956 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Beauchamp et al.
46a : 99%; 1H-NMR (DMSO-d6) δ 1.07 (t, J ) 7.1, 3H,
OCH2CH3), 1.20-1.60 (m, 6H, (CH2)3), 3.44 (t, J ) 6.1, 2H,
NCH2OCH2), 3.66 (m, 2H, OCH2CH3), 5.47 (s, 2H, NCH2O),
7.80 (br s, 2H, NH2), 12.95 (br s, 1H, NH); CIMS m/ z 369 (M
+ 23)+.
Ack n ow led gm en t. Our thanks to Alice Melton and
G. Faye Orr for technical assistance in the chemical
synthesis, to the NMR staff of the Bioanalytical Sciences
division for obtaining spectra and interpretation, and
to Dr. T. Krenitsky for helpful discussions.
46b: 83%; CIMS m/ z 361 (M + 1)+.
46c: 79%; CIMS m/ z 375 (M + 1)+.
Refer en ces
[4-(5-Am in o-6,7-d ih yd r o-7-oxo-3H-1,2,3-tr ia zolo[4,5-d ]-
p yr im id in -3-yl)bu tyl]p h osp h on ic Acid Am m on iu m Sa lt
(42a ) a n d 42b,c. To a suspension of 41 (a -c) (1.5 mmol) and
HMDS (15 mL) in dry CH2Cl2 (15 mL) was added TMSBr (2.29
g, 1.98 mL, 15 mmol). The mixture was stirred overnight at
room temperature. After cooling to 0-5 °C, MeOH was slowly
added. The solvent was evaporated in vacuo, and the resulting
residue was purified by ion exchange column chromatography
to afford compounds 42 (a -c) as analytically pure white solids.
42a : 81%; 1H-NMR (D2O) δ 1.30-1.50 (m, 4H, (CH2)2), 1.83
(m, 2H, PCH2), 4.21 (t, J ) 7.1, 2H, NCH2); 31P-NMR (D2O) δ
25.67; UV λmax nm (ꢀ × 103) (pH 1) 252 (10.5), (pH 7) 252 (9.9),
(pH 13) 277 (9.4); CIMS m/ z 289 (M - NH3 + 1). Anal.
(C8H16N7O4P) C, H, N.
(1) Parks, R. E., J r.; Stoeckler, J . D.; Cambor, C.; Savarese, T. M.;
Crabtree, G. W.; Chu, S.-H. Purine Nucleoside Phosphorylase
and 5′-Methylthioadenosine Phosphorylase: Targets of Chemo-
therapy. In Molecular Actions and Targets for Cancer Chemo-
therapeutic Agents; Sartorelli, A. C., Lazo, J . S., Bertino, J . R.,
Eds.; Academic Press: New York, 1981; pp 229-252.
(2) Sircar, J . C.; Gilbertsen, R. B. Purine Nucleoside Phosphorylase
(PNP) Inhibitors: Potential Selective Immunosuppressive Agents.
Drugs Future 1988, 13, 653-668.
(3) Shewach, D. S.; Chern, J .-W.; Pillote, K. E.; Townsend, L. B.;
Daddona, P. E. Potentiation of 2′-Deoxyguanosine cytotoxicity
by a novel inhibitor of purine nucleoside phosphorylase, 8-amino-
9-benzylguanine. Cancer Res. 1986, 46, 519-523.
(4) Stein, J . M.; Stoeckler, J . D.; Li, S.-Y.; Tolman, R. L.; MacCoss,
M.; Chen, A.; Karkas, J . D.; Ashton, W. T.; Parks, R. E., J r.
Inhibition of Human Purine Nucleoside Phosphorylase by Acyclic
Nucleosides and Nucleotides. Biochem. Pharmacol. 1987, 36,
1237-1244.
(5) Sircar, J . C.; Kostlan, C. R.; Pinter, G. W.; Suto, M. J .; Bobovski,
T. P.; Capiris, T.; Schwender, C. F.; Dong, M. K.; Scott, M. E.;
Bennett, M. K.; Kossarek, L. M.; Gilbertsen, R. B. 8-Amino-9-
substituted Guanines: Potent Purine Nucleoside Phosphorylase
(PNP) Inhibitors. Agents Actions 1987, 21, 253-256.
(6) Halazy, S.; Eggenspiller, A.; Ehrhard, A.; Danzin, C. Phospho-
nate Derivatives of N9-Benzylguanine: A New Class of Potent
Purine Nucleoside Phosphorylase Inhibitors. Bioorg. Med. Chem.
Lett. 1992, 2, 407-410.
(7) Kelley, J . L.; Linn, J . A.; McLean, E. W.; Tuttle, J . V. 9-(Phospho-
no)alkyl)benzyl)guanines. Multisubstrate Analogue Inhibitors of
Human Erythrocyte Purine Nucleoside Phosphorylase. J . Med.
Chem. 1993, 36, 3455-3463.
(8) Tuttle, J . V.; Krenitsky, T. A. Effects of Acyclovir and its
Metabolites on Purine Nucleoside Phosphorylase. J . Biol. Chem.
1984, 259, 4085-4069.
(9) Gilbertsen, R. B.; Sircar, J . C. Enzyme Cascades: Purine
Metabolism and Immunosuppression. In Comprehensive Me-
dicinal Chemistry; Sammes, P. G., Ed.; Pergamon Press: Oxford,
1990; Vol. 2, pp 443-480.
42b : 60%; CIMS m/ z 303 (M - NH3 + 1)+. Anal.
(C9H18N7O4P) C, H, N.
42c: 66%; CIMS m/ z 317 (M - NH3 + 1)+. Anal.
(C10H20N7O4P) C, H, N.
[4-((5-Am in o-6,7-d ih yd r o-7-oxo-3H-1,2,3-tr ia zolo[4,5-d ]-
p yr im id in -3-yl)m eth oxy)bu tyl]p h osp h on ic Acid Am m o-
n iu m Sa lt (47a ) a n d 47b,c. Compounds 47 (a -c) were
prepared from 46 (a -c) following the same procedure for the
synthesis of 42 (a -c).
47a : 73%; 1H-NMR (D2O) δ 1.20-1.50 (m, 6H, (CH2)3), 3.36
(t, J ) 6.2, 2H, NCH2OCH2), 5.49 (s, 2H, NCH2O); 31P-NMR
(D2O) δ 27.21; UV λmax nm (ꢀ × 103) (pH 1) 254 (7.5), (pH 7)
254 (7.5), (pH 13) 278 (6.6); CIMS m/ z 321 (M - NH3 + 1).
Anal. (C9H18N7O5P) C, H, N.
47b : 57%; CIMS m/ z 333 (M - NH3 + 1)+. Anal.
(C10H20N7O5P) C, H, N.
47c: 36%; CIMS m/ z 347 (M - NH3 + 1)+. Anal.
(C11H19N6O5P‚0.6NH3) C, H, N.
(10) Martin, D. W.; Gelfand, E. W. Biochemistry of Diseases of
Immunodevelopment. Annu. Rev. Biochem. 1981, 50, 845-877.
(11) Gilbertsen, R.B.; Ealick, S. E.; Rule, S. A.; Carter, D. C.;
Greenbough, T. J .; Babu, Y. S.; Cook, W. J .; Habash, J .;
Halliwell, J . R.; Stoeckler, J . D.; Parks, R. E. J r.; Chen, S.-F.;
Bugg, C. E. Three Dimensional Structure of Human Eyrthrocytic
Purine Nucleoside Phosphorylase at 3.2 Å Resolution. J . Biol.
Chem. 1990, 265, 1812-820.
(12) Krenitsky, T. A.; Tuttle, J . V.; Miller, W. H.; Moorman, A. R.;
Orr, G. F.; Beauchamp, L. Nucleotide Analogue Inhibitors of
Purine Nucleoside Phosphorylase. J . Biol. Chem. 1990, 265,
3066-3069.
Diet h yl [5-(5-Acet a m id o-6,7-d ih yd r o-7-oxo-2H -1,2,3-
tr ia zolo[4,5-d ]p yr im id in -2-yl)p en tyl]p h osp h on a te (49). A
mixture of 48 (0.422 g, 1.79 mmol), p-TsOH‚H2O (0.034 g, 0.18
mmol), and 10b (2.12 g, 7.16 mmol) was heated at 140 °C
under vacuum for 20 min. The oil was taken in methanol and
purified by flash column chromatography (95:5 CHCl3-MeOH)
to give 49 as a clear oil (0.350 g, 45%): 1H-NMR (DMSO-d6) δ
1.21 (t, J ) 7.0, OCH2CH3), 2.22 (s, 3H, CH3CO), 1.24-1.90
(m, 8H, (CH2)4), 3.53 (m, 2H, NCH2OCH2), 3.95 (m, 4H, OCH2-
CH3), 5.77 (s, 2H, NCH2O).
(13) Halazy, S.; Ehrhard, A.; Danzin, C. 9-(Difluorophosphonoalkyl)
Guanines as a New Class of Multisubstrate Analogue Inhibitos
of Purine Nucleoside Phosphorylase. J . Am. Chem. Soc. 1991,
11, 315-317.
(14) Yamazaki, A.; Okutsu, M.; Yamada, Y. Synthesis of Guanosine
and its Derivatives from 5-Amino-1-â-D-ribofuranosyl-4-imida-
zolecarboxamide. IV. A new route to Guanosine via Cyanamide
derivative. Nucleic Acids Res. 1976, 3, 251-259.
(15) Sznaidman, M.; Beauchamp, L. Unpublished results.
(16) Bzowska, A.; Kulikowska, E.; Shugar, D.; Chen, B. Y.; Lindborg,
B.; J ohansson, N. G. Acyclonucleoside Analog Inhibitors of
Mammalian Purine Nucleoside Phosphorylase. Biochem. Phar-
macol. 1991, 41, 1791-803.
(17) Seela, F.; Steker, H. Synthesis of the â-D-Deoxyribofuranoside
of 6-Amino-1H-Pyrazolo[3,4-d]pyrimidin-4(5H)-one. A new isos-
ter of 2′-Deoxyguanosine. Heterocycles 1985, 23, 2521-2525.
(18) Kjellberg, J .; Liljenberg, M.; J ohansson, N. G. Regioselective
alkylation of 6-(â-Methoxy)Guanine to give the 9-Alkylguanine
derivative. Tetrahedron Lett. 1986, 27, 877-880.
[5-(5-Am in o-6,7-d ih yd r o-7-oxo-2H-1,2,3-tr ia zolo[4,5-d ]-
p yr im id in -2-yl)p en tyl]p h osp h on ic Acid (51). Compound
49 (0.300 g, 0.69 mmol) was deacetylated as described for 31
(a -c) and purified by flash column chromatography (95:5
CHCl3-MeOH) to give 50 as a white residue, which was
immediately treated with TMSBr (0.442 mL, 3.3 mmol) in a
1:1 mixture of CH2Cl2-HMDS (16 mL). The mixture was
stirred at room temperature for 5 h, worked up as described
for 32 (a -c), and purified by ion exchange chromatography
to give 51 as a white solid (0.08 g, 72%): 1H-NMR (D2O) δ
0.91-1.46 (m, 4H, (CH2)2), 3.33 (m, 2H, NCH2OCH2), 5.51 (s,
2H, NCH2O); 31P-NMR (DMSO-d6) δ 23.68; UV λmax nm (ꢀ ×
103) pH 1 ) 271 (9.5), pH 7 ) 239 (8.4), 294 (6.6), pH 13 )
256 (6.4), 299 (8.5). Anal. (C10H17N6O5P‚0.3H2O‚0.6NH3‚
0.5NH4HCO3) C, H, N.
En zym e Assa ys. PNPase was purified from human eryth-
rocytes and assayed using a xanthine oxidase-coupled spec-
trophotometric assay as described previously.8,12 In addition
to enzyme the assay mixtures contained inhibitor, 0.1 mM
inosine, 1.0 mM potassium phosphate buffer, 100 mM Tris-
hydrochloride buffer, 0.1 mM ethylenediaminetetraacetic acid
disodium salt, and 0.2 IU/mL of xanthine oxidase at pH 7.4.
The apparent inhibition constant (Ki′) of a compound was
determined from its ability to inhibit the phosphorolysis of
inosine at 1 mM phosphate and was calculated from fractional
inhibition as described previously.21
(19) Ishido, I.; Hosono, A.; Isome, S.; Maruyama, A.; Sato, T.
Improved fusion method for the synthesis of Purine Nucleosides.
Acid-catalyzed reactions of N7- or N9-acylpurines with acetylated
sugars. Bull. Chem. Soc. J pn. 1964, 37, 1389-1390.
(20) Beauchamp, L. M.; Dolmatch, B. L.; Schaeffer, H. J .; Collins,
P.; Bauer, D. J .; Keller, P. M.; Fyfe, J . A. Modifications on the
Heterocyclic Base Acyclovir: Synthesis and Antiviral Properties.
J . Med. Chem. 1985, 28, 982-987.
(21) Kelley, J . L.; McLean, E. W.; Crouch, R. C.; Averett, D. R.; Tuttle,
J . V. J . Med. Chem. 1995, 38, 1005-1014.
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