Structural, conformational, biochemical, and pharmacological study of some amides derived from 3,7-dimethyl-3,7-diazabicyclo [3.3.1] nonan-9-amine as potential 5-HT3 receptor antagonists

Journal of

MOLECULAR STRUCTURE ELSEVIER

Journal of Molecular Structure 372 (1995) 203-213

Structural, conformational, biochemical, and pharmacological study of some amides derived from 3,7-dimethyl-3,7-diazabicyclo [3.3.1] nonan-9-amine as potential 5-HT3 receptor antagonists M . J . F e r n / m d e z a, R . M . H u e r t a s a, E. G f i l v e z a'*, A . O r j a l e s b, A . B e r i s a b, L. L a b e a g a b, A . G . G a r c i a c, G . U c e d a c, J. S e r v e r - C a r r i 6 d, M . M a r t i n e z - R i p o l l d aDto. de Quimica Orgtinica, Universidad de Alcalgt, 28871 Alcahi de Henares, Madrid, Spain bFAES S.A. Apartado 555, Bilbao, Spah~ CDto. de Farmacologia, Facultad de Medicina, U.A.M. Madrid, Spain dInstituto Rocasolano E.U.L Cristalografia C.S.I.C., Serrano 119, 28006 Madrid, Spain

Received 23 May 1995;accepted in final form 11 July 1995

Abstract A series of amides derived from 3,7-dimethyl-3,7-diazabicyclo [3.3.1] nonan-9-amine have been synthesized and examined by IH and 13C NMR spectroscopy and the crystal structure of 9-(2,4,6-trichlorobenzamido)-3,7-dimethyl3,7-diazabicyclo[3.3.1] nonane hydrochloride (4a.HCI) has been determined by X-ray diffraction. These compounds adopt an almost perfect chair-chair conformation with the N-CH3 groups in equatorial position. This conformation is nearly the same as that observed for compound 4a in the solid state. From binding studies of compounds 4a-c, compound 4b demonstrated the ability to efficiently displace [3H]GR65630 bound to bovine brain area postrema membranes to an extent comparable to MDL 72222. In the von Bezold-Jarish reflex, compound 4b showed significant results at a dose of 25 mg Kg -l . It is shown for the first time that a series of compounds with a bispidine skeleton linked through an amide moiety to several aromatic rings, shows 5-HT3 antagonistic profiles.

1. Introduction In a recent paper [1] we reported the synthesis and structural and conformational study of some amides derived from 3,7-dimethyl-3,7-diazabicyclo [3.3.1] nonan-9-amine, Taking into account that (a) the bispidine system (3,7-diazabicyclo [3.3.1] nonane) is superimposable with the tropane system (see later) (Fig. 1), and (b) the former behaves like a monoacid strong base,

* Corresponding author.

because of the adamantane-like bispidine structure (Fig. 2), we used the bispidine structure to design a novel series of potential 5-HT 3 antagonists.

2. Experimental Crystallographic data for compound 4 a . H C 1 are given in Table 1 [2-7]. All N M R spectra (IH, 13C, COSY-45 and X H C O R D ) were recorded on a Varian UNITY-300 spectrometer in CDCI 3 and/or CD3OD; resolution enhancement was applied to deduce the proton magnetic parameters.

0022-2860/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0022-2860(95)09026-6

204

M.J. Fernandez et al./Journal of Molecular Structure 372 (1995) 203-213

CHa

Female Swiss CD-1 mice (23-28 g, Charles River) were fasted for 15h before the experiments, but water was freely available. Compounds (20mgkg-l) suspended in 0.25% aqueous xanthan gum (Sanofi) solution were given orally. Metoclopramide (Sigma), and MDL 72222 (Research Biochemicals Inc.) were used as reference compounds. One group received only vehicle and was used as a control. Forty-five minutes later, the mice were anaesthetized with urethane (Aldrich) (1.25gkg -j, intraperitoneaUy) and electrocardiogram and heart rate were continuously monitored and recorded (Hugo Sachs Electronik, HSE-571, HSE-567, HSE-WR3310). Fifteen minutes later ( l h after oral treatment) serotonin (Sigma) (0.25mgkg -l) was given intraveneously and changes in heart rate were quantified. Results were computed to show the percent von Bezold-Jarisch reflex inhibition in comparison with the control group (Table 2). The level of statistical significance was determined by Student's test for no-paired data; the significance level was considered to be P < 0.05%.

I

II

II !

u.3

! %

! %1

Fig. 1. Protonated N,N'-dimethylbispidine; the dashed lines shows the molecular superposition of the tropane skeleton.

2.1. Synthesis Compounds 4a-e were prepared as shown in Scheme 1. Reaction of N, N'-dimethylbispidinone with hydroxylamine hydrochloride gave the oxime 2 [8]. Reduction of the oxime with lithium aluminium hydride led to the amine 3. The amides 4a-e were obtained by treatment of the amine with the appropriate acyl chloride [1].

2.2.2. [~H]GR65630 binding [3H]GR65630 (63.74Cimmol -l) was obtained from Dupont. MDL72222 and metoclopramide were obtained from Research Biochemicals Inc. Calf brains were dissected on ice, using a procedure adapted from the method of Glowinski and Iversen [10]. The area postrema was scraped away from the surrounding tissues and placed in cold buffer. Approximately 100 mg wet weight of tissue was dissected per brain. The tissue was homogenized

2.2. Pharmacological nrethods 2.2.1. Seroton&-induced von Bezold-Jarisch reflex & mice Compounds 4a-e were evaluated for antagonism of the von Bezold-Jarisch reflex evoked by serotonin (5-HT) in anaesthetized mice by a modification of the method of Saxena and Lawang [9].

CH3

CHa I

CH3

I ~N H

I ~CH 3

~CH3

Ill

b Fig. 2. Adamantane-like bispidine structure.

M.J. Fern6ndez et al./Journal o f Molecular Structure 372 (1995) 203-213

205

Table 1 Experimental data and structure refinement procedures Crystal data Formula Crystal habit Crystal size (mm) Symmetry Unit cell determination Unit cell dimensions Packing: V(A3); Z D e (gcm-3); M; F(000) #(era- i ) Experimental data Technique

Number of reflections: Measured Independent Observed Range of hkl Value of Rim Standard reflections Absorption correction Solution and refinement Solution Refinement Parameters: Number of variables Degrees of freedom Ratio of freedom H atoms Maximum final shift/error w-scheme Max. thermal value Final A F peak Extinction correction S, unit weight standard deviation Final R and Rw Computer and programs Scattering factors Anomalous dispersion

CI6H21N3OCI4 Colourless prism 0.10 x 0.16 x 0.25 Monoclinic, P21/n Least-squares fit from 61 reflections (/9 < 33 °) a = 8.106 (1), b = 23.276 (2), e = 10.852 (1) .~., /~ = 110.95(1) ° 1912.2 (4); 4 1.44; 413.17; 856 58 Four circle diffractometer, SEIFERT XRD3000S Bisecting geometry Graphite oriented monochromator: Cu K a or/20 scans, scan width: 1.5° Detector apertures 2 x 2 °, up to 0max 65 ° 3396 3118 1656 (2a(1) criterion) - 9 , 9, 0 27, 0 12, (sin 0/A)max 0.58 0.016 2 reflections every 100 reflections. No variation. kV-scan, max. and min. corrections 1.35-0.95 Direct methods Least squars on Fobs with one block 262 1394

6.3 Difference synthesis 0.18 (x of H9) Empirical as to give no trends in (wA2F) vs. (Fo) or (sin 0/A) 0.14 (U22 of CI3) 0.25 e ~-3 None 0.63 0.048, 0.053 VAX 6410, MULTAN80 [2], DIRDIF [3], XRAY76 [4], PESOS [51, CSU [61 Int. Tables for X-Ray Crystallography [7] Int. Tables for X-Ray Crystallography [7]

in ice-cold 50mM Tris-HC1, 0.5mM EDTA, 10mM MgSO4, pH 7.4, and centrifuged at 30 000 g for 15 min. The pellet was resuspended in buffer (in a homogenizer), incubated at 37°C

for 15m in, and then centrifuged at 30 000 g for 15 min, twice (with a resuspension between centrifugations). The final pellet was resuspended in 50mM

M.J. Fernandez et al./Journal o f Molecular Structure 372 (1995) 203-213

206

.ax_

H ~

CH3

CH3 Heq I

I N

4~."'~N~cH3 1 H

O

e

N~ N ~ C H 3

q

,1

i

Hax

I

OH

1

2

CH3 I N

CH3 I N

NHR

4

NH2

4

a

b

c

\oo

R

c' s' .0 ""~+" CI CI

3

.,

el

r

,

H

Scheme 1.

Tris-HCl, 0.5mM EDTA, 10mM MgSO4, 0.1% ascorbate, 10-5M pargyline, and 140mM NaCI, aliquoted and frozen. Assays were performed three times, with sets of tubes in triplicate in competition experiments and in duplicate in saturation experiments, in a 2.0 ml volume containing 2 mg of wet weight tissue (added last). Tubes were incubated at room temperature because total and specific binding of [3H]GR65630 is higher at room temperature than at 37°C [11]. Thus, tubes were incubated for 30 min, filtered on

glass microfibres (Whatman GF/C), and washed with 7 ml of ice-cold buffer three times. The filters were counted in a liquid scintillation analyser (Packard Tri-carb 1500) in 4 ml of aqueous counting scintillation fluid (Beckman Ready Micro), following 12 h of equilibration. Saturation and competition experiments were analysed by a computer program. Protein determinations were performed following the method of Lowry et al. [12] using bovine serum albumin (BSA) as a standard.

M.J. Ferndndez et al./Journal of Molecular Structure 372 (1995) 203-213

207

Table 2 Activities of compounds 4a-e in the inhibition of the yon Bezold-Jarisch reflex induced by serotonin in the mouse Drugs

Dose (mgkg -I)

Inhibition (%)

4a 4b

25 25 10 5 25 25 10 5 1 0.50 0.25

2.48 67.94 8.58 13.48 -4.88 71.11 63.69 66.74 63.03 34.96 2.39

4c Metoclopramide MDL-72222

NS a Sb (p < 0.001) NS NS NS S (p < 0.001) S (p < 0.001) S (p < 0.001) S (p < 0.01) NS NS

a NS not significant, b S significant.

3. Resultsanddiscussion 3.1. Structural study 3.1.1. Description o f the structure o f 4a O n e crystallographically i n d e p e n d e n t molecule

a n d one chloride ion from the a s y m m e t r i c u n i t are s h o w n in Fig. 3 together with the atomic n u m b e r i n g scheme. B o n d lengths a n d angles involving n o n - h y d r o g e n a t o m s are given in Tables 3 a n d 4, respectively. T h e piperidine rings a d o p t a c h a i r - c h a i r

HI02 HI01~HI03

C1 1

o3

'E/

m

"-.

H-0

~

r

11

t

3

o

""'

Ci,I Fig. 3. An ORTEP view of the molecular structure of 4a showing the hydrogen bond and the atomic numbering. Thermal ellipsoidsare drawn at 30% probability level. H atoms are artificially small for clarity.

208

M.J. Ferndndez et al./Journal o f Molecular Structure 372 (1995) 203-213

Table 3 Bond lengths (,~.)

Table 4 Bond angles (deg)

C12-C14 CI3-C16 CI4-C18 O-C12 N1-C9 N1-C12 N3-C2 N3-C4 N3-C10 N7-C6 N7-C8 N7-CI I

1.736 (7) 1.727 (6) 1.732 (7) 1.223 (8) 1.467 (6) 1.329 (7) 1.508 (9) 1.491 (7) 1.479 (7) 1.473 (8) 1.474 (8) 1.471 (7)

C9-N1-C12 C2-N3-C4 C2-N3-C10 C4-N3-CI0 C6-N7-C8 C6-N7-CI I C8-N7-C11 C2-C1-C8 C2-C1-C9 C8-C1-C9 N3-C2-C1 N3-C4-C5

123.3 (5) 111.3 (4) 111.0 (4) 113.1 (4) 110.4 (5) 110.3 (5) 110.5 (5) 112.2 (5) 111.2 (4) 109.0 (4) I 11.0 (4) I 11.0 (4)

CI-C2 C1-C8 C1-C9 C4-C5 C5-C6 C5-C9

1.512 (7) 1.527 (7) 1.517 (7) 1.506 (7) 1.542 (7) 1.544 (9)

C4-C5-C6 C4-C5-C9 C6-C5-C9 N7-C6-C5 N7-C8-C1 NI-C9-CI

112.5 (5) 110.6 (4) 107.4 (5) 111.1 (5) 109.9 (5) 110.2 (4)

C12-C13 CI3-CI4 C13-C18 C14-C15 C15-C16 C16-C17 C17-C18

1.511 (7) 1.392 (8) 1.379 (8) 1.380 (8) 1.382 (10) 1.380 (10) 1.391 (8)

NI-C9-C5 C1-C9-C5 O-C12-NI O-C12-CI3 N1-C12-C13 C12-C13-C14 C12-C13-C18 C14-C13-C18 CI2-C14-C13

113.1 (4) 106.2 (4) 125.5 (5) 119.2 (5) 115.3 (5) 122.0 (5) 121.3 (5) 116.7 (5) 119.9 (5)

c12-c14-c15 c13-c14-c15 c14-C15-ci6 C13-C16-C15 c13-c16-ci7 c15-c16-c17 ci6-c17-c18 c14-c18-c13 ci4-c18-c17 CI3-C18-c17

117.0 (5) 123.2 (6) 117.2 (6) 119.5 (5) 117.8 (5) 122.7(6) 117.4 (6) 119.7(5) 117.4(5) 122.8(6)

conformation. Both rings are flattened at N3 and N7 atoms and puckered at the C9 atom. The best least squares plane in.the ring ( C 1 - C 2 - N 3 - C 4 C5-C9) is defined by C1-C2-C4-C5. N3 and C9 atoms deviate 0.644 (4),~, and -0.748 (6) ,~, respectively, from this plane. In the ring ( C 1 - C 8 N 7 - C 6 - C 5 - C 9 ) the best least squares plane is defined by the C 1 - C 8 - C 6 - C 5 atoms, and N7 and C9 deviate 0.672 (4) A and -0.788 (5).~, respectively. The dihedral angle formed by the planes (C1-C2-C4-C5) and (C1-C8-C6-C5) is 112.3(3) °. The N3 atom is protonated, and the deprotonated Cll atom is linked to N1 through a hydrogen bond of type N 1 - H 1 0 . . . Cll shown in Fig. 3 ( H 1 0 . . . E l l 2.31 (7)]k; N 1 - H I 0 . . . C l l 174 (6)°). This hydrogen bond, together with other rather long hydrogen contacts, mainly determine the crystal packing.

3.1.2. N M R spectra

lH N M R (300MHz) and 13C N M R (75MHz)

spectroscopy was used to provide the required information (Table 5). The unambiguous assignment of all bicyclic proton resonance was achieved by the combined use of 2D N M R techniques (COSY and IH-13C correlation spectra) and double resonance experiments. IH -NMR spectra All the bicyclic proton signals, except the H9 Signal, correspond to two protons. The long range (IV) coupling JH2(4)ax-H6(8)ax was not observed. The Hl(5) signal appears in all cases as a wide singlet (WI/2 ,~ 12 Hz). H 1, H2ax, H2eq (or H5, H4ax and H4eq) and HI, H8ax, H8eq

M . J . F e r n h n d e z et a l . / J o u r n a l o f M o l e c u l a r S t r u c t u r e 3 7 2 ( 1 9 9 5 ) 2 0 3 - 2 1 3 I

"v

"v ~ ~ " 0

~4 -v

u

¢.q

,om. ~

p'..

p..- ~

o

Z

I

U c~ ~4 .v ¢.q

.v I

"I"

~.~

o

~',

f,l

f,l

¢~

~4 .v t"q u

~Z

"-v I

.v ~4 .v ¢.q u

.v I

-7. U ..e ~Q

~

--.

r~

~

.

~,.~ O~

~

"0 ~

f

0~,

~

0

,',

~

(..)

209

210

M.J. Ferndmdezet al./Journalof Molecular Structure372 (1995) 203-213

(or H5, H6ax, H6eq) from a three spin AMX system, the first-order analysis of which led to the protonic parameters given in Table 5. For vicinal coupling constants of these systems, a maximum value of 2Hz was estimated on the basis of the W1/2 of the corresponding doublet peaks. Conformational study From the IH NMR and 13C NMR data of compound 4b, the following general features for the bicyclic system were deduced: (i) this system adopts an almost perfect chair-chair conformation and (ii) the nitrogen methyl groups occupy an equatorial position. These conclusions are supported by the following observations. (i) In the IH NMR spectra, the range of values of Wl/2 of 10-12 Hz for Hl(5) is in good agreement with previously reported values for a chair-chair conformation in related bicyclic systems with the N-substituents in the equatorial position [13,14]. (ii) In all cases, the following relations were found: 3j H2(4)ax-Hl(5)~ 3j H2(4)eq_Hl(5 ) ~ 3 j H6(8)ax-H 1(5) ~ 3j H6(8)eq-H 1(5) ~ 2 Hz. Hence the dihedral angles H2(4)ax-C-C-H 1(5) ~ H2(4)eqC - C - H 1(5) ~ H6(8)ax-C-C-H 1(5) ~ H6(8)eq-CC - H I ( 5 ) ~ 60° according to the Karplus relationship [15]. (iii) The N-CH3 lac chemical shift of compounds 4a-e of about 46 ppm is the same as that found in equatorial N-CH3 substituted piperidines [13]. From the 6 IH and 13C values of the 3,5dichlorophenyl group in 4b a conjugation between this group and the amido moiety can be proposed. The high value of 3j H 9 - N H in CDCI 3 (6.7Hz) accounts for a dihedral angle H 9 - C 9 - N - H close to 180° [16]. Keeping in mind the coplanarity Table 6 IH chemicalshifts(6, ppm) of salts of compound4b and N, N'dimethylbispidine Compound

Solvent

N3-CH3

N7-CH3

4b. HCI 5. HCIO4 5

CD3OD D20 CF3CO2H

2.49 2.42 3.07

2.69 2.42 3.07

between the benzene ring and the amido moiety for thiscompound, it could be noted that the spatial arrangement of the aryl amido moiety is about the same as that found for 4a and 4c [1] in the solid state. In Table 6 we summarize the 1H chemical shifts of N, N'-dimethylbispidine perchlorate in deuterium oxide, N,N'-dimethylbispidine in trifluoroacetic acid [17] and compound 4b in CD3OD. For N, N'-dimethylbispidine perchlorate, Douglass and Ratliff [17] proposed an adamantane-like structure (Fig. 2). Support for this structure was derived from the position of the N-methyl resonance. The observed shift is some 0.6 ppm further upfield than is commonly found when the nitrogen atom carries a full positive charge (as happens on dissolving bispidine in trifluoroacetic acid [17]). However, the same authors admit that this fact does not constitute proof for an adamantane-like structure for this cation, since rapid intramolecular exchange will give an average effect for the N-methyl and the N+-methyl signals (Fig. 2). The observed chemical shifts of the N3-CH 3 and N7-CH 3 protons of 4b. HC1 in CD3OD (Table 6) clearly suggest that both nitrogen atoms carry between them a positive charge. These values can be applied to both an adamantane-like structure (Fig. 2) or a monoprotonated compound where rapid exchange would give an average effect for N3-CH 3 and N7-CH3 (Fig. 2). 3.2. Biological studies 3.2.1. Effects of compounds 4a-c on the binding of [3 H]GR65630 to brain area postrema membranes To test the affinities of compounds 4a-e for 5HT 3 receptors, their ability to displace [3H] GR65630 bound to membranes from the brain stem area postrema [11] was measured. In three duplicate experiments, increasing concentrations of [3H]GR65630 produced a saturation equilibrium isotherm. The Scatchard analysis of the results gave a dissociation constant, K D of 1.3940.5 nM and a maximum number of binding sites, Bmax of 84.32 + 0.5 fmolmg -l of protein (Fig. 4). For displacement experiments, 1 nM [3H]GR65630 was used.

M.J. Fern6nde'_ et al./Journal of Molecular Structure 372 (1995) 203-213

A concentration-displacement curve of [3H] GR65630 binding w~s performed with compounds 4a-c, M D L 72222 and metoclopramide (Fig. 5). the IC50 for compound 4b was 1.37+0.22 ×10-6M and K i = 0 . 8 + 0 . 1 x l0-6M; for 4a 0.95 + 0.12 x 10 -5 M and Ki = 0.55 4- 0.09 ×10 -SM; for 4c 0.36~0.2 x 10- 4 M and Ki = 0.21 + 0.1 x 10-4 M; for M D L 72222 IC50=6.06-t-5.1 x l0-TM and K i = 3 . 5 2 + 2 . 9 ×10-TM, and for metoclopramide, IC50 = 10.8 ×10-SM and Ki.= 5.7 x 10-SM.

K D =1.39 + 0.5 10 "g M 80'

Br~x= 84.32 + 21.9 fmol. mg prot "!

70 60 50

"6 E "0 e.. .i 0 m

40 3O 20" 10"

1

2

211

3

3.2.2. Inhibition of the yon Bezold-Jar&ch reflex From Table 2 it can be clearly seen that compound 4b displays the ability to antagonize the von Bezold-Jarisch reflex in rats to an extent similar to that of metoclopramide. At a dose of 25mgkg -I, 4b causes 67.94% inhibition of the reflex compared with 71. l 1% for metoclopramide at a similar dosage.

4

[ 3H-GR65630 ] (nM)

Fig. 4. Saturable specific binding of ~[H]GR65630 to homogenates of bovine area postrema (2mg wet weight, 174.62/zg protein) using filtration binding methodology. Assays were performed in duplicate; error bars represent S.E.M. of three experiments with separate batches of membranes.

•

MDL 72222 (n=5)

• Metoclopramlde r-I 4b (n=3) •

(n=2)

4a (n=3)

L~ 4c (n=3)

lOO 120

80

"r

O Z ~3

m (J 14.

_z

o

Ul

40

20

~.~~

i'~

o -9

i

i

i

i

-8

-7

-6

-5

A'", -4

Log[Drug|(M)

Fig. 5. Representative competition curves for MDL 72222, metoclopramide, 4a-c, using 3[H]GR65630 (1 nM) as radioligand.

212

M.J. Fern6ndez et al./Journal o f Molecular Structure 372 (1995) 203-213

*"-'H

Fig. 6. Possibilities of N-protonation for the tropane system at physiological pH.

3.3. Discussion

The selective 5-HT 3 receptor antagonists reported to date may be represented by the structure Ar-(carbonyl)-N, in which an aromatic group is linked by a carbonyl-containing moiety to a basic amine. This simple model can be further refined by considering the three-dimensional aspects of the pharmacophore. The carbonyl group is coplanar to an aromatic group, and the interatomic distances in the 5-HT 3 receptor antagonist pharmacophore are in adequate ranges (carbonyl oxygen-an aromatic centre, about 34,~,; carbonyl oxygen-basic nitrogen centre, about 5,~; basic nitrogen centre-aromatic centre, about 7-8 A [18-21]). One of the most important structural factors is the coplanarity between the aromatic ring and the carbonyl moiety. However, according to molecular modelling studies on 1alkyl-2-oxo- 1,2-dihydroquinoline-4-carboxylic acid derivatives, which possess high affinity for the 5-HT 3 receptors, their minimum energy conformations did not have such coplanarity [22]. In the Rizzi Model [23] two regions, one for hydrogen-bond accepting and one for hydrogenbond donating residues were found to be common to ICS 205-930, Zacopride and Ondansetron. An energetically permissible rotation had to be performed on a rotatable bond of the most stable conformer of the thiazole ring, in 3-[2-(guanidinylethyl)-4-thiazolyl]indole, in order to obtain and distance between the two putative receptor residues that was similar to that of the other compounds (7.7A). In the Swain model [24] a basic nitrogen atom coplanar with, and at a distance of 8.6-9.1 A from,

the centre of the aromatic ring, and an atom capable of accepting a hydrogen bond coplanar with, and at a distance of 4-4.5~, from, the aromatic ring and 5-5.2 A from the basic nitrogen, are essential for activity. Bearing in mind that the more stable conformation of compound 4b in solution is rather similar to that found for 4a and 4c [1] in the solid state, and by considering the interatomic distances deduced from the X-ray data in compounds 4a and 4e, it can be assumed that compounds 4a-c satisfy the steric requirements described for the 5-HT 3 pharmacophore. As demonstrated previously, compounds 4a-c at physiological pH adopt a monoprotonated adamantane-like structure. In the case of tropane there are two possibilities of N-protonation at physiological pH (Fig. 6). Carrol et al. [25] demonstrated that protonation takes place as represented in Fig. 6(b). In the compounds 4a-c, the only way of protonation is as shown in Fig. 6(a). Perhaps this could be a reason that explains the poor activity of compounds 4a-c.

Acknowledgment One of us (J. Server-C.) thanks the Conselleria de Educaci6n y Ciencia (Generalitat Valenciana) for a research fellowship to work at the Instituto Rocasolano, CSIC.

References [1] M.J. Fernf.ndez, R.M. Huertas, E. G/dvez, J. Server-

M.J. Fernhndez et al./Journal o f Molecular Structure 372 (1995) 203-213

Carri6, M. Martinez-Ripoll and J. Bellanato, J. Mol. Struct., 355 (1995) 229. [2] P. Main, S.J. Fiske, S.E. Hull, L. Lessinger, G. Germain, J.P. Declercq and M.M. Woolfson, MULTAN90, a system of computer programs for automatic solution of crystal structures from X-ray diffraction data, University of York, U.K., and University of Louvain, Belgium, 1980. [3] P.T. Beursken, W.P. Bosma, H.M. Doesburg, R.O. Gould, T.E.M. Van der Hark, P.A.J. Prick, J.H. Noordik, G. Breurken and V. Parthasaranthi, DIRDIF, Crystallography Laboratory, Tournooiveld, 6525 ED, Nijmegen, The Netherlands, 1982. [4] J.M. Stewart, F.A. Kundell and J.C. Baldwin, The XRAYS0 System, Computer Science Center, University of Maryland, College Park, Maryland, 1980. [5] M. Martinez-Ripoll and F.H. Cano, PESOS, a computer program for the automatic treatment of weighting schemes, Instituto Rocasolano, C.S.I.C., Madrid, Spain, 1975. [6] I. Vickovic, CSU, Crystal Structure Utility, Faculty of Science, University of Zagreb, 41001 Zagreb, Croatia, 1988. [7] International Tables for X-ray Crystallography, vol. IV, Kynoch Press, Birmingham, 1974, (present distributor, C. Reidel, Dordrecht). [8] M.J. Fernandez, R. Huertas, E. G~lvez, J. Sanz-Aparieio, I. Fonseca and J. Bellanato, J. Mol. Struct., 323 (1994) 85. [9] P.R. Saxena and A. Lawang, Arch. Int. Pharmacodyn., 277 (1985) 235. [10] J. Glowinski and L.L. Iversen, J. Neurochem., 13 (1966) 655. [11] K. Miller, E. Weisberg, P. Fletcher and M. Teitler, Synapse, 11 (1992) 58. [12] O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265.

213

[13] E. Galvez, M.S. Arias, J. Bellanato, J.V. Garcia-Ramos, F. Florencio, P. Smith-Verdier and S. Garcia-Blanco, J. Mol. Struct., 127 ~1985) 185. [14] M.S. Arias, E. Ghlvez, J.C. del Castillo, J.J. Vaquero and J. Chicharro, J. Mol. Struct., 156 (1987) 239. [15] C.A.G. Haasnot, F.A.A.M. de Leeuw and C. Altona, Tetrahedron, 36 (1980) 2783. [16] V.F. Bystrov, V.T. Ivanov, T.A. Protnova, S.L. Balashova and Y.A. Ovchinnikov, Tetrahedron, 29 (1973) 873. [17] J.E. Douglass and T.B. Ratliff, J. Org. Chem., 33 (1968) 355. [18] A.W. Schmidt and S. Peroutka, J. Mol. Pharmacol., 36 (1989) 505. [19] M.F. Hibert, R~ Hoffmann, R.C. Miller and A.A. Carr, J. Med. Chem., 33 (1990) 1594. [20] A.W. Schmidt and S. Peroutka, J. Mol. Pharmacol., 38 (1990) 511. [21] S.M. Evans, A. Gades and M. Gall, Pharmacol. Biochem. Behav., 40 (1991) 1033. [22] H. Hayashi, Y. Miwa, I. Miki, S. Ichaikawa, N. Yoda, A. Ishii, M. Kono and F. Suzuki, J. Med. Chem., 35 (1992) 4893. [23] J.P. Rizzi, A.A. Nagel, T. Rosen, T.S. McLean and T. Seeger, J. Med. Chem., 33 (1990) 2721. [24] C.J. Swain, R. Baker, C. Kneen, R. Herbert, J. Moseley, J. Saunders, E.M. Seward, I.G. Stevenson, M. Beer, J. Stanton, K. Watling and R.G. Ball, J. Med Chem., 35 (1992) 1019. [25] F.I. Carrol, P. Abraham, K. Parham, R.C. Griffith, A. Ahmad, M.M. Richard, F.N. Padilla, J.M. Witkin and P.K. Chiang, J. Med Chem., 30 (1987) 809.