M.O. Radwan, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Scheme 1. Synthetic route of SN-5 and SN-6.
Scheme 2. Synthetic route of SN-9.
via 2/4 (Scheme 1). The overall yields of SN-5 and SN-6 were 3.7% and
13%, respectively. SN-7 was obtained by a straightforward reaction
between 2-(bromomethyl)pyridine hydrobromide and 2-aminoetha-
nethiol hydrochloride in the presence of sodium bicarbonate (94%
yield) as reported previously.19 To obtain SN-8, 2-(pyridin-2-yl)-2,3-
dihydrobenzo[d]thiazole was synthesized20 (87% yield), and then its
thiazoline ring was reductively opened with sodium borohydride21
(70% yield), as previously described. SN-9 was synthesized by reductive
amination of 4-(dimethylamino)picolinaldehyde with S-tritylcystea-
mine hydrochloride in the presence of triacetoxyborohydride (18%
yield), followed by removal of the trityl group (22% yield) (Scheme 2).
Whereas the yields of reductive amination products were generally low,
the final compounds were obtained in good purity. Additionally, we
included the previously reported compounds Biotin-SN-1,17 Farnesyl-
SN-1, and Dodecyl-SN-122 in our study to obtain more thorough
structure-activity relationship information (Fig. 1).
observed. SN-2 and SN-3 are expected to generate the same SN-1 by
reductive agents inside cells, and thus, we deduced that SN-3 is a
mixture of large molecules formed by oxidation of thiol groups. To
correctly evaluate the activity of the reductive type of these com-
pounds, DL-dithiothreitol (DTT) (30 μM) was added to the compounds
first, and the experiment was repeated using the same conditions.
As shown in Fig. 2B, SN-3 showed similar activity (85%) to that of
SN-2 (89%), demonstrating that this assay system is valid. Compared
with SN-2, SN-6 had similar (84%) but slightly weaker activity,
showing the small effect of a dimethylamino group on the pyridine ring.
SN-5 did not show clear inhibitory activity. This demonstrates the im-
portance of the nitrogen atom in the pyridine ring. Notably, 2-ni-
trobenzenethiol (NpSH), which is generated from Bis(2-nitrophenyl)
disulfide and is also formed by reductive cleavage of SN-2, SN-5, and
SN-6, did not show activity. SN-7, SN-8, and SN-9 lacked any detectable
activity, implying the importance of the two side chains of the pyridine
ring. Among Biotin-SN-1, Farnesyl-SN-1, and Dodecyl-SN-1, only
Biotin-SN-1 showed activity (78%), although it was weaker than that of
SN-3. This suggests that a long substituent at position 4 of the pyridine
group is not a major obstacle to activity. The reason that Farnesyl-SN-1
and Dodecyl-SN-1 had no activity may be because these compounds
localize to the membrane or to other proteins with a hydrophobic
pocket. In these experiments, SN-2 showed the best activity to suppress
downstream signaling of TRAF6.
signaling was examined with a cellular reporter assay.18,23 HeLa S3
cells co-transfected with 3κB-tk-luc (with a triple κB sequence that
binds to NF-κB joined to a downstream firefly luciferase gene) and
control vector pRL-Luc (with a β-actin promoter joined to a down-
stream renilla luciferase gene) were incubated for 1 day, and SN-1 or its
derivative (10 μM) was added. After 1 h of incubation, cells were sti-
mulated with interleukin-1α (IL-1 α) and further incubated for 3 h.
Cells were lysed, and a dual-luciferase reporter assay was conducted.
The value of luminescence after addition of a substrate of firefly luci-
ferase was normalized to that using a substrate of renilla luciferase to
determine NF-κB activity.
The experimental results can be explained by our previous mole-
cular docking study of SN-1 into the first zinc finger of TRAF6 using the
Molecular Operating Environment.18 Both the pyridyl nitrogen and two
sulfurs bind to an amino acid or zinc of TRAF6, showing their im-
portance (binding score −8.5 kcal/mol). Herein, we predicted that SN-
9 interacts in the same shallow pocket. The ionized sulfur interacts with
zinc and Cys155 by ionic and H-bonds, respectively. One more
The result is shown in Fig. 2A. As previously reported, SN-2 showed
a pronounced inhibitory effect (80%) against NF-κB activation.18 SN-6
showed a similar effect (76%), whereas no activity of SN-3 was
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