Synthesis and characterization of the first fluorescent antagonists for human 5-HT4 receptors

DOI: 10.1021/jm0307887

Source and publish data:

Journal of Medicinal Chemistry p. 2606 - 2620 (2003)

Update date:2022-09-26

Topics:

Authors:

Berque-Bestel, Isabelle

Soulier, Jean-Louis

Giner, Mireille

Rivail, Lucie

Langlois, Michel

Sicsic, Sames

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Article abstract of DOI:10.1021/jm0307887

Fluorescent antagonists for human 5-HT₄ receptors were synthesized based on ML10302 1, a potent 5-HT₄ receptor agonist and on piperazine analogue 2. These molecules were derived with three fluorescent moieties, dansyl, naphthalimide, and NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), through alkyl chains. The synthesized molecules were evaluated in binding assays on the recently cloned human 5-HT_4(e) receptor isoform stably expressed in C6 glial cells with [³H]GR113808 as the radioligand. The affinity values depended upon the basal structure together with the alkyl chain length. The derivatives based on ML10302 were more potent ligands than the derivatives based on piperazine analogue. For ML10302-based ligands, dansyl and NBD derivatives attached through a chain length of one carbon atom 17a and 32, respectively, led to affinities close to the affinity of ML10302. The most potent compounds 17a, 28, and 32 produced an inhibition of the 5-HT stimulated cyclic AMP synthesis in the same cellular system with nanomolar K_b values. Fluorescent properties of 17a, 28, and 32 were more particularly studied. Interactions of the fluorescent ligand 28 with the h5-HT_4(e) receptor were indicated using h5-HT_4(e) receptor transfected C6 glial cell membranes and entire cells. Ligand 28 was also used in fluorescence microscopy experiments in order to label h5-HT_4(e) receptor transfected C6 glial cells, and subcellular localization of these receptors was more precisely determined using confocal microscopy.

Full text of DOI:10.1021/jm0307887

2606

J . Med. Chem. 2003, 46, 2606-2620

Syn th esis a n d Ch a r a cter iza tion of th e F ir st F lu or escen t An ta gon ists for Hu m a n

5-HT₄Recep tor s

Isabelle Berque-Bestel, J ean-Louis Soulier, Mireille Giner, Lucie Rivail, Michel Langlois, and Sames Sicsic*

Biocis, UMR C8076 (CNRS), Faculte´ de Pharmacie, 5, rue J ean-Baptiste Cle´ment, 92296 Chaˆtenay-Malabry, France

Received J anuary 27, 2003

Fluorescent antagonists for human 5-HT₄receptors were synthesized based on ML10302 1, a

potent 5-HT₄receptor agonist and on piperazine analogue 2. These molecules were derived

with three fluorescent moieties, dansyl, naphthalimide, and NBD (7-nitrobenz-2-oxa-1,3-diazol-

4-yl), through alkyl chains. The synthesized molecules were evaluated in binding assays on

the recently cloned human 5-HT_4(e)receptor isoform stably expressed in C6 glial cells with

[³H]GR113808 as the radioligand. The affinity values depended upon the basal structure

together with the alkyl chain length. The derivatives based on ML10302 were more potent

ligands than the derivatives based on piperazine analogue. For ML10302-based ligands, dansyl

and NBD derivatives attached through a chain length of one carbon atom 17a and 32,

respectively, led to affinities close to the affinity of ML10302. The most potent compounds

17a , 28, and 32 produced an inhibition of the 5-HT stimulated cyclic AMP synthesis in the

same cellular system with nanomolar K_bvalues. Fluorescent properties of 17a , 28, and 32

were more particularly studied. Interactions of the fluorescent ligand 28 with the h5-HT_4(e)

receptor were indicated using h5-HT_4(e)receptor transfected C6 glial cell membranes and entire

cells. Ligand 28 was also used in fluorescence microscopy experiments in order to label h5-

HT_4(e)receptor transfected C6 glial cells, and subcellular localization of these receptors was

more precisely determined using confocal microscopy.

In tr od u ction

the allosteric influence of the C-terminal end on the rest

of the isoforms. The elucidation of the structural factors

in this particular phenomenon is a challenge for the

medicinal chemist.

Human 5-HT₄receptors, members of the seven trans-

membrane-spanning G-protein-coupled receptors, are

expressed in a wide variety of tissues: brain, heart,

bladder, gut, and kidney.^1,2They have been implicated

in a number of pathological disorders: irritable bowel

syndrome, gastroparesis, urinary incontinence,²cardiac

atrial arrhythmia, learning, and memorization.³Re-

cently, a large body of evidence has shown an interven-

tion of these receptors in vitro in the secretion of the

nonamyloidogenic soluble form of the amyloid precursor

protein (sAPPR) implicated in Alzheimer disease.⁴Con-

sequently, 5-HT₄receptors constitute a valuable target

for the design of new drugs. At a molecular level, many

5-HT₄receptors were identified as C-terminal splice

variants and one with an extra insertion of 14 amino

acids in the second extracellular loop.^5-7These isoforms

from different species were recently cloned and trans-

fected in different cells allowing their pharmacological

characterization,^8,9and the study of molecules as ago-

nists or antagonists of serotonin.^10,11,3To date, no

information is available on the specific role of the

different isoforms and, in pathological disorders, on the

possible implication of the variability in tissue distribu-

tion. None of the agonists or antagonists showed any

specifity for any given 5-HT₄receptor isoforms. How-

ever, some agonists presented variable efficiency strongly

dependent on the considered isoform. This observation

seems to reside in a specific pattern of expression of the

different 5-HT₄receptor isoforms in a given tissue which

may confer to the cell a unique mechanism rather than

Fluorescent receptor ligands have proven to be useful

tools for the investigation of the interactions of different

receptors with their ligands in complement to classical

methods such as radioligand binding and site-directed

mutagenesis.¹²Indeed, they offer a multiplicity of infor-

mation such as the mechanism of ligand binding,^13,14

the movement and internalization of receptors in living

cells,^15,16the distances between ligands and fluores-

cently labeled amino acids,^17,18the physical nature of

the binding pocket,^19,20and the visualization of labeled

receptors.^21,22In addition, such ligands provide an

attractive alternative to radioligands in receptor studies,

circumventing several drawbacks associated with radio-

activity such as high costs, potential health hazards, and

waste disposal problems.

To our knowledge, no fluorescent agonists or antago-

nists for 5-HT₄receptors have been described in the

literature. However, fluorescent ligands for these recep-

tors should represent valuable tools for the visualization

and the study of binding and activation mechanisms of

the isoforms in different cell types and different tissues.

Moreover, the discovery of potent and selective 5-HT₄

receptor ligands implies high throughput drug screening

which should be envisaged with fluorescent ligands by

the suitable detection method of fluorescent polariza-

tion.^23,24

Here we report the design, synthesis, and physical

characterization of the first fluorescent 5-HT₄antago-

nists and their pharmacological properties for the

* To whom correspondence should be addressed. Phone:

33(0)146835737, fax: 33(0)146835740, sames.sicsic@cep.u-psud.fr.

Published on Web 05/14/2003

Fluorescent Antagonists for Human 5-HT₄Receptors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2607

phores dansyl (5-(dimethylamino)-naphthalene-1-sul-

fonyl), NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), and naph-

thalimide (6-amino-1,3-dioxo-1H,3H-benzo(de)isoquino-

linyl), which are known to have different λ_exc(315 nm

for the dansyl moiety, and 450-470 nm for the others).

Compound 9 was prepared as described in Scheme 1.

In the first step, the weakly basic aromatic amine 3²⁶

was alkylated through reductive amination with N-Boc-

6-aminohexanal 4²⁷in the presence of sodium tri-

acetoxyborohydride in dichloroethane according to a

previously reported method.²⁸Saponification of the

resulting compound 5 with LiOH in H₂O/dioxane af-

forded the desired acid 6 with a good yield. After

activation of 6 with 1,1′-carbonyldiimidazole (CDI) in

dry THF, addition of commercial piperidineethanol to

the isolated imidazolide in the presence of KOH pro-

vided compound 7. Classical deprotection of 7 afforded

the corresponding amine hydrochloride 8, which upon

condensation with dansyl chloride in CH₂Cl₂in the

presence of diisopropylethylamine at low temperature

gave the desired fluorescent compound 9.

Preparation of compounds 17a -d is outlined in

Scheme 2. The not commercially available alcohol 10d

was prepared by reduction of 4-(3-ethoxycarbonyl-

propyl)piperidine-1-carboxylic acid benzyl ester²⁹with

NaBH₄and MeOH in dry THF. The bromo compounds

11a -d ³⁰were obtained by conversion of the correspond-

ing alcohols 10a -d ^30,31with CBr₄/PPh₃in anhydrous

THF. Condensation of 11a -d with di-tert-butylimino-

carbonate potassium salt³²in dry DMF at 40 °C gener-

ated compounds 12a -d . Classical deprotection with

ammonium formate and Pd/C 10% in MeOH provided

secondary amines 13a -d . Coupling these amines with

2-bromoethyl 4-amino-5-chloro-2-methoxybenzoate 14^11a

in the presence of diisopropylethylamine at reflux of dry

CH₃CN yielded 15a -d . Classical deprotection with

MeOH/HCl afforded 16a -d with good yield except for

16a . Finally the target fluorescent molecules 17a -d

were synthesized by condensation of 16 with dansyl

chloride in the presence of triethylamine as previously

described.

An analogous route was used to synthesize compound

20 (Scheme 3). Condensation of tert-butyl N-(4-piperi-

dine)carbamate with 14 in the presence of diisopropyl-

ethylamine at reflux of dry CH₃CN afforded 18. De-

protection gave amine hydrochloride 19, which upon

coupling with dansyl chloride in the presence of tri-

ethylamine in CH₂Cl₂at low temperature yielded the

desired fluorescent derivative 20.

The fluorescent compounds 23a -g were synthesized

as indicated in Scheme 4. Compounds 21a -g were

obtained by condensation of amine 2^11bwith respective

N-protected amino acids activated with BOP. Classical

deprotection with MeOH/HCl gave the very hygroscopic

amine hydrochlorides 22a -g with moderate yields.

Compounds 23a -g were obtained as previously indi-

cated in Scheme 2.

F igu r e 1.

human 5-HT_4(e)receptor stably transfected in C6 glial

cells. Some of them were tested for the labeling of these

C6 glial cells. Spectrofluorimetric determination of the

fluorescence bound to receptors in C6 glial cell mem-

branes and entire C6 glial cells labeled with fluorescent

antagonists was performed. We also report some results

of visualization of fluorescent ligand-labeled cells using

confocal microscopy imaging techniques.

Design a n d Syn th esis of F lu or escen t Liga n d s.

The design of fluorescent ligands of the 5-HT₄receptors

was based on our previously reported agonist ML10302^11a

1 and its piperazine analogue, 2-[N-piperazin-1-yl]ethyl

4-amino-5-chloro-2-methoxy-benzoate 2 (Figure 1).^11b

Previous mutagenesis and molecular modeling studies

performing docking of different 5-HT₄receptor ligands

have shown that the aromatic moiety of the pharma-

cophore was buried into the transmembrane domain of

the receptor in a relatively small pocket.²⁵On the

contrary, the basic amino group was located in a large

favorable region into which voluminous substituents

could be accommodated. Consequently, the piperidine

or piperazine moiety could represent a favorable position

to introduce fluorescent groups. Nevertheless, for these

compounds two points of attachment appeared chemi-

cally obvious: the aromatic amino nitrogen and the

atom in the 4-position of the piperidine or piperazine

ring. Thus, for the fluorescent analogues of ML10302,

these two positions were studied. For the molecules

derived from the first point of attachment (the aromatic

amino nitrogen), the fluorescent group was fixed through

an aminohexane linker (compound 9, Scheme 1) or

directly (compound 33, Scheme 6). Similarly, for the

molecules derived from substitution on the 4-position

of the piperidine ring, the fluorescent group was at-

tached directly (compound 20, Scheme 3) or through an

aliphatic chain of one carbon atom (compound 17a ,

Scheme 2; compound 28, Scheme 5; compound 32,

Scheme 6) to four carbon atoms (compounds 17b-d ,

Scheme 2; compound 29, Scheme 5). For the molecules

derived from the piperazine compound 2 (compounds

23a -g, Scheme 4), the fluorescent moiety was attached

on the secondary amino nitrogen through an aliphatic

carbonyl chain containing one to ten carbon atoms.

Whatever the derivative, the length of the linker was

modified in order to introduce molecular flexibility for

a possible more convenient positioning of the ligand into

the 5-HT₄receptor. Finally, we have introduced three

known fluorescent labels possessing different structures

and fluorescence properties. We selected fluorescent

groups with relatively small molecular volume to intro-

duce a weak perturbation on the affinity. Moreover, they

are known to be strongly sensitive to the polarity of the

medium with higher fluorescence in hydrophobic envi-

ronment, and they possess different absorbance and

fluorescence properties which is interesting in order to

diversify fluorescence experiments. We used the fluoro-

The fluorescent compounds 27b and 28 were prepared

according to Scheme 5. In a first step, molecules 25a ,b

were prepared by reaction of the corresponding anhy-

drides 24a or 24b³³with 1-Boc-4-aminomethylpiperi-

dine³⁴in ethanol under reflux. After deprotection,

amines 26a ,b were condensed with 14 in dry CH₃CN

or CH₃CN/CH₂Cl₂/DMF at reflux in the presence of

2608 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13

Berque-Bestel et al.

Sch em e 1^a

Reagents and conditions: (i) N-Boc-6-aminohexanal 4,²⁷NaBH(OAc)₃, AcOH, dichloroethane, rt, 24 h; (ii) LiOH, dioxane/H₂O 3/1,

reflux, 1 h, 10% KHSO₄; (iii) CDI, THF, piperidineethanol, KOH, reflux, 24 h; (iv) MeOH/HCl 3 N, 0 °C to rt, 2 h; (v) dansyl-Cl, CH₂Cl₂,

DIEA, -10 °C to 0 °C, 1.5 h.

Sch em e 2^a

Reagents and conditions: (i) PPh₃, CBr₄, THF, rt, 24 h; (ii) KNBoc₂,³²DMF, 40 °C, 24 h; (iii) ammonium formate, Pd/C 10%, MeOH,

reflux, 4-5 h; (iv) 2-bromoethyl 4-amino-5-chloro-2-methoxybenzoate 14,^11aDIEA, CH₃CN, reflux, 24 h; (v) MeOH/HCl 4N, rt, 1-4 h; (vi)

dansyl-Cl, CH₂Cl₂, NEt₃, -10 to 0 °C, 6 h; (vii) NaBH₄, then MeOH, 2 h, 70-80 °C.

Sch em e 3^a

deprotection, compound 31 was alkylated with 14 in dry

DMF and diisopropylethylamine at 40 °C for 3 days to

give the desired product 32 with a very low yield.

Compound 33 was prepared under drastic conditions by

reaction of NBD chloride with ML10302 in n-butanol

with KI at 120 °C for 12 h, with a very low yield.

Biologica l Resu lts

The affinity of labeled compounds was evaluated

routinely by binding assays on the human 5-HT_4(e)

receptor stably expressed in C6 glial cells, using

[³H]GR113808 as the radioligand.

Reagents and conditions: (i) 14,^11aDIEA, CH₃CN, reflux, 24

h; (ii) MeOH/HCl 4 N, rt,1-4 h; (iii) dansyl-Cl, CH₂Cl₂, NEt₃, 10

°C to 0 °C, 6 h.

As expected, compounds 9 and 33, where the fluores-

cent label was attached through the aromatic amino

group of compound 1, were found with no affinity for

the h5-HT_4(e)receptor. On the contrary, compounds

17a -d , 20, 27b, 28, 29, and 32 in which the fluorescent

group was fixed on the carbon atom in the 4-position of

the piperidine ring of ML10302 were found to bind to

the h5-HT_4(e)receptor. These compounds presented a

weak to good affinity for this receptor (Table 1). Simi-

larly, compounds 23a -g in which the fluorescent group

was fixed on the nitrogen atom of the piperazine ring

presented an affinity for the h5-HT_4(e)receptor.

diisopropylethylamine giving derivatives 27a ,b. Reduc-

tion of the nitro group of 27a by catalytic hydrogenation

was performed using a rapid and mild method with

ammonium formate and Raney Ni.³⁵Compound 28 was

easily yielded, avoiding the hydrogenolysis of the sensi-

tive reducible chlorine group of the aryl moiety. Product

29 was obtained by the same way as described for

preparation of compounds 25a ,b.

Fluorescent compounds 32 and 33 containing the

NBD moiety were prepared as indicated in Scheme 6.

Condensation of 4-chloro-7-nitro-2,1,3-benzoxadiazole

(NBD chloride) with 1-Boc-4-aminoethyl-piperidine in

THF at 0 °C afforded 30 with a good yield. After

The influence of the chain size was clearly demon-

strated for dansyl-labeled molecules 17-20 derived from

Fluorescent Antagonists for Human 5-HT₄Receptors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2609

Sch em e 4^a

Reagents and conditions: (i) BOP, HOOC(CH₂)_nNHBoc, NEt₃, CH₃CN, 0 °C to rt, 24 h; (ii) MeOH/HCl 4 N, rt, 2-3 h; (iii) dansyl-Cl,

CH₂Cl₂, NEt₃, -10 °C to 0 °C, 6 h.

Sch em e 5^a

Reagents and conditions: (i) 1-Boc-4-aminomethylpiperidine, EtOH, reflux, 24 h; (ii) MeOH/HCl 4 N, rt, 6 or 24 h; (iii) 14, DIEA,

CH₃CN or CH₃CN/CH₂Cl₂/DMF 30/10/10, reflux, 24 h; (iv) ammonium formate, Ra Ni, MeOH/dioxane 50/50, rt, 1 h; (v) 16d , EtOH, NEt₃,

reflux, 24 h.

1. In this series, the direct attachment of the fluorescent

group to the 4-piperidine ring (compound 20) was

deleterious for the affinity (K_i) 264 ( 100 nM), since

a link with one carbon atom (compound 17a ) is sufficient

to restore a good affinity (K_i) 7 ( 5 nM) compared to

the affinity of the reference molecule ML10302 (K_i)

1.07 ( 0.5 nM).^11aHowever, the chain length increase

of one more carbon atom (compound 17b) affects the

affinity of one order (K_i) 77 ( 24 nM), and modifying

this chain length (17c and 17d ) did not change signifi-

cantly this effect. This result confirmed the prediction

of molecular modeling studies:²⁵the large favorable

region of the receptor seemed located around the basic

amino group. Thus a fluorescent group can be allowed

near this moiety but with a relative flexibility to permit

a convenient positioning into the receptor. With a

distance of two, three, or four carbons, the fluorescent

label could leave the large pocket, going into a less

favorable area which results in a decrease of affinity.

In the piperazine series (compounds 23) where the

labeled dansyl group was attached on the secondary

amino nitrogen through an aliphatic carbonyl chain, we

observed that the minimal chain length of two carbon

atoms (compound 23a , n ) 1) induced a dramatic loss

of affinity, compared to the affinity of molecules with

bigger chain length (23b-g). Nevertheless, a fairly good

affinity was restored for a minimum chain length of

eight carbon atoms: K_i) 31 ( 9 nM for compound 23f

(n ) 7) and 96 ( 29 nM for compound 23g (n ) 10). For

a chain length of three to six carbon atoms, (compounds

23b-e), the affinity was one order lower than compound

23f. The general loss of affinity in this series could be

explained by the relative rigidity brought by the amido

junction of the chain with the piperazine moiety. Indeed,

the piperidine compounds 17a -d , more flexible for an

equivalent chain length, possessed higher affinities. The

optimal chain size for the family of piperazine ligands

appeared to be over eight carbon atoms (n g 7), which

would mean that the influence of the rigidity is canceled

for this length or that the fluorescent group is situated

outside the membrane. In this last case, these ligands

might not be really of interest for the study of the

binding site of 5-HT₄receptors.

For naphthalimide labeled series, two different amino

groups on the 4-position of the 1,8-naphthalimide ring

were studied. Compound 28 possessed a primary amino

2610 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13

Berque-Bestel et al.

Sch em e 6^a

Reagents and conditions: (i) 1-Boc-4-aminomethylpiperidine, THF, 0 °C, 2.5 h; (ii) MeOH/HCl 4 N, rt, 6 h; (iii) 14,^11aDIEA, DMF, 30

°C, 3 days; (iv) ML10302, KI, n-butanol, 120 °C, 12 h.

group on this position, since compounds 27b and 29

possessed a tertiary dimethylamino group. For com-

pound 28, the good affinity obtained confirms that a link

of one carbon atom is favorable to affinity (K_i) 64 (

12 nM). Comparison of affinities between compound 29

(K_i) 59 ( 30 nM) and compound 27b (K_i) 181 ( 62

nM) would indicate that a chain length of four carbon

atoms was necessary for restoring the affinity obtained

for primary amino group naphthalimide compound 28.

Nevertheless, compared to the dansyl label, the size of

the naphthalimide label is apparently deleterious for

affinity since compound 28 had a K_ione order higher

than that of compound 17a .

This hypothesis was validated by the affinity obtained

for NBD labeled ligand 32 (K_i) 7.5 ( 3 nM). Indeed,

with the small NBD group and a chain length of one

carbon atom, the affinity of the molecule 17a was

restored.

Despite the steric bulkiness brought by the fluores-

cent label, most of the synthesized ligands presented

an interesting affinity for the h5-HT_4(e)receptor with

two molecules at a nanomolar level, allowing these

molecules for further fluorescent experiments. It is

interesting to note that for each family of ligands,

dansyl-, naphthalimide-, NBD-, a lead (17a , 28, and 32,

respectively) could be selected.

The pharmacological profile of the labeled molecules

was determined on the activity of the second messenger

adenylate cyclase. Production of cAMP was measured

in C6 glial cells stably expressing the human 5-HT_4(e)

receptor, using a radioimmunoassay technique as previ-

ously reported.^11bIn contrast to the homologous ligand

5-HT, which stimulates the synthesis of cAMP, all the

labeled derivatives produced an antagonist effect on the

5-HT-stimulated cAMP synthesis. The competitive an-

tagonist properties of the best compounds in each series

17a , 28, and 32 were evaluated on the 5-HT concentra-

tion-effect curves. In presence of a concentration of 10

K_iof each compounds (17a , 28, and 32), the curves were

shifted to the right (Figure 2). These three labeled

compounds were found to be potent antagonists of the

h5-HT_4(e)receptor, with nanomolar K_B, as calculated by

Schild equation (Table 1).

F lu or escen ce P r op er ties of Liga n d s. For dansyl

ligands, excitation and emission maxima were 315 and

540 nm, respectively. These values were 440/450 and

538 nm, respectively, for naphthalimide ligands, and

470 and 545 nm, respectively, for NBD ligands. The

investigation of changes in properties of environment-

sensitive fluorescent labeled ligands upon interaction

with their receptors is a valuable approach to character-

ize binding sites. Thus, it is well-known that the

quantum yields of dansyl- and NBD-fluorescent groups

are dependent upon the polarity of their environment

with higher quantum yields in less polar solvents. We

found similar results for our dansyl and NBD ligands

(17a and 32) with, respectively, a 16-fold and a 6-fold

increase in quantum yield by going from aqueous to 60%

dioxane-water (v/v) HEPES buffers. This phenomenon

was accompanied by a blue-shift of the emission maxi-

mum respectively from 540 to 524 nm and 545 to 539

nm.

We observed the same phenomenon with naphthal-

imide fluorescent groups³⁶which are less used in the

literature than dansyl and NBD labels. For example,

for compound 28, the decrease in polarity (from aqueous

buffer to 60% dioxane in HEPES) induced a 5-fold

increase in quantum yield accompanied by a blue-shift

of the emission maxima from 538 to 530 nm (Figure 3).

These results show that the fluorescent naphthalimide

ligands as well as dansyl and NBD ligands will consti-

tute good probes for evaluating the nature of the binding

site and thus valuable tools for the study of the ligand-

receptor interactions.

In ter a ction of F lu or escen t Liga n d s w ith th e

Hu m a n 5-HT_4(e)Recep tor . The three leads (17a , 28,

and 32) were evaluated on h5-HT_4(e)receptor stably

transfected C6 glial cell membranes and on transfected

entire C6 glial cells cultivated on well plates.

Interactions of the fluorescent ligands with the h5-

HT_4(e)receptor were indicated by fluorescence emission

of ligands bound to receptor in a suspension of mem-

Fluorescent Antagonists for Human 5-HT₄Receptors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2611

Ta ble 1. Biological Properties of Fluorescent Compounds upon the h5-HT_4(e)Receptor

branes from stably transfected C6 glial cells. Small

increases in fluorescence were observed for ligands 17a

and 32 with a few differences between total and

nonspecific fluorescence. The naphthalimide labeled

ligand 28 gave the best results. Figure 4 shows typical

fluorescence curves representing the autofluorescence

of cell membranes alone (3), the total observed fluores-

cence due to binding of ligand 28 with the receptor (1),

and the nonspecific fluorescence determined in the

presence of a 1000-fold excess of antagonist GR113808

(2). A clearly higher intensity of ligand-induced cellular

fluorescence in cell membranes incubated in the absence

of the 5-HT₄antagonist GR113808 compared to the

fluorescence measured in its presence was observed at

520 nM. The nonspecific binding of 28 in membranes

was about 5% of the total fluorescence. In the inset, the

comparison between the intensity of naphthalimide

F igu r e 2. Concentration-effect curves for 5-HT on adenylyl

cyclase activity in C6 glial cells stably transfected with the

h5-HT_4(e)receptor isoform: 9, 5-HT alone; 2, 5-HT + 32 (100

nM); (, 5-HT + 28 (640 nM); 4, 5-HT + 17a (70 nM).

F igu r e 3. The influence of the polarity of the medium on the

fluorescence of compound 28 was investigated by addition of

dioxane (indicated in % v/v) in HEPES buffer (50 mM, pH 7.4)

measured at a λ_excof 450 nM.

2612 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13

Berque-Bestel et al.

ligands bound was about 3% for compound 28, 25% for

dansyl compound 17a , and 37% for NBD compound 32.

It is interesting to note that the results obtained on

entire cells compared to those obtained on cell mem-

branes are similar and even better. Indeed, the work

and the fluorescence measures on entire cells cultivated

on well plates are much more easy to perform and

permit, notably, the observation and studies of binding

in real time.

F lu or escen t La belin g of C6 Glia l Cells Exp r ess-

in g th e Hu m a n 5-HT_4(e)Recep tor . These labeling

experiments were realized using confocal microscopy

(Figure 6).

Ligand 28, previously shown as the best fluorescent

labeled ligand, was used for the labeling of C6 glial cells

expressing the h5-HT_4(e)receptor. The experiments were

done at a λ_excof 458 nm. Cells were incubated with

ligand 28 in the presence or absence of GR113808 and

observed at 515 nm. A clear labeling of cells was

observed as shown in Figure 6. Panel b illustrates

labeling of transfected cells. The cells were labeled but

labeling intensity varied from one cell to another. The

nonspecific labeling (panel c), obtained with an excess

of GR113808, was relatively low and not different than

the natural autofluorescence of cells (panel a). Panel d

shows the same cells observed in transmitted light.

Fluorescent labeling was mainly around the cells;

however, a few labelings could be observed in the

cytoplasmic area maybe resulting from internalization

process, even if the ligands are antagonists³⁷or because

of the presence of 5-HT₄receptors in maturation. But

further experiments should be performed for the un-

derstanding of this phenomena.

F igu r e 4. Spectrofluorimetric determination of the fluores-

cence of compound 28 bound to receptors in C6 glial cells stably

transfected with the h5-HT_4(e)receptor isoform. Excitation at

450 nm. 1 ) total fluorescence in the presence of the fluores-

cent ligand (60 nM); 2 ) nonspecific fluorescence in the

presence of the fluorescent ligand (60 nM) and GR113808 (2

µM); 3 ) autofluorescence of the cells in absence of ligand.

The inset shows the total fluorescence (1) and the nonspecific

fluorescence (2) after correction for the autofluorescence of cells

and the fluorescence of 28 (60 nM) in solution in HEPES buffer

(50 mM, pH 7.4) (4).

The same experiments realized on wild-type C6 glial

cells revealed no fluorescence intensity increase in the

presence of fluorescent ligand 28, showing that the

binding of this compound is specific of the h5-HT_4(e)

receptor.

Taken together, these results show that compound 28

constitutes a novel fluorescent probe for h5-HT₄recep-

tors. It should be noted that all these experiments were

also evaluated on cells or cell membranes after an

overnight treatment of 5 mM sodium butyrate in order

to increase receptor expression.³⁸Nevertheless, the

results were not better particularly because of the

dramatic increase of the autofluorescence of the cells

without any fluorescent ligand.

F igu r e 5. Spectrofluorimetric determination of the fluores-

cence of compounds 28, 17a , and 32 bound to receptors in C6

glial cells stably transfected with the h5-HT_4(e)receptor isoform

and cultivated on well plates (λ_exc) 450 nM). Cells were

incubated alone (autofluorescence), with the fluorescent ligand

(K_i), supplemented (nonspecific binding) or not (total binding)

with 2 µM of GR113808, in HEPES buffer (50 mM, pH 7.4).

fluorescence emission, corrected for the autofluorescence

of cells, when the antagonist ligand 28 was bound to

the h5-HT₄receptor and the fluorescence intensity of

28 in solution at the same concentration (K_i), reveals

an increase about 5-fold accompanied by a blue-shift

emission maximum from 540 to 520 nM. It indicates

that the fluorophore in receptor-bound state was in a

less polar environment than the unbound state. This

result shows that ligand 28 represents an interesting

candidate for further fluorescence experiments despite

an affinity one order lower than the reference molecule

ML10302.

Binding of the fluorescent ligands 17a , 28, and 32 to

the h5-HT_4(e)receptor was also measured on transfected

C6 glial cells cultivated on well plates with the same

procedure as for cell membranes. Results are given in

Figure 5. Naphthalimide compound 28 induced the

largest difference between total and nonspecific fluo-

rescence. Indeed, the nonspecific binding of fluorescent

Con clu sion

We have reported here the synthesis of the first

fluorescent ligands of 5-HT₄receptors on the basis of

the structural framework of ML10302 1 and of its

piperazine analogue 2. High affinity compounds for the

human 5-HT_4(e)receptor isoform were obtained, showing

antagonist profile in adenylyl cyclase assay using C6

glial cells stably expressing this receptor isoform. The

more potent fluorescent compound (28), possessing a

naphthalimide label, was shown to be a good molecular

probe for labeling the h5-HT_4(e)receptor expressed in

cellular systems. Studies are now in course to evaluate

this new fluorescent probe for the labeling of other h5-

HT₄receptor isoforms on different cellular systems, as

well as different tissues, and to perform binding studies.

Fluorescent Antagonists for Human 5-HT₄Receptors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2613

F igu r e 6. Labeling of C6 glial cells with 60 nM of compound 28 was observed on confocal microscopy. C6 glial cells were incubated

with 60 nM of 28 (panel b) and without (panel a) or with 2 µM of GR113808 (panel c). Panel c shows nonspecific labeling, and

panel d corresponds to the same cells as panel c, observed in transmitted light.

saturated aqueous NaCl (30 mL). The AcOEt layer was dried

(MgSO₄) and concentrated. The crude product was chromato-

graphed on silica gel (petroleum ether/AcOEt 70:30) to give

1.38 g (79%) of 5 as a colorless oil. R_f(petroleum ether/AcOEt

70:30) 0.44; H NMR (200 MHz): δ 7.8 (s, 1H), 6.12 (s, 1H),

4.66 (bs, 1H), 4.5 (bs, 1H), 3.9 (s, 3H), 3.8 (s, 3H), 3.15 (m,

Moreover, this probe possesses an original naphthal-

imide fluorescent group, which appeared as a good label,

for the design of fluorescent agonist ligands in order to

visualize and finely study the internalization process

of h5-HT₄receptors.

4H), 1.7 (m, 2H), 1.43 (m, 15H). Anal. (C₂₀H₃₁ClN₂O₅) C, H,

Exp er im en ta l Section

4-({6-[(ter t-Bu t oxyca r b on yl)a m in o]h e xyl}a m in o)-5-

ch lor o-2-m eth oxyben zoic Acid (6). To a solution of 5

(1.36 g, 3 mmol) in dioxane/H₂O (3:1) mixture (30 mL) was

added at room-temperature lithium hydroxyde (0.34 g, 8.1

mmol). The reaction was then heated under reflux for 1 h and

after cooling extracted with Et₂O (30 mL). The aqueous layer

was acidified to pH 2 with 10% aqueous KHSO₄, and the

precipitate formed was extracted with AcOEt (50 mL). The

organic phase was dried (MgSO₄) and evaporated to give 1.18

g (92%) of 6 as a colorless oil. R_f(petroleum ether/AcOEt 50:

Ch em istr y. Melting points were determined on a Koffler

melting point apparatus. NMR spectra were performed on a

Bruker AMX 200 (¹H, 200 MHz; ¹³C, 50 MHz) or a Bruker

AVANCE 400 (¹H, 400 MHz; ¹³C, 100 MHz). Unless otherwise

stated, CDCl₃was used as solvent. Chemical shifts δ are in

ppm, and the following abbreviations are used: singlet (s),

doublet (d), doublet doublet (dd), triplet (t), multiplet (m),

broad triplet (bt), and broad singlet (bs). Elemental analyses

(C, H, N) were performed at the microanalysis Service of the

Faculty of Pharmacy at Chaˆtenay-Malabry (France) and were

within 0.4% of the theorical values unless otherwise stated.

Mass spectra were obtained using a Bruker Esquire electro-

spray ionization apparatus.

Ma ter ia ls. Tetrahydrofuran (THF) distilled from sodium/

benzophenone, acetonitrile, dimethylformamide, and usual

solvents were purchased from SDS (Paris, France). Liquid

chromatography was performed on Merck silica gel 60 (70/30

mesh), and TLC was performed on silica gel, 60F-254 (0.26

mm thickness) plates. N-Piperidineethanol, dansyl chloride,

4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD chloride), 4-nitro-

1,8-naphthalic anhydride 24a , Boc-Gly-OH, Boc-â-Ala-OH,

Boc-4-aminobutyric acid, Boc-5-aminovaleric acid, Boc-6-amino-

caproic acid, Boc-8-aminocaprylic acid, and Boc-11-amino-

undecanoic acid were purchased from commercial sources.

4-Amino-5-chloro-2-methoxybenzoate 3, N-Boc-6-aminohex-

anal 4, benzyl 4-(hydroxymethyl)piperidine-carboxylate 10a ,³¹

benzyl 4-(2-hydroxyethyl)piperidine-carboxylate 10b,³¹benzyl

4-(3-hydroxypropyl)piperidine-carboxylate 10c,³¹potassium

salt of di-tert-butyliminocarbonate,³²and 4-N,N-dimethyl-

aminonaphthalic anhydride 24b³³were prepared according to

methods reported in the literature. Benzyl 4-(4-hydroxybutyl)-

piperidinecarboxylate 10d was obtained from benzyl 4-(4-

ethoxy-4-oxobutyl)piperidinecarboxylate as previously de-

scribed in the literature.²⁹

50) 0.31; H NMR (200 MHz): δ 8 (s, 1H), 6.12 (s, 1H), 4.84

(bt, 1H), 4.55 (bs, 1H), 4.03 (s, 3H), 3.21(m, 2H), 3.12 (m, 2H),

1.72 (m, 2H), 1.43 (m, 15H). Anal. (C₁₉H₂₉ClN₂O₅), C, H, N.

2-(P ip er id in -1-yl)et h yl 4-({6-[(ter t-Bu t oxyca r b on yl)-

a m in o]h exyl}a m in o)-5-ch lor o-2-m eth oxyben zoa te (7). To

a solution of 6 (3 g, 7 mmol) in anhydrous THF (75 mL) was

added 1,1′-carbonyldiimidazole (1.82 g, 11.2 mmol). The mix-

ture was heated under reflux for 3 h. After cooling and

concentration, the crude product was quickly chromatographed

on silica gel (CH₂Cl₂/MeOH 95:5) to afford 2.93 g (87%) of a

colorless oil (R_f: 0.44) which was immediatly treated with

piperidineethanol (0.98 g, 7.6 mmol) and KOH (2.32 g, 41.4

mmol) in dry THF (120 mL). The reaction mixture was stirred

under reflux overnight, concentrated, and diluted with CH₂Cl₂

(100 mL). After washing with saturated aqueous NaCl, the

organic phase was dried (MgSO₄) and concentrated in vacuo,

and the residue chromatographed on silica gel (CH₂Cl₂/MeOH

90:10) to give 1.73 g (48%) of 7 as a colorless oil. R_f(CH₂Cl₂/

MeOH 90:5) 0.51; ¹H NMR (200 MHz): δ 7.82 (s, 1H), 6.11 (s,

1H), 4.67 (bt, 1H), 4.5 (bs, 1H), 4.37 (t, J ) 6.1 Hz, 2H), 3.88

(s, 3H), 3.21 (m, 2H), 3.12 (m, 2H), 2.72 (t, J ) 6.1 Hz, 2H),

2.51 (m, 4H), 1.72 (m, 2H), 1.61 (m, 2H), 1.44 (m, 19H). Anal.

(C₂₆H₄₂ClN₃O₅), C, H, N.

2-(P ip er id in -1-yl)et h yl 4-[(6-Am in oh exyl)a m in o)]-5-

ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (8). To a solu-

tion of 7 (0.6 g, 1.17 mmol) in MeOH (20 mL) was added 20

mL of 3 N HCl/MeOH solution at 0 °C. The reaction was stirred

at room temperature for 2 h. After addition of anhydrous Et₂O

(20 mL), the hydrochloride salt of 8 precipitated as an

hygroscopic white powder 0.49 g (94%).¹H NMR (200 MHz):

free base δ 7.81 (s, 1H), 6.11 (s, 1H), 4.67 (bt, 1H), 4.36 (t, J )

6.1 Hz, 2H), 3.88 (s, 3H), 3.20 (m, 2H), 2.72 (t, J ) 6.1 Hz,

2H), 2.7 (m, 2H), 2.53 (m, 4H), 1.72 (m, 2H), 1.65-1.3 (m, 14H).

Anal. (C₂₁H₃₄ClN₃O₃), C, H, N.

Meth yl 4-({6-[(ter t-Bu toxyca r bon yl)a m in o]h exyl}a m -

in o)-5-ch lor o-2-m eth oxyben zoa te (5). Ester 3 (0.9 g, 3.94

mmol), aldehyde 4²⁷(1.8 g, 7.78 mmol), and glacial acetic acid

(1.43 mL) were mixed in 1,2-dichloroethane (30 mL). Then,

sodium triacetoxyborohydride (2.65 g, 12.5 mmol) was added

to the above solution, and the reaction mixture was stirred at

room temperature under argon atmosphere for 24 h. After

concentration, the product was taken up with AcOEt (30 mL),

washed with saturated aqueous NaHCO₃(3 × 30 mL) and with

2614 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13

Berque-Bestel et al.

2-(P ip er id in -1-yl)et h yl 5-Ch lor o-4-{[6-({[5-(d im et h yl-

am in o)-1-n aph th yl]su lfon yl}am in o)h exyl]am in o}-2-m eth -

oxyben zoa te (9). Amine 8 (0.14 g, 0.29 mmol) was suspended

in anhydrous CH₂Cl₂(10 mL) under argon. The solution was

cooled between -10 °C and 0 °C, then treated with DIEA (0.41

mmol) and solid dansyl chloride (92 mg, 0.34 mmol). Yellow

green reaction mixture was stirred for 1.5 h. After concentra-

tion, the residue was purified by chromatography on silica gel

(CH₂Cl₂/MeOH 98:2) to afford 96 mg (51%) of 9 as a pale green

Ben zyl 4-{3-[Bis(ter t-bu toxyca r bon yl)a m in o]p r op yl}-

p ip er id in eca r boxyla te (12c). CH₂Cl₂/Et₂O/MeOH 50:45:5;

R_f(CH₂Cl₂/ Et₂O/MeOH 50:45:5) 0.97; 92% yield; yellow oil;¹H

NMR (200 MHz): δ 7.32 (s, 5H), 5.12 (s, 2H), 4.15 (m, 2H),

3.58 (t, J ) 7 Hz, 2H), 2.74 (m, 2H), 1.83-1.45(m, 23H), 1.45-1

(m, 4H). Anal. (C₂₆H₄₀N₂O₆‚0.75H₂O), C, H, N.

Ben zyl 4-{4-[Bis(ter t-b u t oxyca r b on yl)a m in o]b u t yl}-

piper idin ecar boxylate (12d). CH₂Cl₂/ Et₂O 80:20; R_f(CH₂Cl₂/

Et₂O 80:20) 0.82; 86% yield; yellow oil; H NMR (200 MHz):

oil. R_f(CH₂Cl₂/MeOH 98:2) 0.25; H NMR (400 MHz): δ 8.54

δ 7.35 (s, 5H), 5.13 (s, 2H), 4.12 (m, 2H), 3.55 (t, J ) 7 Hz,

2H), 2.73 (m, 2H), 1.75-1.5 (m, 27H), 1.25-1 (m, 2H). Anal.

(C₂₇H₄₂N₂O₆‚0.125H₂O), C, H, N.

(d, J ) 8.5 Hz, 1H), 8.29 (d, J ) 8.6 Hz, 1H), 8.24 (dd, J ) 1

Hz and J ) 7.2 Hz, 1H), 7.81 (s, 1H), 7. 54 (m, 2H), 7.18 (d, J

) 7.5 Hz, 1H), 6.80 (s, 1H), 4.65 (bt, 1H), 4.59 (bt, 1H), 4.37 (t,

J ) 6.1 Hz, 2H), 3.87 (s, 3H), 3.10 (m, 2H), 2.90 (m, 2H), 2.88

(s, 6H), 2.72 (t, J ) 6.1 Hz, 2H), 2.50 (m, 4H), 1.60 (m, 4H),

1.53 (m, 2H), 1.44 (m, 4H), 1.25 (m, 4H). ¹³C NMR (50 MHz):

δ 164.7, 160.9, 152.1, 148.3, 134.9, 132.6, 130.4, 129.9, 129.7,

129.5, 128.3, 123.3, 118.8, 115.2, 109.8, 107.5, 94.2, 62.0, 57.5,

56.2, 54.8, 45.4, 43.0, 30.9, 29.5, 28.8, 26.4, 25.9, 25.8, 24.2.

ESI: m/z 645.4 (M + H⁺), 667.3 (M + Na⁺). Anal. (C₃₃H₄₅Cl-

N₄O₅S), C, H, N.

Syn th esis of Am in o Com p ou n d s 13a -d : Gen er a l P r o-

cedu r e. 4-{[Bis(ter t-bu toxycar bon yl)am in o]m eth yl}piper i-

d in e (13a ). A solution of 12a (1.37 g, 3 mmol), ammonium

formate (0.77 g, 12.2 mmol), and 10% Pd /C (0.25 g) in MeOH

(30 mL) was heated to reflux for 4-5 h. The reaction mixture

was filtered through Celite and concentrated in vacuo to give

a residue which was taken up by CH₂Cl₂(30 mL). The solution

was washed with 20% aqueous NH₄OH (0.5 mL), dried

(MgSO₄), and evaporated to give 0.77 g (82%) of a pale yellow

oil used without further purification in next step. ¹H NMR (200

MHz): δ 3.53 (d, J ) 6.6 Hz, 2H), 3.12 (m, 2H), 2.60 (m, 2H),

1.83-1.55 (m, 3H), 1.50 (s, 18H), 1.31-1 (m, 2H).

4-{2-[Bis(t er t -b u t oxyca r b on yl)a m in o]e t h yl}p ip e r i-

d in e (13b). 96% yield: yellow oil; ¹H NMR (200 MHz): δ 3.55

(t, J ) 7.5 Hz, 2H), 3.03 (m, 2H), 2.55 (m, 2H), 1.75-1.60 (m,

2H), 1.50 (m, 21H), 1.25-1.05 (m, 2H).

4-{3-[Bis(ter t-b u t oxyca r b on yl)a m in o]p r op yl}p ip er i-

d in e (13c). 86% yield: yellow oil;¹H NMR (200 MHz): δ 4.29

(bs, 1H), 3.50 (t, J ) 7.5 Hz, 2H), 3.12 (m, 2H), 2.55 (m, 2H),

1.75-1.40 (m, 5H), 1.51 (m, 18H), 1.31-1.08 (m, 4H).

4-{4-[Bis(t er t -b u t oxyca r b on yl)a m in o]b u t yl}p ip e r i-

d in e (13d ). 89% yield: yellow oil;¹H NMR (200 MHz): δ 3.51

(t, J ) 7.5 Hz, 2H), 3.13 (m, 2H), 2.52 (m, 2H), 2.05 (bs, 1H),

1.77-1.58 (m, 2H), 1.50 (m, 21H), 1.48-1.16 (m, 4H), 1.14-

0.8 (m, 2H).

Syn th esis of Br om o Com p ou n d s 11a -d : Gen er a l P r o-

ced u r e. Ben zyl 4-(Br om om eth yl)p ip er id in eca r boxyla te

(11a ). Triphenylphosphine (22.1 g, 84 mmol) was added in

portions to a solution of carbon tetrabromide (26.6 g, 80 mmol)

and alcohol 10a (10 g, 40 mmol) in anhydrous THF (300 mL)

at 0 °C. The reaction mixture was stirred at room temperature

for 24 h. The solution was filtered and concentrated to give

an oil which was purified by chromatography on silica gel

(CH₂Cl₂) to afford 10 g (80%) of 11a as a beige solid. R_f(CH₂Cl₂)

0.43; mp 64 °C; H NMR (200 MHz): δ 7.35 (s, 5H), 5.13 (s,

2H), 4.20 (m, 2H), 3.35 (d, J ) 6.2 Hz, 2H), 2.77 (m, 2H), 1.92-

1.63 (m, 3H), 1.31-1.05 (m, 2H). Anal. (C₁₄H₁₈BrNO₂), C, H,

Ben zyl 4-(2-Br om oeth yl)p ip er id in eca r boxyla te (11b).

R_f(CH₂Cl₂) 0.97; 80% yield; pale yellow oil; ¹H NMR (200

MHz): δ 7.30 (s, 5H), 5.11 (s, 2H), 4.20 (m, 2H), 3.48 (t, J )

6.6 Hz, 2H), 2.80 (m, 2H), 1.87-1.65 (m, 5H), 1.27-1.00 (m,

2H). Anal. (C₁₅H₂₀BrNO₂‚0.25H₂O), C, H, N.

Ben zyl 4-(3-Br om op r op yl)p ip er id in eca r boxyla te (11c).

R_f(CH₂Cl₂) 0.44; 86% yield; pale yellow oil; ¹H NMR (200

MHz): δ 7.35 (s, 5H), 5.14 (s, 2H), 4.17 (m, 2H), 3.41 (t, J )

6.6 Hz, 2H), 2.77 (m, 2H), 1.98-1.74 (m, 2H), 1.72-1.55 (m,

2H), 1.51-1.40 (m, 3H), 1.38-1 (m, 2H). Anal. (C₁₆H₂₂BrNO₂‚

0.25H₂O), C, H, N.

Syn th esis of Com p ou n d s 15a -d , 18: Gen er a l P r oce-

d u r e. 2-(4-{[Bis(ter t-b u t oxyca r b on yl)a m in o]m et h yl}p i-

p er id in -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoa te

(15a ). A mixture of 14 (0.89 g, 2.9 mmol), N-Boc-protected

amine 13a (0.7 g, 2.23 mmol), and DIEA (0.81 mL, 4.96 mmol)

in dry CH₃CN (30-40 mL) was refluxed for 24 h under argon.

The resulting mixture was concentrated in vacuo and taken

up in CH₂Cl₂. The organic layer was washed with brine, dried

(MgSO₄), and concentrated. Column chromatography (CH₂Cl₂/

Et₂O 50:50 then CH₂Cl₂/MeOH 95:5) gave 0.93 g (59%) of 15a

Ben zyl 4-(4-Br om obu tyl)p ip er id in eca r boxyla te (11d ).

R_f(CH₂Cl₂) 0.6; 77% yield; pale yellow oil; ¹H NMR (200

MHz): δ 7.42 (s, 5H), 5.12 (s, 2H), 4.19 (m, 2H), 3.42 (t, J )

6.6 Hz, 2H), 2.75 (m, 2H), 1.92-1.73(m, 2H), 1.72-1.52 (m,

2H), 1.50-1.32 (m, 5H), 1.12-1 (m, 2H). Anal. (C₁₇H₂₄BrNO₂),

C, H, N.

as a beige solid. R_f(CH₂Cl₂/MeOH 95:5) 0.59; mp 126 °C; H

NMR (200 MHz): δ 7.76 (s, 1H), 6.27 (s, 1H), 4.42 (bs, 2H),

4.36 (t, J ) 6.1 Hz, 2H), 3.83 (s, 3H), 3.47 (d, J ) 6.1 Hz, 2H),

2.96 (m, 2H), 2.74 (t, J ) 6.1 Hz, 2H), 2.12 (m, 2H), 1.65 (m,

2H), 1.81 (m, 1H), 1.49 (s, 18H), 1.31 (m, 2H). Anal. (C₂₆H₄₀

Syn th esis of Boc-P r otected Com p ou n d s 12a -d : Gen -

er a l P r oced u r e. Ben zyl 4-{[Bis(ter t-bu toxyca r bon yl)-

a m in o]m eth yl}p ip er id in eca r boxyla te (12a ). To a vigor-

ously stirred slurry of well dried, finely ground potassium salt

of di-tert-butyl iminodicarbonate (1.6 g, 6.3 mmol) in dry DMF

(40 mL) was added dropwise under argon a solution of 11a

(2.15 g, 6.9 mmol) in dry DMF (20 mL). The reaction mixture

was heated at 40 °C for 24 h. After evaporation, the residue

was taken up with CH₂Cl₂(40 mL), the organic phase was

washed with aqueous NaHCO₃10%, dried (MgSO₄), and

evaporated. Purification by chromatography on silica gel

(CH₂Cl₂/Et₂O 95:5) afforded 1.37 g (49%) of 12a as a pale

yellow oil. R_f(CH₂Cl₂/Et₂O 95:5) 0.42; ¹H NMR (200 MHz): δ

7.35 (s, 5H), 5.11 (s, 2H), 4.13 (m, 2H), 3.52 (t, J ) 7 Hz, 2H),

2.75 (m, 2H), 1.81(m, 1H), 1.75-1.45(m, 20H), 1.30-1.02 (m,

2H). Anal. (C₂₄H₃₆N₂O₆‚0.75H₂O), C, H, N.

ClN₃O₇‚0.5H₂O), C, H, N.

2-(4-{2-[Bis(ter t-bu toxycar bon yl)am in o]eth yl}piper idin -

1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoa te (15b).

CH₂Cl₂/Et₂O 50:50 then CH₂Cl₂/MeOH 95:5; R_f(CH₂Cl₂/MeOH

95:5) 0.12; mp 116.5 °C; 84% yield; beige solid;¹H NMR (200

MHz): δ 7.79 (s, 1H), 6.27 (s, 1H), 4.47 (bs, 2H), 4.35 (t, J )

6.1 Hz, 2H), 3.82 (s, 3H), 3.57 (m, 2H), 2.96 (m, 2H), 2.70 (t, J

) 6.1 Hz, 2H), 2.20 (m, 2H), 1.7-1.67 (m, 3H), 1.49 (m, 20H);

1.37-1.25 (m, 2H). Anal. (C₂₇H₄₂ClN₃O₇‚0.5H₂O), C, H, N.

2-(4-{3-[Bis (ter t-b u t oxyca r b on yl)a m in o]p r op yl}p i-

p er id in -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoa te

(15c). CH₂Cl₂/Et₂O 50:50 then CH₂Cl₂/MeOH 90:10; R_f(CH₂Cl₂/

MeOH 90:10) 0.60; mp 120 °C; 45% yield; white solid;¹H NMR

(200 MHz): δ 7.82 (s, 1H), 6.28 (s, 1H), 4.41 (bs, 2H), 4.33 (t,

J ) 6.1 Hz, 2H), 3.84 (s, 3H), 3.48 (m, 2H), 2.95 (m, 2H), 2.70

(t, J ) 6.1 Hz, 2H), 2.05 (m, 2H), 1.8-1.5 (m, 3H), 1.5 (m, 20H),

1.35-1.1 (m, 4H). Anal. (C₂₈H₄₄ClN₃O_{7 .}H₂O), C, H, N.

2-(4-{4-[Bis (ter t-bu toxycar bon yl)am in o]bu tyl}piper idin -

1-yl)et h yl 4-Am in o-5-ch lor o-2-m et h oxyb en zoa t e (15d ).

CH₂Cl₂/Et₂O 50:50 then CH₂Cl₂/MeOH 95:5 R_f(CH₂Cl₂/MeOH

95:5) 0.58; 71% yield; pale brown oil;¹H NMR (200 MHz): δ

7.79 (s, 1H), 6.28 (s, 1H), 4.48 (bs, 2H), 4.35 (t, J ) 6.1 Hz,

Ben zyl 4-{2-[Bis(ter t-b u t oxyca r b on yl)a m in o]et h yl}-

p ip er id in eca r boxyla te (12b). R_f(CH₂Cl₂/Et₂O 95:5) 0.25;

74% yield; yellow oil; ¹H NMR (200 MHz): δ 7.35 (s, 5H), 5.12

(s, 2H), 4.17 (m, 2H), 3.62 (t, J ) 7 Hz, 2H), 2.78 (m, 2H),

1.81-1.57(m, 3H), 1.50 (m, 20H), 1.25-1.05 (m, 2H). Anal.

(C₂₅H₃₈N₂O₆‚0.25H₂O), C, H, N.

Fluorescent Antagonists for Human 5-HT₄Receptors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2615

2H), 3.81(s, 3H), 3.52 (m, 2H), 2.95 (m, 2H), 2.71 (t, J ) 6.1

Hz, 2H), 2.05 (m, 2H), 1.69-1.49 (m, 5H), 1.49 (bs, 18H), 1.42-

1.12 (m, 6H). Anal. (C₂₉H₄₆ClN₃O₇‚0.75H₂O), C, H, N.

2-{4-[2-({[5-(Dim e t h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)et h yl]p ip er id in -1-yl}et h yl 4-Am in o-5-ch lor o-2-

m eth oxyben zoa te (17b). CH₂Cl₂then CH₂Cl₂/Et₂O/iPrOH

50:45:5; R_fCH₂Cl₂/Et₂O/iPrOH 50:45:5) 0.17; 88% yield; pale

green foam; ¹H NMR (200 MHz): δ 8.55 (m, 1H), 8.25 (m, 2H),

7.79 (s, 1H), 7.56 (m, 2H), 7.19 (m, 1H), 6.28 (s, 1H), 4.47 (bt,

1H,), 4.26 (bs, 2H), 4.30 (t, J ) 6.1 Hz, 2H), 3.83 (s, 3H), 2.95

(m, 2H), 2.86 (s, 6H), 2.80 (bd, 2H), 2.64 (t, J ) 6.1 Hz, 2H),

1.85 (m, 2H), 1.41-1.15 (m, 4H), 1.15-0.95 (m, 3H). ¹³C NMR

(50 MHz): δ 164.5, 160.3, 152.1, 147.9, 134.7, 133.3, 130.5,

129.7, 128.4, 123.2, 118.7, 115.3, 109.9, 98.3, 62.1, 56.9, 56.1,

53.8, 45.4, 42.9, 40.7, 35.9, 32.4, 31.8. Anal. (C₂₉H₃₇ClN₄O₅S‚

0.5H₂O), C, H, N.

2-{4-[3-({[5-(Dim e t h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)p r op yl]p ip er id in -1-yl}eth yl 4-Am in o-5-ch lor o-2-

m eth oxyben zoa te (17c). CH₂Cl₂then CH₂Cl₂/Et₂O/MeOH

50:40:10; R_fCH₂Cl₂/Et₂O/MeOH 50:40:10) 0.20; 59% yield; pale

green foam; ¹H NMR (200 MHz): δ 8.55 (m, 1H), 8.29 (m, 2H),

7.75 (s, 1H,), 7.53 (m, 2H,), 7.11 (m, 1H,), 6.28 (s, 1H), 4.57

(bt, 1H), 4.42 (bs, 2H), 4.27 (t, J ) 6.1 Hz, 2H), 3.80 (s, 3H),

2.82 (m, 10H), 2.62 (t, J ) 6.1 Hz, 2H), 1.92 (m, 2H), 1.65 (m,

2H), 1.45-1.15 (m, 3H), 1.1-0.8 (m, 4H). ¹³C NMR (50 MHz):

δ 164.5, 160.3, 152.1, 148.0, 134.9, 133.3, 130.4, 129.6, 128.4,

123.2, 118.7, 115.2, 109.9, 98.3, 62.1, 56.9, 56.1, 54.0, 45.4, 43.4,

34.9, 33.1, 31.9, 26.7. Anal. (C₃₀H₃₉ClN₄O₅S‚0.5H₂O), C, H, N.

2-{4-[(t er t -Bu t oxyca r b on yl)a m in o]p ip e r id in -1-yl}-

eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoa te (18). CH₂Cl₂/

Et₂O 50:50 then CH₂Cl₂/Et₂O/MeOH 50:45:5; R_f(CH₂Cl₂/Et₂O/

MeOH 50:45:5) 0.44; 39% yield; white foam solid; mp 88-89

°C; ¹H NMR (200 MHz): δ 7.80 (s, 1H), 6.27 (s, 1H), 4.36 (bs,

1H), 4.43 (bs, 2H), 4.35 (t, J ) 6.1 Hz, 2H), 3.83 (s, 3H), 3.49

(m, 1H), 2.93 (m, 2H), 2.72 (t, J ) 6.1 Hz, 2H), 2.22 (m, 2H),

1.95 (m, 2H), 1.65 (m, 2H), 1.44 (s, 9H). Anal. (C₂₀H₃₀ClN₃O₅‚

0.25H₂O), C, H, N.

Syn th esis of Com p ou n d s 16a -d , 19: Gen er a l P r oce-

d u r e. 2-[4-(Am in om et h yl)p ip er id in -1-yl]et h yl 4-a m in o-

5-ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (16a ). 15a

(0.91 g, 1.67 mmol) was dissolved in MeOH (25-30 mL), and

20 mL of MeOH/HCl 4 N was added. After 1-4 h at room

temperature, 0.37 g (54%) of 16a was obtained as a very

hygroscopic beige powder after precipitation with Et₂O or

iPr₂O. H NMR (200 MHz): free base δ 7.77 (s, 1H), 6.23 (s,

1H), 4.58 (bs, 2H), 4.29 (t, J ) 6.1 Hz, 2H), 3.76 (s, 3H), 2.97

(m, 2H), 2.75 (t, J ) 6.1 Hz, 2H), 2.5 (d, J ) 6.1 Hz, 2H), 2.04

(m, 2H), 1.63 (m, 2H), 1.21 (m, 5H). Anal. (C₁₆H₂₄ClN₃O₃‚2HCl‚

2H₂O), C, H, N.

2-[4-(2-Am in oeth yl)piper idin -1-yl]eth yl 4-Am in o-5-ch lo-

r o-2-m eth oxyben zoa te Hyd r och lor id e (16b). Very hygro-

scopic pearly amorphous solid; 71% yield; ¹H NMR (200

MHz): free base δ 7.80 (s, 1H), 6.27 (s, 1H), 4.45 (bs, 2H), 4.38

(t, J ) 6.1 Hz, 2H), 3.82 (s, 3H), 2.95 (m, 2H), 2.71 (m, 4H),

2-{4-[4-({[5-(Dim e t h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)b u t yl]p ip er id in -1-yl}et h yl 4-Am in o-5-ch lor o-2-

m eth oxyben zoa te (17d ). CH₂Cl₂then eluent system A; R_f

(system A) 0.14; Yield ) 70%; pale green foam; ¹H NMR (200

MHz): δ 8.47 (m, 1H), 8.22 (m, 2H), 7.75 (s, 1H), 7.51 (m, 2H),

7.09 (m, 1H), 6.28 (s, 1H), 4.55 (bt, 1H), 4.40 (bs, 2H), 4.29 (t,

J ) 6.1 Hz, 2H), 3.80 (s, 3H), 2.88 (m, 10H), 2.60 (t, J ) 6.1

Hz, 2H), 1.95 (m, 2H), 1.8-0.85 (m, 11H). ¹³C NMR (50 MHz):

δ 164.4, 160.2, 151.9, 147.8, 134.8, 133.2, 130.3, 129.8, 129.6,

129.5, 128.2, 123.0, 118.6, 115.0, 109.8, 109.6, 98.2, 62.0, 56.9,

2.07 (m, 2H), 1.66 (m, 2H), 1.45-0.6 (m, 7H). Anal. (C₁₇H₂₆

ClN₃O₃‚2HCl‚0.75H₂O), C, H, N.

2-[4-(3-Am in op r op yl)p ip er id in -1-yl]et h yl 4-Am in o-5-

ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (16c). Very

hygroscopic beige amorphous solid; 77% yield; H NMR (200

MHz): free base δ 7.77 (s, 1H), 6.24 (s, 1H), 4.53 (bs, 2H), 4.47

55.9, 54.0, 45.3,43.2, 35.7, 35.1, 32.0, 29.6, 23.5. Anal. (C₃₁H₄₁

ClN₄O₅S), C, H, N.

(t, J ) 6.1 Hz, 2H), 3.78 (s, 3H), 2.94 (m, 2H), 2.67 (m, 4H),

2.12 (m, 2H), 1.63 (m, 2H), 1.50-0.85 (m, 9H). Anal. (C₁₈H₂₈

ClN₃O₃‚2HCl‚1.25H₂O), C, H, N.

2-[4-({[5-(Dim eth ylam in o)-1-n aph th yl]su lfon yl}am in o)-

p ip er id in -1-yl]et h yl 4-Am in o-5-ch lor o-2-m et h oxyb en z-

oa te (20). CH₂Cl₂then eluent system A; R_f(system A) 0.5;

2-[4-(4-Am in ob u t yl)p ip er id in -1-yl]et h yl 4-Am in o-5-

ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (16d ). Very

94% yield; pale green foam; H NMR (200 MHz): δ 8.54 (m,

hygroscopic beige amorphous solid; 61% yield; H NMR (200

1H), 8.22 (m, 2H), 7.74 (s, 1H), 7.55 (m, 2H), 7.17 (m, 1H),

6.26 (s, 1H), 4.58 (bd, 1H), 4.43 (bs, 2H), 4.27 (t, J ) 6.1 Hz,

2H), 3.79 (s, 3H), 2.89 (s, 6H), 2.72 (m, 2H), 2.64 (t, J ) 6.1

Hz, 2H), 2.11 (m, 2H), 1.65 (m, 3H), 1.5-1.22 (m, 2H). ¹³C NMR

(50 MHz): δ 164.4, 160.2, 151.9, 147.9, 135.8, 133.2, 130.4,

129.9, 129.5, 129.2, 128.3, 123.1, 118.7, 115.1, 109.8, 109.4,

98.2, 61.8, 56.4, 55.9, 51.9, 45.3, 32.7. Anal. (C₂₇H₃₃ClN₄O₅‚

0.75H₂O), C, H, N.

MHz): free base δ 7.78 (s, 1H), 6.27 (s, 1H), 4.52 (bs, 2H), 4.37

(t, J ) 6.1 Hz, 2H), 3.81 (s, 3H), 2.94 (m, 2H), 2.72 (t, J ) 6.1

Hz, 2H), 2.65 (t, J ) 6.8 Hz, 2H), 2.08 (m, 2H), 1.61 (m, 2H),

1.50-0.95 (m, 11H). Anal. (C₁₉H₃₀ClN₃O₃‚2HCl‚1.25H₂O), C,

H, N.

2-(4-Am in op ip er id in -1-yl)et h yl 4-Am in o-5-ch lor o-2-

m eth oxyben zoa te Hyd r och lor id e (19). Hygroscopic white

powder; 85% yield; H NMR (200 MHz): free base δ 7.78 (s,

Syn th esis of P ip er a zin yl Com p ou n d s 21a -g: Gen er a l

Procedure.2-(4-{2-[(tert-Butoxycarbonyl)amino]acetyl}piper-

azin -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoate (21a).

A solution of Boc-gly-OH (0.6 g, 3.42 mmol) and BOP (1.81 g,

4.1 mmol) in dry CH₃CN (40 mL) was treated at 0 °C with

NEt₃(1.21 g, 11.9 mmol). The mixture was stirred for 0.5 h,

and then hydrochloride salt of 2 (1.32 g, 3.42 mmol) was added.

The reaction was continued at room temperature for 24 h. The

solvent was removed and the residue taken up in CH₂Cl₂(80

mL). The organic layer was washed with 10% aqueous KHSO₄

(80 mL), saturated NaHCO₃(80 mL), and brine, dried (MgSO₄),

and concentrated. The residue was purified by chromatography

on silica gel (eluent system A) to afford 0.86 g (65%) of 21a as

a pale yellow oil which was used without further purification

1H), 6.25 (s, 1H), 4.50 (bs, 2H), 4.33 (t, J ) 6.1 Hz, 2H), 3.80

(s, 3H), 2.93(m, 2H), 2.70 (t, J ) 6.1 Hz, 2H), 2.68 (m, 1H),

2.18 (m, 2H), 1.82 (m, 2H), 1.41 (m, 2H), 1 (bs, 2H). Anal.

(C₁₅H₂₂ClN₃O₃‚2HCl‚0.5H₂O), C, H, N.

Syn th esis of Da n syl Com p ou n d s 17a -d , 20: Gen er a l

P r oced u r e. 2-{4-[({[5-(Dim et h yla m in o)-1-n a p h t h yl]su l-

fon yl}am in o)m eth yl]piper idin -1-yl}eth yl 4-Am in o-5-ch lo-

r o-2-m eth oxyben zoa te (17a ). To a cold solution (-10 °C and

0 °C) of anhydrous CH₂Cl₂(20-30 mL) under argon, contain-

ing 16a (0.1 g, 0.24 mmol) and NEt₃(0.168 mL), was added

dansyl chloride (0.078 g, 0.29 mmol) in several portions (0.168

mL). The yellow green reaction mixture was stirred between

-10 °C and 0 °C for 6 h. Then the solvent was evaporated,

and the residue was purified by chromatography (CH₂Cl₂then

eluent system A CH₂Cl₂/Et₂O/MeOH 50:45:5) to give 93 mg

(67%) of 17a as a pale green foam. R_f(system A) 0.24; ¹H NMR

(200 MHz): δ 8.55 (m, 1H), 8.23 (m, 2H), 7.74 (s, 1H), 7.56

(m, 2H), 7.12 (m, 1H), 6.26 (s, 1H), 4.6 (bt, 1H), 4.42 (bs, 2H),

4.27 (t, J ) 6.1 Hz, 2H), 3.82 (s, 3H), 2.89 (s, 6H), 2.87 (m,

2H), 2.73 (m, 2H), 2.64 (t, J ) 6.1 Hz, 2H), 1.85 (m, 2H), 1.7-

1.45 (m, 3H), 1.27-0.95 (m, 2H). ¹³C NMR (50 MHz): δ 164.4,

160.2, 151.9, 147.7, 134.6, 133.2, 130.3, 129.8, 129.5, 128.3,

123.1, 118.5, 115.1, 109.7, 98.2, 61.9, 56.7, 55.9, 53.3, 48.7, 45.3,

35.7, 29.5. Anal. (C₂₈H₃₅ClN₄O₅S), C, H, N.

in the next step. R_f(system A) 0.22; H NMR (200 MHz): δ

7.80 (s, 1H), 6.31 (s, 1H), 5.5 (bs, 1H), 4.55 (bs, 2H), 4.4 (t, J

) 5.9 Hz, 2H), 3.93 (m, 2H), 3.8 (s, 3H), 3.61 (m, 2H), 3.4 (m,

2H), 2.75 (t, J ) 5.9 Hz, 2H), 2.55 (m, 4H), 1.45 (s, 9H).

2-(4-{3-[(ter t-Bu toxyca r bon yl)a m in o]p r op a n oyl}p ip er -

azin -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoate (21b).

From the reaction of 2 and Boc-â-alanine. Eluent system A;

R_f(system A) 0.48; 99% yield; yellow oil; ¹H NMR (200 MHz):

δ 7.80 (s, 1H), 6.30 (s, 1H), 5.25 (bs, 1H), 4.55 (bs, 2H), 4.35 (t,

J ) 5.9 Hz, 2H), 3.85 (s, 3H), 3.61 (m, 2H), 3.40 (m, 4H), 2.75

(t, J ) 5.9 Hz, 2H), 2.51(m, 6H), 1.44 (s, 9H).

2616 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13

Berque-Bestel et al.

2-(4-{4-[(ter t-Bu toxyca r bon yl)a m in o]bu ta n oyl}p ip er -

azin -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoate (21c).

From the reaction of 2 and Boc-4-aminobutyric acid. Eluent

system A; R_f(System A) 0.25; 88% yield; viscous orange oil;

¹H NMR (200 MHz): δ 7.78 (s, 1H), 6.27 (s, 1H), 4.8 (bs, 1H),

4.48 (bs, 2H), 4.30 (t, J ) 5.9 Hz, 2H), 3.82 (s, 3H), 3.60 (m,

2H), 3.45 (m, 2H), 3.15 (m, 2H), 2.75 (t, J ) 5.9 Hz, 2H), 2.52

(m, 4H), 2.35 (t, J ) 7.2 Hz, 2H), 1.81 (quintet, J ) 7.2 Hz,

2H), 1.41 (s, 9H).

2-(4-{5-[(ter t-Bu toxyca r bon yl)a m in o]p en ta n oyl}p ip er -

azin -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoate (21d).

From the reaction of 2 and Boc-4-aminovaleric acid. Eluent

system A; R_f(system A) 0.23; 61% yield; viscous yellow oil; ¹H

NMR (200 MHz): δ 7.78 (s, 1H), 6.29 (s, 1H), 4.63 (bs, 1H),

4.53 (bs, 2H), 4.35 (t, J ) 5.9 Hz, 2H), 3.82 (s, 3H), 3.61 (m,

2H), 3.45 (m, 2H), 3.08 (m, 2H), 2.74 (t, J ) 5.9 Hz, 2H), 2.51

(m, 4H), 2.32 (t, J ) 7.2 Hz, 2H), 1.75-1.5 (m, 4H), 1.45 (s,

9H).

2-(4-{6-[(ter t-Bu toxyca r bon yl)a m in o]h exa n oyl}p ip er -

azin -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoate (21e).

From the reaction of 2 and Boc-4-aminocaproic acid. Eluent

system A; R_f(system A) 0.53; 45% yield; colorless oil; ¹H NMR

(200 MHz): δ 7.80 (s, 1H), 6.25 (s, 1H), 4.52 (bs, 1H), 4.50 (bs,

2H), 4.35 (t, J ) 5.9 Hz, 2H), 3.80 (s, 3H), 3.61 (m, 2H), 3.45

(m, 2H), 3.10 (m, 2H), 2.75 (t, J ) 5.9 Hz, 2H), 2.51 (m, 4H),

2.30 (t, J ) 7.2 Hz, 2H), 1.75-1.3 (m, 15H).

2-(4-{8-[(ter t-Bu t oxyca r b on yl)a m in o]oct a n oyl}p ip er -

azin -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoate (21f).

From the reaction of 2 and Boc-4-aminocaprylic acid. Eluent

system A; R_f(system A) 0.55; 95% yield; colorless oil; ¹H NMR

(200 MHz): δ 7.78 (s, 1H), 6.28 (s, 1H), 4.53 (bs, 3H), 4.38 (t,

J ) 5.9 Hz, 2H), 3.82 (s, 3H), 3.63 (m, 2H), 3.46 (m, 2H), 3.05

(m, 2H), 2.74 (t, J ) 5.9 Hz, 2H), 2.51 (m, 4H), 2.29 (t, J ) 7.2

Hz, 2H), 1.75-1.20 (m, 19H).

3.60 (m, 2H), 3.45 (m, 2H), 2.71 (m, 4H), 2.5 (m, 4H), 2.30 (t,

J ) 7.2 Hz, 2H), 1.63 (m, 2H), 1.48 (m, 2H), 1.10 (bs, 2H).

ESI: m/z 413.1 (M + H⁺). Anal. (C₁₉H₂₉Cl N₄O₄‚2HCl‚3H₂O),

C, H, N.

2-[4-(6-Am in oh exa n oyl)p ip er a zin -1-yl]eth yl 4-Am in o-

5-ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (22e). Same

procedure as described for 16a . 42% yield; very hygroscopic

white nacre solid; ¹H NMR (200 MHz): free base δ 7.76 (s,

1H), 6.26 (s, 1H), 4.56 (bs, 2H), 4.33 (t, J ) 5.9 Hz, 2H), 3.80

(s, 3H), 3.59 (m, 2H), 3.43 (m, 2H), 2.71 (m, 4H), 2.50 (m, 4H),

2.28 (t, J ) 7.2 Hz, 2H), 1.61 (m, 2H), 1.41 (m, 4H), 1.10 (bs,

2H). ESI: m/z 427.2 (M + H⁺). Anal. (C₂₀H₃₁ClN₄O₄‚2HCl‚

2.5H₂O) C, H, N.

2-[4-(8-Am in ooct a n oyl)p ip er a zin -1-yl]et h yl 4-Am in o-

5-ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (22f). Same

procedure as described for 16a . 48% yield; very hygroscopic

nacre foam; ¹H NMR (200 MHz): free base δ 7.64 (s, 1H), 6.26

(s, 1H), 4.58 (bs, 2H), 4.33 (t, J ) 5.9 Hz, 2H), 3.80 (s, 3H),

3.59 (m, 2H), 3.42 (m, 2H), 2.71 (t, J ) 5.9 Hz, 2H), 2.63 (t, J

) 7.2 Hz, 2H), 2.49 (m, 4H), 2.27 (t, J ) 7.2 Hz, 2H), 1.58 (m,

2H), 1.50-1.2 (m, 8H), 1.21 (bs, 2H). ESI: m/z 455.2 (M +

H⁺). Anal. (C₂₂H₃₅ClN₄O₄‚2HCl‚1.5H₂O), C, H, N.

2-[4-(11-Am in ou n decan oyl)piper azin -1-yl]eth yl4-Am in o-

5-ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (22g). Same

procedure as described for 16a . 66% yield; very hygroscopic

beige foam; ¹H NMR (200 MHz): free base δ 7.75 (s, 1H), 6.25

(s, 1H), 4.62 (bs, 2H), 4.32 (t, J ) 5.9 Hz, 2H), 3.78 (s, 3H),

3.57 (m, 2H), 3.42 (m, 2H), 2.70 (t, J ) 5.9 Hz, 2H), 2.61 (t, J

) 7.2 Hz, 2H), 2.49 (m, 4H), 2.25 (t, J ) 7.2 Hz, 2H), 1.55 (m,

2H), 1.50-1.2 (m, 14H), 1.15 (bs, 2H). ESI: m/z 569.1 (M +

H⁺). Anal. (C₂₅H₄₁ClN₄O₄‚2HCl‚3.5H₂O), C, H, N.

2-{4-[2-({[5-(Dim e t h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)a cetyl]p ip er a zin -1-yl}eth yl 4-Am in o-5-ch lor o-2-

m eth oxyben zoa te (23a ). Same procedure as described for

17a except NEt₃was used instead of DIEA as base. CH₂Cl₂

then eluent system A; R_f(system A) 0.45; 86% yield; pale green

foam; ¹H NMR (200 MHz): δ 8.55 (m, 1H), 8.35 (m, 1H), 8.23

(m, 1H), 7.80 (s, 1H), 7.55 (m, 2H), 7.15 (m, 1H), 6.30 (s, 1H),

5.9 (t, J ) 4.3 Hz, 1H), 4.47 (bs, 2H), 4.36 (t, J ) 6.1 Hz, 2H),

3.81 (s, 3H), 3.71 (d, J ) 4.3 Hz, 2H), 3.54 (m, 2H), 3.27 (m,

2H), 2.88 (s, 6H), 2.75 (t, J ) 6.1 Hz, 2H), 2.5 (m, 4H). ¹³C

NMR (50 MHz, CDCl_3,CD₃OD): δ 165.2, 164.6, 160.3, 151.7,

148.5, 133.9, 132.9, 130.5, 129.8, 129.5, 129.1, 128.4, 122.9,

118.7, 115.3, 109.6, 108.4, 97.9, 61.3, 56.2, 55.7, 52.6, 52.4,

45.2,44.1, 43.5, 41.9. Anal. (C₂₈H₃₄ClN₅O₆S), C, H, N.

2-{4-[3-({[5-(Dim e t h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)p r op a n oyl]piper a zin -1-yl}eth yl 4-Am in o-5-ch lor o-

2-m eth oxyben zoa te (23b). Same procedure as described for

17a except NEt₃was used instead of DIEA as base. CH₂Cl₂/

Et₂O 50:50 then CH₂Cl₂/Et₂O/MeOH 50:47:3; R_f(CH₂Cl₂/Et₂O/

MeOH 50:47:7) 0.45; 92% yield; pale green foam; ¹H NMR (200

MHz): δ 8.52 (m, 1H), 8.24 (m, 2H), 7.80 (s, 1H), 7.55 (m, 2H),

7.20 (m, 1H), 6.29 (s, 1H), 5.84 (m, 1H), 4.47 (bs, 2H), 4.34 (t,

J ) 6.1 Hz, 2H), 3.84 (s, 3H), 3.48 (m, 2H), 3.20 (m, 4H), 2.87

(s, 6H), 2.73 (t, J ) 6.1 Hz, 2H), 2.40 (m, 6H). ¹³C NMR (50

MHz, CDCl_3,CD₃OD): δ 169.4, 164.6, 160.3, 151.7, 148.5,

135.1, 132.9, 130.2, 129.7, 129.4, 128.9, 128.2, 123.0, 118.7,

115.2, 109.6, 108.4, 97.9, 61.3, 56.3, 55.7, 52.7, 52.5, 45.2,44.7,

41.1, 38.8, 32.6. Anal. (C₂₉H₃₆ClN₅O₆S‚1.5H₂O), C, H, N.

2-{4-[4-({[5-(Dim e t h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)bu ta n oyl]p ip er a zin -1-yl}eth yl 4-Am in o-5-ch lor o-

2-m eth oxyben zoa te (23c). Same procedure as described for

17a except NEt₃was used instead of DIEA as base. CH₂Cl₂

then eluent system A; R_f(system A) 0.28; 32% yield; pale green

foam; ¹H NMR (200 MHz): δ 8.52 (m, 1H), 8.24 (m, 2H), 7.80

(s, 1H), 7.51 (m, 2H), 7.16 (m, 1H), 6.28 (s, 1H), 5.45 (m, 1H),

4.48 (bs, 2H), 4.36 (t, J ) 6.1 Hz, 2H), 3.83 (s, 3H), 3.56 (m,

2H), 3.26 (m, 2H), 2.95 (m, 2H), 2.88 (s, 6H), 2.73 (t, J ) 6.1

Hz, 2H), 2.48 (m, 4H), 2.24 (t, J ) 7 Hz, 2H), 1.76 (quintet, J

) 7 Hz, 2H). ¹³C NMR (50 MHz, CDCl_3,CD₃OD): δ 171.1,

164.6, 160.3, 151.7, 148.5, 134.8, 132.9, 129.9, 129.6, 129.4,

129.0, 127.9, 123.0, 118.7, 114.9, 109.5, 108.1, 97.8, 61.2, 56.2,

2-(4-{8-[(ter t-Bu toxycar bon yl)am in o]u n decan oyl}piper -

azin -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth oxyben zoate (21g).

From the reaction of 2 and Boc-11-aminodecanoic acid. CH₂Cl₂/

Et₂O/iPrOH 50:45:5 then eluent system A; R_f(system A) 0.14;

65% yield; colorless oil; H NMR (200 MHz): δ 7.79 (s, 1H),

6.28 (s, 1H), 4.50 (bs, 3H), 4.38 (t, J ) 5.9 Hz, 2H), 3.82 (s,

3H), 3.63 (m, 2H), 3.48 (m, 2H), 3.12 (m, 2H), 2.77 (t, J ) 5.9

Hz, 2H), 2.55 (m, 4H), 2.33 (t, J ) 7.2 Hz, 2H), 1.75-1.1 (m,

25H). Anal. (C₃₀H₄₉ClN₄O₆S‚0.5H₂O), C, H, N.

2-[4-(2-Am in oa cet yl)p ip er a zin -1-yl]et h yl 4-Am in o-5-

ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (22a ). Same

procedure as described for 16a . 79% yield; hygroscopic white

solid; H NMR (200 MHz): free base δ 7.78 (s, 1H), 6.27 (s,

1H), 4.51 (bs, 2H), 4.35 (t, J ) 5.9 Hz, 2H), 3.81 (s, 3H), 3.63

(m, 2H), 3.40 (m, 2H), 3.12 (m, 2H), 2.74 (t, J ) 5.9 Hz, 2H),

2.53 (m, 4H), 1.20 (bs, 2H). ESI: m/z 371.1 (M + H⁺). Anal.

(C₁₆H₂₃ClN₄O₄‚2HCl‚2H₂O), C, H, N.

2-[4-(3-Am in op r op a n oyl)p ip er a zin -1-yl]eth yl 4-Am in o-

5-ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (22b). Same

procedure as described for 16a . 60% yield; hygroscopic white

solid; H NMR (200 MHz): free base δ 7.75 (s, 1H), 6.25 (s,

1H), 4.56 (bs, 2H), 4.32 (t, J ) 5.9 Hz, 2H), 3.79 (s, 3H), 3.58

(m, 2H), 3.42 (m, 2H), 2.95 (t, J ) 6.1 Hz, 2H), 2.71 (t, J ) 5.9

Hz, 2H), 2.49 (m, 4H), 2.41 (t, J ) 6.1 Hz, 2H), 1.0 (bs, 2H).

ESI: m/z 385.2 (M + H⁺). Anal. (C₁₇H₂₅Cl N₄O₄‚2HCl‚1.5H₂O),

C, H, N.

2-[4-(4-Am in obu ta n oyl)p ip er a zin -1-yl]eth yl 4-Am in o-

5-ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (22c). Same

procedure as described for 16a . 57% yield; hygroscopic white

solid; H NMR (200 MHz): free base δ 7.76 (s, 1H), 6.26 (s,

1H), 4.53 (bs, 2H), 4.31 (t, J ) 5.9 Hz, 2H), 3.81 (s, 3H), 3.59

(m, 2H), 3.46 (m, 2H), 2.71 (m, 4H), 2.5 (m, 4H), 2.35 (t, J )

7.2 Hz, 2H), 1.73 (quintet, J ) 7.2 Hz, 2H), 0.86 (bs, 2H). ESI:

m/z 399.2 (M + H⁺). Anal. (C₁₈H₂₇Cl N₄O_4.2HCl‚3.5H₂O), C,

H, N.

2-[4-(5-Am in op en ta n oyl)p ip er a zin -1-yl]eth yl 4-Am in o-

5-ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (22d ). Same

procedure as described for 16a . 53% yield; very hygroscopic

white solid; ¹H NMR (200 MHz): free base δ 7.76 (s, 1H), 6.26

(s, 1H), 4.56 (bs, 2H), 4.33 (t, J ) 5.9 Hz, 2H), 3.80 (s, 3H),

55.6, 52.9, 52.6, 45.1, 44.9, 42.3, 41.2, 29.7, 24.4. Anal. (C₃₀H₃₈

ClN₅O₆S.H₂O), C, H, N.

Fluorescent Antagonists for Human 5-HT₄Receptors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2617

2-{4-[(5-({[5-(Dim et h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)p en ta n oyl]p ip er a zin -1-yl}eth yl 4-Am in o-5-ch lor o-

2-m eth oxyben zoa te (23d ). Same procedure as described for

17a except NEt₃was used instead of DIEA as base. CH₂Cl₂

then eluent system; R_f(sytem A) 0.4; 58% yield; pale green

foam; ¹H NMR (200 MHz): δ 8.53 (m, 1H), 8.24 (m, 2H), 7.80

(s, 1H), 7.55 (m, 2H), 7.18 (m, 1H), 6.29 (s, 1H), 5.06 (m, 1H),

4.46 (bs, 2H), 4.38 (t, J ) 6.1 Hz, 2H), 3.84 (s, 3H), 3.58 (m,

2H), 3.41 (m, 2H), 2.88 (m, 8H), 2.78 (t, J ) 6.1 Hz, 2H), 2.55

(m, 4H), 2.20 (t, J ) 7 Hz, 2H), 1.70-1.40 (m, 4H). ¹³C NMR

(50 MHz, CDCl_3,CD₃OD): δ 171.4, 164.6, 160.3, 151.6, 148.5,

134.9, 132.9, 129.9, 129.7, 129.5, 129.0, 127.9, 122.9, 118.8,

114.9, 109.6, 108.4, 97.9, 61.3, 56.3, 55.7, 53.0, 52.7, 45.2, 42.3,

41.2, 31.9, 28.8, 21.7. Anal. (C₃₁H₄₀ClN₅O₆S‚1.5H₂O), C, H, N.

4.13 (d, J ) 7.2 Hz, 2H), 4.10 (m, 2H), 2.66 (bt, 2H), 2.07 (m,

1H), 1.67 (m, 2H), 1.44 (s, 9H), 1.34 (m, 2H). Anal. (C₂₃H₂₅N₃O₆),

C, H, N.

ter t-Bu tyl 4-{[6-(Dim eth yla m in o)-1,3-d ioxo-1H-ben zo-

[d e]isoqu in olin -2(3H)-yl]m eth yl}p ip er id in e-1-ca r boxyl-

a te (25b). From 24b. CH₂Cl₂then CH₂Cl₂/Et₂O 90:10; R_f

(CH₂Cl₂/Et₂O) 0.21; 83% yield; yellow foam; ¹H NMR (200

MHz): δ 8.54 (dd, J ) 1.17 Hz and J ) 7.29 Hz, 1H), 8.45 (d,

J ) 8.26 Hz, 1H), 8.43 (dd, J ) 1.17 Hz and J ) 8.45 Hz, 1H),

7.64 (dd, J ) 7.29 Hz and J ) 8.45 Hz, 1H), 7.10 (d, J ) 8.26

Hz, 1H), 4.09 (d, J ) 7.2 Hz, 2H), 4.08 (m, 2H), 3.09 (s, 6H),

2.64 (m, 2H), 2.05 (m, 1H), 1.66 (m, 2H), 1.44 (s, 9H,), 1.34

(m, 2H). Anal. (C₂₅H₃₁N₃O₄), C, H, N.

2-(4-{4-[6-(Dim eth yla m in o)-1,3-d ioxo-1H-ben zo[d e]iso-

qu in olin -2(3H)-yl]bu tyl}p ip er id in -1-yl)eth yl 4-Am in o-5-

ch lor o-2-m eth oxyben zoa te (29). From 24b and 16d in the

presence of NEt₃. CH₂Cl₂then CH₂Cl₂/MeOH 90:10; R_f(CH₂Cl₂/

Et₂O) 0.13; 23% yield; yellow foam; ¹H NMR (200 MHz): δ

8.56 (dd, J ) 1.15 Hz and J ) 7.34 Hz, 1H), 8.48 (d, J ) 8.29

Hz,1H), 8.44 (dd, J ) 1.15 Hz and J ) 8.46 Hz, 1H), 7.82 (s,

1H), 7.65 (dd, J ) 7.34 Hz and J ) 8.46 Hz, 1H), 7.16 (d, J )

8.29 Hz, 1H), 6.27 (s, 1H), 4.41 (bs, 2H), 4.36 (t, J ) 5.9 Hz,

2H), 4.16 (t, J ) 7.5 Hz, 2H), 3.83 (s, 3H), 3.10 (s, 6H), 2.96

(m, 2H), 2.72 (t, J ) 5.9 Hz, 2H), 2.08 (m, 2H), 1.80-1.1 (m,

11H). ESI: m/z 607.3 (M + H⁺). Anal. (C₃₃H₃₉ClN₄O₅‚0.5H₂O),

C, H, N.

2-{4-[(6-({[5-(Dim et h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)h exa n oyl]p ip er a zin -1-yl}eth yl 4-Am in o-5-ch lor o-

2-m eth oxyben zoa te (23e). Same procedure as described for

17a except NEt₃was used instead of DIEA as base. CH₂Cl₂

then eluent sytem A; R_f(system A) 0.6; 81% yield; pale green

foam; ¹H NMR (200 MHz): δ 8.54 (m, 1H), 8.26 (m, 2H), 7.80

(s, 1H), 7.53 (m, 2H), 7.17 (m, 1H), 6.29 (s, 1H), 4.82 (m, 1H),

4.45 (bs, 2H), 4.40 (t, J ) 6.1 Hz, 2H), 3.84 (s, 3H), 3.60 (m,

2H), 3.40 (m, 2H), 2.88 (m, 8H), 2.75 (t, J ) 6.1 Hz, 2H), 2.51

(m, 4H), 2.20 (t, J ) 7 Hz, 2H), 1.45 (m, 4H), 1.25 (m, 2H). ¹³

NMR (50 MHz, CDCl_3,CD₃OD): δ 171.6, 164.6, 160.2, 151.6,

148.5, 134.9, 132.9, 129.9, 129.6, 129.4, 128.1, 127.9, 122.9,

118.8, 114.9, 109.5, 108.3, 97.9, 61.3, 56.3, 55.7, 53.1, 52.7, 45.1,

42.4, 41.2, 32.5, 28.9, 25.8, 24.22. Anal. (C₃₂H₄₂ClN₅O₆S‚H₂O),

C, H, N.

6-Nitr o-2-(4-p ip er id in em eth yl)-1H-ben zo[d e]isoqu in o-

lin e-1,3(2H)-d ion e Hyd r och lor id e (26a ). Same procedure

as described for 17a , except that compound 25a was dissolved

in dioxane and reacted for 6 h. 94% yield; pale yellow solid;

¹H NMR (200 MHz): free base δ 8.82 (dd, J ) 1.1 Hz and J )

8.68 Hz, 1H), 8.73 (dd, J ) 0.87 Hz and J ) 7.35 Hz, 1H),

8.69 (d, J ) 8 Hz, 1H), 8.38 (d, J ) 8 Hz, 1H), 7.97 (dd, J )

7.35 Hz and J ) 8.68 Hz, 1H), 4.10 (d, J ) 7.12 Hz, 2H), 3.06

(m, 2H), 2.54 (m, 2H), 2.01 (m, 1H), 1.65 (m, 2H), 1.29 (m,

2H). Anal. (C₁₈H₁₇N₃O_4.HCl), C, H, N.

2-{4-[(8-({[5-(Dim et h yla m in o)-1-n a p h t h yl]su lfon yl}-

a m in o)octa n oyl]p ip er a zin -1-yl}eth yl 4-Am in o-5-ch lor o-

2-m eth oxyben zoa te (23f). Same procedure as described for

17a except NEt₃was used instead of DIEA as base. CH₂Cl₂

then eluent system A; R_f(system A) 0.33; 88% yield; pale green

foam; ¹H NMR (200 MHz): δ 8.53 (m, 1H), 8.26 (m, 2H), 7.80

(s, 1H), 7.53 (m, 2H), 7.18 (m, 1H), 6.29 (s, 1H), 4.60 (m, 1H),

4.46 (bs, 2H), 4.37 (t, J ) 6.1 Hz, 2H), 3.80 (s, 3H), 3.61 (m,

2H), 3.45 (m, 2H), 2.88 (m, 8H), 2.75 (t, J ) 6.1 Hz, 2H), 2.54

(m, 4H), 2.24 (t, J ) 7 Hz), 1.7-1.1 (m, 10H). ¹³C NMR (50

MHz): δ 171.4, 164.4, 160.2, 151.9, 148.0, 134.8, 133.1, 130.2,

129.8, 129.6, 129.4, 128.2, 123.1, 118.7, 115.0, 109.8, 109.3,

98.2, 61.6, 56.5, 55.9, 53.3, 52.9, 45.4, 45.3, 43.1, 41.4, 32.9,

29.3, 28.9, 28.5, 26.1, 24.9. Anal. (C₃₄H₄₆ClN₅O₆S‚0.5H₂O), C,

H, N.

6-(Dim eth yla m in o)-2-(4-p ip er id in em eth yl)-1H-ben zo-

[de]isoqu in olin e-1,3(2H)-dion e Hydr och lor ide (26b). Same

procedure as described for 17a , except reaction time 24 h. 83%

yield; hygroscopic yellow solid; ¹H NMR (200 MHz): free base

δ 8.52 (dd, J ) 1.19 Hz and J ) 7.28 Hz, 1H), 8.43 (d, J )

8.23 Hz, 1H), 8.40 (dd, J ) 1.19 Hz and J ) 8.51 Hz, 1H),

7.61 (dd, J ) 7.28 Hz and J ) 8.51 Hz, 1H), 7.08 (d, J ) 8.23

Hz, 1H), 4.03 (d, J ) 7.12 Hz, 2H), 3.06 (s, 6H,), 3.03 (m, 2H),

2.49 (m, 2H), 2 (m, 1H), 1.63 (m, 2H), 1.28 (m, 2H). Anal.

(C₂₀H₂₃N₃O_{2 .}HCl‚0.25H₂O), C, H, N.

2-(4-{[6-Nitr o-1,3-dioxo-1H-ben zo[de]isoqu in olin -2(3H)-

yl]m eth yl}p ip er id in -1-yl)eth yl 4-Am in o-5-ch lor o-2-m eth -

oxyben zoa te (27a ). Same procedure as described for 15a .

CH₂Cl₂/ Et₂O 50:50 then system eluent A; R_f(system A) 0.48;

13% yield; orange solid; mp 194 °C; ¹H NMR (200 MHz, CDCl₃,

CD₃OD): δ 8.77 (dd, J ) 1.10 Hz and J ) 8.73 Hz, 1H), 8.66

(dd, J ) 1.10 Hz and J ) 7.29 Hz, 1H), 8.62 (d, J ) 8.07 Hz,

1H), 8.35 (d, J ) 8.07 Hz, 1H), 7.93 (dd, J ) 7.29 Hz and J )

8.73 Hz, 1H), 7.73 (s, 1H), 6.24 (s, 1H), 4.29 (t, J ) 5.9 Hz,

2H), 4.07 (d, J ) 7 Hz, 2H), 3.75 (s, 3H), 2.95 (m, 2H), 2.68 (t,

J ) 5.9 Hz, 2H), 2.06 (m, 2H), 1.86 (m, 1H), 1.66 (m, 2H), 1.49

(m, 2H). ESI: m/z 567.1 (M + H⁺). Anal. (C₂₈H₂₇Cl N₄O₇‚

0.5H₂O), C, H, N.

2-{4-[(11-({[5-(Dim et h yla m in o)-1-n a p h t h yl]su lfon yl}-

am in o)u n decan oyl]piper azin -1-yl}eth yl 4-Am in o-5-ch lor o-

2-m eth oxyben zoa te (23g). Same procedure as described for

17a except NEt₃was used instead of DIEA as base. CH₂Cl₂

then eluent system A; R_f(System A) 0.5; 86% yield; pale green

foam; ¹H NMR (200 MHz): δ 8.53 (m, 1H), 8.24 (m, 2H), 7.80

(s, 1H), 7.53 (m, 2H), 7.17 (m, 1H), 6.28 (s, 1H), 4.65 (m, 1H),

4.49 (bs, 2H), 4.39 (t, J ) 6.1 Hz, 2H), 3.82 (s, 3H), 3.64 (m,

2H), 3.48 (m, 2H), 2.88 (m, 8H), 2.79 (t, J ) 6.1 Hz, 2H), 2.58

(m, 4H), 2.28 (t, J ) 7 Hz, 2H), 1.58 (m, 2H), 1.43-1.1 (m,

14H). ¹³C NMR (50 MHz): δ 171.6, 164.4, 160.2, 151.9, 148.1,

134.9, 133.1, 130.2, 129.8, 129.6, 129.4, 128.2, 123.0, 118.7,

115.0, 109.8, 109.2, 98.2, 61.6, 56.5, 55.9, 53.4, 53.0, 45.5, 45.3,

43.2, 41.4, 33.1, 29.4, 29.2, 29.1 29.0, 28.7, 26.2, 25.2. Anal.

(C₃₇H₅₂ClN₅O₆S), C, H, N.

2-(4-{[6-(Dim et h yla m in o)-1,3-d ioxo-1H -b en zo[d e]iso-

qu in olin -2(3H)-yl]m eth yl}p ip er id in -1-yl)eth yl 4-Am in o-

5-ch lor o-2-m eth oxyben zoa te Hyd r och lor id e (27b). Same

procedure as described for 15a except reaction mixture CH₃CN/

CH₂Cl₂/DMF 30:10:10 was used. CH₂Cl₂/MeOH 90:10 then

CH₂Cl₂/Et₂O 50:50. R_f(CH₂Cl₂/Et₂O 50:50) 0.42; 14% yield;

yellow solid. The resulted product was transformed into its

Syn th esis of com p ou n d s 25a , 25b, 29: Gen er a l P r oce-

d u r e. ter t-Bu tyl 4-{[6-Nitr o-1,3-d ioxo-1H-ben zo[d e]iso-

qu in olin -2(3H)-yl]m eth yl}piper idin e-1-car boxylate (25a).

4-Nitro-1,8-naphthalic anhydride 24a (5 g, 0.021 mol) and

1-Boc-4-aminomethylpiperidine (4.28 g, 0.02 mol) in absolute

EtOH (120 mL) was heated under reflux for 24 h. After

concentration, the crude product was chromatographed (EtOAc/

cyclohexane 40:60) and the solid obtained recrystallized from

cyclohexane/EtOAc to provide 6.2 g (71%) of 25a as a yellow

hydrochloride salt with 4 N HCl methanol solution. H NMR

(200 MHz): free base δ 8.53 (dd, J ) 1.15 Hz and J ) 7.34

Hz, 1H), 8.46 (d, J ) 8.29 Hz, 1H), 8.42 (dd, J ) 1.15 Hz and

J ) 8.46 Hz, 1H), 7.77 (s, 1H), 7.63 (dd, J ) 7.34 Hz and J )

8.46 Hz, 1H), 7.09 (d, J ) 8.29 Hz, 1H), 6.25 (s, 1H), 4.45 (bs,

2H), 4.32 (t, J ) 5.9 Hz, 2H), 4.09 (d, J ) 7 Hz, 2H), 3.79 (s,

3H), 3.08 (s, 6H), 2.95 (m, 2H), 2.68 (t, J ) 5.9 Hz, 2H), 2.06

solid; R_f(EtOAc/cyclohexane) 0.92; mp 140 °C; H NMR (400

MHz): δ 8.84 (dd, J ) 0.94 Hz and J ) 8.64 Hz, 1H), 8.73 (dd,

J ) 0.86 Hz and J ) 7.34 Hz, 1H), 8.68 (d, J ) 8 Hz, 1H), 8.40

(d, J ) 8 Hz, 1H), 7.99 (dd, J ) 7.34 Hz and J ) 8.64 Hz, 1H),

2618 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13

Berque-Bestel et al.

(m, 2H), 1.87 (m, 1H), 1.66 (m, 2H), 1.50 (m, 2H). ESI: m/z

565.1 (M + H⁺). Anal. (C₃₀H₃₃ClN₄O₅‚HCl‚1.5H₂O), C, H, N.

buffer (50 mM, pH 7.4). The protein concentration was

determined by the method of Bradford using bovine serum

albumin as the standard.

2-(4-{[6-Am in o-1,3-dioxo-1H-ben zo[de]isoqu in olin -2(3H)-

yl]m eth yl}p ip er id in -1-yl)eth yl 4-a m in o-5-ch lor o-2-m eth -

oxyben zoa te Hyd r och lor id e (28). A suspension of nitro

compound 27a (0.104 g, 0.18 mmol) and Raney nickel (∼ 0.2

g) in MeOH/dioxane 50:50 (12 mL) was stirred with am-

monium formate (0.2 g) at room temperature. After completion

of the reaction (1 h), the mixture was filtered off through

Celite. The organic layer was concentrated, and the residue

was chromatographed on silica gel using CH₂Cl₂/MeOH 80:20

to give an orange solid R_f(CH₂Cl₂/MeOH 80:20) 0.44 which

was transformed into its hydrochloride salt 28 with 4 N HCl

Radioligand binding studies were performed in 500 µL of

HEPES buffer (50 mM, pH 7.4), 20 µL of the studied ligand (7

concentrations), and 20 µL of [³H]GR113808 at a concentration

of 0.2 nM and 50 µL of membranes preparation (100-200 µg

of protein). Nonspecific binding was determined with 10 µM

GR113808. Tubes were incubated at 25 °C for 30 min, and

the reaction was terminated by filtration through Whatman

GF/B Filter paper using the Brandel 48R cell harvester. Filters

were presoaked in a 0.1% solution of polyethylenimine. Filters

were subsequently washed with ice-cold buffer (50 mM Tris-

HCl, pH 7.4) and placed overnight in 4 mL of ready-safe

scintillation cocktail. Radioactivity was measured using a

Beckman model LS 6500C liquid scintillation counter. Binding

data (K_i) were analyzed by computer-assisted nonlinear re-

gression analysis (Prism, Graphpad Software, San Diego, CA).

The data are the results of two or three determinations in

triplicate.

Mea su r em en t of cAMP . C6 glial cells stably transfected

with the human 5-HT_4(e)receptor were grown to confluence

and incubated with serum-free medium for 4 h before the

beginning of the assay. Then the cells were preincubated for

15 min with serum-free medium supplemented with 5 mM

theophylline and 10 µM pargyline. 5-HT (1 µM) and/or

compounds were added and incubated for an additional 15 min

at 37 °C in 5% CO₂. The reaction was stopped by aspiration of

the medium and addition of 50 µL of ice-cold perchloric acid

(20%). After a 30 min period, neutralization buffer was added

(HEPES 25 mM, KOH 2 N) and supernatant was extracted

after centrifugation at 2000g for 5 min, cAMP was quantified

using radioimmunoassay kit (cAMP competitive radio-

immunoassay, Beckman, France). The 5-HT concentration-

effect curve was calculated using seven concentrations (10^-10

to 10^-5) alone or in the presence of compounds.

F lu or escen t La belin g of C6 Glia l Cell Mem br a n es

Exp r essin g th e Hu m a n 5-HT_4(e)Recep tor . Autofluores-

cence: C6 glial cell membranes were incubated alone in

HEPES buffer (50 mM, pH 7.4) for 30 min at 25 °C. Total

fluorescence: C6 glial cell membranes were incubated with

either 60 nM (K_i) of 28 or 7 nM (K_i) of 32 or 7 nM (K_i) of 17a

in HEPES buffer (50 mM, pH 7.4) for 30 min at 25 °C.

Nonspecific fluorescence: C6 glial cell membranes were incu-

bated with 0.2 µM (1000 K_i) of GR113808 in HEPES buffer

(50 mM, pH 7.4) for 30 min at 25 °C, then incubated with

either 60 nM (K_i) of 28 or 7 nM (K_i) of 32 or 7 nM (K_i) of 17a

for 30 min at 25 °C. All the samples were centrifuged at

20 000g for 15 min at 4 °C, washed with HEPES buffer (50

mM, pH 7.4), centrifuged at 20 000g for 15 min at 4 °C. The

same protocole was repeated one more time and the pellet was

resuspended in HEPES buffer for the measure performed at

20 °C. Fluorescence measurements were done on a Perkin-

Elmer fluorimeter LS50B. Excitation and emission slits were

both 15-nm band-pass. Results were analyzed with Prism,

Graphpad Software (San Diego, CA).

F lu or escen t La belin g of C6 Glia l Cells Exp r essin g

Hu m a n th e 5-HT_4(e)Reecep tor . C6 glial cells stably trans-

fected with the h5-HT_4(e)receptor were cultivated on 24 well

plates and eight-well Lab-Tek chamber plates according to the

experiments. They were grown to confluence and incubated

with serum-free medium for 4 h before the beginning of the

assay. Autofluorescence: C6 glial cells were incubated alone

in HEPES buffer (50 mM, pH 7.4) for 30 min at 25 °C. Total

fluorescence: C6 glial cells were incubated with either 60 nM

(K_i) of 28 or 7 nM (K_i) of 32 or 7 nM (K_i) of 17a in HEPES

buffer (50 mM, pH 7.4) for 30 min at 25 °C. Nonspecific

fluorescence: C6 glial cells were incubated with 0.2 µM (1000

K_i) of GR 113808 in HEPES buffer (50 mM, pH 7.4) for 30

min at 25 °C, then incubated with either 60 nM (K_i) of 28 or

7 nM (K_i) of 32 or 7 nM (K_i) of 17a for 30 min at 25 °C. Cells

were then washed twice with HEPES buffer and fluorescence

was observed using a Fluostar plate reader. The data are the

results of two or three determinations in triplicate.

methanol solution (56 mg, 50%); hygroscopic yellow solid; H

NMR (400 MHz, CD₃OD): δ 8.50 (bd, 2H), 8.26 (dd, J ) 1.36

Hz and J ) 8.42 Hz, 1H), 7.79 (s, 1H), 7.62 (t, J ) 8.3 Hz,

1H), 6.87 (d, J ) 8.42 Hz, 1H), 6.45 (s, 1H), 4.55 (m, 2H), 4.12

(d, J ) 6.8 Hz, 2H), 3.80 (s, 3H), 3.71 (m, 2H), 3.50 (m, 2H),

3.05 (m, 2H), 2.24 (m, 1H), 2.03 (m, 2H), 1.76 (m, 2H). ESI:

m/z 537.2 (M + H⁺), 559.2 (M + Na⁺). Anal. (C₂₈H₂₉ClN₄O₄‚

2HCl‚0.5H₂O), C, H, N.

ter t-Bu tyl-4-{[(7-n itr o-2,1,3-ben zoxa d ia zol-4-yl)a m in o]-

m eth yl}p ip er id in e-1-ca r boxyla te (30). A solution of NBD

chloride (2.48 g, 12.4 mmol) in dry THF (120 mL) was slowly

added to a solution of 1-Boc-4-aminomethylpiperidine (9.3 g,

43.4 mmol) in THF (120 mL) at 0 °C for 2.5 h. Then, the solvent

was evaporated, and the residue was chromatographed on

silica gel (CH₂Cl₂/Et₂O 50:50) to give 3.23 g (69%) of 30 as an

orange solid. R_f(CH₂Cl₂/Et₂O 50:50) 0.75; mp > 260 °C; ¹H

NMR (200 MHz): δ 8.46 (d, J ) 8.7 Hz, 1H), 6.43 (bt, 1H),

6.18 (d, J ) 8.7 Hz, 1H), 4.18 (m, 2H), 3.42 (t, J ) 6.3 Hz,

2H), 2.73 (m, 2H), 1.97 (m, 1H), 1.84 (m, 2H), 1.45 (s, 9H),

1.27 (m, 2H). Anal. (C₁₇H₂₃N₅O₅), C, H, N.

7-Nitr o-N-(4-p ip er id in em eth yl)-2,1,3-ben zoxa d ia zol-4-

a m in e Hyd r och lor id e (31). Same procedure as described for

16a , except that compound 30 was dissolved in MeOH/dioxane

50:50 and reacted for 5 h. 85% yield; ¹H NMR (200 MHz): free

base δ 8.43 (d, J ) 8.7 Hz, 1H), 6.13 (d, J ) 8.7 Hz, 1H), 3.34

(d, J ) 6.7 Hz, 2H), 3.10 (m, 2H), 2.58 (m, 2H), 1.95 (m,1H),

1.8 (m, 2H), 1.25 (m, 2H), 0.8 (bs, 1H). Anal. (C₁₂H₁₅N₅O_5.HCl‚

0.25H₂O), C, H, N.

2-(4-{[(7-Nitr o-2,1,3-ben zoxadiazol-4-yl)am in o]m eth yl}-

p ip er id in -1-yl)et h yl 4-Am in o-5-ch lor o-2-m et h oxyb en -

zoa te (32). Same procedure as described for 15a , except that

hydrochloride salt of amine 31 was reacted in dry DMF at 30

°C for 3 days. CH₂Cl₂/acetone 50:50; R_f(CH₂Cl₂/acetone 50:

50) 0.16; mp 182-183 °C (dec); H NMR (200 MHz): δ 8.47

(d, J ) 8.7 Hz, 1H), 7.79 (s, 1H); 6.35 (m, 1H), 6.28 (s, 1H),

6.17 (d, J ) 8.7 Hz, 1H), 4.46 (bs, 2H), 4.39 (t, J ) 5.9 Hz,

2H), 3.83 (s, 3H), 3.39 (m, 2H), 3.05 (m, 2H), 2.76 (t, J ) 5.9

Hz, 2H), 2.20 (m, 2H), 1.80 (m, 3H), 1.46 (m, 2H). ESI: m/z

505.1 (M + H⁺). Anal. (C₂₂H₂₅ClN₆O₆‚0.125H₂O), C, H, N.

2-(P ip er id in -1-yl)eth yl 5-Ch lor o-2-m eth oxy-4-[(7-n itr o-

2,1,3-ben zoxa d ia zo-4-yl)a m in o]ben zoa te (33). A mixture

of 14 (0.17 g, 0.54 mmol), NBD chloride (0.18 g, 0.9 mmol), KI

(8 mg), and n-BuOH (17 mL) was heated at reflux under argon

for 12 h. After cooling, the reaction mixture was diluted with

Et₂O, and the precipitate obtained was filtered, washed several

times with Et₂O, and chromatographed on silica gel (CH₂Cl₂,

then CH₂Cl₂/Et₂O 50:50) to give 80 mg (30%) of 33 as a dark

blue solid; R_f(CH₂Cl₂/Et₂O 50:50) 0.28; H NMR (200 MHz):

δ 8.26 (d, J ) 8.7 Hz, 1H), 7.55 (s, 1H); 6.53 (d, J ) 8.7 Hz,

1H), 5.91 (s, 1H), 4.53 (t, J ) 5.9 Hz, 2H,), 4.31 (bs, 1H), 3.71

(s, 3H), 3.45 (m 2H), 2.46 (t, J ) 5.9 Hz, 2H), 2.12 (m, 2H),

1.52 (m, 6H). ESI: m/z 474.1 (M + H⁺), 496.0 (M + Na⁺).

Biology. Mem br a n e P r ep a r a tion a n d Ra d ioliga n d

Bin d in g Assa ys. Briefly, C6 glial cells stably transfected with

the h5-HT_4(e)receptor, grown to confluence, were incubated

with serum-free medium for 4 h, washed twice with phosphate-

buffered saline (PBS), and centrifuged at 300g for 5 min. The

pellet was used immediately or stored at -80 °C. The pellet

was resuspended in 10 volumes of ice-cold HEPES buffer (50

mM, pH 7.4) and centrifuged at 40 000g for 20 min at 4 °C.

The resulting pellet was resuspended in 15 volumes of HEPES

Fluorescent Antagonists for Human 5-HT₄Receptors

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2619

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receptors by confocal microscopy. Braz. J . Med. Biol. Res. 1998,

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For microscopy observation, cells were fixed with 1%

paraformaldehyde for 15 min, washed with a NH₄Cl (50 mM)-

glycine (20 mM) solution (pH 7.4) and rinsed twice with

HEPES buffer. The cells were observed on a confocal micro-

scope (LSM 510, Zeiss France, Le Pecq, France). Images of cells

were acquired with

Plan-Achromat × 63 1.4 NA oil-

immersion objective (Zeiss) in a median plan with an optical

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Ack n ow led gm en t. We thank Valerie Nicolas for

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J M0307887

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