J. Simonin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1151–1155
1155
Unexpectedly, when we condensed 4-bromobutanoyl chloride
with 2,4-dimethylpyrrole following a literature protocol14 we ob-
tained BODIPY dye 25 as the sole product where two moles of acyl
chloride have reacted with two moles of the pyrrole. The structure
of 25 was confirmed by full spectroscopic characterization and a
crystal structure.
and was not blocked by ondansetron (Fig. 2). These data suggest
nonspecific interactions of this probe with the cells.
In summary, we have designed and synthesized a small library
of fluorescent 5-HT3 receptor ligands. Most notably, novel granise-
tron conjugates 14, 16, 18, 27 and 28 have similar binding affinities
for the human 5-HT3AR as measured for the parent compound 1.
This is in agreement with previous studies which showed that
bulky fluorophores can be appended via short aliphatic linker to
the N1-position of granisetron. Moreover, these high affinity
probes exhibit high quantum yields and emission maxima above
500 nm in polar media. Probes 16 and 18, in particular, show spe-
cific fluorescent labeling of the human 5-HT3AR in live cells. We are
currently focusing our efforts on evaluating our probes in fluores-
cence polarization and flow cytometry applications.
Other than the N1-position on granisetron, we identified the
C7-position as tolerant to substitution in our initial SAR study.13
Therefore we synthesized probes 36–39, where the fluorophores
were conjugated to this latter position via different linkers. The
synthesis of 7-hydroxy granisetron 31 was described previously;
we have, however, found a more practical and scalable route to ac-
cess this intermediate (Scheme 4). The indazole-3-carboxylate 29
was synthesized using a 1,3-dipolar cycloaddition of in situ gener-
ated aryne with a diazo ester.15 Only the C7-substituted regioisom-
er was obtained in this reaction. This was followed by ester
hydrolysis, amide formation with bicyclic amine 30, selective
N1-methylation and methyl ether cleavage. It was crucial to follow
the above order of steps since N-methylation of the indazole ester
29 also yields small amounts of N2-methylated side product which
is extremely difficult to separate from the desired N1-isomer. The
hydroxyl group of 31 was alkylated with protected spacers 8, 10 or
32 and the Boc-group was subsequently cleaved. Finally,
7-(diethylamino)-coumarin-3-carboxylic acid or FITC was coupled
to the spacers to yield probes 36–39.
Acknowledgments
This research was supported by the Swiss National Science
Foundation (SNSF-professorship PP00P2_123536 to ML), the EPSRC
(EP/E042139/1) and the Wellcome Trust (085141 (CNC and JDH)
and 081925/Z/07/Z (SCRL and AJT)). SCRL is a Wellcome Trust Se-
nior Research Fellow in Basic Biomedical Science.
Supplementary data
The photophysical properties of fluorescent granisetron deriva-
tives were measured in MeOH and/or phosphate buffer at pH 7
(Table 1). The granisetron probes that have fluorescein (16, 18
and 21) or BODIPY dyes (6, 14 and 28) appended to N1 show the
highest quantum yields (Uf). The quantum yield of the coumarin-
containing probes 15, 17, 20, 36–38 was low in pH 7 buffer, but
was substantially increased in less polar solvents (e.g., for 15: Uf
(CH2Cl2) = 0.55). The binding affinities of the fluorescent granise-
tron probes for the human 5-HT3AR were determined by competi-
tion binding studies with [3H]granisetron. Probes 14, 16, 18, 27, 28
and 36 exhibited affinities similar to the parent compound granise-
tron (Table 1) and thus could be useful as tracer ligands for fluores-
cence-based binding assays and tools for imaging. In terms of
probe design it appears that optimal binding is obtained if the fluo-
rophore is conjugated to N1 of granisetron via a short (butyl) ali-
phatic spacer.
The utility of granisetron probes 16–18, 26–28 and 36 to fluo-
rescently label the receptor was studied using live cell imaging of
COS-7 cells transiently transfected with human 5-HT3A receptors.
Only probes 16, 18 and 28 gave detectable staining. Probes 16
and 18 produced selective staining for 5-HT3R as demonstrated
by fluorescence at the periphery (plasma membrane) of transfec-
ted cells, whilst this fluorescence was absent in mock transfected
cells (Fig. 2). Furthermore, 5-HT3R staining with these probes
was inhibited by co-incubation with the 5-HT3R antagonist ondan-
Supplementary data (synthesis details and spectral data for all
compounds, crystal structure of 25, HPLC purity assessment for
target compounds and experimental details for photophysical
measurements) associated with this article can be found, in the on-
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setron (OND, 10
lM) (Fig. 2). In contrast, probe 28 produced
intense fluorescence in both 5-HT3A- and mock-transfected cells,