOAc).

¹H NMR (CDCl₃): δ = 8.18 (s, 1 H, H-triazole), 7.99 (d, J = 7.8 Hz, 1 H, NH), 7.89 (d, J = 8.0 Hz, 1 H, CH), 7.73–7.70 (m, 2 H, H-Phth), 7.68–7.65 (m, 2 H, H-Phth), 7.31–7.28 (m, 5 H, H-Ph), 7.13 (d, J = 8.0 Hz, 1 H, CH), 5.32 (s, 2 H, CH₂N), 5.12 (s, 2 H, CH₂N), 5.10 (s, 2 H, OCH₂), 4.86–4.84 (m, 1 H, CH), 4.73–4.71 (m, 1 H, NOCH₂), 4.39–4.36 (m, 1 H, NOCH₂), 3.62 (s, 3 H, OCH₃).

¹³C NMR (CDCl₃): δ = 169.1, 165.9, 163.3, 155.9, 152.5 (Cq), 148.8 (CH), 142.0 (Cq), 134.9, 128.7 (CH), 128.6 (Cq), 128.5, 128.3, 126.3, 123.9, 95.5 (CH), 77.3, 67.8 (OCH₂), 53.1 (CH), 52.5 (CH₂N), 51.9 (OCH₃), 45.3 (NCH₂).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₉H₂₇N₈O₉: 631.1901; found: 631.1891.

• References

• 1 Diederichsen U. Angew. Chem., Int. Ed. Engl. 1996; 35: 445
• 2 Diederichsen U, Schmitt HW. Angew. Chem. Int. Ed. 1998; 37: 302
• 3 Chakraborty P, Diederichsen U. Chem.–Eur. J. 2005; 11: 3207
• 4 McCarthy OK, Schipani A, Buendía AM, Ruiz-Perez LM, Kaiser M, Brun R, Pacanowska DG, Gilbert IH. Bioorg. Med. Chem. Lett. 2006; 16: 3809
• 5 Srivastava R, Ray AK, Diederichsen U. Eur. J. Org. Chem. 2009; 4793
• 6 Geotti-Bianchini P, Crisma M, Peggion C, Bianco A, Formaggio F. Chem. Commun. 2009; 3178
• 7 Geotti-Bianchini P, Moretto A, Peggion C, Beyrath J, Bianco A, Formaggio F. Org. Biomol. Chem. 2010; 8: 1315
• 8 Garner P, Dey S, Huang Y. J. Am. Chem. Soc. 2000; 122: 2405
• 9 Huang Y, Dey S, Zhang X, Sönnichsen F, Garner P. J. Am. Chem. Soc. 2004; 126: 4626
• 10 Geotti-Bianchini P, Beyrath J, Chaloin O, Formaggio F, Bianco A. Org. Biomol. Chem. 2008; 6: 3661
• 11 Yang D, Qu J, Li B, Ng F.-F, Wang X.-C, Cheung K.-K, Wang D.-P, Wu Y.-D. J. Am. Chem. Soc. 1999; 121: 589
• 12 Li X, Wu Y.-D, Yang D. Acc. Chem. Res. 2008; 41: 1428
• 13 Malapelle A, Ramozzi R, Xie J. Synthesis 2009; 888
• 14 Gong YC, Sun HB, Xie J. Eur. J. Org. Chem. 2009; 6027
• 15 Gong Y, Peyrat S, Sun H, Xie J. Tetrahedron 2011; 67: 7114
• 16 Song Z, He X.-P, Chen G.-R, Xie J. Synthesis 2011; 2761
• 17 Peyrat S, Xie J. Synthesis 2012; 44: 1718
• 18 Liu F, Thomas J, Burke Jr TR. Synthesis 2008; 2432
• 19 Katritzky AR, Narindoshvili T. Org. Biomol. Chem. 2008; 6: 3171
• 20 Schwergold C, Depecker G, Di Giorgio C, Patino N, Jossinet F, Ehresmann B, Terreux R, Cabrol-Bass D, Condom R. Tetrahedron 2002; 58: 5675
• 21 Srinivasan A, Stephenson RW, Olsen RK. J. Org. Chem. 1977; 42: 2253
• 22 Decostaire IP, Lelièvre D, Zhang H, Delmas AF. Tetrahedron Lett. 2006; 47: 7057
• 23 Lindsell WE, Murray C, Preston PN, Woodman TA. J. Tetrahedron 2000; 56: 1233
• 24 Haridas V, Sharma YK, Sahu S, Verma RP, Sadanandan S, Kacheshwar BG. Tetrahedron 2011; 67: 1873
• 25 Maisonneuve M, Xie J. Synlett 2009; 2977
• 26 Dueholm KL, Egholm M, Behrens C, Christensen L, Hansen HF, Vulpius T, Petersen KH, Berg RH, Nielsen PE, Buchardt O. J. Org. Chem. 1994; 59: 5767