Fluoro-Unsaturated Nucleosides
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3407
and incubated overnight. The reaction was stopped with stop
solubilization solution (Promega, Madison, WI). The plates
were incubated for 5 h to ensure that the formazan crystals
were dissolved. The plates were read at a wavelength of 570
nm using an ELISA plate reader (Bio-tek instruments, Inc.,
Winooski, VT, model EL 312e). The 50% inhibition concentra-
tion (IC50) was determined from the concentration-response
curve using the median effect method.25
and AI 25899) from the National Institutes of Health
and the Department of Veterans Affairs.
Supporting Information Available:
1H NMR and el-
emental analysis data for compounds 8-32. This material is
References
(1) Richman, D. D.; Fischl, M. A.; Grieco, M. H.; Gottlieb, M. S.;
Volberding, P. A.; Laskin, O. L.; Leedom, J. M.; Groopman, J.
E.; Mildvan, D.; Hirsch, M. S.; Jackson, G. G.; Durack, D. T.;
Phil, D.; Lusinoff-Lehrman, S. The toxicity of azidothymidine
(AZT) in the treatment of patients with AIDS-related complex.
A double-blind, placebo-controlled trial. N. Engl. J. Med. 1987,
317, 192-197.
(2) Strarnes, M. C.; Cheng, Y.-C. Cellular metabolism of 2′,3′-
dideoxycytidine, a compound active against human immunode-
ficiency virus in vitro. J. Biol. Chem. 1987, 262, 988-991.
(3) Lambert, J. S.; Seidlin, M.; Reichman, R. C.; Plank, C. S.;
Laverty, M.; Morse, G. D.; Knupp, C.; McLaren, C.; Pettineli,
C.; Valentine, F. T.; Dolin, R. 2′,3′-Dideoxyinosine (DDI) in
patients with the acquired immunodeficiency syndrome or AIDS-
related complex. A phase I trial. N. Engl. J. Med. 1990, 322,
1333-1340.
(4) Mansuri, M. M.; Hitchcock, M. J. M.; Buroker, R. A.; Bregman,
C. L.; Ghazzouli, I.; Desiderio, J. V.; Starrett, J. E.; Sterzycki,
R. Z.; Martin, J. C. Comparison of in vitro biological properties
and mouse toxicities of 3 thymidine analogs active against
human immunodeficiency virus. Antimicrob. Agents Chemother.
1990, 34, 637-641.
(5) Larder, B. A.; Darby, G.; Richman, D. D. HIV with reduced
sensitivity to zidovudine (AZT) isolated during prolonged therapy.
Science 1989, 243, 1731-1734.
(6) St. Clair, M. H.; Martin, J. L.; Tudor-Williams, G.; Bach, M. C.;
Vavro, C. L.; King, D. M.; Kellam, P.; Kemp, S. D.; Larder, B.
A. Resistance to DDI and sensitivity to AZT induced by a
mutation in HIV-1 reverse transcriptase. Science 1991, 253,
1557-1559.
(7) Richman, D.; Shih, C. K.; Lowy, I.; Rose, J.; Prodanovich, P.;
Goff, S.; Griffin, J. Human immunodeficiency virus type 1
mutants resistant to nonnucleoside inhibitors of reverse tran-
scriptase arise in tissue culture. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 11241-11245.
Adenosine Deaminase Study. Assays were performed at
25 °C in phosphate buffer solution (pH 7.4) with substrate
concentrations in the range 15-100 µM and with 0.15 units
of adenosine deaminase (EC. 3.5.4.4. from calf intestinal
mucosa, purchased from Sigma-Aldrich). The assays were
monitored with a UV spectrometer at 265 nm. Initially,
qualitative assays were performed with D-3′F-d4A 29 (200 µM)
in the presence of 0.24 units of adenosine deaminase for 120
min to determine whether it is a substrate of this enzyme. The
concentration (ct) of each substrate at a certain time (t) was
calculated from the absorbance (At) at that time (t), where it
was assumed that the total change of absorbance (A0 - A∞)
was directly related to the disappearance of the substrate.21
Initial hydrolysis rates for each substrate concentration were
measured manually through graphical curve fitting of the
concentration vs time data for the reaction of each substrate.
From the Lineweaver-Burke plot of these initial rates, Vmax
(maximum velocity) and KM (Michaelis-Menten constant)
were obtained for each substrate and kcat was also calculated
with 0.15 unit of the enzyme (MW ) 33 000) in 2 mL of buffer
solution. The t1/2 values of D-3′F-d4A 25 and adenosine were
also measured at 20 µM with 0.15 unit of the enzyme.
Molecular Modeling Study. (a) Conformational Analy-
sis. The initial conformation of d-3′F-d4C 26 was constructed
by the builder module in Spartan 5.1.1 (Wavefunctions, Inc.
Irvine, CA), and all calculations were performed on a Silicon
Graphics O2 workstation. The initial conformations were
cleaned up and geometry-optimized through quantum me-
chanical ab initio calculations using the RHF/3-21G* basis in
Spartan 5.1.1.
(8) Gulick, R. M.; Mellors, J. W.; Havlir, D.; Eron, J. J.; Gonzalez,
C.; McMahon, D.; Richman, D. D.; Valentine, F. T.; Jonas, L.;
Meibohm, A.; Emini, E. A.; Chodakewitz, J. A. Treatment with
indinavir, zidovudine, and lamivudine in adults with human
immunodeficiency virus infection and prior antiretroviral therapy.
N. Engl. J. Med. 1997, 337, 734-739.
(b) Binding Affinity Study for HIV-1 Reverse Tran-
scriptase. All molecular modeling studies of the enzyme/
substrate complexes were performed using Sybyl 6.7 (Tripos
Associates, St. Louis, MO) on a Silicon Graphics Octane2
workstation. The enzyme site of the enzyme/ligand complex
was built on the basis of the X-ray structure of the covalently
trapped catalytic complex of HIV-1 RT with TTP and primer/
template duplex (PDB entry 1rtd).25 A model of the NRTI
binding site was built, which consisted of residues between
Lys1 and Pro243 in the p66 subunit, and a 7:4 (template/
primer) duplex. The conformationally optimized structure of
D-3′F-d4CTP was used to define the initial Cartesian coordi-
nates. The heterocyclic moiety of the (n + 1)th nucleotide in
the template overhang was modified to the base complemen-
tary to the incoming NRTIs under study, i.e., the adenine
moiety that was in the original X-ray structure (1rtd)27 was
modified to guanine. The inhibitor triphosphates were manu-
ally docked at the active site of the enzyme by adjusting the
torsional angles to those found in the X-ray structure.27
Ga¨steiger-Hu¨ckel charges were given to the enzyme/ligand
complex with formal charges (+2) to the two Mg atoms in the
active site. Then, Kollman all-atom charges were loaded to the
enzyme site using the biopolymer module in Sybyl. Fluorine
parameters were obtained from literature28 and MM2 param-
eters and were entered into the parameter files. To eliminate
local strains resulting from merging inhibitors and/or point
mutations, residues inside 6 Å from the merged inhibitors and
mutated residues were annealed until the energy change from
one iteration to the next was less than 0.05 kcal/mol. The
annealed enzyme/inhibitor complexes were minimized by using
Kollman all-atom force field until the iteration number reached
5000.
(9) De Clercq, E. In search of a selective antiviral chemotherapy.
Clin. Microbiol. Rev. 1997, 10, 674-693.
(10) Schinazi, R. F.; Mellors, J.; Bazmi, H.; Diamond, S.; Garber, S.;
Gallagher, K.; Geleziunas, R.; Klabe, R.; Pierce, M.; Rayner, M.;
Wu, J.-T.; Zhang, H.; Hammond, J.; Bacheler, L.; Manion, D.
J.; Otto, M. J.; Stuyver, L.; Trainor, G.; Liotta, D. C.; Erickson-
Viitanen, S. DPC 817: a cytidine nucleoside analog with activity
against zidovudine- and lamivudine-resistant viral variants.
Antimicrob. Agents Chemother. 2002, 46, 1394-1401.
(11) Lin, T.-S.; Luo, M.-Z.; Liu, M.-C.; Zhu, Y.-L.; Gullen, E.;
Dutschman, G. E.; Cheng, Y.-C. Design and synthesis of 2′,3′-
dideoxy-2′,3′-didehydro-â-L-cytidine (â-L-d4C) and of 2′,3′-dideoxy-
2′,3′-didehydro-â-L-5-fluorocytidine (â-L-Fd4C), two exceptionally
potent inhibitors of human hepatitis B virus (HBV) and potent
inhibitors of human immunodeficiency virus (HIV) in vitro. J.
Med. Chem. 1996, 39, 1757-1759.
(12) Watanabe, K. A.; Su, T. -L.; Klein, R. S.; Chu, C. K.; Matsuda,
A.; Chun, M. W.; Lopez, C.; Fox, J. J. Nucleosides. 123. Synthesis
of antiviral nucleosides. 5-Substituted 1-(2-deoxy-2-halogeno-â-
D-arabinofuranosyl)cytosines and -uracils. Some structure-
activity relationships. J. Med. Chem. 1983, 26, 152-156.
(13) Chu, C. K.; Ma, T. W.; Shanmuganathan, K.; Wang, C. G.; Xiang,
Y. J.; Pai, S. B.; Yao, G. Q.; Sommadossi, J.-P.; Cheng, Y.-C.
Use of 2′-fluoro-5-methyl-â-L-arabinofuranosyluracil as a novel
antiviral agent for hepatitis B virus and Epstein-Barr virus.
Antimicrob. Agents Chemother. 1995, 39, 979-981.
(14) Ma, T. W.; Pai, S. B.; Zhu, Y. L.; Lin, J. S.; Shanmuganathan,
K.; Du, J. F.; Wang, C.; Kim, H. B.; Newton, M. G.; Cheng, Y.-
C.; Chu, C. K. Structure-activity relationships of 1-(2-deoxy-2-
fluoro-â-L-arabinofuranosyl)pyrimidine nucleosides as anti-
hepatitis B virus agents. J. Med. Chem. 1996, 39, 2835-2843.
(15) Choi, Y. Lee, K.; Hong, J. H.; Schinazi, R. F.; Chu, C. K.
Synthesis and anti-HIV activity of L-2′-fluoro-2′,3′-unsaturated
purine nucleosides. Tetrahedron Lett. 1998, 39, 4437-4440.
(16) Lee, K.; Choi, Y.; Gullen, E.; Schlueter-Wirtz, S.; Schinazi, R.
F.; Cheng, Y.-C.; Chu, C. K. Synthesis and anti-HIV and anti-
HBV activities of 2′-fluoro-2′,3′-unsaturated L-nucleosides. J.
Med. Chem. 1999, 42, 1320-1328.
Acknowledgment. This research was supported by
the U.S. Public Health Service funds (Grants AI 32351