J. Debnath et al. / Bioorg. Med. Chem. 18 (2010) 8257–8263
8261
and electrostatic interactions for the designing of nucleosidic
inhibitors of RNase A.
(C). HRMS (ESI+): m/z calculated for C10H11N5O5Na [M+Na]+:
304.0652; found: 304.0660.
3.2.4. 50-O-Trityl-30-tert-butyldimethylsilyl thymidine (5)
3. Experimental section
3.1. Materials
To the stirred solution of compound 4 (1 g, 2.1 mmol) in
anhydrous pyridine (30 ml), tert-butyldimethylsilylchloride (1.2 g
7.9 mmol) and imidazole (0.7 g, 10 mmol) were added and the
mixture was stirred at room temperature under N2 atmosphere
for 10 h (TLC). Saturated aq NaHCO3 solution was added and the
mixture was stirred for another 6 h. The organic portion was ex-
tracted with EtOAc and all organic portions were evaporated under
reduced pressure. Crude residue thus obtained was purified over
silica gel to afford compound 5 (1.2 g, 97%). 1H NMR: (CDCl3): d
0.04 (s, 3H), 0.02 (s, 3H), 0.83 (s, 9H), 1.49 (s, 3H), 2.19–2.26 (m,
1H), 2.31–2.36 (m, 1H), 3.28 (dd, J = 2.4, 10.4 Hz, 1H), 3.46 (dd,
J = 2.8, 10.8 Hz, 1H), 3.97 (d, J = 2.8 Hz, 1H), 4.55 (m, 1H), 6.35 (t,
J = 6.4 Hz, 1H), 7.25–7.43 (m, 15H), 7.64 (s, 1H). 13C NMR (CDCl3):
Bovine pancreatic RNase A, yeast tRNA, 20,30-cCMP, human
serum albumin (HSA) were purchased from Sigma–Aldrich. All
other reagents were purchased from SRL India. Column chromato-
graphic separations were performed using silica gel (60–120 and
230–400 mesh). HPLC separations were performed by Shimadru
Prominence Series HPLC system. Luna 10 lm C18 column (100A)
was used for the separation. Solvents were dried and distilled fol-
lowing the standard procedures. TLC was carried out on pre-coated
plates (Merck silica gel 60, f254), and the spots were visualized with
UV light or by charring the plates dipped in 5% H2SO4–MeOH solu-
tion or 5% H2SO4/vanillin/EtOH or 5% ninhydrine in MeOH solution.
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded
on a Bruker NMR spectrometer (d scale). Acetonitrile was used as a
reference in case of NMRs taken in D2O solvent. UV–vis measure-
ments were made using a Perkin Elmer UV–vis spectrophotometer
(Model Lambda 25). Concentrations of the solutions were deter-
mined spectrophotometrically using the following data: e278.5
d
ꢀ4.9, ꢀ4.7, 11.9, 17.8 (C), 25.7, 41.5 (CH2), 63.0 (CH2), 72.0,
84.8, 86.6, 87.3 (C), 111.0 (C), 127.4, 128.0, 128.6, 135.5, 143.3
(C), 150.2 (C), 154.9 (C).
3.2.5. 30-tert-Butyldimethylsilyl thymidine (6)
Compound 5 (1.25 g, 2 mmol) was stirred with 10% TFA in DCM
(5 ml) for 2 h (TLC) at room temperature. The reaction mixture was
concentrated under vacuo. Crude residue thus obtained was puri-
fied over silica gel to afford compound 6 (0.41 g, 55%). 1H NMR:
(CDCl3): d 0.08 (s, 6H), 0.89 (s, 9H), 1.91 (s, 3H), 2.18–2.24 (m,
1H), 2.32–2.39 (m, 1H), 3.75 (m, 1H), 3.92 (m, 2H), 4.49 (m, 1H),
6.13 (t, J = 6.8 Hz, 1H), 7.36 (s, 1H). 13C NMR (CDCl3): d ꢀ4.9,
ꢀ4.8, 12.4, 17.9 (C), 25.6, 40.4 (CH2), 61.8 (CH2), 71.5, 86.6, 87.5,
110.9 (C), 137.0, 150.4 (C), 164.0 (C).
(RNase A),32 e268 (20,30-cCMP)31 are 9800 and 8500 Mꢀ1 cmꢀ1
respectively. High resolution mass spectra were recorded on
Micromass LCT.
,
3.2. Synthesis of modified nucleosides
3.2.1. 20,30-Isopropylidene adenosine (1)
3.2.6. 50-Carboxy thymidine (7)
To the stirred solution of adenosine (0.25 g, 0.9 mmol) in anhy-
drous DMF (6 ml), 2,2-dimethoxypropane (0.6 ml, 4.5 mmol) and
anhydrous p-toluenesulphonic acid (0.04 g, 0.2 mmol) were added
and the mixture was stirred at 70 °C for 8 h (TLC) under the N2
atmosphere. The reaction mixture was concentrated under vacuo
and the residue was stirred with saturated aq NaHCO3 solution
for another 6 h. The organic portion was extracted with EtOAc
and all the organic portions were evaporated under reduced pres-
sure. Crude residue thus obtained was purified over silica gel to af-
ford compound 2 (0.21 g, 73%). white solid. 1H NMR: (d6-DMSO): d
1.30 (s, 3H), 1.52 (s, 3H), 3.48–3.56 (m, 2H), 4.19 (q, J = 4.4, 6.8 Hz,
1H), 4.94 (dd, J = 2.6 Hz, 1H), 5.23 (t, J = 5.6 Hz, 1H), 5.32 (q, J = 3.2,
6.4 Hz, 1H), 6.10 (d, J = 2.8 Hz, 1H), 7.34 (br s, 2H), 8.13 (s, 1H), 8.32
(s, 1H).
To a stirred solution of compound 6 (0.4 g, 1.1 mmol) in aq KOH
(1 N; 8 ml), K2S2O8 (1.3 g, 4.8 mmol), and RuCl3ꢁ3H2O (0.004 g)
were added and the mixture was stirred vigorously for 3.5 h
(TLC) at room temperature. The reaction mixture was neutralized
by dil. HCl to pH 7. The mixture was concentrated under vacuo.
Crude residue thus obtained was purified over silica gel to afford
compound 7 (0.2 g, 69%). 1H NMR: (d6-DMSO): d 1.75 (s, 3H),
1.84–1.89 (m, 1H), 2.04–2.08 (m, 1H), 4.20 (s, 1H), 4.37 (d,
J = 4 Hz, 1H), 6.28 (q, J = 5.2, 12.8 Hz, 1H), 8.60 (s, 1H), 11.25 (s,
1H). 13C NMR (d6-DMSO): d 12.5, 38.8 (CH2), 74.3, 85.6, 86.4,
109.2 (C), 137.6, 150.7 (C), 164.0 (C), 174.1 (C). HRMS (ESI+): m/z
calculated for C10H12N2O6Na [M+Na]+: 279.0588; found: 279.0593.
3.2.7. 3-tert-Butoxycarbonylamino-N-[5-(5-methyl-2,4-dioxo-
3,4-dihydro-2H-pyrimidin-1-yl)-2-trityloxymethyl-tetrahydro-
furan-3-yl]-succinamic acid benzyl ester (9)
3.2.2. 50-Carboxy-20,30-isopropylidene adenosine (2)
Compound 1 (0.74 g, 2.4 mmol) was dissolved in a ternary
solution mixture of H2O, CH3CN, and CCl4 (3:2:2; 2.6 ml). To this
solution sodium periodate (1 g, 4.6 mmol) and RuCl3ꢁ3H2O
(0.008 g) were added and the mixture was stirred vigorously at
room temperature for 3 h (TLC). The reaction mixture was concen-
trated under vacuo. Crude residue thus obtained was purified over
silica gel to afford compound 2 (0.41 g, 52%). 1H NMR: (d6-DMSO):
d 1.34 (s, 3H), 1.51 (s, 3H), 4.70 (s, 1H), 5.52 (s, 2H), 6.41 (s, 1H),
8.51 (s, 1H), 8.55 (s, 1H), 10.08 (s, 1H).
A mixture of compound 8 (0.54 g, 1.1 mmol) and Boc-Asp(OBzl)–
OH (0.36 g, 1.1 mmol) in anhydrous DMF (10 ml) was cooled to
ꢀ15 °C. Dicyclohexylcarbodiimide (0.23 g, 1.1 mmol) and N-
hydroxysuccinamide (0.13 g, 1.1 mmol) were added and the reac-
tion mixture was stirred for 30 h(TLC) at room temperature under
N2 atmosphere. The reaction mixture was filtered and the residue
was washed with EtOAc. The filtrate was stirred with saturated aq
NH4Cl solution for another 6 h. The organic portion was then ex-
tracted with EtOAc and all the organic portions were evaporated un-
der reduced pressure. Crude residue thus obtained was purified over
silica gel to afford compound 9 (0.53 g, 60%). 1H NMR: (CDCl3): d 1.36
(s, 3H), 1.43 (s, 9H), 2.35–2.45 (m, 2H), 2.69 (dd, J = 5.6, 16.8 Hz, 1H),
2.92 (dd, J = 4, 16.4 Hz, 1H), 3.41 (m, 2H), 3.92 (s, 1H), 4.49 (br s, 1H),
4.73 (m, 1H), 5.09 (m, 2H), 5.70 (d, J = 8.4 Hz, 1H), 6.41 (m, 1H), 7.23–
7.42 (m, 20H), 7.52 (br s, 1H), 7.60 (s, 1H), 9.47 (br s, 1H). 13C NMR
(CDCl3): d 11.6, 28.2, 36.3 (C), 37.9 (C), 50.6, 63.8 (CH2), 66.9 (CH2),
80.8 (C), 84.2, 84.5, 87.4 (C), 111.7 (C), 127.2, 128.0, 128.2, 128.3,
3.2.3. 50-Carboxy adenosine (3)
A solution of compound 2 (0.3 g, 0.9 mmol) in 60% TFA in DCM
(30 ml) was stirred at room temperature for 3 h (TLC). The reaction
mixture was concentrated under vacuo. Crude residue thus
obtained was purified over silica gel to afford compound 3
(0.16 g, 63%). 1H NMR: (D2O): d 3.37 (br s, 1H), 4.12 (s, 2H), 6.07
(d, J = 6 Hz, 1H), 8.06 (s, 1H), 8.53 (s, 1H). 13C NMR (D2O): d 73.9,
74.5, 85.0, 86.6, 119.1, 140.4, 149.2 (C), 152.7, 155.5 (C), 176.5