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Vol. 61, No. 4
inhibited PDE3 with an IC50 of 4µm when cGMP was used as stacking with Phe991.
the substrate, but did not cause >50% inhibition at the 50µm
In conclusion, selective inhibition of cGMP but not of the
screening concentration in the case of cAMP. In addition, the cAMP substrate hydrolysis of PDE3 by replacement of the
bulkier compound 3 inhibited PDE3B when either cAMP or oxygen atom of cyano-2-pyridones by an amino may set the
cGMP were used as substrates, but similar to compound 1 was base for substrate-selective pharmacological modulation of
selective inhibition of cGMP hydrolysis compared with cAMP this important class of drug targets.
hydrolysis with IC50 values of 13.4 and 35.6µm, respectively.
Only compounds 2 and 3 showed modest tumor cell growth
inhibitory potency with IC50 values of 43.0 and 32µm, re-
Experimental
Chemistry All reactions were performed with com-
spectively. Compound 1 did not cause >50% inhibition at the mercially available reagents and were used without further
50µm screening concentration. Relative to compounds 4–7, purification. Solvents were dried by standard methods and
compounds 1–3 displayed the lowest tumor cell growth activ- stored over molecular sieves. All reactions were monitored by
ity and PDE3B (using cAMP as a substrate) inhibitory activ- thin-layer chromatography (TLC) carried on fluorescent pre-
ity. Cyclization of compound 2 by formamide resulted into coated plates and detection of the components was made by
compound 6 that did not inhibit PDE3 when cAMP was used short UV light. Melting points were determined in open capil-
as the substrate and with the third least active to inhibit tumor laries using MEL-TEMP II and Buchi B-540 melting point
cell growth. However, compound 6 inhibited PDE3 with an apparatus and are uncorrected. FT-IR spectra were recorded
IC50 of 10.9µm using cGMP as the substrate. The development on Nicolet Avatar 380 spectrometer. 1H-NMR spectra were
of substrate selective inhibitors to dual acting enzymes as recorded on Varian Mercury VX-300MHz spectrometer. Mass
PDE3 is a new development and opens the horizons towards spectra were obtained with Hewlett Packard GC-MS, model
modulating the level of one substrate rather than the other, 5890, series II at an ionization potential of 70eV. Elemental
which may have safety and/or efficacy consequences for drug analyses were performed by the Microanalytical Unit, Faculty
discovery.
of Science, Cairo University. All values were within 0.4% of
The 2-pyridone derivatives 4 and 5 and the cyclized pyrido- the theoretical ones, unless otherwise indicated. Yields were
pyrimidone derivative 7 inhibited tumor cell growth with IC50 not optimized
as low as 1.34µm for compound 7. These three compounds
General Procedure for the Preparation of 4,6-Diaryl-
effectively inhibited PDE3 with cAMP and cGMP as sub- 2-imino-1,2-dihydropyridine-3-carbonitrile (1–3) The re-
strates. This suggests that inhibition of both cAMP and cGMP spective aromatic ketone (1mmol), together with the respec-
hydrolysis to increase intracellular levels of both signaling tive aromatic aldehyde (1mmol), malononitrile (1mmol),
molecules may be optimal for anticancer activity. Since the and ammonium acetate (8mmol) were dissolved in ethanoll
PDE3 and the growth inhibitory activities are not in paral- (30mL) and put under reflux for 10–12h. The precipitate ob-
lel, thus other phosphodiesterases and/or off targets may be tained was filtered, washed with ethyl alcohol and dried. For
contributing to the anticancer activity, this is confirmed by the the purification purpose, the precipitate was subjected either
inactivity of the positive control milrinone to induce apoptosis to re-crystallization from a mixture of DMF–ethanol (1:10),
to the HT-29 cell line although of its dual inhibition to PDE3.
or to column chromatography on silica gel, eluting with chlo-
Docking of compound 4 and compound 7 to the catalytic roform and/or methylene chloride.
domain of PDE3B (PDB 1SOJ) using MOE software10) showed
2-Amino-6-(3-bromophenyl)-4-(2-methoxyphenyl)-
that the bromophenyl is involved in π–π stacking with the nicotinonitrile (1) Synthesized Using 3-Bromoacetophenone
phenyl of Phe991, while the –C=O and –CN functions of and 2-Methoxybenzaldehyde: Yield 80%, mp 276–274°C; IR
4 are involved indirectly through H2O molecules with mul- (cm−1) 2205 (–C≡N), 3482 (NH); 1H-NMR (DMSO-d6) δ:
tiple interactions including Mg ions, Tyr736, His737, His741, 3.89 (s, 3H, –OCH3), 7.08–7.13 (t, 1H, aromatic), 7.20–7.54
Asp822, Asp937. With regard to compound 7, its bromophenyl (m, 5H, aromatic), 7.63 (d, 1H, aromatic), 7.72–7.75 (d, 1H,
group is also involved in π–π stacking with the phenyl of aromatic), 7.81 (s, 1H, aromatic); MS (electron ionization (EI))
Phe991, while the carbonyl and amino of the newly formed m/z 379 (M+; 100%), m/z 381 (M++2); Anal. (C19H14BrN3O) C,
ring are involved in interaction indirectly through H2O mol- H, N.
ecules with Mg ions, His821, Asp822, Glu851, His854, Thr893
2-Amino-6-(3-bromophenyl)-4-(2-ethoxyphenyl)nicotino-
and Asp937, Fig. 2. It is also worthy to mention that the ligand nitrile (2) Synthesized Using 3-Bromoacetophenone and
(IBMX) in 1SOJ showed 4 interactions with Gln988 through 2-Ethoxybenzaldehyde: Yield 60%; mp 235–237°C; IR
H bonding, with Phe991 through π–π stacking and with (cm−1) 2222 (CN), 2284 (NH); 1H-NMR (DMSO-d6) δ:
Tyr736 and Asp937 through H2O molecules,11) Fig. 2. In our 1.36–1.40 (t, 3H, CH3), 4.12–4.19 (q, 2H, –OCH2), 7.05–7.10
compounds, the higher electronegativity of the oxo substitu- (t, 1H, aromatic), 7.14–7.17 (d, 1H, aromatic), 7.25 (s, 1H,
ents at position 2 on the pyridine rather than the amino, may aromatic), 7.43–7.52 (m, 2H, aromatic), 7.60–7.62 (d, 1H,
explain the stronger ability of cyanopyridones rather than the aromatic), 7.70–7.78 (t, 2H, aromatic), 7.89–7.93 (d, 1H, aro-
aminocyanopyridines to establish indirect hydrogen bonding matic); MS (EI) m/z 393 (M+; 100%), m/z 395 (M++2); Anal.
and the more efficient inhibition of PDE3.
(C20H16BrN3O) C, H, N.
From a conformational point of view, it seems that an al-
2-Amino-6-(4'-bromobiphenyl-4-yl)-4-(2-methoxy-
koxy substituent at the ortho position of the phenyl at position phenyl)nicotinonitrile (3) Synthesized from 1-(4′-Bromo-
4 and a bromo substituent at the ortho position of the phenyl biphenyl-4-yl)ethanone and -2-Methoxybenzaldehyde: Yield
at position 6 will lead to non-coplanarity between the 2 aryls 85%; mp 210–212°C; IR (cm−1) 2213 (–CN–), 3338 (–NH–);
and the pyridone, as in Fig. 3. The enforcement of non- 1H-NMR (DMSO-d6) 3.91 (s, 3H, –OCH3), 6.57 (s, 1H, C-5
coplanarity of the bromophenyl seems essential to allow π–π pyridine), 7.27–7.59 (m, 4H, aromatic), 7.60–7.73 (m, 5H,