Structural analysis of 5-HT3 receptor antagonists: synthesis and pharmacological activity of various aromatic esters or amides derived from tropane and 1,2,6-trisubstituted piperidine

869

Eur J Med Chem (1993) 28,869-880 0 Elsevier, Paris

Structural analysis of 5HT, receptor antagonists: synthesis and pharmacological activity of various aromatic esters or amides derived from tropane and 1,2,64risubstituted piperidine M Langlois’*,

JL Soulierl, D Yang], B Bremontl, C Flora&, V Rampillonl,

IBIOCIS-CNRS, Faculte’ de Pharmacie, 5, rue JB Cl&nent, 92296 Chdtenay-Malabry, ZSANOFI, Centre de Recherche, 38 GB-Piranesi, Milan, 20137 Italy (Received

15 February

A Giudice*

France;

1993; accepted 18 June 1993)

Summary - Preliminary results of a structure-activity relationship in the field of 5-HT, receptor antagonists on the influence of the aromatic ring and steric hindrance around the basic nitrogen atom are reported. The favorable role of the naphthalene moiety substituted by a carbonyl function in position 1 was demonstrated by measuring the biological activity using the inhibition of the specific binding of [sH]BRL 43694 and the inhibition of the Bezold-Jarisch reflex. Several esters and amides of 1,2,6-trisubstituted piperidine derivatives with a suitable fit with the antagonist reference compounds were synthesized. The lack of biological activity of these compounds emphasizes the importance of steric hindrance for binding with the anionic receptor site. These data confirm the major role of the tropane and quinuclidine frameworks in the potency of a number of 5-HT, antagonists, serotonin

/ 5-HT,

antagonists

/ SAR / 1,2,64risubstituted

piperidines

Introduction In the past decade, there have been significant advances in the understanding of the biochemistry and physiology of the neurotransmitter serotonin (5-HT), in particular the discovery of multiple serotonin receptor subtypes and the design of potent specific ligands [ 11. Today, there is much evidence for the existence of 4 groups of serotonin receptors classified as 5-HT,, 5-HT,, 5-HT, and 5-HT,, and several selective agonists and antagonists for these different receptor types have been described [2]. The 5-HT, receptor is a neuronal receptor coupled directly to a cation channel and possesses some of the characteristics of nicotinic and NMDA receptors [3]. They are present within the central and peripheral nervous systems, particularly in enteric and afferent autonomic neurons [4]. Recently, considerable interest has developed in the search for specific antagonists for this receptor due to the numerous pharmacological properties of these compounds which made them very attractive for the treatment of a number of gastrointestinal and brain disorders [5]. Indeed, in various animal paradigms, 5-HT, receptor antagonists have potential anxiolytic [6], promnesic [7] and antipsychotic *Correspondence

and reprints

properties [8] and prevent the behavioural consequences of the withdrawal of drugs of abuse such as benzodiazepines, alcohol and nicotine [9]. 5-HT, antagonists also prevent the emesis induced by oncolytic drugs such as cisplatin and several compounds are on the market and currently in clinical trials for this purpose [lo]. The 5-HT, antagonists most commonly used are: zacopride, ondansetron (GR 38032), tropisetron (ICS 205-930), granisetron (BRL 43694), MDL 72222 and renzapride (fig 1). These structures contain different aromatic chemical moieties such as an indole, an indazole, a benzamide or a benzoate linked to a nitrogen heterocycle such as tropane, granatane, quinuclidine or imidazole. Several structural analyses have been published recently and there is general agreement on the existence of 3 pharmacophore elements for 5-HT, receptor antagonists: an aromatic ring, a carbonyl function or a bioisosteric group and a basic nitrogen atom [ 111. This hypothesis was confirmed by the good superimposition between the energetically stable conformations of the reference compounds and it is possible that these structural elements are involved in the binding with the receptor site. On the other hand, in recent years, a number of serotonin receptor subtypes have been cloned [ 121 and there are several pieces of evidence which implicate the hydroxyl function of a serine or threonine and the

Tropisetron

Zacapride

(KS

205-930)

N kH3

\

I

CH3

Granixtron

MDL

72222

CH3 (BRL

43694)

Ondsnsetron

(CR

38032)

Renwprlde

Fig 1. Chemical structures of the most commonly used 5-HT, antagonists.

carboxylic group of the side chain of an aspartic acid of the protein chain in the binding of ligands with the receptor site. Similar interactions were assumed to exist for the 5-HT, receptor antagonists: a hydrogen bond formed between the hydroxyl group and the carbonyl functions of the molecules and an ionic bond between the carboxylic group and the heterocyclic basic nitrogen atom. The role of the aromatic moiety in the binding with the receptor is less clear and it is possible that there are hydrophobic or charge-transfer interactions. This simple receptor site model was unsuitable for the accurate design of new ligands for the 5-HT, receptor and it became necessary to obtain further information on structural parameters such as the stereochemistry, the steric hindrance or the electronic density of the aromatic system implicated in the binding with the receptor site. We report here the preliminary results of a structure-activity relationship study on the influence of the aromatic moiety and the steric hindrance around the basic nitrogen atom on the affinity and antagonist properties for the 5-HT, receptor of a series of compounds. The most common aromatic rings encountered in the reference molecules are indole or bioisosteric groups [l I] and substituted benzene rings, but no clear rules for the molecular recognition could be established between these different moieties. A reappraisal of the influence of simple aromatic esters of tropine 1 on the affinity for the 5-HT, receptor seemed worthwhile. Indeed, several reference compounds such as tropisetron or zatosetron [13] are derivatives of the tropane framework, having a simple bicyclic heterocycle without a chiral carbon, and display

potent affinity for the 5-HT, receptor. Thus, it was possible that the tropane heterocycle would have a very good fit with part of the receptor site and consequently the variations in the affinity of derivatives of 1 would be related only to the aromatic moiety. In examining the influence of the basic nitrogen heterocycle on 5-HT, receptor antagonist properties, recent studies have emphasized the role of the bicyclic heterocycle such as the already mentioned tropane, the granatane or the quinuclidine and several authors reported the importance of the stereochemistry or the configuration on the potency of the compounds. Thus, in the tropane series, the endo compounds are up to lOOO-fold more active than the corresponding exocompounds [14] and for the quinuclidine derivatives, (S)-zacopride is one order of magnitude more potent than the (R) isomer [ 151. These findings highlight the role of the spatial position of the lone pair of the basic nitrogen, an essential component for binding with the receptor, with regard to the other elements of the pharmacophore: the aromatic moiety and the carbonyl function. With the aim of obtaining further information on the structural requirements of the amino frameworks, we selected 1-naphthoic acid and 4amino-3-chloro-2-methoxybenzoic acid, an acid capable of revealing pharmacological serotoninergic or dopaminergic activity [ 161 to prepare the corresponding esters and amides of a number of 1,2,6trisubstituted-4 hydroxy or amino-piperidine 2 and 3. Chemistry Compounds 1 were easily synthesized from the commercially available aromatic acids or prepared according to several synthetic pathways already described. They were transformed in an acid chloride and condensed with tropine in pyridine or, in the case of quinoline-8 carbonylchloride, in a DMF/CHCl, mixture (scheme 1). In some cases where the reaction

ci Scheme 1. Synthetic 1, 2 and 3.

rivatives

ci

pathways

3

of the ester and amide

de-

failed, condensation was performed using the lithium salt of the alcohol prepared in situ from BuLi in THF (scheme 1; methods C). For picolinic acid, only the condensation with carbonyldiimidazole in DMF proceeded efficiently.

6a n=, 6b n=z

Derivatives 2 and 3 were synthesized from the corresponding amines or alcohols which were novel for a number of them. They were synthesized from the ketones 4, 5, 6a and 6b by a synthetic pathway recently described by ourselves [17]. 5, 6a and 6b were prepared from a Robinson-Schopf cyclization and for 6a and 6b only stereoisomers with two cisjunction hydrogen atoms and one tram with regard to the lone pair of the nitrogen atom were obtained. The stereochemistry was confirmed by comparison with structural data for the coccinelline derivatives which possess such a framework [ 181. For the ketone 4, the only method to obtain a pure compound was via the catalytic hydrogenation of the corresponding pyridone followed by a Swem oxidation (scheme 2). The axial alcohols 7a and 7b (scheme 3) were prepared directly from the ketones by reduction with LS-Selectride which is known to confer such stereoselectivity [19]. 9 and 10 were prepared by reduction of the 1,2,6-trimethyl-4-pyridone which mainly provides the equatorial isomer 10. The alcohols in the mixture were converted into benzoic esters and separated by chromatography, a basic hydrolysis easily yielded the pure compounds 9 and 10. The exe alcohols Sa, 8b and 11 were easily synthesized through NaBH,-reduction of the corresponding ketones (schemes 2, 3). The stereochemistry of each compound was confirmed by ‘H-NMR spectra and Xray crystallographic analysis for 11 [ 171. The equatorial amines 12 and 13b (schemes 3, 4) were synthesized from the reduction of the corresponding oximes by sodium at reflux in n-butano1 or ethanol according to an already reported process [21]. The amino compounds were free of the endo derivatives and their equatorial stereochemistry was confirmed by ‘H-NMR spectra. On the other hand, the methods used to prepare the corresponding endo derivatives as pure compounds failed. Thus

Scheme 2.

6a “=I 6b n=z dH

la n=l 7b n=z

1 NHz 13b n=z

OH

8a n=1 8b n=z

Scheme 3.

Scheme 4.

the reduction of the oxime by AlH,, described for the preparation of N-methyl 3a-aminonortropane [22] led to a 1: 1 inseparable mixture of endo and exe derivatives 14 and 15 for the oxime of ketone 4. Investigations of suitable conditions for the Mitsonubu reaction [23] with phthalimide or triphenyl phosphoryl azide on the pure exe alcohols 10 and Sb were unsuccessful. Few examples [16, 241 of such a reaction have been reported for a nitrogen hetero-

872 cycle. However, the presence of a basic nitrogen atom did not explain the failure of the substitution reaction since we have shown that it was possible to prepare the corresponding amino derivatives from 4-hydroxy1-methylpiperidine or the 3-hydroxyquinuclidine by this process [24]. Only steric hindrance of the axial hydrogens and the conformational rigidity of the frameworks of 8 and 10 could explain the hampering of the en& nucleophilic attack. The esters of 1-naphthoic acid 2 were prepared by methods similar to those described for 1 and the amides 3 were synthesized through the mixed anhydride method using ethyl chloroformate in THF in the presence of Et,N (scheme 1). The only axial amides isolated were prepared from the mixture of amines 14 and 15 synthesized from the oxime reduction of the ketone 4. The endo isomer was easily distinguished from the exe isomer by iH-NMR spectra, the a-proton to the amide nitrogen displayed a well-defined and characterized multiplet with regard to that of the exo isomer. Biological

results and discussion

The affinity of the different synthesized compounds for central SHT, receptors was determined by inhibition of the specific binding of [sH]BRL 43694 [25] or granisetron to membranes of rat posterior cortex using 7-l 1 different concentrations according to conditions previously reported for [sH]zacopride [26]; the results were expressed as IC,, values. The SHT, receptor antagonist activity of the molecules was evaluated in the rat by inhibition of the Bezold-Jarisch reflex [27], a very specific activity of serotonin on cardiac SHT, receptors at the level of the right ventricle which brings about an abrupt and dose-related reduction in the heart rate. The activity of the compounds was given by the ID,, value which is the dose inhibiting 50% of the response to serotonin. The affinity values of the reported compounds were fairly well correlated with activity in the Bezold-Jarisch test except for compound 40. Examination of the results in table I clearly emphasises the favorable role of a 10 7c aromatic system on the potency of the compounds in this homogeneous series of tropine esters. The 1-naphthoic 18 and the 3-indole carboxylic esters 17 (tropisetron) can be regarded as equipotent and had = loo-fold higher affinity than the simple benzoic ester 16 which was devoid of activity in the Bezold-Jarisch test. However, the spatial position of the aromatic ring with regard to the amino framework was an important structural parameter since the 2-naphthoic ester 19 was much less active than 18. The loss of activity may be explained in terms of steric hindrance in the binding site of the receptor for the aromatic moiety.

Thus, when the postulated biological conformations of the molecules were superimposed with regard to the common elements of the pharmacophore, the basic nitrogen atom, the carbonyl function and the proximal aromatic ring by a fit operation with ALCHEMY software (Tripos Inc, St Louis, MO) it was clearly shown that the distal rings of 18 and 19 do not share a common area of interaction with the receptor (fig 2). This operation emphasizes an unfavorable binding of the aromatic ring of 19 with the receptor site. As has already been reported [29], the postulated biologically active conformation of the molecules was similar to the minimal energetically stable conformation and, in the present case, the carbonyl function and the aromatic plane were coplanar. Moreover, the drop in activity observed with more extended systems such as the phenanthrene and anthracene derivatives 20 and 21 indicated a restricted area for the aromatic group receptor site, its limits being inferred from the high potency of the acenaphthene derivative 22, representative of the largest allowed aromatic surface which can bind efficiently with the receptor. On the other hand, the presence of a heterocyclic aromatic nitrogen atom, such as in compounds 23, 24 and 25, contributed to the decrease in potency of the corresponding derivatives with regard to the homocyclic derivatives. These facts support the idea that a charge transfer may be implicated in ligand-receptor interactions at the level of the aromatic moiety. Nevertheless, an electronic influence on the value of the affinity for the 5-HT, receptor was not clear since substituents on the 4 position as different as MeO, F and Br led to potent compounds (26, 27 and 29). On the other hand, the weaker activity of 28 strengthens the evidence for the influence of steric parameters in this part of the receptor. The first part of this study clearly confirms the favorable role of a 10 n: aromatic system on binding of esters with the 5-HT, receptor and in particular with a carbonyl function located in the 1 position such as the 1-naphthoyl moiety or the indole-3-carbonyl group. The border lines of the restricted area for aromatic interactions have been determined and can explain the drop in activity previously reported for some substituted indoles or benzimidazolones [ 14, 301. Compounds 2 and 3 (tables II, III) were prepared with the aim of furthering knowledge on the structural parameters implicated in the interactions of the amino framework with the receptor. Examination of the results shows a significant decrease in the affinity for the 5-HT, receptor compared to the tropane derivatives 18 and BRL 24682 or the quinuclidine ester 40 regardless-of the amino moiety. First of all, the overwhelming importance of the axial stereochemistry was emphasized by the weak activity of compound 30 with regard to 18, and overall the data highlight the dramatic influence of steric parameters on the interac-

873 Table I. 5HT, receptor binding affinity and inhibition of the Bezold-Jarisch reflex for compounds 1. coAC

0 2

1

N

\ CH3

Binding IQ,0

16

329~k9.4

17’

03 :

a7:

21

>500

3*0.9

4.9(3.7-6.4)

23ok9s

176(29-1076)

23

24

:

26

I>

16.4 (1.7-156)

320+95

NT

10.6kO.57

NT

6.03f0.73

NT

20.6*0.8

NT

4.66 fO.58

NT

F

, >

;

: I’ 0

1400~750

>500

27 OMe

12Ok8

58(43-78)

28

:I

22

10*0.86

S-HT-induced bradycardia (IDSO&K&)b

c

19

ab

Binding assays IC5o (nM)’

S-HT-induced bradycardia (ID50 pg/Kg/i~)~

1.22(0.8-1.8)

18

20

assays

(nWa

:

I>

OEt

7.lbO.9d

14.6(10-22)

43

29 Br

aIC50 were determined by measuring the inhibitory effects of the compounds on [3H] BRL 4394 binding; QD,, represents the dose of the antagonists inhibiting 50% of the effect of serotonin (SHT); Cl7 is tropisetron; dnew data with regard to reference

[Ill. tions of the ligands with SHT, receptors. Thus, the important decrease in affinity of the tropane derivatives 37, 38 and 39, which can be superimposed on the active compound 22, is due to the additional steric volume of the substituent in 37 and 38 hampering the efficient hydrogen binding of the hypothetical hydroxyl group of the serine of the receptor protein chain with the carbonyl function. The unfavorable effects of steric hindrance around the basic nitrogen atom were also demonstrated with the different ester or amide derivatives prepared from 1,2,6-trimethylpiperidine (31, 32, 33, 42, 43, 44) or the tricyclic amino framework derived from coccinelline (33, 34, 35, 36) and confirm the recently reported results [20, 281. Thus, the loss of affinity of derivatives 32 and 43,

of which the stereochemistry and the spatial position of the anchorage points with the receptor are similar to those of the endo tropane derivatives 18, BRL 24682 and tropisetron, can be explained by the presence of the bulky volume of the 2 methyls on both sides of the piperidine ring. The view of the fit of the low energy conformations of 18 and 32 particularly emphasizes the influence of the hindered volume around the nitrogen and the impossibility for such a compound to reach the anionic center of the receptor (fig 3). A similar steric effect may explain the weak activity of the tricyclic amino derivatives 33 and 35 in which the 3D-position of the key pharmacophore elements is identical to that of renzapride, a highly potent 5-HT, antagonist.

874

Fig 2. Superimposition of tropisetron and tropine esters of 1 and 2-naphthoic acids 18 and 19. View of the different occupancy of the distal aromatic ring of naphthoic esters.

These data suggest that the receptor site of the amino framework of the 5-I-IT, antagonists is a buried anionic center, such as the carboxylic group of an aspartic side chain in a narrow lipophilic pocket, the optimal volume of which is inferred by the good matching of the tropane and quinuclidine rings. In summary, the data reported herein highlight the importance of steric parameters in the binding of ligands to the 5-HT, receptor site and the unique role of tropane and quinuclidine moieties as suitable amino groups to obtain potent ligands. On the other hand, it has been shown that some potent compounds could be designed from a 10 7c aromatic system such as the 1-naphthalene carbonyl group. Based on these findings, several new series of potent 5-HT, receptor antagonists are currently being developed in our laboratory.

Table II. 5-HT, receptor binding affinity and inhibition of the Bezold-Jarisch reflex for compounds 2.

2

Binding assays ICs,W?

5HT-induced bradycardia(ID501rgl~~i”)b

No

Binding assays ICg#Na

5.HT.induced bradycardiaaD501rg/Kg/i”)b

217k41

>soo

36

106of47

NT

NT

z-500

37

314+&l

NT

672k68

NT

466*57

NT

81OOf560

SSOO

278k91

>500

Wiee footnotes for table I.

40

875 Table III. 5-HT, receptor binding affinity Bezold-Jarisch reflex for compounds 3.

and inhibition

of

Experimental

protocols

Chemistry

Melting points were determined on a Mettler FP 61 apparatus. ‘H- and isC-NMR spectra were recorded with a Brucker AC200E or AM300 spectrometer using tetramethylsilane as internal standard. Chemical shift data are reported in parts per million (6 in ppm) where s, d, dd, t, q and m designate singlet, doublet, doublet of doublets, triplet, quartet and multiplet respectively. Mass spectra were obtained using a Ribermag Rl O- 10 mass spectrometer. Infrared spectra were obtained with a Perkin-Elmer 1420 ratio recording infrared spectrometer. Microanalyses were performed by the CNRS (department of microanalytical services), Vemaison, France. Where analyses are indicated only by symbols of the elements, the results obtained were within f 0.4% of the theoretical values. Most of the acid chlorides were synthesized according to the published procedures: 4-quinoline carbonylchloride [31], S-quinoline carbonylchloride [ 321, 5acenaphthene carbonylchloride [33], 9-anthracene carbonylchloride [34] and 9phenanthrene carbonylchloride [35]. 1 and 2naphthalene carbonylchlorides were purchased from Aldrich-Chimie (Strasbourg). For the other carboxylic acids, the chlorides were prepared as crude materials either by the thionyl chloride method or the oxalyl chloride process. The alcohols 3P-n-butyl-8-methyl-8-azabicyclo[3,2,1]act-3a-ol and 3~,8-dimethyl-8-azabicyclo[3,2,l]oct-8a-o1 were prepared according to a previously described method [36]. 3-Methyl-3-azabicyclo[3,2,l]oct-8P-o1 was synthesized by the Robinson-Schoft cyclization [37]. The reference compound tropisetron 17 was synthesized by Method C (see General procedures).

a.bSee footnotes

for table I; Creference

[43].

(2a,3aa,Sap,7aa)-Decahydrupyrido

(2,1,6

cd) pyrrolizine-2-ol7a

To a solution of 13.4 ml LS-Selectride (1 M in THF) in 10 ml THF, cooled to -78°C under N, was added 1.8 g (11 mmol) of ketone 6a [ 171 in 15 ml THF and stirring was continued for 2 h. The reaction was quenched by adding 1.5 ml aqueous 1 N HCl. The mixture was dissolved in 20 ml ethanol and 10 ml 10 N NaOH. The resulting solution was refluxed for 30 min and then cooled to room temperature. Extraction with diethyl ether and purification by chromatography (Al,O,, Et,O/ MeOH; 15:l) gave 1.56 g (yield: 85%) of 7a. Recrystallization in isopropyl ether yielded colorless crystals, mp: 118°C. ‘H-NMR (200 MHz, CDCl,) 6: 4.05 (t, J = 3 Hz, IH), 3.4 (m, 2H), 3.2 (tt, J = 11 and 4 Hz), 2.4 (m, 2H), 1.5 (m, 4H), 1.7 (m, 4H), 1.4 (m, 2H). ‘-1C-NMR (CDCl,) 6: 66.9 (lC), 60.8 (IQ

46.2

(2C),

36.0

(2C),

(20~.3aa,6a4,9aa)-Dodecahvdrop~rido/2,1 izitz-2-01 7b ’

34.4

(2C),

_

27.4

._

(2C).

.

,h-delquinol_.

Following essentially the same procedure as for 7a, 0.9 g (4.7 mmol) 6b was reduced with LS-Selectride (6.0 ml. 1 M in THF) in THF (15 ml). The crude product was purified by recrystallization from EtOH/hexane to provide 7b (0.55 g, yield: 60%), mp: 158°C. ‘H-NMR (200 MHz, CDCl,) 6: 4.26 (t, J = 3 Hz, lH,,), 3.39 (dd, J = 5, 13 Hz, lH), 2.68 (broad t, .I = 11 Hz, lH), 2.08 (dt, J = 3 Hz, .I = 14 Hz, 2H), 1.8-1.4 (m, lOH), 1.4-1.1 (m, 4H). Fig 3. Superimposition of tropine and 1,2,6trimethyl eridine esters of I-naphthoic acid 18 and 32. View additional volumes of the methyl groups.

pipof the

(2P,3aa,6ap,9acr)-Dodecahydropyrido[2,1,6-de]quinolizin-2-ol8b

To a stirred solution of 6b (1 .O g, 5.2 mmol) in MeOH (10 ml), NaBH, (0.4 g, 10 mmol) was added portionwise. Stirring was

876 continued at room temperature for 30 min. The excess hydride was removed by the addition of hydrochloric acid (1 N). The mixture was concentrated, basified with aqueous NaOH and extracted with ether. After the usual workup, repeated crystallizations from isopropyl ether gave the pure alcohol (0.76 g, yield: 75%), mp: 146°C. tH-NMR (200 MHz, CDCl,) 6: 3.76 (septet, IH) 2.95 (broad d, J = 13 Hz, 2H), 2.60 (tt, J = 2, J = 10 Hz, lH), 1.95-1.65 (m, 4H), 1.6-1.3 (m, lOH), 1.2-1.0 (m, 2H). (2~,3a&5a~,7aa)-Decahydropyrido[2,1,6-cd]pyrrolizin-2-olSa In a similar manner to the previous process, 6a (1.0 g, 6.1 mmol) was reduced with NaBH, (0.5 g, 13.2 mmol) in MeOH (15 ml) to provide 8a (0.7 g, yield: 69%). tH-NMR (200 MHz, CDCl,) 6: 3.77 (tt, J = 4, J = 11 Hz, lH), 3.22-3.08 (m, 3H), 2.55-2.36 (m, 2H), 1.8-1.0 (m, 10H); tsC-NMR (CDCI,): 68.75 (lC), 61.17 (lC), 50.05 (2C), 37.67 (2C), 36.22 (2C), 26.84 (2C). (2p,4~,@) and (2@,4p,@)-I ,2,6-Trimethyl-4-piperidinol9 and 10 A mixture of 1,2,6-trimethyl-4-pyridone [38] (60 g, 0.44 mol), 400 ml absolute ethanol and 9 g Raney nickel were placed in an autoclave and heated to 125°C for 4 h under hydrogen pressure (130-160 atm). After filtration, the solution was evaporated and the residual oil distilled in vacua to provide 43.5 g colorless oil, bpo,osmmHg:72°C (yield: 70%). tH-NMR (200 MHz, CDCl,), 6: 4 (sharp m, H, ), 3.53 (broad m, H,), 2.21 (s, CH,-N), 2.15 (s, CH,-N), l.b9 (dd, 6H). The intensity ratio of the 2 latter signals indicated a 4:l mixture of exo and endo alcohols. The mixture of alcohols was transformed to benzoic esters according to the following process: 16 g (110 mmol) were dissolved in 500 ml dry pyridine and added slowly under stirring to 26 ml benzoyl chloride at 0°C. The mixture was stirred at room temperature for 24 h and the solvent evaporated in vacua. The crude product was washed with Et,0 and purified by chromatography on silica gel (CHCl,/MeOH,95:5)- to provide a compound which was recrvstallized in a mixture of acetone/i-PrOH to urovide 15.9 n (yield: 49%) hydrochloride as a white solid, mp: 254°C. Thi equatorial stereochemistry was assigned by tH-NMR spectra (200 MHz, CDCI,) 6: 5.1 (broad m, lH,,). 11.4 g compound was added to a 10% solution of NaOH in MeOH and the mixture stirred at room temperature for 24 h. The solvent was distilled under reduced pressure, the residual material diluted with water and extracted with a CH,Cl,/Et,O mixture (4 x 300 ml). The organic layer was dried (MgSO,) and evaporated. Distillation of the crude product gave 5.7 g (yield: 99%) of 10 as a colorless oil, bp,,, mmH:- 82’-84’C: ‘H-NMR (200 MHz. CDCl,) 6: 3.58 (broad m. lk). 2.71 (br s. 1H). 2.13 (s, 3H), 2 (m, 2H)‘T 1.83 (m,‘2H), 1.2’(dd,‘2H), I.?)8 (d, 6H). A residual oil was recovered from the previous ester mixture after washing with diethyl ether. It was purified by chromatography under the same conditions as for the equatorial ester. Recrystallization in acetone/i-PrOH mixture gave 1.7 g (yield: 5%) -benzoate. The axial stereochemistry was assigned by tH-NMR (200 MHz) 6: 5.27 (sham m. lH--). The comnound I (1 g) was hydrolyzed according to the prev:ous procedure to provide 0.5 g (yield: 87%) of the alcohol 9 as a light yellow solid. tH-NMR (200 MHz, CDCl,) 6: 4.0 (sharp m, lH,,), 2.55 (m, 2H), 2.16 (s, 3H), 1.6 (m, 4H), 1.06 (d, 6H). \ - ,

I

4.0 g (29 mmol) of ketone 5 was dissolved in 50 ml methanol cooled at 0°C. 2.8 g (74 mmol) NaBH, was added portionwise and the mixture stirred overnight at room temperature. It was diluted with 1 N HCl and, after stirring for 30 min, was

saturated with K,CO, and the mixture extracted with methylene chloride (70 ml x 3). The organic layers were dried over Na,SO,. The solvent was removed under reduced pressure and the residue distilled, bp,,,Z, mmH: 60°C to give 2.8 g of the alcohol 11. tH-NMR (200 MHz, C!DCl,) 6: 3.11 (m, 1H), 2.89 (m, lH), 2.42 (m, lH), 2.26 (s, 3H), 1.81 (m, 2H), 1.62 (m, 2H), 0.99 (d, 3H), 0.94 (d, 3H). IX?NMR (200 MHz, CDCl,) 6: 64.24 (lC, C,), 55.35 (lC, C,), 49.38 (lC, C,), 43.26 (lC, C,), 40.71 (lC, C,), 38.39 (lC, C-N), 20.44 (lC, C,,), 11.11 (lC, G). (2a,4P,6P)-4-Amino-I ,2,6-trimethyl-piperidine 12 From the 4: 1 mixture of trans and cis 1,2,6-trimethyl-4-piperidone 1171, the pure oxime of the trans ketone was isolated by the following process: 4 g (28 mmol) of the mixture of ketones, 2 g (28 mmol) NH,OH. HCl and 2.4 ml piperidine was refluxed in 80 ml ethanol for 1 h. The solvent was evaporated and the solid filtered off and washed with cold ethanol (2.6 g, 58%). It was recrystallized in ethanol to give 1.5 g pure t&s oxime (vield: 35%) characterized bv both doublets of the methyl groups (6: 1.03 (d, 3H, CH,,), 1.18 (d, 3H, CH,,,)). 3.0 -g (20 mmol) oxime was refluxed in 100 ml ethanol. The solution was stirred and 4.58 g sodium was added portionwise to maintain a vigorous reflux. When the addition was completed, the reflux was continued for 2 h, then the solution was evaporated, diluted in water and extracted with CH,Cl, (3 x 100 ml). The solution was dried over Na,SO, and evaporated. The residual oil was distilled to provide 12 as a colorless oil, bp,,, mmHg: 60°C 1.3 g, yield: 50%. tH-NMR (200 MHz. CDCI,) 6: 3.11 (m, IH), 2.89 (m, lH), 2.42 (m, lH), 2.26 (s, 3H), 1.81 (m, 2H), 1.62 (m, 2H), 0.99 (d, 3H), 0.94 (d, 3H). t3C-NMR (CDCl,) 6: 55.6 (lC, C,), 49.06 (lC, C,), 44.9 (2C, C,, C,), 41.8 (lC, C,), 38.9 (lC, C,), 20.8 (lC, C,,), 10 (lC, C,,). (2/$4~x and 4fi,6fi)-4-Amino-1,2,6-trimethyl-piperidine 14 and 15 In a similar manner, 4 (2.8 g, 20 mmol) was converted to the crude oxime (2.8 g) which was added to a mixture of H,SO, (40 mmol) and LiAlH, (80 mmol) in 50 ml THF and allowed to react at 40°C for 3 h. ‘After cooling, a mixture of H,O/THF (1:1) was added carefully. The precipitate was filtered-off and washed with CHCl,. The combined organic nhases were evaporated and distilled to give 1.5 g (yield: 53%), bp,, ,,,,,,” : 65°C which proved to be a mixture of epimers 14 and 15 (1: f) by examination of tH-NMR spectra. It was directly condensed to give the benzamide compounds 41 and 42. (2P,3aa,6ap,9aa)-2-Amino-dodecahydropyrido[2,1,6-de]quinolizine 13b NH,OH.HCl (1.6 g, 23.2 mmol) was added in portions to a stirred solution of 6b (2.5 g. 12.9 mmol) in EtOH (20 ml) containing pyridine (2.0 ml).-The mixture ‘was stirred at room temnerature for 2 h. then ooured into 2 N NaOH (20 ml) and extracted with CHQ,. The usual workup gave the pure oxime (2.1 g, 78%), mp: 232°C. *H-NMR (200 MHz, CDCl,) 6: 3.1 (broad t, 2H), 2.8 (broad t, 2H), 2.6 (t, lH), 2.2 (t. lH), 1.8 (m, 3H), 1.6-1.4 (m, 8H), 1.3-1.1 (m, 2H). 2.1 g (10 mmol) of the previous oxime in amyl alcohol (25 ml) were refluxed and sodium (2.3 g, 0.1 mol) was added portionwise for 1 h. The mixture was poured into water and extracted with CH,Cl,. The organic layer was dried over Na,SO, and concentrated. The residue was distilled bulbto-bulb, bp,.,, m,,,Hg:78°C to give 13b (1.34 g, yield: 70%). tH-NMR (CDCI,): 3.39 (m, lH), 2.88 (broad d, J = 13 Hz, 2H), 2.64 (broad t, J = 11 Hz, lH), 2.1-2.0 (m, 16H). ‘sC-NMR (CDCI,): 56.75 (2C), 50.44 (lC), 48.16 (lC), 34.23 (2C), 32.66 (2C), 30.62 (2C), 19.54 (2C). I

877 Synthesis of the esters 1 and 2: general procedure Method A (compounds 16,20,23,31,32,33,34,35,36,40) To the crude acid chloride (21 mmol) in dry pyridine (30 ml) at 0°C was added slowlv drouwise under stirrina a solution of the alcohol (42 mmol). The reaction mixture was-stirred for 12 h at room temperature and the reaction was complete within this period. The solvent was evaporated in vacua to dryness. The residue was purified directly by chromatography on silica gel (Chromagel 6A CC 70-230 mesh, SDS Paris) by eluting with a mixture of MeOH/CHCl, to provide the compound in a hydrochloride salt form. Endo-8-methyl-8-azahicyclo[3.2.I]octan-3-yl henzoate hydrochloride 16 The compound was synthesized from benzoyl chloride and tropine (yield: 45%) and recrystallized as bright colorless needles from a mixture of i-Pr,O/MeOH, mp: > 260°C. IR (CHCl,) 1700 cm-t (C=O). iH-NMR (200 MHz, CDCl,) 6: 12.3 (broad s, lH), 7.9 (m, 2H), 7.6-7.4 (m, 3H), 5.34 (t, lH), 3.8 (broad s, 2H), 3.2-3.1 (m, 2H), 2.87-2.76 (d, J = 5.1 Hz, 3H), 2.1-2.5 (m, 6H). Anal C,,H2,N0,Cl (C, H, N, Cl). Endo-8-methyl-8-azahicyclo[3.2.l]octan-3-y1 anthracene-9carhoxylate hydrochloride 20 From 9-anthracene carbonylchloride and tropine an amorphous solid (yield: 12%) was obtained from a mixture of i-Pr,O/ MeOH, mp: > 260°C. IR (CHCl,) 1710 cm-t (C=O). tH-NMR (200 MHz, CDCI,) 6: 12.3 (broad s, lH), 8.49 (s, lH), 8-7.85 (m, 4H), 7.5-7.4 (m, 4H), 5.7 (t, lH), 3.4-3.2 (m, 2H), 2.66 and 2.87 (d, J = 4 Hz, 3H), 2.3-l .9 (m, 6H). Anal C,,H2,0,NC1 CC, H, N, Cl). Endo-8-methyl-8-azahicyclo[3.2.I]octan-3-y1 quinoline-$-carboxylate hydrochloride 23 From 4-quinoline carbonylchloride and tropine, pale pink needles (yield: 43%) after-recrystallization from a mixture of acetone and i-PrOH were obtained. mo: > 260°C. IR (CHCl,) 1710 cm-1 (C=O). tH-NMR (200 MHz’, CDCI,) 6: 8.99 (d, J4.4 Hz, d), 8.72 (d, J = 7.4 Hz), 8.13 (d, J = 8.2 Hz, lH), 7.8-7.6 (m, 3H), 5.5 (t, lH), 3.8 (broad s, 2H), 3.5-3.1 (m, 2H), 2.7 (s, 3H), 2.1-2.4 (m, 6H). Anal C,,HZ,NO,Cl (C, H, N, Cl). I -Methyl piperidine-4-yl naphthalene-I -carboxylate hydrochloride 30 From 1-methyl-4-piperidinol and 1-naphthoic acid, 30 was prepared (yield: 46%) as a white solid from a mixture of MeOH and i-Pr,O, mp: 194°C. tH-NMR (200 MHz, CDCl,) 6: 12.3 (broad s), 8.78 (m, 2H), 8.13-7.94 (m, 2H), 7.82 (m, lH), 7.6-7.37 (m, 3H), 5.4-5.17 (broad m, lH), 3.7-3.38 (m, 2H), 3.2-2.4 (m, 7H), 2.29-2.16 (m, 2H). Anal C,,H2,N0,Cl (C, H, N, Cl). I -Methyl-2,Cdimethyl piperidine-4-yl naphthalene-I -carboxylate hydrochloride 31 A 56% yield was obtained from 1-naphthalene carbonylchloride and alcohol 10 as colorless needles by crystallization in a mixture of MeOH and diisopropyl ether, mp: 244°C. IR (CHCl,) 1700 cm-t (C=O). IH-NMR (200 MHz, CDCl,) 6: 12.3 (broad s, lH), 8.8 (d, J = 8.4, IH), 8.07 (dd, J = 1.2 Hz, /=7.1 Hz, lH),7.94(d,J=8.2Hz, lH),7.81 (dd,J= 1.7Hz, .I = 8.6 Hz, lH), 7.58-7.38 (3H, m), 5.12-5.02 (lH, m), 2.3 (3H, s), 2.33-2.25 (2H, m), 2.14-2.05 (2H, m), 1.52-1.7 (2H, m), 1.18 (d, J = 6.2 Hz, 6H). Anal C,,H,,NO,Cl (C, H, N, Cl).

I-Methyl-2,Cdimethyl piperidine-4-yl naphthalene-I-carboxy late hydrochloride 32 The compound was obtained from I-naphthalene carbonylchloride and the alcohol 9 as a white amorphous solid from a mixture of isopropyl ether and MeOH, mp: 220°C. IR (base, CHCl,), C=O. tH-NMR (200 MHz, CDCI,) 6: 12.3 (broad s, 14), 8.86 (d, J = 8.6 Hz, lH), 8.12 (dd, J = 1.2 Hz, / = 7.2 Hz, lH), 8.05 (d, 8.2 Hz, lH), 7.84 (dd, J = 1.7 Hz, J = 7.7 Hz, lH), 7.35-7.61 (3H, m), 5.22-5.4 (lH, m), 3.1 (2H, m), 2.52 (s, 3H), 2.06 (4H, m), 1.35 (d, J = 6.4 Hz, 6H). Anal C,,H,,NO,Cl (C, H, N, Cl). 1-[(2~,3a(x,6a~,9a~+Dodecahydropyrido[2,1 ,h-de]quinolizin2-yl]-naphthoate 33 0.53 g (yield: 69%) was obtained from Sb and 1-naphthalene carbonylchloride after recrystallization from a MeOH/AcOEt mixture, mp: 230°C (dec). tH-NMR (200 MHz, CDCI,) 6: 8.80-7.35 (m, 7H), 5.22 (m, lH,,), 3.11 (broad d, J = 14 Hz, 2H), 2.80 (broad t, J = 12 Hz, lH), 2.08 (q, J = 14 Hz, 2H), 2.0-1.1 (m. 14H). ‘XZ-NMR (CDCl,) 8: 166.07 (C=O). 133.83-124.‘16 (Id C,,), 69.18 (lC), 57:33 (2C), 51.85 (1Cj: 30.43,28.63, 27.56, 17.63. Anal C,,H,,NO,.HCl (C, H, Cl, N). 1-[(2~~,3aa,6a~,9aor)-Dodecahydropyrido[2,1 ,Cde]quinolizin2-yl]-naphthoate 34 1.12 g (yield: 96%) was prepared from 7b (0.59 g, 3.02 mmol) and 1-naphthalene carbonylchloride. The compound was recrystallized from a MeOH/AcOEt mixture, mp: 240°C. tH-NMR (200 MHz, CDCI,) 6: 8.89-7.43 (m, 7H), 5.52 (t, lH,,), 3.91 (broad d, J = 14 Hz, 2H), 3.24 (broad t, J = 12 Hz, lH), 2.61 (m, 2H), 2.30 (m, 2H), 2.09-1.43 (m, 12H). ‘sC-NMR (CDCl,) 6: 165.82 (C=O), 134.20-124.53 (10 C,,), 65.94 (-OC-), 54.04 (2C), 51.19 (lC), 30.40, 27.34, 17.44. Anal C&H,,NO,.HCl (H, N, Cl), C,,,,: 7 1.58, Cfound:70.98. 1-[(2~,3aa,5a~,7ac+Decahydropyrido[2,1,6-cd]pyrrolizin-2yl]-naphthoate 35 1.3 g (yield: 61%) was obtained from 8a (1 .O g, 6 mmol) by recrystallization from a EtOH/AcOEt mixture, mp: 200°C. iH-NMR (200 MHz, CDCI,): 8.73-7.31 (m, 7H), 5.05 (broad t, .l = 12 Hz, IH,,), 3.80 (m, 3H), 2.80 (m, 2H), 2.28 (m, 2H), 2.20-1.96 (m, 6H), 1.66-1.48 (m, 2H). r”C-NMR (CDCl,) 6: 165.74 (C=O), 133.20-124.16 (IO C,,), 67.59, 65.05, 49.59, 35.35, 32.89,23.19. Anal Cz,H2,N0,.HCl (C, H, N). 1-[(2a,3a~~,Sa~,7aa)-Decahydropyrido[2,1 ,h-cdlpyrrolizin-2y/l-naphthoate 36 2.4 g (yield: 81%) was prepared from 7a (1.4 g, 8.5 mmol) and I-naphthalene carbonylchloride and recrystallized from MeOH/ i-Pr,O. mu: > 220°C. iH-NMR (CDCI,) 6: 8.86-7.41 (m. 7H). 5.37 (i, lk,,), 4.02 (m, 2H), 3.74 (m, TH), 3.03-2.78 (ml 2H): 2.41-2.01 (m, 8H), 1.97-1.50 (m, 2H). r”C-NMR (CDCI,) 6: 166.10 (C=O), 134.20-124.93 (10 C,,), 65.99, 65.01, 46.77, 35.30, 30.92, 23.52. Anal C,,H,,NO,.HCl (C, H, Cl, N). 1 -Azabicyclo[2.2.2]octan-3-yl naphthalene-1 -carboxylate hydrochloride 40 From 1-naphthalene carbonylchloride and 3-quinuclidinol (yield: 36%) the compound was isolated as a white amorphous solid from a mixture of i-PrOJMeOH, mp: > 260°C. IR (CHCI,) 1700 cm-r. ‘H-NMR (200 MHz, CDCl,) 6: 8.87 (d, J = 8.45 Hz, lH), 8.18 (dd, J = 1.2, J = 7.3 Hz, lH), 8.08 (d, J = 7.7 Hz, lH), 7.91 (dd, J = 1.6, .I = 7.7 Hz, lH), 7.7-7.4 (m, 3H), 5.44 (m, lH), 3.92 (m, lH), 3.6-3.2 (m, 5H), 2.8-1.8 (m, 5H). Anal C,,H,,O,NCl (C, H, N, Cl).

878 Method B (compounds 18,19,26,27,28,29) The experimental procedure was similar to that of Method A except that the reaction mixture was refluxed for 6 h. After concentration in vacua, the residue was purified by chromatography on silica gel (Chromagel 6A CC 70-230 mesh, SDS Paris) and eluted with a CHClJMeOH mixture to give the esters as hydrochloride salts. Endo-8-methyl-8-azabicyclo[3.2.1 loctan-3- yl naphthalene-I -carboxylate hydrochloride-18 From 1-nauhthalene carbonvlchloride and trouine. the compound \;as prepared (yield:‘62%) after crystallization from a mixture of Et,O/acetone as colorless needles, mp: 258°C. IR (CHCl,) 1700 cm-t. tH-NMR (200 MHz, CDCl3,) 6: 8.9 (d, IH), 8 (d, 2H), 7.85 (d, lH), 7.6-7.4 (m, 3H), 5.4 (t, lH), 3.8 (broad m, 2H), 3.3-3.1 (m, 2H), 2.7 (s, 3H), 2.4-2.1 (m, 6H). Anal C,,HZ2N0,Cl (C, H, N). Endo-8-methyl-Sazabicyclo [3.2.I]octan-3-yl naphthalene-2carboxylate hydrochloride 19 From 2naphthalene carbonylchloride and tropine, the compound was obtained (yield: 54%) as colorless needles after crystallization from CH,CN, mp: > 260°C. IR (CHCl,) 1700 cm-t. tH-NMR (200 MHz, CDCI,) 6: 8.5 (s, lH), 8-7.8 (m, 4H), 7.7-7.5 (m, 2H), 5.4 (lH, t), 3.7 (broad s, 2H), 3.1-3.3 (m, 2H), 2.7 (s, 3H), 2.4-2.1 (m, 6H). Anal C,&NO,Cl K, H, N). Endo-8-methyl-8-azabicyclol3.2.l]octan-3-y1 4$uoronaphthalene-I-carboxylate hydrochloride 26 From tropine and the commercially available 4-fluoro-l-naphthoic acid (Aldrich, Strasbourg), the compound was obtained as white needles from a mixture of petroleum ether and CHCI, (yield: lo%), mp: > 250°C. IR (KBr) 1700 cm-1 (C=O). tH-NMR (200 MHz, CDCl,) 6: 8.9 (m, lH), 8 (m, 2H), 7.5 (m, 2H), 7.1 (m, lH), 5.4 (sharp m, lH), 3.8 (broad s, 2H), 3.1 (m, 2H), 2.7 (s, 3H), 2.1-2.4 (m, 6H). tYF-NMR (200 MHz, D20) 6: 111.3. Anal C,,H,,NO,ClF (C, H, N, Cl). Endo-8-methyl-8-azabicyclo[3.2.I]octan-3-y1 4-methoxy naphthalene-1 -carboxylate hydrochloride 27 From the commercially available 4-methoxy-l-cyanonaphthalene (Aldrich, Strasbourg) hydrolyzed into the pure acid by KOH in methanol at reflux and trouine, the comnound (Geld: 9.3%) was obtained after several crystallizations as beige-crystals from a mixture of isopropyl ether and MeOH, mp: 237°C. IR (KBr) 1685 cm-t (C=O). tH-NMR (200 MHz, CDCl,) 6: 9 (m, lH), 8.2 (dd, J = 8.8, J = 1.3 Hz, lH), 8.0 (d, J = 8.3 Hz, lH), 7.6-7.4 (m, 2H), 6.7 (d, J = 8.4 Hz, lH), 5.4 (sharp m, IH), 4 (s, 3H), 3.7 (broad s, 2H), 3.1 (m, 2H), 2.6 (s, 3H), 2.3-2.1 (m, 6H). Endo-8-methyl-8-azabicyclo[3.2.l]octan-3-yi 4-ethoxynapthalene-I -carboxylate hydrochloride 28 From tropine and 4-ethoxy- 1naphthoic acid prepared by treatment with KOH in ethanol at reflux of 4-methoxy-llcyanonaphthalene [39], the pure compound (yield: 10%) was isolated as white crystals from a mixture of isopropyl ether and MeOH, mp: > 260°C. IR (KBr) 1690 cm-r (C=O). IH-NMR (200 MHz, CDCI,) 6: 9 (m, lH), 8.3 (m, lH), 8 (d, J = 8.3 Hz, lH), 7.5 (m, 2H), 6.7 (d, J = 8.4 Hz, lH), 5.4 (sharp m, lH), 4.2 (q, 2H), 3.8 (broad s, 2H), 3.1 (m, 2H), 2.7 (s, 3H), 2.1-2.4 (m. 6H), 1.5 (t, J = 6.95 Hz, 3H). Anal C,,H,,NO,Cl W, N, Cl).

Endo-8-methyl-8-azabicyclo[3.2.I]octan-3-y1 4-bromo naphthalene-I -carboxylate hydrochloride 29 From the corresponding acid prepared from 4-bromo- 1-acetylnaphthalene 1401 (Aldrich, Strasbourg), the compound was obiained as white crystals from a mixture of isopropyl ether and MeOH (vield: 12%). mm > 260°C. IR (KBr) 1710 cm-t (C=O).~ ‘H-NMR (200 ‘MHi, CDCl,) 6: 8.9 (m, lH), 8.3 (m, lH), 7.8 (s, 2H), 7.6 (m, 2H), 5.4 (sharp m, lH), 3.8 (broad s, 2H), 3.2 (m, 2H), 2.7 (s, 3H), 2.4-2.2 (m, 6H). Anal C,gH,,NOzBrCl (C, H, N, Br). Method C (compounds 21,22,37,38,39) A 1.6 M hexane solution of n-BuLi (7.5 ml, 12 mmol) was added to a cooled (OOC) stirred solution of amino alcohol (11 mmol) under argon. The temperature was maintained at 0°C for 30 min when the addition of the reagent was completed. A solution of the crude acid chloride (10 mmol) in dry THF was added dropwise and the mixture was warmed to 25°C and stirred for 24 h. The solvent was distilled in vacua and the residue treated with 10% NaHCO, solution and extracted with 4 oortions (30 ml) of CHCl,. after which the organic layer was’ dried and evaporated. The crude material was purified by chromatography on neutral alumina with a mixture of MeOH/CHCl, (1:9). The compound was isolated in the basic form or transformed into an oxalate or hydrochloride salt. Endo-8-methyl-8-azabicyclo[3.2.l]octan-3-y1 phenanthrene-9carboxylate hydrochloride 21 From tronine and 9-nhenanthrene carbonvlchloride a yield of 49% was obtained. ‘Bright colorless crystals were obtained from a mixture of i-PrOJMeOH. mn: 214°C. IR (base. CHCl,) 1700 cm-l (C=O). tH-NMR (260 MHz, DMSO-d,) k: 9-S:; (m, 3H), 8.5 (s, lH), 8.16 (d, J = 7 Hz, lH), 7.9-7.6 (m, 4H), 5.3 (s, lH), 3.86 (broad s, 2H), 2.7 (s, 3H), 2.5 (m, 2H), 2.2-2 (m, 6H). Anal C,,H,,O,NCl (C, H, N, Cl). Endo-8-methyl-8-azabicyclo[3.2.l]octan-3-y1 acenaphthene-5 carbo,uylate oxalate 22 From tropine and 5-acenaphthene carbonylchloride, the reaction provided (yield: 24%) a beige amorphous solid from a mixture of i-Pr&/MeOH, mp: 212°C. IR (base, CHCl,) 1690 cm-’ (C=O). tH-NMR (200 MHz. DMSO-d,) 6: 8.64 (d. J = 8.5 Hz,‘lH),‘8.26 (d, J =‘ 7.4 Hz, iH), 7.69 (m, lH), 7.49 (m, 2H), 5.3 (m, lH), 3.92 (broad s, 2H), 2.8 (s, 3H), 2.75-2.5 (m, 2H), 2.5-2.1 (m, 5H). Anal C,,H?,O,N (C, H, N). 1-(3~,8-Dimethyl-8-azabicyclo[3.2.I]oct-3a-y1) naphthoate hydrochloride 37 1.1 g (64 %) of 37 (HCl), was obtained from 3P,8-dimethyl-8azabicvclol3.2.lloct-3a-ol (0.78 .g, 5 mmol) and 1-naphthalene carbonylchloride, mp: 222°C. IH-NMR (200 MHz, CDCl,) 6: 8.83-7.34 (m. 7H). 3.09 (broad s. 2H). 2.55 (d. / = 15 Hz. 2H), 2.20 (S, 3Hj, 1.98 (d, JX= 15 Hz, 2flj, 1.80‘(m, 4H), 1.66 (s, 3H). ‘“C-NMR (CDCl,): 166.78 (C=O), 133.70-124.32 (10 C,,.), 81.21 (-0-CH-), 60.13 (2C), 39.67 (lC), 28.02, 24.99. Anal CZOH,,NO,.HCl (C, H, Cl, N). I-(3~-n-Butyl-8-methyl-8-azabicyclo[3.2.I]oct-3a-y1) naphthoate hydrochloride 38 0.34 g (yield: 35%) of 38 (HCl) was obtained from 30-n-butyl8-methyl-8-azabicyclo[3.2.l]oct-3a-o1 (0.48 g, 2:4 mmbl) and 1-nanhthalene carbonvlchloride. mm 196°C. tH-NMR (200 MHz, CD,OD) 6: 9.OL7.70 (m, ‘7H): 4.12 (broad m, 2H), 3.18 (d, J = 16 Hz, 2H), 2.96 (s, 3H), 2.51-2.32 (m, 8H), 1.51 (m, 4H), 1.04 (t, J = 7 Hz, 3H). “C-NMR (CD,OD) 6: 167.68

879 (C=O), 135.45-125.97 (10 C,,), 8 1.89 (-0-CH-), 64.15 (2C), 40.14, 39.91, 25.89, 24.74, 23.68, 14.38. Anal C&H,,NO,,.HCl CC, H, Cl, N. I -(3-Methyl-3-azahi~yyclo[3.2.l]oct-~~-yl) naphthoate hydrochloride 39 From I-naphthalene carbonylchloride (1.15 g, 6 mmol) and 3methyl-3-azabicyclo[3.2.l]oct-Sp-ol (0.7g, 5 mmol), 39 was obtained (yield: 54%), mp: 261°C (dec). lH-NMR (200 MHz, CDCl,) 8:8.85-7.44 (m, jH), 5.08 (t, / = 5 Hz, lH), 3.34-3.09 (m. 4H). 2.69 (s. 3H). 2.57 (broad m. 2H). 2.51-2.42 (m. 2H). i.O’O-1.51 (m: 2Hj.’ WZ-hMR (CDCj;): 166.70 ’ (c=Oi: 133.67-124.32 (10 C,,), 74.54 (-0-CH-), 55.31 (2C), 45.71 (lC), 36.16 (2C), 24.58 (2C). Endo-R-methyl-8-azahi~y~lo[3.3.l]~~~ta~~-3-y1 quinoline-S-carkuylate oxalate 24 A solution of the crude acid chloride of 8-quinoline carboxylic acid (2.68 g, 14 mmol) in dried DMF (40 ml) was added dropwise at 0°C to a stirred solution of tropine (2.26 g, 16 mmol) in dry CHCI, (50 ml). After strirring for 24 h, the solvent was evaporated in vucuo. The residue was partitioned between CHCl, and a 10% K,CO, solution. The organic solution was decanted, washed with water and dried over Na,SO,. The solvent was removed by distillation to yield a dark brown oil. The compound was purified by column chromatography on silica gel with a mixture of solvents (MeOH/CHCl,, 1:9) to give an orange yellow oil which was converted to an oxalate salt. The compound was recrystallized in a mixture of i-Pro,/ MeOH to provide (yield: 24%) beige needles, mp: 202-204°C. IR (base, CHCI,) 1705 cm-’ (C=O). IH-NMR (200 MHz, D?O) 6: 9.21 (d, 2H), 8.75 (d, J = 7.4 Hz, lH), 8.53 (d, J = 8 Hz, IH), 8.15 (dd, IH), 8 (t, J = 8 Hz, lH), 5.46 (m, lH), 3.9 (s, 2H), 2.76 (s, 3H), 2.2-2.6 (m, 8H). Anal C10H220hN2r 1.5 H,O CC, H, N). E~zdo-8-n~ethyl-K-azahicyclo[3.2.l]octan-3-yl pyridine-2-car-ho.uylate hydrochloride 25 Carbonyldiimidazole (0.79 g, 4.6 mmol) was added in one portion to a solution of picolinic acid (0.5 g, 4 mmol) in 6 ml DMF. The mixture was stirred for 30 min at 25°C. Tropine (0.65 g) in 32 ml DMF was added dropwise to the solution at room temperature and the mixture stirred overnight. The solvent was removed by distillation in vacua and the crude material was taken off with CHCl, (15 ml), washed with a 10% K&O, solution and water, then dried over Na,SO,. After distillation of the solvent, the residue (0.57 g) was purified on neutral alumina with a CH,OH/MeOH (5:95) mixture. The compound was converted to.the hydrochloride’ salt to provide (vield: 4 1%) brinht colorless needles (from a mixture of i-Pro,/ ?H,OH), &p: ; 260°C. IR (CHdl,) 1710 cm-1 (C=O$. ‘H-NMR (200 MHz, D,O) 6: 8.38 (m, lH), 7.9-7.7 (m, 2H), 7.44-7.3 (m, IH), 5.05 (t, lH), 3.7 (broad s, 2H), 2.6 (s, 3H), 2.3 (m, 8H). Anal C,,H,,N20,Cl (C, H, N, Cl). Preparation of the 4-amino-5-chloro-2-methoxy-N-(suhstituted) benzamides 3: general procedure A suspension of 4-amino-5-chloro-2-methoxybenzoic acid (0.84 g, 4.2 mmol) in THF (20 ml) containing triethylamine (0.42 g, 4.2 mmol) was cooled in an ice-bath and ethyl chloroformate (0.43 g, 4.0 mmol) was added dropwise. This was allowed to warm to room temperature and stirred for 1 h. An amine solution (0.78 g, 4.0 mmol) was added. After stirred overnight. it was diluted with ethvl acetate, washed with NaOH (1 N, ?ZO’ml) and water. The organic layer was dried over sodium sulfate and concentrated. The crude benzamide was recrystallized or purified by chromatography.

4-Amino-5-chloro-2-methoxy-N-[(2~,4~,6~)-1,2,6-trimethylpiperidin-4-yll-benzamide 41 From the r&ture of the amines 14 and 1.5 (1 g, 7 mmol), a mixture of amides 41 and 42 was obtained (1.7 g. vield: 67%) and separated by chromatography on neutral alu&&ium oxide with a mixture of CH,Cl,/CHCl, as eluent. 41 was obtained (yield: 20%) after re&ysiallizatibn from CHCl,, mp: 193°C. ‘H-NMR (200 MHz. CDCl,) 6: 8.02 (s. 1H). 7.50 (d. .I = 7 Hz. lH), 6.22\s, lH), 4:32 (s, ?H), 4.01 idroad’t, J = 8 Hz, lH,,): 3.80 (s, OCH,), 2.40-2.20 (m, 2H), 2.26 (s, 3H), 1.99-1.93 (m, 2H), 1.30 (q, J = 12 Hz, 2H), 1.15 (d, .I = 6, 6H). Anal C,,H&1N,02 (C, H, N, Cl). 4-Amino-5-chloro-2-methoxy-N-~(2~,4u,6~)-l.2,6-trimethylpiperidin-4-yl]-benzamide 42. - ’ ’ From the nrevious reaction. 42 was obtained (vield: 22%) after chromatography and recrystallization from ethyl acetate/petroleum ether, mp: 235°C (dec). ‘H-NMR (200 MHz, CDCl,) 6: 8.03 (s, lH), 8.00 (d, J = 7 Hz, lH), 6.25 (s, lH), 4.35 (s, 2H), 4.27 (m, lH, ), 3.88 (s, 3H), 2.36 (m, 2H), 2.28 (s, 3H), 1.801.58 (m, 4Hj, 1.15 (d, ,I = 6 Hz, 6H). Anal C,,H&lN,O, (C, N, Cl), H,,,,: 7.43, Hluund: 6.89. 4-Amino-S-chloro-2-methoxy-N-[(2u,4~,6~)-l,2,6-trimethylpiperidin-4-ylf-benzamide 43 From the amine 12 (2.0 g, 10 mmol), 1.1 g compound 43 was obtained from crystallization in acetone, mp: 163°C. IH-NMR (200 MHz, CDCI,) 6: 8.05 (s, lH), 7.44 (d, lH), 6.22 (s, lH), 4.36 (m, 2H), 4.22 (m, IH), 3.77 (s, 3H), 3.12 (m, lH), 2.55 (m, IH), 2.24 (s, 3H), 1.95 (m, 2H), 1.62 (m, lH), 1.07 (m, lH), 1.03 (d, 3H), 0.97 (d, 3H). Anal C,,HZ,CIN,OI (C, H, N, Cl). 4-Amino-S-chloro-2-methoxy-N-[(2P,3au,6a~,9au)-dode~ahydropyrido[2,1,6-delquinolizin-2-yl]-benzamide 44 From 13b (0.78 8, 4.0 mmol), 1.13 g (yield: 74%) of 44 was obtained after re&ystallization from &hi1 acetate, mp: 196°C. ‘H-NMR (200 MHz. CDCl,) 6: 8.04 (s. 1H). 7.50 rd. J = 7 Hz. lH), 6.23 is, IH), 4.33 (s, I&), 4.11 (bioad’f, / = 1‘2’Hz, lH,,): 3.85 (s, 3H), 3.05 (broad d, J = 13 Hz, 2H), 2.74 (broad t, .I = 10 Hz, lH), 1.88-1.63 (m, 4H), 1.55-1.43 (m, lOH), 1.38-1.18 (m, 2H). “C-NMR (200 MHz, CDCI,): 163.48, 157.22, 146.77, 132.59, 111.95, 111.12, 97.59, 56.59, 55.86, 48.72, 47.98, 34.09, 30.39, 29.03, 19.42. Anal C,,,H&lN,02 (C, H, N). 4-Amino-5-~hloro-2-methnxy-N-(l-methylpiperidin-4-yl]-benzamide 40 This compound was prepared according to [42]. Pharmacological

methods

j-NT, binding assays Male Sprague-Dawley rats from Janvier Laboratory (France) were used. Animals were housed at 22 f l”C, with 55% humidity, on a 12-h light-dark cycle with free access to food and water for 4 d prior to the experiments. [3H]-BRL 43694 (61 Ci/mmol) was purchased from NEN research products. GR 38032F was a generous gift from Glaxo (UK). All other chemicals and reagents were commercially available from Sigma. The membranes were prepared from the rat posterior cortex for binding assays according to the procedure of Hall and Gozlan [41]. The binding of 1.2 nM [3H]-BRL 43694 (Kd = 1.5 nM, B,,, = 30 fmol/mg prot for 5-HT, receptor) was measured using membranes (loo-y1 aliquots equivalent to 0.95 mg protein) suspended in a final volume of 0.5 ml 50 mM Hepes, pH 8.4 and incubated at 25°C for

880 30 min. 7-11 concentrations of each drug were used in triplicate. Non-specific binding was determined by the addition of GR 38032F at 10 PM in duplicate. Total binding was defined in quadriplicate. Bound radioactivity was separated by vacuum filtration through Whatman GF/B glass filters, presoaked in a 0.1% polyethylenimine solution, using a Brandell cell harvester. The filters were then washed twice with 5 ml 50 mM Tris-HCl (pH 8.4) at room temperature and dried. The filters were placed in polyethylene vials to which were added 4 ml scintillation cocktail (Beckman, Ready-Safe) and after equilibration, the radioactivity was determined using liquid scintillation spectrometry. The data were analyzed by a computerassisted curve fitting programme in Lotus 1.2.3. to provide IC,,, K, and r2 values. Inhibition of the Bezold-Jarisch reflex Male Crl: CD(SD)BR rats (Charles River) weighing 280-320 g were fasted for 24 h, then anaesthetized with urethane (1.25 g/kg ip). In order to monitor the Bezold-Jarisch reflex (an abrupt dose-related reduction in cardiac rate, following a rapid iv bolus injection of 5HT, 5-30 pg/kg), the carotid artery was cannulated and connected to a Statham transducer. Heart rate and blood pressure were measured using the pressure transducer signal and a cardiotachometer coupler, and recorded on a Gemini polygraph (Ugo Basile, Italy). Test compounds were dissolved in water and administered iv (0.5 ml/kg), via a cannula placed in the jugular vein. For each agonist, an ED,,, value (ie the dose reducing heart rate by 50%) was calculated from the linear regression of the log dose-response curve. Antagonists were administered iv at various doses 3 min before a dose of 5-HT which, when given alone, reduced the heart rate by = 60%. ID,, values, which represent the dose of the antagonist inhibiting 50% of the effect of serotonin, were calculated from 3 doses (3 animals per dose) by a linear-regression analysis.

References :

3 4

; 10 11

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