L. Li et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1245–1248
1247
pig illeum.7 The removal of a methyl group from the
tertiary amino group in mepyramine, as well as the
replacement of the methyl with an aminoalkyl group,
results in a pronounced decrease in activity (3, 7a–d).
However, compared to the non-labeled building blocks
7a–d, some of the fluorescent probes are more potent H1
blockers, for example, depending on the chain length of
the spacer, suitable fluorophores may confer additional
H1 receptor affinity. Comparing the activities of 5- and
6-carboxyfluorescein-labeled H1 antagonists 11a–d and
12a–d on the ileum there is a spacer-dependent increase
with highest activity residing in the compounds with
more than 2 methylene groups. Though the pA2 or pKB
values of mepyramine were not achieved, a number of
the fluorescent probes listed in Table 1 were found to
have H1 antagonistic activities in the range of commer-
cially available drugs such as pheniramine or diphen-
hydramine (pA2 values in the range of 8.1–9.0). The
derivatives 13–16c and 19c, which have the same chain
length but are labeled with different dyes, were found to
be less active than mepyramine by 1 to 2 orders of
magnitude. Compound 18a, which contains only a short
chain (CH2CH2) connecting the affinity-conferring par-
tial structure and the NBD dye, was found to be about
half as potent as 17c, which contains a long spacer
group ((CH)6NHCO(CH2)5).
receptors may be obtained by this approach, however,
predictions of the best suited fluorophore are difficult.
The fluorescent properties of the H1 antagonists are not
detrimental to the application of the Fura assay. This
gives further support to the idea that it may be possible
to combine fluorescence-based methods for (a) func-
tional studies (e.g., Ca2+ assay) and (b) the determina-
tion of binding constants of GPCR ligands. The
combination of both may be potentially useful in high-
throughput screening to get information on affinities
and agonistic/antagonistic properties of potential drug
candidates in one assay. Moreover, fluorescent probes
such as the fluorescein-labeled mepyramine analogues
may be useful to study the GPCRs in tissue prepara-
tions and on cells.
Acknowledgements
The authors are grateful to the DAAD for a scholarship
to L. Li (DAAD ‘sandwich PhD program’) and to the
Fonds der Chemischen Industrie for grants to A. B. and
G. B.
References and Notes
The H1 antagonistic activities found on the guinea pig
ileum are in rather good agreement with the results from
U373MG human glioblastoma cells for the non-labeled
compounds and for most of the fluorescent ligands.
There are only a few exceptions among the investigated
compounds showing discrepancies between the two
pharmacological test models >0.6 log units, which may
be associated with the distinct incubation periods (15
min (ileum) vs 1 min (U373MG)) or the different acces-
sibility of the H1 receptor (diffusion and/or enrichment
of the compounds in the tissue and the cells). For-
tunately, the Fura assays (ratiometric method, lex: 340
and 380 nm, lem: 510 nm) was not impaired by the
fluorescent H1 antagonist, which have excitation max-
ima at longer wavelengths. This finding is of particular
importance with respect to the development of multi-
parametric cellular assays for the determination of
receptor binding and functional data.
1. McGrath, J. C.; Arribas, S.; Daly, C. J. Trends Pharmacol.
Sci. 1996, 17, 393.
2. Mellentin-Michelotti, J.; Evangelista, L. T.; Swartzman,
E. E.; Miraglia, S. J.; Werner, W. E.; Yuan, P.-M. Anal. Bio-
chem. 1999, 272, 182.
3. Mayer, M.; Li, L.; Kracht, J.; Bernhardt, G.; Buschauer, A.
Arch. Pharm.-Pharm. Med. Chem. 2000, 333 (Suppl. 2), 51.
4. Mayer, M. PhD Thesis, University of Regensburg, 2002.
5. Hill, S. J.; Ganellin, C. R.; Timmerman, H.; Schwartz, J. C.;
Shankley, N. P.; Young, J. M.; Schunack, W.; Levi, R.; Haas,
H. L. Pharmacol. Rev. 1997, 49, 253.
6. Korner, M.; Bouthenet, M. L.; Ganellin, C. R.; Garbarg,
M.; Gros, C.; Ife, R. J.; Sales, N.; Schwartz, J. C. Eur. J.
Pharmacol. 1986, 120, 151.
7. Blakemore, R. C.; Ganellin, C. R.; Garbarg, M.; Gros, C.;
Ife, R. J.; Korner, M.; Ruat, M.; Schartz, J.-C.; Tertiuk, W.;
Theobald, C. J. Eur. J. Med. Chem. 1987, 22, 91.
8. General procedure for preparation of the N0-[o-(fluorescein
- 5(and - 6) - carbonyl)aminoalkyl] - N - (4 - methoxybenzyl)-N0-
methyl-N-(pyridin-2-yl)-1,2-ethanediamines 11a–d and 12a–d.
To the solution of 7a–d (0.02 mmol) in 2 mL anhydrous di-
chloromethane, the solution of 10.0 mg (0.021 mmol) of 5- and
6-carboxyfluorescein succinimidyl ester (commercially avail-
able mixture of 10A/10B with 10A as the main isomer) in 600
mL of anhydrous DMSO was added. The mixture was stirred
at room temperature in the dark for 24 h. After concentration
in vacuo the residue was chromatographed analytically (col-
umn (250ꢂ4 mm, 5 mm) and precolumn: Eurospher 100-C18
(Knauer); Kontron Instruments: HPLC Pump 420; UV-detec-
tor: HPLC Detector 430; Fluorescence detector: Merck-Hita-
chi F1000) eluent: MeOH/0.1% TFA (50/50), flow rate: 1 mL/
min; UV detection: 254 nm, fluorescence detection: lex: 490
nm, lem: 520 nm). Then portions of the crude mixture of iso-
mers (sufficient for pharmacological investigations) were pur-
ified with HPLC on a semipreparative scale (column (500ꢂ16
mm, 7 mm): Nucleosil-100-C18 (Macherey & Nagel), ALTEX
Model 110A pump, Merck-Hitachi UV Monitor 655A-22;
eluent: MeOH/0.1% TFA, 50:50 (11a,c, 12a,c), 55:45 (11b,
12b), 60:40 (11d, 12d), flow rate 6 mL/min; UV detection: 254
Bulky fluorophores are tolerated when linked to the
affinity-conferring partial structure by an appropriate
spacer group. It is conceivable that the various bulky
dyes are not interacting with amino acids of the trans-
membrane H1 receptor domains but are extending into
the extracellular space. The contribution of the fluoro-
phore to the receptor affinity could result from its
interaction with extracellular loop regions.
Conclusions
The results summarized in Table 1 show that rather
potent fluorescence-labeled H1 antagonists are obtained
by connecting normepyramine and fluorescein or NBD
via spacer groups of appropriate length. In principle,
high affinity fluorescent ligands of biogenic amine