Novel potent and selective central 5-HT3 receptor ligands provided with different intrinsic efficacy. 2. Molecular basis of the intrinsic efficacy of arylpiperazine derivatives at the central 5-HT3 receptors

1556

J . Med. Chem. 1999, 42, 1556-1575

Novel P oten t a n d Selective Cen tr a l 5-HT₃Recep tor Liga n d s P r ovid ed w ith

Differ en t In tr in sic Effica cy. 2. Molecu la r Ba sis of th e In tr in sic Effica cy of

Ar ylp ip er a zin e Der iva tives a t th e Cen tr a l 5-HT₃Recep tor s

Andrea Cappelli,*^,‡Maurizio Anzini,^∇Salvatore Vomero,^‡Laura Canullo,^‡Laura Mennuni,^§Francesco Makovec,^§

Edith Doucet,^|Michel Hamon,^|M. Cristina Menziani,^†Pier G. De Benedetti,^†Giancarlo Bruni,

Maria R. Romeo, Gianluca Giorgi,^Xand Alessandro Donati⁾

Dipartimento Farmaco Chimico Tecnologico, Universita` di Siena, Via Banchi di Sotto 55, 53100 Siena, Italy,

Facolta` di Farmacia, Universita` degli Studi di Catanzaro “Magna Graecia”, Complesso Nin`ı Barbieri,

88021 Roccelletta di Borgia, Catanzaro, Italy, Rotta Research Laboratorium S.p.A., Via Valosa di Sopra 7, 20052 Monza, Italy,

Neurobiologie Cellulaire et Fonctionnelle, INSERM U. 288, Faculte´ de Me´dicine Pitie´-Salpeˆtrie´re, 91, Boulevard de l’Hoˆpital,

75634 Paris Cedex 13, France, Dipartimento di Chimica, Universita` degli Studi di Modena, Via Campi 183,

41100 Modena, Italy, Istituto di Farmacologia, Universita` di Siena, Le Scotte 6, 53100 Siena, Italy, Centro Interdipartimentale

di Analisi e Determinazioni Strutturali, Universita` di Siena, Via A. Moro, 53100 Siena, Italy, and Dipartimento di Scienze e

Tecnologie Chimiche e dei Biosistemi, Universita` di Siena, Pian dei Mantellini 44, 53100 Siena, Italy

Received November 3, 1998

Novel 5-HT₃receptor ligands were designed and synthesized with the aim of obtaining deeper

insight into the molecular basis of the intrinsic efficacy of arylpiperazines interacting with the

central 5-HT₃receptor. The newly synthesized compounds and some previously published

compounds belonging to the same class of heteroarylpiperazines were tested for their potential

ability to displace [³H]granisetron from rat cortical membranes. These 5-HT₃receptor binding

studies revealed subnanomolar affinity in several of the compounds under study. The most

active ligands were quipazine derivatives bearing a phenyl group in the 4-position and various

oxygenated alkyl side chains in the 3-position of the quinoline nucleus. Qualitative and

theoretical quantitative structure-affinity relationship studies were carried out, and the

interaction model for the 5-HT₃ligands related to quipazine with their receptor, proposed in

part 1 of the present work, was updated to incorporate the latest data. The potential 5-HT₃

agonist/antagonist activity of 12 selected compounds was assessed in vitro on the 5-HT₃receptor-

dependent [¹⁴C]guanidinium uptake in NG 108-15 cells. Their intrinsic efficacy ranged from

the 5-HT₃full agonist properties of compounds 7a and 8h ,i to those of partial agonists 10a ,d

and antagonists 8b,d ,e, and 9c,d ,h ,i. The comparison between these functional data and those

relative to the previously described compounds suggested that in this class of 5-HT₃ligands

the intrinsic efficacy is modulated in a rather subtle manner by the steric features of the

heteroaryl moiety.

In tr od u ction

It has been suggested that the stimulation of the

5-HT₃receptor in the central nervous system (CNS)

enhances the release of dopamine from rat striatal

slices³and that of cholecystokinin from the cortex and

nucleus accumbens⁴and inhibits the release of acetyl-

choline from the entorhinal cortex.⁵However, the

potential therapeutic interest of the 5-HT₃agonists still

remains to be evaluated because only very recently have

potent and selective 5-HT₃agonists became available.

Among the wide variety of serotonin (5-hydroxy-

tryptamine, 5-HT) receptors so far identified, the 5-HT₃

receptor subtype is the only one that belongs to the

ligand-gated ion channel receptor family.¹Recently,

great interest was developed toward the search for new

5-HT₃receptor antagonists: ondansetron (1), granis-

etron (BRL 43694, 2), and tropisetron (3) (Chart 1) are

in use as antiemetic agents associated with anticancer

chemotherapy. Furthermore, other possible therapeutic

uses in anxiety disorder, Alzheimer’s disease, dementia,

depression, schizophrenia, psychosis, migraine, drug

abuse, and heart arrhythmia are the subjects of intense

investigations.²

Quipazine (4a ) was earlier reported to behave as a

5-HT₃antagonist in peripheral models,⁶but subsequent

studies revealed its 5-HT₃agonist action in the [¹⁴C]-

guanidinium accumulation test applied to NG 108-15

cells.⁷It is noteworthy that the 5-HT₃receptor expressed

by NG 108-15 cells is very similar to that present in

the rat cortex.⁸Thus, quipazine is most probably a

5-HT₃receptor agonist in the CNS. In this respect, we

recently reported on a new class of pyrroloquinoxaline

derivatives 5 related to quipazine which behaved as

5-HT₃receptor agonists both in NG 108-15 cells and on

cortical acetylcholine release in freely moving rats, while

they showed partial agonist or antagonist properties in

the Bezold-J arish reflex test.⁹

^‡Dipartimento Farmaco Chimico Tecnologico, Universita` di Siena.

^∇Universita` degli Studi di Catanzaro “Magna Graecia”.

^§Rotta Research Laboratorium S.p.A.

^|INSERM U. 288.

^†Universita` degli Studi di Modena.

Instituto di Farmacologia, Universita` di Siena.

^XCentro Interdipartimentale di Analisi e Determinazioni Strut-

turali, Universita` di Siena.

⁾Dipartimento di Scienze e Tecnologie Chimiche e dei Biosistemi,

Universita` di Siena.

Published on Web 04/10/1999

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1557

Ch a r t 1

Sch em e 1^a

a

Reagents: (i) pyrrolidine, C₂H₅OC₂H₅, TiCl₄, C₆H₆; (ii) (a)

C₆H₅NCO, CHCl₃, (b) PPA; (iii) POCl₃; (iv) N-methylpiperazine

or morpholine (method A); (v) 1-cyclohexene-1-isocyanate, CH₃CN;

(vi) (a) (CF₃SO₂)O, Na₂CO₃, CH₂Cl₂, (b) N-methylpiperazine

(method B).

nists 6 and have been synthesized and tested for their

potential ability to displace [³H]granisetron from the rat

cortical membranes. The affinity of some previously

published compounds belonging to this class of het-

eroarylpiperazines was reinvestigated by means of the

same test system in order to obtain a more reliable

correlation. The intrinsic efficacy of some selected

compounds has been assessed in vitro on 5-HT₃recep-

tor-dependent [¹⁴C]guanidinium uptake in NG 108-15

cells as a predictive model for the intrinsic efficacy

toward the central 5-HT₃receptor.⁹

Ch a r t 2

The results have been quantitatively rationalized by

making use of theoretical molecular descriptors. The

qualitative and quantitative structure-activity rela-

tionships (QSAR) obtained are discussed in term of (a)

the structural features responsible for the affinity of the

ligands toward the binding site of the central 5-HT₃

receptor, on the basis of the interaction model published

in part 1 of this work,¹²and (b) the influence of the

stereoelectronic parameters of the heteroaryl moiety on

receptor activation-deactivation process (intrinsic ef-

ficacy toward the central 5-HT₃receptor).

In a previous paper we described the synthesis and

the 5-HT₃receptor antagonist properties of a new class

of quipazine derivatives 6,¹⁰and later we tried to

rationalize the differences in the intrinsic efficacy

showed by quipazine and antagonists 6, but some points

still remained unclear.¹¹Moreover, in part 1 of this

work,¹²we described the synthesis and the pharmaco-

logical characterization of high-affinity central 5-HT₃

receptor ligands 7 related to quipazine provided with

different intrinsic efficacy, and we proposed a model for

the interaction of these ligands with their receptor.

We thought the fact that the intrinsic efficacy ap-

peared to be modulated by subtle modifications of the

substituents at the c-edge of the quinoline nucleus of

quipazine to be especially intriguing and interesting.

Therefore, we have devoted the present work to the

understanding of the molecular basis of the intrinsic

efficacy of arylpiperazines interacting with the central

5-HT₃receptor.

Ch em istr y

Preparations of target 4-methyl-1-piperazinyl or mor-

pholino derivatives 8-10 were carried out starting from

the suitable 2(1H)-pyridone derivatives via either im-

idoyl chlorides (method A) or imidoyl triflates (method

B) by reaction with the appropriate amine. While

imidoyl chlorides were isolated and characterized, im-

idoyl triflates were reacted with the appropriate amine

without further purification. Compounds 8a -d ,f,g were

prepared following the procedure summarized in Scheme

1. The required cyclic ketones 11a -d were converted

according to the procedure described by White et al.¹³

New 5-HT₃receptor ligands (Chart 2) have been

designed starting from the previously described antago-

1558 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Cappelli et al.

Sch em e 2^a

Sch em e 3^a

a

Reagents: (i) (a) DPPA, (C₂H₅)₃N, C₆H₆, (b) 50% H₂SO₄; (ii)

(ClCH₂CH₂)₂NCH₃‚HCl, K₂CO₃, diglyme.

a

Reagents: (i) CH₂dCH-COOC₂H₅, (C₂H₅)₃N, Pd(PPh₃)₂Cl₂,

DMF; (ii) (a) NaOH, H₂O, C₂H₅OH, (b) ClCOOCH₂CH(CH₃)₂,

18 which was converted into target 4-methyl-1-piper-

azinyl derivative 8h by method B.

(C₂H₅)₃N, NaN₃, CH₃COCH₃, (c) (CH₃CH₂CH₂CH₂)₃N, Dowtherm

A.

The synthesis of naphthylpiperazine derivative 9b is

shown in Scheme 3. Naphthalenecarboxylic acid 19¹⁸

reacted with diphenyl phosphorazidate (DPPA), and the

resulting acyl azide underwent Curtius rearrangement

to give the corresponding isocyanate derivative which

was promptly hydrolyzed into naphthylamine 20. Reac-

tion of the latter with N-methylbis(2-chloroethyl)amine

(mechlorethamine) hydrochloride in the presence of

potassium carbonate in refluxing diglyme gave 9b in

acceptable yield.

Among the quipazine derivatives 9a ,c-q reported in

Table 2, several compounds were available in our lab

as a result of previous studies (see Table 2),¹⁹while the

synthesis of the remaining compounds was carried out

as follows. 1-Adamantyl 2-aminophenyl ketone (22) was

prepared by ortho lithiation of N-(tert-butoxycarbonyl)-

aniline (21) with tert-butyllithium using the procedure

described by Muchowski et al.²⁰followed by quenching

with methyl 1-adamantanecarboxylate and deprotec-

tion. Compound 22 was then acylated with acetic

anhydride and cyclized into quinolinone 23, which was

converted into piperazinyl derivative 9c via imidoyl

chloride 24 (Scheme 4).

into the corresponding pyrrolidine enamines 12a -d

which were reacted with phenyl isocyanate and then

cyclized into quinolinones 13a -d . The latter compounds

were readily converted into imidoyl chlorides 14a -d by

refluxing in phosphorus oxychloride and finally into the

target compounds 8a -d by method A. For the synthesis

of compound 8d , commercially available trans-1-deca-

lone (11d ) was used as the starting material, but a

diastereomeric mixture (trans/ cis 85:15) of intermediate

1

quinolone 13d was obtained on the basis of H NMR

spectroscopy (most probably an isomerization process

occurred early in the enamine formation step). Thus,

the diastereomeric mixture of 13d was refluxed in

phosphorus oxychloride, and pure trans-14d was iso-

lated by flash chromatography of its diastereomeric

mixture. This compound reacted with N-methylpipera-

zine to give pure trans-8d , the stereochemistry of which

was ascertained by high-field NMR studies (dqfCOSY

and NOESY experiments performed at 600 MHz).

Further studies concerning the three-dimensional struc-

ture of compound 8d in solution are in progress, and

full details will be published elsewhere.

Alternatively, enamines 12a ,c were reacted with

1-cyclohexene-1-isocyanate¹⁴in acetonitrile to give tet-

rahydroquinolinones 15a ,c directly which were con-

verted into the corresponding 4-methyl-1-piperazinyl

derivatives 8f,g by method B. The crystal and molecular

structure of compound 8g was determined by single-

crystal X-ray diffraction, and the structural data ob-

tained were used as input for molecular modeling

studies.

Moreover, piperazinyl derivative 9q was prepared by

method A from the corresponding imidoyl chloride 26,

which was available from 25²¹by reaction with phos-

phorus oxychloride. Lithium aluminum hydride reduc-

tion of 9q gave alcohol 9i which was transformed into

tetrahydrofuranyl derivative 9j (Scheme 5). Finally,

compounds 9f,l,m were synthesized in an analogous

fashion from alcohol derivatives 9e,k , respectively.

The comparison between the ¹H NMR spectra of

homologue alcohol derivatives 9e,i revealed interesting

differences concerning the OH protons. In fact, in the

spectra of higher homologue 9i the OH proton showed

about the same resonance frequency (4.60 ppm) both

in CDCl₃and in DMSO-d₆, while in the case of the lower

homologue 9e a signal attributable to its OH proton

could be detected only in the spectrum performed in

DMSO-d₆. This observation could be explained by as-

suming that the OH proton of compound 9i could be

engaged in hydrogen bonding (perhaps intramolecular)

more stably than that of 9e. To test this hypothesis the

Compounds 8h ,i,k were prepared from the corre-

sponding pyridone derivative available by Curtius rear-

rangement of the suitable acryloyl azide, followed by a

thermal cyclization of the formed isocyanate according

to the procedure developed by Eloy et al.¹⁵The synthesis

of 8h is exemplified in Scheme 2. Thus, vinyl triflate

16¹⁶underwent palladium-catalyzed Heck olefination

reaction¹⁷with ethyl acrylate to give R,â,γ,δ-dienoic

ester 17. The latter was saponified, converted into the

corresponding R,â,γ,δ-dienoyl azide, and finally heated

at 220 °C in Dowtherm A to obtain the cyclized product

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1559

Sch em e 4^a

Sch em e 6^a

a

Reagents: (i) (a) (CH₃)₃CLi, THF, (b) methyl 1-adamantane-

a

Reagents: (i) (a) BrCH₂COBr, CH₂Cl₂, (b) PPh₃, C₆H₆; (ii)

carboxylate, THF, (c) HCl, H₂O, C₂H₅OH; (ii) (a) (CH₃CO)₂O, (b)

CH₃ONa, C₂H₅OH; (iii) POCl₃; (iv) N-methylpiperazine.

NaH, DMF; (iii) POCl₃; (iv) N-methylpiperazine.

Sch em e 5^a

apolar solvents such as CDCl₃. The molecular structures

of compounds 9e,i as determined by single-crystal X-ray

diffraction were used as input for molecular modeling

studies.

Simple piperazinylindenoquinoline derivative 10a

was obtained via imidoyl chloride 30 from indenoquin-

olinone 29 (method A), as shown in Scheme 6. Though

the synthesis of the latter compound was earlier de-

scribed to involve decarboxylation of carboxylic acid 32²²

(see Scheme 7), we found that this compound could be

obtained in a more direct manner by intramolecular

Wittig reaction of phosphonium salt 28 available from

27 by bromoacetylation followed by reaction of the

formed bromoacetamide with triphenylphosphine. Fi-

nally, substituted piperazinylindenoquinoline deriva-

tives 10b-d were prepared by standard procedures

from ester 31²²and acid 32²²(Scheme 7).

Com p u ta tion a l Meth od s

Geom etr y Op tim iza tion . Conformational analysis

of the N4-protonated form of some simple arylpiperazine

derivatives was recently performed,¹²and the resulting

AM1²³absolute minimum structure of quipazine was

considered in this study as the starting geometry. The

substituents were assembled within the Chemnote

module of the program Quanta96.²⁴Substituents show-

ing flexible chains were considered in their extended

conformations. The three-dimensional structure of ligands

8g and 9e,i was solved by X-ray crystallography in our

laboratory. The protonated structures of the compounds

studied were fully optimized by means of molecular

orbital calculations (AM1), by means of MOPAC 6.0

(QCPE 455) program.

a

Reagents: (i) POCl₃; (ii) N-methylpiperazine; (iii) LiAlH₄, THF;

(iv) 2,3-dihydrofuran, PTSA, CH₂Cl₂.

crystal and molecular structures of these two homologue

alcohol derivatives were determined by single-crystal

X-ray diffraction, and in both cases only intermolecular

hydrogen bonding was found. However, it is possible for

a competition between the engagement of intramolecu-

lar and intermolecular hydrogen bonding to exist in the

case of compound 9i, and the intermolecular hydrogen

bonding could be preferred in the solid state, while the

intramolecular one could be preferred in solution in

Molecu la r Su p er im p osition . Compounds 9e,i-

l,n ,q, and 10a , the most structurally different ligands

showing high affinity for the 5-HT₃receptor, were

chosen for the construction of the reference supermol-

1560 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Cappelli et al.

Sch em e 7^a

a

Reagents: (i) NaOH, H₂O, C₂H₅OH; (ii) POCl₃; (iii) n-C₄H₉OH, POCl₃; (iv) (n-C₃H₇)₂NH, CH₂Cl₂; (v) N-methylpiperazine.

ecule and were superimposed, by a rigid fit procedure,

on their common piperazine nucleus. Therefore, the

volume of the supermolecule reflects all the original

contributions of the three-dimensional shape features

permitted by the receptor.

The other compounds were superimposed on the

analogous ligand present in the supermolecule or on its

structurally closest compound by using the same match-

ing procedure. All the compounds were considered in

their protonated form and energy minimum conforma-

tion.

descriptor scales, r_full) 0.99; significant intercorrelation

level, r_sig) 0.80. As a final result, the program lists the

10 correlations found with the highest squared correla-

tion coefficient and the 10 correlations found with the

highest F-test values. The quantitative structure-

affinity relationship models reported in this paper were

selected on the basis of the best statistical parameters

and the largest diversity in the physical information

content of the descriptors involved.

Resu lts

The size and shape descriptor V_inis the intersection

van der Waals volume of the ligands considered with

respect to the resultant van der Waals volume of the

reference supermolecule. The Quanta96 molecular mod-

eling software was used for molecular comparisons,

matching, and computation of van der Waals volumes.

Sta tistica l Tr ea tm en t of Da ta . The MOPAC output

files were loaded into the CODESSA program²⁵along

with the experimental binding affinities, and a large

number of global as well as fragment descriptors

(constitutional, topological, electrostatic, geometrical,

and quantum-chemical) were generated for each com-

pound. The molecular size and shape parameters were

added as external descriptors.²⁶

Qu a lita tive Str u ctu r e-Affin ity Rela tion sh ip s.

The newly synthesized compounds and some previously

published compounds belonging to this class of het-

eroarylpiperazines were tested for their potential ability

to displace [³H]granisetron specifically bound to 5-HT₃

receptor in rat cortical membrane, in comparison with

reference compounds quipazine (2a ) and granisetron,

following well-established protocols.²⁹The results of the

binding studies along with the binding values reported

in the literature³⁰for N-methylquipazine (NMQ, 4b)

(which are included for comparison) are summarized in

Tables 1 and 2.

To obtain information on the possible role of the

different groups of the molecule of the previously

described antagonist 6a , its structure was modified by

using strategies similar to that described in part 1 of

the present work.¹²Thus, the deletion of the fused

benzene ring of the quinoline nucleus of compounds 6a ,b

seems to have little effect (compare 6a with 8h and 6b

with 8i), while its saturation in compounds 6a and 8c

produces a dramatic decrease in the 5-HT₃receptor

binding affinity (compare 6a with 8f and 8c with 8g).

It is interesting to note that in the small subseries of

compounds without the quinoline benzo ring the shifting

of the pyridine nitrogen atom produces a decrease in

the affinity of about 1 order of magnitude (compare

The search for the best correlation equation was

achieved by means of the heuristic method implemented

in the CODESSA program.²⁷It accomplishes a pre-

selection of descriptors on the basis of their statistical

significance. Default values for control parameters and

criteria were used:²⁸minimum squared correlation

coefficient for the one-parameter correlation to be

considered significant, R²

) 0.1; t-test value for the

min

descriptor to be considered significant in one-parameter

correlation, t₁) 1.5; t-test value for the descriptor to

be considered significant in multiparameter correlation,

t₂) 3.0; highest pair correlation coefficient of two

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1561

Ta ble 1. Effects of Modification of the Heteroaryl Moiety of Quipazine on 5-HT₃Receptor Affinity and Intrinsic Efficacy: Binding

Affinities for the Central 5-HT₃Receptor and Effects on [¹⁴C]Guanidinium Accumulation in NG 108-15 Cells of Compounds 4

and 6-8

1562 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Ta ble 1 (Continued)

Cappelli et al.

a

Each value is the mean ( SEM of three determinations and represents the concentration giving half-maximum inhibition of

[³H]granisetron specific binding to rat cortical membranes. A, pure agonist; PA, partial agonist; Ant, pure antagonist. ^cThe quantitative

b

effects of the compounds on the [¹⁴C]guanidinium accumulation in NG 108-15 cells were expressed as EC₅₀values in the case of both pure

d

and partial agonists and as IC₅₀values in the case of pure antagonists. Compounds were tested in triplicate for each concentration. See

ref 7. ^eSee ref 30. ^fSee ref 10. See ref 12. See ref 31.

g

h

compound 8i with 8j), and the replacement of the

terminal protonable piperazine nitrogen with an oxygen

atom as in compound 8k leads to a dramatic decrease

in the affinity (compare 8i with 8k ). The latter struc-

ture-affinity relationship (SAFIR) trend was observed

also in the previously reported antagonist 6a (compare

6a with 8a ) and partial agonist 7b (see part 1 of the

present work).¹²

Finally, the modification of the phenyl group in the

4-position of the quinoline nucleus of 6a reveals the full

bioisosterism between the phenyl group and the thienyl

group of compound 8b and the importance of the

aromaticity in the interaction with the receptor (com-

pare 6a with 8d ).

one, while the new K_ivalues of compounds 6b and 9a

significantly differ from those previously described.¹⁰In

particular, the apparent affinity of compound 9a was

found to be 6.3-fold higher than that reported earlier

and very similar to that reported for NMQ. This finding

prompted us to reconsider some earlier observations^10,11,31

and stimulated the design of new derivatives closely

related to compound 9a . It was in this subseries of

compounds that the most interesting results were

obtained. In fact, it was found that a large variety of

substituents was tolerated in the 3-position of the

quinoline nucleus of compound 9a , and these substitu-

ents appeared to be capable of operating a fine-tuning

of the 5-HT₃receptor affinity.

On the other hand, the binding studies performed on

the previously published compounds allow some com-

parisons to be made. The new apparent affinity of

compound 6a is very similar to the previously reported

Thus, the introduction of a methyl group, a hy-

droxymethyl group, or a hydroxyethyl group in the

3-position of the quinoline nucleus of compound 9a

(leading to compounds 9d ,e,i, respectively) increases the

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1563

Ta ble 2. Effects of Modification of the Heteroaryl Moiety of Quipazine Derivatives on 5-HT₃Receptor Affinity and Intrinsic Efficacy:

Binding Affinities for the Central 5-HT₃Receptor and Effects on [¹⁴C]Guanidinium Accumulation in NG 108-15 Cells of Compounds 9

and 10

compd

9a ^d

9b

9c

Y

R

R′

K_i(nM) ( SEM^a

intrinsic efficacy^b

EC₅₀or IC₅₀^c(nM)

N

C₆H₅

H

CH₃

5.4 ( 0.3

1100 ( 110

80 ( 12

2.4 ( 0.3

0.98 ( 0.07

3.6 ( 0.2

29 ( 4.2

8.8 ( 1.3

0.4 ( 0.03

0.8 ( 0.04

0.6 ( 0.03

0.7 ( 0.1

1.9 ( 0.04

0.9 ( 0.03

4.1 ( 1.3

12 ( 0.2

0.6 ( 0.04

1.8 ( 0.2

3.9 ( 0.04

78 ( 4.4

CH

N

1-adamantyl

C₆H₅

Ant

80

67

9d

9e^d

9f

C₆H₅

CH₂OH

CH₂OTHF^e

CH₂On-C₄H₉

CH₂OCOC₆H₅

CH₂CH₂OH

CH₂CH₂OTHF^e

CH₂CH₂OH

CH₂CH₂OTHF^e

CH₂CH₂OTHP^g

CHdCH₂

9g^d

9h ^d

9i

Ant

9

10

9j

9k ^f

9l

2-F-C₆H₄

2-OH-C₆H₄

C₆H₅

9m

9n ^f

9o^d

9p ^d

9q

COOC₂H₅

C₆H₅

COOn-C₄H₉

CH₂COOC₂H₅

H

COOC₂H₅

COOn-C₄H₉

CON(n-C₃H₇)₂

10a

10b

10c

10d

granisetron

5-HT

PA

12

3.0 ( 0.2

0.35 ( 0.06

120 ( 34

≈300

a

Each value is the mean ( SEM of three determinations and represents the concentration giving half-maximum inhibition of

[³H]granisetron specific binding to rat cortical membranes. A, pure agonist; PA, partial agonist; Ant, pure antagonist. ^cThe quantitative

b

effects of the compounds on the [¹⁴C]guanidinium accumulation in NG 108-15 cells expressed as EC₅₀values in the case of both pure and

d

partial agonists and as IC₅₀values in the case of pure antagonists. Compounds were tested in triplicate for each concentration. See ref

19a. ^eTHF, 2-tetrahydrofuranyl. ^fSee ref 19b. THP, 2-tetrahydropyranyl.

g

affinity to a variable extent. In fact, methyl derivative

9d shows an affinity slightly higher than that shown

by 9a , while hydroxymethyl derivative 9e is more potent

than 9d , and hydroxyethyl derivative 9i showing sub-

nanomolar affinity is about 1 order of magnitude more

potent than 9a .

Additional steric hindrance appears to be tolerated

depending on the nature of the group involved. Linear

alkyl groups such as n-butyl in compound 9g are less

well-tolerated than cyclic substituents such as those

present in compounds 9h ,f,j,l,m . It is noteworthy that

the replacement of the hydroxyl hydrogen atoms of

compounds 9e,i,k with the bulkier tetrahydrofuranyl

group to give 9f,j,l produces negligible change in the

affinity. Similarly, the presence of the fluorine atom in

the 2-position of the phenyl group in the 4-position of

the quinoline nucleus has negligible effects (compare 9i

with 9k and 9j with 9l).

The introduction of a carboxyethyl group in 9a leading

to compound 9o has negligible effect on the 5-HT₃

receptor affinity, while the corresponding acetic ester

derivative 9q is about 1 order of magnitude more potent

than 9a . The elongation of the ethyl ester side chain of

compound 9o to n-butyl as in compound 9p leads to a

decrease in the affinity similar to that observed in ether

9g. This SAFIR trend is paralleled also in the confor-

mationally constrained compounds 10b,c. The SAFIR

analysis in this subseries of compounds reveals the

bioisosterism between the ethyl ester group of compound

10b and the N,N-dipropylcarboxamido group of 10d .

The replacement of the phenyl group of compound 9a

with a 1-adamantyl group as in compound 9c leads to

a 15-fold decrease in the affinity, while the replacement

of its quinoline nitrogen atom with a sp²carbon atom

as in compound 9b appears to be detrimental.

Qu a n t it a t ive St r u ct u r e-Affin it y R ela t ion sh ip

An a lysis. A quantitative rationalization of this SAFIR

study can be obtained by means of theoretical molecular

descriptors. The molecular determinants which better

rationalize the variation in the 5-HT₃binding affinity

data values are reported in Table 3, together with the

cologarithmic form of the experimental data values of

the ligands considered. A quantitative overview of the

collinearities existing between the theoretical descrip-

tors of Table 3 is shown in Table 4, where the respective

intercorrelation coefficients are given.

More than 50% variation in the 5-HT₃binding affinity

is captured by the average information content molec-

ular descriptor (AIC(1)),³²as shown by the following

equation:

pK_i) -5.14((2.14) + 3.75((0.61)AIC(1) (1)

R²) 0.559; n ) 32; F ) 37.98; s²) 0.493;

R²) 0.501

cv

where R is the correlation coefficient, n is the number

of compounds, F is the value of the Fisher ratio, s is

the standard deviation, and R_cvis the cross-validated

correlation coefficient; the numbers in parentheses are

1564 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Cappelli et al.

Ta ble 3. 5-HT₃Binding Affinities and Theoretical Descriptors of the Protonated Form of the Ligands Reported in Tables 1 and 2

∑f_a′-S^H

∆E_HOMO-LUMO

(eV^-1

compd

pK_i

V_in/V_mol

AIC(1)

ABIC(1)

f_a-FNSA-1

(eV^-1

)

P(N)

Pσ-π(N-H)⁺

6a

6b

8b

8c

8d

8e

8f

8g

8h

8i

8.38

7.34

8.24

7.72

7.54

8.52

5.64

5.57

7.8

7.54

6.3

8.27

5.96

7.1

8.62

9

8.4

7.54

8.05

9.4

9.01

9.22

9.15

8.72

9.08

8.39

7.92

9.22

8.74

8.41

7.1

0.9467

0.9358

0.9571

0.9561

0.9375

0.9263

0.9047

0.9072

0.9455

0.9526

0.9406

0.9652

0.9402

0.8877

0.997

1

3.5616

3.4793

3.6515

3.68

3.1322

3.6684

3.1611

3.354

0.6218

0.6134

0.647

0.461

0.022

7.0732

6.963

1.2313

1.2118

1.2325

1.2298

1.2284

1.235

0.000076

0.000059

0.000061

0.000071

0.000068

0.000059

0.000086

0.000089

0.000038

0.000071

0.000050

0.000046

0.000039

0.000066

0.000028

0.000045

0.000049

0.000804

0.000813

0.000033

0.000037

0.000027

0.000053

0.000167

0.000054

0.000840

0.000849

0.000043

0.000059

0.000060

0.000061

0.000051

0.5022

0.4669

0.5057

0.4594

0.7142

0.7872

0.8893

0.8867

0.8422

0.5867

0.9474

0.644

0.0125

0.018

0.0

0.046

0.029

0.0015

0.0

0.0355

0.034

0.054

0.049

0.048

0.05

0.052

0.046

0.051

0.04

0.053

0.045

0.011

0.046

0.054

0.032

0.029

0.03

6.9197

6.5274

7.3523

7.6263

7.3036

6.3321

7.3342

7.1295

6.847

7.3404

6.9641

7.4769

7.4005

7.6242

7.7154

7.5415

7.7826

7.53

7.4378

7.5707

7.7654

7.4611

7.3374

7.6969

7.6763

7.6649

7.1143

7.2873

7.2827

7.2984

0.6488

0.5324

0.6534

0.5419

0.5801

0.6114

0.6416

0.608

0.6217

0.5108

0.5226

0.612

0.6404

0.5995

0.5834

0.5911

0.6309

0.587

1.355

1.3481

1.3562

1.3283

1.2732

1.24

3.3771

3.5026

3.3194

3.4722

2.8679

3.1354

3.4716

3.6505

3.5973

3.5005

3.5598

3.6477

3.5607

3.8042

3.7329

3.6384

3.7617

3.6412

3.5973

3.6715

3.4829

3.6396

3.589

8j

9a

9b

9c

9d

9e

9f

9g

9h

9i

1.2396

1.2382

1.2328

1.2331

1.2376

1.2363

1.2377

1.2378

1.2384

1.2338

1.2384

1.2362

1.2344

1.2348

1.2343

1.2416

1.2353

1.2349

1.2372

0.5254

0.5708

0.487

0.9834

0.9032

0.9066

1

0.436

0.5347

0.4675

0.4443

0.4194

0.5514

0.486

0.4848

0.5274

0.5784

0.4897

0.6014

0.4779

0.5303

0.4365

9j

9k

9l

0.658

1

0.6222

0.5936

0.6567

0.6243

0.6018

0.6216

0.627

0.6267

0.6028

0.5546

9m

9n

9o

9p

9q

10a

10b

10c

10d

0.9385

1

0.9783

0.927

1

0.9674

0.902

0.922

8.52

3.3876

0.031

the 95% confidence intervals of the regression coefficient

and the intercept. The average information content,

defined on the basis of Shannon’s information theory,³²

encodes the main structural characteristics of the

ligands and explains the portion of the biological

response elicited by nonspecific interactions (dispersion

forces).

The branching ratio and the constitutional diversity

of the different substituents introduced in the 3-position

of the quinoline nucleus are very well-explained by this

index when the subset of 17 more congeneric compounds

(9a -q) is considered:

eroaryl moiety and the suitable amino acids of the

receptor, combined with the averaged bonding informa-

tion content (ABIC(1)) index,³²furnishes a very good

QSAR model explaining 75% of the variation in the

5-HT₃binding affinity of the 32 ligands considered:

pK_i) -4.57((1.55) + 18.94((2.49)ABIC(1) +

34.97((5.01)Σf_a′-S^H(4)

R²) 0.757; n ) 32; F ) 45.23; s²) 0.281;

R²) 0.705

cv

where Σf_a′-S^His the sum of the electrophilic superde-

localizability³³on the atoms of either the benzene ring

of the quinoline nucleus or the saturated ring in the

analogous position.

Omission of compound 10d significantly improves the

equation stability, as underlined by the cross-validated

correlation coefficient:

pK_i) -4.08((1.56) + 3.52((0.48)AIC(1) (2)

R²) 0.811; n ) 17; F ) 64.56; s²) 0.416;

R²) 0.782

cv

Moreover, the inclusion of the molecular orbital index

σ-π bond order for the (N-H)⁺pair³³in this correlation

leads to:

pK_i) -5.79((1.36) + 20.82((2.18)ABIC(1) +

35.74((4.26)Σf_a′-S^H(5)

pK_i) -4.22((0.98) + 3.62((0.27)AIC(1) -

918.8((187.0)Pσ-π (N-H)⁺(3)

R²) 0.83; n ) 31; F ) 68.31; s²) 0.202;

R²) 0.793; omitted 10d

cv

R²) 0.931; n ) 17; F ) 94.12; s²) 0.06;

R²) 0.898

Other significant QSAR models are obtained by

cv

means of the molecular descriptor V_in/V_mol

:

Therefore the propensity of the protonated nitrogen

atom to act as a proton donor toward a nucleophilic site

of the receptor is explicitly considered in this correlation.

Interestingly, the role of specific interactions (π-π or

π charge) between the fused benzene ring of the het-

pK_i) -12.47(3.44) + 21.47((3.60)V_in/V_mol(6)

R²) 0.542; n ) 32; F ) 35.57; s²) 0.511;

R²) 0.482

cv

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1565

Ta ble 4. Correlation Matrix of the Experimental Binding Affinities and Molecular Descriptors of Table 3

∑f_a′-S^H

(eV^-1

∆E

HOMO-LUMO

pK_i

V_in/V_mol

AIC(1)

ABIC(1)

f_a-FNSA-1

)

(eV^-1

)

P(N)

Pσ-π(N-H)⁺

pK_i

1.00

0.73

0.75

0.59

-0.67

0.52

0.58

-0.60

-0.03

V_in/V_mol

AIC(1)

ABIC(1)

f_a-FNSA-1

∑f_a′-S^H

∆E

1.00

0.52

0.57

1.00

0.86

1.00

-0.32

-0.18

0.05

-0.20

-0.07

-0.26

-0.63

1.00

-0.50

-0.40

0.83

0.24

0.16

1.00

0.76

-0.63

0.32

0.24

-0.29

-0.30

0.32

-0.45

0.12

1.00

-0.35

0.37

HOMO-LUMO

P(N)

1.00

Pσ-π(N-H)⁺

-0.13

1.00

This index describes the size and shape similarity with

respect to a reference supermolecule.^12,26,34It is assumed

that the volume obtained by superimposing the most

structurally different ligands which show the highest

affinities might reflect the overall shape and the con-

formational freedom of the 5-HT₃receptor binding site.

The sign of the regression coefficient in the equation

indicates that the higher the molecular volume shared

by a generic ligand (V_in) and the supermolecule the

higher the binding affinity. Thus, in this series of

compounds the binding affinity is modulated by the

molecular shape of the heteroaryl moiety through the

optimization of dispersive interactions.

According to the rationale behind these molecular

descriptors, which have been devised to account for the

strict complementary requirements toward receptor

binding, a significant improvement in the statistical

parameters is obtained by considering only the ligands

which show high affinity (pK_i) 7 ÷ 9.4) toward the

5-HT₃receptor:

F igu r e 1. Calculated versus experimental 5-HT₃binding

affinities according to eq 10.

f_a-FNSA-1 is calculated according to the following

formula: PNSA-1/TFSA, where PNSA-1 is the partial

negative surface area of the pyridine ring and TFSA is

the total fragment surface area of the pyridine ring. This

index may be thought to rationalize the electrostatic

component of the binding adjustments which modulates

the binding affinity after the main docking has been

accomplished and which helps to overcome the main

drawback of the V_in/V_moldescriptor which considers

equally active all the ligands whose V_incoincides with

pK_i) -6.47((2.09) + 15.42((2.18)V_in/V_mol(7)

R²) 0.658; n ) 28; F ) 49.95; s²) 0.160;

R²) 0.608; 8d ,f,j, and 9b omitted

cv

The addition of the valency-related bond order de-

scriptor for the nitrogen atom of the quinoline ring

(P(N))³³as a second parameter improves the predictivity

of the model for the whole set of compounds carrying a

nitrogen atom in position 1 of the quinoline ring:

V

_mol, irrespective of their size.³⁴

Finally, the predictivity of the model is increased by

considering as third parameter, the energy difference

between the lowest unoccupied (LUMO) and the highest

occupied (HOMO) molecular orbitals (∆E_HOMO-LUMO):³³

pK_i) 3.97((4.26) + 17.14((2.49)V_in/V_mol

-

9.77((2.32)P(N) (8)

pK_i) -11.59((2.79) + 16.02((2.33)V_in/V_mol

2.91((0.59)f_a-FNSA-1 +

-

R²) 0.771; n ) 30; F ) 45.44; s²) 0.219;

R²) 0.683; 8j and 9b omitted

0.81((0.25)∆E_HOMO-LUMO(10)

cv

R²) 0.840; n ) 32; F ) 49.07; s²) 0.191;

This index renders the model fit to describe the pro-

pensity of the quinoline nitrogen to give hydrogen-

bonding interactions with the receptor, as shown by the

negative value of the regression coefficient.

The involvement of the fractional negative partial

surface area³⁵(f_a-FNSA-1) of the pyridine ring of the

heteroayl moiety in the following equation leads to a

QSAR model with analogous statistical parameters:

R²) 0.784

cv

This molecular descriptor, being related to the polariz-

ability of the molecules, expresses the potential ability

to establish attractive short-range interactions with the

receptor. The comparison between the experimental

binding affinities and those calculated by eq 10 is shown

in Figure 1. The latter equation can be considered, on

the basis of its statistical indices and predictive power,

the best QSAR equation to describe the variation in the

5-HT₃binding affinity in the whole set of ligands

considered.

pK_i) -6.45((2.66) + 17.30((2.65)V_in/V_mol

-

3.59((0.43)f_a-FNSA-1 (9)

R²) 0.779; n ) 32; F ) 51.27; s²) 0.255;

Selectivity. Three compounds, representative of the

various structural subclasses, were tested for their

R²) 0.714

cv

1566 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Cappelli et al.

Ta ble 5. 5-HT Receptor Binding Profile of Compounds 8b, 9h , a n d 10a (K_i(nM) (SEM)^a

compd

5-HT₃

5-HT_1A

5-HT_1B

5-HT_2A

5-HT_2C

5-HT_transporter

8b

5.7 ( 0.7

12000 ( 1200

(2100)

4100 ( 1200

(470)

24000 ( 5100

(13000)

3600 ( 800

(2000)

1500 ( 510

(260)

7600 ( 1200

(860)

3100 ( 31

420000 ( 51000

(74000)

31 ( 2.1

(3.5)

>10000

(>5600)

190^b

4600 ( 1300

(810)

1100 ( 340

(130)

1600 ( 370

(890)

31 ( 2.9

(17)

(540)

9h

8.8 ( 1.3

1.8 ( 0.2

1.8 ( 0.3

>10000

(>1100)

13000 ( 3400

(7200)

1800 ( 480

(1000)

10a

640 ( 82

(360)

4a (quipazine)

310^b

(170)

(110)

granisetron

5-HT

8-OH-DPAT

mesulergine

2c (6-nitroquipazine)

0.35 ( 0.06

120 ( 34

7.3 ( 1.2

1.2 ( 0.13

1.1 ( 0.098

740 ( 120

0.73 ( 0.083

58 ( 3.2

0.12 ( 0.01

a

Each value is the mean ( SEM of three determinations. Each value in parentheses is the selectivity ratio calculated as the ratio of

b

the K_ivalue for the indicated site over that for 5-HT₃receptors. See ref 46.

possible binding to several 5-HT receptor subtypes

interacting with quipazine derivatives (5-HT_1A, 5-HT_1B

Finally, compounds 8b,d ,e and 9c,d ,h ,i showed an-

tagonist properties with IC₅₀values ranging from 9 to

250 nM.

,

5-HT_2A, 5-HT_2C) and to the 5-HT transporter in com-

parison with reference compounds by means of well-

established protocols.³⁶From the results of these bind-

ing studies, summarized in Table 5, it appeared that

compounds 10a and 8b are very selective, indeed

showing selectivity ratios higher than those of quipa-

zine, while the binding profile of 9h appears to be

characterized by the residual affinity for 5-HT_2Creceptor

subtype, so that the latter compound can be considered

a mixed 5-HT_3/2Creceptor ligand.

Discu ssion

Ar ylp ip er a zin e In ter a ction s a t th e Cen tr a l 5-HT₃

Recep tor . In the already published part 1 of this

work,¹²a three-component model for the interaction of

central 5-HT₃ligands related to quipazine with their

receptor was proposed, which involves (1) a charge-

assisted hydrogen bond between the protonated termi-

nal piperazine nitrogen atom and a negatively charged

carboxylic amino acid residue in the receptor, (2) a

hydrogen-bonding interaction between the heterocyclic

nitrogen atom and a suitable H-bond donor in the

receptor, and (3) a specific interaction between an

aromatic ring and a suitable amino acid residue in the

receptor.

In the development of a comprehensive model for the

interaction of central 5-HT₃receptor ligands related to

quipazine showing different intrinsic efficacy with their

receptor, it is of central importance to ascertain the

existence of a common mode of binding of these arylpip-

erazines to their receptor. This task has been ac-

complished through the comparative analysis of the

SAFIR trends involving the key pharmacophoric points

of the different subseries of compounds belonging to this

class of heteroarylpiperazines,³⁷as well as through the

derivation of predictive and interpretative QSAR mod-

els.^12,38

For example, in the case of our heteroarylpiperazine

antagonists, the differences in the affinity observed

between 6a and 8a and the one shown by the couple of

closely related compounds 9a ,b support the key role

played by the terminal piperazine protonatable nitrogen

(first pharmacophoric point) and that of the quinoline

nitrogen atom (second pharmacophoric point), respec-

tively. Moreover, it is worth noting that the quinoline

benzo ring plays a key role in the interaction with the

receptor (third pharmacophoric point), as shown by the

difference in affinity between 6a and 8f and between

8c and 8g.

On the other hand, compounds 8i,h apparently lack

the third pharmacophoric point and show relatively high

affinity and agonist properties at the central 5-HT₃

receptor. In this compound subseries, the low affinity

shown by compounds 8j,k shows the importance of the

two remaining pharmacophoric points and suggests that

F u n ction a l Activity. The measurements of [¹⁴C]-

guanidinium uptake in the presence of substance P (SP)

appears to be a rapid and reliable method to assess the

functional properties of 5-HT₃agents in NG 108-15

hybridoma cells.⁷Owing to the similarities existing

between 5-HT₃receptors present in NG 108-15 cells and

those of rat cortex,⁸relevant inferences can be drawn

from this pharmacological model about the functional

properties of the ligands of the central 5-HT₃receptor.⁹

Accordingly, the potential 5-HT₃agonist/antagonist

activity of some selected compounds was assessed in

vitro on the 5-HT₃receptor-dependent [¹⁴C]guanidinium

uptake in NG 108-15 cells. The results of these func-

tional studies are summarized in Tables 1 and 2. The

previously reported intrinsic efficacy data of compounds

6a and 7b,c and new data for the previously described

compounds 7a and 8e are included for comparative

purposes.

In the presence of substance P (10 µM) compounds

7a and 8h ,i increased in a concentration-dependent

manner the uptake of [¹⁴C]guanidinium into NG 108-

15 cells with EC₅₀values of about 10, 7, and 30 nM,

respectively, showing themselves to act as 5-HT₃ago-

nists. The maximum uptake increase was similar to that

of 5-HT and was antagonized by ondansetron (1).

Moreover, these compounds were unable to inhibit the

effects of 5-HT (1 µM).

On the other hand, compound 10a appeared to act as

a partial agonist with an EC₅₀value of about 12 nM

since it stimulated [¹⁴C]guanidinium uptake in the

presence of substance P (10 µM), up to a level equal to

about two-thirds of that reached with 5-HT (1 µM) plus

substance P (10 µM), while in the presence of the latter

two agents 10a produced a decrease in the accumulation

of [¹⁴C]guanidinium. Similarly, the closely related com-

pound 10d behaved as a partial agonist, but it displayed

a potency and intrinsic efficacy lower than that of 10a .

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1567

the assumed pharmacophoric model probably works also

in these central 5-HT₃receptor agonists. On the whole,

the SAFIR analysis clearly suggests a common mode of

binding at the central 5-HT₃receptor of these het-

eroarylpiperazine derivatives. Further support to this

conclusion is provided by the results obtained by a

quantitative rationalization of the binding affinity data

values for these ligands.

perpendicular to quinoline) showing very close 5-HT₃

receptor affinities fails to show a clear preference of the

receptor for a particular conformation of the phenyl.

Therefore, if an aromatic specific interaction involves

this aromatic moiety in the interaction with the recep-

tor, it appears to lack any evident directionality.

The high affinity shown by the derivative 9a allowed

the substitution on the 3-position of the quinoline

nucleus of 9a to be explored. The SAFIR trends analyzed

above suggest that in the area of the receptor binding

site interacting with the groups present in the 3-position

of the quinoline nucleus of quipazine or of compound

9a there might exist a pocket mainly composed of

lipophilic amino acid residues in which a hydrogen-

bonding interaction may be involved with the ligand

oxygen atoms. The limited dimensions of this pocket can

be inferred from the reduced affinity of the n-butyl

derivatives 9g,p and 10c.

Very good QSAR models rationalizing the main

structural characteristics of this subset of ligands are

obtained (eqs 2 and 3) by means of the average informa-

tion content index (AIC(1)). This index, which reflects

the branching ratio and constitutional diversity of the

various substituents introduced in the 3-position of the

quinoline nucleus, accounts for more than 80% of the

variation in the binding affinity of the subset considered.

Molecu la r Ba sis of th e In tr in sic Effica cy of

Ar ylp ip er a zin e Der iva tives a t th e Cen tr a l 5-HT₃

Recep tor s. In the already published part 1 of this

work,¹²the interactions in the area of the receptor

binding site interacting with the c-edge of the quinoline

nucleus of quipazine were assumed to be of crucial

importance in modulating the intrinsic efficacy at the

central 5-HT₃receptor. The comparison between the

functional data available for the newly synthesized

compounds (reported in Tables 1 and 2 and described

in the relative section) with those relative to the

previously described ones allows the structural deter-

minants responsible for the intrinsic efficacy of the

quipazine derivatives to be more comprehensively de-

fined.

The molecular descriptors involved in the QSAR

models obtained codified for the three-component phar-

macophore previously presented. In fact, the long-range

interaction component is explicitly considered in eq 3

by the σ-π bond order for the (N-H)⁺pair showing the

propensity of the protonated nitrogen to act as a proton

donor and form charge-reinforced hydrogen-bonding

interactions. The modulation in the binding affinity data

values supposed to be due to a hydrogen-bonding

interaction between the heterocyclic nitrogen atom and

a suitable H-bond donor of the receptor is accounted for

by the valency-related bond order descriptor P(N) (eq

7). Moreover, the partial negative surface area of the

heteroaryl moiety pyridine ring (f_a-FNSA-1) rational-

izes the electrostatic component of this H-bonding

interaction (eqs 9 and 10). Finally, the aromatic specific

interaction with the receptor is represented by the sum

of the electrophilic superdelocalizability on the atoms

of the benzene ring of either the quinoline nucleus or

the saturated ring in analogous position (eqs 4 and 5).

It is worth noting that the most appreciable contribution

to the rationalization of the binding affinity in this set

of ligands is given by theoretical descriptors (AIC(1),

ABIC(1), V_in/V_mol, and ∆E_HOMO-LUMO) which encode the

potential for dispersion-type interactions with nonspe-

cific receptive surfaces. In particular, eqs 6-10, which

involve the size and shape descriptor V_in/V_mol, suggest

that optimization of the binding affinity is obtained by

maximizing the amount of bulk in one ligand in the

region (delimited by V_sup) assumed to represent a map

of the target 5-HT₃binding site (see QSAR section).

In the refinement of the original interaction model,

our attention was particularly focused on the receptor

area interacting with the c-edge (positions 3 and 4) of

the quinoline nucleus of quipazine since interactions of

crucial importance in modulating the intrinsic efficacy

at the central 5-HT₃receptor appear to be involved in

this area. The effects of the occupancy of this receptor

area are indeed very complex. In fact, the fusion of a

monocyclic system on the c-edge enhances the affinity

to a variable extent depending on the nature of the

system involved, and in this first stage the saturated

ring, as it occurs in the case of compound 7b, is better

recognized than the aromatic one of compound 7a . On

the other hand, the presence of an additional ring

decreases the affinity (compare 7b with 6a , 8b,d and

7a with 6b). Also in this second stage the nature of the

system involved affects the affinity, but in this case an

aromatic ring is better tolerated than a saturated one

(compare 6a with 8d ). This preference for aromaticity

is also supported by the comparison between 9a ,c.

On the whole, the intrinsic efficacy appears to be

rather subtly modulated by the steric features of the

heterocyclic moiety, as shown in Figures 2 and 3.

In particular, given the plane xy coincident with the

quinoline nucleus, the intrinsic efficacy appears to be

modulated by the steric hindrance on the z-axis of the

substituents on the c-edge of the quinoline nucleus. In

fact, among the compounds showing almost the same

projection in the xy-plane, compound 7a possesses a

benzo ring (low occupancy on the z-axis) on the c-edge

and is a full agonist, while 7c possesses a rigid bicyclic

system (higher occupancy on the z-axis) and shows

antagonist properties. Finally, 7b possessing a cyclo-

hexane ring condensed on the c-edge (intermediate

occupancy joined to a certain degree of mobility) shows

partial agonist properties (Figure 2). Furthermore, the

intrinsic efficacy appears to be modulated also by the

molecular geometry of the substituent on faces c, d, and

e of the quinoline nucleus. In fact, compounds 7a , 10a ,

and 8e³¹show planar heteroaryl moieties and quite

similar 5-HT₃receptor affinities, but the differences in

their heteroaryl moieties (Figure 3) are reflected in the

intrinsic efficacy, the compounds being full agonist,

As far the spatial requirements of this aromatic group

are concerned, the new data imply a revision of some

earlier conclusions.^10,11,31The comparison of compounds

10a (phenyl ring coplanar with quinoline), 6a (35°

deviation from the coplanarity), and 9a ,d (phenyl ring

1568 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Cappelli et al.

F igu r e 2. Superposition of agonist 7a (red), partial agonist 7b (yellow), and antagonist 7c (blue) showing the occupancy in

planes xy and xz.

F igu r e 3. Superposition of agonist 7a (red), partial agonist 10a (yellow), and antagonist 8e (blue) showing the occupancy in

planes xy and xz.

partial agonist, and antagonist, respectively. Finally, the

intrinsic efficacy appears to be related to the presence

of the quinoline benzo ring as the ring deletion in

antagonist 6a yields the full agonist 8h .

of intrinsic efficacies were discovered starting from

quipazine and previously published 5-HT₃receptor

antagonists 6. The interaction model for the quipazine-

related 5-HT₃ligands with their receptor, proposed in

the already published part 1 of this work,¹²has been

revised and refined to incorporate the latest data on the

basis of qualitative and quantitative structure-affinity

relationship studies.

Con clu sion s

Novel central 5-HT₃receptor ligands provided with

nanomolar or subnanomolar affinity and a full range

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1569

chromatography (Al₂O₃), and the elution with n-hexane-ethyl

acetate (65:35) gave the pure target 4-methyl-1-piperazinyl

derivatives.

The physical information carried by the theoretical

molecular descriptors involved in the QSAR models

obtained confirms the fundamental role of the proton-

ated terminal piperazine nitrogen for the recognition

step (electrostatic interaction) and the key role of the

heterocyclic nitrogen atom for a correct docking (hydro-

gen-bonding interaction). Moreover, a fine-tuning of the

binding affinity is accomplished by the optimization of

short-range intermolecular interactions and dispersion

contributions of the various molecular regions. In fact,

both strength and specificity arise from the cumulation

and interplay of many weak interactions established by

the receptor with the quinoline benzo ring, the cyclic

system on the c-edge, and/or the substituent in the

3-position of the quinoline nucleus of the ligands.

7,8-Dih ydr o-6-m or ph olin oben zo[k]ph en an th r idin e (8a).

The title compound was prepared by method A from 14a and

morpholine (reaction time 17 h) to obtain a white crystalline

1

solid (0.29 g, 92%): mp 165-166 °C. H NMR (CDCl₃): 2.80

(m, 4H), 3.35 (t, J ) 4.5, 4H), 3.92 (t, J ) 4.4, 4H), 7.38 (m,

4H), 7.59 (t, J ) 7.1, 1H), 7.92 (m, 2H), 8.34 (d, J ) 8.7, 1H).

Anal. (C₂₁H₂₀N₂O) C, H, N.

7,8-Dih yd r o-6-(4-m et h yl-1-p ip er a zin yl)t h ien o[2,3-k ]-

p h en a n th r id in e (8b). The title compound was prepared by

method A from 14b and N-methylpiperazine (reaction time

22 h) to obtain a pale-yellow oil which crystallized on standing

(0.31 g, 92%): mp 147-149 °C. ¹H NMR (CDCl₃): 2.39 (s, 3H),

2.64 (t, J ) 4.4, 4H), 2.88-3.02 (m, 4H), 3.35 (t, J ) 4.7, 4H),

7.27 (d, J ) 5.1, 1H), 7.38 (t, J ) 7.8, 1H), 7.59 (t, J ) 7.3,

1H), 7.71 (d, J ) 5.3, 1H), 7.92 (d, J ) 8.2, 1H), 8.31 (d, J )

8.4, 1H). Anal. (C₂₀H₂₁N₃S) C, H, N.

7-(4-Meth yl-1-p ip er a zin yl)-6H-[1]ben zoth iop yr a n o[3,4-

c]qu in olin e (8c). The title compound was prepared by method

A from 14c and N-methylpiperazine (reaction time 21 h) to

obtain a pale-yellow oil which crystallized on standing (0.33

g, 95%): mp 140-141 °C. ¹H NMR (CDCl₃): 2.40 (s, 3H), 2.65

(t, J ) 4.4, 4H), 3.35 (t, J ) 4.7, 4H), 3.88 (s, 2H), 7.36 (m,

3H), 7.61 (m, 2H), 7.84 (m, 1H), 7.95 (d, J ) 8.7, 1H), 8.13 (d,

J ) 8.6, 1H). Anal. (C₂₁H₂₁N₃S‚0.25H₂O) C, H, N

Finally, the comparison between the functional data

available for the new ligands and those relative to the

previously described compounds has allowed a compre-

hensive definition of the structural determinants re-

sponsible for the intrinsic efficacy of the quipazine

derivatives in terms of steric hindrance and molecular

geometry of the heteroaryl moiety of these arylpipera-

zines.

tr a n s-6-(4-Meth yl-1-p ip er a zin yl)-7,8,8a ,9,10,11,12,12a -

octa h yd r oben zo[k]p h en a n th r id in e (8d ). The title com-

pound was prepared by method A from 14d and N-methylpip-

erazine (reaction time 23 h) to obtain a pale-yellow oil which

crystallized on standing (0.30 g, 89%): mp 156-157 °C. ¹H

NMR (CDCl₃): 1.21 (m, 2H), 1.52 (m, 3H), 1.75-2.04 (m, 5H),

2.37 (s, 3H), 2.49-2.72 (m, 6H), 2.90 (m, 2H), 3.25 (m, 2H),

3.53 (m, 2H), 7.30 (m, 1H), 7.50 (t, J ) 7.9, 1H), 7.83 (m, 2H).

Anal. (C₂₂H₂₉N₃) C, H, N.

1,2,3,4,7,8-Hexa h yd r o-6-(4-m eth yl-1-piper a zin yl)ben zo-

[k]p h en a n th r id in e (8f). The title compound was prepared

by method B from 15a and N-methylpiperazine (reaction time

4 h) to obtain a colorless oil which crystallized on standing

(0.22 g, 66%): mp 150-151 °C. ¹H NMR (CDCl₃): 1.72 (m,

2H), 1.92 (m, 2H), 2.35 (s, 3H), 2.57 (t, J ) 4.7, 4H), 2.66 (s,

4H), 2.91 (q, J ) 6.1, 4H), 3.18 (t, J ) 4.7, 4H), 7.27 (m, 3H),

7.59 (m, 1H). Anal. (C₂₂H₂₇N₃) C, H, N.

Exp er im en ta l Section

Melting points were determined in open capillaries on a

Gallenkamp apparatus and are uncorrected. Microanalyses

were carried out using a Perkin-Elmer 240C elemental ana-

lyzer. Merck silica gel 60 (70-230 or 230-400 mesh) and

Merck aluminum oxide 90 II-III (70-230 mesh) were used

for column chromatography. Merck TLC plates, silica gel 60

1

F

₂₅₄, were used for TLC. H NMR spectra were recorded with

a Bruker AC 200 spectrometer in the indicated solvents (TMS

as internal standard): the values of chemical shifts are

expressed in ppm and coupling constants (J ) in Hz. Mass

spectra (EI, 70 eV) were recorded either on a VG 70-250S

spectrometer (Centro di Analisi e Determinazioni Strutturali,

Universita` di Siena) or on a Varian Saturn 3 (Dipartimento

Farmaco Chimico Tecnologico, Universita` di Siena). NMR

spectra and elemental analyses were performed in the Dipar-

timento Farmaco Chimico Tecnologico, Universita` di Siena.

7-(4-Meth yl-1-p ip er a zin yl)-9,10,11,12-tetr a h yd r o-6H-[1]-

ben zoth iop yr a n o[3,4-c]qu in olin e (8g). The title compound

was prepared by method B from 15c and N-methylpiperazine

(reaction time 2 h) to obtain a white crystalline solid (0.29 g,

P r epa r a tion s of Ta r get 4-Meth yl-1-p ip er a zin yl or Mor -

p h olin o Der iva tives 8-10. Meth od A. A mixture of the

appropriate imidoyl chloride (1.0 mmol) with N-methylpip-

erazine or morpholine (5 mL) was heated at 130-140 °C under

argon for a suitable time (2-44 h), and the reaction progress

was monitored by TLC. When the chloro derivative disap-

peared from the chromatogram, the reaction mixture was

poured into ice-water (250 mL) and extracted with chloroform

(4 × 15 mL). The organic layer was washed with water (2 ×

30 mL), dried over sodium sulfate, and evaporated under

reduced pressure. Purification of the residue by flash chro-

matography with ethyl acetate-triethylamine (8:2) as the

eluent (or n-hexane-ethyl acetate (8:2) in the case of mor-

pholino derivatives) gave the pure target compounds.

1

82%): mp 184-185 °C. H NMR (CDCl₃): 1.63 (m, 2H), 1.90

(m, 2H), 2.36 (s, 3H), 2.58 (t, J ) 4.2, 4H), 2.81 (t, J ) 5.9,

2H), 2.91 (t, J ) 6.4, 2H), 3.14 (t, J ) 4.7, 4H), 3.74 (s, 2H),

7.24 (m, 2H), 7.50-7.62 (m, 2H). Anal. (C₂₁H₂₅N₃S) C, H, N.

5,6-Dih yd r o-4-(4-m eth yl-1-p ip er a zin yl)ben z[f]isoqu in -

olin e (8h ). The title compound was prepared by method B

from 18 and N-methylpiperazine (reaction time 3 h) to obtain

an oil which crystallized on standing (0.21 g, 75%): mp 116-

1

118 °C. H NMR (CDCl₃): 2.37 (s, 3H), 2.60 (t, J ) 4.5, 4H),

2.81 (s, 4H), 3.20 (t, J ) 4.7, 4H), 7.30 (m, 4H), 7.70 (m, 1H),

8.26 (d, J ) 5.4, 1H). Anal. (C₁₈H₂₁N₃) C, H, N.

4-(4-Meth yl-1-p ip er a zin yl)ben z[f]isoqu in olin e (8i). The

title compound was prepared by method A from 4-chlorobenz-

[f]isoquinoline^15band N-methylpiperazine (reaction time 9 h)

to obtain an oil which crystallized on standing (0.26 g, 94%).

An analytical sample was obtained by recrystallization from

Meth od B. To a mixture of the appropriate pyridones (1.0

mmol) and anhydrous Na₂CO₃(0.26 g, 2.45 mmol) in CH₂Cl₂

(20 mL) cooled to -60 °C was added trifluoromethanesulfonic

anhydride (0.5 mL, 2.97 mmol). The resulting mixture was

allowed to stir at the same temperature for 10 min and at 0

°C for 1 h and then filtered, and the filtrate was concentrated

under reduced pressure. The residue was diluted with 5 mL

of N-methylpiperazine, and the resulting mixture was heated

at 130-140 °C under argon for a suitable time (the reaction

progress was monitored by TLC). The reaction mixture was

then poured into ice-water, and the precipitate was extracted

with CH₂Cl₂(5 × 10 mL). The combined organic extracts were

washed with brine, dried over sodium sulfate, and evaporated

under reduced pressure. The residue was purified by column

1

n-hexane: mp 92-94 °C. H NMR (CDCl₃): 2.44 (s, 3H), 2.74

(t, J ) 4.7, 4H), 3.48 (t, J ) 4.8, 4H), 7.68 (m, 2H), 7.78 (d, J

) 9.0, 1H), 7.88-8.06 (m, 3H), 8.40 (d, J ) 5.8, 1H), 8.62 (m,

1H). Anal. (C₁₈H₁₉N₃) C, H, N.

4-(4-Meth yl-1-p ip er a zin yl)ben zo[h ]qu in olin e (8j). The

title compound was prepared by method A starting from

4-chlorobenzo[h]quinoline¹²and N-methylpiperazine (reaction

time 44 h) to obtain an oil which crystallized on standing (0.24

g, 86%). An analytical sample was obtained by recrystallization

from n-hexane: mp 154-156 °C. ¹H NMR (CDCl₃): 2.43 (s,

1570 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Cappelli et al.

3H), 2.74 (t, J ) 4.6, 4H), 3.28 (t, J ) 4.7, 4H), 7.04 (d, J )

5.0, 1H), 7.63-7.78 (m, 3H), 7.86-7.96 (m, 2H), 8.83 (d, J )

5.0, 1H), 9.26 (m, 1H). Anal. (C₁₈H₁₉N₃) C, H, N.

4-Mor p h olin oben z[f]isoqu in olin e (8k ). The title com-

pound was prepared by method A from 4-chlorobenz[f]-

isoquinoline^15band morpholine (reaction time 22 h) to obtain

a white crystalline solid (0.23 g, 87%): mp 143-145 °C. ¹H

NMR (CDCl₃): 3.42 (t, J ) 4.7, 4H), 4.00 (t, J ) 4.7, 4H), 7.68

(m, 2H), 7.79 (d, J ) 9.0, 1H), 7.90-8.09 (m, 3H), 8.41 (d, J )

5.9, 1H), 8.62 (m, 1H). Anal. (C₁₇H₁₆N₂O) C, H, N.

(11d )) obtained from Aldrich (10 mmol), freshly distilled

anhydrous ethyl ether (40 mL), and pyrrolidine (3.3 mL, 39.5

mmol). To the resulting solution was added, over a 20-30-

min period, TiCl₄(0.55 mL, 5.0 mmol) in 10 mL of dry benzene.

The temperature was kept between 0 and 10 °C during the

addition. When the TiCl₄addition was complete, the mixture

was allowed to stir at room temperature for 19 h. The reaction

mixture was then filtered, the solvent was removed under

reduced pressure, and the corresponding enamine was used

immediately without further purification.

4-(1-Ad a m a n t yl)-2-(4-m et h yl-1-p ip er a zin yl)q u in olin e

(9c). The title compound was prepared by method A starting

from 24 and N-methylpiperazine (reaction time 3 h) to obtain

an oil which crystallized on standing (0.31 g, 86%): mp 133-

135 °C. ¹H NMR (CDCl₃): 1.86 (s, 6H), 2.18 (s, 3H), 2.25 (s,

6H), 2.36 (s, 3H), 2.55 (t, J ) 4.9, 4H), 3.75 (t, J ) 5.0, 4H),

6.90 (s, 1H), 7.18 (m, 1H), 7.46 (m, 1H), 7.73 (m, 1H), 8.37 (d,

J ) 7.9, 1H). Anal. (C₂₄H₃₁N₃) C, H, N.

3-Met h yl-2-(4-m et h yl-1-p ip er a zin yl)-4-p h en ylq u in o-

lin e (9d ). The title compound was prepared by method A

starting from 2-chloro-3-methyl-4-phenylquinoline³⁹and N-

methylpiperazine (reaction time 5 h) to obtain an oil which

crystallized on standing (0.23 g, 72%): mp 133-134 °C. ¹H

NMR (CDCl₃): 2.13 (s, 3H), 2.38 (s, 3H), 2.63 (t, J ) 4.8, 4H),

3.39 (t, J ) 4.7, 4H), 7.21 (m, 4H), 7.48 (m, 4H), 7.89 (d, J )

8.4, 1H). Anal. (C₂₁H₂₃N₃‚0.25 H₂O) C, H, N.

Gen er a l P r oced u r e for th e P r ep a r a tion of Qu in olin -

on es 13a -d . The suitable enamine 12a -d (10 mmol) was

dissolved into 10 mL of CHCl₃, and the resulting solution was

cooled at 0 °C. To this was added a solution of phenyl

isocyanate (1.1 mL, 10.1 mmol) in CHCl₃(10 mL) dropwise.

When the addition was complete, the reaction mixture was

stirred for 2 h at room temperature, the solvent was removed

under reduced pressure, and the residue was refluxed for 1 h

in ethanol (40 mL). The solvent was then removed, and the

residue was partitioned between dichloromethane and 1 N

HCl. The organic layer was washed with water, dried over

sodium sulfate, and concentrated under reduced pressure.

Purification by chromatography of the residue (n-hexane-

ethyl acetate (65:35) as the eluent) gave an oil which crystal-

lized on standing or by treatment with n-hexane-ethyl ether.

To the material so obtained was added PPA (20 g), and the

resulting mixture was heated at 150 °C with stirring for 10-

50 min. Then, the cooled brown mass was decomposed with

ice-water, and the precipitate was extracted with CHCl₃(3

× 40 mL). The combined organic extracts were washed with

water, dried over sodium sulfate, and concentrated under

reduced pressure. Purification of the residue by washing with

ethyl acetate or by flash chromatography gave the pure title

compounds as white solids.

Eth yl 2-(4-Meth yl-1-p ip er a zin yl)-4-p h en yl-3-qu in olin e-

a ceta te (9q). The title compound was prepared by method A

starting from 26 and N-methylpiperazine (reaction time 8 h)

to obtain an oil which crystallized on standing (0.31 g, 79%):

1

mp 109-110 °C. H NMR (CDCl₃): 1.14 (t, J ) 7.0, 3H), 2.36

(s, 3H), 2.59 (t, J ) 4.6, 4H), 3.29 (t, J ) 4.8, 4H), 3.61 (s, 2H),

4.01 (q, J ) 7.0, 2H), 7.23 (m, 4H), 7.49 (m, 3H), 7.58 (m, 1H),

7.94 (d, J ) 8.2, 1H). Anal. (C₂₄H₂₇N₃O₂) C, H, N.

2-(4-Methyl-1-piperazinyl)indeno[1,2,3-de]quinoline (10a).

The title compound was prepared by method A starting from

30 and N-methylpiperazine (reaction time 2 h) to obtain an

oil which crystallized on standing (0.29 g, 96%): mp 94-96

°C.¹H NMR (CDCl₃): 2.39 (s, 3H), 2.61 (t, J ) 5.1, 4H), 3.89

(t, J ) 5.0, 4H), 7.30-7.48 (m, 3H), 7.58 (m, 3H), 7.83 (m, 2H).

Anal. (C₂₀H₁₉N₃‚0.25H₂O) C, H, N.

7,8-Dih yd r ob en zo[k ]p h en a n t h r id in -6(5H )-on e (13a ).

This compound was prepared in 21% yield starting from

enamine 12a and was purified by flash chromatography (ethyl

acetate as the eluent): mp 246-248 °C (lit.²¹mp 250-252

°C).¹H NMR (CDCl₃): 2.86 (m, 4H), 7.19-7.50 (m, 6H), 7.86

(m, 1H), 8.17 (d, J ) 8.3, 1H), 11.56 (br s, 1H).

7,8-Dih ydr oth ien o[2,3-k]ph en an th r idin -6(5H)-on e (13b).

This compound was prepared in 11% yield starting from

enamine 12b and was purified by flash chromatography (ethyl

acetate as the eluent): mp 227-229 °C. ¹H NMR (CDCl₃): 3.02

(m, 4H), 7.27 (m, 2H), 7.38 (d, J ) 7.5, 1H), 7.51 (t, J ) 7.9,

1H), 7.63 (d, J ) 5.5, 1H), 8.19 (d, J ) 7.9, 1H), 11.02 (br s,

1H). HR-MS: m/z calcd for (C₁₅H₁₁NOS) 253.0561, found

253.0552.

Eth yl 2-(4-Meth yl-1-p ip er a zin yl)in d en o[1,2,3-d e]qu in -

olin e-1-ca r boxyla te (10b). The title compound was prepared

by method A starting from 34 and N-methylpiperazine (reac-

tion time 2 h and 30 min) to obtain a yellow oil which

crystallized on standing (0.33 g, 88%): mp 139-140 °C.¹H

NMR (CDCl₃): 1.46 (t, J ) 7.0, 3H), 2.36 (s, 3H), 2.57 (t, J )

4.7, 4H), 3.61 (t, J ) 4.7, 4H), 4.53 (q, J ) 7.1, 2H), 7.31 (t, J

) 7.5, 1H), 7.44 (t, J ) 7.4, 1H), 7.62 (m, 3H), 7.79 (d, J ) 7.3,

1H), 7.97 (d, J ) 7.6, 1H). Anal. (C₂₃H₂₃N₃O₂) C, H, N.

n -Bu tyl 2-(4-Meth yl-1-piper azin yl)in den o[1,2,3-de]qu in -

olin e-1-ca r boxyla te (10c). The title compound was prepared

by method A starting from 35 and N-methylpiperazine (reac-

tion time 1 h) to obtain a yellow oil which crystallized on

standing (0.37 g, 92%): mp 95-97 °C.¹H NMR (CDCl₃): 0.97

(t, J ) 7.2, 3H), 1.48 (m, 2H), 1.80 (m, 2H), 2.37 (s, 3H), 2.58

(t, J ) 4.8, 4H), 3.61 (t, J ) 4.8, 4H), 4.47 (t, J ) 6.8, 2H),

7.32 (t, J ) 7.2, 1H), 7.44 (t, J ) 7.3, 1H), 7.63 (m, 3H), 7.79

(d, J ) 7.4, 1H), 7.96 (d, J ) 7.6, 1H). Anal. (C₂₅H₂₇N₃O₂) C,

H, N.

N,N-Dip r op yl-2-(4-m eth yl-1-p ip er a zin yl)in d en o[1,2,3-

d e]qu in olin e-1-ca r boxa m id e (10d ). The title compound was

prepared by method A starting from 36 and N-methylpipera-

zine (reaction time 2 h and 30 min) to obtain a yellow oil (0.39

g, 91%). ¹H NMR (CDCl₃): 0.45 (t, J ) 7.3, 3H), 1.02-1.55

(m, 5H), 1.82 (m, 2H), 2.34 (s, 3H), 2.54 (m, 4H), 2.99 (m, 2H),

3.38 (m, 3H), 3.76 (m, 3H), 7.28 (t, J ) 7.4, 1H), 7.40 (t, J )

7.3, 1H), 7.63 (m, 3H), 7.77 (d, J ) 7.4, 1H), 7.84 (d, J ) 7.6,

1H). Anal. (C₂₇H₃₂N₄O) C, H, N.

6,8-Dih yd r o-7H-[1]ben zoth iop yr a n o[3,4-c]qu in olin -7-

on e (13c). This compound was prepared in 53% yield starting

from enamine 12c and was purified by washing with ethyl

acetate: mp 264-265 °C. ¹H NMR (CDCl₃): 3.92 (s, 2H), 7.23

(m, 1H), 7.36 (m, 2H), 7.50 (m, 2H), 7.64 (m, 1H), 7.80 (m,

1H), 7.98 (d, J ) 8.3, 1H), 11.98 (br s, 1H).

7,8,8a,9,10,11,12,12a-Octah ydr oben zo[k]ph en an th r idin -

6(5H)-on e (13d ). This compound was obtained from enamine

12d in 78% yield as a white solid. Recrystallization from ethyl

acetate-methanol gave a diastereomeric mixture (trans/ cis

88:12) of 13d : mp 295-297 °C (lit.⁴⁰mp 285-288 °C). ¹H NMR

(CDCl₃) (only the resonances of the major diasteromer are

given): 1.07-1.92 (m, 10H), 2.58 (m, 3H), 2.88 (m, 1H), 7.17

(t, J ) 7.4, 1H), 7.26 (d, J ) 7.5, 1H), 7.40 (t, J ) 7.5, 1H),

7.62 (d, J ) 8.1, 1H), 10.57 (br s, 1H). Anal. (C₁₇H₁₉NO) C, H,

N.

Gen er a l P r oced u r e for th e P r ep a r a tion of Im id oyl

Ch lor id es 14a -d . A mixture of the appropriate quinolinone

derivative (1.0 mmol) and POCl₃(5 mL) was refluxed for 1 h.

Then, the cooled reaction mixture was poured into ice-water,

and the precipitate was extracted with CH₂Cl₂(3 × 30 mL).

The combined extracts were washed with water, dried over

sodium sulfate, and concentrated under reduced pressure to

obtain the title compounds which were purified by flash

chromatography when necessary.

P r ep a r a tion of En a m in es 12a -d . To a 100-mL, three-

necked flask fitted with

a condenser, thermometer, and

dropping funnel (under argon) were added the appropriate

cyclic ketone (R-tetralone (11a ), 4-keto-4,5,6,7-tetrahydrothia-

naphthene (11b), thiochroman-4-one (11c), trans-1-decalone

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1571

6-Ch lor o-7,8-dih ydr oben zo[k]ph en an th r idin e (14a). The

title compound was prepared from 13a and was purified by

flash chromatography with n-hexane-ethyl acetate (8:2) as

the eluent to obtain a colorless oil which crystallized on

standing to give a white crystalline solid (0.23 g, 86%): mp

96-97 °C, (lit.²¹mp 80-81 °C; lit.⁴¹mp 95-96 °C).¹H NMR

(CDCl₃): 2.89 (m, 2H), 3.06 (m, 2H), 7.37-7.42 (m, 3H), 7.56

(m, 1H), 7.69 (m, 1H), 7.91 (m, 1H), 8.07 (d, J ) 8.0, 1H), 8.42

(d, J ) 8.5, 1H).

6-Ch lor o-7,8-dih ydr oth ien o[2,3-k]ph en an th r idin e (14b).

This compound was prepared in 98% yield starting from 13b

and was revealed pure by GC-MS, so it was used in the

subsequent step without further purification.¹H NMR (CDCl₃):

3.05 (m, 2H), 3.23 (m, 2H), 7.31 (d, J ) 5.1, 1H), 7.56 (m, 1H),

7.69 (m, 2H), 8.03 (d, J ) 8.8, 1H), 8.41 (d, J ) 8.5, 1H). MS:

m/z 271 (M⁺, 100).

5,6-Dih yd r oben z[f]isoqu in olin -4(3H)-on e (18). A mix-

ture of 17 (0.46 g, 2.0 mmol) in ethanol (30 mL) with 3 N NaOH

solution (10 mL) was stirred at room temperature for 2 h. The

organic solvent was then removed under reduced pressure; the

residue was poured into ice-water and acidified with 2 N HCl

solution. The precipitate was extracted with ethyl acetate (3

× 30 mL), and the organic layer was washed with brine (2 ×

100 mL), dried over sodium sulfate, and concentrated under

reduced pressure. The residue was dissolved into acetone (10

mL), and triethylamine (0.33 mL, 2.38 mmol) was added. To

the resulting mixture cooled at 0 °C was added dropwise a

solution of isobutyl chloroformate (0.31 mL, 2.38 mmol) in

acetone (8 mL). The reaction mixture was stirred at 0 °C for

30 min, and a solution of sodium azide (0.18 g, 2.76 mmol) in

5 mL of water was slowly added keeping the temperature

below 10 °C. When the addition was complete, the reaction

mixture was stirred for 1 h at room temperature and then

poured into ice-water, and the precipitate was extracted with

dichloromethane (4 × 10 mL). The combined organic extracts

were dried over sodium sulfate and slowly added to a mixture

of tributylamine (1.0 mL, 4.2 mmol) in Dowtherm A (20 mL)

preheated at 200-220 °C into a distillation apparatus which

allowed the distillation of the dichloromethane during the

addition. When the addition was complete, the solvent was

removed under reduced pressure and the residue was purified

by flash chromatography. Elution with ethyl acetate-metha-

nol (85:15) gave pure 18 (46 mg, 12%). ¹H NMR (CDCl₃): 2.88

(s, 4H), 6.72 (d, J ) 6.9, 1H), 7.31 (m, 4H), 7.64 (m, 1H), 12.19

(br s, 1H). HR-MS: m/z calcd for (C₁₃H₁₁NO) 197.0841, found

197.0840.

3-Am in o-1-p h en yln a p h th a len e (20). To a mixture of 19¹⁸

(0.25 g, 1.0 mmol) in dry benzene (10 mL) with triethylamine

(0.14 mL, 1.0 mmol) cooled at 0-5 °C was added DPPA (0.22

mL, 1.0 mmol). The resulting mixture was stirred at room

temperature for 3 h, heated to reflux for 4 h, and cooled, and

20 mL of 50% sulfuric acid was added. After overnight stirring

at room temperature, the reaction mixture was poured into

ice-water, made alkaline with concentrated NH₄OH, and

extracted with chloroform (3 × 20 mL). The combined extracts

were washed with water, dried over sodium sulfate, and

concentrated under reduced pressure. Purification of the

residue by flash chromatography with n-hexane-ethyl acetate

(65:35) gave pure 20 (0.18 g, 82%) as a pale-yellow oil which

was used immediately in the subsequent step. ¹H NMR

(CDCl₃): 3.86 (br s, 2H), 6.90 (d, J ) 2.0, 1H), 7.01 (d, J )

2.2, 1H), 7.16 (t, J ) 7.1, 1H), 7.32-7.48 (m, 6H), 7.64 (d, J )

8.1, 1H), 7.70 (d, J ) 8.4, 1H).

7-Ch lor o-6H-[1]ben zoth iop yr a n o[3,4-c]qu in olin e (14c).

This compound was prepared starting from 13c and was

purified by flash chromatography with n-hexane-ethyl acetate

(8:2) as the eluent to obtain a colorless oil which crystallized

on standing (0.26 g, 92%): mp 139-140 °C. ¹H NMR (CDCl₃):

4.08 (s, 2H), 7.35-7.45 (m, 2H), 7.55 (m, 1H), 7.64-7.76 (m,

2H), 7.85 (m, 1H), 8.08 (d, J ) 8.9, 1H), 8.23 (d, J ) 8.0, 1H).

MS: m/z 283 (M⁺, 100).

tr a n s-6-Ch lor o-7,8,8a ,9,10,11,12,12a -oct a h yd r ob en zo-

[k]p h en a n th r id in e (14d ). This compound was prepared

starting from a diastereomeric mixture (trans/ cis 85:15) of 13d

and was isolated by flash chromatography with n-hexane-

dichloromethane (6:4) as a colorless oil which crystallized on

standing (0.076 g, 28%): mp 86-87 °C. ¹H NMR (CDCl₃):

1.04-1.95 (m, 10H), 2.63 (br d, J ) 13.3, 1H), 2.76-2.91 (m,

3H), 7.48 (m, 1H), 7.62 (m, 1H), 7.89 (d, J ) 8.5, 1H), 7.98 (d,

J ) 8.9, 1H). MS: m/z 271 (M⁺, 100). Anal. (C₁₇H₁₈ClN) C, H,

N.

1,2,3,4,7,8-H exa h yd r ob en zo[k ]p h en a n t h r id in -6(5H )-

on e (15a ). To a solution of 1-cyclohexene-1-isocyanate¹⁴(1.0

g, 8.1 mmol) in dry acetonitrile (10 mL) cooled at 0 °C was

added enamine 12a (2.0 g, 10 mmol) dissolved into 10 mL of

dry acetonirile, and the resulting solution was stirred for 48

h at room temperature. The solvent was then removed, and

the residue was partitioned between chloroform and 1 N HCl.

The organic layer was washed with water, dried over sodium

sulfate, and concentrated under reduced pressure. Treatment

of the residue with methanol-ethyl acetate gave 15a as white

crystals (0.51 g, 25%). An analytical sample was obtained from

methanol as colorless prisms: mp 268-270 °C. ¹H NMR

(CDCl₃): 1.70 (m, 2H), 1.87 (m, 2H), 2.75 (m, 8H), 7.29 (m,

3H), 7.65 (m, 1H), 11.96 (br s, 1H). Anal. (C₁₇H₁₇NO) C, H, N.

3-(4-Meth yl-1-p ip er a zin yl)-1-p h en yln a p h th a len e (9b).

A mixture of 20 (0.14 g, 0.64 mmol), N-methylbis(2-chloro-

ethyl)amine hydrochloride (0.18 g, 0.93 mmol), K₂CO₃(0.26

g, 1.88 mmol), and diglyme (15 mL) was heated to reflux for

21 h under argon atmosphere. The cooled reaction mixture was

poured into ice-water, and the solution was made basic with

50% NaOH solution and extracted with chloroform (4 × 20

mL). The organic layer was dried over sodium sulfate and

concentrated under reduced pressure, and the residue obtained

was purified by flash chromatography with ethyl acetate-

triethylamine (8:2) to obtain 9b (43 mg, 22%) as a brownwish

6,8,9,10,11,12-Hexa h yd r o-7H-[1]ben zoth iop yr a n o[3,4-

c]qu in olin -7-on e (15c). This compound was prepared in 22%

yield from enamine 12c employing the same procedure de-

scribed for 15a and was purified by treatment with ethyl

acetate-methanol: mp 288 °C dec. ¹H NMR (CDCl₃): 1.64 (m,

2H), 1.87 (m, 2H), 2.62 (t, J ) 5.6, 2H), 2.78 (t, J ) 6.5, 2H),

3.80 (s, 2H), 7.24 (m, 2H), 7.55 (m, 1H), 7.65 (m, 1H), 12.01

(br s, 1H).

Eth yl (E)-3-(3,4-Dih yd r on a p h th -1-yl)p r op en oa te (17).

To a slurry of Pd(PPh₃)₂Cl₂(0.12 g, 0.17 mmol) in dry DMF

(15 mL) was added a solution of 3,4-dihydro-1-[[(trifluorometh-

yl)sulfonyl]oxy]naphthalene¹⁶(16) (2.1 g, 7.5 mmol), ethyl

acrylate (1.95 mL, 18.0 mmol), and triethylamine (3.6 mL, 26

mmol) in dry DMF (5 mL). The resulting mixture was heated

at 75-80 °C in an argon atmosphere for 3 h, cooled to room

temperature, and poured into ice-water. The precipitate was

extracted with dichloromethane (3 × 30 mL), and the combined

extracts were washed with water (2 × 100 mL), dried over

sodium sulfate, and concentrated under reduced pressure.

Purification of the residue by flash chromatography with

n-hexane-ethyl acetate (9:1) as the eluent gave 17 as a pale-

yellow oil (0.47 g, 27%). ¹H NMR (CDCl₃): 1.30 (t, J ) 7.1,

3H), 2.33 (m, 2H), 2.72 (t, J ) 7.9, 2H), 4.22 (q, J ) 7.1, 2H),

6.24 (d, J ) 15.3, 1H), 6.43 (t, J ) 4.5, 1H), 7.18 (m, 3H), 7.35

(m, 1H), 7.61 (d, J ) 15.4, 1H).

1

oil. H NMR (CDCl₃): 2.37 (s, 3H), 2.63 (t, J ) 4.9, 4H), 3.35

(t, J ) 4.8, 4H), 7.14 (d, J ) 2.0, 1H), 7.21 (d, J ) 2.1, 1H),

7.35-7.47 (m, 7H), 7.72 (m, 2H). HR-MS: m/z calcd for

(C₂₁H₂₂N₂) 302.1783, found 302.1787. Anal. (C₂₁H₂₂N₂) C, H,

N.

1-Ad a m a n tyl 2-Am in op h en yl Keton e (22). A 1.7 M

solution of tert-butyllithium in pentane (11.3 mL, 19.2 mmol)

was added in a dropwise manner to a solution of N-(tert-

butoxycarbonyl)aniline (21)²⁰(1.5 g, 7.8 mmol) in anhydrous

THF (10 mL, argon atmosphere) maintained at -65 °C. After

15 min at -65 °C, the solution was allowed to warm to -20

°C where it was maintained for 2 h. A solution of methyl

1-adamantanecarboxylate (1.5 g, 7.7 mmol) in anhydrous THF

(10 mL) was then added, and the resulting mixture was stirred

at -15 °C for 1 h and at room temperature for 30 min. The

reaction mixture was poured into ice-water and extracted

1572 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Cappelli et al.

with chloroform (3 × 30 mL). The combined extracts were

washed with water, dried over sodium sulfate, and concen-

trated under reduced pressure. The residue was purified by

flash chromatography with n-hexane-ethyl acetate (8:2) as

the eluent to give an oil which crystallized on standing. This

product was dissolved into ethanol (60 mL), and 3 N HCl (30

mL) was added under stirring. The resulting mixture was

heated to reflux for 1 h, and the organic solvent was removed

under reduced pressure. The residue was diluted with water,

made alkaline with solid Na₂CO₃, and extracted with dichlo-

romethane (3 × 30 mL). The combined extracts were washed

with water, dried over sodium sulfate, and concentrated under

reduced pressure. Purification of the residue by flash chro-

matography with n-hexane-ethyl acetate (8:2) as the eluent

gave 22 (0.57 g, 29%) as an oil which crystallized on standing.

An analytical sample recrystallized from n-hexane melted at

143-144 °C. ¹H NMR (CDCl₃): 1.76 (s, 6H), 2.07 (s, 9H), 5.08

(br s, 2H), 6.66 (m, 2H), 7.17 (t, J ) 8.0, 1H), 7.68 (d, J ) 7.9,

1H). Anal. (C₁₇H₂₁NO) C, H, N.

4-(1-Ad a m a n tyl)qu in olin -2(1H)-on e (23). A mixture of 22

(0.22 g, 0.86 mmol) in acetic anhydride (4.0 mL) was stirred

at room temperature for 4 h and then poured into ice-water.

The precipitate was extracted with chloroform (3 × 25 mL),

and the organic layer was washed with water, dried over

sodium sulfate, and concentrated under reduced pressure to

give an oil which crystallized on standing. This product was

dissolved into absolute ethanol (10 mL), and a 30% solution

of sodium methoxide in methanol (0.8 mL, 4.3 mmol) was

added under stirring. The resulting mixture was heated to

reflux for 8 h under an argon atmosphere, cooled to room

temperature, and poured into ice-water. The aqueous mixture

was acidified with 6 N HCl, and the precipitate was collected

by filtration, washed in sequence with water and n-hexane-

diethyl ether, and finally dried to give 23 (0.17 g, 71%) as a

white crystalline solid. An analytical sample was obtained by

recrystallization from methanol-chloroform: mp 276 °C dec.

¹H NMR (CDCl₃): 1.86 (s, 6 H), 2.21 (s, 9H), 6.67 (s, 1H), 7.19

(m, 1H), 7.40 (m, 2H), 8.35 (d, J ) 8.4, 1H), 11.44 (br s, 1H).

Anal. (C₁₉H₂₁NO) C, H, N.

was maintained at room temperature for 6 h. The solution was

then diluted with 30 mL of dichloromethane, washed with a

1% solution of Na₂CO₃, dried over sodium sulfate, and con-

centrated under reduced pressure. Purification of the residue

by flash chromatography with ethyl acetate-triethylamine (8:

2) as the eluent gave 9j (0.18 g, 88%) as an oil which

1

crystallized on standing: mp 102-103 °C. H NMR (CDCl₃):

1.78 (m, 4H), 2.38 (s, 3H), 2.64 (t, J ) 4.5, 4H), 2.89 (t, J )

7.6, 2H), 3.37 (m, 5H), 3.68 (m, 3H), 4.90 (d, J ) 3.1, 1H), 7.23

(m, 4H), 7.49 (m, 4H), 7.89 (d, J ) 8.4, 1H). Anal. (C₂₆H₃₁N₃O₂)

C, H, N.

2-(4-Meth yl-1-p ip er a zin yl)-4-p h en yl-3-[[(2-tetr a h yd r o-

fu r a n yl)oxy]m eth yl]qu in olin e (9f). The title compound was

prepared in 94% yield starting from 3-hydroxymethyl-2-(4-

methyl-1-piperazinyl)-4-phenylquinoline^19a(9e) using the same

procedure described for the preparation of 9j (reaction time

24 h at room temperature). Compound 9f was obtained as an

1

oil which crystallized on standing: mp 140-141 °C. H NMR

(CDCl₃): 1.83 (m, 4H), 2.38 (s, 3H), 2.64 (m, 4H), 3.44-3.85

(m, 6H), 4.03 (d, J ) 9.5, 1H), 4.61 (d, J ) 9.8, 1H), 4.74 (br s,

1H), 7.25 (m, 4H), 7.51 (m, 4H), 7.86 (d, J ) 8.5, 1H). Anal.

(C₂₅H₂₉N₃O₂) C, H, N.

4-(2-F lu or op h en yl)-2-(4-m eth yl-1-p ip er a zin yl)-3-[2-[(2-

tetr a h yd r ofu r a n yl)oxy]eth yl]qu in olin e (9l). The title com-

pound was prepared in 92% yield starting from 4-(2-fluorophen-

yl)-3-(2-hydroxyethyl)-2-(4-methyl-1-piperazinyl)quinoline^19b(9k)

using the same procedure described for the preparation of 9j

(reaction time 6 h at room temperature). Compound 9l was

1

obtained as a pale-yellow oil. H NMR (CDCl₃): 1.76 (m, 4H),

2.38 (s, 3H), 2.64 (t, J ) 4.7, 4H), 2.91 (m, 2H), 3.36 (m, 5H),

3.69 (m, 3H), 4.92 (t, J ) 3.2, 1H), 7.20 (m, 5H), 7.52 (m, 2H),

7.90 (d, J ) 8.3, 1H). MS: m/z 435 (M⁺, 12). Anal. (C₂₆H₃₀

FN₃O₂) C, H, N.

-

4-(2-F lu or op h en yl)-2-(4-m eth yl-1-p ip er a zin yl)-3-[2-[(2-

tetr a h yd r op yr a n yl)oxy]eth yl]qu in olin e (9m ). The title

compound was prepared in 91% yield by reaction of 4-(2-

fluorophenyl)-3-(2-hydroxyethyl)-2-(4-methyl-1-piperazinyl)-

quinoline^19b(9k ) with 3,4-dihydro-2H-pyran following the same

procedure described for the preparation of 9j (reaction time

19 h at room temperature). Compound 9m was obtained as a

4-(1-Ad a m a n tyl)-2-ch lor oqu in olin e (24). This compound

was prepared in 89% yield starting from 23 using the same

procedure described for the preparation of imidoyl chlorides

14a -d and was used in the subsequent step without further

1

pale-yellow oil. H NMR (CDCl₃): 1.42-1.66 (m, 6H), 2.38 (s,

3H), 2.64 (t, J ) 4.7, 4H), 2.95 (m, 2H), 3.44 (m, 6H), 3.68 (m,

2H), 4.41 (t, J ) 5.1, 1H), 7.22 (m, 5H), 7.50 (m, 2H), 7.91 (d,

J ) 8.3, 1H). MS: m/z 450 (M + 1, 29). Anal. (C₂₇H₃₂FN₃O₂)

C, H, N.

1

purification. H NMR (CDCl₃): 1.89 (s, 6H), 2.23 (s, 3H), 2.30

(s, 6H), 7.30 (s, 1H), 7.51 (m, 1H), 7.68 (m, 1H), 8.06 (m, 1H),

8.60 (d, J ) 8.5, 1H).

[[(9-Oxoflu or en -1-yl)a m in oca r b on yl]m et h yl]t r ip h en -

ylp h osp h on iu m Br om id e (28). To a solution of 1-amino-9-

fluorenone (27) (0.57 g, 2.9 mmol) in dichloromethane (20 mL)

cooled at 0-5 °C was added a solution of bromoacetyl bromide

(0.31 mL, 3.5 mmol) in 10 mL of dichloromethane. After the

addition was over, the resulting mixture was stirred overnight

at room temperature, and 50 mL of a staturated solution of

NaHCO₃was added with vigorous stirring and cooling in ice-

water. The organic layer was separated, washed with water,

dried over sodium sulfate, and evaporated under reduced

pressure. The residue was dissolved into 50 mL of dry benzene,

and 0.94 g (3.6 mmol) of triphenylphosphine was added. The

resulting mixture was heated to reflux under argon atmo-

sphere for 9 h. The precipitate formed was collected by

filtration, washed with dry benzene and n-hexane in sequence,

and dried under reduced pressure to give pure 28 (1.42 g, 85%)

as a yellow solid: mp 229 °C dec. ¹H NMR (CDCl₃): 5.70 (d, J

) 13.8, 2H), 7.26-7.80 (m, 16H), 7.88-7.99 (m, 6H), 10.93 (br

s, 1H). Anal. (C₃₃H₂₅BrNO₂P) C, H, N.

Eth yl 2-Ch lor o-4-p h en yl-3-qu in olin ea ceta te (26). The

title compound was prepared in 94% yield starting from ethyl

1,2-dihydro-2-oxo-4-phenyl-3-quinolineacetate²¹(25) using the

same procedure described for the preparation of imidoyl

chlorides 14a -d and was used in the subsequent step without

1

further purification: mp 110-111 °C. H NMR (CDCl₃): 1.21

(t, J ) 7.0, 3H), 3.70 (s, 2H), 4.14 (q, J ) 7.0, 2H), 7.26-7.52

(s, 7H), 7.69 (m, 1H), 8.05 (d, J ) 8.4, 1H).

3-(2-Hyd r oxyeth yl)-2-(4-m eth yl-1-p ip er a zin yl)-4-p h en -

ylqu in olin e (9i). To a suspension of lithium aluminum

hydride (0.5 g, 13.2 mmol) in anhydrous THF (10 mL) cooled

at 0-5 °C was added a solution of 9q (0.86 g, 2.2 mmol) in

anhydrous THF (10 mL). After stirring for 30 min at 0-5 °C

and 30 min at room temperature, the hydride was hydrolyzed

by addition of water and the inorganic material was filtered

off and washed with tetrahydrofuran. The filtrate was washed

with brine, dried over sodium sulfate, and evaporated to give

pure 9i as a white solid (0.75 g, yield 98%). Crystallization

from ethyl acetate gave an analytical sample: mp 165-166

1

°C. H NMR (CDCl₃): 2.38 (s, 3H), 2.68 (t, J ) 4.4, 4H), 2.91

In d en o[1,2,3-d e]qu in olin -2(3H)-on e (29). A mixture of 28

(0.72 g, 1.2 mmol) in dry DMF (10 mL) with NaH (0.089 g, 3.7

mmol) was stirred at room temperature for 3 h under argon

atmosphere. The reaction mixture was then poured into ice-

water and acidified with 3 N HCl. The precipitate was collected

by filtration, washed in sequence with water, ethyl acetate,

and n-hexane, and dried under reduced pressure to give pure

29 (0.25 g, 95%) as a yellow solid: mp 288-289 °C (lit.²²mp

(t, J ) 5.7, 2H), 3.35 (t, J ) 4.8, 4H), 3.64 (m, 2H), 4.60 (br s,

1H), 7.24 (m, 4H), 7.52 (m, 4H), 7.95 (d, J ) 8.4, 1H). Anal.

(C₂₂H₂₅N₃O) C, H, N.

2-(4-Met h yl-1-p ip er a zin yl)-4-p h en yl-3-[2-[(2-t et r a h y-

d r ofu r a n yl)oxy]eth yl]qu in olin e (9j). To a stirred solution

of 9i (0.17 g, 0.49 mmol) in dichloromethane (10 mL) with 2,3-

dihydrofuran (0.15 mL, 2.0 mmol) was added 0.11 g (0.58

mmol) of p-toluenesulfonic acid monohydrate, and the mixture

1

277-279 °C). H NMR (CDCl₃): 7.07 (s, 1H), 7.13 (d, J ) 8.4,

Novel Central 5-HT₃Receptor Ligands

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1573

1H), 7.35-7.58 (m, 4H), 7.79 (d, J ) 7.3, 1H), 7.86 (d, J ) 7.2,

1H), 9.85 (br s, 1H).

(5) Å³, Z ) 4, D_c) 1.24 g/cm³. A total of 6567 unique reflections

(R_int) 0.018) were collected at 22 °C of which 4549 were

observed with F > 4σ(F). The final refinement converged to R

) 0.048 and wR2 ) 0.113 for F²> 2σ(I). Minimun and

maximum heights in the last ∆F map were -0.25 and 0.26 eÅ

2-Ch lor oin d en o[1,2,3-d e]qu in olin e (30). This compound

was prepared in 82% yield starting from 30 using the same

procedure described for the preparation of imidoyl chlorides

14a -d and was purified by flash chromatography with dichlo-

romethane as the eluent to obtain a colorless oil which

crystallized on standing: mp 124-125 °C (lit.²²mp 121-122

°C). ¹H NMR (CDCl₃): 7.36-7.53 (m, 2H), 7.70-7.90 (m, 6H).

2-Ch lor oin d en o[1,2,3-d e]qu in olin e-1-ca r boxylic Acid

Ch lor id e (33). This compound was prepared in 66% yield

starting from ethyl 2,3-dihydro-2-oxoindeno[1,2,3-de]quinoline-

1-carboxylic acid²²(32) using the same procedure described

for the preparation of imidoyl chlorides 14a -d and was

purified by flash chromatography with dichloromethane as the

eluent. ¹H NMR (CDCl₃): 7.39 (t, J ) 7.8, 1H), 7.53 (t, J )

7.6, 1H), 7.82 (m, 5H). MS: m/z 299 (M⁺, 24).

Eth yl 2-Ch lor oin d en o[1,2,3-d e]qu in olin e-1-ca r boxyl-

a te (34). This compound was prepared in 83% yield starting

from ethyl 2,3-dihydro-2-oxoindeno[1,2,3-de]quinoline-1-car-

boxylate²²(31) using the same procedure described for the

preparation of imidoyl chlorides 14a -d and was purified by

flash chromatography with dichloromethane as the eluent to

obtain a yellow solid: mp 122-123 °C. ¹H NMR (CDCl₃): 1.50

(t, J ) 6.9, 3H), 4.62 (q, J ) 7.2, 2H), 7.36 (t, J ) 7.2, 1H),

7.49 (m, 1H), 7.71-7.88 (m, 5H). Anal. (C₁₈H₁₂ClNO₂) C, H,

N.

-3

.

9e: C₂₁H₂₃N₃O (MW 333.42), a single crystal, dimensions

0.2 × 0.2 × 0.1 mm, was used for data collection; monoclinic;

space group P2₁/ c (No. 14); a ) 6.470(2), b ) 9.328(3), c )

30.739(7) Å; â ) 94.75(2)°; V ) 1848.8(9) Å³; Z ) 4, D_c) 1.20

g/cm³. A total of 2395 unique reflections (R_int) 0.062) were

collected at 22 °C. The final refinement converged to R ) 0.067

and wR2 ) 0.092 for F²> 2σ(I). Minimum and maximum

-3

heights in the last ∆F map were -0.20 and 0.17 eÅ

.

9i: C₂₂H₂₅N₃O (MW 347.4), a single crystal, dimensions 0.2

× 0.3 × 0.2 mm, was used for data collection; triclinic; space

group P1h (No. 2); a ) 7.734(2), b ) 12.364(2), c ) 21.504(3) Å;

R ) 103.25(1), â ) 94.08(1), γ ) 105.70(1)°; V ) 1907.3(6) Å³,

Z ) 4, D_c) 1.21 g/cm³. A total of 4933 unique reflections (R_int

) 0.043) were collected at 22 °C of which 4549 were observed

with F > 4σ(F). The final refinement converged to R ) 0.062

and wR2 ) 0.102 for F²> 2σ(I). Minimun and maximum

-3

heights in the last ∆F map were -0.20 and 0.32 eÅ

.

Full crystallographic details will be given elsewhere.

In Vitr o Bin d in g Assa ys. Binding assays were performed

as described in refs 36a (5-HT_1Areceptors), 36b (5-HT_1B

receptors), 36c (5-HT_2Areceptors), 36b (5-HT_2Creceptors), 29

(5-HT₃receptors), and 36d (5-HT transporter).

Male Wistar rats (Charles River, Calco, Italy) were killed

by decapitation, and their brains were rapidly dissected into

n -Bu t yl 2-Ch lor oin d en o[1,2,3-d e]q u in olin e-1-ca r b ox-

yla te (35). To a mixture of 2,3-dihydro-2-oxoindeno[1,2,3-de]-

quinoline-1-carboxylic acid²²(32) (0.36 g, 1.4 mmol) in 1-bu-

tanol (20 mL) was added POCl₃(1.5 mL). The resulting

mixture was refluxed for 1 h and then concentrated under

reduced pressure. The residue was treated with ice-water,

and the precipitate was collected by filtration, washed with

water, and dried under reduced pressure to give a yellow solid.

A mixture of this solid in POCl₃(5 mL) was refluxed for 1 h,

then cooled, and poured into ice-water. The precipitate was

extracted with dichloromethane (3 × 30 mL), and the combined

extracts were washed with water, dried over sodium sulfate,

and concentrated under reduced pressure. The oil residue was

purified by flash chromatography with dichloromethane as the

eluent to obtain 35 (0.29 g, 61%) as a yellow crystalline solid:

various areas (hippocampus for 5-HT_1A; striatum for 5-HT_1B

;

pre-frontal cortex for 5-HT_2A; cortex for 5-HT_2C; cortex and

hippocampus for 5-HT₃; forebrain for 5-HT transporter).

Tissues were homogenized (Polytron PTA 10TS) in ice-cold Tris

HCl, 50 mM, at the appropriate pH value (or 50 mM Hepes

buffer, pH 7.4, for 5-HT₃receptors) and centrifuged according

to the procedures indicated in the above-cited references. The

pellet obtained was finally resuspended in the appropriate

incubation buffer (Tris HCl, 50 mM, pH 7.4, for 5-HT_1A

receptors; Tris HCl, 50 mM, pH 7.7, containing 10 µM

pargyline, 4 mM CaCl₂, and 0.1% ascorbic acid for 5-HT_1Band

5-HT_2Creceptors; Tris HCl, 50 mM, pH 7.7, for 5-HT_2A

receptors; Hepes HCl, 50 mM, pH 7.4, for 5-HT₃receptors; Tris

HCl, 50 mM, pH 7.4, containing 120 mM NaCl and 5 mM KCl

for 5-HT transporter) just before the binding assay. Membrane

preparations were used the day of the experiment or stored

at -80 °C (for 5-HT transporter binding assay).

[³H]8-OH-DPAT (s.a. 137 Ci/mmol, for 5-HT_1A; NEN) and

[³H]ketanserin (s.a. 80.9 Ci/mmol, for 5-HT_2A; NEN) binding

were assayed in a final incubation volume of 0.5 mL. Tissue

concentrations and [³H]ligand final concentrations were 4 mg

of tissue/sample and 0.5 nM and 8 mg of tissue/sample and

0.5 nM, respectively.

[³H]Mesulergine (s.a. 86 Ci/mmol, for 5-HT_2C; Amersham)

and [³H]5-HT (s.a. 30 Ci/mmol, for 5-HT_1B; NEN) binding were

assayed in a final incubation volume of 1.0 mL. Tissue

concentration and [³H]ligand final concentration were 23 mg

of tissue/sample and 1.3 nM ([³H]mesulergine binding) and 14

mg of tissue/sample and 2.3 nM ([³H]5-HT binding).

[³H]Granisetron (s.a. 81 Ci/mmol, for 5-HT₃; NEN) and [³H]-

paroxetine (s.a. 29.7 Ci/mmol, for 5-HT transporter; NEN)

binding were assayed in final incubation volumes of 1.0 and

2.0 mL, respectively. Tissue concentration and [³H]ligand final

concentration were 20 mg of tissue/sample and 0.5 nM ([³H]-

granisetron binding) and 2 mg of tissue/sample and 0.1 nM

([³H]paroxetine binding). The specific binding of the tritiated

ligands was defined as the difference between total binding

and nonspecific binding in the presence of 10 µM 5-HT (for

5-HT_1Aand 5-HT_1B), 3 µM ketanserin (for 5-HT_2A), 1 µM

mianserine (for 5-HT_2C), 100 µM 5-HT (for 5-HT₃), and 1 µM

6-nitroquipazine (for 5-HT transporter); it represented 80%,

62%, 90%, 70%, 70%, and 75%, respectively, of the total

binding.

1

mp 104-105 °C. H NMR (CDCl₃): 0.99 (t, J ) 7.3, 3H), 1.52

(m, 2H), 1.85 (m, 2H), 4.57 (t, J ) 6.8, 2H), 7.36 (t, J ) 7.3,

1H), 7.49 (t, J ) 7.2, 1H), 7.72-7.88 (m, 5H). Anal. (C₂₀H₁₆

ClNO₂) C, H, N.

-

2-Ch lor o-N,N-d ip r op ylin d en o[1,2,3-d e]qu in olin e-1-ca r -

boxa m id e (36). To a solution of 33 (0.16 g, 0.53 mmol) in

dichloromethane (20 mL) cooled at 0-5 °C was added dipro-

pylamine (0.3 mL, 2.2 mmol). The resulting mixture was

stirred at room temperature for 2 h, then washed with water,

dried over sodium sulfate, and concentrated under reduced

pressure to give pure 36 (0.19 g, 98%) as yellow crystals: mp

1

152-153 °C. H NMR (CDCl₃): 0.61 (t, J ) 7.4, 3H), 1.09 (t,

J ) 7.4, 3H), 1.25-1.57 (m, 2H), 1.89 (m, 2H), 3.16 (m, 2H),

3.41 (m, 1H), 3.92 (m, 1H), 7.34 (t, J ) 8.0, 1H), 7.47 (t, J )

7.2, 1H), 7.71-7.89 (m, 5H). Anal. (C₂₂H₂₁ClN₂O) C, H, N.

X-r a y Cr ysta llogr a p h y. Single crystals of 8g and 9e,i were

submitted to X-ray data collection on a Siemens P4 four-circle

diffractometer with graphite monochromated Mo KR radiation

(λ ) 0.71069 Å). The ω/2θ scan technique was used. The

structures were solved by direct methods, and the refinements

were carried out by full-matrix anisotropic least-squares of F²

against all reflections. The hydrogen atoms were located on

Fourier difference maps and included in the structure-factor

calculations with a common isotropic temperature factor.

Atomic scattering factors including f ′ and f " were taken from

ref 42. Structure solution and refinement were carried out

using the SHELX-97 package,⁴²while molecular graphics was

performed by the SHELXTL PC program.⁴³

8g: C₂₁H₂₅N₃S (MW 351.5), a single crystal, dimensions 0.3

× 0.2 × 0.1 mm, was used for data collection; triclinic; space

group P1h (No. 2); a ) 10.3890(10), b ) 13.613(2), c ) 14.1350-

(10) Å; R ) 75.39(1), â ) 78.78(1), γ ) 79.10(1)°; V ) 1876.8-

1574 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9

Cappelli et al.

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4801.

Incubations were stopped by rapid filtration under vacuum

through Whatman GF/B filters presoaked in assay buffer or

poly(ethylenimine) at 0.1% (for 5-HT₃) or 0.5% (for 5-HT

transporter). Filters were immediately rinsed with 12 mL (3

× 4 mL) of ice-cold buffer using a Brandel M-24R cell

harvester, dried, and immersed into vials containing 8 mL of

Ultima Gold MV (or Filter Count) (Packard) for the measure-

ment of trapped radioactivity with a TRI-CARB 1900TR (or

300C) (Packard) liquid scintillation spectrometer, at a counting

efficiency of about 60%. Competition experiments were ana-

lyzed by the “Allfit” program⁴⁴to obtain the concentration of

unlabeled drug that caused 50% inhibition of ligand binding

(IC₅₀). Apparent affinity costants (K_i) were derived from the

IC₅₀values according to the Cheng and Prusoff equation.⁴⁵The

K_d(nM) values of the radiolabeled ligands obtained in satura-

tion isotherms were 2.3 for [³H]8-OH-DPAT binding; 2.5 for

[³H]5-HT binding; 0.5 for [³H]ketanserin binding; 2.0 for [³H]-

mesulergine binding; 0.6 for [³H]granisetron binding; 0.09 for

[³H]paroxetine binding.

Mea su r em en t of [¹⁴C]Gu a n id in iu m Up ta k e in NG 108-

15 Cells. This procedure has been described by Emerit et al.⁷

Briefly, mouse neuroblastoma × rat glioma hybrid cells of the

NG 108-15 clone were grown in Dulbecco’s modified Eagle’s

medium supplemented with the appropriate nutrients⁷for 2

days. The cell layer in each culture dish (35 mm) was then

washed twice with 1.5 mL of buffer A (145 mM NaCl, 5.4 mM

KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 2.0 mM Na₂HPO₄, 20 mM

glucose, 20 mM HEPES, pH adjusted to 7.4 with NaOH) and

covered with 1 mL of buffer B (same composition as buffer A

except that [NaCl] was reduced to 135 mM and 10 mM

guanidinium was added) containing 0.20-0.25 mCi of [¹⁴C]-

guanidinium (s.a. 59 mCi/mmol; Service des Mole´cules Mar-

que´es at CEA, 91191 Gif-surYvette, France) and, where

indicated, 1 µM 5-HT, 10 µM substance P, and/or eight

different concentrations of each drug to be tested. After 10 min

at 37 °C, the assay was stopped by aspiration of the medium,

and the cell layer was washed three times with 1.5 mL of ice-

cold buffer C (same composition as buffer A except that NaCl

was replaced by choline chloride). The cells were then dissolved

into 0.5 mL of 0.4 M NaOH, and the resulting extracts were

transferred to scintillation vials. The culture dishes were

further rinsed with 0.5 mL of 1 M HCl and then 0.5 mL of 0.4

M NaOH, which were added to the vials. Each mixture (1.5

mL) was supplemented with 10 mL of the scintillation fluid

Aquasol (New England Nuclear, Les Ulis, France) for radio-

activity counting at 50% efficiency. All assays were performed

in triplicate.

Under these conditions, [¹⁴C]guanidinium accumulation in

NG 108-15 cells was 4-5 times higher in the presence of both

1 µM 5-HT and 10 µM substance P than in their absence (basal

uptake). 5-HT₃receptor antagonists (zacopride, ondansetron,

tropisetron, etc.) completely prevented the stimulatory effect

of 5-HT (with substance P) (see ref 7 for details).

Ack n ow led gm en t . Thanks are due to Italian

MURST (40% and 60% funds) and to French INSERM

and DRET for financial support. M.C.M. and P.G.D.B.

are especially grateful to Prof. Mati Karelson for the

CODESSA Program. Prof. Stefania D’Agata D’Ottavi’s

careful reading of the manuscript is also acknowledged.

Unlabeled granisetron was a generous gift of Smith

Kline Beecham Pharmaceuticals.

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J M981112S

Article Doi

Novel potent and selective central 5-HT3 receptor ligands provided with different intrinsic efficacy. 2. Molecular basis of the intrinsic efficacy of arylpiperazine derivatives at the central 5-HT3 receptors

DOI: 10.1021/jm981112s

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Article abstract of DOI:10.1021/jm981112s

Full text of DOI:10.1021/jm981112s

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