3. Experimental
3.1. General Methods and Materials
All reagents were from standard commercial sources and of analytic grade. All reaction were performed under argon atmosphere except specified otherwise. Dry solvents were directly acquired from commercial sources. Precoated Merck Silica Gel F254 plates were used for TLC. Spots were examined under ultraviolet light at 254 nm and further visualized by sulfuric acid-anisaldehyde spray. Column chromatography was performed on silica gel (200–400 mm, 60 Å). 1H, 13C, and 31P-NMR spectra were recorded in CDCl3, DMSO-d6, or D2O on a Varian Mercury 300 MHz spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts are given in parts per million (ppm δ), δ relative to residual solvent peak or TMS for 1H and 13C and to external D3PO4 for 31P. Structural assignment was confirmed with COSY, HSQC and HMBC. Exact mass measurements were performed on a LCT Premier XE orthogonal time-of flight spectrometer with API-ES source (Waters, Zellik, Belgium) Waters LCT Premier XETM Time of flight (TOF) instrument. Samples were infused in a CH3CN/H2O (1:1) mixture at 10 mL/min.
Diethyl ((E)-2-((3aR,5R,6R,6aR)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][
1,
3]
dioxol-5-yl)-vinyl)phosphonate (
7): NaIO
4 (0.52 g, 2.22 mmol) was added to a stirred solution of compound
6 in CH
2Cl
2 (15 mL) and H
2O (10 mL) and the reaction mixture was stirred for 0.5 h at rt. The mixture was then filtrated and the filtrate was extracted with CH
2Cl
2. The combined organic layers were concentrated
in vacuo to give the crude aldehyde, which was used in the next step without further purification. To a stirred suspension of NaH (0.31 g, 7.81 mmol) in dry THF (10 mL) was added tetraethyl methylene bisphosphonate (1.41 g, 4.88 mmol) under argon. After 0.5 h the mixture was cooled in an ice bath and a solution of the crude aldehyde in THF (10 mL) was added. The reaction mixture was allowed to warm to rt and stirred overnight. The crude mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried (Na
2SO
4) and concentrated
in vacuo. Silica gel column chromatography (CH
2Cl
2/MeOH 97:3) afforded compound
7 as a colorless oil (0.77 g, 84.2%).
1H-NMR (300 MHz, CDCl
3) δ: 1.28–1.35 (m, 6H, OCH
2C
H3), 1.36, 1.60 (2s, 6H, C(CH
3)
2), 3.53 (dd,
J = 9.2, 4.2 Hz, 1H, H-3'), 3.98–4.16 (m, 4H, OC
H2CH
3), 4.55–4.66 (m, 3H, H-2',H-4', OCH
2Ph), 4.70–4.77 (m, 1H, OCH
2Ph), 5.77 (d,
J = 3.8 Hz, 1H, H-1'), 5.96–6.12 (m, 1H, CH=CH), 6.74–6.90 (m, 1H, CH=CH), 7.28–7.42 (m, 5H, Ph).
13C-NMR (75 MHz, CDCl
3) δ: 16.47, 16.56 (OCH
2CH
3), 26.63, 26.93 (C(
CH
3)
2), 61.97 (t,
J = 4.7 Hz, O
CH
2CH
3), 72.57 (O
CH
2Ph), 77.68 (d,
J = 9.4 Hz, C-4'), 78.04 (C-2'), 81.93 (d,
J = 1.7 Hz, C-3'), 104.15 (C-1'), 113.48 (C(CH
3)
2), 118.45 (d,
J = 186.0 Hz, C-6'), 128.18 , 128.30, 128.68, 137.19 (Ph), 148.00 (d,
J = 6.1 Hz, C-5').
31P-NMR (CDCl
3) δ: 17.49. HRMS (ESI): Calculated for C
20H
30O
7P [(M+H)
+], 413.1724; found, 413.1754.
Diethyl (2-((3aR,5R,6R,6aR)-6-hydroxy-2,2-dimethyltetrahydrofuro[2,3-d][
1,
3]
dioxol-5-yl)ethyl) phosphonate (
8): To a solution of compound
7 (0.91 g, 2.22 mmol) in MeOH was added 10% Pd/C (0.5 g). The reaction mixture was stirred for 2 h under hydrogen atmosphere at rt and filtered. The filtrate was evaporated under vacuum and the residue purified by silica gel column chromatography (CH
2Cl
2/MeOH 97:3) to yield compound
8 as a colorless oil (0.58 g, 81.5%).
1H-NMR (300 MHz, CDCl
3) δ: 1.29–1.35 (m, 6H, OCH
2C
H3), 1.36, 1.56 (2s, 6H, OC(CH
3)
2), 1.79–2.12 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.62 (ddd,
J = 10.3, 8.7, 5.3 Hz, 1H, H-3'), 3.77 (ddd,
J = 8.6, 6.7, 4.2 Hz, 1H, H-4'), 4.0–4.23 (m, 4H, OC
H2CH
3), 4.56 (dd,
J = 5.0, 4.1 Hz, 1H, H-2'), 5.77 (d,
J = 4.1 Hz, 1H, H-1);
13C-NMR (75 MHz, CDCl
3) δ: 16.43, 16.50 (OCH
2CH
3), 20.61 (d,
J = 141.7 Hz, C-6'), 24.99 (d,
J = 5.0 Hz, C-5'), 26.40, 26.52 (C(
CH
3)
2), 61.67 (d,
J = 6.63 Hz, O
CH
2CH
3), 75.55 (C-3'), 78.76 (C-2'), 79.49 (d,
J = 15.8 Hz, C-4'), 103.65 (C-1'), 112.52 (
C(CH
3)
2);
31P-NMR (CDCl
3) δ: 31.66. HRMS (ESI): calculated for C
13H
26O
7P [(M+H)
+], 325.1411; found, 325.1429.
(3R,4R,5R)-5-(2-(Diethoxyphosphoryl)ethyl)tetrahydrofuran-2,3,4-triyl triacetate (9): A solution of compound 8 (0.45 g, 1.38 mmol) and 60% AcOH (10 mL) was heated at 80 °C under argon for 16 h. When the starting material was consumed completely, the reaction mixture was evaporated in vacuo and co-evaporated twice with AcCN. The residue was dissolved in dry pyridine (12 mL) at 0 °C under argon followed by dropwise addition of acetic anhydride (1.56 mL, 16.52 mmol). The reaction mixture was slowly warmed to rt and stirred overnight. Then it was concentrated and co-evaporated with toluene. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH 95:5) to give 9 (0.47 g, 83.2%) as a low melting solid (α:β anomeric ratio = 41:9). α anomer: 1H-NMR (300 MHz, CDCl3) δ: 1.22–1.30 (m, 6H, OCH2CH3), 1.63–1.94 (m, 4H, H-5'a, H-5′b, H-6'a and H-6'b), 1.99, 2.02, 2.04 (s, 9H, 3COCH3), 3.96–4.08 (m, 4H, OCH2CH3), 4.08–4.17 (m, 1H, H-4'), 5.09 (dd, J = 7.0, 4.9 Hz, 1H, H-3'), 5.25 (d, J = 4.9 Hz, 1H, H-2'), 6.06 (s, 1H, H-1'). 13C-NMR (75 MHz, CDCl3) δ: 16.49, 16.57 (OCH2CH3), 20.58, 20.60 (COCH3), 21.80 (d, J = 143.3 Hz, C-6'), 21.15 (COCH3), 27.27 (d, J = 4.4 Hz, C-5'), 61.73, 61.82 (OCH2CH3), 73.27 (C-3'), 74.65 (C-2'), 81.27 (d, J = 18.2 Hz, C-4'), 98.24 (C-1'), 169.19, 169.52, 169.87 (CO). 31P-NMR (CDCl3) δ: 30.82. β anomer: 1H-NMR (300 MHz, CDCl3) δ: 1.32 (t, J = 7.0 Hz, 6H, OCH2CH3), 1.72–1.98 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 2.06, 2.10, 2.11 (s, 9H, 3 COCH3), 4.01–4.17 (m, 4H, OCH2CH3), 4.20–4.29 (m, 1H, H-4'), 5.02 (dd, J = 6.8, 3.7 Hz, 1H, H-3'), 5.19 (dd, J = 6.8, 4.6 Hz, 1H, H-2'), 6.36 (d, J = 4.6 Hz, 1H, H-1'). 13C-NMR (75 MHz, CDCl3) δ: 16.54, 16.62 (OCH2CH3), 20.43, 20.76, 21.17 (COCH3), 21.81 (d, J = 141.83 Hz, C-6'), 26.72 (d, J = 4.4 Hz, C-5'), 61.81, 61.88 (OCH2CH3), 69.95 (C-2′), 72.02 (C-3'), 83.06 (d, J = 17.1 Hz, C-4'), 93.95 (C-1'), 169.46, 169.80, 170.17 (CH3CO). 31P-NMR (CDCl3) δ: 30.57. ESI-HRMS for [C16H27O10P+H]+ calcd, 411.1420; found, 411.1436. HRMS (ESI): calculated for C16H28O10P [(M+H)+], 411.1415; found 411.1436.
3.2. General Procedures for the Silyl-Hilbert-Johnson Reaction to Synthesize Products 10a to 10g
Method 1: To a suspension of the appropriate quinazoline-2,4-(1H,3H)-dione (1.5 eq.) in dry acetonitrile (50 eq.) was added BSTFA (4 eq.) under argon. The solution was heated at 65 °C for 2 h. After cooling, a solution of triacetate 9 (1 eq.) in dry acetonitrile (50 eq.) and trimethylsilyl triflate (1.5 eq.) were added. The solution was stirred for 2 h at rt. The reaction was quenched with saturated aqueous NaHCO3 (50 mL for 1 mmol triacetate 9) and extracted with dichloromethane (50 mL for 1 mmol triacetate 9). The combined organic layer was washed with saturated aqueous NaHCO3 (3 × 50 mL for 1 mmol triacetate 9), dried over anhydrous MgSO4 and evaporated. The crude mixture was dissolved in MeOH (50 eq.) and 5.4 N sodium methoxide (8 eq.) in MeOH was added dropwise. After 1 h, the mixture was neutralized with acetic acid (10 eq.) and evaporated. Purification of the residue by silica-gel column chromatography (CH2Cl2/MeOH 97:3→95:5) gave the pure product as a white foam.
Method 2: To a suspension of appropriate quinazoline-2,4-(1H,3H)-dione (1.5 eq.) in hexamethyl-disilazane (50 eq.) was added trimethylsilyl chloride (0.7 eq.) and pyridine (10 eq.) under argon. The mixture was stirred at 130 °C overnight. The reaction mixture was evaporated to dryness under high vacuum. To the obtained residue a solution of triacetate 9 (1 eq.) in dry acetonitrile (50 eq.) was added under nitrogen followed by trimethylsilyl triflate (1.5 eq.). The solution was stirred for 2 h at rt. and the work up was similar as described in method 1.
Diethyl-(2-((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)-3,4-dihydroxy tetrahydrofuran-2-yl)ethyl)phosphonate (10a): Following Method 1 reaction between 9 (0.47 g, 1.14 mmol) and 4a (0.28 g, 1.71 mmol) yielded compound 10a (0.22 g, 50%). 1H-NMR (300 MHz, CDCl3) δ: 1.27 (dt, J = 18.3, 7.1 Hz, 6H, OCH2CH3), 1.79–2.15 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 2.15–2.36 (m), 4.45 (br s), 4.97 (br s, 2OH, NH), 3.88–3.97 (m, 1H, H-4'), 3.97–4.15 (m, 4H, OCH2CH3), 4.59 (t, J = 6.7 Hz, 1H, H-3'), 4.80–4.89 (m, 1H, H-2'), 5.96 (d, J = 2.0 Hz, 1H, H-1'), 7.19–7.27 (m, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.59–7.67 (m, 1H), 8.14 (d, J = 7.7 Hz, 1H, Ph). 13C-NMR (75 MHz, CDCl3) δ: 15.99–16.20 (m, OCH2CH3), 20.69 (d, J = 141.8 Hz, C-6'), 25.02 (d, J = 4.4 Hz, C-5'), 61.66 (dd, J = 12.2, 6.6 Hz, OCH2CH3), 71.95 (C-3'), 72.50 (C-2'), 82.32 (d, J = 17.7 Hz, C-4'), 91.68 (C-1'), 114.27,115.92, 123.41, 128.37, 135.12 and 140.98 (Ph), 149.74 (C-2), 162.04 (C-4). 31P-NMR (CDCl3) δ: 32.08. HRMS (ESI): calculated for C18H24N2O8P [(M–H)−], 427.1276; found, 427.1284.
Diethyl-(2-((2R,3S,4R,5R)-5-(6-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl)ethyl)phosphonate (10b): Following Method 2 reaction between 9 (0.34 g, 0.84 mmol) and 4b (0.18 g, 1.01 mmol) yielded compound 10b (133.3 mg, 61.3%). 1H-NMR (300 MHz, CDCl3) δ: 1.29 (dt, J = 17.9, 7.1 Hz, 6H, OCH2CH3), 1.80–2.15 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.89–3.98 (m, 1H, H-4'), 3.98–4.16 (m, 4H, OCH2CH3), 4.29, 5.01, 10.31 (br s, 2OH, NH), 4.60 (t, J = 6.2 Hz, 1H, H-3'), 4.83 (dd, J = 6.2, 2.3 Hz, 1H, H-2'), 5.90 (d, J = 2.6 Hz, 1H, H-1'), 7.33–7.42 (m, 1H, Ph), 7.43–7.50 (m, 1H, Ph), 7.78–7.85 (m,1H, Ph). 13C-NMR (75 MHz, CDCl3) δ: 16.57 (d, J = 5.53 Hz, OCH2CH3), 21.14 (d, J = 140.1 Hz, C-6'), 25.52 (t, J = 5.3 Hz, C-5'), 62.14 (dd, J = 13.82, 6.6 Hz, OCH2CH3), 72.36 (C-3'), 73.01 (C-2'), 82.87 (d, J = 17.1 Hz, C-4'), 92.40 (C-1'), 114.10–114.57 (m, Ph), 116.86 (d, J = 7.2 Hz, Ph), 117.68–117.98 (m, Ph), 122.86–123.38 (m, Ph), 137.76 (Ph), 149.83 (C-2), 156.99–160.41 (m, Ph), 161.39 (C-4). 31P-NMR (CDCl3) δ: 32.71. HRMS (ESI): Calculated for C18H23FN2O8P [(M–H)−], 445.1182; found, 445.1188.
Diethyl-(2-((2R,3S,4R,5R)-5-(6-chloro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)-3,4-dihydroxytetra- hydrofuran-2-yl)ethyl)phosphonate (10c): The reaction between 9 (0.34 g, 0.84 mmol) and 4c (0.25 g, 1.26 mmol) as described for method 1 to yield compound 10c (0.18 g, 45.1%). 1H-NMR (300 MHz, CDCl3) δ:1.28 (dt, J = 16.69, 7.03 Hz, 6H, OCH2CH3), 1.79–2.14 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.92 (q, J = 5.8 Hz, 1H, H-4'), 3.97–4.15 (m, 4H, OCH2CH3), 4.55 (t, J = 6.7 Hz, 1H, H-3'), 4.85 (dd, J = 6.15, 2.3 Hz, 1H, H-2'), 4.96, 10.49 (br s OH, NH), 5.89 (d, J = 2.6 Hz, 1H, H-1'), 7.40 (d, J = 9.4 Hz, 1H, Ph), 7.57 (dd, J = 9.1, 2.6 Hz, 1H, Ph), 8.04 (d, J = 2.3 Hz, 1H, Ph). 13C-NMR (75 MHz, CDCl3) δ: 16.44–16.63 (m, OCH2CH3), 21.13 (d, J = 141.0 Hz, C-6'), 25.48 (d, J = 4.4 Hz, C-5'), 62.13 (dd, J = 11.6, 6.6 Hz, OCH2CH3), 72.42 (C-3'), 72.80 (C-2'), 82.84 (d, J = 17.1 Hz, C-4'), 92.25 (C-1'), 116.51, 117.55, 128.08, 129.76, 135.46 and 139.87 (Ph), 149.85 (C-2), 161.33 (C-4). 31P-NMR (CDCl3) δ: 32.68. HRMS (ESI): calculated for C18H23ClN2O8P [(M−H)−], 461.0886; found, 461.0899.
Diethyl-(2-((2R,3S,4R,5R)-5-(6-methyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)-3,4-dihydroxytetra- hydrofuran-2-yl)ethyl)phosphonate (10d): The reaction between 9 (0.25 g, 0.61 mmol) and 4d (0.16 g, 0.91 mmol) as described for method 1 to yield compound 10d (125.2 mg, 46.5%). 1H-NMR (300 MHz, CDCl3) δ:1.28 (dt, J = 19.0, 7.1 Hz, 6H, OCH2CH3), 1.81–2.18 (m, 4H, H-5'a, H-5′b, H-6'a and H-6'b), 2.37 (s, 3H, CH3), 3.88–3.97 (m, 1H, H-4'), 3.97–4.16 (m, 4H, OCH2CH3), 4.28 (br s, 1H, OH), 4.56–4.68 (m, 1H, H-3'), 4.80–4.88 (m, 1H, H-2'), 4.98 (d, J = 5.0 Hz, 1H, OH), 5.94 (d, J = 2.3 Hz, 1H, H-1'), 7.34 (d, J = 8.8 Hz, 1H, Ph), 7.40–7.48 (m, 1H, Ph), 7.94 (s, 1H, Ph), 10.28 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δ: 15.78–16.15 (m, OCH2CH3), 20.51 (d, J = 141.0 Hz, C-6'), 19.95 (CH3), 24.83 (d, J = 4.4 Hz, C-5'), 61.26–61.77 (m, OCH2CH3), 71.76 (C-3'), 72.55 (C-2'), 82.18 (d, J = 17.1 Hz, C-4'), 91.52 (C-1'), 113.98, 115.56, 127.99, 133.17, 135.95 and 138.69 (Ph), 149.56 (C-2), 161.96 (C-4). 31P-NMR (CDCl3) δ: 32.84. HRMS (ESI): calculated for C19H26N2O8P [(M−H)−], 441.1432; found, 441.1427.
Diethyl-(2-((2R,3S,4R,5R)-5-(6-methoxy-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)ethyl)phosphonate (10e): The reaction between 9 (0.25 g, 0.61 mmol) and 4e (0.18 g, 0.91 mmol) as described for method 1 to yield compound 10e (122.7 mg, 44.0%). 1H-NMR (300 MHz, CDCl3) δ:1.21–1.35 (m, 6H, OCH2CH3), 1.79–2.17 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.86 (s, 3H, OCH3), 3.89–3.97 (m, 1H, H-4'), 3.98–4.15 (m, 4H, OCH2CH3), 4.63 (t, J = 6.7 Hz, 1H, H-3'), 4.85 (dd, J = 6.0, 2.5 Hz, 1H, H-2'), 5.04 (br s, OH), 5.92 (d, J = 2.3 Hz, 1H, H-1'), 7.20–7.25 (m, 1H, Ph), 7.39 (d, J = 9.7 Hz, 1H, Ph), 7.59 (d, J = 3.2 Hz, 1H, Ph), 10.27 (br s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δ: 16.42–16.64 (m, OCH2CH3), 21.09 (d, J = 141.0 Hz, C-6'), 25.42 (d, J = 4.4 Hz, C-5'), 56.02 (OCH3), 62.08 (dd, J = 14.9, 6.6 Hz, OCH2CH3), 72.33 (C-3'), 73.08 (C-2'), 82.78 (d, J = 17.1 Hz, C-4'), 92.19 (C-1'), 109.63, 116.27, 117.12, 124.35 and 135.41 (Ph), 149.94 (C-2), 155.95 (Ph), 162.30 (C-4). 31P-NMR (CDCl3) δ: 32.82. HRMS (ESI): calculated for C19H26N2O9P [(M−H)−], 457.1381; found, 457.1390.
Diethyl-(2-((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydrothieno[3,2-d]pyrimidin-1(2H)-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)ethyl)phosphonate (10f): The reaction between 9 (0.25 g, 0.61 mmol) and 4f (0.15 g, 0.91 mmol) as described for method 1 to yield compound 10f (198.8 mg, 75.2%). 1H-NMR (300 MHz, CDCl3) δ:1.20–1.35 (m, 6H, OCH2CH3), 1.79–2.15 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.92 (q, J = 6.1 Hz, 1H, H-4'), 3.97–4.14 (m, 4H, OCH2CH3), 4.50 (t, J = 6.3 Hz, 2H, H-3', OH), 4.80 (dd, J = 5.9, 2.9 Hz, 1H, H-2'), 4.95 (br s, 1H, OH), 5.81 (d, J = 3.2 Hz, 1H, H-1'), 7.13 (d, J = 5.3 Hz, 1H, H-6), 7.73 (d, J = 5.6 Hz, 1H, H-7), 10.33 (br s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δ: 16.43–16.62 (m, OCH2CH3), 21.15 (d, J = 141.0 Hz, C-6'), 25.54 (d, J = 4.4 Hz, C-5'), 62.12 (dd, J = 14.4, 6.6 Hz, OCH2CH3), 72.39 (C-3'), 72.71 (C-2'), 83.14 (d, J = 17.1 Hz, C-4'), 93.87 (C-1'), 114.38 (C-4a), 116.94 (C-6), 135.47 (C-7), 146.87 (C-7a), 151.09 (C-2), 158.53 (C-4). 31P-NMR (CDCl3) δ: 32.63. HRMS (ESI): calculated for C16H22N2O8PS [(M−H)−], 433.0840; found, 433.0838.
Diethyl-(2-((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydrobenzo[g]quinazolin-1(2H)-yl)-3,4-dihydroxytetra- hydrofuran-2-yl)ethyl)phosphonate (10g): The reaction between 9 (0.25 g, 0.61 mmol) and 4g (0.19 g, 0.91 mmol) as described for method 1 to yield compound 10g (125.0 mg, 43.1%). 1H-NMR (300 MHz, CDCl3) δ:1.22 (t, J = 7.0 Hz, 6H, OCH2CH3), 1.77–2.07 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.81 (d, J = 4.4 Hz, 1H, H-4'), 3.98 (quin, J = 7.1 Hz, 4H, 2 × OCH2CH3), 4.13–4.26 (m, 1H, H-3'), 4.65 (br s, 1H, H-2'), 5.05 (d, J = 6.2 Hz, 1H, OH), 5.21–5.32 (m, 1H, OH), 6.11 (d, J = 4.1 Hz, 1H, H-1'), 7.54 (t, J = 7.3 Hz, 1H, Ph), 7.66 (t, J = 7.3 Hz, 1H, Ph), 7.91 (s, 1H, Ph), 8.06 (d, J = 8.2 Hz, 1H, Ph), 8.14 (d, J = 8.2 Hz, 1H, Ph), 8.75 (s, 1H, Ph), 11.68 (br s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δ: 16.24, 16.31 (OCH2CH3), 20.74 (d, J = 140.1 Hz, C-6'), 25.63 (d, J = 3.9 Hz, C-5'), 60.92 (dd, J = 6.1, 2.8 Hz, OCH2CH3), 70.12 (C-2'), 72.01 (C-3'), 82.07 (d, J = 17.1 Hz, C-4'), 91.20 (C-1'), 111.46, 116.35, 125.85, 127.59, 128.34, 129.05, 129.36, 129.52, 135.99 and 136.04 (Ph), 149.80 (C-2), 161.65 (C-4). 31P-NMR (CDCl3) δ: 32.08. HRMS (ESI): calculated for C22H26N2O8P [(M−H)−], 477.1432; found, 477.1419.
3.3. General Procedure for the Deprotection of 10a–g to 3a–g
The appropriate phosphonic ester 10 (1 eq.) was dissolved in dry CH2Cl2 (50 eq.) and treated with TMSBr (20 eq.) at 0 °C under argon. After stirring overnight at rt, the mixture was quenched with 7 N NH3/MeOH (20 eq.) and evaporated. The residue was dissolved in H2O (15 mL for 0.1 mmol phosphonic ester) and washed with CH2Cl2 (3 × 10 mL for 0.1 mmol phosphonic ester). The aqueous layer was evaporated and purified with RP high-performance liquid chromatography (HPLC, Phenomenex Luna C-18, H2O/0.1% HCOOH in CH3CN, 90:10→0:100 in 23 min, flow 17.5 mL/min) to give the desired product after lyophilization of the appropriate fractions.
(2-((2R,3S,4R,5R)-5-(2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)-ethyl) phosphonic acid (3a): Deprotection of compound 10a (0.13 g, 0.30 mmol) with TMSBr (0.91 g, 5.95 mmol) according to the general procedure yielded compound 3a as a white power (32.2 mg, 29.1%). 1H-NMR (300 MHz, D2O) δ: 1.79–2.21 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.93–4.06 (m, 1H, H-4'), 4.34 (t, J = 6.9 Hz, 1H, H-3'), 4.86 (dd, J = 6.2, 4.4 Hz, 1H, H-2'), 6.09 (d, J = 3.8 Hz, 1H, H-1'), 7.35 (t, J = 7.5 Hz, 1H, Ph), 7.48 (d, J = 8.5 Hz, 1H, Ph), 7.71–7.81 (m, 1H, Ph), 8.00 (d, J = 7.9 Hz, 1H, Ph). 13C-NMR (75 MHz, D2O) δ: 22.89 (d, J = 135.8 Hz, C-6'), 25.82 (d, J = 3.9 Hz, C-5'), 71.04 (C-2'), 72.22 (C-3'), 82.47 (d, J = 17.7 Hz, C-4'), 90.85 (C-1'), 115.11, 115.72, 124.53, 128.05, 136.31 and 140.37 (Ph) 150.95 (C-2), 164.12 (C-4). 31P-NMR (D2O) δ: 29.02. HRMS (ESI): calculated for C14H16N2O8P [(M−H)−], 371.0650; found, 371.0651.
(2-((2R,3S,4R,5R)-5-(6-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)-3,4-dihydroxytetrahydro-furan-2-yl)ethyl)phosphonic acid (3b): The reaction between compound 10b (286.4 mg, 0.54 mmol) and TMSBr (1.65 g, 10.79 mmol) as described above, yielded compound 3b as a white powder (15.7 mg, 14.8%). 1H-NMR (300 MHz, DMSO-d6) δ:1.50–2.04 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.65–3.76 (m, 1H, H-4'), 4.04 (t, J = 6.7 Hz, 1H, H-3'), 4.49 (dd, J = 6.4, 4.7 Hz, 1H, H-2'), 5.98 (d, J = 4.4 Hz, 1H, H-1'), 6.66 (br s, 4H, 4OH), 7.47–7.55 (m, 1H, Ph), 7.55–7.64 (m, 1H, Ph), 7.71 (dd, J = 8.2, 2.9 Hz, 1H, Ph), 11.79 (br s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 23.81 (d, J = 137.3 Hz, C-6'), 26.41 (C-5'), 69.88 (C-2'), 72.11 (C-3'), 82.49 (d, J = 17.7 Hz, C-4'), 90.77 (C-1′'), 112.63–113.19 (m, C-5), 117.91 (d, J = 7.7 Hz, C-4a), 118.12 (d, J = 7.7 Hz, C-8), 122.31–122.85 (m, C-7), 136.87 (d, J = 1.7 Hz, C-8a), 149.73 (C-2), 156.06–159.37 (m, C-6), 160.85 (d, J = 2.2 Hz, C-4). 31P-NMR (CDCl3) δ: 26.92. HRMS (ESI): calculated for C14H15FN2O8P [(M−H)−], 389.0556; found, 389.0560.
(2-((2R,3S,4R,5R)-5-(6-chloro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)-3,4-dihydroxytetrahydro-furan-2-yl)ethyl)phosphonic acid (3c): The reaction between compound 10c (0.14 g, 0.31 mmol) and TMSBr (0.94 g, 6.11mmol) as described above, yielded compound 3c as a white powder (49.0 mg, 39.5%). 1H-NMR (300 MHz, D2O) δ:1.80–2.17 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.87–3.98 (m, 1H, H-4'), 4.28 (t, J = 7.0 Hz, 1H, H-3'), 4.76 (d, J = 3.8 Hz, 1H, H-2'), 5.88 (d, J = 3.8 Hz, 1H, H-1'), 7.25 (d, J = 9.4 Hz, 1H, Ph), 7.50 (dd, J = 9.1, 2.3 Hz, 1H, Ph), 7.56–7.61 (m, 1H, Ph). 13C-NMR (75 MHz, D2O) δ: 22.69 (d, J = 135.8 Hz, C-6'), 25.63 (d, J = 3.3 Hz, C-5'), 71.29 (C-2'), 72.21 (C-3'), 82.29 (d, J = 18.3 Hz, C-4'), 90.98 (C-1'), 116.62, 116.96, 126.91, 129.32, 135.69 and 138.75 (Ph), 150.20 (C-2), 162.11 (C-4). 31P-NMR (D2O) δ: 30.04. HRMS (ESI): calculated for [C14H15ClN2O8P [(M−H)−], 405.0260; found, 405.0266.
(2-((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-methyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)tetrahydro-furan-2-yl)ethyl)phosphonic acid (3d): The reaction between compound 10d (0.11 g, 0.25 mmol) and TMSBr (0.77 g, 5.01mmol) as described above, yielded compound 3d as a white powder (39.6 mg, 41.0%). 1H-NMR (300 MHz, D2O) δ:1.82–2.13 (m, 4H, H-5'a, H-5′b, H-6'a and H-6'b), 2.20 (s, 3H, CH3), 3.86–3.97 (m, 1H, H-4'), 4.28 (t, J = 7.0 Hz, 1H, H-3'), 4.73 (m, 1H, H-2'), 5.86 (d, J = 3.2 Hz, 1H, H-1'), 7.12 (d, J = 8.5 Hz, 1H, Ph), 7.31–7.44 (m, 2H, Ph). 13C-NMR (75 MHz, D2O) δ: 19.62 (CH3), 22.76 (d, J = 133.5 Hz, C-6'), 25.66 (d, J = 2.8 Hz, C-5′), 71.32 (C-2'), 72.26 (C-3'), 82.13 (d, J = 17.7 Hz, C-4'), 90.82 (C-1′), 114.83, 114.96, 127.30, 134.51, 136.96 and 137.79 (Ph), 150.46 (C-2), 163.38 (C-4). 31P-NMR (D2O) δ: 29.86. HRMS (ESI): calculated for C15H18N2O8P [(M−H)−], 385.0806; found, 385.0819.
(2-((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-methoxy-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)tetrahydro-furan-2-yl)ethyl)phosphonic acid (3e): The reaction between compound 10e (0.11 g, 0.23 mmol) and TMSBr (0.70 g, 4.58 mmol) as described above, yielded compound 3e as a white powder (48.9 mg, 53.1%). 1H-NMR (300 MHz, D2O) δ:1.78–2.18 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.86 (s, 3H, CH3), 3.93–4.01 (m, 1H, H-4'), 4.32 (t, J = 6.8 Hz, H-3'), 4.84 (dd, J = 6.6, 4.2 Hz, 1H, H-2'), 6.04 (d, J = 4.0 Hz, 1H, H-1'), 7.29–7.35 (m, 1H, Ph), 7.37–7.43 (m, 2H, Ph). 13C-NMR (75 MHz, D2O) δ: 22.99 (d, J = 126.1 Hz, C-6'), 25.51 (C-5'), 55.90 (CH3), 70.98 (C-2'), 72.21 (C-3'), 82.46 (d, J = 18.2 Hz, C-4'), 90.83 (C-1'), 109.87, 116.65, 116.92, 123.80, 134.38 and 155.46 (Ph), 150.68 (C-2), 163.60 (C-4). 31P-NMR (D2O) δ: 28.81. HRMS (ESI): calculated for C15H18N2O9P [(M−H)−], 401.0755; found, 401.0755.
(2-((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydrothieno[3,2-d]pyrimidin-1(2H)-yl)-3,4-dihydroxytetra- hydrofuran-2-yl)ethyl)phosphonic acid (3f): The reaction between compound 10f (0.18 g, 0.41 mmol) and TMSBr (1.24 g, 8.13 mmol) as described above, yielded compound 3f as a white powder (75.6 mg, 49.2%). 1H-NMR (300 MHz, DMSO-d6) δ:1.48–2.04 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.67–3.77 (m, 1H, H-4'), 4.01 (t, J = 6.4 Hz, 1H, H-3'), 4.41 (dd, J = 6.4, 5.3 Hz, 1H, H-2'), 5.91 (d, J = 5.0 Hz, 1H, H-1'), 6.53 (br s, 2H, 2OH), 7.25 (d, J = 5.6 Hz, 1H, H-6), 8.12 (d, J = 5.3 Hz, 1H, H-7), 11.62 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 23.78 (d, J = 135.8 Hz, C-6', 26.48 (C-5'), 70.41 (C-2'), 71.94 (C-3'), 83.00 (d, J = 17.7 Hz, C-4'), 90.78 (C-1′), 113.85 (C-4a), 118.02 (C-6), 135.77 (C-7), 145.17 (C-7a), 150.77 (C-2), 157.91 (C-4). 31P-NMR (DMSO-d6) δ: 28.07. HRMS (ESI): calculated for C12H14N2O8PS [(M−H)−], 377.0214; found, 377.0197.
(2-((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydrobenzo[g]quinazolin-1(2H)-yl)-3,4-dihydroxytetrahydro-furan-2-yl)ethyl)phosphonic acid (3g): The reaction between compound 10g (0.32 g, 0.56 mmol) and TMSBr (1.73 g, 11.30 mmol) as described above. The working up as follows: After stirring overnight, the reaction mixture was evaporated and co-distilled with toluene. The residue was dissolved with 7 N NH3/MeOH (10 mL) and stirred at room temperature for 3 h. The reaction mixture was concentrated and purified with RP high-performance liquid chromatography (HPLC, Phenomenex Luna C-18, H2O/0.1% HCOOH in CH3CN, 90:10→0:100 in 23 min, flow 17.5 mL/min) to give the compound 3g as a white power (50.0 mg, 21.0%). 1H-NMR (300 MHz, DMSO-d6) δ:1.52–2.14 (m, 4H, H-5'a, H-5'b, H-6'a and H-6'b), 3.75–3.85 (m, 1H, H-4'), 4.16 (t, J = 6.6 Hz, 1H, H-3'), 4.63 (dd, J = 6.4, 4.1 Hz, 1H, H-2'), 5.21 (br s, 2H, 2OH), 6.10 (d, J = 4.1 Hz, 1H, H-1'), 7.49–7.58 (m, 1H, Ph), 7.61–7.70 (m, 1H, Ph), 7.93 (s, 1H, Ph), 8.05 (d, J = 8.2 Hz, 1H, Ph), 8.13 (d, J = 8.2 Hz, 1H, Ph), 8.74 (s, 1H, Ph), 11.67 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 23.54 (d, J = 137.1 Hz, C-6'), 26.14–26.32 (m, C-5'), 69.83 (C-2'), 71.82 (C-3'), 82.23 (d, J = 17.4 Hz, C-4'), 90.86 (C-1'), 111.25, 116.09, 125.56, 127.35, 128.06, 128.75, 129.11, 129.22, 135.70 and 135.74 (Ph), 149.54 (C-2), 161.38 (C-4). 31P-NMR (DMSO-d6) δ: 26.39. HRMS (ESI): calculated for C18H18N2O9P [(M−H)−], 421.0806; found, 421.0795.
3.4. Procedures for Phospholipase C assay
Stable cell lines for study of the human (h) P2Y2, were generated by retroviral expression of the receptor in 1321N1 human astrocytoma cells, which do not natively express P2YRs. Agonist-induced [3H]inositol phosphate production was measured in cells plated at 20,000 cells/well on 96-well plates two days prior to assay. Sixteen hours before the assay, the inositol lipid pool of the cells was radiolabeled by incubation in 100 μL of serum-free inositol-free Dulbecco’s modified Eagle’s medium, containing 1.0 μCi of [3H]myo-inositol. No changes of medium were made subsequent to the addition of [3H]inositol. On the day of the assay, cells were challenged with 25 μL of a five-fold concentrated solution of receptor agonists in 100 mM Hepes (N-(2-hydroxyethyl)-piperazine-N''-2-ethanesulfonic acid), pH 7.3 in HBSS, containing 50 mM LiCl for 30 min at 37 °C. Incubations were terminated by aspiration of the drug-containing medium and addition of 90 μL of ice-cold 50 mM formic acid. After 30 min, supernatants were neutralized with 30 μL of 150 mM NH4OH and applied to Dowex AG1-X8 anion exchange columns. Total inositol phosphates were eluted and radioactivity was measured using a liquid scintillation counter. Antagonist activities were conducted in a similar manner using a maximally efficatious dose of agonist with variable amount of antagonist.