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H. Jin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3989–3992
dictate that conformer A may be more favorable and the
average DE is 1.7 kcal/mole for conformer A.
The synthetic schemes described above have allowed us
to examine a variety of structures based on a pre-orga-
nized pyrolloquinoline tri-cyclic core and evaluate their
inhibitory activities against HIV integration in both
the strand transfer and cytopathic anti-HIV assays.
We have discovered novel, highly organized and potent
HIV integrase inhibitors from this class of compounds.
Furthermore, our lead compound 22 is more soluble
and more potent than the clinically efficacious com-
pound 1 when tested under the physiologically relevant
conditions. The identification of a compound demon-
strating in vivo pharmacological properties for further
development as a clinically useful HIV inhibitor is the
subject of our ongoing research program and results will
be published in due course.
5. See Refs. 9a for IC50 and 9b (MT-4 cell) for EC50.
Compound 2 was prepared from commercially available
8-hydroxyl-quinoline-7-carboxylic acid and 4-fluoroben-
zylamine by standard amide-forming conditions similar to
reaction b in Scheme 4. Compound 3 was obtained from
triflate 15 in Scheme 3 according to condition b.
6. Chen, X, Tsiang, M. and coworkers, manuscript in
preparation.
7. Chen, J. M.; Chen, X.; Fardis, M.; Jin, H.; Kim, C. U. and
Schacherer, L. N. PCT. Int. Patent WO2004035577;
Chem. Abstr. 2004, 140, 375156.
8. (a) For two other series of tricyclic HIV integrase
inhibitors: (a) Fardis, M. et al., Bioorg. Med. Chem.
Lett. 2006, accepted for publication; (b) Metobo, S.
et al., Bioorg. Med. Chem. Lett. 2006, accepted for
publication.
9. (a) Strand transfer assay modified from a previous
report (Hazuda, et al., Nucleic Acid Res. 1994, 22, 1121)
Biotinylated donor DNA was bound to Reacti-Bind
High Binding Capacity Streptavidin-coated white plates.
DIG-tagged target DNA with anti-DIG antibody-con-
jugated horseradish peroxidase detection was used; (b)
For antiviral assay, 50 ll of 2X test concentration of 5-
fold serially diluted drug in culture medium was added
to each well of a 96-well plate (nine concentrations) in
triplicate. MT-2 or MT-4 cells were infected with HIV-1
IIIB at an moi of 0.01 for 3 h. Fifty microliters of
infected cell suspension in culture medium (ꢀ1.5 · 104
cells) was then added to each well containing the drug
dilutions. The plates are incubated at 37 ꢁC for 5 days.
One hundred microliters of CellTiter-GloTM Reagent
(catalog # G7571, Promega Biosciences, Inc., Madison,
WI) was then added to each well. Cell lysis was allowed
to complete by incubating at room temperature for
10 min. Chemiluminescence was then read. For the
cytotoxicity assay, the protocol is identical to that of
the antiviral assay, except that uninfected cells and a 3-
fold serial dilution of drugs were used; (c) The effect of
compounds binding to serum protein components was
evaluated by determining the antiviral EC50 in MT-2
cells in 10% FBS in the presence or absence of serum
concentrations of HSA (35 mg/ml) or a1-AGP (1.5 mg/
ml). From the EC50 data in the presence of each
individual protein, the EC50 resulting from the combined
effect of both proteins (as in serum) can be calculated.
The derivation of the appropriate equation for this
calculation can be made through competitive binding
assumptions .
Acknowledgments
We thank Michael Mish and Will Watkins for critical
reading of the manuscript and useful suggestions, Vahid
Zia and the formulation group for measuring solubility,
Gregg Jones, Magdeleine Hung, Wei Huang, Ameneh
Zeynalzadegan Xiaoping Qi, and Fang Yu for deter-
mining biological activities, Swami Swaminathan for
discussion and suggestions in modeling work, and Mat-
thew Wright for general comments.
References and notes
1. Mugavero, M. J.; Hicks, C. B. Drug Discovery Today:
Therapeutic Strategies 2004, 1, 529.
2. Esposito, D.; Craigie, R. Adv. Virus Res. 1999, 52, 319.
3. (a) Little, S.; Drusano, D.; Schooley, R. Haas, D.; Kumar,
P.; Hammer, S.; McMahon, D.; Squires, K.; Asfour, R.;
Richman, D.; Chen, J.; Saah, A.; Leavitt, R.; Hazuda, D.
J.; Nguyen, B. Y. Protocol 004 Study Team, Abstracts of
Papers, # 161, 12th Conference on Retroviruses and
Opportunistic Infections, Boston, MA, February 22–25,
2005; Foundation of Retrovirology and Human Health,
Alexandria, VA, 2005; (b) Hazuda, D. J.; Anthony, N. J.;
Gomez, R. P.; Jolly, S. M.; Wai, J. S.; Zhuang, L.; Fisher,
T. E.; Embry, M.; Guare, J. P., Jr.; Egbertson, M. S.;
Vacca, J. P.; Huff, J. R.; Felock, P. J.; Witmer, M. V.;
Stillmock, K. A.; Danovich, R.; Grobler, J.; Miller, M. D.;
Espeseth, A. S.; Jin, L.; Chen, I.-W.; Lin, J. H.; Kassahun,
K.; Ellis, J. D.; Wong, B. K.; Xu, W.; Pearson, P. G.;
Schleif, W. A.; Cortese, R.; Emini, E.; Summa, V.;
Holoway, M. K.; Young, S. D. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 11233.
4. Since molecular mechanics force-field methods do differ
(based on the parameterization of each specific method)
and may produce different results for specific molecular
systems, we decided to utilize several methods (Ref.: 1,
Sybyl v.6.9, Tripos, Molecular Modeling Software, St.
Louis MI; and 2, MacroModel, Schrodinger Inc., Molec-
ular Modeling Software) in order to evaluate consistency
in the results. The lowest energy structures for conformer
A and B was computed using six different molecular
mechanics methods. The energy difference (DE) between A
and B was computed from each method. All six methods
10. (a) Verschueren, W. G.; Dierynck, I.; Surleraux, D. L. N.
G.; Daeyaert, F. F. D. PCT. Int. Patent WO2004096807;
Chem. Abstr. 2004, 141, 410963; (b) Verschueren, W. G.;
Dierynck, I.; Amssoms, K. I. E.; Hu, L.; Boonants, P. M.
J. G.; Pille, G. M. E.; Daeyaert, F. F. D.; Hertogs, K.;
Surleraux, D. L. N. G.; Wigerinck, P. B. T. P. J. Med.
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1
11. The characterization of compound 22: H NMR (CDCl3)
9.08 (dd, 1H), 8.15 (s, 1H), 8.06 (dd, 1H), 7.76 (dd, 4H),
7.55 (dd, 1H), 7.30 (m, 8H), 7.07 (dd, 2H), 4.95 (d,
J = 15 Hz, 1H), 4.70 (d, J = 15 Hz, 1H), 4.42 (d,
J = 15 Hz, 1H), 4.14 (d, J = 15 Hz, 1H), 3.94–3.79 (m,
4H), 3.41 (m, 2H), 2.99 (m, 2H.) MS: 588 (M+1).
12. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
J. Adv. Drug Deliv. Rev. 1997, 23, 3.