Syntheses of fluorescent imidazoquinoline conjugates as probes of Toll-like receptor 7

Article abstract of DOI:10.1016/j.bmcl.2010.09.093

Toll-like receptor (TLR)-7 agonists show prominent immunostimulatory activities. The synthesis of a TLR7-active N¹-(4-aminomethyl)benzyl substituted imidazoquinoline 5d served as a convenient precursor for the covalent attachment of fluorophore

Full text of DOI:10.1016/j.bmcl.2010.09.093

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6384–6386
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses of fluorescent imidazoquinoline conjugates as probes
of Toll-like receptor 7
Nikunj M. Shukla a, Cole A. Mutz a, Rehman Ukani a, Hemamali J. Warshakoon a, David S. Moore b,
Sunil A. David a,
⇑
a Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66047, United States
b Microscopy and Analytical Imaging Laboratory, University of Kansas, Lawrence, KS 66045, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Toll-like receptor (TLR)-7 agonists show prominent immunostimulatory activities. The synthesis of a
TLR7-active N1-(4-aminomethyl)benzyl substituted imidazoquinoline 5d served as a convenient precur-
sor for the covalent attachment of fluorophores without significant loss of activity. Fluorescence micros-
copy experiments show that the fluorescent analogues are internalized and distributed in the endosomal
compartment. Flow cytometry experiments using whole human blood show differential partitioning into
B, T, and natural killer (NK) lymphocytic subsets, which correlate with the degree of activation in these
subsets. These fluorescently-labeled imidazoquinolines will likely be useful in examining the trafficking
of TLR7 in immunological synapses.
Received 9 September 2010
Revised 14 September 2010
Accepted 15 September 2010
Available online 21 September 2010
Keywords:
Imidazoquinoline
Toll-like receptor
TLR7
Ó 2010 Elsevier Ltd. All rights reserved.
Fluorescent probe
Flow cytometry
Toll-like receptors (TLRs) are pattern recognition receptors that
recognize specific molecular patterns present in molecules that are
broadly shared by pathogens, but are structurally distinct from
host molecules.1 There are 10 TLRs in the human genome.2 The li-
gands for these receptors are highly conserved microbial molecules
such as lipopolysaccharides (LPS) (recognized by TLR4), lipopep-
tides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5),
single stranded RNA (TLR7 and TLR8), double stranded RNA
(TLR3), CpG motif-containing DNA (recognized by TLR9), and pro-
filin present on uropathogenic bacteria (TLR 11).3 The activation
of TLRs by their cognate ligands leads to activation of innate
immune effector mechanisms, including the production of pro-
inflammatory cytokines, up-regulation of MHC molecules and
co-stimulatory signals in antigen-presenting cells, resulting in
amplification of specific adaptive immune responses involving
both T- and B-cell effector functions.4–6 Thus, TLR stimuli serve
to link innate and adaptive immunity4 and can therefore be
exploited as powerful adjuvants in eliciting both primary and
anamnestic immune responses.
these requirements.7 Continuing work on characterizing the
immunostimulatory activities of TLR7 agonists show, as expected,
clear involvement of plasmacytoid dendritic cells,10 but we have
also observed a set of accessory cell-independent direct responses
in CD4+ and CD8+ T and CD3ÀCD56+ natural killer (NK) lympho-
cytes (to be published elsewhere). We are specifically desirous of
examining the uptake, intracellular distribution, and trafficking of
the imidazoquinoline in immunological synapses,11,12 and it
became necessary to develop probes of TLR7 that are fluores-
cently-labeled.
Our earlier SAR study on the TLR7-agonistic activities had
exhaustively explored C2 and C4 substituents on the imidazoquin-
oline scaffold (Fig. 1),13 but proved unsuccessful in identifying po-
tential positions that would tolerate the introduction of bulky aryl
groups without compromising activity.
NH2
4
3
N
5
Our point of departure in the systematic evaluation of TLR ago-
nists as vaccine adjuvants7–9 focuses on identifying chemotypes
which are strongly immunostimulatory, and yet devoid of domi-
nant pro-inflammatory cytokine-inducing activities;7 the TLR7-
agonistic imidazoquinolines have thus far seemed ideal in meeting
N
8
2
R1
6
7
1
N
R2
9
Figure 1. General structure of 1H-imidazo[4,5-c]quinolin-4-amine. The C2 and N1
substituents that were found to correspond to optimal TLR7-agonistic activity were
n-butyl and benzyl, respectively (Ref. 13).
⇑
Corresponding author. Tel.: +1 785 864 1610; fax: +1 785 864 1961.
E-mail address: sdavid@ku.edu (S.A. David).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.093

N. M. Shukla et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6384–6386
6385
NH2
N
N
Cl
N
Cl
N
Cl
i
ii
iii
N
NO2
NH2
NO2
N
NH
NH
Cl
R
R
R
C4H9
1
2a-2d
3a-3d
4a-4d
NH2
N
iv
R1 = Boc for 2,3, and 4
R1 = H for 5
N
R =
N
R
HN
NH
R1
C4H9
R1
5c, 5d
a
b
c
d
Scheme 1. Syntheses of N1-substituted imidazoquinolines. Reagents: (i) RNH2, Et3N, CH2Cl2; (ii) Pt/C, H2, Na2SO4, EtOAc; (iii) (a) C4H9COCl, Et3N, THF; (b) NH3/MeOH, 150 °C;
(iv) HCl/dioxane.
NH2
N
N
NH2
N
N
i
N
N
C4H9
C4H9
NH2
NCS
iv
F
5d
6
ii
iii
N
B
S
N
NH2
N
F
N
NH2
N
N
OH
N
N
N
C4H9
O
O
N
NH2
N
C4H9
O
HO
O
O
S
H
HN
N
S
N
N
OH
N
H
N
C4H9
N
H
O
H
O
S
7
8
9
NH
N
H
Scheme 2. Syntheses of fluorescent analogues of 5d. Reagents: (i) CS2, Et3N, DMAP, (Boc)2O, CH2Cl2; (ii) fluorescein isothiocyanate, Et3N, MeOH; (iii) rhodamine B
isothiocyanate, Et3N, CH2Cl2; (iii) BODIPYÒ TR cadaverine, pyridine.
3
2
5d
7
8
1
0
9
Neg. Ctrl.
10-9
10-8
10-7
10-6
10-5
Compound Concentration (M)
Figure 3. Murine J774 cells treated with 100 nM of 9. An overlay of phase-contrast
and epifluorescence images is depicted. An excitation filter at 562 nm and a long-
pass emission filter (601–800 nm) were used.
Figure 2. Activities of 5d, 7, 8, and 9 in reporter gene assays using human TLR7.
to maximal activity.13 The N1-naphthylenemethyl-substituted
compound 4a was inactive, and the N1-biphenyl-4-methyl com-
pound 4b was weakly active (EC50: 396 nM); the N1-(4-amino-
Our attention subsequently turned to exploring the effect of
varying substituents at N1, while holding the C2-n-butyl and C4-
NH2 groups constant since these have been shown to correspond

6386
N. M. Shukla et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6384–6386
3000
2500
2000
1500
1000
500
Our earlier immunoprofiling of the TLR7-agonistic imidazo-
quinolines had shown a very prominent activation of B- and
NK-cells, but minimal activation of T cells,7 and we asked if a pos-
sible reason could be differential uptake of the TLR7 agonist in
lymphocytic subsets. Flow cytometric analysis of the FITC-labeled
7 in experiments employing whole human blood indeed show a
prominent uptake of 7 in CD3ÀCD56+ NK and CD3ÀCD56À B lym-
phocytes as compared to CD3+CD56À T lymphocytes (Fig. 4).
The syntheses of fluorescent imidazoquinoline analogues that
retain TLR7-agonistic activity are expected to be useful probes in
examining their potential immunostimulatory and adjuvantic
properties.
5
10
B Lymphocytes
T Lymphocytes
NK Lymphocytes
4
3
10
10
10
2
1
10
4
5
3
2
1
10
CD56-APC
10
10
10
10
Acknowledgment
0
This work was supported by NIH/NIAID contract HHSN27
2200900033C.
0
1
2
3
4
5
Concentration of 7 (µg/mL)
Figure 4. Uptake of 7 in lymphocytic subsets as examined by flow cytometry.
Whole human blood was incubated with graded concentrations of 7 for 30 min,
lymphocytes stained with cell surface markers (anti-CD3-phycoerythrin [PE], and
anti-CD56-PE-allophycocyanin). Erythrocytes were lysed, and 105 total events were
acquired per sample.
Supplementary data
Supplementary data (experimental methods and characteriza-
tion of compounds (1H, 13C, and mass spectra)) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2010.09.093.
methyl)benzyl substituted analogue 5d was considerably more ac-
tive (EC50: 20 nM; Scheme 1) than its N1-(3-aminomethyl)benzyl
regioisomer 5c (EC50: 110 nM). The free primary amine on the N1
substituent of 5d was covalently coupled directly to commer-
cially-available fluorescein isothiocyanate and rhodamine B isothi-
ocyanate (Scheme 2). Conversely, the amine on 5d was converted
first to the isothiocyanate 6, allowing the subsequent coupling of
amine-bearing fluorophores, such as the bora-diazaindacene dye,
BODIPY-TR-cadaverine (Scheme 2). All three fluorescent conju-
gates retain TLR7-agonistic activity, although their potencies are
slightly attenuated relative to the parent compound, 5d (Fig. 2);
the EC50 values of 7, 8, and 9 are, respectively, 247 nM, 115 nM,
and 108 nM.
Incubation of murine macrophage J774.A1 cells with 8 or 9, fol-
lowed by intravital epi- and confocal fluorescence microcopy
showed prominent perinuclear localization, which is consistent
with the expected endosomal distribution of TLR7.14 Shown in
Figure 3 is a representative epifluorescence micrograph of J774
cells treated with 9 at 100 nM concentration.
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