Accepted Manuscript Synthesis and structure-activity relationship studies in serotonin 5-HT4 receptor ligands based on a benzo[de][2,6]naphthridine scaffold Federica Castriconi, Marco Paolino, Germano Giuliani, Maurizio Anzini, Giuseppe Campiani, Laura Mennuni, Chiara Sabatini, Marco Lanza, Gianfranco Caselli, Francesca De Rienzo, Maria Cristina Menziani, Maria Sbraccia, Paola Molinari, Tommaso Costa, Andrea Cappelli PII:

S0223-5234(14)00429-2

DOI:

10.1016/j.ejmech.2014.05.015

Reference:

EJMECH 6971

To appear in:

European Journal of Medicinal Chemistry

Received Date: 3 December 2013 Revised Date:

30 April 2014

Accepted Date: 3 May 2014

Please cite this article as: F. Castriconi, M. Paolino, G. Giuliani, M. Anzini, G. Campiani, L. Mennuni, C. Sabatini, M. Lanza, G. Caselli, F. De Rienzo, M.C. Menziani, M. Sbraccia, P. Molinari, T. Costa, A. Cappelli, Synthesis and structure-activity relationship studies in serotonin 5-HT4 receptor ligands based on a benzo[de][2,6]naphthridine scaffold, European Journal of Medicinal Chemistry (2014), doi: 10.1016/ j.ejmech.2014.05.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AC C EP TE D

O

N O

H3C O

X N

7a

N N

O

Relative intrinsic activity (5-HT = 1)

R 8a-g 8d

0.4

RI PT

0.6

8c

0.8

8b

8a

7a

RS-23597

0.2

SC

0.0

M AN U

N Cisapride

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Synthesis and structure-activity relationship studies in serotonin 5-

RI PT

HT4 receptor ligands based on a benzo[de][2,6]naphthridine scaffold

Federica Castriconi,a Marco Paolino,a Germano Giuliani,a Maurizio Anzini,a Giuseppe Campiani,a

SC

Laura Mennuni,b Chiara Sabatini,b Marco Lanza,b Gianfranco Caselli,b Francesca De Rienzo,c Maria

a

M AN U

Cristina Menziani,c Maria Sbraccia,d Paola Molinari,d Tommaso Costa,d and Andrea Cappelli.a,*

Dipartimento di Biotecnologie, Chimica e Farmacia and European Research Centre for Drug Discovery and Development, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy b

c

TE D

Rottapharm Madaus, Via Valosa di Sopra 9, 20052 Monza, Italy

Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Modena e Reggio Emilia,

Dipartimento di Farmacologia, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy

AC C

d

EP

Via Campi 183, 41100 Modena, Italy

* Corresponding author. E-mail address: [email protected]. Phone: +39 0577 234320. Fax: +39 0577 234333.

1

ACCEPTED MANUSCRIPT

Abbreviations: 5-HT, 5-hydroxytryptamine; GPCRs, G-protein coupled receptors; 5-HT4R, 5-HT4 receptor; IBS, irritable bowel syndrome; GERD, gastroesophageal reflux disease; CV, cardiovascular; AD, Alzheimer’s disease; sAPPα, soluble amyloid precursor protein; BRET, bioluminescence resonance

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energy transfer; GFP, green fluorescent protein.

SC

ABSTRACT

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A small series of serotonin 5-HT4 receptor ligands has been designed from flexible 2methoxyquinoline compounds 7a,b by applying the conformational constraint approach. Ligands 7a,b and the corresponding conformationally constrained analogues 8a-g were synthesized and their interactions with the 5-HT4 receptor were examined by measuring both binding affinity and the ability

TE D

to promote or inhibit receptor–G protein coupling. Ester derivative 7a and conformationally constrained compound 8b were demonstrated to be the most interesting compounds showing a nanomolar 5-HT4R affinity similar to that shown by reference ligands cisapride (1) and RS-23,597-190 (4). The result was

EP

rationalized by docking studies in term of high similarity in the binding modalities of flexible 7a and conformationally constrained 8b. The intrinsic efficacy of some selected ligands was determined by

AC C

evaluating the receptor–G protein coupling and the results obtained demonstrated that the nature and the position of substituents play a critical role in the interaction of these ligands with their receptor.

2

ACCEPTED MANUSCRIPT 1. Introduction Serotonin (5-hydroxytryptamine, 5-HT) is one of the most important monoamine neurotransmitters in the human body. 5-HT is primarily found in the gastrointestinal tract, in platelets, and in the peripheral and central nervous systems [1]. 5-HT is involved in many physiological, behavioral, and cognitive

RI PT

functions [1]. The many physiological effects of 5-HT are mediated by seven receptor subfamilies (from 5-HT1 to 5-HT7), which belong to the G-protein coupled receptors (GPCRs) superfamily, with the exception of the 5-HT3 receptor, which is a ligand-gated ion channel [2]. Since its discovery in mouse

SC

colliculi neuronal cells and in the guinea pig ileum, the 5-HT4 receptor (5-HT4R) has stimulated a great deal of interest [3,4], and has been taken into consideration as a promising target for drug design. 5-

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HT4Rs are especially expressed in the central nervous system [5], heart [6], intestine [7], adrenal cortex [8], and the bladder [9], and their stimulation regulates many important physiological features such as the increase of acetylcholine levels in the prefrontal cortex, the power of stimulated theta oscillation in the hippocampus [10], the initiation of the intestinal peristaltic reflex [7], and the increase of Ca2+ and

TE D

pacemaker currents in the atrium [11].

A great number of 5-HT4R ligands has been developed by medicinal chemists (representative compounds 1-6 are reported in Fig. 1) [12], but the clinical use of these drug molecules has met some

EP

difficulties. For instance, the 5-HT4R agonist cisapride (1) was approved to treat symptomatic nocturnal heartburn due to gastroesophageal reflux disease (GERD) and used off-label in the treatment of diabetic

AC C

gastroparesis and peptic ulcer disease [13]. The agonist tegaserod (2) was approved for treatment of women showing constipation predominant irritable bowel syndrome (IBS-C) and of patients showing chronic idiopathic constipation (CIC) [13]. However, the therapeutic use of these 5-HT4R agonists was complicated by the occurrence of severe adverse cardiovascular (CV) events. Thus, they have been withdrawn from the United States market because of their CV effects due to off-target binding (i. e. 5HT1R and 5-HT2R subfamilies expressed in the CV system as well as hERG channels) [13]. Owing to the assumed etiology, new 5-HT4R agonists are being developed based on the hypothesis that a selective

3

ACCEPTED MANUSCRIPT interaction with 5-HT4Rs could produce safer prokinetic agents [13].

F

Cl

N

N N

O H3C

N H

K+ O-

N H tegaserod (2)

O

O Cl O

N

H2N

PRX-03140 (3)

O

N

RS-23,597-190 (4)

O

O

H2N

O CH3

O CH3 ML 10302 (5)

N

M AN U

Cl

O

N

SC

S

N H

N H

O

OCH3 O CH3 cisapride (1)

H2N

NH

RI PT

O

H

Cl

H2N

O CH3

N

RS-67,333 (6)

TE D

Fig. 1. Structures of some relevant serotonin 5-HT4R ligands.

EP

Recent studies suggested that 5-HT4R agonists could have a role in the treatment of Alzheimer’s disease (AD) [14, 15], an irreversible and progressive brain disease characterized by cognitive decline,

AC C

loss of episodic memory, and behavioral and physical disability. Thus, 5-HT4R agonists may represent a new class of drugs potentially useful for treating cognitive deficits associated with AD [14]. In particular, PRX-03140 (3), a partial 5-HT4R agonist developed by Epix Pharmaceuticals, was found to be capable of improving cognitive processes by increasing the release of acetylcholine and of a soluble amyloid precursor protein (sAPPα), which has potent neuroprotective and neurotrophic properties [14]. In recent years, the interest in the development of new serotoninergic agents led us to design a large number of ligands showing different affinity profiles, selectivity, and pharmacological properties [16-

4

ACCEPTED MANUSCRIPT 19]. In particular, we have discovered potent ligands for 5-HT3 or 5-HT1A receptors possessing quinoline bicyclic aromatic system in their structure [16-19]. Interestingly, the quinoline moiety has been used in the design of interesting 5-HT4R ligands described in the literature [1, 20-22]. Thus, in pursuing our large program focused on the medicinal chemistry of 5-HT receptor ligands, the aromatic

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moieties of reference 5-HT4R ligands 3 and 4 were replaced with the 2-methoxyquinoline one to afford compounds 7a,b. In this first design step, the fused benzene ring of 7a,b was assumed to play the role of either the chlorine atom of reference ligand 4 or the fused thiophene moiety of 3. In a subsequent design

SC

step, flexible compounds 7a,b were in turn transformed into the conformationally constrained derivatives 8a-g (Fig. 2) in order to obtain information on the interaction of these ligands with their

K + O-

O Cl

O

H2N

N

S

O CH3

EP

X

O

7a,b

N H

N

PRX-03140 (3)

X

N

N O

N 8a-g R

AC C

H3C

N

N

N

O

O

TE D

RS-23,597-190 (4)

O

M AN U

receptor.

Fig. 2. Design of 5-HT4R ligands 7a,b and 8a-g from reference ligands 3 and 4. For the specific structure of compounds 7a,b and 8a-g see Table 1.

The present paper reports on the synthesis and the preliminary pharmacological characterization of 5HT4R ligands 7a,b and 8a-g together with the computational study of the ligand-receptor interaction of the most interesting compounds of the series. 5

ACCEPTED MANUSCRIPT

2. Results and discussion. 2.1. Chemistry Flexible target compounds 7a,b were prepared according to the reaction sequence shown in Scheme

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1. Commercially available acid 9 was alkylated with 1,3-dibromopropane to afford bromoderivative 10, which was then reacted with piperidine to afford expected ester derivative 7a. Quinoline-4-carboxylic acid 9 was used also in the synthesis of amide derivative 7b by reaction with 1-(3in

the

presence

of

BOP

[(benzotriazol-1-

SC

aminopropyl)piperidine

yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, Castro’s reagent] [23] as the coupling

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agent.

Scheme 1. Synthesis of flexible target compounds 7a,b. O

OH

O

O

Br

i O CH3

9

N 10

iii

ii

N N

N

O

O CH3

N

N

7a

O CH3

AC C

7b

O H

EP

O

O CH3

TE D

N

Reagents: (i) BrCH2CH2CH2Br, DBU, THF; (ii) piperidine, DIPEA, CH3CN; (iii) 1-(3aminopropyl)piperidine, BOP, TEA, DMF.

The conformationally constrained target compound 8a was prepared starting from commercially available 4-methylisatin 11 (Scheme 2), which was reacted with acetic anhydride to give N-acylisatin 12

6

ACCEPTED MANUSCRIPT and then submitted to intramolecular aldol condensation in the presence of sodium hydroxide to afford quinoline-4-carboxylic acid 13. This latter was converted into 2-chloro-5-methylquinoline-4-carbonyl chloride (14) by reaction with thionyl chloride in the presence of a catalytic amount of DMF. The dichloro intermediate 14 was reacted with sodium methoxide in methanol to give methyl ester 15, which

RI PT

was first brominated with N-bromosuccinimide (NBS) in the presence of dibenzoyl peroxide as a radical initiator, and finally reacted with 1-(3-aminopropyl)piperidine to afford target compound 8a.

O

O i

O N 12 O

11 ii

OH

O

Cl

iii N H

O

N 14

TE D

13

Cl

iv

O

O

N

N

O

CH3

N

8a

O CH3

AC C

15

O CH3

EP

v, vi N

M AN U

O N H

O

SC

Scheme 2. Synthesis of conformationally constrained target compound 8a.

Reagents: (i) (CH3CO)2O; (ii) NaOH, H2O; (iii) SOCl2, DMF; (iv) NaH, CH3OH; (iii) NBS, dibenzoyl peroxide, CCl4; (iv) 1-(3-aminopropyl)piperidine, C2H5OH.

On the other hand, dichloro intermediate 14 was reacted with ethanol in the presence of TEA to give ethylester 1611 (Scheme 3), which, after radical bromination with NBS and dibenzoyl peroxide as a radical initiator, was reacted with 1-(3-aminopropyl)piperidine to obtain target compound 8c. 7

ACCEPTED MANUSCRIPT

Scheme 3. Synthesis of conformationally constrained target compound 8c. O

OH

O

Cl

i O

N 14 ii

O

N

N

O

O

SC

13

Cl

RI PT

N H

iii, iv N

Cl

Cl

8c

16

M AN U

N

Reagents: (i) SOCl2, DMF; (ii) TEA, C2H5OH; (iii) NBS, dibenzoyl peroxide, CCl4; (iv) 1-(3-

TE D

aminopropyl)piperidine, C2H5OH.

The structure of compound 8c was confirmed by crystallographic studies (Fig. 3) and used as an input

AC C

EP

in the molecular modeling studies.

Fig. 3. Structure of target compound 8c found by crystallography. Ellipsoids enclose 50% probability.

8

ACCEPTED MANUSCRIPT

Target compounds 8b,d-g were prepared starting from 8c by means the reaction sequence shown in Scheme 4. In particular, chloroderivative 8c was reacted with pyrrolidine to obtain target compound 8e. However, we observed that in our conditions the nucleophilic aromatic substitution leading to 8e was with

the

spontaneous

oxidation

of

its

angular

methylene

group

so

that

RI PT

combined

benzo[de][2,6]naphthyridin-4,6-dione derivative 8f was isolated from the reaction mixture. On the other hand, benzo[de][2,6]naphthyridin-4,6-dione derivative 8d was obtained by oxidation of 8c with

SC

selenium dioxide. Compound 8d was then reacted with sodium methoxide in methanol to obtain 8b or

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with piperidine to afford 8g.

Scheme 4. Synthesis of conformationally constrained target compound 8b,d-g.

N

O

O

N

O

i N

Cl

N

O

N

Cl

N

O

8b

EP

8d

iv

N

O

N 8e: X = CH2 8f: X = C=O

AC C

iii

N

O

ii

8c

X

N

TE D

N

N

O

N

N

N

N

O

N

8g

Reagents: (i) SeO2, 1,4-dioxane; (ii) NaH, CH3OH; (iii) pyrrolidine, C2H5OH; (iv) piperidine, C2H5OH.

9

ACCEPTED MANUSCRIPT

The

structure

of

benzo[de][2,6]naphthyridin-4,6-dione

derivative

8g

was

confirmed

by

SC

RI PT

crystallography (Fig. 4).

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Fig. 4. Structure of target compound 8g as determined by crystallography. Ellipsoids enclose 50% probability.

2.2. Binding Assays and Structure-Activity Relationships (SARs).

The receptor interactions of the target compounds 7a,b and 8a-g were examined by measuring both

TE D

binding affinity and the ability to promote or inhibit receptor–G protein coupling. Affinities were assessed as activity in inhibiting the specific binding of [3H]GR113808 (i. e. [1-[2(methylsulfonylamino)ethyl]-4-piperidinyl]methyl 1-methyl-1H-indole-3-carboxylate, 17) to 5-HT4R in

EP

guinea pig striatum membranes [24] and were compared with those of the reference ligands 1 and 4.

AC C

The results of binding studies are summarized in Table 1.

10

ACCEPTED MANUSCRIPT Table 1. 5-HT4R interaction features of target compounds 7a,b and 8a-g. X

N

X

O

N O

N H3C

N

7a,b

O

8a-g R effect on 5-HT4R-G protein coupling

inhibitory activity IC50 (nM) ± SEMb

O

52 ± 11

antagonist

203 ± 14

7b

NH

> 1000

NTc

8a

CH2

OCH3

283 ± 82

antagonist

8b

C=O

OCH3

44 ± 11

antagonist

110 ± 10

8c

CH2

Cl

563 ± 114

partial agonist

195 ± 25

8d

C=O

Cl

105 ± 21

antagonist

453 ± 118

8e

CH2

pyrrolidino

> 1000

NTc

NTc

8f

C=O

pyrrolidino

> 1000

NTc

NTc

8g

C=O

piperidino

344 ± 101

NTc

NTc

partial agonist

49 ± 11

SC

7a

R

M AN U

X

RI PT

affinity Ki (nM) ± SEMa

compd

1

20 ± 5.5

4

19 ± 3.5

TE D

a

N

NTc

916 ± 21

AC C

EP

Each value is the mean ± SEM of 3 independent determinations performed in duplicate and represents the apparent affinity constant assessed in [3H]17 (final concentration 0.2 nM) specific binding assay to guinea pig striatum membranes. bIC50 values were computed from the inhibition curves on the BRET signal induced by 100 nM 5-HT, as shown in Fig. 6. Each value is the mean ± SEM of 3 independent determinations. cNT = not tested.

Flexible quinoline derivatives 7a,b showed remarkable differences in 5-HT4R affinity, which are related to the chemical nature of the bond linking the side chain with the quinoline moiety. In fact, while the ester derivative 7a showed a Ki value in the nanomolar range, the corresponding secondary amide derivative 7b was at least two orders of magnitude less active. It is noteworthy, that between the reference ligands 3 and 4 used as templates in the design of our compounds 7a,b, compound 4 (Ki = 19

11

ACCEPTED MANUSCRIPT nM) is an ester derivative similar to 7a (Ki = 52 nM), while compound 3 (Ki = 36) [25] is a secondary amide derivative. The similarity in Ki values between 7a and the reference ligand 4 suggests that these two ligands establish similar interactions with the 5-HT4R binding site (i. e., the 2-methoxyquinolin-4-yl moiety of 7a is virtually equivalent to the 4-amino-5-chloro-2-methoxyphenyl group of 4). The

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transformation of ligand 7b into the corresponding conformationally constrained derivatives 8a,b (through the insertion of a methylene or a carbonyl group bridging position 5 of the quinoline nucleus with the amide nitrogen atom) enhanced 5-HT4R affinity to the nanomolar range (Ki = 283 and 44 nM,

SC

respectively); thus, the benzo[de][2,6]naphthyridin-4,6-dione derivative 8b was equipotent with flexible ester derivative 7a. These results could be easily rationalized by assuming that conformationally

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constrained derivatives 8a,b represented the bioactive conformation of ester 7a (Fig. 5) that could not be populated by amide derivative 7b for steric reasons. However, the higher affinity shown by 8b with respect to 8a suggested a potential role of the second carbonyl group in the interaction with 5-HT4R

TE D

binding site and led to consider the use of molecular modeling techniques to obtain a reliable insight.

O

H3CO

O

N

N

N N H H steric hindrance

N

7a Ki = 52 nM

EP

O

H3CO

7b Ki > 1000 nM

O

H3CO

AC C

N

O N

N

H3CO

N N

8a Ki = 283 nM

N O

8b Ki = 44 nM

Fig. 5. Effects of the conformational constraint on the 5-HT4R affinity of ligands 7a,b and 8a,b.

In ligands 8c,d, chlorine atom replacement of the methoxy group present in the conformationally constrained derivatives 8a,b produced minor changes of 5-HT4R affinity. In contrast, the replacement with bulkier cyclic amine residues (such as pyrrolidine in 8e,f or piperidine in 8g) had deleterious 12

ACCEPTED MANUSCRIPT effects on the interaction with the 5-HT4R binding site. To assess ligand effects on 5-HT4R activation, we used a cell-free bioluminescence resonance energy transfer (BRET) assay of receptor-G protein interaction. In this system receptor binding to endogenous Gαs subunits results in reduced distance between the receptor C-terminal and the N-terminal region of

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the Gβγ subunit. This causes enhanced BRET emission between the bioluminescent Renilla luciferase donor fused to the C-terminus of 5-HT4R and the fluorescent Renilla GFP acceptor that is genetically tethered to the N-terminus of the Gβ subunit. Cell membranes prepared from neuroblastoma cells co-

SC

expressing luminescent 5-HT4R and fluorescent Gβ1 were used for these studies.

Although RS-23,597 (i.e. ligand 4) was initially reported to behave as a pure antagonist at 5-HT4R

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[26], subsequent work based on preparations with a more efficiently coupled receptor [27-29] has shown that this ligand is instead a partial agonist. In agreement with such findings, we found that ligand 4 exhibited small but detectable intrinsic activity in promoting G protein coupling (Fig. 6A and Table

AC C

EP

TE D

1).

13

EP

TE D

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SC

RI PT

ACCEPTED MANUSCRIPT

AC C

Fig. 6. Functional activity of 5-HT4R ligands 7a and 8a-d with respect to reference ligands cisapride and 4 (RS-23597). A: All ligands are tested at 10 µM . The BRET ratio measured for each ligand was divided for that measured in the presence of 10 µM serotonin, after subtraction of the baseline recorded in the absence of ligand. Data are means (± SEM) of 3 determinations. B. Concentration-response curves for inhibition of 5-HT4R-Gβ coupling induced by 100 nM serotonin. Data points were averaged from 3 independent experiments with SEM given in the error bars.

14

ACCEPTED MANUSCRIPT

Interestingly, among the newly synthesized analogs, only compound 8c maintained a similar level of partial agonism as in 4. Thus, it is clear that the substitution of the 4-amino-5-chloro-2-methoxyphenyl moiety of ligand 4 with the 2-methoxyquinolin-4-yl group of 7a (Fig. 7) is sufficient to abolish this

RI PT

residual intrinsic activity of the molecule. Moreover, the additional structural constraints imposed in derivatives 8a,b did not further alter this null level of agonism. Conversely, it is equally interesting to note that the level of compound 4 intrinsic activity was restored by the methoxy to chlorine replacement

SC

from 8a to 8c, but not in the corresponding couple of imide derivatives 8b/8d (Fig. 7). Considering the negligible effects of chlorine-methoxy exchange observed on ligand affinity, it is conceivable to think

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that this substituent plays a far more important effect on receptor activation than on ligand recognition, although such an effect is crucially dependent on the other structural properties of the molecular scaffold. Along this line, we may speculate that 8c represents the minimal constrained configuration of 4 that can still allow the chlorine substituent to adopt the required orientation for exerting a positive role

break this requirement.

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on receptor activation. Conversely, the additional presence of the secondary carbonyl group in 8d may

Using the BRET assay we also studied the functional antagonism of the ligands by examining their

EP

concentration-dependent ability to inhibit receptor-G protein coupling induced by a sub-maximal concentration of serotonin (Fig. 6B). The measured inhibitory constants (IC50) were consistent with the

AC C

Ki values observed in the binding assay (taking into account the competitive effect of the fixed concentration of agonist present in the functional assay). These experiments also confirmed that only the curves of compounds 4 and 8c reached a plateau at a lower level of coupling activity in inhibiting 5-HT stimulation, consistent with their partial agonistic effect, whereas all the other ligands inhibited coupling activity to baseline levels.

15

ACCEPTED MANUSCRIPT

benzene to quinoline replacement H3CO

O O

H2N

N

RS-23,597-190 (4) Ki = 19 nM partial agonist - antagonist

conformational constraint O

N N

H3CO

N

N N

O

8b Ki = 44 nM antagonist

SC methoxy to chlorine replacement

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O

N

8a Ki = 283 nM antagonist

methoxy to chlorine replacement

O

N N

N

7a Ki = 52 nM antagonist

O

Cl

O N

O CH3

H3CO

O

RI PT

Cl

N O

8d Ki = 105 nM antagonist

Cl

N

N

N

8c Ki = 563 nM partial agonist

2.3. Docking Studies.

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Fig. 7. Structure-activity relationships in 5-HT4R ligands 7a and 8a-d with respect to reference ligand 4.

EP

A remarkable feature of aminergic GPCRs (encompassing the 5-HT4R, ß1-AR, ß2-AR, M2R, M3R and D3R) is their ability to bind amine ligands showing different shapes and sizes. Notwithstanding

AC C

their differences, the ligands bind in a pocket located in the extracellular side of the transmembrane (TM) region and show similarities in the amino acid residues involved in the interactions (i. e. the residues lying within 4 Å distance of any ligand atom). In particular, across the subfamily of the aminergic GPCRs, there are topologically equivalent residues from TM4, TM5, TM6 and TM7 that contact the diverse ligands (i. e. residues at positions 3.28, 3.32, 3.33, 3.36; 5.42, 5.46 and 5.47; 6.48, 6.51, 6.52, 6.55; 7.35, 7.39 and 7.43). Among them, residues at positions 3.32, 3.33, 3.36, 6.48, 6.51 and 7.39 make consensus contacts with many diverse ligands across the entire rhodopsin-like GPCR family

16

ACCEPTED MANUSCRIPT [30]. All the above mentioned residues are listed in Table 2 together with the corresponding amino acid residues in the 5-HT4R. The docking experiments reported in this study have been guided by the spatial

AC C

EP

TE D

M AN U

SC

RI PT

position of these determinant residues in the ligand binding site.

17

ACCEPTED MANUSCRIPT

Table 2. Residues constituting the ligand binding pocket of the aminergic GPCR family, corresponding residues in the 5-HT4R, and their interactions with ligands 7a, 7b, and 8b in different docking poses (poseI, poseII, and poses III.1 and III.2).

R96

3.32

H-bond e

V101

X

3.36

T104

Xe

C196

e

5.43

S197

5.46

A200

5.47

F201

6.48 6.51 6.52

W272 F275 F276

X

X

e

X

e

π-π e

X

7.35

W294

Xe

L298

e

Y302

H-bond

H-bond

e

X

≈H-bond

X

x

f

X

pose I

H-bond X

e

X

e

H-bond

Xe

X

e

X

e

X

e

X

e

X

e

T

d

T

d

x

≈H-bond or π-π xf X

e

X

e

e

f

b

X

e

X

e

X

e

T

X

e

X

e

π-π b

Xe

Xe

X

e

e

X

e

X

X

e

Xe e

H-bond

e

X

e

X

b

H-bond

e

X

x

f

Xe

f

x

X

e

X

e

X

e

X

e

T

d

d

≈H-bond or  xf X

e

X

e

H-bond

e

X

H-bondb X X

e

X

T

c

H-bond

e

e

Xe

X X

e

X b

X X

e

π-π c

H-bondb

e

X

pose III.2 Xe

e

H-bondb Xe

H-bondb

Xe

e

Xe

X

Xe

Xe

X

x

x

H-bond

H-bond

e

H-bondb Xe

b

π-π

xf

pose III.1 Xe

X

X

f

e

pose II

b

Xe

f

x

pose I

b

X

H-bondb

H-bond

π-π

H-bond

X

H-bond Xe

b

pose III.2 Xe

b

Xe

H-bond

e

pose III.1 Xe

b

X

Xe

H-bond Xe

c

H-bond

X

b

d

b

X

H-bondb

e

pose I Xe

b

e

H-bondb c

Xf π-π

pose III.2 Xe b

xf

H-bond

N279

7.43

b

X

b

6.55

7.39

pose III.1 Xe

Xe b

3.33 5.42

a

D100

pose II

SC

3.28

pose I

7b

M AN U

Residue in h5-HT4Ra

TE D

Residue positiona

7a

RI PT

8b

X

b

H-bond

X

X

e

X

e

X

e

X

e

T

d

d

Xe Xe d

T

T

H-bond

≈H-bond

X

FAR!

H-bond

≈H-bondc

Xe

Xe

Xe

xf

Xe

Xe

X

e

X

e

e

f

x

X

e

Xe

X

e

X

e

f

f

X

e

X

X

x

x

b

e

AC C

EP

Residues are reported in bold or italics according to the probability (frequence) to be found in the ligand binding pocket of the aminergic receptor family, residues that make consensus contacts with ligands across the class A GPCRs are grey highlighted [30] bH-bond = the residue forms a Hbond with the ligand; c≈H-bond = the residue establishes a non optimal H-bond interaction with the ligand; dT = the residue interacts with the ligand through an edge to face aromatic contact; eX = the residue is located within 4 Å distance of any ligand atom; fx = the residue is located within 5 Å distance of any ligand atom.

18

ACCEPTED MANUSCRIPT In order to rationalize the above discussed SAR data, the most potent newly synthesized ligands 7a and 8b have been docked in several different poses, the most significant ones (i. e. pose I, pose II and pose III) being reported in Table 2. In addition, the detrimental effect of the –NH substitution in the flexible quinoline derivatives 7b has also been investigated.

RI PT

Poses I and II have been built according to the binding modalities described for ligand 17 by LópezRodríguez and co-workers [31], and by Rivail et al. [32], respectively. Although dated, these two papers describe, to our knowledge, the only docking studies performed directly on computational models of the

SC

5-HT4R receptor. The 5-HT4Rs in these two studies were built on two different X-ray structures of the same bovine rhodopsin (pdb 1F88 and 1HZX were used by López-Rodríguez and coworkers [31] and

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Rivail et al. [32], respectively), which explains their similar ligand docking features. However, belonging to early 2000s, these receptor structures are not inclusive of all the latest structural information and data, i.e. many 3D structures of aminergic GPCRs, which could be used to update and ameliorate the 5-HT4R models, have been published only recently. The receptor model used in the present work was built by

TE D

exploiting as a template the structure of the β1-AR from turkey (pdb id 2Y00), published in 2011 [33]. This shows a 41% sequence similarity to 5-HT4R, which is much higher than that (19%) of bovin Rhodopsin used in the previous studies; the resulting three-dimensional 5-HT4R model is thus

EP

structurally different from the models used in previous papers. Therefore, a third pose (III) of the ligands into the active site has been attempted according to the structural binding modalities of a few ligands

AC C

(dobutamine, carazolol, eticlopride) complexed with the β-1 adrenergic receptor from turkey (pdb codes 2Y00, 2YCW) [33, 34] and human dopamine D3 receptor (pdb code 3PBL) [35], which have high structural similarities to the 5-HT4R. The ligands 7a, 7b, and 8b have been docked manually into the binding site of the receptor; all the rotatable angles have been rotated when necessary to obtain a better fit of the ligands into the binding site and to optimize the interaction with the receptor residues, forming the consensus contact network. Therefore, several almost equivalent poses have been obtained for slightly different combinations of

19

ACCEPTED MANUSCRIPT rotatable angles; the most representative results being listed in Table 2.

M AN U

SC

RI PT

The binding modalities of ligand 8b (pose I, pose II and pose III) are shown in Fig. 8

TE D

Fig. 8. Binding poses I, II and III of ligand 8b. The ligand is drawn in thick stick and the binding residues in thin stick. H-bonds are shown with yellow dashed lines. Color code: grey: Carbon atoms; blue: Nitrogen atoms; red: Oxygen atoms; yellow: Sulfur atoms; white: Hydrogen atoms.

EP

Pose I reproduces, as much as possible, the pose proposed by Rivail et al. [32], where the ligand interacts with S1975.43 through its carbonyl oxygen and with the D1003.32 side chain through its cationic

AC C

head. In addition, here, the aromatic bicycle is sandwiched between F2015.47 and F2766.52. Interestingly, the three ligands 8b, 7a and 7b bind identically in this pose (see Table 2). In binding pose II, which reproduces a modified version of the pose suggested by Rivail et al. [32], as indicated by López-Rodríguez and al. [31], the ligand is moved toward the residues in the TM2 helix, although it is still capable of contacting both D1003.32 and S1975.43 (which were considered to be key contacts [31, 32]). Here, ligand 8b forms charge reinforced H-bond with D1003.32 through its protonated amine; a second H-bond is established between the ligand methoxy oxygen atom and the hydroxyl side 20

ACCEPTED MANUSCRIPT chain of S1975.43; a third H-bond might be established between the sulphydryl group of C1965.42 and one of the two carbonyl oxygens of the ligand (the one of X group); F2766.52 stabilizes the complex through T-shaped interactions with the aromatic side of the ligand. In addition, N2796.55 might establish H-bond interaction with the second carbonyl oxygen or π−π interactions with the ligand aromatic portion. The

RI PT

interaction mode of 7a is highly similar, with the only difference that N2796.55 tends to establish H-bond with its ester oxygen. Molecule 7b shows some binding difference: in particular, the N2796.55 side chain is not in the ligand binding sphere, being repelled by the amidic NH group. Interestingly, no side chain

SC

of the consensus residues belonging to TM7 are in the immediate proximity of the ligand.

As previously mentioned, key points for poses I and II are residues D1003.32 and Ser1975.43. However,

M AN U

differently from D1003.32, S1975.43 is not one of the most representative consensus binding residues [30], although it is the only Ser present in the TM5 of the 5-HT4R. In other receptors (β-1 and β-2 ADRs and D3R), TM5 contains three Ser (at position 5.42, 5.43 and 5.46), which are located in the same region of the receptor sequence and space. Of these Ser residues, only S5.42 and S5.46 are part of the consensus

TE D

binding region in the majority of the class A GPCRs. In 5-HT4R, position 5.42 and 5.46 are occupied by Cys and Ala, respectively. For all these reasons, C1965.42 appears to be part of the ligand binding network with a higher probability than S1975.43. Interestingly, in poses III.1 and III.2, which have been

EP

built according to known three-dimensional structures of a few aminergic receptor structures docked with ligands, C1965.42 appears to be more involved in binding than S1975.43. Poses III.1 and III.2 are

AC C

very similar and both characterized by three H-bond interactions. The first one is established between the ligand cationic head and D1003.32 side chain, the second one involves the second carbonyl oxygen (the one of X group in Table 1) and T1043.36 side chain, and the last one involves the methoxy oxygen (R substituent) and the side chain of N2796.55. Moreover, the aromatic ring of F2766.52 establishes an edge to face (or T-shaped) aromatic interactions with the ligand. Interestingly, ligand 7a shows an interaction network that is nearly identical to that shown by ligand 8b (see Table 2, poses III.1 and III.2 for the two ligands), thus giving reason of the similar binding affinity shown by the two molecules. On the contrary,

21

ACCEPTED MANUSCRIPT ligand 7b shows different binding modalities: in pose III.1, T1043.36 is H-bonded to the amide carbonyl group, while in pose III.2, a rotation of the amide plan allows a more favorable interaction between the amide NH (instead of the carbonyl O) and the T1043.36 hydroxyl chain. Similarly, due to local modification of the ligand conformation, the H-bond between N2796.55 and the OCH3 substituent is also

RI PT

lost. The charge-reinforced H-bond between the ligand cationic head and the D1003.32 carboxyl group is instead maintained.

To date, it is not possible to discriminate between the three discussed poses: they all seem reasonable in

SC

the light of the available structural and modeling information. As a whole, ligand 7a and 8b show similar binding modalities, as expected by their similar experimental Ki values. On the other side, the presence

M AN U

of the amidic group in the ligand 7b confers alternative binding modes which might, although to a small extent, contribute to explain the difference in the experimental binding affinity.

3. Conclusion

TE D

Flexible quinoline derivatives 7a,b were designed by replacing the aromatic moieties of reference piperidinopropyl 5-HT4R ligands 3 and 4 with the 2-methoxyquinoline one. The nanomolar 5-HT4R affinity shown by ester 7a suggested that its 2-methoxyquinoline moiety is virtually equivalent to the 4-

EP

amino-5-chloro-2-methoxyphenyl one of 4 and stimulated its transformation into the conformationally constrained derivatives 8a-g in order to obtain information on the interaction of these ligands with their

AC C

receptor. A short series of conformationally constrained benzo[de][2,6]naphthridine derivatives (8a-g) was designed by tacking into account the potential role of different substituents (i. e. OCH3, Cl, pyrrolidino, piperidino, C=O) in the interaction with the binding pocket. The binding data demonstrated that constrained benzo[de][2,6]naphthyridin-4,6-dione derivative 8b was equipotent with flexible ester derivative 7a, reference compounds 4, and cisapride. This result was easily rationalized by docking studies in term of high similarity in the binding modalities of flexible 7a and conformationally constrained 8b. The intrinsic efficacy of some selected ligands was determined by measuring the ability

22

ACCEPTED MANUSCRIPT to promote or inhibit receptor–G protein coupling and the structure-activity relationship analysis suggested that the nature and the position of the substituents play a key role in modulating both affinity

AC C

EP

TE D

M AN U

SC

RI PT

and intrinsic efficacy.

23

ACCEPTED MANUSCRIPT 4. Experimental 4.1. Chemistry 4.1.1. General procedures All chemicals used were of reagent grade. Yields refer to purified products and are not optimized.

RI PT

Melting points were determined in open capillaries on a Gallenkamp apparatus and are uncorrected. Merck silica gel 60 (230-400 mesh) was used for column chromatography. Merck TLC plates, silica gel 60 F254 were used for TLC. 1H NMR spectra were recorded by means of both a Bruker AC 200 or a

SC

Bruker DRX 400 AVANCE spectrometers in the indicated solvents (TMS as internal standard); the values of the chemical shifts are expressed in ppm and the coupling constants (J) in Hz. The purity of

M AN U

compounds 7a,b and 8a-g was assessed by RP-HPLC and was found to be higher than 95%. An Agilent 1100 Series system equipped with a Zorbax Eclipse XDB-C8 (4.6 x 150 mm, 5 µm) column was used in the HPLC analysis with acetonitrile-methanol-(1% acetic acid-water) (40:30:30) as the mobile phase at a flow rate of 0.5 mL/min. UV detection was achieved at 254 nm.

TE D

Mass spectra were recorded on either a Thermo LCQ-Deca or an Agilent 1100 LC/MSD. Highresolution mass spectrometry (HRMS) measurements were carried out on a Thermo LTQ Orbitrap instrument. Operating conditions for the ESI source were as follows: spray voltage + 4.2 kV; capillary

EP

temperature 275 °C; sheath gas (nitrogen) flow rate, ca. 0.75 L/min. Methanolic solutions of the different

AC C

compounds (ca. 1 x 10-4 M) have been introduced via direct infusion at a flow rate of 5 µL/min.

4.1.2. 3-Bromopropyl 2-methoxyquinoline-4-carboxylate (10). A mixture of commercially available compound 9 (Aldrich, USA, 0.20 g, 0.98 mmol) in dry THF (15 mL) containing 1,3-dibromopropane (1.0 mL, 9.85 mmol) was heated under reflux for 10 min and DBU (0.15 mL, 0.98 mmol) was then added dropwise. The reaction mixture was heated under reflux for 5 h and then concentrated under reduced pressure. The residue was partitioned between dichloromethane and water. The organic layer was dried over sodium sulfate and evaporated under reduced pressure.

24

ACCEPTED MANUSCRIPT Purification of the residue by flash chromatography with dichloromethane as the eluent gave pure compound 10 (0.22 g, yield 69%) as a yellow oil. 1H NMR (400 MHz, CDCl3): 2.36 (m, 2H), 3.55 (t, J = 6.4, 2H), 4.09 (s, 3H), 4.56 (t, J = 6.1, 2H), 7.40-7.48 (m, 2H), 7.66 (t, J = 7.7, 1H), 7.89 (d, J = 8.4, 1H),

RI PT

8.57 (d, J = 8.4, 1H). MS (ESI): m/z 324, 326 (M+H+).

4.1.3. 3-(Piperidin-1-yl)propyl 2-methoxyquinoline-4-carboxylate (7a).

A mixture of bromide 10 (0.20 g, 0.62 mmol) in dry acetonitrile (10 mL) containing piperidine (0.092

SC

mL, 0.93 mmol) e DIPEA (0.22 mL, 1.26 mmol) was heated under reflux for 4 h. The reaction mixture was then concentrated under reduced pressure and the residue was partitioned between dichloromethane

M AN U

and water. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography with ethyl acetate as the eluent gave pure compound 7a (0.16 g, yield 79%) as a colorless oil. 1H NMR (400 MHz, CDCl3): 1.38-1.50 (m, 2H), 1.54-1.64 (m, 4H), 1.94-2.08 (m, 2H), 2.35-2.53 (m, 6H), 4.08 (s, 3H), 4.45 (t, J = 6.5, 2H), 7.40 (s, 1H), 7.41-7.48 (m,

TE D

1H), 7.61-7.67 (m, 1H), 7.88 (d, J = 8.4, 1H), 8.58 (d, J = 8.5, 1H). 13C NMR (100 MHz, CDCl3): 24.4, 26.0, 26.2, 53.7, 54.7, 55.8, 64.5, 115.1, 122.0, 125.1, 125.6, 127.8, 129.8, 138.2, 147.6, 161.7, 165.9.

EP

HRMS (ESI): m/z calculated for [C19H24N2O3 + H+] requires 329.1860, found 329.1862.

4.1.4. 2-Methoxy-N-[3-(piperidin-1-yl)propyl]quinoline-4-carboxamide (7b).

AC C

A mixture of commercially available compound 9 (0.25 g, 1.23 mmol) in dry DMF (10 mL) containing BOP (0.54 g, 1.22 mmol), TEA (0.52 mL, 3.7 mmol), and 1-(3-aminopropyl)piperidine (0.29 mL, 1.82 mmol) was stirred at room temperature for 3 h. The reaction mixture was then concentrated under reduced pressure and the residue was partitioned between dichloromethane and water. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography with ethyl acetate/triethylamine (9:1) as the eluent gave pure compound 7b (0.22 g, yield 55%) as a yellow oil, which crystallized on standing. An analytical sample was obtained

25

ACCEPTED MANUSCRIPT by recrystallization from n-hexane-diethyl ether (mp 84-85 °C). 1H NMR (400 MHz, CDCl3): 1.23-1.31 (m, 6H), 1.76-1.82 (m, 2H), 2.35 (br s, 4H), 2.49-2.52 (m, 2H), 3.62 (q, J = 5.7, 2H), 4.07 (s, 3H), 6.98 (s, 1H), 7.40 (t, J = 7.0, 1H), 7.63 (t, J = 7.2, 1H), 7.85 (d, J = 8.4, 1H), 8.18 (d, J = 8.4, 1H), 8.70 (br s, 1H).

13

C NMR (100 MHz, CDCl3): 24.0, 24.1, 25.8, 41.1, 53.5, 54.6, 59.0, 110.9, 121.8, 124.6, 125.5,

RI PT

127.5, 129.9, 145.6, 147.3, 161.7, 166.8. HRMS (ESI): m/z calculated for [C19H25N3O2 + H+] requires 328.2020, found 328.2020.

SC

4.1.5. 1-Acetyl-4-methylindoline-2,3-dione (12).

A mixture of commercially available compound 11 (ChemCollect, GmbH, 2.0 g, 12.4 mmol) in acetic

M AN U

anhydride (3.0 mL) was heated under reflux for 3 h and cooled to room temperature. The precipitate was collected by filtration, washed with diethyl ether, and dried under reduced pressure to obtain compound 12 as a yellow solid (1.6 g, yield 64%, mp 143-144 °C). 1H NMR (200 MHz, DMSO-d6): 2.52 (s, 3H),

TE D

2.55 (s, 3H), 7.14 (d, J = 7.4, 1H), 7.59 (t, J = 7.9, 1H), 8.07 (d, J = 8.0, 1H).

4.1.6. 5-Methyl-2-oxo-1,2-dihydroquinoline-4-carboxylic acid (13). A mixture of compound 12 (1.6 g, 7.87 mmol) in water (10 mL) containing NaOH (0.63 g, 15.8

EP

mmol) was heated under reflux for 3 h. The reaction mixture was then cooled to 0 °C and cautiously acidified with 3N HCl to pH 1. The precipitate was collected by filtration, washed with acetone, and

AC C

dried under reduced pressure to obtain compound 13 as an off-white solid (1.4 g, yield 88%). 1H NMR (200 MHz, DMSO-d6): 2.46 (s, 3H), 6.46 (s, 1H), 7.02 (d, J = 7.2, 1H), 7.21 (d, J = 8.0, 1H), 7.41 (t, J = 7.7, 1H), 11.94 (br s, 1H). MS (ESI, negative ions): m/z 202 (M-H+).

4.1.7. Methyl 2-methoxy-5-methylquinoline-4-carboxylate (15). A mixture of compound 13 (1.0 g, 4.92 mmol) in SOCl2 (10 mL) containing a catalytic amount of DMF (5 drops) was heated under reflux for 3 h. The excess of SOCl2 was then evaporated under reduced

26

ACCEPTED MANUSCRIPT pressure and the yellow residue obtained (compound 14) was dissolved in dry methanol (20 mL). To this solution, NaH was added cautiously (0.47 g, 19.6 mmol) and the resulting mixture was refluxed for 4 h. The reaction mixture was then evaporated under reduced pressure and the residue was diluted with ethyl acetate and washed with water. The organic layer was dried over sodium sulfate and evaporated under

RI PT

reduced pressure. Purification of the residue by flash chromatography with petroleum ether-ethyl acetate (9:1) as eluent gave compound 15 as a white solid (0.93 g, yield 82%, mp 65-66 °C). 1H NMR (200

1H), 7.74 (d, J = 8.4, 1H). MS (ESI): m/z 232 (M+H+).

M AN U

4.1.8. Ethyl 2-chloro-5-methylquinoline-4-carboxylate (16).

SC

MHz, CDCl3): 2.52 (s, 3H), 3.97 (s, 3H), 4.05 (s, 3H), 6.88 (s, 1H), 7.20 (d, J = 7.0, 1H), 7.51 (t, J = 7.6,

A mixture of compound 13 (0.28 g, 1.38 mmol) in SOCl2 (10 mL) containing a catalytic amount of DMF (5 drops) was heated under reflux for 3 h. The excess of SOCl2 was then evaporated under reduced pressure and the yellow residue obtained (compound 14) was dissolved in ethanol (10 mL), and TEA

TE D

(5.0 mL) was added. The resulting mixture was stirred at room temperature under inert atmosphere for 30 min. The reaction mixture was concentrated under reduced pressure and the residue was diluted with dichloromethane and washed with water. The organic layer was dried over sodium sulfate and

EP

evaporated under reduced pressure. Purification of the residue by flash chromatography with petroleum ether-ethyl acetate (8:2) as the eluent gave pure 16 as a pale yellow oil (0.27 g, yield 78%), which

AC C

crystallized on standing. An analytical sample was obtained by recrystallization from petroleum ether by slow evaporation (mp 70-71 °C). 1H NMR (200 MHz, CDCl3): 1.43 (t, J = 7.3, 3H), 2.60 (s, 3H), 4.48 (q, J = 7.2, 2H), 7.34 (s, 1H), 7.40 (d, J = 7.0, 1H), 7.64 (t, J = 7.7, 1H), 7.92 (d, J = 8.4, 1H). MS (ESI): m/z 250 (M+H+).

27

SC

RI PT

ACCEPTED MANUSCRIPT

4.1.9.

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Fig. 9. Structure of compound 16 as determined by crystallography. Ellipsoids enclose 50% probability.

2-Methoxy-5-[3-(piperidin-1-yl)propyl]-5,6-dihydro-4H-benzo[de][2,6]naphthyridin-4-one

(8a).

A mixture of compound 15 (0.25 g, 1.08 mmol) in CCl4 (10 mL) with NBS (0.19 g, 1.08 mmol) and

TE D

dibenzoyl peroxide (0.024 g, 0.099 mmol) was refluxed for 4 h. After cooling to room temperature, the succinimide was filtered-off and the filtrate was concentrated under reduced pressure to obtain the corresponding bromide intermediate as a yellow oil, which crystallized on standing. A mixture of the

EP

bromide in EtOH (10 mL) containing 1-(3-aminopropyl)piperidine (0.52 mL, 3.24 mmol) was heated

AC C

under reflux for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was partitioned between dichloromethane and water. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography, with ethyl acetate-triethylamine (8:2) as the eluent, gave pure compound 8a as a light brown solid (0.21 g, yield 57%). An analytical sample was obtained by recrystallization from ethyl acetate by slow evaporation (mp 88-89 °C). 1H NMR (200 MHz, CDCl3): 1.21-1.57 (m, 6H), 1.94 (m, 2H), 2.40 (m, 6H), 3.68 (t, J = 7.2, 2H), 4.06 (s, 3H), 4.97 (s, 2H), 7.19 (d, J = 6.8, 1H), 7.53 (s, 1H), 7.59 (t, J = 7.9, 1H), 7.74 (d, J =

28

ACCEPTED MANUSCRIPT 8.6, 1H). HRMS (ESI): m/z calculated for [C20H25N3O2 + H+] requires 340.2020, found 340.2020.

4.1.10. 2-Methoxy-5-[3-(piperidin-1-yl)propyl]-4H-benzo[de][2,6]naphthyridine-4,6(5H)-dione (8b). To a solution of compound 8d (44 mg, 0.12 mmol) in dry CH3OH (10 mL) was added cautiously NaH

RI PT

(9.0 mg, 0.375 mmol). The resulting mixture was heated under reflux for 2 h and then concentrated under reduced pressure. The residue was partitioned between ethyl acetate and water, and the organic layer was washed with water, dried over sodium sulfate, and concentrated under reduced pressure.

SC

Purification of the residue by flash chromatography, with ethyl acetate-triethylamine (9:1) as the eluent, gave pure compound 8b as a yellow solid (30 mg, yield 71%, mp 162-163 °C). 1H NMR (400 MHz,

M AN U

CDCl3): 1.32-1.38 (m, 2H), 1.44-1.49 (m, 4H), 1.85-1.97 (m, 2H), 2.35 (br s, 4H), 2.44 (t, J = 7.3, 2H), 4.12 (s, 3H), 4.18 (t, J = 7.2, 2H), 7.79 (s, 1H), 7.83 (t, J = 7.9, 1H), 8.14 (d, J = 8.4, 1H), 8.35 (d, J = 7.4, 1H).

13

C NMR (100 MHz, CDCl3): 24.4, 25.0, 25.9, 39.2, 54.2, 54.4, 56.8, 115.8, 119.9, 122.8,

127.5, 130.4, 132.6, 144.4, 145.8, 162.8, 163.4. HRMS (ESI): m/z calculated for [C20H23N3O3 + H+]

TE D

requires 354.1812, found 354.1815.

4.1.11. 2-Chloro-5-[3-(piperidin-1-yl)propyl]-5,6-dihydro-4H-benzo[de][2,6]naphthyridin-4-one (8c).

EP

To a solution of compound 16 (0.25 g, 1.0 mmol) in CCl4 (10 mL) with NBS (0.18 g, 1.0 mmol) and dibenzoyl peroxide (0.024 g, 0.099 mmol) was refluxed for 4 h. After cooling to room temperature, the

AC C

succinimide was filtered-off and the filtrate was concentrated under reduced pressure to obtain the corresponding bromide intermediate as a yellow oil, which crystallized on standing. A mixture of the bromide in EtOH (10 mL) containing 1-(3-aminopropyl)piperidine (0.48 mL, 3.0 mmol) was heated under reflux for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was partitioned between dichloromethane and water. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography, with ethyl acetate-triethylamine (9:1) as the eluent, gave pure compound 8c as a pale yellow oil, which crystallized

29

ACCEPTED MANUSCRIPT on standing (0.17 g, yield 49%). An analytical sample was obtained by recrystallization from n-hexanediethyl ether by slow evaporation (mp 91-92 °C). 1H NMR (400 MHz, CDCl3): 1.37-1.41 (m, 2H), 1.501.56 (m, 4H), 1.91-1.98 (m, 2H), 2.33-2.41 (m, 6H), 3.71 (t, J = 7.2, 2H), 5.04 (s, 2H), 7.41 (d, J = 7.2, 1H), 7.73 (t, J = 7.9, 1H), 7.93 (d, J = 8.5, 1H), 8.01 (s, 1H). 13C NMR (100 MHz, CDCl3): 24.3, 25.9,

RI PT

46.3, 50.9, 54.5, 56.3, 120.1, 121.6, 122.9, 126.4, 128.9, 130.5, 135.3, 147.4, 152.5, 159.7. HRMS (ESI): m/z calculated for [C19H22ClN3O + H+] requires 344.1524, found 344.1526.

SC

4.1.12. 2-Chloro-5-[3-(piperidin-1-yl)propyl]-4H-benzo[de][2,6]naphthyridine-4,6(5H)-dione (8d). A mixture of compound 8c (0.10 g, 0.29 mmol) and SeO2 (0.16 g, 1.44 mmol) in 1,4-dioxane (10 mL)

M AN U

was heated under reflux for 2 h and cooled to room temperature. The reaction mixture was diluted with a saturated NaHCO3 solution and extracted with diethyl ether. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. Purification of the residue by flash chromatography, with ethyl acetate-triethylamine (9:1) as the eluent, gave pure compound 8d (0.065 g, yield 62%) as a

TE D

yellow solid melting at 105-107 °C. 1H NMR (400 MHz, CDCl3): 1.41 (m, 2H), 1.61 (m, 4H), 2.05 (m, 2H), 2.50 (m, 6H), 4.23 (t, J = 7.2, 2H), 7.99 (t, J = 7.9, 1H), 8.27 (s, 1H), 8.36 (d, J = 8.5, 1H), 8.56 (d,

EP

J = 7.3, 1H). HRMS (ESI): m/z calculated for [C19H20ClN3O2 + H+] requires 358.1317, found 358.1317.

4.1.13. 5-[3-(Piperidin-1-yl)propyl]-2-(pyrrolidin-1-yl)-5,6-dihydro-4H-benzo[de][2,6]naphthyridin-

AC C

4-one (8e).

A mixture of compound 8c (0.12 g, 0.35 mmol) in ethanol (10 mL) with pyrrolidine (0.15 mL, 1.8 mmol) was refluxed in a nitrogen atmosphere for 5 h. The reaction mixture was then concentrated under reduced pressure and the residue was partitioned between dichloromethane and water. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography with ethyl acetate-triethylamine (9:1) as the eluent gave pure compounds 8e (0.037 g, yield 28%). An analytical sample of 8e was obtained by recrystallization from n-hexane-

30

ACCEPTED MANUSCRIPT diethyl ether by slow evaporation (mp 107-108 °C). 1H-NMR (200 MHz, CDCl3): 1.41 (m, 2H), 1.57 (m, 4H), 1.93-2.06 (m, 6H), 2.43 (m, 6H), 3.67 (m, 6H), 4.93 (s, 2H), 6.97 (m, 1H), 7.42-7.61 (m, 3H). HRMS (ESI): m/z calculated for [C23H30N4O + H+] requires 379.2492, found 379.2493.

5-[3-(Piperidin-1-yl)propyl]-2-(pyrrolidin-1-yl)-4H-benzo[de][2,6]naphthyridine-4,6(5H)-

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4.1.14. dione (8f).

The above flash chromatography purification of 8e with ethyl acetate-triethylamine (9:1) as the eluent

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gave (as a less polar fraction) pure compounds 8f (0.017 g, yield 12%) as an orange solid. An analytical sample of 8f was obtained by recrystallization from n-hexane by slow evaporation (mp 142-143 °C). 1H

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NMR (200 MHz, CDCl3): 1.37 (m, 2H), 1.55 (m, 4H), 1.92-2.08 (m, 6H), 2.48 (m, 6H), 3.68 (m, 4H), 4.17 (t, J = 7.2, 2H), 7.62-7.73 (m, 2H), 7.99 (d, J = 8.5, 1H), 8.12 (d, J = 7.3, 1H). HRMS (ESI): m/z calculated for [C23H28N4O2 + H+] requires 393.2285, found 393.2285.

2-(Piperidin-1-yl)-5-[3-(piperidin-1-yl)propyl]-4H-benzo[de][2,6]naphthyridine-4,6(5H)-

dione (8g).

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4.1.15.

A mixture of compound 8d (22 mg, 0.061 mmol) in EtOH (5.0 mL) with piperidine (0.030 mL, 0.30

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mmol was heated under reflux for 3 h. The reaction mixture was then concentrated under reduced pressure and the residue was partitioned between dichloromethane and water. The organic layer was

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dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue by flash chromatography with ethyl acetate-triethylamine (9:1) as the eluent gave pure compound 8g (23 mg, yield 93%) as a yellow solid. Recrystallization from ethyl acetate by slow evaporation gave an analytical sample as orange needles melting at 152-153 °C. 1H-NMR (400 MHz, CDCl3): 1.40-1.71 (m, 12H), 2.03 (m, 2H), 2.61 (m, 6H), 3.83 (m, 4H), 4.19 (t, J = 7.2, 2H), 7.69 (t, J = 7.9, 1H), 7.90-7.94 (m, 2H), 8.12 (d, J = 7.3, 1H). HRMS (ESI): m/z calculated for [C24H30N4O2 + H+] requires 407.2442, found 407.2443.

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ACCEPTED MANUSCRIPT 4.2. X-Ray crystallography Single crystals of compounds 8c,g and 16 were submitted to X-ray data collection by using a Xcalibur Sapphire3 (Oxford Diffraction, UK) four-circle diffractometer at 293K, equipped with a graphite monochromated Mo-Kα radiation (λ = 0.71069 Å). The structure was solved by direct methods

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implemented in the SHELXS-97 program [36]. Data refinement was carried out by full-matrix anisotropic least-squares on F2 for all reflections for non-H atoms by means of the SHELXL-97 program [37]. Crystallographic data (excluding structure factors) for the structure in this paper have been

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deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 950600 (8c), CCDC 950601 (8g), and CCDC 950599 (16). Copies of the data can be obtained, free of

033; or e-mail: [email protected]).

4.3. Molecular Modeling

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charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; (fax: + 44 (0) 1223 336

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4.3.1. Model choice, ligand setup and docking procedure

The three-dimensional structure of human 5-HT4R (h5-HT4R) was retrieved from the database GPCRDB (http://www.gpcr.org/7tm/) that collects, combines, validates and stores large amounts of heterogenous

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data on G protein-coupled receptors (GPCRs).

The selected structure was built using the sequence of the h5-HT4R isoform B (UniProtKB accession

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code: Q13639-1) [38] and the three-dimensional structure of the β-adrenergic receptor from turkey (ADRB1) as template. (pdb code: 2y00) [33]. Notwithstanding all the modelled three-dimensional structures available on the web show highly similar structural features in the active site region, the ADRB1 structure was indicated by the BLAST similarity search [39] to be the best template for the h5HT4R [BLAST score: 211 bits (536); Expectation value: 7e-55; Identities = 126/311 (40%); Positives = 178/311 (57%), Gaps = 20/311 (6%)]. The three-dimensional structures of ligands 7a,b, and 8b were built starting from the

32

ACCEPTED MANUSCRIPT experimental X-ray data of 8c, and were afterward minimized using the automatic algorithm implemented in the MAESTRO suite (Schrödinger) [40]. The compounds were then docked manually into the active site. Different poses were tried exploiting the knowledge about similar ligands in the same receptor [31] or in other GPCR receptors (i.e.

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PDB code: 2Y00, 2YCW, 3PBL). All the relevant torsion angles were treated as rotatable during the docking process, thus allowing a search of the conformational space and a better fitting to the active site. The complexes underwent a fast minimization procedure (automatic minimization algorithm

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implemented in the MAESTRO suite), which minimizes the energy of the selected atoms with the

4.4. In vitro pharmacological studies 4.4.1. Binding assays

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OPLS_2005 force field [41].

Male Dunkin-Hartley guinea pigs (Charles River Italia, Calco, CO, Italy) weighing 300-400 g were

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used. Animal care and handling throughout the experimental procedures were in accordance with the European Communities Council Directive of 22 September 2010 (2010/63 UE). Animal were sacrificed by decapitation, brains were rapidly removed and striata nuclei dissected and

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used for binding assay preparation according to Grossman et al. with slight modification [24]. Crude membranes were diluted in the binding buffer (Hepes 50 mM, pH 7.4) in order to obtain the final protein

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concentration. The incubation was performed in 24-well polystyrene plate at 37 °C for 30 min. The bound radioligand was separated by rapid filtration on glass fiber Unifilter GF/B 24w plate pre-treated with polyethyleneimine 0.1% in buffer. Filtrates were washed two times with 2 mL cold binding buffer, plates were dried for 30 min at room temperature, then 0.2 mL/well MICROSCINT-20 (Perkin Elmer Life and Analytical Sciences) and radioactivity measured after at least 2 h of stabilization. The specific binding of [3H]17 (final concentration 0.2 nM), defined as the difference between the total binding and the nonspecific binding determined in the presence of 30 µM 5-HT, represented about 70-80% of the

33

ACCEPTED MANUSCRIPT total binding. Competition experiments were analyzed by the GraphPad Prism software (version 6 for Windows) to obtain the concentration of unlabelled drug that caused 50% inhibition of [3H]17 specific binding (IC50). Apparent affinity constants (Ki) were derived from the IC50 values according to the Cheng and Prusoff

4.4.2. BRET recording of 5-HT4R-G protein coupling

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equation [Ki = IC50/(1+L/Kd)] [42].

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The preparation of retroviral vectors coding for Rluc-tagged human 5-HT4 receptor and RGFP-fused to Gβ1, and the transduction of SH-5YSY human neuroblastoma cells were done using procedures

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described previously [43]. The 5-HT4R-Rluc fusion protein cDNA was engineered by replacing the stop codon of the receptor with a sequence encoding a 13-mer linker peptide (RTEEQKLISEEDL) and cloning it in frame with the Rluc sequence into the retroviral expression vector pQIXN (Clontech). Cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and F-12, with 10% (v/v) fetal calf

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serum, 100 µg/mL hygromycin B and 400 µg/mL G418 in a humidified atmosphere of 5% CO2 at 37°C. Enriched membranes from transfected cells were obtained by differential centrifugation [43] and stored in aliquots at -80 °C before use. Receptor/Gβ1 interactions were measured in membrane preparations (5

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µg of proteins in a total volume of 100 µL phosphate-buffered saline) placed into white plastic 96-well plates (Packard Opti-plate); BRET recording and computations methods were described previously [43].

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The ligand concentration yielding 50% inhibition of coupling (IC50) were computed from concentrationinhibition curves obtained using 8 log-spaced concentrations of the ligands in the presence of 100 nM serotonin. The data were analyzed by nonlinear curve fitting with a general 4-parametrs logistic function [43].

Acknowledgments. The authors are grateful to Dr. Francesco Berrettini (CIADS, Università di Siena) for the X-ray data collection, to Dr. Laura Salvini (Toscana Life Sciences, Siena) for HRMS measurements, and to Prof. Gianluca Giorgi for structure refinements and fruitful discussions. This work 34

ACCEPTED MANUSCRIPT was partly supported by MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) - PRIN

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(Programmi di ricerca di Rilevante Interesse Nazionale).

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Highlights A new series of 5-HT4 receptor ligands has been designed and synthesized.

• • •

Two compounds showed a 5-HT4R affinity similar to reference ligand cisapride. The intrinsic efficacy was assessed by evaluating receptor–G protein coupling. The substituent properties play a critical role in ligand-receptor interactions.

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The SAR data have been rationalized by docking studies.

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•