Bioconjugate Chemistry
ARTICLE
the use of radioactive materials. 5-(SAENTA)-X8-fluorescein
(SAENTA-fluor, SF, 1) is an example of an ENT1 fluorescent
probe.20 As an analogue of nitrobenzylmercaptopurine riboside
(NBMPR, 2), it has been used in several studies for nonradioactive
assays of compounds’ ENT1 inhibitory activities.22ꢀ26
SIL-10AD VP auto injector, a sample cooler kept at 4 °C, and an
SPD-M10A VP diode array detector. Chromatograms were
acquired and analyzed with the Schimadzu Class-vp 7.3 SP1
software running on a Dell computer.
2-Diethanolamino-4,8-diheptamethyleneimino-6-chloro-
pyrimido[5,4-d]pyrimidine (6). To a solution of 2,4,6,8-tetra-
chloropyrimido[5,4-d]pyrimidine (TCPP, 5) (0.54 g, 2 mmol)
in anhydrous THF (20 mL), heptamethyleneimine (1.06 mL,
8.4 mmol) was added. The reaction was stirred in an iceꢀwater
bath for 20 min, and then water (150 mL) was added to precipitate
the reaction intermediate. After drying over a P2O5 desiccator, the
intermediate was dissolved in dimethylsulfoxide (DMSO) (10 mL)
with diethanolamine (3 mL, 30 mmol). The reaction was stirred at
120 °C for 3 days. The product, compound 6 (445 mg, 45% overall
yield in 2 steps), was purified by flash silica gel chromatography
(hexane/acetone = 5/1). Mp: 122ꢀ124 °C; MS (ESI) m/z 492
(M þ H)þ, 514 (M þ Na)þ. 1H NMR (DMSO-d6) δ 4.737 (t,
2H, exchanged with D2O, 2 ꢁ OH, J = 5 Hz), 3.868 (m, 8H, 2 ꢁ
N(CH2CH2CH2)2CH2), 3.589 (t, 8H, N(CH2CH2OH)2, J =
5 Hz), 1.796 (s, 8H, 2 ꢁ N(CH2CH2CH2)2CH2), 1.534 (s, 8H,
2 ꢁ N(CH2CH2CH2)2CH2), 1.495 (s, 4H, 2 ꢁ N(CH2CH2-
CH2)2CH2). Anal. (C24H38ClN7O2) C, H, N.
2-Diethanolamino-4,8-diheptamethyleneimino-6-[(N-dibenzy-
laminoethyl)-ethanolamino]-pyrimido[5,4-d]pyrimidine (7).
Compound 6 (370 mg, 0.75 mmol) was heated with 2-(2-
dibenzylamino-ethylamino)-ethanol (3 g, 10 mmol) at 150 °C
overnight. The product, compound 7 (130 mg, 23%), was
purified by flash silica gel chromatography (hexane/acetone =
2/1) as a yellow sticky solid. MS (ESI) m/z 762 (M þ Na)þ. 1H
NMR (DMSO-d6) δ 7.306 - 7.251 (m, 10H, Ar), 7.181 (m, 4H,
2 ꢁ CH2), 4.698 (s, 2H, disappeared after addition of D2O, 2 ꢁ
OH), 4.606 (s, 1H, disappeared after addition of D2O, OH),
4.007ꢀ3.677 (br s, 8H, 2 ꢁ N(CH2CH2CH2)2CH2), 3.575
(s, 12H, N(CH2CH2OH)2, NCH2CH2OH), 3.520 (d, 4H,
NCH2CH2ON, J = 6.5 Hz), 1.726 (br s, 8H, 2 ꢁ N(CH2CH2
CH2)2CH2), 1.496 (br d, 12H, 2 ꢁ N(CH2CH2CH2)2CH2). LC
retention time 5.804 min.
In our attempts to develop new ENT fluorescent probes, we
have concentrated on the best compound we identified from a
structureꢀactivity relationship study of a series of dipyridamole
(DP, 3) analogues that we synthesized recently as ENT1 and
ENT2 inhibitors.23,27 This compound, the 8-membered ring
dipyridamole analogue, compound 4 (8MDP) had Ki values of
0.97 nM and 90.8 nM against hENT1 and hENT2, respectively,27
indicating much higher binding affinities than DP, which had Ki
values of 14.5 nM and 308 nM against hENT1 and hENT2,
respectively.27 In the current study, we attached a fluorescent
reporter group to a modified 8MDP to obtain a new fluorescent
probe, which was successfully used in flow cytometric analysis of
ENT1 protein expression as well as the measurement of relative
affinities of ENT1 ligands.
2-Diethanolamino-4,8-diheptamethyleneimino-6-[(N-6-(fluore-
scein-6-carboxamido)-hexanoylaminoethyl)- ethanolamino]-
pyrimido[5,4-d]pyrimidine (8). The benzyl groups on com-
pound 7 (100 mg, 0.13 mmol) were removed by hydrogenation
in the presence of a catalytic amount of 10% PdꢀC in methanol.
After filtration and evaporation, the residue was reacted with
commercially available 6-(fluorescein-5-carboxamido)hexanoic
acid succinimidyl ester (10 mg, 17 μmol) in dimethylformamide
(DMF) (3 mL) for 2 h. The solvent was removed under reduced
pressure, and the residue was purified by sephadex LH-20 gel
filtration with MeOH as the elution solvent to obtain the product
8 (7.2 mg, 41%) as a sticky gum. MS (ESI) m/z 1029 (M ꢀ H)ꢀ.
LC retention time 11.368 min.
’ EXPERIMENTAL PROCEDURES
Chemistry. Thin-layer chromatography (TLC) was con-
ducted on silica gel plates (Analtech). Compounds were visua-
lized by UV light (254 and 365 nm). 1D NMR spectra were
recorded on a Varian Inova 500 MHz NMR instrument, using
CDCl3 or (CD3)2SO as solvents and tetramethylsilane (TMS) as
an internal standard. Flash column chromatography was per-
formed on Fisher silica gel (170ꢀ400 mesh). Melting points
were determined using a Fisher-Johns melting point apparatus
and were reported uncorrected. Mass spectra were obtained on a
Bruker-HP ESQUIRE Ion Trap LC/MS system. Elemental
analyses were performed by Atlantic Microlab Inc., Norcross,
GA. All solvents and reagents were purchased from Aldrich or
other major chemical companies and used without further
purification. All reactions were carried out under argon gas.
Analytic high performance liquid chromatography (HPLC) was
performed on a 150 mm ꢁ 2.00 mm Phenomenex Luna C18
column (3-μm particle size), protected by a Phenomenex
SecurityGuard guard column (cartridge 4 mm ꢁ 2.00 mm
internal diameter); and gradient CH3CN/H2O; 0 to 100 (15
min) with detection at 224 nm. The HPLC system consisted of
an automated system with two Shimadzu LC-10AD VP liquid
chromatography pumps, an SCL-10A VP system controller, a
Biological Testing. [3H]Uridine Uptake Experiments with
K562 Cells Used to Determine ENT1 Inhibitory Activities of Test
Compounds. Human chronic myelogenous leukemia K562 cells
(American Type Culture Collection (ATCC), Manassas, VA)
were maintained in RPMI 1640 medium containing 10% fetal
bovine serum and amikacin (60 mg/Liter), at 37 °C in 5%
CO2 and 95% air atmosphere. All experiments were done in
sodium free uptake buffer (20 mM Tris/HCl, 3 mM K2HPO4,
1 mM MgCl2 6H2O, 2 mM CaCl2, 5 mM glucose, and 130 mM
3
N-methyl-D-glucamine/HCl, pH 7.4). 5 ꢁ105 K562 cells/sample
were harvested and washed once with sodium free uptake buffer
and resuspended in 50 μL of the same buffer. Uptake buffer
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dx.doi.org/10.1021/bc2000758 |Bioconjugate Chem. 2011, 22, 1221–1227