7164 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Fattorusso et al.
W. J.; Byrnes, V. W.; Emini, E. A. L-743,726 (DMP-266): a novel,
highly potent nonnucleoside inhibitor of the human immuno-
deficiency virus type 1 reverse transcriptase. Antimicrob. Agents
Chemother. 1995, 39, 2602-2605.
method, the yield, after removal of the nonadherent cells, was
105 macrophages per well. Test was conducted as described
elsewhere.22 Macrophages obtained with this method are >95%
pure as detected by nonspecific esterase activity. C8166 is a
CD4+ T-cell line containing an HTLV-I genome of which only
the tat gene is expressed.23
2. Virus. A laboratory lymphocyte-tropic strain of HIV-1
(HIV-1-IIIB) was used to infect C8166, while macrophages
were infected with a laboratory monocyte-tropic strain of
HIV-1 (HTLV-III-Ba-L, also called HIV-Ba-L).24,25 Titration
to determine the infectivity was performed in human mac-
rophages as previously described. The titer of the virus stocks,
expressed as 50% tissue culture infectious dose (TCID50), was
determined as previously described.26
3. Antiviral Agent. Zidovudine (AZT) was purchased from
Sigma Chimica. Efavirenz was a gift from Dr. Massimo
Pregnolato, University of Pavia, Italy.
4. Toxicity. Chemicals toxicity in C8166 was evaluated with
a procedure involving a colorimetric assay (MTT assay) that
monitors the ability of viable, but not dead, cells to reduce
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) to a blue formazan product, which can be measured
spectrophotometrically.27,28
(8) (a) Campiani, G.; Morelli, E.; Fabbrini, M.; Nacci, V.; Greco, G.;
Novellino, E.; Ramunno, A.; Maga, G.; Spadari, S.; Caliendo, G.;
Bergamini, A.; Faggioli, E.; Uccella, I.; Bolacchi, F.; Marini, S.;
Coletta, M.; Nacca, A.; Caccia, S. Pyrrolobenzoxazepinone
Derivatives as Non-Nucleoside HIV-1 RT Inhibitors: Further
Structure-Activity Relationship Studies and Identification of
More Potent Broad-Spectrum HIV-1 RT Inhibitors with Antiviral
Activity. J. Med. Chem. 1999, 42, 4462-4470, and references
therein. (b) Watkinson, J. G.; Watson, W.; Yates, B. L. The effects
of ortho substituents on reactivity. I. The alkaline hydrolysis of
substituted ethyl phenylacetates. J. Chem. Soc. 1963, 5437-
5444. (c) Pitre`, D.; Fumagalli, L., Lorenzotti, E. Radiological
contrast media. XXI. Iodo derivatives of aryl[3-(N-alkylacyl-
amino)phenoxy]acetic acids. Il Farmaco-Ed. Sc. 1972, 27, 408-
418. (d) Guanti, G.; Banfi, L.; Riva, R. Regioselective synthesis
of 1,8-dihydroxytetralins through a tandem reduction/intra-
molecular hydroxyalkylation of 4-(3-hydroxyphenyl)alkanoates.
Tetrahedron 1994, 50, 11945-11966. (e) Epstein, J. W.; Bra-
bander, H. J.; Fanshawe, W. J.; Hofmann, C. M.; McKenzie, T.
C.; Safir, S. R.; Osterberg, A. C.; Cosulich, D. B.; Lovell, F. M.
1-Aryl-3-azabicyclo[3.1.0]hexanes, a new series of nonnarcotic
analgesic agents. J. Med. Chem. 1981, 24, 481-490.
(9) (a) Maga, G.; Ramunno, A.; Nacci, V.; Locatelli, G. A.; Spadari,
S.; Fiorini, I.; Baldanti, F.; Paolucci, S.; Zavattoni, M.; Bergamini,
A.; Galletti, B.; Muck, S.; Hubsher, U.; Giorgi, G.; Guiso, G.;
Caccia, S.; Campiani, G. The Stereoselective Targeting of a
Specific Enzyme-Substrate Complex Is the Molecular Mecha-
nism for the Synergic Inhibition of HIV-1 Reverse Transcriptase
by (R)-(-)-PPO464. J. Biol. Chem. 2001, 48, 44653-44662. (b)
Locatelli, G. A.; Campiani, G.; Cancio, R.; Morelli, E.; Ramunno,
A.; Gemma, S.; Hubscher, U.; Spadari, S.; Maga, G. Effects of
drug resistance mutations L100I and V106A on the binding of
pyrrolobenzoxazepinone nonnucleoside inhibitors to the human
immunodeficiency virus type 1 reverse transcriptase catalytic
complex. Antimicrob Agents Chemother. 2004, 48, 1570-
1580.
(10) Jacobo-Molina, A.; Ding, J.; Nanni, R. G.; Clark, A. D., Jr.; Lu,
X.; Tantillo, C.; Williams, R. L.; Kamer, G.; Ferris, A. L.; Clark,
P.; Hizi, A.; Hughes, S. H.; Arnold, E. Crystal structure of human
immunodeficiency virus type 1 reverse transcriptase complexed
with double-stranded DNA at 3.0 A resolution shows bent DNA.
Proc Natl Acad Sci U.S.A. 1993, 90, 6320-6324.
5. Assay of Antiviral Activity. Antiviral activity of the
tested compounds in acutely infected C8166 cultures was
performed following an already described procedure.29 The
assay to evaluate anti-HIV drug efficacy in acutely infected
mature macrophages has been previously described.33 The
antiviral activity of the compounds was assessed by measuring
HIV-p24 antigen production in the supernatants of infected
cultures as previously described24 by using a commercially
available HIV-antigen kit.
6. Immunofluorescence Virus Binding Assay. Calcula-
tion of the 50% effective dose (ED50) and 50% inhibitory dose
(ID50) was performed. The ED50 and ID50 values were calcu-
lated from pooled values in the effective dynamic range of the
antiviral and toxicity assays (5-95%) using the median effect
equation as previously described.30
Synergy Calculations. The multiple drug effect analysis
of Chou and Talalay31 was used to calculate combined drug
effects.
(11) (a) Wohrl, B. M.; Krebs, R.; Thrall, S. H.; Le Grice, S. F.;
Scheidig, A. J.; Goody, R. S. Kinetic analysis of four HIV-1
reverse transcriptase enzymes mutated in the primer grip region
of p 66. Implications for DNA synthesis and dimerization. J. Biol.
Chem. 1997, 272, 17581-44662. (b) Ghosh, M.; Williams, J.;
Powell, M. D.; Levin, J. G.; Le Grice, S. F. Mutating a conserved
motif of the HIV-1 reverse transcriptase palm subdomain alters
primer utilization. Biochemistry 1997, 36, 5758-5768. (c) Pele-
mans, H.; Esnouf, R.; De Clerq, E.; Balzarini, J. Mutational
Analysis of Trp-229 of Human Immunodeficiency Virus Type 1
Reverse Transcriptase (RT) Identifies This Amino Acid Residue
as a Prime Target for the Rational Design of New Non-
Nucleoside RT Inhibitors Mol. Pharmacol. 2000, 57, 954-960.
(d) Pelemans, H.; Esnouf, R.; Min, K.; Parniak, M.; De Clerq,
E.; Balzarini, J. Mutation at Amino Acid Positions 63, 189, and
396 of Human Immunodeficiency Virus Type 1 Reverse Tran-
scriptase (RT) Partially Restore the DNA Plymerase Activity of
a TRP229TYR Mutant RT. Virology 2001, 287, 143-150.
(12) Ding, J.; Das, K.; Hsiou, Y.; Sarafianos, S. G.; Clark, A. D., Jr.;
Jacobo-Molina, A.; Tantillo, C.; Hughes, S. H.; Arnold, E.
Structure and functional implications of the polymerase active
site region in a complex of HIV-1 RT with a double-stranded
DNA template-primer and an antibody Fab fragment at 2.8 A
resolution. J. Mol. Biol. 1998, 284, 1095-111.
(13) Huang, H.; Chopra, R.; Verdine, G. L.; Harrison S. C. Structure
of a Covalently Trapped Catalytic Complex of HIV-1 Reverse
Transcriptase: Implications for Drug Resistance. Science 2004,
306, 1901-1902.
(14) Campiani, G.; Ramunno, A.; Fiorini, I.; Nacci, V.; Morelli, E.;
Novellino, E.; Goegan, M.; Mennini, T.; Sullivan, S.; Zisterer,
D. M.; Williams, C. D. Synthesis of New Molecular Probes for
Investigation of Steroid Biosynthesis Induced by Selective
Interaction with Peripheral Type Benzodiazepine Receptors
(PBR). J. Med. Chem. 2002, 45, 4276-4281.
Acknowledgment. This work has been partially
supported by the ISS Programma Nazionale di Ricerca
sull’ AIDS (Contract 30C.70) and by the CNR Target
Project on Biotechnology to S.S.
Supporting Information Available: Experimental pro-
cedures for compounds 6b,c, 7a-e, 8b-d, 10d-e, 11b-e,
12b-e, 13e-g,i, 14b, 15b,c,e-i, 21b, molecular modeling and
toxicity tests, Figures S1 and S2, and a table with elemental
analyses of compounds described. This material is available
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