Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 879
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Cyclin-dependent kinase inhibitors: useful targets in cell cycle
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Formation of Novel Functionalized 1-Aza-9-oxafluorenes. Het-
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Comparison of in vitro anticancer drug-screening data generated
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Sausville, E. A.; Meijer, L.; Kunick, C. Paullones, a Series of
Cyclin-Dependent Kinase Inhibitors: Synthesis, Evaluation of
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Med. Chem. 1999, 42, 2909-2919.
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bazoles as antitumor active compounds: evaluation of the target
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point of the picrate was 125-126 °C (from ethanol) (ref 7, mp
126-129 °C).
3-Acetyl-6-h yd r oxy-4-p h en ylben zo[4.5]fu r o[2.3-b]p yr i-
d in e (5b). Yield 0.40 (72%); mp 240-244 °C.
Eth yl 6-Hydr oxy-4-m eth ylben zo[4.5]fu r o[2.3-b]pyr idin e-
3-ca r boxyla te (5c). Yield 0.34 g (69%); mp 249-253 °C.
3-Acetyl-6-h yd r oxy-4-m eth ylben zo[4.5]fu r o[2.3-b]p yr i-
d in e (5d ). Yield 0.31 g (71%); mp 289-294 °C.
3-Acetyl-4-p h en ylp yr id in e (7b). Yield 0.06 g (18%); mp
(picrate) 142-144 °C (ref 7, mp 145-148 °C).
Eth yl 4-Meth ylp yr id in e (7c). Yield 0.05 g (19%); mp
(picrate) 126-131 °C (ref 7, mp 128-134 °C).
3-Acetyl-4-m eth ylp yr id in e (7b). Yield 0.05 g (20%); mp
(picrate) 136-138 °C (ref 7, mp 137-140 °C).
Eth yl 6-Acetoxy-4-ph en ylben zo[4.5]fu r o[2,3-b]pyr idin e-
3-ca r boxyla te (5e). An amount of 0.04 g (0.1 mmol) of
compound 5a was stirred overnight in 50 mL of dried acetic
anhydride containing 5 drops of dried pyridine base. Then the
mixture was evaporated to dryness in vaccuum, leaving a
colored solid that was stirred in ether, filtered off, and
recrystallized leading to 0.03 g (80%) of acetylation product
5e: mp 313-319 °C; 1H NMR (CDCl3) δ 9.04 (s, 1 Η), 7.60 (d,
J ) 9.0 Hz, 1 H), 7.55-7.36 (m, 5 H), 7.17 (dd, J ) 9.0/2.4 Hz,
1 H), 6.60 (d, J ) 2.5 Hz, 1 H), 4.13 (q, J ) 7.0 Hz, 2 H), 2.23
(s, 3 H), 1.03 (t, J ) 7.0 Hz, 3 H); MS (EI) m/z 375 (M+). Anal.
(C22H17NO5) C, H, N.
En zym e In h ibition Assa ys.11,17 Kinase activities were
assayed at 30 °C at a final ATP concentration of 15 µM. Blank
values were subtracted and activities were calculated as
picomoles of phosphate incorporated for a 10 min incubation.
CDK1/cyclin B was extracted from M phase starfish (Mar-
thasterias glacialis) oocytes and purified as described. Kinase
activity was assayed in a homogenization buffer containing
60 mM â-glycerophosphate, 15 mM p-nitrophenyl phosphate,
25 mM Mops (pH 7.2), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT,
1 mM sodium vanadate, and 1 mM phenyl phosphate with 1
mg histone H1/mL in the presence of 15 µM [γ-32P]ATP (3000
Ci/mmol; 1 mCi/mL) in
a final volume of 30 µL. After
incubation, 25 µL aliquots of the supernatant where spotted
on phosphocellulose filter paper, which was washed five times
with 10 mL of phosphoric acid per liter of water. Then
scintillation counting was carried out. CDK5/p25 was recon-
stituted by mixing equal amounts of recombinant mammalian
CDK5 and p25 as glutathione-S-transferase (GST) fusion
proteins and purified as described. Its activity was assayed in
the same homogenization buffer as used for CDK1/cyclin B
inhibition.
F lu or escen ce Up ta k e Assa y. Cells from the L5178Y
mouse T-cell lymphoma parental cell line and from its mul-
tidrug-resistant subline L5178YvMDR were adjusted to a
concentration of 2 × 106/mL in serum-free McCoys 5A medium
from which 0.5 mL was incubated at room temperature with
the test compounds. Then rhodamine 123 with 5.2 µM as the
final concentration was added and incubation continued for
20 min at 37 °C. After it was washed twice with PBS, the
fluorescence of 1 × 104 cells was measured by flow cytometry.
(18) Hilgeroth, A.; Molna´r, J .; De Clercq, E. Mit molekularer Sym-
metrie zu neuen Wirkstoffen: Hydroxymethyl-substituierte 3,9-
Diazatetraasterane als erste eigensta¨ndige Klasse symme-
trischer MDR-Modulatoren. Angew. Chem. 2002, 114, 3772-
3775. Hilgeroth, A.; Molna´r, J .; De Clercq, E. Using Molecular
Symmetry to Form New Drugs: Hydroxymethyl-Substituted 3,9-
Diazatetraasteranes as the First Class of Symmetric MDR
Modulators. Angew. Chem., Int. Ed. 2002, 41, 3623-3625.
Ack n ow led gm en t. The work was financially sup-
ported within the framework of the COST B16 Action
of the EU.
Refer en ces
(1) Fischer, P. M.; Lane, D. P. Inhibitors of cyclin-dependent kinases
as anti-cancer therapeutics. Curr. Med. Chem. 2000, 7, 1213-
1245.
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