University of Groningen

The 2-aminotetralin system as a structural base for new dopamine- and melatonin-receptoragentsCopinga, Swier

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CHAPTER 6

4-ARYL-2-AMIDOTETRALINS: NONINDOLIC MELATONIN-RECEPTOR

ANTAGONISTS AND AGONISTS*

The neurohonnone melatonin (N-acetyl-5-methoxytryptamine, I), synthesized and secreted primarily by the pineal gland and the retinas, appears to be an important modulator of a variety of physiological processes, like chronobiological rhythms (e.g. circadian activity) and neuroendocrinologicaVphysiological functions (e.g. sexual maturation, seasonal reproduction and retinal physiology) (see 4.2). At present, it is clear that the various actions of melatonin are mediated by specific melatonin receptors, which are not only localized in discrete regions of the central nervous system, but also in peripheral organs (for reviews, see ref. 1-6; see 4.4). The radioligand 2-[1251]iodo- melatonin is an useful probe for the localization of these specific melatonin receptors [7- 1 11. Likewise, indolic melatonin-receptor agonists (see 4.5), such as N-acyl-5- methoxytryptamines (2-4) and halogenated analogues of melatonin (5-9) [4,9,11-201, and melatonin-receptor partial agonists and antagonists (see 4.5), such as N- acyltryptamines (10,ll) and N-acetyl-2-aryltryptamines (12-14) [4,15,2 1-28], can be used for the preliminary pharmacological characterization of these specific melatonin receptors. However, a significant problem in further elucidating the mode of action of melatonin is the lack of potent and selective melatonin-receptor agonists and antagonists from other chemical classes than melatonin itself.

0CH3 0CH3 0CH3

v O\i\-R 0 b'$-cH3 0 "$, / N ' c - ~ ~ 3

Chart 6.1 Chemical structures of melatonin (1) and indolic melatonin-receptor agonists (2-9).

* This chapter is partially based on: Copinga S, Van Brummelen G, Drijfhout WJ, Tepper PG, Grol CJ, Dubocovich ML (Submitted for Publication) 4-Aryl-2-arnidotetralins: nonindolic melatonin-receptor agents.

Chart 6.2 Chemical structures of indolic melatonin-receptor partial agonists and antagonists (10-14). 12: N-acetyl-2-benzyltryptamine = N-0774 = luzindole 13: 2-benzylmelatonin = N-0745 = 5-methoxyluzindole

Recently, this problem has been solved partially by the introduction of benzo- [blthtophenic (15) and naphthalenic (16-19) bioisosteres of melatonin as melatonin- receptor agonists [17,18,29-321. Additionally, the recent development of 8-methoxy-2- amidotetralins (20-23) and 2-amidotetralins (24-27) as conformationally restricted, nonindolic melatonin-receptor agents has contributed to the solution of this problem [27,33-361. All these nonindolic melatonin-receptor agonists and partial agonists can be used to study the interaction of melatonin with its receptors.

Chart 6.3 Chemical structures of nonindolic melatonin-receptor agonists and partial agonists (15-27).

However, there is still a need for potent and selective melatonin-receptor antagonists, chemically unrelated to melatonin. Analogous to the idea behind the development of the only competitive melatonin-receptor antagonist luzindole (12), i. e. to produce a competitive melatonin-receptor antagonist by the appendage of a bulky lipophllic group to the 2-position of the indole nucleus of the melatonin-receptor antagonist or partial agonist N-acetyltryptamine (10) [23], we prepared two series of 2- amidotetralins with a lipophilic group attached to the 4-position of the semi-rigid tetralin system as potential melatonin-receptor antagonists, i.e. the 4-phenyl-2- amidotetralins 28-30 and the 4-benzyl-2-amidotetralins 3 1-33 [33].

Chart 6.4 Chemical structures of the 4-aryl-2-amidotetralins 28-33

The key intermediate in the first synthetic route, by which we prepared the 4- phenyl-2-amidotetralins 28-30, was 4-phenyl-2-tetralone (35), as outlined in Scheme 6.1. This 2-tetralone 35 was prepared according to a method previously described by Fine and Stem [37]. The method included as the fust step a base-catalyzed condensation between phenyl-2-propanone and benzaldehyde, followed by a dehydration. The product of this step was the a,P-unsaturated ketone trans-1,4-diphenyl-3-buten-2-one (34) in good yield. The second step of this method involved the ring closure of this ketone 34

Scheme 6.1 Reagents: (a) NaOH, A; (b) AlCl3, CS2; (c) PhCH2NH2, p-TsOH.H2O; (d) NaCNBH3; (e) Pd-on-C (lo%)), H2; (f) (RC0)20, CH3COONa; (g) RCOCl, NaOH.

to 4-phenyl-2-tetralone (35). The ring closure was performed via an intramolecular Friedel-Crafts alkylation reaction with the Lewis acid aluminum chloride in carbon disulfide. Because of the low yield of this ring closure (170/0), we tried two other solvents, namely dichloromethane and 1,1,2,2-tetrachloroethane, for this reaction. In both solvents the ring closure did not proceed at all. Instead of a Lewis acid we also used a proton acid (polyphosphoric acid) to bring about this ring closure. This reaction was performed at two different temperatures, namely 50 "C and 90 "C. Under both circumstances the ring closure failed.

4-Phenyl-Ztetralone (35) was converted to 4-phenyl-2-aminotetralin (37) by a well known sequence of reactions, namely a condensation with benzylamine, a reduction and a catalytic debenzylation. Because the catalyhc reduction of the imine, resulting fiom the condensation, with platinum(1V) oxide under an atmosphere of hydrogen and ethanol as solvent failed, this reduction was carried out chemically with sodium cyanoborohydride and a mixture of methanol and tetrahydrofuran (1 : 15) as solvent. The preparation of 4-phenyl-2-(acetamido)tetralin (28) and 4-phenyl-2-@ropionamido)- tetralin (29) from 4-phenyl-2-aminotetralin (37) was conducted by the use of the appropriate anhydride in the presence of sodium acetate and the biphasic medium waterlethylacetate. The preparation of 4-phenyl-2-(ch1oroacetamido)tetralin (30) from 4-phenyl-2-aminotetralin (37) was conducted by the use of chloroacetyl chloride in the presence of sodium hydroxide and the biphasic medium waterldichloromethane according to the Schotten-Baumann procedure.

The overall yields for the 4-phenyl-2-amidotetralins 28-30, prepared according to this synthetic route (Scheme 6. l), were very low [e.g. 1.6% for 4-phenyl-2-(acetamid0)- tetralin (28)]. With the aid of 1~ NMR-spectroscopy (Table 6.1) and single-crystal X- ray crystallography (Figure 6.1) we were able to show that this synthetic route leads to the preparation of the cis-isomers of the 4-phenyl-2-amidotetralins 28-30.

Table 6.1 IH NMR spectral data (200 MHz) of the 4-phenyl-2-arnidotetralins 28-30 in CDC13. and, not determined.

chemical shifts (6, ppm) compound

H ~ a x Hleq H2ax Wax Weq H4ax

28 2.79 3.23 4.36 1.79 2.41 4.24 29 2.80 3.23 4.38 1.79 2.42 4.24 30 2.88 3.26 4.39 1.87 2.45 4.26

coupling constants (J, Hz) compound

Jlax,leq Jlax,2ax Jleq,2ax J2ax,3ax J2ax,3eq J3ax,3eq J3ax,4ax J3eq,4ax

28 -15.8 11.0 4.5 12.0 nda -12.0 11.9 5.6 29 -15.8 10.7 3.7 12.0 nd -12.0 11.7 5.8 30 -15.8 11.0 5.2 12.1 nd -12.1 11.9 5.9

Figure 6.1 Molecular structure and atom numbering scheme of 4-phenyl-2-(propionamido)tetralin (29).

Due to the very low overall yields we tried to find a more optimal synthetic route to prepare the 4-phenyl-2-amidotetralins 28-30. It appeared to us that the synthetic route, as outlined in Scheme 6.2, is an alternative to the first synthetic route. In this second synthetic route, by which we prepared the 4-phenyl-2-amidotetralins 28-30, the crucial intermediate was 4-phenyl-1-tetralone (41). The first step en route to this 1-tetralone 41 was a Friedel-Crafts acylation reaction between benzene and succinic anhydride with aluminum chloride as the Lewis acid, yielding 3-benzoylpropionic acid (38) (85%). The next step was the reductive ring closure of this propionic acid 38 to y-phenyl-y- butyrolactone (39). Hydrogenation with palladium-on-carbon as catalyst and methanol as solvent did not proceed well [38]. Varying the solvent (dioxane instead of methanol), the pressure of hydrogen (3 atrn. instead of 1 atrn.) and the addition of a base (ammonia or triethylamine instead of no base) did not result in the desired lactone 39 or 4- hydroxy-4-phenylbutyric acid. This butyric acid could undergo spontaneously ring closure on standing. However, the reductive ring closure, described previously by Meyer and Vaughan [39], using sodium borohydride to reduce 3-benzoylpropionic acid (38) to 4-hydroxy-4-phenylbutytlc acid and hydrochloric acid to destroy the formed borates and to support the intramolecular lactone formation of y-phenyl-y-butyrolactone (39) succeeded very well (81%). Subsequently, this lactone 39 was converted to 4,4- diphenylbutync acid (40) trough a Friedel-Crafts alkylation reaction with aluminum chloride as the Lewis acid. The last step in this sequence to get the important intermediate 4-phenyl-1-tetralone (41) was the cyclization of this butync acid 40 via an intramolecular Friedel-Crafts acylation reaction. The use of polyphosphoric acid (PPA) had the advantage that it not only produced the protons, required for the reaction, but it also withdrew the water, formed in the reaction. The often occurring aromatization in this sort of circumstances did not take place. Since the reacting butyric acid 40 is completely symmetric, only one product was formed, namely 4-phenyl- 1-tetralone (41), in very good yield (87%).

NOTs (yoH~$-$k / / 0

44 4547 28-30

Scheme 6.2 Reagents: (a) AICl3; (b) NaBQ; (c) AICl3, CgHg; (d) PPA, A; (e) NH20H; (f) p-TSCI; (g) (CH3)3COK, HCI (36%); (h) (RC0)20, CH3COONa; (i) RCOCI, (C2H5)3N; (j) Pd- on-C (lo%), Ha, CH3COOH / HClO4 (70%).

The transformation of the 1-tetralone 41 to the 2-amino-1-tetralone 44 included the so-called Neber rearrangement. This Neber rearrangement uses as reactant an 0- tosyloxime due to the good leaving-group ability of the tosylate. Thus, 4-phenyl-1- tetralone (41) was converted first to 4-phenyl-1-tetralone 0-p-toluenesulfonyloxime (43). This conversion involved the formation of oxime 42 using hydroxylamine, followed by 0-tosylation using p-toluenesulfonylchloride. Neber rearrangement of 0- tosyloxime 43 by utilizing potassium t-butoxide as proton acceptor afforded 4-phenyl-2- amino-1 -tetralone (44), isolated as its hydrochloride salt, in a fairly good yield (57%). Subsequently, this Zamino-1-tetralone 44 was converted to the 4-phenyl-2-amido-1- tetralones 45-47 by the use of the appropriate anhydride in the presence of sodium acetate and the biphasic medium waterlethylacetate or the appropriate acid chloride in the presence of triethylamine and the monophasic medium dichloromethane. Ultimately, the 4-phenyl-2-amidotetralins 28-30 were prepared from these 2-amido-1-tetralones 45- 47 via a catalyhc reduction with palladium-on-carbon as catalyst under an atmosphere of hydrogen.

1~ NMR-spectroscopy revealed that this synthetic route leads also to the preparation of the cis-isomers of the 4-phenyl-2-amidotetralins 28-30. The overall yields for the 4-phenyl-2-amidotetralins 28-30, prepared according to this synthetic route (Scheme 6.2), were low [e.g. 3.2% for 4-phenyl-2-(acetamid0)tetralin (28)l. Nevertheless, although three more reaction steps are involved, these yields are considerable higher than those for the same 4-phenyl-2-amidotetralins 28-30, prepared via the intermediate 4-phenyl-2-tetralone (35) (Scheme 6.1).

The synthetic route, by which we prepared the 4-benzyl-2-amidotetralins 31-33, is outlined in Scheme 6.3. This synthetic route is very similar to the above-described second synthetic route, by which we prepared the 4-phenyl-2-amidotetsalins 28-30 (Scheme 6.2). The key intermediate in this synthetic route is 4-benzyl-1-tetralone (50). The synthesis of this 1-tetralone 50 included three steps. The first step was the formation of 6-chloro-y-valerolactone (48) from diethyl malonate and epichlorohydnn in reasonable yield (54%). Subsequently, this y-valerolactone 48 was converted very

NOTs

50 ' 53 '

Scheme 6.3 Reagents: (a) Na, C2H50H, HCI (36%), 4 (b) AIC13, C6H6; (c) PPA, A; (d) NH20H; (e) p-TsC1; (f) (CH3)3COK, HC1(36%); (g) (RC0)20, CH3COONa; (h) Pd-on-C (lo%)), H2, CH3COOH I HClO4 (70%).

efficiently to 4,5-diphenylvaleric acid (49) trough Friedel-Crafts alkylation reactions (86%). The last step to obtain the crucial intermediate 4-benzyl-1-tetralone (50) was the cyclization of this valeric acid 49 via an intramolecular Friedel-Crafts acylation reaction with aluminum chloride as Lewis acid (75%).

The 4-benzyl-2-amidotetralins 31-33 were prepared from 4-benzyl- 1-tetralone (50) by the same sequence of reactions, as used for the transformation of 4-phenyl-l- tetralone (41) to the 4-phenyl-2-amidotetralins 28-30 (see 6.2.1), i.e. a Neber rearrangement, an amide formation and a catalytic reduction. With the aid of 1~ NMR- spectroscopy (Table 6.2) and single-clystal X-ray crystallography (Figure 6.2) we were able to show that this synthetic route leads to the preparation of the trans-isomers of the 4-benzyl-2-amidotetralins 31-33.

Figure 6.2 Molecular structure and atom numbering scheme of 4-benzyl-2-(acetarmdo)tetralin (31).

Table 6.2 IH NMR spectral data (200 or 300 MHz) of the 4-benzyl-2-amidotetralins 31-33 in CDCI3. %bs, obscured. bnd, not determined.

chemical shifts (6, ppm) compound

H lax Hleq H2ax H3ax H3eq H4eq

31 2.83 3.08 4.48 1.62 1.90 obsa 32 2.81 3.03 4.46 1.57 1.86 obs 33 2.78 3.03 4.46 1.62 1.88 obs

coupling constants (J, Hz) compound

6.3 PHARMACOLOGICAL EVALUATION OF 4-ARYL-2-AMIDOTETRALINS

The 4-aryl-2-amidotetralins 28-33 were evaluated for their in vztro affinities at melatonin receptors by examining their abilities to compete for 2-[1251]-iodomelatonin binding to chicken retinal membranes (Table 6.3). This radioligand binding assay was conducted essentially as reported by Dubocovich and Takahashi [9].

Table 6.3 Pharmacological evaluation of the 4-aryl-2-amidotetralins 28-33 as compared with some indolic as well as nonindolic melatonin-receptor agents. acornpetition for 2-[1251]iodo- melatonin binding to chicken retinal membranes by various concentrations (0.1 nM - 0.1 rnM) of the test compounds. b ~ ; values were calculated from I C ~ O values obtained from competition curves by the method of Cheng and Prusoff [40]. Results are mean values of at least three independent determinations in d~plicate.~Inhibition by various concentrations of the test compounds (1 pM - 1 pM) of the calciumdependent release of [3~dopamine from rabbit retina. d ~ ~ 5 0 values were determined graphically from concentration-effect curves. eThe maximal inhibition of the calciumdependent [3HIdoPamine release obtained with 1 pM of the test compound. f~ntagonism by various concentrations of the test compounds (0.1 nM - 1 pM) of the inhibition, induced by various concentrations of melatonin (0.1 nM - 10 nM), of the calciumdependent release of [3~dopamine from rabbit retina. gML, melatonin (1); DA, dopamine. h ~ B values were calculated from concentratlon-effect curves using the method of Arunlakshana and Schild [4 11. 'See also ref. 35,36,42. JNAT, N-acetyltryptamine (10). k ~ e e also ref. 9,15,23,25,27. IND, not determined. mLUZ, luzindole, N-acetyl-2- benzyltryptamine (12). "See also ref. 23,25,27. ONE, not effective.

competition for inhibition of antagonism of ML-induced 2-[125~]iodomelatonin bindinga [3~]dopamine overflowC inhibition of [ 3 ~ ~ ~ overflowfZ

% max. comp. Ki, nMb ICSO, n ~ d inhibitione Kg, nMh

~ ~ g , i 0.57 0.02 80 --

Additionally, these 4-aryl-2-arnidotetralins 28-33 were evaluated for their in vitro potencies at melatonin receptors by examining their abilities to inhibit the calcium- dependent release of [ 3 ~ ] d o ~ a m i n e from rabbit retina via activation of presynaptic melatonin heteroreceptors andfor to antagonize the melatonin-induced Inhibition of the [ 3 ~ ] d o ~ a m i n e release in this assay by blockade of these presynaptic receptors (Table 6.3) (see 4.4.3). These in vitro bioassays were performed as described by Dubocovich [15,23,33].

Evaluation of the cis-4-phenyl-2-amidotetralins 28-30 for their in vitro affrnities and potencies at melatonin receptors revealed that these agents possess moderate melatonin-receptor affinities (120 nM I Ki I 500 nM), whereas they are unable to inhibit the calcium-dependent retinal release of dopamine as melatonin (1) and 8- methoxy-2-amidotetralins 20,21, and 23 can do. Moreover, it was shown that the cis-4- phenyl-2-amidotetralins 28-30 possess the ability to effectively antagonize the melatonin-induced inhibition of the calcium-dependent release of dopamine fiom rabbit retina (1.0 nM I Kg I 3.1 nM). In comparison with the affinities of the 2-amidotetralins 24, 25, and 27 at melatonin receptors, the cis-4-phenyl-2-amidotetralins 28-30 show the same order of melatonin-receptor affinities [acetarnide (24128) < propionamide (25129) < chloroacetarnide (27/30)]. However, whereas the 2-amidotetralins 24, 25, and 27 act as melatonin-receptor partial or full agonists in the dopamine-release assay, the cisd- phenyl-2-amidotetralins 28-30 act as melatonin-receptor antagonists in this assay. Hence, the cis-appendage of a lipophilic phenyl group to the 4-position of the tetralin system of 2-amidotetralins has no influence on their melatonin-receptor affinities, but, in contrast, is totally detrimental to their melatonin-receptor potencies. This agrees very well with the development of the competitive melatonin-receptor antagonist luzindole (1 2) from the melatonin-receptor partial agonist N-acetyltryptarnine (1 0) [23]. Comparing the melatonin-receptor antagonist properties of the cis-4-phenyl-2-amido- tetralins 28-30 (1.0 nM 5 Kg I 3.1 nM) and luzindole (12) (Kg = 20 nM), it is evident that the cis-4-phenyl-2-amidotetralins 28-30 are about 10 times more potent melatonin- receptor antagonists than luzindole (12).

Evaluation of the trans-4-benzyl-2-amidotetralins 31-33 for their in vitro affrnities at melatonin receptors revealed that these agents possess moderate melatonin-receptor &ties (85 nM I Ki I 200 nM). In addition, it was shown that these agents possess the ability to inhibit the calcium-dependent retinal release of dopamine. However, these agents are less potent than melatonin and the conformational restricted 8-methoxy-2- amidotetralins 20, 21, and 23 in this assay. The properties of the transd-benzyl-2- amidotetralins 31-33 are quite comparable with those of the 2-amidotetralins 24, 25, and 27. Thus, the trans-appendage of a lipophilic benzyl group to the 4-position of the tetralin system of 2-amidotetralins does not give rise to major changes in melatonin-

receptor affinity and potency, i.e. the trans-4-benzyl-2-amidotetralins 30-33 act as melatonin-receptor agonists of moderate amity and potency.

In summary, the cis-4-phenyl-2-amidotetralins 28-30 are nonindolic melatonin- receptor antagonists of moderate affinity, which are, however, about 10 times more potent than the indolic melatonin-receptor antagonist luzindole (12). In contrast, the trans-4-benzyl-2-amidotetralins 31-33 are nonindolic melatonin-receptor agonists of moderate affinity and potency, similar to the 2-amidotetralins 24, 25, and 27.

Melting points were determined in open glass capillaries on an Electrothermal hgital melting-point apparatus and are uncorrected. IR spectra were recorded on a Philips PU 9706 spectrophotometer or on a Beckman AccuLab 2 spectrophotometer, and only the important absorptions are given. IH NMR spectra were recorded on a 60 MHz Hitachi Perkin-Elmer R-24 B spectrometer, on a 200 MHz Varian Gemini 200 spectrometer, or on a 300 MHz Varian VXR-300 spectrometer. Chemical shifts are reported in 6 units (parts per million) relative to (CH3)4Si as an internal standard or via 6 CDC13 (7.24) or (CD3)2SO (2.49). Chemical-ionisation (CI) mass spectra, using NH3 as reactant gas, were obtained with a Finnegan 3300 system. Elemental analyses and single-crystal X-ray analyses for new subtances were performed at the Department of Chemistry, University of Groningen. Where elemental analyses are indicated, obtained results were within 0.4% of the theoretical values, except where noted. All mentioned yields are unoptimized.

Trans-!, 4-diphenyl- 3-buten-2-one (34) Phenyl-2-propanone (38.6 g, 0.29 mol) and benzaldehyde (30.5 g, 0.29 mol) were added to a well stirred solution of NaOH (3.1 g) in H20 (1.4 I), which was kept at 60 OC. The reaction mixture was mechanically stirred for 22 h at the same temperature. After cooling to room temperature, the reaction mixture was decanted, the remaining yellow precipitate dissolved in CHC13, and the H20 layer extracted with CHC13. After combining the CHC13 layers, the organic layer was washed with H20 ( 3 4 and a saturated aqueous solution of NaCl (lx) and dried over MgS04. Removal of the solvent under reduced pressure yielded 60.2 g (0.270 mol, 94%) of crude trans-l,4-diphenyl-3-buten-2-one (34) as an oil, whlch solidified on standing. Recrystallization of this crude product from MeOHhexane yielded 26.5 g (0.12 mol, 41%) of truns-1,4diphenyl-3-buten-2-one (34) as light yellow crystals: mp 69-72 OC ([37] mp 73-76 OC, MeOH; [43] mp 68-70 OC; [44] 66-69 OC); IR (cm-l, KBr) 1655 (C=O); IH NMR (60 MHz, CDC13) 6 3.9 (s, 2H, CH2), 6.75 (d, IH, CHPh, J = 16.5 Hz), 7.3 (s, 5H, ArH), 7.4 (s, 5H, ArH), 7.65 (d, lH, COCH, J = 16.5 Hz); MS (CI with NH3) m/z 223 (M+l), 240 (M+18).

4-Phenyl-2-tetralone (35) To a suspension of anhydrous, finely powdered AICl3 (30.4 g, 0.23 mol) and dry CS2 (380 ml) was added dropwise under an atmosphere of nitrogen and vigorously stirring a solution of the 3-buten-2-one 34 (25.0 g, 0.11 mol) in dry CS2 (270 ml). After stirring for 10 min at room temperature, the reaction mixture was refluxed for 2 h (T = * 45 OC). Subsequently the reaction mixture was poured carefully into

a strongly stirred solution of ice (I kg) and 36% HCI (100 ml). After stining for 0.5 h, the H20 layer was decanted and extracted with Et2O (3 x 250 ml). After combining the organic layers, the resulting organic layer was washed with H z 0 (2 x 500 rnl) and evaporated under reduced pressure. The resulting residue was dissolved in Et20 (250 ml) and this Et20 layer was washed with 10% NaOH (3 x 150 rnl) and subsequently with a saturated aqueous solution of NaCl(2 x 100 ml) and dried over MgS04. After in vacuo evaporation of the volatiles, a dark brown oil was afforded. The crude oil was purified by vacuum distillation to yield 4.2 g (19 mmol, 17%) of 4-phenyl-2-tetralone (35) as a light yellow oil: bp 115-125 OC (0.01 mbar) [[37] bp 132-136 OC (0.12 mmHg)]; IR (cm-l, neat) 1705 (C=O); IH NMR (60 MHz, CDC13) 6 2.9 (d, 2H, CHz), 3.6 (s, 2H, CH2), 4.4 (t, lH, CH), 6.9-7.5 (m, 9H, ArH); MS (CI with NH3) m/z 223 (M+ l), 240 (M+18).

4-Phenyl-2-(N-benzylamino)tetralin (36) Under an atmosphere of nitrogen, a solution of 2-tetralone 35 (4.20 g, 18.9 mmol), benzylamine (2.0 g, 19 mmol), and a couple of crystals of p-toluenesulfonic acid monohydrate in dry benzene (35 ml) was refluxed for 1.5 h under continuous removal of H20 using a Dean and Stark apparatus. The volatiles were removed under reduced pressure and the residue was dissolved in dry MeOH (3 rnl) and dry THF (45 ml). The pH of the resulting solution was brought down to 4 with dry ethereal HCI and a precipitate appeared. To this stirred solution was added in small portions, under an atmosphere of nitrogen, NaBH3CN (1.55 g, 24.7 mmol). The reaction mixture was heated to 40 OC. When after a while the pH of the reaction mixture rose, the pH was brought down again to 4 with dry ethereal HC1. After stirring for 20 h at room temperature, the reaction mixture was poured into H20 (50 ml) and this two layer system was made basic by using NaHC03. After separation of the two layers, the H20 layer was extracted with Et20 (3 x 25 ml) and the Et20 layers were combined, decolourized with charcoal, and dried over MgS04. Evaporation of the Et2O under reduced pressure yielded crude 4-phenyl-2-(N-benzyl- amino)tetralin (36) as an oil. Conversion of the crude amine to its HCI salt and recrystallization from EtOH/Et20 yielded 2.91 g (8.3 mmol, 44'Yo) of the HCI salt of 4-phenyl-2-(N-benzy1amino)tetralin (36): mp 204.5-206.3 OC; IR (cm-l, KBr) 2850-2300 (NH2+); MS (CI with NH3) rn/z 314 (M+l) (M: free amine).

4-Phenyl-2-aminotetruiin (37) 4-Phenyl-2-(N-benzy1amino)tetralin (36) (0.38 g, 1.2 mmol) was dissolved in absolute EtOH (10 ml), 10% Pd-on-C catalyst (0.25 g) was added, and the solution was debenzylated in a Parr hydrogenation flask for 21 h at 35 "C under a H2 pressure of 3 atmospheres. After filtering off the catalyst, the volatiles were removed under reduced pressure to give a brown oil. This oil was taken up in Et20 and precipitated as its HCI salt by dry ethereal HCI. The yield was 0.18 g (0.7 mmol, 57%) of the HCI salt of 4-phenyl-2- aminotetralin (37) as a white solid: mp 237-238 "C dec; IR (cm-l, KBr) 3200-2400 (NH3+), 2040 (NH3+); MS (C1 with NH3) m/t 224 (M+l) (M: free mine).

4-Phenyl-2-(acetamido)tetralin (28) Acetic anhydride (0.40 ml, 4.2 mmol) was added dropwise at room temperature to a well stirred mixture of 4-phenyl-2-aminotetralin hydrochloride (37-HC1) (0.18 g, 0.7 mmol), NaOAc (0.36 g), EtOAc (5 ml), and H20 (2 ml). After 24 h of stirring and diluting the mixture with Hz0 (3 ml), the phases were separated and the H z 0 layer was extracted twice with EtOAc (10 ml). Subsequently the EtOAc layers were combined and washed with saturated aqueous solutions of NaHC03 (3 x 10 ml) and NaCl (1 x 10 ml) and then dned over MgS04. Evaporation of the solvent under reduced pressure yielded 0.17 g (0.6 mmol, 93%) of 4-phenyl-2-(acetamido)tetralin (28) as a white solid: IR (cm-l, KBr) 3275 (NH), 1635 (C=O: arnide I), 1545 (C=O: amide 11); NMR (200 MHz, CDC13) 6 1.79 (q, lH, CCH,CN, J = 12.0 Hz, 12.0 Hz, 1 1.9 Hz), 1.97 (s, 3H, COCH3), 2.4 1 (m, lH, CC%CN), 2.79 (dd, IH, ArCH,CN, J =

15.9 Hz, 11.0 Hz), 3.23 (dd, lH, ArCHe CN, J = 15.7 Hz, 4.5 Hz), 4.24 (dd, lH, ArCHaxPh, J = 11.8 Hz, 5.6 Hz), 4.36 (m, lH, CHaxCN), 5.96 (bd, lH, NH), 6.77-736 (m, 9H, ArH); MS (CI with NH3) d z 266 (M+l); Anal. (C18H19NO) C, H, N.

4-Phenyl-2-(propionamido)letralin (29) Propionic anhydride (0.30 ml, 2.3 mmol) was added dropwise at room temperature to a well stirred mixture of 4-phenyl-2-aminotetralin hydrochloride (37HCl) (0.10 g, 0.4 mmol), NaOAc (0.20 g), EtOAc (5 ml), and H z 0 (2 ml). After 18 h of stirring and diluting the mixture with H z 0 (3 ml), the phases were separated and the H20 layer was extracted twice with EtOAc (10 ml). Subsequently the EtOAc layers were combined and washed with saturated aqueous solutions of NaHC0-j (3 x 10 ml) and NaCl (1 x 10 ml) and then dried over MgS04. Evaporation of the solvent under reduced pressure yielded an oil. After addition of a small amount of Et20 to this oil, the solution was placed into liquid nitrogen and crystalli- zation took place. Filtration yielded 0.06 g (0.2 mmol, 56%) of 4-phenyl-2-(propionamido)-tetralin (29) as white crystals: IR (cm-l, KBr) 3290 (NH), 1640 (C=O: amide I), 1550 (C=O: amide 11); 1~ NMR (200 MHz, CDC13) 6 1.15 (t, 3H, CH3), 1.79 (q, lH, CCH,CN, J = 12.0 Hz, 12.0 Hz, 1 1.9 Hz), 2.15 (q, 2H, COCH2), 2.42 (m, lH, CC CN), 2.80 (dd, lH, ArCHaxCN, J = 15.8 Hz, 10.7 Hz), 3.23 (dd,

Hy ,,, CN, J = 15.9 Hz, 3.7 "ss H z , 4.24 (dd, lH, ArCHaxPh, J = 11.4 Hz, 5.8 Hz), 4.38 (m, lH, CH,CN), .57 (bd, lH, NH), 6.78-7.36 (m, 9H, ArH); MS (CI with NH3) m/z 280 (M+l). Single-crystal X-ray analysis of 4-phenyl-2-(propionamido)tetralin (29): Reasonable crystals of 4-phenyl-2-(propionamido)tetralin (29) were grown from EtOH by slow evaporation of the solvent. A crystal, having approximate dunensions of 0.45 x 0.40 x 0.35 mm3, was mounted on a glass fiber in a random orientation. Cell constants and orientation matrix for the data collection were obtained from least-squares refinements, using the angular settings of 25 reflections (13.0 ' < 8 < 17.0'). The monoclinic cell parameters and volume are: a = 9.745 A (I), b = 14.489 A (I), c =

11.698 A (l), P = 106.50' (1) and V = 1583.7 A3. For Z = 4 and F.W.= 279.39 the calculated density (dcalcd) is 1.17 &m3. The data collection was performed at 293 K with Mo Ku radiation (h = 0.71073 A) on a Nonius CAD4F difiactometer equipped with a graphite monochromator and interfaced to a VAX-730, using the 8/28 scan method. The intensities of three standard reflections, which were measured every 3 h, were used to control drift in the primary beam and counting system and also possible decrease of the crystal quality. From a total of 50 16 reflections (I0 < 8 5 3 1 "), 2 132 reflections with I > 3 0 0 were used in the refinements. Scaling factors, Lorentz and polarization corrections, and empirical absorption corrections were applied to the data. The linear absorption coefficient (k) is 0.7 cm-l: minimum and maximum transmission coefficient of 0.994 and 1.000, respectively. The structure was partly solved by direct methods using SDPIPDP computer software. The positions of the other atoms were located from succeeding Fourier difference synthesis maps. Block-diagonal least-squares of F, with unit weight, converged to a final R = 0.097 and Rw = 0.099 (w = l), respectively, including 25 1 variable parameters, using anisotropic temperature factors for the non-hydrogen atoms and fixed isotropic temperature factors (B = 5.0 A2) for the hydrogen atoms. In the final refinements the hydrogen atoms were riding on their corresponding atoms at a distance of 0.97 i\. The molecular structure as well as the atom numbering scheme are given in Figure 6.1.

4-Phenyl-2-(ch1oroacetamido)tetralin (30) Chloroacetyl chloride (0.13 ml, 1.6 mmol) was added dropwise at room temperature to a vigorously stirred mixture of 4-phenyl-2-aminotetralin hydrochloride (37.HC1) (0.13 g, 0.5 mmol), CH2CI2 (6 ml) and 10% NaOH (1.4 ml). After 3 h of stining at room temperature, the reaction mixture was poured into H20 (10 ml) and the phases were separated. Subsequently the Hz0 layer was extracted with CH2C12 (2 x 15 ml) and the combined organic layers were washed with a saturated aqueous solution of NaHC03 (3 x 15 ml) and H20 (1 x 15 ml), dried over MgS04 and then evaporated under reduced pressure to yield an oil. After addition of a small amount of Et20 to this oil, the solution was placed into liquid nitrogen

and crystallization took place. Filtration yielded 0.01 g (0.03 mmol, 6%) of 4-phenyl-2-(chloroacet- amido)tetralin (30) as white crystals: IR (cm-l, KBr) 3300 (NH), 1640 (C=O: amide I), 1540 (C=O: amide 11); IH NMR (200 MHz, CDC13) 6 1.87 (q, IH, CCH,CN, J = 12.1 Hz, 12.1 Hz, 12.0 Hz), 2.45 (m, lH, CCH, CN), 2.88 (dd, lH, ArCHaxCN, J = 15.8 Hz, 11.0 Hz), 3.26 (ddd, lH,

CN, J = 15.8 ?Iz, 5.2 Hz, 2.0 Hz), 4.05 (d, 2H, COCH2CI), 4.26 (dd, IH, ArCHaxPh, I = 1 1.8 gk), 4.39 (m, lH, CH=CN), 6.58 (M, IH, NH), 6.80-7.37 (m, 9H, ArH); MS (CI with NH3) m/z 300 (M[CI=35]+1), 302 (M[C1=37]+1), 317 (M[C1=35]+18), 319 (M[C1=37]+18).

3-Benzoylpropionic Acid (38) To a vigorously stirred solution of succinic anhydride (34.0 g, 0.34 mol) in dry benzene (200 ml, 2.2 mol) was added at once 100.0 g of anhydrous, finely powdered AIC13 (0.75 mol). Slowly a reaction became apparent, hydrogen chloride evolved and the reaction mixture became hot. The hydrogen chloride was removed by a constant stream of nitrogen and absorbed in H20. The reaction mixture was gently refluxed for 2 h. After cooling to room temperature, Hz0 (150 ml) and 36% HCI (50 ml) were added dropwise and a white precipitate was formed. Removal of the benzene layer by azeotropic distillation gave rise to the separation from the reaction mixture of a brown oil, which solidified. After cooling by ice, the solid was collected by suction filtration, washed with cold aqueous 2N HCI (100 ml) and H20, until the pH of the filtrate was neutral. The crude acid was boiled for 15 min in a 15% aqueous solution of Na2C03 (250 ml). This hot solution was filtered to remove the small amount of AI(OH)3. After decolourization of this filtrate with charcoal, 36% HCl (70 ml) was added slowly and a white precipitate appeared. This mixture was cooled by ice and the acid was collected by suction filtration, washed with cold H20 and dried in a vacuum desiccator for several nights. The yield of 3-benzoylpropionic acid (38) as a white powder was 51.5 g (0.29 mol, 85%): mp 116-1 17 OC ([45] mp 116.5 OC, H20; [46] mp 114- 116 "C, acetondhexane); IR (cm-I, KBr) 3400-2300 (OH), 1690 (C=O); IH NMR (60 MHz, CDCI3) 6 2.8 (t, 2H, CH2), 3.4 (t, 2H, CH2), 7.3-8.1 (m, SH, ArH), 11.0 (s, IH, OH).

y-Phenyl-y-butyrolactone (39) Sodium borohydride (23.0 g, 0.61 mol) was added pomonwise to a mechanically stirred and ice-cooled solution of propionic acid 38 (65.0 g, 0.36 mol) in 96% EtOH (600 ml) under an atmosphere of nitrogen. Subsequently aqueous 3N HCI was added dropwise until pH 2 was reached. After evaporation of the volatiles under reduced pressure, a white solid was left and this solid was dissolved in Et20 (500 ml), washed with a saturated aqueous solution of NaHCOj (2 x 500 ml) and a saturated aqueous solution of NaCl (1 x 500 ml) and dried over Na2S04. After in vacuo evaporation of the Et20, a pale yeHow oil remained. This oil was almost pure y-phenyl-y-butyrolactone (39) (47.5 g, 0.29 mol, 81%): IR (cm-l, NaCl) 1780 (GO); NMR (60 MHz, CDC13) 6 5.4 (t, IH, CH), 7.3 (s, SH, ArH). Because this oil crystallized very slowly on standing [mp 35-36 OC ([38] mp 38 OC; [39] mp 34-36 OC; [47] mp 37 "C; [48] mp 37-38 "C), the oil was used in the next reaction.

4,4-Diphenylbutyric Acid (40) To a mechanically stirred solution of y-butyrolactone 39 (50.0 g, 0.3 1 mol) in sodium-dried benzene (500 ml) was added slowly 50.0 g of anhydrous, finely powdered AICl3 (0.37 mol). During the addition the temperature of the reamon mixture rose to approximately 40 OC and was kept at this height by using an ice-bath. Evolved hydrogen chloride was removed by a constant stream of nitrogen and absorbed in H20. After addition of the whole amount AlCl3, the reaction mixture was refluxed for 4.5 h and subsequently poured into a mixture of ice (800 g) and 36% HCI (50 ml). The formed whitebrown solid was collected

by suction filtration. From the filtrate the benzene was separated and evaporated under reduced pressure. The H20 layer was extracted with CH2CI2 (3 x 400 ml). These CH2CI2 layers were combined, evaporated under reduced pressure and the residue was combined with the residue of the benzene layer. The combined residue was dissolved in Et20 (600 ml) and this Et20 layer was washed with a saturated aqueous solution of NaCl(300 ml) and dried over Na2S04. After in vacuo evaporation of the Et20, the residue was a pale brown solid. The combined solids were purified by vacuum distillation (12 mmHg, 160-167 OC) to yield 48.7 g (0.20 mol, 65%) of 4,4diphenylbutyric acid (40) as pale pink crystals: mp 106-108 OC; IR (cm-l, KBr) 3400-2500 (OH), 1725 (C=O); IH NMR (60 MHz, CDC13) 6 2.4 (m, 4H, CH2CH2), 3.9 (t, 1H, CH), 7.3 (s, 10H, ArH), 10.8 (bs, lH, OH).

4-Phenyl-1 -tefralone (41) Butync acid 40 (20.0 g, 83 mmol) was added to mechanically stirred and heated at 50 OC polyphosphoric acid (85% P205) (200 g). After complete dissolution, the temperature of the reaction mixture was increased to 90 OC and after 15 min it was seen that the reaction, which could be followed by Si02 thin- layer chromatography (eluent: light petroleum ether/EtOAc = 411; spot of tetralone at Rf 0.60) was completed. Then very carefully a small amount of ice was added. A sudden increase in temperature was prevented by cooling with an ice-bath. More ice (500 g) was added and the mixture was stirred for 1 h. A pink precipitate was formed. The aqueous reaction mixture was extracted with Et20 (3 x 200 ml). The combined Et20 layers were washed with aqueous 20% NaHC03 (3 x 200 ml) and a saturated aqueous NaCl solution (1 x 200 ml) and dried over Na2S04. Most of the pink colour could be removed by decolourization of this Et2O layer with charcoal. Evaporation under reduced pressure of the Et2O yielded 16.0 g (72 mmol, 87%) of 4-phenyl-1-tetralone (41) as an almost white solid: mp 74-75 OC; IR (cm-1, KBr) 1690 (C=O); IH NMR (60 MHz, CDC13) 6 2.0-3.0 (m, 4H, CHZCH~), 4.3 (t, lH, CH), 6.9-8.2 (m, 9H, ArH); MS (CI with NH3) m/z 223 (M+l), 240 (M+18). The 1-tetralone 41 thus obtained was used in the next reaction without firther purification.

4-Phenyl-I-tetralone Oxime (42) H2NOH.HCl (26.6 g, 0.38 mol) and NaOAc (26.6 g, 0.32 mol) were added to a solution of 1-tetralone 41 (20.0 g, 90 mmol) in absolute EtOH (200 ml). After refluxing for 2.5 h, the reaction mixture was poured into H z 0 (600 ml) and a white precipitate was formed. The precipitate was collected by suction filtration and washed with cold H20, until the pH of the filtrate was neutral. Crude 4-phenyl-1-tetralone oxime (42) was purified by column chromatography on silica gel 60 (Merck) using a mixture of CH2C12 and MeOH (6011) as the eluent. Recrystallization from absolute EtOH yielded 16.0 g (67 mmol, 74%) of 4-phenyl-1-tetralone oxime (42): mp 118-1 19 "C; IR (cm-l, KBr) 3400-3000 (=NOH), 1595 (C=N); IH NMR (60 MHz, CDC13) 6 2.2 (m, 2H, CH2), 2.9 (t, 2H, CH2), 4.2 (t, IH, CH), 6.9-8.2 (m, 9H, ArH), 9.8 (s, lH, OH).

4-Phenyl-1 -tetralone 0-p-Toluenesulfonyloxime (43) To a solution of oxime 42 (2.10 g, 8.8 mmol) in dry pyridine (30 ml) was added slowly at 0 "C a solution of p-toluenesulfonyl chloride (6.60 g, 34.6 mmol) in dry pyridine (I5 ml). After stirring for 2 h at 0 OC and for 16 h at room temperature, the reaction mixture was carefully poured into vigorously stirred ice and a light yellow precipitate appeared. After stirring for 2 h, the precipitate was collected by suction filtration and washed with ice-cold H20 and EtOH. Recrystallization from EtOAc yielded 3.40 g (8.7 mmol, 98%) of 4-phenyl-1-tetralone 0-p-toluenesulfonyloxime (43): mp 122-124 OC; IR (cm-l, KBr) 1590 (Ar, C=N), 1360 (S02), I180 (S02); IH NMR (60 MHz, CDC13) 6 2.1 (m, 2H, CH2), 2.5 (s, 3H, CH3), 2.9 (t, 2H, CH2), 4.1 (t, lH, CH), 6.9-8.2 (m, 13H, ArH).

4-Phenyl-2amino-I-tetralone (44) Under an atmosphere of nitrogen potassium tert-butoxide (0.90 g, 8.0 mmol) was dissolved at 0 OC in very dry EtOH (12 ml). To this cooled solution a solution of 0-p-tosyloxime 43 (2.01 g, 5.1 mmol) in dry toluene (50 ml) was added dropwise. After stirring for 2 h at 0 OC and for 2 h at 10 OC, the reaction mixture was filtrated and the residue was washed with Et20 (3 x 5 ml). To the filtrate 36% HCl(2.5 ml) was added dropwise and a white precipitate was formed. After stining for 16 h at room temperature, the HCl salt was collected by suction filtration and washed with ice-cold acetone (2 x 5 ml). The yield was 0.79 g (2.9 mmol, 57%) of the HCl salt of 4-phenyl-2-amino-1-tetralone (44): mp 181-183 OC; IR (cm-l, KBr) 3300-2500 ( ~ ~ - 3 9 , 1700 (C=O); MS (CI with NH3) m/z 238 (M+l) (M: free amine).

Acetic anhydride (3.8 ml, 40 mmol) was added dropwise at room temperature to a well stirred mixture of 4-phenyl-2-amino-1-tetralone hydrochloride (44.HCI) (2.00 g, 7.3 mmol), NaOAc (4.8 g), EtOAc (25 ml), and H20 (5 ml). After 2 h of stining, diluting the mixture with H20 (10 ml) and another 0.5 h of stirring, the phases were separated and the EtOAc layer was washed with H20 (3 x 15 ml) and a saturated aqueous solution of NaCI. The residue, yielded by evaporation of the solvent under reduced pressure, was dissolved in toluene (30 ml) and H20 was removed from this solution by azeotropic distillation for 0.5 h. After in vacua evaporation of the toluene, the residual green oil crystallized slowly. To these crystals dry Et2O (I5 ml) was added and this mixture was suction filtrated. After washing with dry Et20 (2 x 10 ml), the yield was 1 .OO g (3.6 mmol, 49%) of 4-phenyl-2-acetamido-1-tetralone 45 as a white solid: IR (cm-l, KBr) 3260 (NH), 1700 (C=O), 1630 (C=O: amide I), 1550 (C=O: amide 11). The acetamide 45 thus obtained was used in the next reaction without further purification

4-Phenyl-2-propionamido-1-tetralone (46) Propionyl chloride (0.5 ml, 5.8 mmol) was added dropwise at room temperature to a well stirred solution of 4-phenyl-2-amino-1-tetralone hydrochloride (44.HC1) (1.02 g, 3.7 mmol) and triethylamine (1.5 ml, 10.8 mmol) in CH2C12 (50 ml). After stirring for 1 h, the organic layer was washed with Hz0 (3 x 50 ml) and a saturated aqueous NaCl solution (1 x 50 ml) and dried over MgS04. Evaporation of the volatiles under reduced pressure yielded 1.0 1 g (3.4 mmol, 92%) of 4-phenyl-2-propionamido-1-tetralone (46) as a red oil: IR (cm-l, NaCI) 3320 (NH), 1695 (GO), 1640 (C=O: amide I), 1520 (C=O: amide 11). The propionarnide 46 thus obtained was used in the next reaction without further purification.

4-Phenyl-2-chloroacetamido-1 -tetralone (47) This chloroacetarnide was prepared from 4-phenyl-2-amino-1-tetralone hydrochloride (44-HC1) (1.98 g, 7.2 mmol) and chloroacetic anhydride (6.3 g, 37 mmol) by essentially the same procedure as described for the preparation of acetamide 45 from 4-phenyl-2-amino-1-tetralone hydrochloride (44-HC1) and acetic anhydride. The yield was 0.98 g (3.1 mmol, 43%) of 4-phenyl-2-chloroacetarnido-1-tetralone (47) as a white solid: IR (cm-1, KBr) 3320 (NH), 1700 (C=O), 1640 (C=O: amide I), 1530 (C=O: amide 11). The chloroacetamide 47 thus obtained was used in the next reaction without further purification.

4-Phenyl-2-(acetamido)terralin (28) In a mixture of 100% acetic acid (1 1 ml) and 70% perchloric acid (0.5 ml) acetamide 45 (0.70 g, 2.5 mmol) was dissolved. After adding 10% Pd-on-C catalyst (0.12 g) under an atmosphere of nitrogen, the reaction mixture was placed under a Hg pressure of 3 atmospheres for 16 h in a Parr hydrogenation apparatus. After filtering off the catalyst and washing with absolute EtOH, the volatiles were removed under reduced pressure and the residue was dissolved in H20. Subsequently the solution was neutralized with NaHC03 and extracted with CH2C12 (3 x 30 ml). The combined CH2C12 layers were washed with a saturated aqueous NaCl solution (1 x 50 ml) and dried over MgS04. Then the CH2C12 was evaporated

under reduced pressure and the residue was dissolved in dry Et20 (15 rnl). From this yellow solution white c~ystals were formed slowly. After suction filtration and washing with dry Et20, crude 4-phenyl-2- (acetarnid0)tetralin (28) was further purified by column chromatography on silica gel 60 (Merck) using a mixture of CH2C12 and MeOH (911) as the eluent. The yield was 0.27 g (1.0 mmol, 41%) of 4-phenyl-2- (acetamid0)tetralin (28) as a white solid: mp 155-157 OC; IR (cm-l, KBr) 3275 (NH), 1635 (C=O: amide I), 1545 (C=O: amide 11); IH NMR (300 MHz, CDC13) 6 1.80 (q, IH, CCHaxCN, J = 11.9 Hz [3x]), 1.97 (s, 3H, COCH3), 2.42 (m, lH, CC CN), 2.80 (dd, lH, ArCHaxCN, J = 15.8 Hz, 11.0 Hz), 3.24

(dd7 lH7 CN, J = 15.8 Hz, 4.5 k H z , 4.25 (dd, IH, ArCHaxPh, J = 11.8 Hz, 5.6 Hz), 4.37 (m,

lH, CHaxCN), .6 (bd, IH, NH), 6.78-7.38 (m, 9H, ArH); MS (CI with NH3) m/z 266 (M+l); Anal. (C18H19NO) C, H, N.

4-Phenyl-2-@ropionamido)tetralin (29) This propionamide was prepared from propionamide 46 (0.60 g, 2.0 mmol) by essentially the same procedure as described for the preparation of acetamide 28 from acetamide 45. The yeld was 0.22 g (0.8 -01, 38%) of 4-phenyl-2-(propionamido)tetralin (29) as a white solid: mp 160-162 OC; IR (cm-l, KBr) 3290 (NH), 1640 (C=O: amide I), 1550 (C=O: amide 11); NMR (300 MHz, CDC13) 6 1.14 (t, 3H, CH3), 1.77 (q, IH, CCH,CN, J = 11.7 Hz [3x]), 2.16 (q, 2H, COCH2), 2.41 (m, lH, CCbqCN), 2.77(dd,lH,ArCH,CN,J=15.4Hz,ll.OHz),3.21(ddd,lH,ArC CN,J=16.1Hz,5 .1Hz,1 .5 Hz), 4.21 (dd, lH, ArCH,Ph, J = 11.4 Hz, 5.5 Hz), 4.37 (m, lH, CH, "? N), 5.40 (bd, lH, NH), 6.77- 7.34 (m, 9H, ArH); MS (CI with NH3) m/z 280 (M+l); Anal. (ClgH21NO) C, H, N.

4-Phenyl-2-(chloroacetamido)tetralin (30) This chloracetamide was prepared from chloroacetarnide 47 (0.66 g, 2.1 rnrnol) by essentially the same procedure as described for the preparation of acetamide 28 from acetamide 45. The yield was 0.16 g (0.5 mmol, 25%) of 4-phenyl-2-(ch1oroacetamido)tetralin 30 as a white solid: mp 127-129 "C; IR (cm-l, KBr) 3300 (NH), 1640 (C=O: amide I), 1540 (C=O: amide 11); IH NMR (300 MHz, CDC13) 6 1.86 (q, IH, CCH,CN, J = 12.1 Hz, 12.1 Hz, 12.0 Hz), 2.43 (m, lH, CCHeqCN), 2.87 (dd, IH, ArCHaxCN, J =15.9Hz,ll.2Hz),3.24(ddd,lH,ArC CN,J=15.7Hz,5.0Hz,1.9Hz),4.04(d,2H,COCH2CI), % 4.24 (dd, IH, ArCHaxPh, J = 11.7 Hz, 5.9 ), 4.37 (m, lH, CH,CN), 6.54 (bd, IH, NH), 6.78-7.34 (m, 9H, ArH); MS (CI with NH3) d z 300 (M[Cl=35]+1), 302 (M[C1=37]+1), 317 (M[C1=35]+18), 319 (M[Cl=37]+18); Anal. (C 1gHlgNOCI) C, H, N.

GChloro- y-valerolactone (48) To a well stirred solution of sodium (23.0 g) in very dry EtOH (300 ml) diethyl malonate (320 g, 2.00 mol) was added dropwise at 35 OC. During the addition a precipitate appeared, which dissolved at k 50 O

C. After cooling the reaction mixture to 5 OC, 1-chloro-2,3-epoxypropane (92.5 g, 1.00 mol) was added dropwise, while the reaction temperature was kept below 10 OC. When the addition was completed, the temperature of the reaction mixture was raised slowly to room temperature. After a while suddenly a precipitate appeared and the temperature ofthe reaction mixture increased to 60 OC. After stirring for 18 h at 50 OC, the reaction mixture was cooled to A 30 OC and a mixture of 36% HCI (100 ml) and EtOH (200 ml) was added dropwise. Subsequently the EtOH was &stilled off, Hz0 (500 ml) was added at room temperature and the mixture was stirred vigorously. The formed oil was separated and the H20 layer was extracted with Et2O (500 ml). After addition of the oil to the Et20 layer, the Et2O layer was washed with a saturated aqueous solution of NaCl and dried over NaS04. The residual oil, which remained after in vacuo evaporation, was taken up into 36% HCl (600 ml) and refluxed for 2 h.

under reduced pressure and the residue was dissolved in dry Et2O (15 ml). From this yellow solution white crystals were formed slowly. After suction filtration and washing with dry Et20, crude 4-phenyl-2- (acetarnid0)tetralin (28) was further purified by column chromatography on silica gel 60 (Merck) using a mixture of CH2CI2 and MeOH (911) as the eluent. The yield was 0.27 g (1.0 mmol, 41%) of 4-phenyl-2- (acetamid0)tetralin (28) as a white solid: mp 155-157 "C; IR (cm-l, KBr) 3275 (NH), 1635 (C=O: amide I), 1545 (C=O: amide 11); IH NMR (300 MHz, CDC13) 6 1.80 (q, lH, CCHaxCN, J = 11.9 Hz [3x]), 1.97 (s, 3H, COCH3), 2.42 (m, lH, CC CN), 2.80 (dd, lH, ArCHaxCN, J = 15.8 Hz, 1 1.0 Hz), 3.24 (dd, lH,ArCH, CN, J = 15.8Hz,4.5Hz,4.25 k (dd, lH,ArCHaxPh, J = 11.8 Hz, 5.6Hz),4.37 (m, IH, CHaxCN), 9.6 (bd, IH, NH), 6.78-7.38 (m, 9H, ArH); MS (CI with NH3) d z 266 (M+l); Anal. (C18Hl9NO) C, H, N.

4-Phenyl-2-(propionarnido)tetralin (29) This propionamide was prepared from propionamide 46 (0.60 g, 2.0 mmol) by essentially the same procedure as described for the preparation of acetamide 28 from acetamide 45. The veld was 0.22 g (0.8 -01, 38%) of 4-phenyl-2-(propionamido)tetralin (29) as a white solid: mp 160-162 "C; IR (cm-l, KBr) 3290 (NH), 1640 (C=O: amide l), 1550 (C=O: amide 11); NMR (300 MHz, CDC13) 6 1.14 (t, 3H, CH3), 1.77 (q, lH, CCH,CN, J = 11.7 Hz [3x]), 2.16 (q, 2H, COCH2), 2.41 (m, lH, CC%CN), 2.77(dd,lH,ArCH,CN,J=15.4Hz,ll.OHz),3.21(ddd,lH,ArC CN,J=16.1Hz,5.1Hz,1.5 Hz), 4.21 (dd, lH, ArCH,Ph, J = 11.4 Hz, 5.5 Hz), 4.37 (m, lH, CH, "2 N), 5.40 (bd, lH, NH), 6.77- 7.34 (m, 9H, ArH); MS (CI with NH3) m/z 280 (M+l); Anal. (C19H21NO) C, H, N.

4-Phenyl-2-(ch1oroacetamido)tetralin (30) This chloracetamide was prepared from chloroacetarnide 47 (0.66 g, 2.1 mmol) by essentially the same procedure as described for the preparation of acetamide 28 from acetamide 45. The yield was 0.16 g (0.5 mmol, 25%) of 4-phenyl-2-(chloroacetamido)tetralin 30 as a white solid: mp 127-129 "C; IR (cm-l, KBr) 3300 (NH), 1640 (C=O: amide I), 1540 (C=O: amide 11); IH NMR (300 MHz, CDC13) 6 1.86 (q, IH, CCHaxCN, J = 12.1 Hz, 12.1 Hz, 12.0 Hz), 2.43 (m, lH, CCH,qCN), 2.87 (dd, lH, ArCH,CN, J =15.9Hz,11.2Hz),3.24(ddd,lH,ArC CN,J=15.7Hz,5.0Hz,1.9Hz),4.04(d,2H,COCH2Cl), "ea, 4.24 (dd, lH, ArCHaxPh, J = 1 1.7 Hz, 5.9 ), 4.37 (m, lH, CH,CN), 6.54 (bd, lH, NH), 6.78-7.34 (m, 9H, ArH); MS (CI with NH3) d z 300 (M[C1=35]+1), 302 (M[C1=37]+1), 3 17 (M[C1=35]+18), 3 19 (M[CI=37]+18); Anal. (ClgHlgNOCI) C, H, N.

GChloro- y-valerolactone (48) To a well stirred solution of sodium (23.0 g) in very dry EtOH (300 ml) diethyl malonate (320 g, 2.00 mol) was added dropwise at 35 OC. During the addition a precipitate appeared, which dissolved at * 50 " C. After cooling the reaction mixture to 5 OC, I-chloro-2,3-epoxypropane (92.5 g, 1.00 mol) was added dropwise, while the reaction temperature was kept below 10 OC. When the addition was completed, the temperature of the reaction mixture was raised slowly to room temperature. After a while suddenly a precipitate appeared and the temperature of the reaction mixture increased to * 60 OC. After stining for 18 h at 50 OC, the reaction mixture was cooled to 30 OC and a mixture of 36% HCI (100 ml) and EtOH (200 ml) was added dropwise. Subsequently the EtOH was distilled off, H z 0 (500 ml) was added at room temperature and the mixture was stirred vigorously. The formed oil was separated and the H20 layer was extracted with Et20 (500 ml). After addition of the oil to the Et20 layer, the Et2O layer was washed with a saturated aqueous solution of NaCl and dried over NaS04. The residual oil, which remained after in vacuo evaporation, was taken up into 36% HCI (600 ml) and refluxed for 2 h.

Subsequently the volatiles were evaporated under reduced pressure and the yielded residue was taken up into Et2O (600 ml). The Et20 layer was washed with a saturated aqueous solution of NaCl and dried over NaS04. After in vacuo evaporation, the remaining oil was heated for 1 h at 140 OC under vacuum (15 mmHg). During the heating C02 evolved. The residue was purified by vacuum distillation to yield 72.0 g (0.54 mol, 54%) of 6-chloro-y-valerolactone (48) as an oil: bp 121-125 "C (15 mmHg) [[49] bp 64 OC (0.01 rnmHg)]; IR (cm-l, NaCI) 1775 (C=O).

4,5-Diphenylvaleric Acid (49) To a well stirred solution of y-valerolactone 48 (10.0 g, 74 mmol) in sodium-dried benzene (125 ml) was added portionwise over a period of 45 min at room temperature 30.0 g of anhydrous, finely powdered AlCl3 (225 mmol). During the addition the temperature of the brown reaction mixture rose to approximately 45 OC and hydrogen chloride evolved, which was removed by a constant stream of nitro- gen and absorbed in H20. After addition of the whole amount AlCl3, the temparature of the reaction mixture was increased to 60 OC and the reaction mixture was stirred for 1 h at this temperature and subsequently overnight at 40 OC. To the pulpy mixture was added benzene (50 ml) and carefully 250 g ice, resulting in a redbrownish precipitate. Then aqueous 36% HCI (25 ml) was added dropwise and the reaction mixture was stirred vigorously for 45 min. After separation of the H20 layer and extraction of this layer with Et20 (100 ml), the benzene layer and the Et20 layer were combined. The resulting organic layer was extracted with H20 (3 x 100 ml) and a saturated aqueous solution of NaCl (100 ml) and dried over MgS04 and a small amount of charcoal. In vacuo evaporation yielded 16.3 g (64 mmol, 86%) of crude 4,5-diphenylvaleric acid (49) as a redbrownish solid: IR (cm-l, KBr) 1710 (C=O). The valeric acid 49 thus obtained was used in the next reaction without further purification.

4-Benzyl-1 -tetralone (50) A mixture of valeric acid 49 (10.0 g, 39 mmol) and 100 g of polyphosphoric acid (85% P205) was stirred mechanically at 60 OC. The reaction was followed by Si02 thin-layer chromatography (eluent: light petroleum ether / EtOAc = 4/1; spot of tetralone at Rf 0.44). After 1 h, when the reaction was completed, to the hot, dark brown reaction mixture was added carefully, portionwise 300 g ice. After the first portions of ice the temperature of the reaction mixture rose to + 95 OC. After the addition of ice was completed, the reaction mixture was stirred vigorously for another 15 min. After cooling to room temperature, the reaction mixture was extracted with Et2O (3 x 150 ml). The combined Et20 layers were washed with a 5% aqueous solution of NaHC03 (200 ml) and a saturated aqueous solution of NaCl(150 ml) and dried over NaS04. After removal of the solvent under reduced pressure, the residual oil was purified by vacuum distillation to yield 7.0 g (30 mmol, 75%) of 4-benzyl-I-tetralone (50) as a colour- less, viscous oil: bp 150-164 OC (0.001 mbar); IR (cm-l, NaCI) 1670 (C=O); I H NMR (60 MHz, CDC13) 6 1.7-3.5 (m, 7H, 3 x CH2 + CH), 6.5-8.5 (m, 9H, ArH); MS (CI with NH3) m/z 237 (M+l), 254 (M+18).

4-Benzyl-1-tetralone Oxime (51) This oxime was prepared from I-tetralone 50 (63.3 g, 0.27 mol) by essentially the same procedure as described for the preparation of oxime 42 from 1-tetralone 41. The only difference was that this oxime 51 was recrystallized from absolute EtOH after collection by suction filtration without purification by column chromatography. The yield was 53.8 g (0.2 1 mol, 80%) of 4-benzyl-1 tetralone oxime (51) as white crystals: mp 143-144 "C; IR (cm-l, KBr) 3400-3000 (=NOH), 1590 (C=N); MS (CI with NH3) m/t 252 (M+l).

4-Benzyl-1-tetralone 0-p-Toluenesulfonyloxime (52) This 0-p-tosyloxime was prepared from oxirne 51 (48.5 g, 0.19 mol) by essentially the same procedure as described for the preparation of 0-p-tosyloxime 43 fiom oxime 42. The yleld was 65.0 g (0.16 mol, 83%) of 4-benzyl-1-tetralone 0-p-toluenesulfonyloxime (52) as cubical, white crystals: mp 159-160 "C; IR (cm-l, KBr) 1590 (Ar, C=N), 1370 (S02), 1180 (SO2); IH NMR (60 MHz, CDC13) 6 1.8 (m, 2H, CH2), 2.4 (s, 3H, CH3), 2.7-3.1 (m, 5H, 2 x CH2 + CH), 7.0-8.3 (m, 13H, ArH).; MS (CI with NH3) d z 406 (M+l), 252 (M-171+18).

4-Benzyl-2amino-1-tetralone (53) The HCI-salt of this 2-amino-1-tetralone was prepared from 0-p-tosyloxime 52 (20.0 g, 49 mmol) by essentially the same procedure as described for the preparation of the HCI-salt of 2-amino-1-tetralone 44 from 0-p-tosyloxime 43. The yield was 12.6 g (44 mrnol, 89%) of the HCI salt of 4-benzyl-2-amino-l- tetralone (53.HCI) as white crystals: mp 180 OC dec; IR (cm-l, KBr) 3250-2500 (NH~+), 1690 (C=O); MS (CI with NH3) 252 (M+1) (M: free amine).

4-Benzyl-2-acetamido-I-tetralone (54) This acetamide was prepared from 4-benzyl-2-amino-I-tetralone hydrochloride (53.HC1) (8.03 g, 27.9 mmol) and acetic anhydride (16.7 ml, 0.18 mol) by essentially the same procedure as described for the preparation of acetamide 45 fiom 4-phenyl-2-amino-I-tetralone hydrochloride (44.HC1) and acetic anhydride. The yield was 6.51 g (22.2 mmol, 80%) of 4-benzyl-2-acetamido-1-tetralone (54) as colourless needles: mp 142-143 OC; IR (cm-l, KBr) 3300 (NH), 1695 (C=O), 1630 (C=O: amide I), 1535 (C=O: amide 11); MS (CI with NH3) 294 (M+ 1).

4-Benzyl-2-propionamido-1 -tetralone (55) This propionamide was prepared from 4-benzyl-2-amino-1-tetralone hydrochloride (53-HC1) (3.37 g, 11.7 mmol) and propionic anhydride (8.9 rnl, 0.07 mol) by essentially the same procedure as described for the preparation of acetamide 45 from 4-phenyl-2-amino-I-tetralone hydrochloride (44.HCI) and acetic anhydride. The yield was 2.65 g (8.6 mmol, 74%) of 4-benzyl-2-propionamido-1-tetralone (55) as a white solid: mp 114-115 OC; IR (cm-l, KBr) 3305 (NH), 1695 (C=O), 1640 (C=O: amide I), 1540 (C=O: amide 11); MS (CI with NH3) 308 (M+l).

4-Benzyl-2-chloroacetamido-1-tetralone (56) This chloroacetamide was prepared from 4-benzyl-2-amino-1-tetralone hydrochloride (53.HCI) (0.98 g, 3.4 mmol) and chloroacetic anhydride (3.4 g, 0.02 mol) by essentially the same procedure as described for the preparation of acetamide 45 from 4-phenyl-2-amino-1-tetralone hydrochloride (44.HC1) and acetic anhydride. The yield was 0.95 g (2.9 mmol, 85%) of 4-benzyl-2-chloroacetamido-1-tetralone (56) as light yellow solid: mp 121-123 OC; IR (cm-l, KBr) 3300 (NH), 1700 (C=O), 1660 (C=O: amide I), 1550 (C=O: amide 11); MS (CI with NH3) m/z 328 (M[CI=35]+1), 330 (M[CI=37]+1), 345 (M[C1=35]+18), 347 (M[C1=37] +18).

4-Benzyl-2-(acetamido)tetralin (31) This acetamide was prepared from acetamide 54 (2.06 g, 7.0 mmol) by essentially the same procedure as described for the preparation of acetamide 28 from acetamide 45. The only difference was that this acetamide 31 was not purified by column chromatography after suction filtration. The yield was 1.33 g (4.8 -01, 68%) of 4-benzyl-2-(acetamido)tetralin (31) as a white solid: mp 168-169 OC; IR (cm-l, KBr) 3280 (NH), 1625 (C=O: amide I), 1530 (C=O: amide 11); IH NMR (200 MHz, CDC13) 6 1.62 (m, lH, CCHaxCN), 1.90 (m, lH, CC CN), 1.98 (s, 3H, COCH3), 2.60 (dd, IH, PhCHCAr, J = 16.2 Hz, 9.8 Hz), 2.83 (dd, lH, J = 13.5 Hz, 10.6 Hz), 3.08 (dd, lH, ArCH,-,CN, J = 13.6 Hz,

4.6 Hz), 3.23 (m, 2H, PhCHCAr + Arc CPh), 4.48 (m, lH, CHaxN), 5.59 (bd, 1 H, NH), 7.09-7.37 (m, 9H, ArH); MS (CI with NH3) d z 280 % +I); Anal. ( C I ~ H ~ ~ N O ) C, H, N. Single-crystal X-ray analysis of 4-benzyl-2-(acetamido)tetralin (31): Reasonable crystals of 4-benzyl-2-(acetamid0)tetralin (31) were grown from EtOH by slow evaporation of the solvent. A crystal, having approximate dimensions of 0.25 x 0.15 x 0.45 mm3, was mounted on a glass fiber in a random orientation. Cell constants and orientation matrix for the data collection were obtained from least-squares refinements, using the angular settings of 25 reflections (8.2' 5 9 5 14.3"). The triclinic cell parameters and volume are: a = 4.869 A (4), b = 11.533 (3), c = 14.162 A (3), a =

79.64' (2), P = 86.56' ( S ) , y = 85.83' (4) and V = 779.3 A3. For Z=2 and F.W.= 279.39 the calculated density (dcalcd) is 1.19 &m3. The data collection was performed at room temperature with Mo K a radiation (k= 0.71073 A) on a Nonius CAD4F diffractometer equipped with a graphite monochromator and interfaced to a PDP 11/23, using the 8/28 scan method. The intensities of three standard reflections, which were measured every 3h, were used to control drift in the primary beam and counting system and also possible decrease of the crystal quality. From a total of 3329 reflections (1' 2 8 I 28'), 2344 reflections with I > 3 0 0 were used in the refinements. Scaling factors, Lorentz and polarization corrections were applied to the data. The linear absorption coefficient is 0.679 cm-l. No absorption corrections were made. The structure was partly solved by direct methods using SDPIPDP computer software. The positions of the other atoms were located from succeeding Fourier difference synthesis maps. Block-diagonal least-squares of F, with unit weight, converged to a final R = 0.069 and Rw =

0.070 respectively, using anisotropic temperature factors for the non-hydrogen atoms and fixed isotropic temperature factors (B = 5.0 A2) for the hydrogen atoms. In the final refinements the hydrogen atoms were constraint to their corresponding atoms at a distance of 0.96 A. The molecular structure as well as the atom numbering scheme are given in Figure 6.2.

4-Benzyl-2-(propionamido)tetralin (32) This propionamide was prepared from propionamide 55 (1.48 g, 4.8 mmol) by essentially the same procedure as described for the preparation of acetamide 28 from acetamide 45. The only difference was that this propionamide 32 was not purified by column chromatography after suction filtration. The yield was 0.82 g (2.8 mmol, 58%) of 4-benzyl-2-(propionarnido)tetralin (32) as a white solid: mp 149-150 OC; IR (cm-l, KBr) 3300 (NH), 1630 ( 0 : amide I), 1540 (C=O: arnide 11); IH NMR (300 MHz, CDC13) 6 1.13 (t, 3H, CH3, J = 7.7 Hz), 1.57 (m, lH, CCHaxCN), 1.86 (m, lH, CCH, CN), 2.16 (q, 2H, COCH2, J = 7.7 Hz), 2.54 (dd, lH, PhCHCAr, J = 16.1 Hz, 9.5 Hz), 2.81 (dd 1% ArCHaxCN, J = 13.7 Hz, 10.8 Hz), 3.03 (dd, lH, Arc CN, J = 13.7 Hz, 4.6 Hz), 3.18 (m, 2H, PhCHCAr+ k ArC%CPh), 4.46 (m, lH, CHaxN), 5.29 ( 4 lH, NH), 7.04-7.37 (m, 9H, ArH); MS (CI with NH3) m/z 294 (M+1); Anal. (C20H23NO) C, H, N.

4-Benzyf-2-(chloroacetamido)tetralin (33) This chloroacetamide was prepared from chloroacetamide 56 (0.56 g, 1.7 mmol) by essentially the same procedure as described for the preparation of acetamide 28 from acetamide 45. The only difference was that this chloroacetarnide 33 was not purified by column chromatography after suction filtration. The yield was 0.35 g (1.1 mmol, 65%) of 4-benzyl-2-(chloroacetamido)tetralin (33) as a white solid: mp 152- 153 "C; IR (cm-l, KBr) 3300 (NH), 1640 (C=O: amide I), 1540 (C=O: amide 11); 1~ NMR (300 MHz, CDC13) 6 1.62 (m, lH, CCH,CN), 1.88 (m, lH, CCH,qCN), 2.61 (dd, lH, PhCHCAr, J = 16.1 Hz, 10.3Hz),2.78(dd,1H,ArCH;U,CN,J=13.7Hz,10.8Hz),3.03(dd,1H,ArC CN,J=13.7&,4.6 HSR Hz), 3.18 (m, 2H, PhCHCAr + ArCHeqCPh), 4.01 (s, 2H, COCH2C1), 4.46 (m, 1 , CH,N), 6.43 (bd, lH, NH), 7.04-7.36 (m, 9H, ArH); MS (CI with NH3) d z 314 (M[C1=35]+1), 316 (M[C1=37]+1), 331 (M[C1=35] +18), 333 (M[C1=37]+18); Anal. (C19H20NOC1) C, H, N.

6.6.1 DETERMINATION OF MELATONIN-RECEPTOR AFFINITIES BY COMPETITION FOR 2 - [ 1 2 5 ~ ] ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 1 ~ BINDING TO CHICKEN RETINAL MEMBRANES

Tissue Preparation Chickens (4-6 weeks old) maintained in a controlled lighting regime (14 h light/lO h dark) were decapitated during the light phase. Retinas were dissected free of pigment epithelium and homogenized in ice-cold buffer containing 50 mM TrisHCl (pH 7.5 at 25 OC) and 0.1% ascorbic acid with a Brinkmann Polytron PT-5 at settings 5 for 10 sec. The homogenate was centrifuged at 50000g for 10 min at 4 OC. The obtained pellet was rehomogenized and centrifuged for a second time. The final pellet was resus- pended by homogenization at a concentration of 500 ~g protein/ml and aliquots frozen until use.

2-[1251]1odomelatonin Binding Assay For competition experiments 2-[125~]lodomelatonin (synthesized by Takahashi, N~kaido and Dubocovich by a modification of the method of Vakkuri et al. [3,4]; specific activity: 1800-2175 Ci/mmol; stable for 60 days; purity >95%) was diluted in Tris.HC1 buffer containing 0.01% bovine serum albumin, and the compounds to be tested were dissolved in 1 mM HCI containing 0.1% bovine serum albumin. Binding ( i

duplicate) was initiated by adhtion of 220 pl of chicken retinal membranal suspension to tubes con- taining 20 p1 of appropriate test compound concentrations or vehicle, and 20 pl of 2-[125~]iodomelatonin dilution (final concentration: f 50 pM (30-60 pM)). The tubes were incubated at 25 OC for 1 h in the dark. Reactions were terminated by addition of 5 ml of ice-cold Tris.HC1 buffer, and the contents were immediately filtered over glass-fiber filters (Schleicher & Schuell no. 30) soaked in 0.5% (vlv) poly- ethylenimine solution. Each filter was washed twice with 5 ml of the ice-cold buffer. Radioactivity was determined in a gamma counter. Non-specific binding was defined as binding in the presence of 3 pM 6- chloromelatonin (donated by Clemens, Eli Lilly Laboratories, Indianapolis, USA). Specific binding of 2- [1251]iodomelatonin was calculated by subtracting non-specific binding from total binding and expressed as W m g of protein. El values of the test compounds were calculated from ICsO values by the method of Cheng and Prusoff [40].

6.6.2 DETERMINATION OF INHIBITION OF CALCIUM-DEPENDENT RELEASE OF [~H]DOPAMINE FROM RABBIT RETINA

Tissue Preparation Albino rabbits (2.5-3.5 kg) maintained on a 14 to 10 h lightdark cycle were lulled by decapitation during the light phase. All experimental procedures were carried out in the light. Rabbit eyes were enucleated and the cornea, lens and vitreous humor were removed carehlly. The remaining eye cup was everted and placed in a vial containing 5 rnl of Krebs' solution containing 1.3 mM CaC12 and gassed with 95% 02- 5% C02. The rabbit retina was detached by gentle shaking during which the retina fragmented into several pieces. These pieces were used in the [3~]dopamine release experiments.

( 3 ~ ] ~ ~ p a r n i n e Release Experiments Retinal pieces were incubated for 20 min at 37 O C in the presence of 0.1 pM [3~dopamine (specific activity: h 30 Ci/mmol; New England Nuclear, USA). Thereafter, the pieces of retina were washed in 5 ml of Krebs' solution at 37 OC. The tissue from each retina was divided into four approximately equal portions and placed in four individual cyliidrical plastic tubes with a thii nylon mesh on the bottom. These plastic tubes were then transferred to individual glass supehsion chambers containing platinum

electrodes 30 mm apart. The tissue samples were superfused with Krebs' solution, prewarmed at 37 OC, at a rate of 1 mumin until the spontaneous outflow of radioactivity had levelled off (about 60 min). Tritium release, i.e. release of [3~dopamine, was elicited by field stimulation at 3 Hz for 2 min (20 mA, 2 msec duration). Field stimulations were applied in each experiment at either 60 (S1) or 100 (S2) rnin after the end of the incubation with [3~dopamine . In all experiments S-sulpiride (0.1 pM) was added to the superfusion medium 40 min before S 1 and maintained until the end of the experiment. Melatonin, or a test compound, was added to the perfusion medium 20 rnin after S1 and was present throughout the remainder of the experiment. Four-minute samples of the superfusate were collected before, during and after the period of stimulation. The outflow of radioactivity in each sample was expressed as the percentage of the total tissue radioactivity present at the beginning of each sample collection [15]. The overflow of transmitter, also termed "stimulation-evoked release of [3~dopamine" or "calcium- dependent release of [3~dopamine", was the percentage of total tissue radioactivity released above the spontaneous levels during and after the period of stimulation [15]. Results are expressed as the ratio S2/Sl obtained between the percentage of total tissue radioactivity released above spontaneous levels during the second (S2) and first (S1) periods of field stimulation within the same experiment. The ratio S2/S1 obtained in the absence of a test compound is approximately one. The IC50 value of a test com- pound is the concentration of the test compound required to get 50% inhibition of the maximal inhibition of the stimulation-evoked release of [3~dopamine, that can be reached with the test compound. The ICsO values were determined graphically from concentration-effect curves.

6.6.3 DETERMINATION OF ANTAGONISM OF MELATONIN-INDUCED INHIBITION OF CALCIUM-DEPENDENT RELEASE OF ME HI DOPA MINE FROM RABBIT RETINA

Tissue Preparation The same procedure was followed as for the determination of the inhibition of the calciumdependent release of [ 3 ~ d o p a m i n e from rabbit retina (see 6.6.2).

[ 3 ~ ] ~ o p a m i n e Release Experiments The [3~]dopamine release experiments were executed as described for the determination of the Inhibition of the calciumdependent release of [3~dopamine from rabbit retina (see 6.6.2). With this exception that a test compound was added to the pehs ion medium 40 min before S1 and subsequently melatonin 20 min after S 1. Both compounds were present throughout the remainder of the experiment. The dissociation constant (KB) of a test compound, which acts as an antagonist, was calculated from concentrationeffect curves using the method of Arunlakshana and Schild [41].

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