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Synlett 2006(18): 2997-3000  
DOI: 10.1055/s-2006-948176
LETTER
© Georg Thieme Verlag Stuttgart · New York

A Facile Synthesis of Fluorophores Based on 5-Phenylethynyluracils

Robert H. E. Hudson*, Joanne M. Moszynski
Department of Chemistry, The University of Western Ontario, London, ON, N6A 5B7, Canada
Fax: +1(519)6613022; e-Mail: robert.hudson@uwo.ca;
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Publication History

Received 23 March 2006
Publication Date:
04 August 2006 (online)

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Abstract

Compact, fluorescent uracil aglycones and derivatives suitable for incorporation into the oligonucleotide mimic peptide nucleic acid (PNA) have been prepared by Sonogashira/Castro-Stephens coupling to monosubstituted phenylacetylenes. Cyclic 6-(phenyl)furo[2,3-d]pyrimidin-2(3H)-ones were accessed by the Ag+-catalyzed cyclization of the 5-alkynyluracil precursors. Although this reaction was sluggish, it gave quantitative chemical yields. Electron-rich alkynes, such as p-methoxyphenylethyne, ­cyclize much more rapidly than electron-deficient alkynes. Adjustment of the reaction conditions permitted the synthesis of p-nitrophenylfuranouracil in excellent yield.

Key words

annulations - silver(I)-catalyzed cyclization - furano­uracils - 5-phenylethynyluracil - nucleobases

    References and Notes

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13

Compound 1: uracil (134 mmol, 15.0 g) was suspended in 800 mL of MeOH. Then, I2 (81 mmol, 20.4 g) and CAN (67 mmol, 36.7 g) were added. The reaction was refluxed for 16 h, cooled, and a white precipitate was collected by filtration. The filtrate was evaporated, and then co-evaporated with 2:1 EtOH-H2O to remove iodine (2 × 90 mL). Both product fractions were then combined and recrystallized from 1:1 EtOH-H2O to obtain 30.6 g (89% yield) of 2. Translucent needles; mp >280 °C (dec.). HRMS (EI): m/z calcd for C4H3IN2O2: 237.9239; found: 237.9246. 1H NMR (DMSO-d 6): 11.41 (s, 1 H), 11.17 (s, 1 H), 7.88 (s, 1 H).

14

General Procedure for Cross-Coupling.
5-Iodouracil compound 1 or 2 (1 mmol) was stirred in anhydrous DMF (3 mL). Then, CuI (0.2 mmol) was added and the solvent was deoxygenated. Pd(PPh3)4 (0.1 mmol), Et3N (2 mmol) and alkyne (1.5-3 mmol) were added sequentially. The reaction mixture was then stirred overnight at r.t. The completed reaction was extracted with a sat. EDTA solution against CH2Cl2, evaporated, and dried under vacuum. The residue was then suspended in a minimum amount of CH2Cl2 and precipitated with Et2O. Silica gel chromatography using gradient elution with MeOH-CH2Cl2 mixtures was used to purify the 5-alkynyluracils 3a-c and 4a-c. All 1H NMR spectra were recorded at 400.09 MHz in DMSO-d 6.
Compound 3a: white solid; elution at 8% MeOH-CH2Cl2; mp >280 °C (dec.). HRMS (EI): m/z calcd for C12H8N2O2: 212.0586; found: 212.0594. 1H NMR: δ = 11.41 (br s, 1 H) overlapping with 11.36 (br s, 1 H), 7.88 (s, 1 H), 7.43-7.47 (m, 2 H), 7.37-7.41 (m, 3 H).

Compound 3b: white solid; elution at 15% MeOH; mp >270 °C (dec.). HRMS (EI): m/z calcd for C13H10N2O3: 242.0691; found: 242.0695. 1H NMR: δ = 11.40 (s, 1 H), 11.31 (br s, 1 H), 7.84 (s, 1 H), 7.37 (d, 2 H, J = 8.8 Hz), 6.96 (d, 2 H, J = 8.9 Hz), 3.77 (s, 3 H).
Compound 3c: light orange solid; elution from 15-30% MeOH; mp 282-286 °C (dec.). HRMS (EI): m/z calcd for C12H7N3O4: 257.0437; found: 257.0440. 1H NMR: δ = 11.54 (br s, 1 H), 11.51 (s, 1 H), 8.24 (d, 2 H, J = 9.1 Hz), 8.03 (s, 1 H), 7.68 (d, 2 H, J = 8.9 Hz).
Compound 4a: white crystals; elution from silica at 2% MeOH; mp 193-196 °C. HRMS (EI): m/z calcd for C16H14N2O4: 298.0954; found: 298.0498. 1H NMR: δ = 11.85 (s, 1 H), 8.55 (s, 1 H), 7.45-7.48 (m, 2 H), 7.40-7.43 (m, 3 H), 4.55 (s, 2 H), 4.17 (q, 2 H, J = 7.0 Hz), 1.21 (t, 3 H, J = 7.0 Hz).
Compound 4b: white solid; elution at 2.5% MeOH-CH2Cl2; mp 196-197 °C. HRMS (EI): m/z calcd for C17H16N2O5: 328.1059; found: 328.1068. 1H NMR: δ = 11.82 (s, 1 H), 8.14 (s, 1 H), 7.40 (d, 2 H, J = 8.8 Hz), 6.96 (d, 2 H, J = 8.6 Hz), 4.54 (s, 2 H), 4.15 (q, 2 H, J = 7.1 Hz), 3.77 (s, 3 H), 1.20 (t, 3 H, J = 7.2 Hz).
Compound 4c: pale yellow solid; elution at 3% MeOH-CH2Cl2; mp >245 °C (dec.). HRMS (EI): m/z calcd for C16H13N3O6: 343.0804; found: 343.0810. 1H NMR: δ = 11.95 (s, 1 H), 8.32 (s, 1 H), 8.25 (d, 2 H, J = 8.9 Hz), 7.71 (d, 2 H, J = 8.8 Hz), 4.57 (s, 2 H), 4.17 (q, 2 H, J = 7.0 Hz), 1.21 (t, 3 H, J = 7.1 Hz).

15

Compound 2: 1 (63.3 mmol, 15 g) was suspended in 100 mL DMF, to which anhyd K2CO3 (63.0 mmol, 8.7 g) was added. Then, BrCH2CO2Et (63.1 mmol, 7.0 mL) was added dropwise under N2. The reaction was stirred for 16 h, filtered to remove salts and then the solvent was evaporated. The residue was then cooled in an ice bath and acidified with
4 M HCl (65 mL). The resulting precipitate was then filtered, washed with Et2O and dried. The crude product was recrystallized from 1:1 EtOH-H2O to produce 10.6 g (85% yield) of pure product. White crystals; mp 170-172 °C. HRMS (EI): m/z calcd for C8H9IN2O4: 323.9607; found: 323.9724. 1H NMR: δ = 11.82 (s, 1 H), 8.20 (s, 1 H), 4.49 (s, 2 H), 4.13 (q, 2 H, J = 7.1 Hz), 1.19 (t, 3 H, J = 7.1 Hz).

19

General Procedure for Annulation.
The 5-alkynyluracil was suspended in deoxygenated acetone, and 5 mol% AgNO3 was added. While the original procedure called for 0.047 mmol of alkyne and 0.009 mmol catalyst; scale-up was found to be effective up to 2 mmol of starting alkynyl-uracil. The reaction mixture was stirred in the dark for >24 h and monitored by TLC (5-15% MeOH-CH2Cl2) until completion reaction. The completed reaction was washed with H2O against CH2Cl2, evaporated and dried under vacuum to provide the phenylfuranouracils 5a-c and 6a-c. As the N1-unsubstituted furanouracils 5a-c are quite water-soluble, silver nitrate removal caused a significant loss of product for these cases, accounting for the lower yields. All 1H NMR spectra were recorded at 400.09 MHz in DMSO-d 6.
Compound 5a: AgNO3 (5 mol%), r.t., 96 h; white solid; mp >350 °C (dec.). HRMS (EI): m/z calcd for C12H8N2O2: 212.0586; found: 212.0574. 1H NMR: δ = 8.34 (s, 1 H), 7.82 (m, 2 H), 7.51-7.40 (m, 3 H), 7.22 (s, 1 H).
Compound 5b: AgNO3 (5 mol%), 82 h. Reaction is quantitative as measured by 1H NMR, however, significant losses were encountered during the work-up and purification. Chromatography on an elemental sulfur column or through a plug of EDTA·2Na+ was performed, but neither adsorbed the catalyst. In situ generation of Cl- by addition of chlorotrimethylsilane, or addition of brine was unsuccessful for removal of silver ion. The reaction mixture was subsequently purified by silica gel chromatography (elution at 30-40% MeOH), with some of the very polar compound remaining on the column, thereby reducing the yield. Pale yellow solid; mp >285 °C (dec.). HRMS (EI): m/z calcd for C13H10N2O3: 242.0691; found: 242.0693. 1H NMR: δ = 8.28 (s, 1 H), 7.75 (d, 2 H, J = 8.9 Hz), 7.05 (d, 2 H, J = 9.1 Hz), 7.04 (s, 1 H), 3.81 (s, 3 H).
Compound 5c: AgNO3 (15 mol%), reflux(acetone), 14 d, 80% yield. Alternatively, Ag2SO4 (1 equiv), 1:1 dioxane-water, 50 °C, 6 d, isolated by precipitation caused by the addition of one volume of H2O: 88% yield. Yellow-orange solid; mp >320 °C (dec.). 1H NMR: δ = 8.52 (s, 1 H), 8.32 (d, 2 H, J = 9.1 Hz), 8.07 (d, 2 H, J = 9.4 Hz), 7.57 (s, 1 H). Note, the exchangeable proton was not observed. HRMS (EI): m/z calcd for C17H16N2O5: 257.0437; found: 257.0445.
Compound 6a: AgNO3 (5 mol%), 60 h. 1H NMR: δ = 8.65 (s, 1 H), 7.84 (d, 2 H, J = 7.1 Hz), 7.51 (t, 2 H, J = 7.1 Hz), 7.44 (t, 1 H, J = 7.3 Hz), 7.37 (s, 1 H), 4.79 (s, 2 H), 4.16 (q, 2 H, J = 7.1 Hz), 1.21 (t, 3 H, J = 7.1 Hz). White solid; mp >310 °C (dec.). HRMS (EI): m/z calcd for C16H14N2O4: 298.0954; found: 298.1023.
Compound 6b: AgNO3 (5 mol%), 48 h. Off-white powder; mp >300 °C (dec.). HRMS (EI): m/z calcd for C17H16N2O5: 328.1059; found: 328.1069. 1H NMR: δ = 8.56 (s, 1 H), 7.76 (d, 2 H, J = 8.6 Hz), 7.17 (s, 1 H), 7.05 (d, 2 H, J = 8.6 Hz), 4.77 (s, 2 H), 4.17 (q, 2 H, J = 7.0 Hz), 3.81 (s, 3 H), 1.21 (t, 3 H, J = 7.0 Hz).
Compound 6c: AgNO3 (10 mol%), 8 d. Orange powder; mp 310-314 °C (dec.). HRMS (EI): m/z calcd for C16H13N3O6: 343.0804; found: 343.0799. 1H NMR: δ = 8.79 (s, 1 H), 8.34 (d, 2 H, J = 9.1 Hz), 8.08 (d, 2 H, J = 8.9 Hz), 7.69 (s, 1 H), 4.81 (s, 2 H), 4.17 (q, 2 H, J = 7.1 Hz), 1.22 (t, 3 H, J = 7.1 Hz).

20

The conversion of 3a-c → 4a-c was conveniently monitored in the 1H NMR spectra by observation of the appearance of the characteristic signal for the proton on the furan ring [δ(ppm) in DMSO-d 6: 5a: 7.22; 5b: 7.04; 5c: 7.57; 6a: 7.37; 6b: 7.17; 6c: 7.69], disappearance of the resonance corresponding to the N3-imino proton (ca. 11.4-11.8 ppm) and a uniform downfield shift for the resonances associated with the phenyl ring and H6 of the uracil ring.

22

Fluorescence and UV measurements for N1-substituted compounds (4a,b, 6a,b) were done in MeOH that had been degassed by bubbling with N2 for 5 min and at a concen-tration of 2.5 µM. Although the N1-unsubstituted com-pounds (3a,b, 5a,b) are soluble in water, these were also examined in degassed MeOH at 2.5 µM for comparison purposes and the difficulty with fully deoxygenating the water. Fluorescence excitation and emission spectra were determined with at least 3 replicates with a 1 min rest between scans. For comparison of intensities, excitation was done at λ = 350 nm, and emission data from the maxima were used. At this time, we have no evidence for the occurrence of photochemistry during the course of these measurements. Extinction coefficients were determined for the wavelength of interest using at least three data points.