December 2007
1743
48 h. The mixture was concentrated under reduced pressure to furnished
product. Pure products were obtained under recrystalliztion with methanol.
transfer reactions were performed in triplicate in 96-well plates using
0.25 pmol of labeled U5A/U5B-2 double-stranded DNA substrate, 12 pmol
of ST1/ST2 3ꢀ-biotinylated target DNA and 2 pmol of integrase in buffer A
3,3ꢀ-(4-(4-Methoxybenzoyl)oxy)-benzylidene)bis-4-hydroxycoumarin (3):
1
Yield: 60%, a white powder, mp 192—193 °C. H-NMR (CDCl3) d: 8.05— in a final volume of 40 ml. Radiolabeled reaction products were bound to
7.08 (m, 16H, ArH), 6.37 (s, 1H, CH), 3.85 (s, 3H, CH3). IR (KBr) cmꢄ1
3622, 1736, 1663. MS m/z: 563 (MHꢂ). Anal. Cacld for C33H22O9: C, 70.46, buffer supplemented with 0.025% tween 20 and 10 mg/ml BSA) and quanti-
H, 3.94. Found: C, 70.16, H, 3.93. fied on a beta radiation counter. Inhibition in the presence of drugs is ex-
:
Streptavidincoated magnetic beads (DynaL), washed twice in buffer B (PBS
4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-N-p-tolylbenzamide pressed as the fractional product in percent of the control without drug.
(4): Yield: 52%, a white powder, mp ꢅ270 °C. 1H-NMR (DMSO-d6) d:
10.05 (s, 1H, NH), 7.81—7.09 (m, 16H, ArH), 6.31 (s, 1H, CH), 3.93 (s,
Acknowledgments This study was supported by research grants from
National Science Council (NSC 94-2320-B-016-027). We thank Ms. Shu-
3H, CH3). IR (KBr) cmꢄ1: 3400, 3332, 1653. MS m/z: 562 (MHꢂ). Anal.
Cacld for C33H23NO8: C, 70.58, H, 4.13, N, 2.49. Found: C, 70.70, H, 4.38, Yun Sun and Ching-Wei Lu, Instrumentation Center, NSC for their help in
N, 2.51. obtaining the mass spectra and elemental analysis.
3,3ꢀ-(4-(2-(4-Acetoxy)-ethoxy)-benzylidene)bis-4-hydroxycoumarin (7):
Yield: 65%, a white powder, mp 228—229 °C. 1H-NMR (DMSO-d6) d: References
7.84—6.79 (m, 12H, ArH), 6.24 (s, 1H, CH), 4.53 (s, 2H, CH2OCO), 4.25
(s, 2H, ArOCH2), 2.25 (s, 3H, CH3). IR (KBr) cmꢄ1: 3437, 1752, 1712. MS
m/z: 515 (MHꢂ). Anal. Cacld for C29H22O9: C, 67.70, H, 4.31. Found: C,
67.34, H, 3.99.
3,3ꢀ-(4-(2-(4-Methylbenzoyloxy)-ethoxy)-benzylidene)bis-4-hydroxy-
coumarin (8): Yield: 78%, a white powder, mp 225—226 °C. 1H-NMR
(DMSO-d6) d: 7.83—6.78 (m, 16H, ArH), 6.23 (s, 1H, CH), 4.52 (t,
Jꢁ5.1 Hz, 2H, CH2CH2), 4.24 (t, Jꢁ5.1 Hz, 2H, CH2CH2), 2.34 (s, 3H,
CH3). IR (KBr) cmꢄ1: 3412, 1712, 1653. MS m/z: 591 (MHꢂ). Anal. Cacld
for C35H26O9: C, 71.18, H, 4.44. Found: C, 71.34, H, 4.12.
1) Vandamme A. M., Van Vaerenbergh K., De Clercq E., Antiviral Chem.
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2) Pani A., Loi A. G., Mura M., Marceddu T., La Colla P., Marongiu M.
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(1996).
3,3ꢀ-(4-(Bis[2-(acetoxy)ethyl]amino)-benzylidene)bis-4-hydroxycoumarin
(12): Yield: 47%, a yellow powder, mp 211—212 °C. H-NMR (DMSO-d6)
7) Farnet C. M., Wang B., Lipford J. R., Bushman F. D., Proc. Natl. Acad.
Sci. U.S.A., 93, 9742—9747 (1996).
1
d: 7.81—6.55 (m, 12H, ArH), 6.13 (s, 1H, CH), 4.08 (t, Jꢁ5.2 Hz, 4H,
8) Fesen M. R., Pommier Y., Leteurtre F., Hiroguchi S., Yung J., Kohn K.
W., Biochem. Pharmacol., 48, 595—608 (1994).
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Chemother., 39, 320—324 (1995).
CH2O), 3.50 (t, Jꢁ5.2 Hz, 4H, NCH2), 1.96 (s, 6H, CH3). IR (KBr) cmꢄ1
:
3433, 1736, 1232. MS m/z: 600 (MHꢂ). Anal. Cacld for C33H29NO10: C,
66.10, H, 4.88 , N, 2.34. Found: C, 66.49, H, 4.73, N, 2.27.
3,3ꢀ-(4-(Bis-(2-(4-methylbenzoyloxy)-ethyl)-amino)-benzylidene)-bis-4-
hydroxycoumarin (13): Yield: 67%, a dark red powder, mp 175—176 °C. 10) Fesen M. R., Kohn K. W., Leteurtre F., Pommier Y., Proc. Natl. Acad.
1H-NMR (DMSO-d6) d: 7.84—6.71 (m, 20H, ArH), 6.19 (s, 1H, CH), 4.36
Sci. U.S.A., 90, 2399—2403 (1993).
(t, Jꢁ5.6 Hz, 4H, CH2O), 3.71 (t, Jꢁ5.6 Hz, 4H, NCH2), 2.33 (s, 6H, CH3). 11) Dupont R., Jeanson L., Mouscadet J. F., Cotelle P., Bioorg. Med.
IR (KBr) cmꢄ1: 3633, 1707, 1271. MS m/z: 752 (MHꢂ). Anal. Cacld for
C45H37NO10: C, 71.89, H, 4.96, N, 1.86. Found: C, 71.60, H, 4.62, N, 1.89.
3,3ꢀ-(4-(Bis-(2-(4-bromobenzoyloxy)-ethyl)-amino)-benzylidene)-bis-4-
Chem. Lett., 11, 3175—3178 (2001).
12) Hazuda D. J., Felock P., Witmer M., Wolfe A., Stillmock K., Grobler J.
A., Espeseth A., Gabryelski L., Schleif W., Blau C., Miller M. D., Sci-
ence, 287, 646—650 (2000).
1
hydroxycoumarin (14): Yield: 78%, a pink powder, mp 222—223 °C. H-
NMR (DMSO-d6) d: 7.85—6.72 (m, 20H, ArH), 6.19 (s, 1H, CH), 4.37 (t, 13) Cooper D., Gatell J., Rockstroh J., Katlama C., Yeni P., Lazzarin A.,
Jꢁ5.5 Hz, 4H, CH2O), 3.72 (t, Jꢁ5.2 Hz, 4H, NCH2). IR (KBr) cmꢄ1: 3477,
1713, 1271. MS m/z: 882 (MHꢂ). Anal. Cacld for C43H31NO10Br2: C, 58.59,
H, 3.54, N, 1.59. Found: C, 58.51, H, 3.32, N, 1.56.
Chen J., Isaacs R., Teppler H., Nguyen B., Results of BENCHMRK-1,
a Phase III Study Evaluating the Efficacy and Safety of MK-0518, a
Novel HIV-1 Integrase Inhibitor, in Patients with Triple-class Resistant
Abstracts/30687.htm
3,3ꢀ-(3,5-Di-((4-methylbenzoyl)oxy)-benzylidene)bis-4-hydroxycoumarin
1
(16): Yield: 46%, a white powder, mp ꢅ270 °C. H-NMR (DMSO-d6) d:
7.95—6.93 (m, 19H, ArH), 6.38 (s, 1H, CH), 2.35 (s, 6H, CH3). IR (KBr) 14) Steigbigel R., Kumar P., Eron J., Schechter M., Markowitz M.,
cmꢄ1: 3477, 1730, 1658. MS m/z: 681 (MHꢂ). Anal. Cacld for C41H28O10: C,
72.35 , H, 4.15. Found: C, 72.57, H, 4.42.
Loufty M., Zhao J., Isaacs R., Nguyen B., Teppler H., Results of
BENCHMRK-2, a Phase III Study Evaluating the Efficacy and Safety
of MK-0518, a Novel HIV-1 Integrase Inhibitor, in Patients with
conference.org/2007/Abstracts/30688.htm
4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)phenyl-4ꢀ-(bis(4-hy-
droxy-2-oxo-2H-chromen-3-yl)methyl)benzoate (18): Yield: 75%, a white
1
powder, mp ꢅ270 °C. H-NMR (DMSO-d6) d: 7.93—7.02 (m, 24H, ArH),
6.34, 6.28 (s, 1H each, CH). IR (KBr) cmꢄ1: 3445, 1674, 1271. MS m/z: 867 15) Mazumder A., Wang S., Neamati N., Nicklaus M., Sunder S., Chen J.,
(MHꢂ). Anal. Cacld for C51H30O14: C, 70.67, H, 3.49. Found: C, 70.37, H,
Milne G. W., Rice W. G., Jr., Burke T. R., Pommier Y., J. Med. Chem.,
39, 2472—2481 (1996).
3.71.
HIV-1 Integrase Inhibitory Assay. Oligonucleotides Oligonucleotides 16) Zhao H., Neamati N., Hong H., Mazumder A., Wang S., Sunder S.,
were purchased from Eurogentec and further purified on 18% acrylamide/
urea denaturing gel. U5B: GTGTGGAAAATCTCTAGCA; U5B-2: GTG-
Milne G. W., Pommier Y., Jr., Burke T. R., J. Med. Chem., 40, 242—
249 (1997).
TGGAAAATCTCTAG; U5A: 5ꢀ-ACTGCTAGAGATTTTCCACAC; ST1: 17) Mao P. C. M., Mouscadet J. F., Leh H., Auclair C., Hsu L. Y., Hetero-
AGTGAATTAGCCCTTGGTCA-biotine; ST2: 5ꢀ-TGACCAAGGGCTAAT- cycles, 55, 1263—1270 (2001).
TCACT-biotine; U5B and U5B-2 were radiolabeled using T4 polynucleotide 18) Mao P. C.-M., Mouscadet J.-F., Leh H., Auclair C., Hsu L. Y., Chem.
kinase for respectively 3ꢀ-processing and strand transfer reactions. Pharm. Bull., 50, 1634—1637 (2002).
HIV-1 Integrase Assays Wild-type HIV-1 integrase was purified as de- 19) Long Y.-Q., Jiang X.-H., Dayam R., Sanchez T., Shoemaker R., Sei S.,
scribsed previously.15,16) 3ꢀ-Processing assay was performed in a reaction
volume of 20 ml containing 0.025 pmol of labeled U5A/U5B double-
stranded DNA substrate and 1 pmol of integrase in buffer A [20 mM Hepes
(pH 7.2), 10 mM MgCl2, 25 mM NaCl, 1 mM DTT]. Products were separated
on a 18% acrylamide/urea denaturing gel and quantified on a phosphoim-
ager using Image Quant software (Amersham Pharmacia Biotech). Strand
Neamati N., J. Med. Chem., 47, 2561—2573 (2004).
20) Taddeo B., Haseltine W. A., Farnet C. M., J. Virol., 68, 8401—8405
(1994).
21) Mekouar K., Mouscadet J. F., Desmaele D., Subra F., Leh H., Savoure
D., Auclair C., d’Angelo J., J. Med. Chem., 41, 2846—2857 (1998).