N. M. Shukla et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2211–2214
2213
quinoline 11. Amidation of 11 with 22, followed by cyclization
using NaOH afforded the C-2-substituted imidazoquinoline scaf-
fold 12. Reaction of 12 with excess of the 2,2-dimethyloxirane in
the presence of DBU afforded the N-3 substituted imidazoquinoline
13, which after deprotection yielded 16. Subsequent steps, similar
to the ones described above afforded the 3H-regioisomer 15. Com-
parison of the 1H NMR spectra of 9 and 15 showed, as expected,
differences in the chemical shifts of the substituents on the imid-
azole ring; significant differences were also seen for the C9 pro-
tons, probably due to through-space effects of the substituents
(Supplementary data).
cin and normocin. Stable expression of secreted alkaline phospha-
tase (sAP) under control of NF- B/AP-1 promoters is inducible by
the TLR7 agonists, and extracellular sAP in the supernatant is pro-
j
portional to NF-jB induction. HEK-Blue-7 cells were incubated at a
density of ꢀ105 cells/mL in a volume of 80
lL/well, in 384-well,
flat-bottomed, cell culture-treated microtiter plates until conflu-
ency was achieved, and subsequently graded concentrations of
stimuli were added. sAP was assayed spectrophotometrically using
an alkaline phosphatase-specific chromogen (present in HEK-
detection medium as supplied by the vendor) at 620 nm. The
TLR7 agonistic potency of 9 was indistinguishable from that of
the commercially available gardiquimod (Fig. 1), while 15 was de-
void of agonistic activity (Fig. 1).
TLR7 agonism and antagonism: A reporter gene assay using
TLR7-dependent NF-
B was quantified using HEK-Blue-cells as previously described
by us.18 HEK293 cells were stably transfected with plasmids
encoding TLR7 as well as an NF- B reporter gene coupled to se-
jB induction was used. The induction of NF-
j
None of the precursors of 9 displayed any significant agonistic
activities (data not shown). Antagonistic activities were examined
by incubating HEK-Blue-7 cells with graded concentrations of test
compounds in the presence of 250 ng/mL of gardiquimod. 15 dis-
j
creted alkaline phosphatase (sAP) (InvivoGen, San Diego, CA), and
were maintained in HEK-BlueTM Selection medium containing zeo-
played weak TLR7 antagonism (IC50
: 25 lM, Fig. 2), which
prompted us to carefully examine all its precursors. The des-amino
compound 16 obtained after deprotecting 13 was found to be max-
0.6
0.4
imally antagonistic with an IC50 of 7.5 lM. Compound 16, fortu-
itously, also crystallized with ease in MeOH, the structure of
which is shown in Fig. 2.
Compound 16, to our knowledge, is the first small molecule
with TLR7-antagonistic activity, and may therefore be useful as a
lead for the syntheses of focused libraries toward the generation
of more potent small-molecule receptor antagonists of TLR7.
Acknowledgements
We gratefully acknowledge the expertise of Dr. Victor Day,
Director of the KU Small-Molecule X-Ray Crystallography
Laboratory.
IC50 Values:
0.2
15 (25 μM)
Supplementary data
16 (7.5 μM)
The crystal structure of 16 has been deposited at the Cambridge
Crystallographic Data Centre (Accession number: CDC 718787).
Synthetic procedures, 1H, 13C NMR, mass-spectral data of target
compounds and precursors, and 1H NMR spectra overlays of 9
and 15. Supplementary data associated with this article can be
10-7
10-6
10-5
10-4
Compound Concentration (M)
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Figure 2. Top: TLR7-antagonistic activities of the 3H regioisomer 15, and of its des-
amino precursor 16. Bottom: Crystal structure of 16 as the di-TFA salt. The locations
of all the hydrogens were determined from a difference Fourier and refined as
individual isotropic atoms.