Mohamed Ettaoussi
email: m.ettaoussi@yahoo.fr

Basile Pérès

Aïcha Errazani

Jean A Boutin

Daniel-Henri Caignard

Philippe Delagrange

Patricia Melnyk

Pascal Berthelot

Saïd Yous
email: said.yous@univ-lille2.fr

Synthesis and pharmacological evaluation of dual ligands for melatonin (MT1/MT2) and serotonin 5-HT2C receptor subtypes (II)

Keywords: Agomelatine, Melatonin receptors, MT2-selectivity, Serotonin 5-HT2C receptor. 4

R 1 = CH 2 F Ki (nM): MT 1 = 0.12 MT 2 = 0.21 5-HT 2C = 708 Ki (nM): MT 1 = 7.8 MT 2 = 1.1 5-HT 2C = 242 Ki (nM): MT 1 = 4.7 MT 2 = 0.6 5-HT 2C = 187 16a: R 3 = C 2 H 5 Ki (nM): MT 1 = 3.8 MT 2 = 0.6 5-HT 2C = 130 Ki (nM): MT 1 = 13.4 MT 2 = 0.77 5-HT 2C = 130 Ki (nM): MT 1 = 3.9 MT 2 = 0.06 5-HT 2C = 391 R 3 = CH=CH 2 Ki (nM): MT 1 = 0.15 MT 2 = 0.13 5-HT 2C = 1000 A new series of agomelatine analogues dual ligands for melatonin and serotonin were synthesized issued from combination of and C-3 modulations. Synthesized series conserved submicromolar melatonin binding affinities.

RESEARCH HIGHLIGHTS:

-New agomelatine analogues dual ligands for melatonin and serotonin were reported -Combination of and C-3 modulations led to the improvement of serotonin binding affinity -Synthesized series conserved melatonin binding affinities; The allyl 7b and ethyl 16a represented the more interesting compounds of this series.

-Methyl thiourea 12h represent one of the highest MT2 selective derivative nowadays reported

Introduction

Melatonin or N-acetyl-5-methoxytryptamine is mainly produced and secreted by the pineal gland during the period of darkness following a circadian rhythm [1]. This neurohormone is the endogenous ligand for MT1 and MT2, two receptors belonging to the family of G protein-coupled receptors (GPCRs) and primarily expressed in the central nervous system (CNS) [2] and widely distributed in other tissues [3,4]. Melatonin interacts also with a low binding affinity site called MT3 and characterized as the hamster homologue of the human enzyme quinone reductase 2 [5]. Once secreted, melatonin plays an important role in modulating various physiological processes upon activation of its binding sites [6][7][8].

Since its isolation, then characterization by Aaron Lerner in 1958 [9], melatonin was extensively studied during the 20 th century in order to clarify its physiological and behavioural implications. Over the years were discovered and proved its biological implications. However, since the development of ramelteon and agomelatine (Chart 1) the interest of the scientific community is growing up significantly and special attention has been paid for the design and synthesis of selective ligands. The elucidation of the exact role of the two melatonin receptor subtypes remains a real challenge but may provide great potential in the comprehension of their biological functions and the development of specific ligands.

Melatonin was demonstrated to be involved in the regulation and re-establishment of circadian rhythms and sleep promoting [10]. Moreover, it is also suggested to be involved in a variety of other physiopathological processes. In fact, experimental evidence suggests its implication in type 2 diabetes [11], in depression [12][13][14], cancer [15], in Alzheimer and Parkinson diseases [16][17]. Furthermore, other studies showed the involvment of this neurohormone in the modulation of immunity, cardiovascular responses and in blood pressure control [18].

Our research group started working, two decades ago, on the discovery of new tools for the study of the melatoninergic system. Hence, a multicomponent research program was initiated and consisted in the synthesis of new ligands selective or not for one or the other of the melatonin binding sites MT1, MT2 and MT3. A substantial work was done in terms of SAR and many interesting ligands were discovered [19][20][21][22]. In one of the investigated areas, the aim was to design and synthesize new molecules with a similar receptoriel profil as agomelatine, a melatonin MT1/MT2 agonist and serotonin 5-HT2C antagonist. To hit this objective, we previousely described the synthesis of new analogues issued from the modulation of different sites of agomelatine [START_REF] Ettaoussi | [END_REF]24]. Accordingly, the modulation of the C-3 position of the naphthalene ring [START_REF] Ettaoussi | [END_REF] and the -position of the amide side chain [24] have been shown to possess positive effects on binding affinities at both (MT1, MT2) and 5-HT2C.

In the current work, we describe the synthesis and pharmacological results of compounds issued from the combination of these two modulations of agomelatine using βhydroxymethyl-agomelatine as a lead.

Results and Discussion

2.1. Chemistry. Synthetic routes to the designed compounds are depicted in Schemes 1-4.

The acetamide 4 was resynthesized from cyanoester 1 [24] through two synthetic routes (Scheme 1). First, by a catalytic hydrogenation over Raney nickel in acetic anhydride [25] leading to the desired amidoester 2a and two unsaturated isomers (E,Z)-2b. The treatment of compound 2a with a mixture of lithium aluminum hydride furnished the desired compound 4 in a global yield of 56% [26]; The structures of isomers E-2b and Z-2b were determined by analytical studies using 1 H, 13 C and ROESY 1D NMR analyses. Z-2b identification was based on the presence of dipolar interactions between the ethylenic proton of the lateral chain and the naphthalenic proton H8 (see supporting information). The first strategy gave only access to acetamide derivative while the second method allowed preparation of different amides and areas as was previousely described by us [24].

This later method was realized in two steps, a one-pot reduction of nitrile and ester by treatment with a mixture of LiAlH4/AlCl3 followed by N-acylation of the resulting primary amine into the desired acetamide 4.

After preparation of lead compound 4, we moved to the synthesis of compounds issued from C-3 modulation. First, the access to vinyl and allyl derivatives 7a and 7b was achieved by C-3 selective bromination of compound 4 in acetic acid at 70 °C. The obtained ester 5 was then submitted to Stille cross-coupling reaction in the presence of vinyl and allyl tin derivatives to afford prodrugs 6a and 6b [27]. Finally, saponification of these later esters by sodium hydroxide in a hydro-alcoholic mixture furnished the desired compounds 7a and 7b in good yields (Scheme 2). To prepare methylcyclopropyl 12a we have adopted a strategy based on direct acylation of compound 4 (Scheme 2). By using Friedel-Crafts acylation conditions and cyclopropanoyl chloride, we obtained the intermediate ester 8 in a low yield (15%). Saponification of this later in methanol in the presence of sodium hydroxide furnished quantitatively acetamidoalcohol 9. Next, attempts to selectively reduce the ketone group onto methylene (compound 12a) using triethylsilane [28] were unsuccessful. This method figures out the difficulty of access to the desired compound by direct acylation of the amidolalcohol 4. To overcome this synthetic difficulty, we developed a new strategy permiting also the access to different amides and ureas (12a-j and 13a-c) and depicted in Scheme 3. This strategy consists in direct acylation of cyanoester 1 under Friedel-crafts conditions followed by one-step reduction of nitrile, ketone and ester functions using a mixture of LiAlH4 and AlCl3 in dry diethyl ether. The formed primary amines 11a and 11b were then N-acylated following the Schotten-Baumann conditions to afford compounds 12a-d and 13a-b. Otherwise, fluoroacetamides 12e and 13c were prepared from amine bases 11a and 11b as previously described by us using ethyl fluoroacetate in 2,2,2-trifluoethanol. Finally, ureas and thioureas (12f-j) were prepared from the amine hydrochloride 11a by treatment with the corresponding isocyanates or isothiocyanates [29]. Finally, a selective chemical reduction of the methyl ester into hydroxymethyl using lithium aluminum hydride afforded the desired compounds 16a and 16b in good yields (Scheme 4). It is worth nothing that synthesis of compounds 16a-16b starting from compounds 1 and 4 was tried and abandoned because of low yields.

1 10a, R 3 = COc-C 3 H 5 10b, R 3 = COc-C 4 H 7 11a, R 3 = CH 2 c-C 3 H 5 11b, R 3 = CH 2 c-C 4 H 7 12a, R 3 = CH 2 c-C 3 H 5 12b, R 3 = CH 2 c-C 3 H 5 , R 2 = CH 3 , R 2 = C 2 H 5 12c, R 3 = CH 2 c-C 3 H 5 12d, R 3 = CH 2 c-C 3 H 5 , R 2 = c-C 3 H 5 , R 2 = c-C 4 H 7 12e, R 3 = CH 2 c-C 3 H 5 13a, R 3 = CH 2 c-C 4 H 7 , R 2 = CH 2 F , R 2 = CH 3 13b, R 3 = CH 2 c-C 4 H 7 13c, R 3 = CH 2 c-C 4 H 7 , R 2 = C 2 H 5 , R 2 = CH 2 F 12f, X= O 12g, X= O , R 2 = NHC 2 H 5 , R 2 = NHC 3 H 7 12h, X= S 12i, X= S , R 2 = NHCH 3 , R 2 = NHC 2 H 5 11a d 12j, X= S, R 2 = NHC 3 H 7 R 3 R 3 R 3 a Reagents: a) R3Cl, AlCl3, DCM, 0°C; b) (i) LiAlH4, AlCl3,

Scheme 4. Synthesis of compounds 16a-16b

O O NHCOCH 3 O O O NHCOCH 3 O OH O NHCOCH 3 a b c 2a 14a, R 3 = COCH 3 14b, R 3 = COC 2 H 5 15a, R 3 = C 2 H 5 15b, R 3 = C 3 H 7 16a, R 3 = C 2 H 5 16b, R 3 = C 3 H 7 R 3 R 3 R 3
a Reagents: a) AlCl3, DCM/DMF, R3Cl, 0°C; b) Et3SiH, TFA, rt; c) LiAlH4, THF, 0°C.

Biological Results. Newly synthesized compounds were assayed at human MT1 and MT2 receptors stably transfected in Chinese Hamster Ovarian (CHO) cells, using 2-

[ 125 I]iodomelatonin as radioligand [START_REF] Audinot | Naunyn-Schmiedeberg's[END_REF]. Serotoninergic 5-HT2C binding affinity was performed using Chinese Hamster Ovarian (CHO) cell lines stably expressing the human 5-HT2C receptors. Tables 123report the chemical structures, melatonin (MT1, MT2) and serotonin 5-HT2C binding affinities of these compounds.

As was previously stated, the introduction of a hydroxymethyl group in the -position of the acetamide lateral chain and the modulation of C-3 position of agomelatine improved both the melatoninergic and serotoninergic 5-HT2C binding affinities [START_REF] Ettaoussi | [END_REF]24]. Hence, compound 4 was chosen as a lead for new modulations in order to prepare new "dual" ligands for melatonin and serotonin receptors. In the current study we report the synthesis and biological results of a new series of derivatives issued from C-3 position modulation of 4.

Accordingly, the introduction of aliphatic chains such as vinyl (7a), allyl (7b), ethyl (16a) and propyl (16b) showed in general a positive effect on serotoninergic 5-HT2C binding affinity and the conservation of melatoninergic binding affinities (Tables 12). This observation was in agreement with our previous findings. In fact, the insertion of an allyl (7b) or an ethyl group (16a) produced an improvement of binding affinities at both melatonin (MT1, MT2) and serotonin 5-HT2C receptor subtypes. These two compounds represented the more interesting ligands of this series. Besides, the vinyl 7a exhibited a good binding affinity at melatonin receptor subtypes but a lower binding affinity at 5-HT2C. On contrary, the propyl 16b showed the conservation of both serotonin 5-HT2C and melatonin MT2 affinities with a decrease of the MT1 binding affinity.

Table 1. Binding affinities of compounds 4-7b The introduction, in the C-3 position of naphthalene cycle, of a methyl cyclopropyl (12a) or a methyl cyclobutyl (13a), had no change in MT2 and 5-HT2C binding affinities in comparison with the parent ligand (Table 2). However, we noticed a decrease in the MT1 binding affinity resulting in an MT2 selectivity of about 79-fold for 12a and 19-fold for 13a, respectively. Otherwise, the acetamide functions of 12a and 13a were substituted with other amides (12b-12d and 13b-13c), fluoroacetamide (12e) or ureas and thioureas (12f-12j). The acetamide 12a replacement by a propylamide (12b) or a cyclopropylamide (12c) has produced a decrease of melatonin MT1 and serotonin 5-HT2C binding affinities. In addition, the MT2 selectivity was fallen by 55 and 27-fold respectively in comparison with 12a.

O NHCOCH 3 O NHCOCH 3 Agomelatine R 3 OR 1 Compound R1 R3 Ki (nM) MT1 [I95] (n) Ki (nM) MT2 [I95] (n) Ki (nM)5-HT2[I95](n) Agomelatine - - 0.
Furthermore, the bulky cyclobutanamide 12d displayed a selectivity of 91-fold toward the MT2 receptor subtype while the fluoroacetamide 12e conserved the same good binding affinity towards MT2 and 5-HT2C receptors as the lead compound 4 and displayed a MT2 selectivity of about 153-fold. acetamide was then substituted by ureas (12f-12g) and thioureas (12h-12j). Hence, this modulation has led to a decrease of melatonin and serotonin 5-HT2C binding affinities (Table 2). The same effect was observed with thioureas but the methyl thiourea 12h who exhibited one of the highest MT2 selectivity nowadays reported. Furthermore, propyl thiourea 12j displayed one of the good serotonin 5-HT2C binding affinities of this series. Finally, the acetamide function of compound 13a was also submitted to modulation. Even its replacement by a propylamide (13b) had shown no noticed evolution, its substitution with a fluoroacetamide (13c) produced a lost of the MT2 affinity and a decrease of both MT1 and 5-HT2C binding affinities. Regarding the intrinsic activity, we investigated the EC50 and Emax at melatonin MT1 and MT2 receptor subtypes of synthesized compounds. The obtained results are illustrated in

Table 3. Functional activities of synthesized compounds

Compound h-MT1 h-MT2 EC50 (nM) I95 (n) Emax (%)±ESM(n) EC50 (nM) [I95] (n) Emax (%) ± ESM (n) Agomelatine 1.

Conclusions

We report in this paper the design and synthesis of a series of agomelatine analogues. The current strategy issued from the modulation of agomelatine β-acetamide position and C-3 position of the naphthalene ring led to compounds with different pharmacological profiles. In fact, insertion of aliphatic chains produced a positive effect on serotonin 5-HT2C binding affinity and the conservation of melatonin binding affinities. The allyl 7b and ethyl 16a exhibited the most interesting results of this series. However, the introduction of sterically hindered groups (methyl cycloalkyls) resulted in a decrease of MT1 binding affinity leading to an MT2 selectivity and confirming the findings previously obtained with C-3 modulation. At melatonin receptors, all the synthesized compounds showed a partial agonist activity at MT1 and full or partial agonist activities at MT2.

Experimental Section

Chemistry: Melting points were determined on a Büchi SMP-20 capillary apparatus and are uncorrected. IR spectra were recorded on a Vector 22 Bruker spectrometer. 1 H and 13 C NMR spectra were recorded on an AC300 Bruker spectrometer. Chemical shifts are reported in ppm units (parts per million) relative to (CH3)4Si. Elemental analyses for new compounds were performed by CNRS Laboratories (Vernaison, France) and the obtained results were within 0.4% of the theoretical values. Compounds purities were verified with analytical HPLC method and established to be > 95%.

Methyl 3-Acetylamino-2-(7-methoxy-naphthalen-1-yl) propanoate (2a).

Compound 1 (4.0 g, 39.2 mmol) was dissolved in acetic anhydride (200 mL) and the mixture was hydrogenated over Raney Ni (4.0 g) under pressure (30 bars) at 60 °C for 3 h. After filtration on celite, the solvent was concentrated and the residue was dissolved in water (100 mL) then extracted with ethyl ether. The organic phase was washed with a solution of sodium hydrogenocarbonate (1 M), dried over MgSO4, and evaporated under reduced pressure to give

Methyl (Z/E) 3-Acetylamino-2-(7-methoxy-naphthalen-1-yl) acrylate (2b).

After compound 2 recrystallisation, the filtrate was purified on a silica gel column eluted with a mixture acetone-cyclohexane (3:7) and gave the two isomers Z-2b and E-2b as white solids (8% yield (dd, 1H, J = 2.0, J = 8.9), 7.32 (m, 1H), 7.42 (m, 1H), 7.80 (d, 1H, J = 8.9), 7.86 (m, 1H), 8.52 (d, 1H, J = 12.2); 13 C NMR (CDCl3):  23. 5, 52.0, 55.4, 102.9, 112.1, 119.1, 123.5, 128.6, 128.8, 128.9, 129.5, 130.2, 132.8, 134.2, 158.4, 167.6, 167.7; IR (neat, cm -1 ): 3286 (NH), 1718 (C=O), 1636 (C=O). 9, 52.8, 55.3, 104.0, 109.1, 118.3, 123.1, 128.2, 128.8, 129.0, 129.9, 132.3, 134.3, 138.6, 157.8, 168.4, 169.8; IR (neat, cm -1 ): 3336 (NH), 1723

E-2b:

(C=O), 1683 (C=O).

3-Hydroxy-2-(7-methoxy-naphthalen-1-yl)propylamine hydrochloride (3).

To a suspension of LiAlH4 (3.04 g, 80 mmol) in dry diethyl ether (300 mL) at 0 °C, was added dropwise a solution of AlCl3 (10.6 g, 80 mmol) in dry diethyl ether (200 mL). After 10 mn under stirring, a solution of 1 (5.1 g, 20 mmol) in dry diethyl ether (200 mL) was added.

The reaction mixture was stirred for additional 30 mn, and hydrolyzed with a solution of NaOH (10 g, 250 mmol). After filtration and evaporation of the filtrate, the residue was dissolved in water and extracted with dichloromethane. The organic phase was washed with water, dried over MgSO4, filtered and evaporated under reduced pressure. The oily residue was then treated with HCl gas and recrystallized from acetonitrile. White solid (65% yield);

N-[3-Hydroxy-2-(7-methoxy-naphthalen-1-yl)propyl]acetamide (4)

. Route 1: Compound 2a (6.02 g, 20 mmol) was dissolved in dry tetrahydrofuran (65 mL) and cooled at 0 °C in an ice-bath, then aluminum lithium hydride (1 g, 26 mmol) was added portionwise. After 1 h stirring, the reaction mixture was hydrolyzed with 1 mL of water, 1 mL of an aqueous solution of NaOH (15%) and 3 mL of water. The mineral formed was filtered and the filtrate was washed, dried over MgSO4 and evaporated under reduced pressure to lead to a white solid after recrystallization from acetonitrile (80% yield).

Route 2. To a suspension of 3 (5.35 g, 20 mmol) in 100 mL of a mixture of H2O/AcOEt (1:1) was added potassium carbonate (8.28 g, 60 mmol) and the reaction mixture was cooled to 0 °C in an ice-bath. Acetyl chloride (1.85 mL, 26 mmol) was added and the mixture stirred for 15 mn. The two phases were separated and the organic layer was washed with a solution of HCl (1 M) and water, dried over MgSO4, filtered and evaporated under reduced pressure. The desired product was collected as a white solid after recrystallization from acetonitrile (80% yield). Mp 136-138 °C; MS (APCI, pos. 30 V) m/z: [M+H] + , 274.16; 1 H NMR (DMSO-d6):  1.76 (s, 3H), 3.33-7.37 (m, 2H), 3.65-3.85 (m, 3H), 3.96 (s, 3H), 4.73 (br s, 1H), 7.17 3-Acetylamino-2-(7-methoxy-3-vinyl-naphthalen-1-yl)propyle acetate (6a). Compound 5 (1.5 g, 3.8 mmol) and triphenylphosphine palladium tetrakis (0.219 g, 0.19 mmol) were dissolved in 22 mL of dry toluene under argon atmosphere, then vinyltributyltin (5.7 mmol) was added dropwise cautiously and the mixture was refluxed for 2 h. After cooling, the mixture was poured into 50 mL of an aqueous solution of KF 10% and the solid formed was filtered and washed with ethyl acetate. The organic phase was washed, dried over MgSO4 and evaporated under reduced pressure to give the desired product as a white solid after purification on silica gel column eluted with a mixture of ethyl acetate/cyclohexane (8:2).

Recrystallized from toluene (30% yield); Mp 122-124 °C; MS (APCI, pos. 30 V) m/z: 42. 3, 55.7, 63.1, 103.1, 113.9, 118.5, 122.1, 125.2, 129.4, 130.8, 132.3, 133.7, 137.1, 137.5, 158.0, 170.1; IR (neat, cm -1 ): 3279, 3210, 1650; Anal. (C18H21NO3) C, H, N.

N-[3-Hydroxy-2-(3-allyl-7-methoxy-naphthalen-1-yl)propyl]acetamide (7b).

This compound was prepared from compound 6b according to the procedure described for 7a.

Recrystallized from acetonitrile as a white solid (80% yield) 40.1, 41.6, 42.2, 55.6, 63.1, 102.8, 116.3, 118.2, 125.4, 126.2, 129.6, 130.2, 132.3, 134.7, 136.9, 138.1, 157.5, 170.1; IR (neat, cm -1 ): 3254 (NH), 3172 (C=O), 1636 (C=O); Anal.

(C19H23NO3) C, H, N.

Preparation of compound 8. To a solution of compound 4 (3.0 g, 11.0 mmol) in 50 mL of dry dichloromethane at 0 °C, was added cautiously aluminum chloride (8.8 g, 66.0 mmol) then cyclopropanoyle chloride (3.3 mL, 36.4 mmol). The reaction mixture was stirred at room temperature for 1 h, poured into water (100 mL) and the organic phase was washed with brine, dried over MgSO4 and evaporated under reduced pressure. The yellow oil obtained was then purified on a silica gel column eluted with a mixture of acetone-cyclohexane (3:7) to lead to compound 8. Synthesis of ureas and thioureas 12f-j. To a suspension of compound 11a (6.2 mmol) in dichloromethane (60 mL) at 0 °C, were added respectively triethylamine (12.4 mmol) and the corresponding isocyanate or isothiocyanate (6.3 mmol). The mixture was left to stir for 2 h and the solid formed was filtered, washed with diethyl ether and recrystallized to lead to the desired product. 4, 16.2, 34.6, 42.2 (2C), 43.2, 55.6, 63.6, 102.8, 118.1, 125.0, 126.4, 129.6, 130.1, 132.2, 136.7, 136.8, 157.3, 158.7; IR (neat, cm 11.8, 12.4, 23.7, 41.6 (2C), 42.1, 43.1, 55.5, 63.6, 102.8, 118.0, 125.0, 126.3, 129.5, 130 9, 23.1, 28.8, 41.6, 42.3, 55.7, 63.2, 102.8, 118.1, 124.5, 126.0, 129.6, 130.2, 132.3, 136.7, 138.7, 157.3, 170.1; IR (neat, cm -1 ): 3260 (NH), 2959-2852 (OH), 1634 (C=O); Anal. (C18H23NO3) C, H, N. 10.5, 16.0, 23.3, 28.9, 41.9, 42.5, 55.6, 63.1, 102.6, 118.2, 124.0, 125.9, 129.2, 130.0, 132.3, 136.7, 138.3, 157.3, 170 [ 35 S]GTPS binding assay was performed according to published methodology [START_REF] Audinot | Naunyn-Schmiedeberg's[END_REF]. Briefly, membranes from transfected CHO cells expressing MT1 and MT2 receptor subtypes and compounds were diluted in binding buffer (20 mM HEPES,pH 7.4, 100 mM NaCl, 3 µM GDP, 3 mM MgCl2, and 20 µg/mL saponin). Incubation was started by the addition of 0.2 nM [ 35 S]GTPS to membranes (20 µg/mL) and drugs, and further followed for 1 h at room temperature. Reaction was stopped by rapid filtration through GF/B filters followed by three successive washes with ice-cold buffer.

N-[3-Acetylamino-2-(3-cyclopropanecarbonyl-7-methoxy-naphthalen-1-yl)propyl

N-[3-Hydroxy-2-(3-propyl-7-methoxy-naphthalen-1-yl)propyl]acetamide (16b

Usual levels of [ 35 S]GTPS binding (expressed in dpm) were for CHO-MT2 membranes: 2000 for basal activity, 8000 in the presence of melatonin 1 µM and 180 in the presence of GTPS 10 µM which defined the non-specific binding. Data from the dose-response curves (seven concentrations in duplicate) were analyzed by using the program PRISM (Graph Pad Software Inc., San Diego, CA) to yield pEC50 (pEC50 = -Log10 (EC50) with EC50 the effective concentration 50%) and Emax (maximal effect) for agonists.

Assays for 5-HT2C receptor subtypes. Serotonin 5-HT2C competition binding assay was determined according to the protocol previously reported in the literature [32]. Membranes from CHO cell lines, stably expressing the human 5-HT2C(VSV) receptors were incubated for 60 min at 37 °C in binding buffer (50 mM Tris-HCl buffer (pH 7.4), containing 10 mM MgCl2 and 0.1% BSA) containing the radioligand [ 3 H]mesulergine (1 nM). Non-specific binding was defined in the presence of 10 µM mianserine. Reaction was terminated by rapid filtration through GF/B filters presoaked in 0.1% (v/v) polyethylenimine. Filters were washed three times with 1 mL of ice-cold 50 mM Tris-HCl buffer (pH 7.4). Residual radioactivity on filters was measured using a TopCount scintillation counter (PerkinElmer Life Sciences) after addition of Microscint 20. Isotherms were analyzed using non-linear regression to yield IC50.

Affinities are expressed as Ki.

1 .

 1 Melatonin and some of its analogues.

Scheme 1 .

 1 Scheme 1. Synthesis of compound 4

Scheme 2 .a

 2 Scheme 2. Synthesis of compounds 7a-b and 9

Scheme 3 .

 3 Scheme 3. Synthesis of compounds 12a-j and 13a-c

  Et2O, 0°C, (ii) HCl(g), Et2O; c) K2CO3, EtOAc/H2O then (i) R2COCl, 0°C-rt for 12a-d, 13a-b, (ii) FCH2CO2Et, TFE, reflux for 12e and 13c; d) Et3N, R2CX, DCM, 0°C. Finally, C-3 alkylated derivatives (16a-16b) were prepared from intermediate 2a previously described in Scheme 1. Direct acylation of compound 2a in the presence of aluminum chloride and acetyl or propanoyl chloride provided intermediates 14a-b. Selective reduction of the ketone with triethylsilane in TFA furnished C-3 alkylated derivatives 15a-b.

  Mp 161-163 °C; MS (APCI, pos. 30 V) m/z: [M+H] + , 300.14; 1 H NMR (CDCl3):  1.91 (s, 3H), 3.73 (s, 3H), 3.88 (s, 3H), 6.92 (d, 1H, J = 2.0), 7.21 (dd, 1H, J = 2.0, J = 8.9), 7.32(m, 1H), 7.44 (m, 1H), 7.71 (d, 1H, J = 11.0), 7.80 (d, 1H, J = 8.9), 7.87 (m, 1H), 10.83 (d, 1H, J = 11.0);13 C NMR (CDCl3):  23.

  Mp 164-166 °C. MS (APCI, pos. 30 V) m/z: [M+H] + , 232.14; 1 H NMR (DMSO-d6):  3.20 (m, 1H), 3.41 (m, 1H), 3.69-3.72 (m, 2H), 3.92-4.02 (m, 4H), 5.31 (br s, 1H), 7.22 (dd, 1H, J = 2.0, J = 9.1), 7.33 (m, 1H), 7.46 (m, 1H), 7.51 (d, 1H, J = 2.0), 7.68 (m, 1H), 7.88 (d, 1H, J = 9.1), 7.99 (br s, 3H); IR (neat, cm -1 ): 3330-2800; Anal. (C14H18ClNO2) C, H, N.

  (dd, 1H, J = 2.0, J = 9.1),7.32 (m, 1H), 7.46 (m, 1H), 7.62 (d, 1H, J = 2.0),7.71 (m, 1H), 7.83 (d, 1H, J = 9.1), 7.96 (br s, 1H); IR (neat, cm -1 ): 3280 (NH), 3199 (OH), 1645 (C=O)To a heated solution of 4 (2.73 g, 10 mmol) in glacial acetic acid (40 mL) at 70 °C, was added dropwise dibromine (0.55 mL,11 mmol). The reaction mixture was stirred for 4 h at this temperature then poured into water (80 mL) and extracted twice with 100 mL of diethyl ether. The organic phase was washed with NaHCO3 (1 M) and water, dried over MgSO4, filtered and evaporated under reduced pressure to furnish the desired product as a white solid (60% yield), after recrystallization from toluene. Mp 150-152 °C; 1 H NMR (DMSO-d6):  1.76 (s, 3H), 1.96 (s, 3H),3.28 (m, 1H), 3.50 (m, 1H), 3.88-4.10 (m, 4H), 4.40-4.43 (m, 2H), 7.24 (dd, 1H, J = 2.0, J = 9.1), 7.53 (d, 1H, J = 2.0), 7.69 (s, 1H), 7.83 (d, 1H, J = 9.1), 8.04 (s, 1H), 8.12 (br s, 1H); IR (neat, cm -1 ): 3395 (NH), 1718 (C=O), 1673 (C=O); Anal. (C18H20BrNO4) C, H, N.

  Recrystallized from acetonitrile as a white solid (15% yield); Mp 108-110 °C; MS (APCI, pos. 30 V) m/z: [M+H] + , 410.25; 1 H NMR (DMSO-d6):  0.72-0.78 (m, 8H), 1.46-1.54 (m, 2H), 1.78 (s, 3H), 3.30(m, 1H), 3.50 (m, 1H), 3.95 (s, 3H), 3.99 (m, 1H), 4.35 (m, 1H), 4.46 (m, 1H), 7.16 (dd, 1H, J = 9.0, J = 2.0),7.33 (m, 1H), 7.44 (m, 1H),N.

13 C

 13 Recrystallized from toluene as a white solid (72% yield); Mp 116-118 °C; MS (APCI, pos. 30 V) m/z: [M+H] + , 357.19; 1 H NMR (DMSO-d6):  0.20-0.25 (m, 2H), 0.45-0.49 (m, 2H), 0.91-0.96 (m, 3H), 1.05 (m, 1H), 2.61 (d, 2H, J = 6.6), 2.97-3.00 (m, 2H), 3.38-3.42 (m, 2H), 3.66-3.72 (m, 3H), 3.91 (s, 3H), 4.82 (br s, 1H), 5.85 (br s, 2H), 7.13 (dd, 1H, J = 9.0, J = 2.4), 7.33 (m, 1H), 7.53 (d, 1H, J = 2.4), 7.56 (m, 1H), 7.75 (d, 1H, J = 9.0); NMR (DMSO-d6):  5.1 (2C), 12.

  -1 ): 3368-3291 (NH), 3074-2821 (OH), 1653 (C=O); Anal. (C21H28N2O3) C, H, N. Recrystallized from toluene-cyclohexane (1:1) as a white solid (30% yield); Mp 92-94 °C; MS (APCI, pos. 30 V) m/z: [M+H] + , 371.22; 1 H NMR (DMSO-d6):  0.22-0.25 (m, 2H), 0.45-0.50 (m, 2H), 0.78-0.83 (m, 3H), 1.05 (m, 1H), 1.31-1.34 (m, 2H), 2.62 (d, 2H, J = 6.6), 2.91-2.95 (m, 2H), 3.42-3.45 (m, 2H), 3.66-3.69 (m, 3H), 3.91 (s, 3H), 4.82 (br s, 1H), 5.80 (br s, 1H), 5.90 (br s, 1H), 7.13 (dd, 1H, J = 9.0, J = 2.4), 7.34 (d, 1H, J = 1.6), 7.52 (d, 1H, J = 2.4), 7.56 (d, 1H, J = 1.6), 7.76 (d, 1H, J = 9.0); 13 C NMR (DMSO-d6):  5.0 (2C),

  0; IR (neat, cm -1 ): 3258 (NH), 2959-2849 (OH), 1633 (C=O); Anal. (C19H25NO3) C,H, N. Pharmacology: Reagents and chemicals. 2-[ 125 I]Iodomelatonin (2200 Ci/mmol) was purchased from NEN (Boston, MA). Other drugs and chemicals were purchased from Sigma-Aldrich (Saint Quentin, France) and used without further purification.Assays for MT1 and MT2 receptor subtypes. 2-[ 125 I]Iodomelatonin competition binding assay conditions were essentially as previously described[START_REF] Audinot | Naunyn-Schmiedeberg's[END_REF]. Briefly, binding was initiated by addition of membrane preparations from transfected CHO cells stably expressing the human melatonin MT1 or MT2 diluted in binding buffer (50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl2) to 2-[ 125 I]iodomelatonin (20 pM for MT1 and MT2 receptors expressed in CHO cells) and the tested drug. Non-specific binding was defined in the presence of 1 µM of melatonin. After 120 min incubation at 37 °C, reaction was stopped by rapid filtration through GF/B filters presoaked in 0.5% (v/v) polyethylenimine. Filters were washed three times with 1 mL of ice-cold 50 mM Tris-HCl buffer (pH 7.4).Data from the dose-response curves (seven concentrations in duplicate) were analysed using the program PRISM (Graph Pad Software Inc., San Diego, CA) to yield IC50 (inhibitory concentration 50). Affinities are expressed as pKi (pKi = -Log10 (Ki)) with Ki = IC50/1 + ([L]/KD), where[L] is the concentration of radioligand used in the assay and KD, the dissociation constant of the radioligand characterizing the membrane preparation.

Table 2 .

 2 Binding affinities of compounds 9-16b

					OH
					NHCXR 2
				O
					R 3
	Compd.	R2	R3	X	Ki (nM) MT1 [I95] (n)	Ki (nM) MT2 [I95] (n)	Ki (nM) 5-HT2C [I95] (n)
	9	CH3	COc-C3H5	O	116 [53;254] (2)	81 [61;108] (2)	> 10000 (2)
	12a	CH3	CH2c-C3H5	O	174 [81;376] (3)	2.2[1.3;3.6] (2)	220 [100;500](2)
	12b	C2H5	CH2c-C3H5	O	100 [46;215] (3)	1.8 [1.4;2.3](2)	670 [340;1300] (2)
	12c	c-C3H5	CH2c-C3H5	O	217 [173;273] (2)	8.1 [4.6;14] (2)	1060 [1060;1060](2)
	12d	c-C4H7	CH2c-C3H5	O	1040 [724;1490](2)	11.4 [6.3;20.7](3)	990 [480;2020] (2)
	12e	CH2F	CH2c-C3H5	O	338 [210;544] (2)	2.2[2.0;2.5](2)	300 [150;620] (3)
	12f	NHC2H5	CH2c-C3H5	O	763 [69;84] (2)	15.9 [13.7;18.3] (2)	790 [460;1370] (3)
	12g	NHC3H7	CH2c-C3H5	O	2360 (1)	25.5 [19.5;33.2] (2)	990 [440;2250] (2)
	12h	NHCH3	CH2c-C3H5	S	867 [45;169] (2)	0.48 [39.6;57.5] (2)	260 [110;610] (3)
	12i	NHC2H5	CH2c-C3H5	S	2800 (1)	70.5 [51.5;96.4] (2)	350 [170;750] (3)
	12j	NHC3H7	CH2c-C3H5	S	4870 (1)	143 [69;298](2)	160 [110;230] (2)
	13a	CH3	CH2c-C4H7	O	199 [189;209] (2)	10.2 [6.1;16.9] (2)	1110 [520;2250](2)
	13b	C2H5	CH2c-C4H7	O	163 [126;211] (2)	0.81 [0.65;1.01] (2)	1000 [670;1480](2)
	13c	CH2F	CH2c-C4H7	O	229 [131;398] (2)	> 10000 (2)	580 [390;870] (2)
	16a	CH3	C2H5	O	13.4 [5.8;31.2] (3)	0.77 [0.47;1.29] (2)	130 [120;150] (2)
	16b	CH3	C3H7	O	59 [31;112](3)	1.1 [0.6;2.0](2)	210 [160;280](2)

Ki (nM)

values are geometric mean values (with 95% confidence limits shown in brackets) of at least n separate experiments performed in duplicate.

Table 3

 3 and revealed the obtention of compounds with different pharmacological profiles. In

	fact, several compounds showed a partial agonist activity profile at MT1 and MT2 receptors
	(compounds 7b, 12c, 12d, 12i, 12j, and 16b) while other compounds (7a, 12a, 12b, 12f, 13a-
	c, and 16a) behaved as partial agonist at MT1 and full agonist at MT2 receptor subtype.

  .1, the organic phase was dried over MgSO4 and evaporated under reduced pressure to give the desired product as a white solid; Recrystallized from toluene (44% yield); Mp 120-122 °C; MS (APCI, pos. 30 V) m/z: [M+H] + , 302.16; 1 H NMR (DMSO-d6):  1.22-1.26(m, 3H), 1.76

	(s, 3H), 2.70-2.73 (m, 2H), 3.39-3.42 (m, 2H), 3.73-3.76 (m, 3H), 3.91 (s, 3H), 4.71 (br s,
	1H), 7.13 (dd, 1H, J = 9.0, J = 2.4), 7.31 (d, 1H, J = 2.0), 7.48 (d, 1H, J = 2.0), 7.54 (d, 1H, J
	= 2.4), 7.74 (d, 1H, J = 9.0), 7.91 (br s, 1H); 13 C NMR (DMSO-d6):  15.

Acknowledgement

The authors would like to gratefully acknowledge the Region Nord Pas de Calais (France), the Ministère de la Jeunesse, de l'Education Nationale et de la Recherche (MJENR) and the Fonds Européens de Développement Régional (FEDER) for funds allowed for the 300 MHz NMR facilities.

= 9.0, J = 2.4), 7.79 (d, 1H, J = 2.0), 7.81 (d, 1H, J = 2.4), 8.08 (d, 1H, J = 9.0), 8.31 (br s, 1H), 8.59 (d, 1H, J = 2.0); IR (neat, cm -1 ): 3365 (NH), 1734 (C=O), 1663 (C=O), 1649 (C=O).

Methyl N-[3-acetylamino-2-(3-ethyl-7-methoxy-naphthalen-1-yl)]propionate (15a).

To a solution of 14a (2.0 g, 5.8 mmol) in 20 mL of trifluoroacetic acid (TFA) was added dropwise triethylsilane (2.4 mL, 15.0 mmol) and the mixture was stirred at room temperature for 12 h. The solvent was evaporated under reduced pressure and the white

Methyl N-[3-acetylamino-2-(3-propyl-7-methoxy-naphthalen-1-yl)]propionate (15b).

Prepared from 14b according to the procedure described for compound 15a; Recrystallized from cyclohexane as a white solid (98% yield); Mp 76-80 °C; MS (APCI, pos. 30 V) m/z:

[M+H] + , 344.20; 1 H NMR (DMSO-d6):  0.89-0.96 (m, 3H), 1.60-1.65 (m, 2H), 1.81 (s, 3H), 2.63-2.68 (m, 2H), 3.50-3.56 (m, 2H), 3.66 (s, 3H), 3.93 (s, 3H), 4.65 (m, 1H), 7.10 (d, 1H, J = 2.0), 7.16 (dd, 1H, J = 9.0, J = 2.4), 7.56 (d, 1H, J = 2.0), 7.70 (d, 1H, J = 2.4), 7.78 (d, 1H, J = 9.0), 8.24 (br s, 1H); IR (neat, cm -1 ): 3393 (NH), 1742 (C=O), 1632 (C=O).

N-[3-Hydroxy-2-(3-ethyl-7-methoxy-naphthalen-1-yl)propyl]acetamide (16a).

Compound 15a (2.0 g, 6.1 mmol) was dissolved in dry THF (200 mL), stirred and cooled at 0 °C. Aluminum lithium hydride (0.5 g, 12.1 mmol) was then added portionwise cautiously and the mixture was allowed to stir at room temperature for 6 h. 2 mL of water were added followed by the addition of 2 mL of a NaOH solution (1N). The mixture was washed with water and


Elodie Landagaray

Mohamed Ettaoussi
email: m.ettaoussi@yahoo.fr

Romain Duroux

Jean A Boutin

Daniel-Henri Caignard

Philippe Delagrange

Patricia Melnyk

Pascal Berthelot

Saïd Yous
email: said.yous@univ-lille2.fr

R = Ch

Nhcoch

O Ch

Ch

N H Nhcoch

Melatoninergic Ligands : Design, Synthesis and Pharmacological Evaluation of Novel Series of Naphthofuranic Derivatives

Keywords: Melatonin, Naphthofuranic derivatives, MT 1, MT 2, Partial agonist O

Following our research for new melatoninergic ligands, herein we report the design, synthesis and biological evaluation of new series of naphthofuranic derivatives as MT 1 and MT 2 ligands. Binding affinity results of the prepared compounds revealed good binding affinities at both MT 1 and MT 2 receptors. Particularly, compound 6a behaves as MT 1 partial agonist and MT 2 full agonist and exhibits an excellent binding affinity at MT 2 (Ki = 0.09 nM). Moreover, lateral chain displacement from position 1 to 2 of the furane core has no effect on the binding affinity at MT 1 and MT 2 , while elongation of this side chain, lead to decreased melatoninergic binding affinities.

Introduction

The neurohormone melatonin (Figure 1) was discovered and characterized as N-acetyl-5methoxytryptamine more than fifty years ago by Aaron Lerner. 1 Its biosynthetic route was then established starting from the L-tryptophan acid following a circadian rhythm with high plasmatic concentration at night and low circulating levels during the day time. Melatonin is mainly secreted in humans by the pineal gland located in the hypothalamic suprachiasmatic nucleus (SCN), 2 but it is also synthesized by other regions of the CNS and by other tissues and cells such as the retina, 3 skin, bone marrow, lymphocytes, 4 and gastrointestinal tract. 5 Melatonin seems to play a major role in various physiological processes including, modulation of hormones secretion, 6 regulation of sleep-wake cycle 7 and cardiovascular functions, 8 pain perception, 9 immune system and core body temperature control. 10 Furthermore, melatonin was shown to be involved in several pathological processes such as sleep disturbances and insomnia, 11 cancer and inflammation, 12 neurodegenerative diseases, 13 diabetes, 14 depression and anxiety. 15 The endogenous role and mechanism of action of melatonin have not yet been fully elucidated.

The therapeutic potential of melatonin and most of its physiological effects are mainly mediated via activation of two of its receptors belonging to the superfamily of G-proteincoupled receptors (GPCRs) and named MT 1 and MT 2 . Both MT 1 and MT 2 have been cloned revealing that these two receptors are coupled to G αi proteins (Morgan et al., 1994). 16 These receptors exhibit a sub-nanomolar binding affinity for melatonin. 17 A third binding site of melatonin named MT 3 was characterized as the human quinone reductase 2 and displays a low binding affinity for melatonin. 18 Later it was shown that these three melatoninergic binding sites were localized in different compartments of the human body. 19 In the central nervous system in SCN, cortex, pars tuberalis, 20 and peripherally in kidney, adipocytes, retina, blood vessels, gut, testes, bone marrow cells and human lymphocytes among others. 21 In order to provide a better understanding of the melatoninergic system and its physiological roles, during the last decades, much of the research was focused on the discovery of new analogs of melatonin. Moreover, the characterization of melatonin receptor-mediated functions requires potent and selective ligands for MT 1 and MT 2 . Nowadays, a lot of work has been done in this area, 22 however despite the large number of high affinity non-selective ligands, pronounced receptor subtype selective ligands especially for MT 1 remain a real challenge. Accordingly, after more than fifty years of research only few melatoninergic compounds are marketed or under clinical trials. Hence, circadin, ramelteon, agomelatine and tasimelteon constitute the only melatonin analogs that are commercialized up to now (Figure 1). 23 For decades, our lab was involved in a large research program that consists in the pharmacomodulation of melatonin by replacing the indole scaffold by different bioisostere nucleus such as benzofurane, benzothiophene, isoquinoline, phthalazine, and naphthalene among others. In parallel, other modulations targeting different positions of the aromatic nucleus, the lateral acetamide chain and the methoxy group were performed. 24,25 The substitution of the indole of melatonin with naphthalene and benzofurane nucleus was of major interest. First, agomelatine, the naphthalenic analogue of melatonin has showed a very good binding affinity at both MT 1 and MT 2 . Furthermore, this ligand is the only of the bioisosteric analogues of melatonin owning a serotonergic 5-HT 2C binding affinity. Secondly, the benzofuran analogue of melatonin, the N-acetyl-2-(5-methoxybenzo [b]furan -3-yl) ethylamine (Chart 1), is a good ligand for the melatonin receptors and is metabolically more stable than melatonin. 26 In order to study the effect of the fusion of these two scaffolds on both the melatoninergic affinity and activity, herein, we report the synthesis and pharmacological evaluation of a new series of naphthofuranic derivatives as new MT 1 /MT 2 ligands. The new obtained naphthofuranic derivatives were then submitted to a series of additional modulations applied to the lateral chain and the acetamide function (Chart 1). We report hereafter the synthetic strategies and biological results of the prepared compounds.

Results and discussion

2.1. Chemistry. The synthetic strategy applied in order to prepare key amines 3 and 6 is shown in scheme 1 starting from 7-methoxy-beta-naphthol. Hence, two main routes were performed starting from the acid 1 issued from the condensation of 7-methoxynaphthol with ethyl 4-chloro-3-oxobutanoate in sulfuric acid according to the Pechmann procedure [START_REF] Pechmann | [END_REF] followed by the transformation of the resulted chromenone into naphthofuranic derivative 1 by treatment with sodium hydroxide. 28a Route 1 consists of azide 2 preparation from 1 by treatment with 1-ethyl-3-(3-dimethylaminopropyl) In Scheme 2 is outlined the synthetic sequence used for the preparation of amines 10 and 15.

First, formylation of 2,7-dimethoxynaphthalene according to Vilsmeier-Haack conditions furnished aldehyde 7. 29 Selective demethylation via aluminum chloride treatment of the 2methoxy resulted into naphthol 7. O-alkylation of this later compound with the appropriate alkyl bromide in the presence of potassium carbonate led to derivatives 8 and 11. Submission of 8 and 11 to a base-catalysed cyclisation reaction led to naphthofuranic derivatives 9 and 12.

Reduction of the ester 12 by treatment with lithium aluminum hydride provided the alcohol 13. Activation of compound 13 with thionyl chloride in the presence of pyridine followed by nucleophilic substitution with sodium cyanide gave the nitrile 14 in a good yield. Finally, chemical reduction of nitriles 9 and 14, by a mixture of lithium aluminum hydride and aluminum chloride gave the desired amines 10 and 15.

Pharmacology

In this paper, we describe the design and synthesis of a new series of naphthofuranic derivatives as melatonin MT 1 and MT 2 ligands. The new synthesized derivatives are composed of two position isomers, differently substituted at position 1 or 2 of the naphthofuranic nucleus. To determine their binding affinities and functional activities, the synthesized compounds were assayed at human MT 1 and MT 2 melatonin receptors stably transfected in Chinese Hamster Ovarian (CHO) cells, using 2-[ 125 I]iodomelatonin as radioligand. [START_REF] Audinot | Naunyn-Schmiedeberg's[END_REF] In tables 1-2, are shown the chemical structures and obtained biological results.

Results and Discussion.

The binding affinity results of the prepared acetamides 3a and 6a revealed that this type of structure owns a good melatoninergic binding affinity at both MT 1 and MT 2 receptors, and a very good affinity at MT 2 with regard to both melatonin and agomelatine (Table 1). Particularly, compound 6a exhibits an excellent binding affinity and a selectivity of 31 fold to MT 2 receptor subtype than to MT 1 . Modulation of the acetamide group of both 3a and 6a has led to the synthesis of cyclopropanamides (3b, 6b), isopropylamide (3c), and fluoroacetamide (3d) derivatives with lower binding affinity at both MT 1 and MT 2 . Similarly, substitution of the acetamide by an ethylurea (6c) produced the decrease of the melatoninergic binding affinity. Furthermore and in order to fine tune the effect of the lateral chain position on the melatoninergic binding affinity, compounds 10a and 15a, isomers of 3a and 6a, were synthesized and biologically evaluated. From the obtained results (Table 1), we can conclude that the lateral chain displacement from position 1 to 2 in this naphthofuranic series has no effect on the binding affinity at MT 1 and MT 2 . Interestingly, replacement of the acetamide group by bulky amides (10b, 15b) and fluoroacetamide (10c) in this series (isomers 2) has no significant effect on binding affinity in comparison with isomers 1. However, its replacement by ureas and thioureas lead to the decrease of the binding affinity especially at MT 1 producing the appearance of a weak MT 2 selectivity (Table 1). Finally, as an observed general rule herein, the elongation of the side chain, lead to decreased melatoninergic binding affinities, despite its position in position 1 or 2 of the naphthofuranic ring. Additional docking investigations are necessary to study the conformation of these two isomers in the pocket of the binding site.

Table 1. MT1 and MT2 binding affinity data of synthesized analogues of melatonin. and6a-c 10a-i and 15a-c Finally, with regard to the agomelatine profile, the prepared compounds were tested for their binding affinities at serotonin 5-HT 2C receptor subtype and revealed a binding affinity at the micromolar range for most of the compounds (data not shown).

O O NH n CH 3 R X O NH O n X R CH 3 3a-d
O CH 3 O NHCOCH 3 S21767 Compound n X R K i (nM) [ I95] (n) h-MT 1 h-MT 2 Amides Melatonin - - - 0.

Table 2. Intrinsic activity of synthesized compounds

Compound 4 [0.7; 2.5

h-MT 1 h-MT 2 EC 50 (nM) [I95] (n) E max (%) ± ESM (n) EC 50 (nM) [I95] (n) E max (%) ± ESM (n) Agomelatine 1.

Conclusions

In conclusion we report herein the synthesis and binding affinity results of new series of naphthofuran derivatives as melatonin ligands. Besides acetamides showed good binding affinities at both MT 1 and MT 2 receptor subtypes in comparison with the other amides and ureas. Most of the synthesized compounds displayed a weak MT 2 selectivity except 6b and 6c. Furthermore, no noticeable difference in terms of binding affinities was observed between naphthofuran isomers 1 and 2. The mixture was stirred for 2 h at room temperature and concentrated in vacuo. Et 2 O (10 mL)

was added and the mixture was cooled at 0 °C to add an aqueous solution of ammonia 30% (10 mL). 14). To a solution of 13 (4.57 g, 20 mmol) in CH 2 Cl 2 (50 mL) at room temperature were added pyridine (0.49 mL, 6 mmol)

and SOCl 2 (1.45 mL, 20 mmol). The mixture was stirred during 5 hours. Water (20 mL) and DCM (20 mL) were added then, addition of NaCN (4.90 g, 100 mmol) followed by TBABr (16.12 g, 50 mmol). After 16 h of stirring at room temperature, the layers were separated and the aqueous solution was extracted twice with DCM. The combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (petroleum ether/DCM 1/1) to afford 14 (42%) as a yellow solid; mp 84-86 °C ; 169.88, 158.96, 154.11, 143.79, 130.93, 129.55, 126.21, 125.70, 120.40, 119.38, 116.84, 110.52, 103.32, 55.80, 34.51, 23.03 ; IR (υ, cm -1 , KBr) : 3287 (NH), 1644 (CO) ; LC-MS : m/z = 270 (MH + ).

N-((8-Methoxynaphtho[2,1-b]furan-1-yl)methyl)cyclopropanecarboxamide (3b).

Recrystallized from acetonitrile (53%) as a white solid ; mp 250-252 °C ; 173.33, 158.59, 154.18, 143.97, 130.87, 129.49, 126.13, 125.80, 120.47, 119.22, 116.91, 110.60, 103.49, 55.69, 34.63, 13.96, 6.78 (2C) ; IR (υ, cm -1 , KBr) : 3282 (NH), 1632 (CO) ;

LC-MS : m/z = 296 (MH + ). (br s, 1H), 7.99 (s, 1H), 7.94 (d, 1H, 9.0 Hz), 7.77 (d, 1H, 9.0 Hz), 7.60 (d, 1H, 9.0 Hz), 7.44 (d, 1H, 2.4 Hz), 7.15 (dd, 1H, 9.0 Hz and 2.4 Hz), 4.70 (d, 2H, 4.8 Hz), 3.89 (s, 3H), 2.43 (mt, 1H) ; 1.01 (mt, 6H) ; 13 C NMR (300 MHz, DMSO-d 6 ) δ : 176.67, 158.57, 154.06, 143.65, 130.82, 129.58, 126.09, 125.67, 119.51, 116.71, 110.50, 103.52, 103.34, 55.74, 34.32, 34.22, 20. 169.85, 158.50, 153.80, 142.30, 131.03, 129.80, 125.86, 125.63, 120.61, 119.69, 116.22, 110.58, 103.17, 55.79, 39.28, 25.85, 23.07 ;

N-((8-Methoxynaphtho[2,1-b]furan-1-yl)methyl)isobutyramide (3c

IR (υ, cm -1 , KBr) : 3296 (NH), 1628 (CO) ; LC-MS : m/z = 284 (MH + ).

N-(2-(8-Methoxynaphtho[2,1-b]furan-1-yl)ethyl)cyclopropanecarboxamide (6b).

Recrystallized from toluene (83%) as a white solid ; mp 160-161 °C ; 173.18, 158.55, 153.86, 142.39, 131.10, 129.84, 125.96, 125.77, 120.63, 119.82, 116.29, 110.61, 103.20, 55.80, 39.28, 26.00, 14.18, 6.68 (2C) ; IR (υ, cm -1 , KBr) : 3288 (NH), 1634 (CO) ; LC-MS : m/z = 310 (MH + ). 1, 158.3, 152.9, 152.8, 130.3, 128.9, 125.3, 125.0, 122.7, 116.5, 109.6, 103.4, 102.6, 55.4, 37.2, 23.2 9, 154.8, 152.8, 131.0, 129.4, 125.7, 125.4, 123.5, 117.3, 110.4, (d, 2H, 5.7 Hz), 3.93 in PBS containing EDTA 2 mM and centrifuged at 1000 x g for 5 min (4 °C). The resulting pellet was suspended in TRIS 5 mM (pH 7.5), containing EDTA 2 mM and homogenized using a Kinematica polytron. The homogenate was then centrifuged (95 000g, 30 min, 4 °C) and the resulting pellet suspended in 75 mM TRIS (pH 7.5), 12.5 mM MgCl 2 and 2 mM EDTA. Aliquots of membrane preparations were stored at -80 °C until use.

N-((8-Methoxynaphtho[2,1-b]furan-2-yl)methyl)acetamide (10a

5.2.3. Binding Assays. 2-[ 125 I]iodomelatonin binding assay conditions were essentially as previously described. 30 Briefly, binding was initiated by addition of membrane preparations from stable transfected HEK or CHO cells diluted in binding buffer (50 mM Tris-HCl buffer, [ 35 S] GTPS binding assay was performed according to published methodology. 30 Briefly, membranes from transfected CHO cells expressing MT 1 or MT 2 receptor subtype and compounds were diluted in binding buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 3 µM GDP, 3 mM MgCl 2, and 20 µg/mL saponin). Incubation was started by the addition of 0.2 nM [ 35 S]GTPS to membranes (20 µg/ml) and drugs, and further followed for 1 h at room temperature. For experiments with antagonists, membranes were pre-incubated with both the melatonin (3 nM) and the antagonist for 30 min prior the addition of [ 35 S]GTPS. Non specific binding was defined using cold GTPS (10 µM). Reaction was stopped by rapid filtration through GF/B filters followed by three successive washes with ice cold buffer.

Scheme 1 .

 1 Scheme 1. Synthesis of amines 3 and 6.

Scheme 2 .

 2 Scheme 2. Synthesis of compounds 10 and 15.

( 1 .

 1 Melting points were determined on a Buchi SMP-20 capillary apparatus and are uncorrected. FT-IR spectra were recorded on a Thermo Nicolet Avatar 320 FT-IR spectrometer.1 H and 13 C NMR spectra were recorded on an AMX 300 Bruker or DPX 300 Advance spectrometer. Chemical shifts are reported in ppm (parts per million) relative to that of (CH 3 ) 4 Si and J values are reported in hertz (Hz). Reactions and resulted products were monitored via TLC on Merck precoated silica gel 60 F-254. LC-MS spectra were performed on a LC-Surveyor MSQ spectrometer and were recorded in the APCI positive mode (Toho Bioscience column, TSK gel Super ODS, 4.6 mm DI x 5.0 cm) eluted in a gradient of H ) in dichloromethane (DCM) (20 mL) and some drops of DMF were added sodium azide (0.22 g, 4.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (0.88 g, 4.4 mmol). The mixture was stirred for 3 h at room temperature and washed with a saturated aqueous solution of NaHCO 3 and water. The organic layer was dried over MgSO 4 , filtered and evaporated under vacuo. The resulting solid was recrystallized from acetonitrile to give 2 (52%) as a yellow solid; mp 114-116 °C ; 1 H NMR (300 MHz, CDCl 3 ) δ : 7.87(d, 1H, 9.0 Hz), 7.72 (s, 1H), 7.68(d, 1H, 8.7 Hz), 7.58 (d, 1H, 2.4 Hz), 7.51 (d, 1H, 8.7 Hz), 7.16 (dd, 1H, 9.0 Hz and 2.4 Hz), 4.07 (s, 2H), 4.00 (s, 3H) ; IR (, cm -1 , KBr) : 2131 (N 3 ), 1703 (CO) ; LC-MS : m/z = 282 (MH + ). 44 g, 5 mmol) in toluene (25 mL) was refluxed for 1h. Then, 12M solution of HCl(10 mL) was added and the mixture was refluxed for an additional 30 min. The solvent was evaporated and the resulting solid was recrystallized from acetonitrile to afford 3 (80%) as a white solid; mp 246-248 °C ; 1 H NMR (300 MHz, DMSO-d 6 ) δ : 8.73 (br s, 3H), 8.13 (s, 1H), 8.00(d, 1H, 8.7 Hz), 7.82 (d, 1H, 9.0 Hz), 7.64(d, 1H, 8.7 Hz), 7.46 (d, 1H, 2.4 Hz), 7.22 (dd, 1H, 9.0 Hz and 2.4 Hz), 4.63 (m, 2H), 3.99 (s, 3H) ; IR (υ, cm -1 , KBr) : 3427-2605 (NH 3 + Cl -) ; LC-MS : To a solution of 1 (1.05 g, 4 mmol) in DCM (40 mL) and some drops of DMF at 0 °C was added oxalyl chloride (0.62 mL, 6 mmol).

  To a solution of 12 (9.46 g, 35 mmol) in THF (150 mL) was added LiAlH 4 (2.7 g, 70 mmol) at 0 °C. The mixture was stirred at room temperature during 3 h and hydrolyzed by addition of an aqueous solution of sodium hydroxide 10%. The resulting mineral solid was filtered and washed with THF. The filtrate was concentrated under reduce pressure and partitioned between CH 2 Cl 2 and waterThe organic layer was dried over MgSO 4 , filtered and concentrated under reduced pressure to afford 13 (79%) as a white solid ; mp 112-114 °C ; 1 H NMR (300 MHz, CDCl 3 ) δ : 7.85(d, 1H, 9.0 Hz), 7.66(d, 1H, 9.0 Hz), 7.50 (d, 1H, 9.0 Hz), 7.40 (d, 1H, 2.7 Hz), 7.15 (dd, 1H, 9.0 Hz and 2.7 Hz), 7.14 (s, 1H), 4.87 (s, 2H), 3.99 (s, 3H), 1.93 (br s, 1H) ; IR (υ, cm -1 , KBr) : 3261 (OH) ; LC-MS : m/z = 229 (MH + ).

  31 g, 8 mmol) in Et 2 O (20 mL) at 0 °C was added a solution of AlCl 3(1.06 g, 8 mmol) in ether (20 mL). After 5 minutes of stirring, 14 (0.79 g, 4 mmol) in DCM(10 mL) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature during 1 h. An aqueous solution of sodium hydroxide 10% was added carefully and the formed mineral was filtered and washed with ether. The filtrate was washed with water and dried over MgSO 4 , filtered and concentrated under reduced pressure to afford 15 (72%) as a yellow solid ; mp 64-66 °C ; 1 H NMR (300 MHz, CDCl 3 ) δ : 7.82(d, 1H, 9.0 Hz), 7.60(d, 1H, 9.0 Hz), 7.46(d, 1H, 9.0 Hz), 7.37(d, 1H, 2.4 Hz), 7.12 (dd, 1H, 9.0 Hz and 2.4 Hz), 6.93 (s, 1H), 3.98 (s, 3H), 3.16 (mt, 2H), 3.02 (mt, 2H), 1.69 (br s, 2H) ; IR (υ, cm -1 , KBr) : 3358 (NH 2 ); LC-MS :m/z = 242 (MH + ).General procedure for synthesis of amides 3a-c, 6a-b, 10a-b and 15a-b. To a solution of corresponding amine 3, 6, 10 or 15(1 mmol) in EtOAc(30 mL) and water (10 mL) were added K 2 CO 3(3 mmol) and the corresponding acid chloride (1.5 mmol) at 0 °C. The mixture was stirred at room temperature during 2 h and the layers were separated. The organic one was washed with a 1M solution of HCl and water, dried over MgSO 4 , filtered and concentrated under reduced pressure.

  pH 7.4 containing 5 mM MgCl 2 ) to 2-[ 125 I]-iodomelatonin (25 or 200 pM for MT 1 and MT 2 receptors, respectively, expressed in HEK cells or 20 pM for MT 1 and MT 2 receptors expressed in CHO cells) and the tested drug. Nonspecific binding was defined in the presence of 1 µM melatonin. After 120 min incubation at 37 °C, reaction was stopped by rapid filtration through GF/B filters presoaked in 0.5% (v/v) polyethylenimine. Filters were washed three times with 1 ml of ice-cold 50 mM Tris-HCl buffer, pH 7.4.Data from the dose-response curves (7 concentrations in duplicate) were analysed using the program PRISM (Graph Pad Software Inc., San Diego, CA) to yield IC 50 (inhibitory concentration 50). Results are expressed as K i = IC 50 / 1 + ([L]/K D ), where[L] is the concentration of radioligand used in the assay and K D , the dissociation constant of the radioligand characterising the membrane preparation.33

  Usual levels of [ 35 S]GTPγs binding (expressed in dpm) were for CHO-MT 1 or MT 2 membranes: 2000 for basal activity, 8000 in the presence of melatonin 1 µM and 180 in the presence of GTPγS 10 µM which defined the non specific binding. Data from the doseresponse curves (7 concentrations in duplicate) were analyzed by using the program PRISM (Graph Pad Software Inc., San Diego, CA) to yield EC 50 (Effective concentration 50 %) and E max (maximal effect) for agonists. Antagonist potencies are expressed as K B = IC 50 / 1 + ([Ago]/EC 50 ago), where IC 50 is the inhibitory concentration of antagonist that gives 50% inhibition of [ 35 S] GTPS binding in the presence of a fixed concentration of melatonin ([Ago]) and EC 50 ago is the EC 50 of the molecule when tested alone. I max (maximal inhibitory effect) was expressed as a percentage of that observed with melatonin at 3 nM for MT 2 receptor.

-d, 6a-c, 10a-i and 15a-c was

	3 , DCM, rt, 86% ; c)
	K 2 CO 3 , BrCH 2 A, acetone, rt, 100% for 8 and 97% for 11 ; d) K 2 CO 3 , DMF, 60 °C, 75% for 9
	and 70% for 12 ; e) LiAlH 4 , THF, rt, 79% ; f) (i) SOCl 2 , pyridine, DCM, rt ; (ii) NaCN,
	TBABr, DCM/H 2 O, rt, 42% ; g) LiAlH 4 , AlCl 3 , Et 2 O/DCM, 0 °C to rt, 90% for 10 and 72%
	for 15.
	Synthesis of the desired final amides, ureas, and thioureas 3acarried out as illustrated in Scheme 3. Hence, N-acylated compounds

3a-c, 6a-b, 10a-b and 15a-b were

	3d and 10c were prepared by reaction of the corresponding free amine with ethyl
	fluoroacetate in 2,2,2-trifluoroethanol. Urea 10d and thiourea 10g were obtained from 10 by
	treatment with potassium cyanate and potassium thiocyanate respectivly. Otherwise, the
	reaction of 10 with N-methylphenylcarbamate, synthesized as previously described in the
	literature, 31 furnished methyl urea 10e. Finally, alkylureas (6c, 10f, 15c) and alkythioureas
	(10h, 10i) were prepared from the corresponding amines by treatment with alkyl isocyanate or
	alkylisothiocyanate in dry dichloromethane.
	Scheme 3. Synthesis of amides, ureas and thioureas.
	O CH 3	1	O NH 2 n 2		a,b,c,d or e	O CH 3	1	H N n O 2	X R
	Compound Isomer n	X	R	Conditions	Yield (%)
	3a		1	1	O	CH 3	a		68
	3b		1	1	O	c-C 3 H 5	a		53
	3c		1	1	O	i-C 3 H 7	a		55
	3d		1	1	O	CH 2 F	b		40
	6a		1	2	O	CH 3	a		72
	6b		1	2	O	c-C 3 H 5	a		83
	6c		1	2	O	NHC 2 H 5	e		84
	10a		2	1	O	CH 3	a		79
	10b		2	1	O	c-C 3 H 5	a		80
	10c		2	1	O	CH 2 F	b		45
	10d, 10g		2	1 O, S	H	c		62
	10e		2	1	O	NHCH 3	d		40
	10f		2	1	O	NHC 2 H 5	e		65
	10h		2	1	S	NHCH 3	e		63
	10i		2	1	S	NHC 2 H 5	e		72
	15a		2	2	O	CH 3	a		74
	15b		2	2	O	c-C 3 H 5	a		53
	15c		2	2	O	NHC 2 H 5	e		54
	obtained from the corresponding amines by reaction with the appropriate acid
	chlorides according to a variant of the Schotten-Baumann procedure. 30 Fluorinated derivatives

Reagents: a) RCOCl, K 2 CO 3

, EtOAc/H 2 O, 0 °C to rt ; b) FCH 2 COOEt, CF 3 CH 2 OH, reflux ; c) KNCO, H 2 O/HCl, rt for 10d and KNCS, dioxane/THF, 60 °C for 10g ; d) N-methylphenylcarbamate, DMSO, 60 °C ; e) R 1 NCX, DCM, rt.

  The intrinsic activity evaluation results' are shown in Table2and reveal that most of the synthesized derivatives exhibit a partial agonist activity. Besides, acetamide 3a acts a partial agonist at both MT 1 and MT 2 with EC 50 of 4.8 and 1.4 nM respectively. More interestingly, the acetamide 6a behaves as MT 1 partial agonist and MT 2 full agonist with EC 50 of 4.6 and 0.7 nM respectively, hence representing one of the most interesting compounds of this series.

							23 [ 0.21; 0.26 ](136) 0.52 [0.4; 0.58 ] (136)
			Agomelatine -	-	-	0.12 [ 0.12; 0.12 ](2)	0.21 [0.08; 0.57 ] (2)
			S21767	-	-	-	0.15 [0.15] (1)	0.34 [0.34] (1)
			3a	1 O	CH 3	4.6 [2.2; 9.7] (2)	0.7 [0.5; 1.1] (2)
			6a	2 O	CH 3	2.8 [2.1; 3.8] (2)	0.09 [0.04; 0.2] (2)
			10a	1 O	CH 3	2.1 [1.9; 2.3] (2)	0.4 [0.4; 0.4] (2)
			15a	2 O	CH 3	10 [9; 10] (2)	1.7 [1.0; 2.6] (2)
			3b	1 O c-C 3 H 5	20 [16; 24] (2)	15 [14; 17] (2)
			6b	2 O c-C 3 H 5	23.6 [107; 522] (2)	13.3 [7.5; 23.2] (2)
			10b	1 O c-C 3 H 5	1.9 [1.7; 2.3] (2)	0.6 [0.5; 0.7] (2)
			15b	2 O c-C 3 H 5	73 [67; 79] (2)	11 [11; 11] (2)
			3c	1 O i-C 3 H 7	158 [116; 217] (2)	24 [11; 57] (2)
			3d	1 O	CH 2 F	9.9 [6.1; 16] (3)	2.1 [0.9; 5.2] (2)
			10c	1 O	CH 2 F	5.7 [5.6; 5.7] (2)	0.7 [0.5; 1.0] (2)
			10d	1 O	NH 2	nd	nd
	Ureas	10e 6c 10f	1 O NHCH 3 2 O NHC 2 H 5 1 O NHC 2 H 5	nd 157 [99; 249] (2) 17 [16; 18 ] (2)	nd 226 [104; 493] (3) 4.2 [4.2; 4.2] (2)
			15c	2 O NHC 2 H 5	210 [191; 230] (2)	46 [39; 54] (2)
	Thio	ureas	10g 10h 10i	1 S 1 S NHCH 3 NH 2 1 S NHC 2 H 5	nd 43 [30; 48] (2) 140 [120; 155] (2)	nd 0.8 [0.5; 1.1] (2) 7 [4; 11] (2)

  The mixture was extracted with EtOAc, washed with water, dried over MgSO 4 and 0.97 mL, 7.2 mmol). The mixture was stirred at 0 °C for 1h and at room temperature for an additional hour, evaporated in vacuo and poured into water. The mixture was acidified with a 3M solution of HCl, and extracted with EtOAc. The organic After concentration of the filtrate, the residue was partitioned between CH 2 Cl 2 and water. The organic layer was dried over MgSO 4 , filtered, concentrated under reduced pressure and recrystallized from methanol to give 11 (97%) as a white solid ; mp 115-117 °C ; 1 H NMR

	MgSO 4 , filtered and concentrated under reduced pressure to afford 8 (100%) as a white solid ;
	mp 136-138 °C ; 1 H NMR (300 MHz, CDCl 3 ) δ : 10.84 (s, 1H), 8.76 (d, 1H, 2.4 Hz), 8.01 (d,
	1H, 9.0 Hz), 7.68 (d, 1H, 9.0 Hz), 7.13 (dd, 1H, 9.0 Hz and 2.4 Hz), 7.09 (d, 1H, 9.0 Hz), 4.99
	layer was washed with an aqueous solution of K 2 CO 3 and water, dried over MgSO 4 and (s, 2H), 3.97 (s, 3H) ; IR (υ, cm -1 , KBr) : 2133 (CN), 1670 (CO) ; LC-MS : m/z = 242 (MH + ). (300 MHz, CDCl 3 ) δ : 11.00 (s, 1H), 8.86 (d, 1H, 2.4 Hz), 7.97 (d, 1H, 9.0 Hz), 7.68 (d, 1H,
	concentrated under reduced pressure. The resulting solid was recrystallized from toluene to 8-Methoxynaphtho[2,1-b]furan-2-carbonitrile (9). To a solution of 8 (12.06 g, 50 mmol) in 9,0 Hz), 7.10 (dd, 1H, 2.4 Hz and 9.0 Hz), 6.97 (d, 1H, 9.0 Hz), 4.87 (s, 2H), 4.30 (q, 2H,
	afford 5 (92%) as a yellow solid ; mp 163-164 °C ; 1 H NMR (300 MHz, DMSO-d 6 ) δ : 8.09 dimetylformamide (150 mL) was added potassium carbonate (27.64 g, 200 mmol). The 7.2 Hz), 3.99 (s, 3H), 1.31 (t, 3H, 7.2 Hz) ; IR (υ, cm -1 , KBr) : 1773 (COOEt), 1658 (CHO) ;
	(s, 1H), 8.00 (d, 1H, 9.0 Hz), 7.83 (d, 1H, 8.7 Hz), 7.61-7.66 (m, 2H), 7.22 (dd, 1H, 9.0 Hz mixture was stirred for 16 h at 60 °C and poured into cold water. The resulting solid was dried LC-MS : m/z = 289 (MH + ).
	and 2.4 Hz), 4.57 (s, 2H), 3.99 (3H, s) ; IR (υ, cm -1 , KBr) : 2237 (CN) ; LC-MS : m/z = 238 and recrystallized from toluene to afford 9 (75%) as a white solid ; mp 151-153 °C ; 1 H NMR Ethyl 8-methoxynaphtho[2,1-b]furan-2-carboxylate (12). To a solution of 11 (14.41 g, 50
	(MH + ). (300 MHz, CDCl 3 ) δ : 7.89 (s, 1H), 7.87 (d, 1H, 9.0 Hz), 7.85 (d, 1H, 9.0 Hz), 7.51 (d, 1H, mmol) in dimetylformamide (150 mL) was added potassium carbonate (27.64 g, 200 mmol).
	9.0 Hz), 7.42 (d, 1H, 2.4 Hz), 7.24 (dd, 1H, 9.0 Hz et 2.4 Hz), 4.01 (s, 3H) ; IR (υ, cm -1 , KBr)
	: 2219 (CN) ; LC-MS : m/z = 224 (MH + ).
	95 (d, 1H, 8.7 Hz), 7.84 (s, 1H), 7.73 (d, 1H,
	9.0 Hz), 7.62 (d, 1H, 2.4 Hz), 7.56 (d, 1H, 9.0 Hz), 7.16 (dd, 1H, 8.7 Hz and 2.4 Hz), 5.75
	evaporated in vacuo. The resulting solid was recrystallized from acetonitrile to give 4 (78%) (br s, 2H), 3.93 (s, 3H), 3.09 (m, 2H), 2.99 (m, 2H); IR (υ, cm -1 , KBr) : 2941-2835 (NH 2 ) ; 203 °C ; 1 H NMR (300 MHz, CDCl 3 ) δ : 7.82 (d, 1H, 9.0 Hz), 7.61 (d, 1H, 9.0 Hz), 7.47 (1H,
	as a brown solid ; mp 208-210 °C ; 1 H NMR (300 MHz, CDCl 3 ) δ : 7.86 (d, 1H, 9.0 Hz), LC-MS : m/z = 242 (MH + ). d, 9.0 Hz), 7.37 (d, 1H, 2.7 Hz), 7.13 (dd, 1H, 9.0 Hz and 2.7 Hz), 6.98 (s, 1H), 4.06 (s, 2H),
	7.72-7.69 (m, 2H), 7.56 (d, 1H, 2.4 Hz), 7.52 (d, 1H, 8.7 Hz), 7.16 (dd, 1H, (1-Formyl-7-methoxynaphthalen-2-yloxy)acetonitrile (8). To a solution of 2-hydroxy-7-3.97 (s, 3H), 1.68 (br s, 2H); IR (υ, cm -1 , KBr): 2941-2835 (NH 2 );
	9.0 Hz et 2.4 Hz), 5.57 (br s, 2H), 4.00 (s, 2H), 3.97 (s, 3H) ; IR (υ, cm -1 , KBr) : 3386-3177 methoxy-naphthalene-1-carbaldehyde (7) (10.11 g, 50 mmol) in acetone (230 mL) were added LC-MS : m/z = 228 (MH + ).
	(NH 2 ), 1654 (CO) ; LC-MS : m/z = 256 (MH + ). K 2 CO 3 (13.82 g, 100 mmol) and bromoacetonitrile (4.53 mL, 65 mmol). The mixture was Ethyl 2-(1-formyl-7-methoxynaphthalen-2-yloxy)acetate (11). A mixture of 7 (10.11 g,

2-(8-Methoxynaphtho[2,1-b]furan-1-yl)acetonitrile (5).

To a solution of 4

(1.54 g, 6 mmol

) in THF (60 mL) were added carefully at 0 °C triethylamine

(1.69 mL, 12 mmol

) and trifluoroacetic anhydride (2-(8-Methoxynaphtho[2,1-b]furan-1-yl)ethylamine (

6

). To a suspension of LiAlH 4 (0.44 g, 12 mmol) in Et 2 O (20 mL) at 0 °C was added a solution of AlCl 3

(1.59 g, 12 mmol)

in Et 2 O (20 mL). After 5 minutes of stirring, 5

(1.43 g, 6 mmol

) in CH 2 Cl 2 (5 mL) was added dropwise at 0 °C. Then, the resulting mixture was stirred at room temperature during 1 h. An aqueous solution of sodium hydroxide 10% was added carefully and the mineral solid was filtered and washed with Et 2 O. The filtrate was washed with water and dried over MgSO 4 , filtered and concentrated under reduced pressure to afford 6 (74%) as a yellow solid ; mp 88-90 °C ; 1 H NMR (300 MHz, DMSO-d 6 ) δ : 7.stirred at reflux for 5 h. After filtration and evaporation of the filtrate, the residue was partitioned between CH 2 Cl 2 and water. The organic layer was washed with water, dried over

(8-Methoxynaphtho[2,1-b]furan-2-yl)methylamine

(10)

. To a suspension of LiAlH 4

(1.14 g, 30 mmol)

in Et 2 O (15 mL) at 0 °C was added a solution of AlCl 3 (4 g, 30 mmol) in Et 2 O (15 mL). After 5 min of stirring, a solution of 9 (3.35 g, 15 mmol) in DCM (10 mL) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature during 1 h. An aqueous solution of sodium hydroxide 10% was added carefully and the mineral solid was filtered and washed with Et 2 O. The filtrate was washed with water and dried over MgSO 4 , filtered and concentrated under reduced pressure to afford 10 (90%) as a white solid ; mp 201-50 mmol), potassium carbonate (13.82 g, 100 mmol) and ethyl bromoacetate (7.21 mL, 65 mmol) in acetone (230 mL) was refluxed for 5 h. Then, the mixture was filtered. This mixture was stirred for 16 h at 60 °C, then poured into cold water and the resulting solid was filtered, dried and recrystallized from acetonitrile to give 12 (70%) as a white solid ; mp 67-69 °C ; 1 H NMR (300 MHz, CDCl 3 ) δ : 8.00 (s, 1H), 7.87

(d, 1H, 9

.0 Hz), 7.80

(d, 1H, 9

,0 Hz), 7.57

(d, 1H, 9.0 Hz), 7.48 (d, 1H, 2.4 Hz), 7.19 (dd, 1H, 9

.0 Hz and 2.4 Hz), 4.49 (q, 2H, 7.2 Hz), 4.00 (s, 3H),

1.48 (t, 3H, 7.2 Hz)

; IR (υ, cm -1 , KBr) : 1735 (CO) ; LC-MS : m/z = 271 (MH + ).

  (32 mmol). The mixture was refluxed during 12 h then, hydrolyzed and extracted with Et 2 O. The organic layer was washed with a 1M solution of HCl and water, dried over MgSO 4 , filtered and concentrated under reduced pressure.

	2-Fluoro-N-((8-methoxynaphtho[2,1-b]furan-1-yl)methyl)acetamide (3d). Recrystallized
	from cyclohexane (40%) as a white solid ; mp 152-153 °C ; 1 H NMR
	(300 MHz, DMSO-d 6 ) δ : 8.70 (br s, 1H), 8.00 (s, 1H), 7.95 (d, 1H, 9.0 Hz), 7.77 (d, 1H,
	9.0 Hz), 7.60 (d, 1H, 9.0 Hz), 7.48 (d, 1H, 2.6 Hz), 7.12 (dd, 1H, 9.0 Hz and 2.6 Hz), 4.90 (d,
	2H, 37.8 Hz), 4.80 (mt, 2H), 3.89 (s, 3H) ; IR (υ, cm -1 , KBr) : 3439 (NH), 1657 (CO) ;
	LC-MS : m/z = 288 (MH + ).
	2-Fluoro-N-((8-methoxynaphtho[2,1-b]furan-2-yl)methyl)acetamide (10c). Recrystallized
	from acetonitrile (45%) as a white solid ; mp 204-206 °C ; 1 H NMR (300 MHz, CDCl 3 ) δ :
	7.84 (d, 1H, 9.0 Hz), 7.66 (d, 1H, 9.0 Hz), 7.48 (d, 1H, 9.0Hz), 7.38 (d, 1H,
	2.7 Hz), 7.14 (dd, 1H, 9.0 Hz et 2.7 Hz), 7.13 (s, 1H), 6.81 (br s, 1H), 4.89 (d, 2H, 47.1 Hz),
	4.77 (d, 2H, 5.7 Hz), 3.98 (s, 3H) ; 13 C NMR (300 MHz, DMSO-d 6 ) δ : 168.3 (d,
	J C-F 73.8 Hz), 158.
	; IR (υ, cm -1 , KBr) : 3281 (NH), 1625 (CO) ; LC-

MS : m/z = 270 (MH + ). fluoroacetate

N 1 -((8-Methoxynaphtho[2,1-b]furan-2-yl)methyl)urea (10d). To

	104.1,
	104.0, 80.9 (d, J C-F 714.0 Hz), 56.3, 36.6 ; IR (υ, cm -1 , KBr) : 3310 (NH), 1651 (CO) ;
	LC-MS : m/z = 288 (MH + ).
	a solution of 10 (0.45 g,
	2 mmol) in water (10 mL) and 1M solution of HCl (1 mL) was added potassium cyanate (0.2
	g, 2.4 mmol). The mixture was stirred at room temperature during 2 h then, hydrolyzed and
	extracted with EtOAc. The organic layer was washed with water, dried over MgSO 4 , filtered
	and concentrated under reduced pressure. The resulting solid was treated with diethyl ether to
	afford 10d (62%) as a white solid ; mp 227-229 °C ; 1 H NMR (300 MHz, DMSO-d 6 ) δ : 7.89

(d, 1H, 9.0 Hz), 7.68 (d, 1H, 9.0 Hz), 7.63 (d, 1H, 2.4 Hz), 7.54 (d, 1H, 9

.0 Hz), 7.30 (s, 1H), 7.12 (dd, 1H, 9.0 Hz and 2.4 Hz), 6.74 (br s, 1H), 5.73 (br s, 2H),

4,42

Ki (nM) values are geometric mean values (with 95% confidence limits shown in brackets) of at least (n) separate experiments performed in duplicate. nd: not determined.

Acknowledgement

The authors would like to gratefully acknowledge the Region Nord Pas de Calais (France), the Ministère de la Jeunesse, de l'Education Nationale et de la Recherche (MJENR) and the Fonds Européens de Développement Régional (FEDER) for funds allowed for NMR facilities.


Emmanuel Paradis

Gerald Guedon

Roger Pradel

RH: Vole Survival Rates -Paradis et al. ESTIMATION OF SEX-AND AGE-RELATED SURVIVAL RATES IN A MICROTINE POPULATION

Keywords: capture-recapture, individual variations, Mediterranean area, Microtus duodecimcostatus, parsimony, Pine vole, probabilistic models, survival rate

Little is known about the sex-and age-related survival rates of microtine rodents. Thus, we used probabilistic models and recent software developments to estimate survival rates in a Mediterranean pine vole (Microtus duodecimcostatus) population studied by capture-recapture. Goodness-of-fit tests revealed that erratics (voles caught only once) were captured during every trapping-session. Removal of the erratics from the data was necessary for application of the Jolly-Seber model. The model selected according to parsimony criteria, which assumes higher survival rate for juvenile males than juvenile females. Survival rates were higher among adults of both sexes than among juveniles, but there was no influence of sex on adult survival rate.

These variations of survival rates can be related to demographic traits of Mediterranean pine vole (relative abundance in different habitats, local population dynamics). Our data also suggest that the regional abundance of the Mediterranean pine vole is determined by "source-sink dynamics".

In general, management of wild populations requires knowledge about population parameters, particularly survival rate of the individuals [START_REF] Eberhardt | Assessing the dynamics of wild populations[END_REF]. The development of stochastic models of capture-recapture in the 1960's provided an analytical tool to population ecologists for the estimation of survival rates [START_REF] Cormack | Estimates of survival from the sighting of marked animals[END_REF][START_REF] Jolly | Explicit estimates from capture-recapture data with both death and immigration-stochastic models[END_REF][START_REF] Seber | A note on the multiple recapture census[END_REF]. Recent developments have made these methods more flexible, more powerful, and more available to field ecologists [START_REF] Seber | A review of estimating animal abundance[END_REF][START_REF] Clobert | A general approach to survival rate estimation by recaptures or resightings of marked birds[END_REF], Pollock et al. 1990), and allowed differential estimation of survival rates for the individuals of a population relative to individual features [START_REF] Pradel | Recent developments for the analysis of capture-recapture multiple data sets. An example concerning two Blue Tit populations, Parus caeruleus[END_REF][START_REF] Lebreton | Modeling survival and testing biological hypotheses using marked animals: a unified approach with case studies[END_REF]). This improvement is of particular interest for ecologists who face the problem of variability in survivorship within and among populations under study.

Ignoring such variability can lead to wrong conclusions about the dynamics of a population [START_REF] Johnson | The role of heterogeneity in animal population dynamics[END_REF].

Several rodent species are agricultural pests. The Mediterranean pine vole occurs in some cultivated areas of the Mediterranean region where it sometimes causes dramatic agricultural damage. When a population is driven to extinction by snap-trapping or poisoning, recolonization of the area occurs rapidly, and undisturbed populations persist for many years. These observations indicate that migration may be critical in the population dynamics of the Mediterranean pine vole; undisturbed populations seem to function as source populations, and snap-trapping or poisoning artificially create sink populations. In this type of metapopulation, local population dynamics of source populations and dispersal are important in determining the regional abundance of the species (Pulliam 1988, Hanski and[START_REF] Hanski | Metapopulation dynamics: brief history and conceptual domain[END_REF]. Theoretical as well as empirical considerations indicate that dispersal is male-biased in populations of mammalian species such that females are mainly recruited in situ [START_REF] Greenwood | Mating systems, philopatry and dispersal in birds and mammals[END_REF][START_REF] Dobson | Competition for mates and predominant juvenile male dispersal in mammals[END_REF][START_REF] Pusey | Sex-biased dispersal and inbreeding avoidance in birds and mammals[END_REF]. So, we could predict that males in source populations have a lower apparent survival rate than females. Here we test this hypothesis in a population of Mediterranean pine voles by studying variations of apparent survival rates between individuals. We could have attempted to measure dispersal directly, but this is quite difficult, and statistics for the analysis of dispersal patterns are not yet fully developed [START_REF] Tonkyn | Models of multiple dispersers from the nest: predictions and inference[END_REF]. Because estimations of survival rates are motivated by testing biological hypotheses, modeling survival rates and fitting parsimonious models are appropriate analytical tools [START_REF] Lebreton | Modeling survival and testing biological hypotheses using marked animals: a unified approach with case studies[END_REF]. We thank J.-P. Airoldi, J. D. Nichols, and an anymous reviewer for their helpful comments on an early draft of the manuscript, and F. Catzeflis for encouraging us. We are indebted to Mr. Giammatei for allowing us access to his property. We also thank various people for their assistance in the field.

METHODS

The Mediterranean pine vole has a very narrow geographic distribution including Spain, Portugal, and southern France (where we studied a population in an apple orchard [43°39' N, 4°11' E]). Trapping-sessions were conducted every 3 months (Feb, May, Aug, and Nov) during 1989-1990 using Longworth traps baited with apple. Traps were distributed on a 1-ha area, along 6 parallel 5-× 100-m strips divided into 25-m 2 quadrats. Up to 2 trap stations were arranged in each square, and each trap station contained 1, 2, or 3 traps oriented in the axis of a previously dug tunnel. In such habitats, home ranges have an elliptical shape [START_REF] Pagano | Seasonal variations in movements and habitat use by pine and meadow voles[END_REF]Madison 1981, Fitzgerald and[START_REF] Fitzgerald | Social organization of a free-ranging population of pine voles, Microtus pinetorum[END_REF], so we spaced the strips regularly on the area (14 m between the edges of 2 strips) in such a way as to catch most of the animals. Traps were checked every 6 hours over a 42-hour period. When captured, animals were marked by toeclipping and ear-marking, reproductive condition was noted (males: testes scrotal or abdominal; females: pregnant, lactating, and vulva open or closed), and their mass was determined. We distinguished 2 age classes using a body-mass criterion: adults (> 17 g), and juveniles (≤ 17 g).

We used programs RELEASE 2.4 [START_REF] Burnham | Design and analysis methods for fish survival experiments based on releaserecapture[END_REF]) and SURGE 4.1 [START_REF] Pradel | User's manual for program SURGE version 4[END_REF] to perform analysis of the data. RELEASE was used to estimate goodness-of-fit to test the validity of the assumptions made for the application of the Jolly-Seber model. These assumptions are: 1) the probability of capture is the same for all individuals (but may vary with time), 2) the survival rate is the same for all individuals (but may vary with time), 3) emigration is permanent [START_REF] Begon | Abuses of mathematical techniques in ecology: applications of Jolly's capture-recapture method[END_REF]. We incorporated the results of the tests performed by RELEASE into a SURGE model. SURGE fits a probabilistic model chosen by the user to the capture-recapture data (histories of capture). The model computed with SURGE is specified by the user who chooses the variables influencing survival and/or recapture rates. For instance, survival and/or recapture rates can be age-dependent, and/or time-dependent, or constant over age and time, or influenced by a climatic variable, or related to some other environmental feature. The Jolly-Seber model is only a particular case of these models, for which both survival and recapture rates are time-dependent. Several data sets can be analyzed simultaneously, allowing incorporation of individual features (e.g., sex, genotypes, nest location, etc.) in estimation of survival and/or recapture rates. A large class of models can be computed that incorporate many sources of variation (age, time, sex, external factors, interactions between factors, etc). Maximum likelihood estimation of parameters (survival and recapture rates) is performed by program SURGE. Selection of models is the "critical" step of modeling capture-recapture data: we took into account both parsimony criteria and the biological significance of the models [START_REF] Pradel | Recent developments for the analysis of capture-recapture multiple data sets. An example concerning two Blue Tit populations, Parus caeruleus[END_REF][START_REF] Lebreton | Modeling survival and testing biological hypotheses using marked animals: a unified approach with case studies[END_REF]). We compared models by likelihood ratio tests (χ 2 -tests). Mean life-span for animals grown as adults is estimated as follows (provided they have a constant survival rate):

-I/ln φ where: I = time between two occasions of capture, and φ = estimated adult survival rate.

Raw data were structured into 4 data sets: male juveniles, female juveniles, male adults, and female adults (age determined at first capture).We analysed each data set separately or in association with ≥ 1 other data set.

RESULTS

We trapped the population during 8 sessions (Feb 1989-Nov 1990) and caught 1,449 voles. The 4 data sets consisted of: 524 adult males, 531 adult females, 200 juvenile males, and 194 juvenile females. Fifteen animals were killed by the traps (for each data set: 5, 4, 4, and 2, respectively). We incorporated this mortality caused by the trapping into the SURGE analysis.

Goodness-of-fit Tests

Tests performed with male and female adult data sets led to the rejection of the Jolly-Seber model (χ 2 20 = 88.683, P < 0.0005, and χ 2 23 = 70.426, P < 0.0005, respectively).

Detailed analysis of the intermediate tests [START_REF] Burnham | Design and analysis methods for fish survival experiments based on releaserecapture[END_REF] showed that unmarked animals captured and marked at a given time have a lower probability of being recaught subsequently than animals marked before; but if they are re-caught, their subsequent recapture probability is not different from that of previously marked animals. This result led us to remove first captures from both data sets, so that animals caught only once are not taken into account in the corrected data. RELEASE tests performed with the corrected data showed complete homogeneity (χ 2 13 = 16.574, P = 0.220 and χ 2 13 = 13.275, P = 0.427, for males and females, respectively). This result can be interpreted as being due to the capture of erratics (those caught only once), or to an increased mortality due to toe-clipping.

Tests performed with the male juvenile data led to the acceptance of the Jolly-Seber model (χ 2 16 = 17.001, P = 0.386). Data on female juveniles were insufficient to perform chi square-tests. However, application of Fisher's exact tests also led to an acceptance of the Jolly-Seber model. Lack of disappearance of newly caught animals versus those previously caught (as observed for the adults) led us to reject the hypothesis of a lethal effect of the marking technique that was used and to assume that some erratic adults were continuously trapped in our population. To fit a model to the data, we used the corrected data with first captures removed for adults. [START_REF] Boutin | Estimating survival rates of snowshoe hares[END_REF] also addressed this problem of animals with single capture, which substantially decreased the Jolly-Seber estimates of survival rates in their study of snowshoe hares (Lepus americanus). However, they didn't provide goodness-of-fit tests of the Jolly-Seber model to their data.

Age and Sex Effects on Survival and Recapture Probabilities

Although the Jolly-Seber model was accepted for the 4 data sets, we considered the possibility of an age effect in the analyses because it is a biologically reasonable

hypothesis. An age effect can be masked by the time effect. A preliminary survey analysis of the juvenile data sets actually showed a trend of increasing survival with age.

First, we analyzed the data sets of adults separately to detect the influence of absolute age or time on survival rates and recapture rates. For both data sets, the hypothesis that survival and recapture rates were constant over absolute age and time was accepted (χ 2 24 = 21.032, P = 0.6, and χ 2 24 = 21.602, P = 0.6, for males and females, respectively).

"Absolute age" does not refer to the real age of the individuals, but to the time since first capture [START_REF] Clobert | A general approach to survival rate estimation by recaptures or resightings of marked birds[END_REF]; however, a high level of non-significance of the tests evidences a homogeneity of survival and recapture rates within a absolute cohort.

Second, we constructed a model to analyze the 4 age and sex data sets together. The time required for the Mediterranean pine vole to reach adulthood is about 2.5 months [START_REF] Pascal | Recherche de critères d'âge chez le campagnol provençal Pitymys duodecimcostatus (De Sélys-Longchamps, 1839)[END_REF]). We assumed that juveniles become adults during the 3 months between 2 trapping sessions (only 1 animal out of 388 retained juvenile characteristics between 2 periods), so the survival rate changed after the second capture for animals caught as juveniles and was assumed to be the same as for animals caught as adults.

Potential sources of variation for survival and recapture rates were sex, age class, and the interactions between these 2 factors (Table 1). We graphically present the selection process (Fig. 1). The survival rate of adults is greater than that of juveniles (Table 2);

for juveniles, survival rate is greater among males than among females. The recapture rate is not influenced by sex or age class (Table 2). The estimated adult mean life span is 7.75 months.

DISCUSSION

There are 2 main applications for probabilistic models: estimating population sizes and estimating survival rates. The first application in small mammal capture-recapture studies was discussed by [START_REF] Jolly | The problem of unequal catchability in markrecapture estimation of small mammal populations[END_REF], Nichols and[START_REF] Nichols | Estimation methodology in contemporary small mammal capture-recapture studies[END_REF][START_REF] Boonstra | Demography of Microtus pennsylvanicus in southern Ontario: enumeration versus Jolly-Seber estimation compared[END_REF]. Estimation of survival rates in small mammal populations is not yet as widespread. [START_REF] Nichols | Active-seasonal dynamics of a population of Zapus hudsonius in Michigan[END_REF], [START_REF] Boonstra | Demography of Microtus pennsylvanicus in southern Ontario: enumeration versus Jolly-Seber estimation compared[END_REF] and [START_REF] Nichols | Estimation of recruitment from immigration versus in situ reproduction using Pollock's robust design[END_REF] used the Jolly-Seber model; however, significance of time-dependence was not tested in these studies. Anyway, simulation and field studies have demonstrated that Jolly-Seber estimators perform better than estimators based on enumeration methods [START_REF] Nichols | Estimation methodology in contemporary small mammal capture-recapture studies[END_REF][START_REF] Boonstra | Demography of Microtus pennsylvanicus in southern Ontario: enumeration versus Jolly-Seber estimation compared[END_REF][START_REF] Boutin | Estimating survival rates of snowshoe hares[END_REF].

Our study further emphasized the need for flexibility in the analysis of capturerecapture data. The demonstration of a difference in survival between juveniles and adults and between females and males among juveniles was made possible by the use of parsimonious models without time effect, and the simultaneous treatment of different data sets corresponding to different categories of individuals sharing some parameters.

In this context, the biologically relevant question is addressed without interference from background noise (here time effect) and with all the information available. As a consequence, the tests are more powerful.

The results of the goodness-of-fit tests to the Jolly-Seber model with the uncorrected adult data sets (resident individuals with erratics) could have been interpreted within the framework provided by [START_REF] Brownie | Estimation of time-specific survival rates from tag-resighting samples: a generalization of the Jolly-Seber model[END_REF], where individuals have a lower survival after the first observation than after reobservation (it is unimportant whether the first observation differs from reobservations because physical capture takes place only at this time as occurs in Brownie-Robson model, or because toe-clipping occurs at this moment as in our study). Yet the results of the goodness-of-fit tests to the Jolly-Seber model with the uncorrected juvenile data sets led us to reject this assumption, and to infer the presence of erratics in the adult data sets. In this situation, use of the Brownie-Robson model would be incorrect. It is noteworthy that the goodness-of-fit test for the Brownie-Robson model (which is exactly one of the tests performed by RELEASE [test 3.SR, [START_REF] Burnham | Design and analysis methods for fish survival experiments based on releaserecapture[END_REF]) tests for a difference of destiny (subsequently recaught or not) between the marked individuals and the unmarked ones.

So, the hypothesis of a decrease in survival after the first capture is not the only possible biological interpretation.

Our analyses allow us to draw conclusions about the structure of the studied population without prior knowledge of social behavior. This is possible because sources of heterogeneity in the data can be related to individual features (residence time, age, or sex). The low juvenile survival rate in our population of Mediterranean pine vole must be related to high mortality and/or dispersal for juveniles both sexes. An increase of survival rate with maturation seems to be a pattern common to birds and mammals [START_REF] Loery | Age-specificity of blackcapped chickadee survival rates: analysis of capture-recapture data[END_REF]. We must note that voles became adults during the 3 months between the 2 trapping sessions [START_REF] Pascal | Recherche de critères d'âge chez le campagnol provençal Pitymys duodecimcostatus (De Sélys-Longchamps, 1839)[END_REF]). The adult survival rate is not influenced by absolute age, this apparently excludes phenomena like senescence or increased survivorship with age.

Our most surprising result was the higher survival rate of males among juveniles.

Some reviews [START_REF] Greenwood | Mating systems, philopatry and dispersal in birds and mammals[END_REF][START_REF] Dobson | Competition for mates and predominant juvenile male dispersal in mammals[END_REF][START_REF] Pusey | Sex-biased dispersal and inbreeding avoidance in birds and mammals[END_REF] showed that dispersal by male juveniles is predominant among mammalian species, and some very recent studies [START_REF] Boonstra | Natal philopatry and breeding systems in voles (Microtus spp.)[END_REF][START_REF] Boonstra | Similarity of residence times among Microtus littermates: importance of sex and maturation[END_REF][START_REF] Ims | Kinship and origin effects on dispersal and space sharing in Clethrionomys rufocanus[END_REF][START_REF] Ims | Determinants of natal dispersal and space use in grey-sided voles, Clethrionomys rufocanus: a combined field and laboratory experiment[END_REF][START_REF] Sandell | Natal dispersal in relation to population density and sex ratio in the field vole, Microtus agrestis[END_REF] support this fact about voles. With the method we used, dispersal and mortality cannot be distinguished, so it was not possible to address the question of philopatry. However, dispersal in small mammal species may be more complex than previously suggested (e.g., [START_REF] Jones | Density-related changes in survival of philopatric and dispersing kangaroo rats[END_REF][START_REF] Jones | Philopatry, dispersal, and habitat saturation in the banner-tailed kangaroo rat, Dipodomys spectabilis[END_REF][START_REF] Sandell | Natal dispersal in relation to population density and sex ratio in the field vole, Microtus agrestis[END_REF][START_REF] Sandell | Adult philopatry and dispersal in the field vole Microtus agrestis[END_REF]. We must also be aware that method issues can affect our perceptions of dispersal. For instance, some studies have focused on distances moved by dispersers without measuring mortality rates (cf. [START_REF] Jones | Density-related changes in survival of philopatric and dispersing kangaroo rats[END_REF]. Recapture probability (as estimated by probabilistic models) is sometimes very low (e.g., [START_REF] Lefebvre | Comparison of open and closed models for cotton rat population estimates[END_REF], and we believe that this must be taken into account in studies of population ecology of small mammals.

RESEARCH IMPLICATIONS

Our analysis of data was directed by the use of parsimony in the models and the introduction of heterogeneity only when necessary. This rule was advantageous because parsimonious models have fewer problems of identifiability of parameters [START_REF] Sandland | Estimation of survival in marked populations with possibly dependent sighting probabilities[END_REF], more accurate estimation of the parameters can be achieved [START_REF] Jolly | Mark-recapture models with parameters constant in time[END_REF], and it is possible to test biological significance of the models via likelihood ratio tests [START_REF] Lebreton | Modeling survival and testing biological hypotheses using marked animals: a unified approach with case studies[END_REF].

Our results seem to confirm that the regional abundance of the Mediterranean pine vole is determined by "source-sink" dynamics [START_REF] Pulliam | Sources, sinks, and population regulation[END_REF] In our case, the software we used (SURGE) gives only a relative deviance (deviance up to a constant). This is sufficient to compare the models and choose the one with the lower AIC. For further technical details see [START_REF] Lebreton | Modeling survival and testing biological hypotheses using marked animals: a unified approach with case studies[END_REF]. n.s. = P > 0.05, ** = 0.01 < P < 0.025, *** = P < 0.001.

a

  Models consider different patterns of variation in survival probabilities (ϕ) and recapture probabilities (p) between four categories of individuals.b Male adults are taken as reference; (a) is the juvenile effect, (s) the female effect, (a.s) is an effect limited to female juveniles.

Fig. 1 .

 1 Fig. 1. Modeling of capture-recapture data for Mediterranean pine vole. Model D is selected on the basis of a minimal Akaike's information criterion (AIC) and compared with model B and model E by likelihood ratio tests (χ 2 -tests). The AIC is a practical criterion to compare different models fitted to the same data. It is a compromise between fit and parsimony as shown by its formula: AIC = Dev + 2np, where Dev is a measure of fit (the deviance), np is the number of identifiable parameters in the model.

Table 1 .

 1 . The colonizers are adults without established home ranges that appeared as erratics in our study. Most of the juveniles born in a source population die or disperse before breeding. So, most of reproductive output is due to resident individuals in the source populations. Description of the models used to study Mediterranean pine vole population, southern France, 1989-90.

	Model a	Notation b	Description
	A	ϕa+s+a.s, pa+s+a.s	All 4 categories have different survival and
			recapture probabilities.
	B	ϕa+s+a.s, p	All 4 categories have different survival
			probabilities; recapture rate is constant.
	C	ϕa+s, p	Age and sex effects on survival are
			cumulative; recapture rate is constant.
	D	ϕa+a.s, p	Adults of both sexes have the same survival
			probabilities, female juveniles and male
			juveniles differ in survival between them
			and from adults; recapture rate is constant.
	E	ϕa.s, p	Same survival for all groups except female
			juveniles; recapture rate is constant.

Table 2 .

 2 Survival rates and recapture rates of Mediterranean pine voles estimated using model D (Table1), southern France, 1989-90. Estimates apply to the whole population.

	Age class	Sex	Survival rate	Recapture rate
			φ	SE	p	SE
	Ad	M	0.679	0.022 a	0.575	0.030 b
		F	0.679	0.022	0.575	0.030
	Juv	M	0.358	0.048	0.575	0.030
		F	0.212	0.038	0.575	0.030
	a Estimates apply to both males and females.

b