J. Med. Chem. 2010, 53, 7095–7106 7095 DOI: 10.1021/jm1007177
Development of Molecular Probes for the Human 5-HT6 Receptor Henar Vazquez-Villa,† Juan A. Gonz alez-Vera,† Bellinda Benham u,† Alma Viso,§ Roberto Fern andez de la Pradilla,§ ‡ ‡ ,† ,† Elena Junquera, Emilio Aicart, Marı´ a L. L opez-Rodrı´ guez,* and Silvia Ortega-Gutierrez* † Departamento de Quı´mica Org anica I, and ‡Departamento de Quı´mica Fı´sica, Facultad de Ciencias Quı´micas, Universidad Complutense de Madrid, E-28040 Madrid, Spain, and §Instituto de Quı´mica Org anica General, Consejo Superior de Investigaciones Cientı´ficas, Juan de la Cierva 3, E-28006 Madrid, Spain
Received June 15, 2010
In this work we report the synthesis of a set of labeled ligands targeting the human 5-HT6 receptor (h5-HT6R). Among the synthesized compounds, fluorescent probe 10 (Ki = 175 nM and Φf = 0.21) and biotinylated derivative 15 (Ki = 90 nM) deserve special attention because they enable direct observation of the h5-HT6R in cells. Thus, they represent the first molecular probes for 5-HT6R visualization. These results are the starting point for introducing a variety of tags in these or other 5-HT6R ligand scaffolds aimed at the development of optimized probes with tailored profiles in terms of fluorescence, affinity, or selectivity.
Introduction Fluorescence-based molecular imaging facilitates visualization of specific biomolecules in their native environments. Current advances in this field include the development of fluorescent small molecules that allow detection of metabolites and ions,1,2 imaging of glycan- and lipid-containing proteins,3-5 and profiling of enzyme activities.6 However, extension of these strategies to G-protein-coupled receptors (GPCRsa), which are the target of about the 50% of current marketed drugs, has proved challenging. Changes in levels and expression pattern of GPCRs have been associated with a variety of disease states including central nervous system (CNS) related pathologies and cancer.7-9 Therefore, the ability to directly monitor GPCRs in the context of cells, tissues, and organisms would undoubtedly augment our understanding of their physiological significance and provide new clinical tools boosting drug development. Although antibodies against GPCRs enable their visualization in vitro or ex vivo, they have in vivo limited use. In addition, generation of GPCR antibodies is difficult because of their structure, poor immunogenicity, and low receptor density.10 These technical challenges have hampered the general availability of GPCR antibodies and are associated with the restricted utility in many experimental formats of some of the available ones. Thus, the development of complementary methods for GPCR imaging is of great importance. Moreover, the introduction of alternative tags other than fluorophores to obtain additional information in a highthroughput manner comparable to activity-based protein profiling approaches in the field of enzymes is still lacking.6 *For M.L.L.-R.: phone, þ34 913944239; fax, þ34 913944103; e-mail,
[email protected]. For S.O.-G.: phone, þ34 913944894; fax, þ34 913944103; e-mail,
[email protected]. a Abbreviations: 5-HT, serotonin; ACN, acetonitrile; a.u., arbitrary units; Ds, dansyl; DIPEA, N,N-diisopropylethylamine; Phth, phthalimide; BINAP, (()-2,20 -bis(diphenylphosphino)-1,10 -binaphthalene; Em, emission; Ex, excitation; Φf, fluorescence quantum yield; GPCR, G-protein-coupled receptor; h5-HT6R, type 6 of the human 5-HT receptor; HOBt, 1-hydroxybenzotriazole; I, intensity; SAR, structureactivity relationship. r 2010 American Chemical Society
With this broad objective in mind, we have initiated a project aimed at the search of fluorescent probes that enable direct visualization of GPCRs in complex biological systems. Although some GPCR fluorescent ligands have been recently described, their suitability for application in cell systems has proved challenging.11 Ideally, excellent probes would be ligands with high affinity (i.e., Ki < 100 nM) and with fluorescence emission intensity as high as possible. In this work, we have focused our efforts on the 5-HT6 receptor (5-HT6R), one of the most recent additions to the serotonin family of GPCRs. This receptor has been involved in the pathogenesis of CNS diseases related to cognitive or eating disorders,12,13 and several 5-HT6R ligands have entered clinical trials although none of them is on the market yet. In this regard, availability of agents able to directly monitor the 5-HT6R, which to our knowledge has not been described so far, would aid the therapeutic validation of this receptor for the proposed or even new indications. In this work we report the synthesis of a set of probes 1-15 based on the potent 5-HT6R antagonist developed by SmithKlineBeecham SB-27104614,15 (16, pKi = 8.9; pA2 = 8.7, Figure 1). Among the synthesized compounds, fluorescent probe 10 (Ki = 175 nM and fluorescence quantum yield, Φf, of 0.21) and biotinylated derivative 15 (Ki = 90 nM) deserve special attention, as they enable direct observation of the h5-HT6R in cells. These results support the validity of our approach and represent the starting point for further probe optimization with tailored profiles in terms of tag incorporation, affinity, and selectivity, experiments that are underway in our laboratory. Results and Discussion Initially we based our structural exploration on the scaffold of 16, in which we have studied different structural possibilities for the introduction of the fluorescent tag aiming to have a minimal influence on the biological activity of the parent compound. Accordingly, we considered small sets of probes where the tag was introduced (a) in the piperazine ring, (b) in Published on Web 09/16/2010
pubs.acs.org/jmc
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Figure 1. Labeled derivatives of the h5-HT6R antagonist 16.
Scheme 1. Synthesis of Dansylated Derivatives 1-3a
a
Reagents and conditions: (a) DsCl (1.5 equiv), Et3N (2 equiv), CH2Cl2, room temp, 12 h, 66-74%; (b) H2 (40 psi), Pd/C (8% wt), EtOH/H2O (200:1), room temp, 16 h, 94-96%; (c) 5-chloro-3-methylbenzo[b]thiophene-2-sulfonyl chloride (1.5 equiv), pyridine (2 equiv), CH2Cl2, room temp, 12 h, 62-66%; (d) NaI (2 equiv), ACN, reflux, 32 h, 79-89%; (e) TFA (20 equiv), CH2Cl2, room temp, 10 h, 99%.
the sulfonamide moiety, or (c) in the methoxy group (Figure 1). As tag, a dansyl ((5-dimethylamino)naphthalene1-sulfonyl, Ds) group was originally selected by virtue of its synthetic versatility, small size, relatively large Stokes shift, the high degree of sensitivity of its emission wavelength and quantum yield to environmental changes, and its suitability for use in cellular environments.16,17 In all cases, the tag was
attached either directly or through a variable length aliphatic spacer that may provide some flexibility to the fluorophore in order to minimally affect probe-receptor affinity. Synthesis of Probes Based on the Scaffold of 16. We prepared the first series of labeled compounds by attaching a dansyl group to the nitrogen of the piperazine ring of 16 either directly or through a spacer (Figure 1). The reaction
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Scheme 2. Synthesis of Dansylated Derivatives 4-6a
a Reagents and conditions: (a) DsCl (1.5 equiv), DIPEA (2 equiv), DMAP cat., CH2Cl2, room temp, 12 h, 85%; (b) TFA (20 equiv), CH2Cl2, room temp, 6 h, 81-97%; (c) NaH (1.8 equiv), DMF, 90 °C, 16 h, 95-96%; (d) N2H4 3 H2O (15 equiv), EtOH, 50 °C, 5 h, 86-98%; (e) DsCl (1.5 equiv), Et3N (3 equiv), CH2Cl2, room temp, 16 h, 73-76%.
Scheme 3. Synthesis of Dansylated Derivative 7a
a
Reagents and conditions: (a) DEAD (2 equiv), PPh3 (2 equiv), THF, reflux, 34 h, 60%; (b) 1-Boc-piperazine (3 equiv), Pd(OAc)2 (10 mol %), BINAP (15 mol %), Cs2CO3 (1.5 equiv), toluene, 100 °C, 24 h, 21%; (c) H2 (40 psi), Pd/C (8% wt), EtOH/H2O (100:1), room temp, 16 h, 99%; (d) 5-chloro-3methylbenzo[b]thiophene-2-sulfonyl chloride (1.5 equiv), pyridine (2 equiv), CH2Cl2, room temp, 12 h, 80%; (e) TFA (20 equiv), CH2Cl2, room temp, 10 h, 88%.
of 1-(2-methoxy-5-nitrophenyl)piperazine 17 with dansyl chloride yielded intermediate 18. Hydrogenation of the nitro group in 18 afforded the aniline derivative 19 that was then reacted with 5-chloro-3-methylbenzo[b]thiophene-2-sulfonyl chloride to afford the target compound 1. Compound 2, in which the dansyl group is attached to the piperazine through a hexamethylene chain, was prepared following a similar procedure starting with alkylation of 17 with N-Boc-6-bromohexylamine. Further increase of the spacer length (compound 3) was achieved by direct alkylation of 16 with the dansylated bromooctylamine 24 (Scheme 1). A second series of labeled ligands derived from 16 was synthesized by linking the dansyl group to the nitrogen atom of the sulfonamide group through alkyl spacers of variable length (Figure 1). Compound 4, where the dansyl group is directly attached to the core structure, was prepared by reaction of the derivative 25 with dansyl chloride in the presence of DIPEA. Compounds 5 and 6, in which the fluorophore is
linked to the sulfonamide nitrogen through four and eight carbon alkyl chains, respectively, were prepared following an alkylation sequence (Scheme 2). Thus, reaction of 25 with the corresponding N-(bromoalkyl)phthalimide afforded the alkylated products 27 and 28. Subsequent removal of the phthalimide followed by reaction with dansyl chloride led to the protected intermediates 31 and 32 that were finally transformed into the target compounds 5 and 6 by cleavage of the Boc group with TFA in dichloromethane. Finally, we also explored whether it was possible to substitute the methoxy group of 16 by a dansyl tag attached through a spacer (see Figure 1). This compound was prepared following the synthetic route shown in Scheme 3. Mitsunobu reaction of 2-bromo-4-nitrophenol with the dansyl tagged alcohol 33 yielded compound 34. Buchwald-Hartwig coupling of 34 with 1-Boc-piperazine led to 35 that was then transformed to the corresponding aniline derivative 36. Its reaction with the sulfonyl chloride derivative followed by
7098 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 Table 1. 5-HT6R Affinity and Fluorescence Values for Probes 1-7
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Scheme 4. Synthesis of Compounds 8 and 9a
a Reagents and conditions: (a) DsCl (1 equiv), pyridine (2 equiv), CH2Cl2, room temp, 15 h, 82%; (b) TFA (20 equiv), CH2Cl2, room temp, 6 h, 83-93%; (c) DsCl, DIPEA (2 equiv), DMAP cat., CH2Cl2, room temp, 12 h, 50%.
Table 2. 5-HT6R Affinity and Fluorescence Values for Probes 8-12
The values are the mean ( SEM from two to four independent experiments performed in triplicate. b Fluorescence values were determined at 10 μM in binding assay buffer (50 mM Tris-HCl, 0.5 mM MgSO4, pH 7.4) at 25 °C using an excitation slit of 2.5 nm. Excitation wavelength (λex) was 350 nm for all the compounds except for 3 (λex = 338 nm). c Emission slit of 5 nm. a
cleavage of the Boc protecting group rendered the final compound 7. Biological Data and Fluorescent Properties of Probes 1-7. Table 1 shows the affinity values for the h5-HT6R and the fluorescent properties of the probes 1-7. Affinity values were determined by competition binding assays on membranes from h5-HT6R stably expressing HEK-293 EBNA cells, using [3H]LSD as radioligand. The fluorescent properties of compounds 1-7 were determined in 10 μM solutions of the corresponding compound in the buffer solution used for the binding assays. In general, the introduction of a dansyl group at any of the positions of 16 was detrimental for the h5-HT6R affinity, which remained between moderate (compounds 1, 2, and 6) and low (compounds 3, 5, and 7). Only derivative 4 conserved high affinity (Ki = 27 nM), though in this case with a negligible fluorescence. The highest fluorescence values were shown by derivatives 2, 3, and 6. Taken together, all these data indicate that modification of 16 by attachment of a fluorophore is not likely to yield a fluorescent high-affinity 5-HT6R ligand. Therefore, we envisioned the possibility of replacing the benzothiophenesulfonyl moiety of 16 for the dansyl group. According to our previous pharmacophore model and structure-affinity relationship (SAR) studies,18 this modification
a The values are the mean ( SEM from two to four independent experiments performed in triplicate. b Fluorescence values were determined at 10 μM in binding assay buffer (50 mM Tris-HCl, 0.5 mM MgSO4, pH 7.4 at 25 °C) using an excitation slit of 2.5 nm. λex was 350 nm for 8 and 9, 340 nm for 10 and 11, and 338 nm for 12. c Emission slit of 5 nm.
was likely to keep high 5-HT6R affinity and we were confident in that this new scaffold would be fluorescent. Dansyl-Based Probes. Initially, we synthesized an analogue of 16 in which the dansyl group replaces the benzothiophenesulfonyl group (Scheme 4). Thus, reaction of N-Boc1-(5-amino-2-methoxyphenyl)piperazine 38 with dansyl chloride in the presence of pyridine yielded the expected compound 8 after deprotection of the piperazine ring by treatment with TFA. Although this compound showed a high 5-HT6R affinity (Ki = 8 nM), it was not fluorescent (Table 2). Not even the introduction of a second dansyl moiety in the sulfonamide group (derivative 9), obtained
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Scheme 5. Synthesis of Compounds 10-12a
a
Reagents and conditions: (a) Br(CH2)nNHBoc, NaI (2 equiv), ACN, reflux, 32 h, 38-89%; (b) TFA (20 equiv), CH2Cl2, room temp, 10 h, 99%; (c) DsCl (1.5 equiv), Et3N (3 equiv), CH2Cl2, room temp, 12 h, 74-79%; (d) H2 (40 psi), Pd/C (8% wt), EtOH/H2O (200:1), room temp, 16 h, 94%; (e) DsCl (1.5 equiv), pyridine (2 equiv), CH2Cl2, room temp, 16 h, 65-71%; (f) 24, NaI (2 equiv), ACN, reflux, 32 h, 45%.
Scheme 6. Synthesis of Compounds 13 and 14a
a
Reagents and conditions: (a) dapoxyl chloride (1.1 equiv), pyridine (2 equiv), CH2Cl2, room temp, 16 h, 84%; (b) TFA (20 equiv), CH2Cl2, room temp, 6 h, 91-99%; (c) Lissamine rhodamine B sulfonyl chloride (1 equiv), Et3N (5 equiv), THF, 80 °C, 24 h, 52%.
under the same conditions as 8 when diisopropylethylamine was used as base, allowed for obtaining significant fluorescence. Therefore, we focused our efforts on establishing whether the introduction of a second dansyl group attached to the piperazine by a methylene spacer of variable length would render simultaneous high 5-HT6R affinity and fluorescence. Compounds 10 and 11 were synthesized following a route analogous to that used for compound 2 (see Scheme 1), attaching the second dansyl group to the aromatic ring in the last step of the sequence, whereas derivative 12, with the longest spacer (n = 8), was synthesized by direct alkylation of the piperazine nitrogen of the parent compound 8 with the tagged chain 24 (Scheme 5). Determination of the 5-HT6R affinity and fluorescence (Table 2) of the synthesized compounds showed that 10, with a Ki value of 175 nM, a fluorescence emission intensity (Iem) above 1000 a.u. for excitation and emission slits of 2.5 and 5 nm and a high Stokes shift, exhibited the best profile in
Table 3. 5-HT6R Affinity and Fluorescence Values for Compounds 13 and 14
fluorescenceb compd 13 14
R1
R2 R3 Ki a (nM) λex c (nm) λem d (nm) Iem (a.u.)
dapoxyl H lissamine H
H 523 ( 22 H >1000
370 570
588
e 550
a The values are the mean ( SEM from two to four independent experiments performed in triplicate. b Fluorescence values were determined at 10 μM in binding assay buffer (50 mM Tris-HCl, 0.5 mM MgSO4, pH 7.4 at 25 °C). Wavelengths for the maximal absorption and emission. c Excitation slit of 2.5 nm. d Emission slit of 5 nm. e Nonfluorescent compound.
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Scheme 7. Synthesis of Biotin-Tagged Compound 15a
a Reagents and conditions: (a) NaI (2 equiv), ACN, reflux, 36 h, 81%; (b) TFA (20 equiv), CH2Cl2, room temp, 6 h, 83%; (c) D-(þ)-biotin (0.83 equiv), HOBt (0.2 equiv), DCC (1.1 equiv), DMAP (1 mol %), DMF, room temp, 24 h, 82%.
Figure 2. Labeling of COS7 cells transiently transfected with h5-HT6R with fluorescent compound 10. Cells were incubated in the presence of 10 (5 μM) for 10 min, washed, fixed, mounted, and then observed by confocal microscopy (A). To assess specificity, nontransfected cells were labeled under the same conditions (B) or transfected cells were incubated with 10 in the presence of an excess (50 μM) of 16 (C). Scale bar represents 25 μm. Preparations were visualized under a SP2 Leica confocal microscope with a 63 objective with constant laser beam and photodetector sensitivity and are representative of two or three independent experiments.
Figure 3. Labeling of COS7 cells transiently transfected with h5-HT6R with biotinylated compound 15. Cells were fixed, incubated in the presence of 15 (5 μM) for 10 min followed by streptavidin-Alexa Fluor 488 conjugate, mounted, and then observed by fluorescence microscopy. Nuclei were counterstained with Hoechst 33258 (A). To assess specificity, nontransfected cells were labeled under the same conditions (B) or in the absence of probe (C). Scale bar represents 25 μm. Preparations were visualized under a Zeiss Axioplan2 fluorescence microscope. Images are representative of two independent experiments.
terms of affinity and fluorescence and therefore could be a candidate to assess its potential as a probe for 5-HT6R visualization in cellular systems. In spite of this encouraging data, we notice that even the best values obtained for simultaneous fluorescence and affinity (compound 10, Ki=175 nM; Iem > 1000 a.u., Table 2) are only moderate, so it was conceivable that further optimization of the probes would be highly desirable for their use in complex biological settings. However, the extensive structural variations addressed in derivatives 1-12 suggest that dansyl labeling of 16 does not allow for high affinity and fluorescent 5-HT6R ligands. Therefore, we envisioned the possibility of incorporating other
fluorophores or a versatile biotin subunit. Considering that substitution of the benzothiophenesulfonyl group in 16 for a dansyl moiety (compound 8) kept 5-HT6R affinity [Ki (8) = 8 nM], we tried to replace it for dapoxyl (4-{5-[4-(dimethylamino)phenyl]-1,3-oxazol-2-yl}benzenesulfonyl) or lissamine rhodamine B (2-(chlorosulfonyl)-5-[6-(diethylamino)-3-(diethyliminio)-3Hxanthen-9-yl]benzenesulfonate), fluorescent moieties with higher extinction coefficients than dansyl group. Dapoxyl-labeled derivative 13 was synthesized by treatment of N-Boc-1-(5amino-2-methoxyphenyl)piperazine 38 with dapoxyl chloride followed by cleavage of the Boc protecting group under standard conditions. On the other hand, reaction of 38 with
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lissamine rhodamine B sulfonyl chloride (mixture of isomers) and subsequent deprotection of the piperazine ring yielded probe 14 as a mixture of isomers (Scheme 6). Unfortunately, both compounds exhibited poor 5-HT6R affinity and low fluorescence (Table 3). These results made us consider the piperazine ring as an attachment point of the biotin moiety. Thus, N-alkylation of 16 with N-Boc-6-bromohexylamine led to compound 43. Cleavage of the Boc protecting group followed by coupling reaction with biotin afforded the tagged compound 15 (Scheme 7) which was characterized as 5-HT6R ligand (Ki = 90 nM). Cell Visualization. On the basis of all these results, we have selected two probes to carry out visualization experiments, dansyl-based derivative 10, with the best balance between 5-HT6R affinity and fluorescence (Ki=175 nM; Iem>1000 a.u. for excitation and emission slits of 2.5 and 5 nm; λex = 340 nm; λem = 495 nm; Φf = 0.21), and the biotinylated probe 15 (Ki = 90 nM), which can be visualized using fluorophore-conjugated streptavidin. To assess whether these probes kept the binding profile of the parent compound 16, we selected some representative metabotropic serotonin receptors (5-HT1A, 5-HT5A, and 5-HT7) for which 16 displayed moderate affinity.15 Both compounds 10 and 15 (at 1 μM) did not significantly bind (radioligand displacement
