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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–8
Structurally Modified Benzimidazoles as Sirtuin Inhibitors
Archiv der Pharmazie
Ethyl 2-(4-chlorophenyl)-1-phenyl-1H-benzo[d]imidazole-
5-carboxylate (10)
Ethyl 2-(4-(dimethylamino)phenyl)-1-phenyl-1H-benzo[d]-
imidazole-5-carboxylate (16)
Yield: 81%; 1H NMR (500 MHz, CDCl3): d 1.43 (3H, t, J ¼ 7.1 Hz),
4.42 (2H, q, J ¼ 7.1 Hz), 7.25 (1H, d, J ¼ 9 Hz), 7.28 (2H, d,
J ¼ 9 Hz), 7.40–7.60 (5H, m), 7.54 (2H, d, J ¼ 9 Hz), 8.00 (1H, dd,
J ¼ 1.5 Hz, 9 Hz), 8.52 (1H, s). 13C NMR (125 MHz, CDCl3): 14.40,
61.64, 110.28, 111.80, 116.26, 122.70, 122.97, 124.65, 125.67,
127.91, 129.20, 130.35, 131.18, 137.50, 140.76, 142.71, 152.68,
167.48. ESI-MS: m/z 377.1 [MþH]þ. Anal. calcd. for
Yield: 94%; 1H NMR (500 MHz, CDCl3): d 1.43 (3H, t, J ¼ 7.1 Hz),
2.97 (6H, s), 4.41 (2H, q, J ¼ 7.1 Hz), 6.58 (2H, d, J ¼ 9 Hz), 7.18
(1H, d, J ¼ 9 Hz), 7.35–7.60 (5H, m), 7.46 (2H, d, J ¼ 9 Hz), 7.92
(1H, dd, J ¼ 1.5 Hz, 9 Hz), 8.56 (1H, s). 13C NMR (125 MHz,
CDCl3): 14.79, 40.43, 61.22, 110.04, 111.74, 116.39, 121.56,
124.65, 125.67, 127.91, 129.21, 130.42, 130.99, 137.49, 140.76,
142.71, 151.55, 155.13, 167.57. ESI-MS: m/z 386.1 [MþH]þ.
Anal. calcd. for C24H23N3O2: C, 74.78%; H, 6.01%; N, 10.90%.
Found: C, 74.76%; H, 6.02%; N, 10.90%.
C
22H17N2O2Cl: C, 70.12%; H, 4.55%; N, 7.43%. Found: C,
70.10%; H, 4.52%; N, 7.54%.
Ethyl 2-(3-chlorophenyl)-1-phenyl-1H-benzo[d]imidazole-
5-carboxylate (11)
Ethyl 2-(3-(dimethylamino)phenyl)-1-phenyl-1H-benzo[d]-
imidazole-5-carboxylate (17)
Yield: 66%; 1H NMR (500 MHz, CDCl3): d 1.43 (3H, t, J ¼ 7.1 Hz),
4.42 (2H, q, J ¼ 7.1 Hz), 6.80–7.40 (9H, m), 7.56 (1H, s), 7.89 (1H,
dd, J ¼ 1.5 Hz, 9 Hz), 8.50 (1H, s). ESI-MS: m/z 377.1 [MþH]þ.
Anal. calcd. for C22H17N2O2Cl: C, 70.12%; H, 4.55%; N, 7.43%.
Found: C, 69.96%; H, 4.85%; N, 7.34%.
Yield: 42%; 1H NMR (500 MHz, CDCl3): d 1.42 (3H, t, J ¼ 7.1 Hz),
2.98 (6H, s), 4.42 (2H, q, J ¼ 7.1 Hz), 6.79 (1H, dd, J ¼ 1.5 Hz,
9 Hz), 6.93 (1H, s), 7.00–7.50 (8H, m), 7.90 (1H, dd, J ¼ 1.5 Hz,
9 Hz), 8.53 (1H, s). ESI-MS: m/z 386.2 [MþH]þ. Anal. calcd. for
C
24H23N3O2: C, 74.78%; H, 6.01%; N, 10.90%. Found: C,
74.66%; H, 6.18%; N, 10.87%.
Ethyl 2-(2-chlorophenyl)-1-phenyl-1H-benzo[d]imidazole-
5-carboxylate (12)
Ethyl 2-(2-(dimethylamino)phenyl)-1-phenyl-1H-benzo[d]-
imidazole-5-carboxylate (18)
Yield: 80%; 1H NMR (500 MHz, CDCl3): d 1.44 (3H, t, J ¼ 7.1 Hz),
4.43 (2H, q, J ¼ 7.1 Hz), 6.90–7.45 (10H, m), 7.87 (1H, dd,
J ¼ 1.5 Hz, 9 Hz), 8.50 (1H, s). ESI-MS: m/z 377.1 [MþH]þ. Anal.
calcd. for C22H17N2O2Cl: C, 70.12%; H, 4.55%; N, 7.43%.
Found: C, 70.04%; H, 4.56%; N, 7.50%.
Yield: 49%; 1H NMR (500 MHz, CDCl3): d 1.43 (3H, t, J ¼ 7.1 Hz),
3.00 (6H, s), 4.42 (2H, q, J ¼ 7.1 Hz), 6.84 (1H, d, J ¼ 9 Hz), 6.90–
7.50 (9H, m), 7.89 (1H, dd, J ¼ 1.5 Hz, 9 Hz), 8.52 (1H, s). ESI-MS:
m/z 386.2 [MþH]þ. Anal. calcd. for C24H23N3O2: C, 74.78%; H,
6.01%; N, 10.90%. Found: C, 74.65%; H, 6.22%; N, 10.84%.
Ethyl 2-(4-hydroxyphenyl)-1-phenyl-1H-benzo[d]-
imidazole-5-carboxylate (13)
Biological assays
Yield: 77%; 1H NMR (500 MHz, CDCl3): d 1.43 (3H, t, J ¼ 7.1 Hz),
4.42(2H,q,J ¼ 7.1 Hz),7.19(2H,d,J ¼ 9 Hz),7.28(1H,d,J ¼ 9 Hz),
7.35–7.60 (5H, m), 7.47 (2H, d, J ¼ 9 Hz), 7.99 (1H, dd, J ¼ 1.5 Hz,
9 Hz), 8.56 (1H, s). 13C NMR (125 MHz, CDCl3): 14.26, 61.05,
118.95, 120.87, 122.54, 124.79, 126.95, 127.26, 128.08, 129.17,
130.34, 132.96, 136.15, 140.28, 143.03, 158.69, 167.00. ESI-MS:
m/z 359.1 [MþH]þ. Anal. calcd. for C22H18N2O3: C, 73.73%; H,
5.06%; N, 7.82%. Found: C, 73.56%; H, 5.16%; N, 7.88%.
SIRT1 in vitro assay
SIRT1 peptide substrate (3.3 mM) derived from the human
p53 sequences, 66.7 mM NADþ, 50 mM of the interesting
compounds (all final concentrations), and 0.5 mg of human
recombinant SIRT1 (GenBank Accession Number: NM_012238;
with amino acids 193–741 and a glutathione S-transferase
(GST) tag at its N terminus) were incubated for 45 min at
37°C in an incubator. Stop solution (50 mL) consisting of
nicotinamide and SIRT1 developer (mixed proportionally
according to the manufacturer’s protocol) was then added
and the resulting mixture was incubated for a further 10 min
at 37°C. The final concentration of DMSO in each well was
maintained at 1.0% v/v. Fluorescence was measured at 490 nm
(excitation) and 520 nm (emission) and the percentage of
enzyme inhibition was calculated with Eq. (1)
Ethyl 2-(3-hydroxyphenyl)-1-phenyl-1H-benzo[d]-
imidazole-5-carboxylate (14)
Yield: 82%; 1H NMR (500 MHz, CDCl3): d 1.43 (3H, t, J ¼ 7.1 Hz),
4.42 (2H, q, J ¼ 7.1 Hz), 6.86 (1H, dd, J ¼ 1.5 Hz, 9 Hz), 7.01 (1H,
s), 7.10–7.60 (8H, m), 8.01 (1H, dd, J ¼ 1.5 Hz, 9 Hz), 8.54 (1H, s).
ESI-MS: m/z 359.2 [MþH]þ. Anal. calcd. for C22H18N2O3: C,
73.73%; H, 5.06%; N, 7.82%. Found: C, 73.70%; H, 5.04%; N,
7.85%.
%Enzyme inhibition ¼ ðFluorI ꢂ FluorSÞ=FluorI ꢁ 100%
ð1Þ
where FluorI is the initial activity per well (reading of the well
containing all reagents except for the test compound after
deducting the background reading) and FluorS is the activity
of the sample well (reading of the well containing all
reagents including the test compound after deducting the
background reading). Blank wells contained all reagents
except for the test compounds and the SIRT1 human
recombinant enzyme.
Ethyl 2-(2-hydroxyphenyl)-1-phenyl-1H-benzo[d]-
imidazole-5-carboxylate (15)
Yield: 83%; 1H NMR (500 MHz, CDCl3): d 1.44 (3H, t, J ¼ 7.1 Hz),
4.44 (2H, q, J ¼ 7.1 Hz), 6.85–7.45 (10H, m), 7.87 (1H, dd,
J ¼ 1.5 Hz, 9 Hz), 8.52 (1H, s). ESI-MS: m/z 359.2 [MþH]þ. Anal.
calcd. for C22H18N2O3: C, 73.73%; H, 5.06%; N, 7.82%. Found:
C, 73.67%; H, 5.09%; N, 7.77%.
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