1252
W. Zhou et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1249±1252
been identi®ed in cell-based assays.11 The resulting
structure±activity relationship is helping to guide the
lead development through synthesis and evaluation of
additional focused libraries. Details of these ongoing
eorts will be reported in due course.
(D2O, 200 Mz): d 58.96. ES±MS (negative mode): calcd
for C13H21N3O6PS, 378 (M); found m/z 378.
Acknowledgements
We thank Dr. Japhet Lyaku for helpful suggestion, and
Jennifer Abbott for editorial assistance.
Experimental Procedure for Assembly of the Library
Each alcohol (30 mmol), and each of the nucleoside ami-
dites 5a±f (20 mmol) were added sequentially to a series of
conical microtubes (2 mL, Ultident Scienti®c) containing
1H-tetrazole solution in acetonitrile (1 mL, 100 mmol).
The mixture was shaken in a platform shaker at room
temperature for 5 min. Then 3H-BD (40 mmol) was
added as a solid, and the contents shaken for another 5
min. The acetonitrile was evaporated in a Speed Vac.
Ethyl acetate (0.8 mL) was then added, followed by
aqueous sodium bicarbonate (0.4 mL). Following thor-
ough mixing of the phases, the organic layer containing
the intermediate thiophosphate triester 8 was separated
and evaporated to dryness. Aqueous ammonium hydrox-
ide (28%, 1 mL) was added to the residue in each micro-
tube. The tightly capped tubes were heated at 55ꢀC for 3
h. The aqueous ammoniacal solution was concentrated to
dryness in a Speed Vac. The contents were dissolved in
water (0.8 mL), and extracted with chloroform (2 Â 0.4
mL). The aqueous layer was evaporated to dryness in a
Speed Vac to obtain the library 1. Each member was
obtained as a white solid. Quantitation was achieved on
the basis of A260 units, and the yields of product 1 were
found to range from 75 to 85% starting from 5a±f.
References and Notes
1. For an excellent treatise on nucleic acids chemistry, see:
Sanger, W. Principles of Nucleic Acids Structure; Springer-
Verlag: New York, 1984.
2. For selected reviews on molecular diversity and combina-
torial methods to drug discovery, see: (a) Nefzi, A.; Ostresh, J.
M.; Houghten, R. A. Chem. Rev. 1997, 97, 449. (b) Gallop, M.
A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon, E.
M. J. Med. Chem. 1994, 37, 1233. (c) Gordon, E. M.; Barrett,
R. W.; Dower, W. J.; Fodor, S. P. A.; Gallop, M. A. J. Med.
Chem. 1994, 37, 1385. (d) Thompson, L. A.; Ellman, J. A.
Chem. Rev. 1996, 96, 555.
3. Bleczinski, C. F.; Richert, C. J. Am. Chem. Soc. 1999, 121,
10889.
4. Zhou, W.; Roland, A.; Jin, Y.; Iyer, R. P. Tetrahedron Lett.
2000, 41, 441.
5. For selected methods of library assembly using solution-
phase approach, see: (a) Boger, D. L.; Tarby, C. M.; Myers, P.
L.; Caporale, L. H.; J. Am. Chem. Soc. 1996, 118, 2109. (b) An,
H.; Haly, B. D.; Fraser, A. S.; Guinosso, C. J.; Cook, P. D. J.
Org. Chem. 1997, 62, 5156. (c) Cheng, S.; Comer, D. D.; Wil-
liams, J. P.; Myers, P. I., Boger, D. L. J. Am. Chem. Soc. 1996,
118, 2567. (d) Carell, T.; Wintner, E. A.; Bashir-Hashemi, A.;
Rebek Jr, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 2059.
6. Gayo, L. M. Biotechnol. Bioeng. 1998, 61, 95.
7. Barone, A. D.; Tang, J.-Y.; Caruthers, M. H. Nucl. Acids
Res. 1984, 12, 4051.
8. For methods of preparation of phosphoramidite derivatives,
see: Iyer, R. P.; Beaucage, S. L. In Comprehensive Natural
Products Chemistry; Kool, E. T., Ed.; Elsevier Science: Lon-
don, 1999; Vol. 7, pp 105±152.
NMR and MS data of the representative library mem-
1
bers: 1a-i. H NMR (D2O, 500 MHz): d 8.44 (d, 1H,
J=9.2 Hz), 8.21 (s, 1H), 6.44±6.47 (m, 1H), 4.24 (s, 1H),
3.96±4.06 (m, 2H), 3.55±3.64 (m, 2H), 2.81±2.88 (m, 1H),
2.57±2.62 (m, 1H), 1.30±1.35 (m, 2H), 1.04±1.09 (m, 2H),
0.67 (t, 3H, J=7.5 Hz, J=7.3 Hz) ppm. 31P NMR (D2O,
200 MHz): d 58.97, 58.64 ppm. ES±MS (negative mode):
calcd for C14H21N5O5PS, 402 (M); found m/z, 402. 1b-i:
1H NMR (D2O, 500 MHz): d 8.05 (d, 1H, J=7.7 Hz), 6.24
(t, 1H, J=6.6 Hz), 6.10 (d, 1H, J=7.7 Hz), 4.50±4.53 (m,
1H), 4.19 (s, 1H), 4.03±4.12 (m, 2H), 3.84±3.88 (m, 2H),
2.40±2.45 (m, 1H), 2.24±2.29 (m, 1H), 1.51±1.57 (m, 2H),
1.25±1.33 (m, 2H), 0.83 (t, 3H, J=7.5 Hz). 31P NMR
9. Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L. J. Am.
Chem. Soc. 1990, 112, 1253.
10. For a selected review, see: Houghten, R. A.; Pinilla, C.;
Appel, J. R.; Blondelle, S. E.; Dooley, C. T.; Eichler, J.; Nefzi,
A.; Ostresh, J. M. J. Med. Chem. 1999, 42, 3743.
11. The cell-based assays helped to validate the biological
relevance of the libraries in terms of having `drug-like' attri-
butes. The antiviral screen results will be reported in a separate
account in due course.