S. Ravichandran et al. / Journal of Molecular Graphics and Modelling 49 (2014) 38–46
39
QRMNH2 (98%): 1H NMR (500 MHz; DMSO-d6): ı 9.38 (s, 1H),
8.11 (d, J = 7.6 Hz, 1H), 7.79 (t, J = 7.5 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H),
7.46 (t, J = 7.4 Hz, 1H), 7.43 (s, 1H), 7.37 (d, J = 7.4 Hz, 1H), 7.19 (t,
J = 7.7 Hz, 1H), 6.70 (d, J = 7.3 Hz, 1H), 5.31 (s, 2H).
model was generated with a subset of 14 training compounds and
a test set of 6 compounds. Of the twenty compounds used, six were
from the initial study, naringenin and daidzein were purchased
and the remaining twelve quercetin analogs were synthesized and
screened against SIRT6. Herein we report and discuss the refine-
ment of this model.
QRMNO2 (59%): 1H NMR (500 MHz; CDCl3): ı 9.09 (s, 1H), 8.63
(d, J = 7.9 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 7.9 Hz, 1H), 7.76
(t, J = 7.7 Hz, 1H), 7.72 (t, J = 8.1 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.45
(t, J = 7.5 Hz, 1H), 7.33 (s, 1H).
2. Materials and methods
QRMBr (87%): 1H NMR (500 MHz; CDCl3): ı 8.38 (t, J = 1.7 Hz,
1H), 8.25–8.20 (m, 2H), 7.72 (ddd, J = 8.6, 6.9, 1.7 Hz, 1H), 7.60–7.57
(m, 2H), 7.43–7.37 (m, 2H), 7.19 (s, 1H).
2.1. Materials
1-Ethyl-3-(3-methylaminopropyl)carbodiimide (EDC), gluter-
aldehyde, hydroxylamine hydrochloride, nicotinamide adenine
dinucleotide (NAD+), dithiothreitol (DTT) potassium phosphate
dibasic, sodium cyanoborohydride, and sodium phosphate
monobasic, quercetin, vitexin, naringenin, kaempferol, apigenin
were obtained from Sigma–Aldrich Chemical Co. (Milwaukee, WI).
Solutions were prepared using purified water from a Millipore
MilliQ system (Millipore Corporation, Bedford, MA).
QROOH (32%): 1H NMR (500 MHz; DMSO-d6): ı 9.78 (s, 1H),
8.99 (s, 1H), 8.14 (dd, J = 8.0, 1.3 Hz, 1H), 7.76 (ddd, J = 8.4, 7.1, 1.4 Hz,
1H), 7.62 (d, J = 8.4 Hz, 1H), 7.48–7.44 (m, 2H), 7.35 (td, J = 7.8, 1.3 Hz,
1H), 6.99 (d, J = 8.3 Hz, 1H), 6.93 (t, J = 7.5 Hz, 1H).
QRPOMe (64%): 1H NMR (500 MHz; CDCl3): ı 8.26–8.24 (m, 3H),
7.70 (ddd, J = 8.7, 6.9, 1.7 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.41 (t,
J = 7.5 Hz, 1H), 7.06 (d, J = 9.0 Hz, 2H), 6.95 (s, 1H), 3.90 (s, 3H).
6OHQR (23%): 1H NMR (500 MHz; DMSO-d6): ı 9.97 (s, 1H),
9.41 (s, 1H), 8.19 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 9.0 Hz, 1H), 7.55 (t,
J = 7.5 Hz, 2H), 7.48 (t, J = 7.5 Hz, 1H), 7.37 (d, J = 2.9 Hz, 1H), 7.26 (dd,
J = 9.0, 2.9 Hz, 1H).
2.2. General procedure
6MeOQR (55%): 1H NMR (500 MHz; CDCl3): ı 8.25 (d, J = 7.4 Hz,
2H), 7.57 (d, J = 3.0 Hz, 1H), 7.55–7.51 (m, 3H), 7.47 (t, J = 7.3 Hz, 1H),
7.31 (dd, J = 9.2, 3.1 Hz, 1H), 7.03 (s, 1H), 3.92 (s, 3H).
Solvents and reagents were obtained from commercial suppli-
ers and were used without further purification. NMR experiments
were run on a Bruker Avance 500 equipped with a BBI probe
and Z-gradients. Spectra were acquired at 300 K, using deuterated
dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3) as
the solvent. Chemical shifts for 1H and 13C spectra were recorded
in parts per million using the residual nondeuterated solvent as
the internal standard (for DMSO-d6, 2.50 ppm 1H, and for CDCl3,
7.26 ppm 1H). Data are reported as follows: chemical shift (ppm),
multiplicity (indicated as br, broad signal; s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet, and combinations thereof), cou-
pling constant (J, Hz), and integrated intensity. All final compounds
displayed ≥95% purity as determined by NMR analysis.
6MeQR (71%): 1H NMR (500 MHz; CDCl3): ı 8.25 (d, J = 7.5 Hz,
2H), 8.03 (s, 1H), 7.55–7.45 (m, 5H), 7.05 (s, 1H), 2.48 (s, 3H).
2.3. Frontal displacement chromatography
The SIRT6 (CT)-OT column was prepared as previously described
[12]. The column was attached to the chromatographic system
Series 1100 Liquid Chromatography/Mass Selective Detector (Agi-
lent Technologies, Palo Alto, CA, USA) equipped with a vacuum
de-gasser (G1322 A), a binary pump (1312 A), an autosampler
(G1313 A) with a 20 L injection loop, a mass selective detec-
tor (G1946 B) supplied with atmospheric pressure ionization
electrospray and an on-line nitrogen generation system (What-
man, Haverhill, MA, USA). The chromatographic system was
interfaced to a 250 MHz Kayak XA computer (Hewlett-Packard,
Palo Alto, CA, USA) running ChemStation software (Rev B.10.00,
Hewlett-Packard). In the chromatographic studies, the mobile
phase consisted of ammonium acetate (10 mM, pH 7.4):methanol
(90:10, v/v) containing 0.2 mM NAD+ and 5 M quercetin delivered
at 0.05 mL min−1 at room temperature. Then 10 M concentration
of each of the polyphenols was placed in the mobile phase and the
change in retention volume was obtained to rank the compounds in
order of affinity based on the displacement of quercetin. Quercetin
was monitored in the negative ion mode using single ion monitor-
ing at m/z = 301.00 [MW−H]− ion for quercetin, with the capillary
voltage at 3000 V, the nebulizer pressure at 35 psi, and the drying
gas flow at 11 L/min at a temperature of 350 ◦C.
The target quercetin derivatives were synthesized using the
Frederique’s method [13].
2.2.1. Synthesis of the chalcones (step 1)
Substituted 2ꢀ-hydroxyacetophenone (10 mmol) and the substi-
tuted aldehyde (10 mmol) were dissolved in ethanol (100 ml) and
potassium hydroxide (1.12 g, 20 mmol) was added and the mixture
was stirred at 50 ◦C for 15 h. After this time the pH was adjusted to
1–2 by the addition of HCl(c) and the precipitate formed was filtered
under vacuum to obtain the target chalcone as a yellow solid.
2.2.2. Synthesis of the flavanols (step 2)
The chalcone was dissolved in 100 ml of methanol and 5 ml
of 30% hydrogen peroxide and sodium hydroxide (1.2 g, 30 mmol)
were added and the mixture was stirred at room temperature for
15 h. HCl(c) was added until pH 1 and precipitate formed was fil-
tered under vacuum to obtain the title compound as a solid.
QRMCH3 (76%): 1H NMR (500 MHz; CDCl3): ı 8.31 (dd, J = 8.0,
1.5 Hz, 1H), 8.11 (d, J = 6.8 Hz, 2H), 7.75 (td, J = 7.8, 1.5 Hz, 1H), 7.65
(d, J = 8.5 Hz, 1H), 7.49–7.45 (m, 2H), 7.34 (d, J = 7.5 Hz, 1H), 7.11 (s,
1H), 2.52 (s, 3H).
2.4. Molecular modeling
QRPOH (34%): 1H NMR (500 MHz; DMSO-d6): ı 10.10 (s, 1H),
9.35 (s, 1H), 8.14–8.10 (m, 3H), 7.78 (td, J = 7.7, 1.5 Hz, 1H), 7.74 (d,
J = 8.2 Hz, 1H), 7.46 (t, J = 7.4 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H).
QRPCF3 (67%): 1H NMR (500 MHz; CDCl3): ı 8.38 (d, J = 8.3 Hz,
2H), 8.26 (dd, J = 8.0, 1.1 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.76–7.72
(m, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.23 (s, 1H).
QRMOH (43%): 1H NMR (500 MHz; DMSO-d6): ı 9.71 (s, 1H),
9.56 (s, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.81 (ddd, J = 8.5, 7.0, 1.4 Hz, 1H),
7.74 (d, J = 8.4 Hz, 1H), 7.69 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.47 (t,
J = 7.5 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 6.90 (d, J = 7.9 Hz, 1H).
Molecular structures used in modeling were either downloaded
from the PubChem database or modified from their close analogs
using Discovery Studio (version 3.5; Accelrys Inc., San Diego, CA,
Studio. Using the experimental elution times and our previously
published pharmacophore model as guidance [12], we created a
training set that consisted of 14 flavonoid molecules (see Table 1,
Fig. 1 and Supplementary Table S3) and a test set (see Table 2, Fig. 2