A Facile Synthesis of Fluorophores Based on 5-Phenylethynyluracils

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.

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

References and Notes

1a Woo J. Meyer RB. Gamper HB. Nucleic Acids Res.  1996,  24:  2470 

1b Berry DA. Jung K.-Y. Wise DS. Sercel AD. Pearson WH. Mackie H. Randolph JB. Somers RL. Tetrahedron Lett.  2004,  45:  2457 

2 Synthesis of 5-ethynyluracil was achieved in 36% yield: Perman J. Sharma RA. Bobek M. Tetrahedron Lett.  1976,  28:  2427 

3a One example: 5-(o-aminophenylethynyl)uracil, 90%. See: Arcadi A. Cacchi S. Marinelli F. Tetrahedron Lett.  1989,  30:  2581 

3b One example: 5-(phenylethynyl)uracil, 61%. See: Farina V. Hauck SI. Synlett  1991,  157 

3c Two examples, failed thermal reaction but 65% of 5-(phenylethynyl)uracil under microwave irradiation. See: Petricci E. Radi M. Corelli F. Botta M. Tetrahedron Lett.  2003,  44:  9181 

4a Robins MJ. Barr PJ. Tetrahedron Lett.  1981,  22:  421 

4b Hudson RHE. Li G. Tse J. Tetrahedron Lett.  2002,  43:  1381 

5a Sonogashira K. Tohda Y. Hagihara N. Tetrahedron Lett.  1975,  4467 

5b Stephens RD. Castro CE. J. Org. Chem.  1963,  28:  3313 

6 A preliminary report has appeared in conference proceedings: Hudson RHE. Dambenieks AK. Moszynski JM. Proc. SPIE - Int. Soc. Optical Eng.  2005,  103 

Although 5-phenylethynyluracils (ref. 3), 5-phenylethynyl-uridine and 5-phenylethynyl-2′-dexoyuridine have been previously synthesized no description of their luminescent properties has appeared:

7a Rai D. Johar M. Manning T. Agrawal B. Kunimoto DY. Kumar R. J. Med. Chem.  2005,  48:  7012 

7b Angell A. McGuigan C. Garcia Sevillano L. Snoeck R. Andrei G. De Clercq E. Balzarini J. Bioorg. Med. Chem. Lett.  2004,  14:  2397 

7c Aucagne V. Berteina-Raboin S. Guenot P. Agrofoglio LA. J. Comb. Chem.  2004,  6:  717 

7d The fluorescence of 5-tolylethynyl-2′-dexoyuridine has been recognized: Esho N. Davies B. Lee J. Dembinski R. Chem. Commun.  2002,  332 

Intrinsically fluorescent uracils have been noted for substantially larger ethynyl-linked aromatic chromophores:

8a Hurley DJ. Seaman SE. Mazura JC. Tor Y. Org. Lett.  2002,  4:  2305 

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9 Yoshimoto K. Xu C.-Y. Nishizawa S. Haga T. Satake H. Teramae N. Chem. Commun.  2003,  2960 

10a Barr PJ. Jones S. Serafinowski P. Walker RT. J. Chem. Soc., Perkin Trans. 1  1978,  1263 

10b Valdivia V. Hernandez A. Rivera A. Sartillo F. Loukaci A. Fourrey J.-L. Quintero L. Tetrahedron Lett.  2005,  46:  6511 

11 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 

12 Asakura J.-I. Robins MJ. J. Org. Chem.  1990,  55:  4928 

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).

16 Robins MJ. Barr PJ. J. Org. Chem.  1983,  48:  1854 

17 Hudson RHE. Dambenieks AK. Viirre RD. Synlett  2004,  1400 

18 Aucagne V. Amblard F. Agrofoglio LA. Synlett  2004,  2406 

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.

21 McGuigan C. Brancale A. Barucki H. Srinivasan S. Jones G. Pathirana R. Carangio A. Blewett S. Luoni G. Bidet O. Jukes A. Jarvis C. Andrei G. Snoeck R. De Clercq E. Balzarini J. Antiviral Chem. Chemother.  2001,  12:  77 

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.