Synthesis and Structural, Biochemical, and Pharmacological Study of 30-Acyloxy-3a-methoxycarbonyltropane Derivatives

Synthesis and Structural, Biochemical, and Pharmacological Study of 3&AcyloxyS~~methoxycarbonyltropane Derivatives E. GALvEz*', M. L. IZQUIERDO*, C. BURGOS*,M. S. ARIAS*,J. SANZ-APARICIO~, 1. FONSECA*, F. G A G d , G. BALDOMINOS~, P. L ~ P E z AND ~ , J. C. PRIETO~ Received December 10,1991,from the 'Departamento de Quimica Orgdnica, Campus Universitario de Alcal4, 28871 Alcald de Hepares, rtamepto de Fisiologia y Madrid, the 'lnstituto Rocasolano, Departamento de Rayos X, C.S.I.C., Serrano 119, 28006 Madrid, the " e Farmacologia, Cam us Universitario de Alpld, 28871 Alcal4 de Henares, Madrid, and the Departamento d?Biou,mica, Campus Universitario de Alcald, 28871 A& / de Henares, Madrid, Spain. Accepted for publication December 7,1992. Abstract 0 A series of 3&acyloxy-3~~methoxycarbonyltropanes were synthesized and studied by 'H and I3C NMR spectroscopy, and the crystal structure of 3amethoxycarbonyl-3p-pyridincarbonyloxytropane (5d) was determined by X-ray diffraction. In CDCI, solution, compounds 5%-f display the same preferred conformation. The pyrrolidine and piperidine rings adopt an envelope conformation flattened at N-8 and a distorted chair conformation puckered at N-8 and flattened at C3, respectively,with the N-substituent in the equatorial position with respect

to the piperidine ring. The pharmacological profile of one of these compounds makes it an adequate candidate for the design of novel GABA, antagonist agents.

Previous papers13 reported the synthesis, 'H NMR, lSC NMR, and X-ray diffraction data, and the pharmacological study of several anticholinergic esters derived from tropane, granatane, and camphidine skeletons. In a research program aimed at the development of new antagonists of the B receptor of yaminobutyric acid (GABAB),we have synthesized a new series of esters derived from 3P-hydroxy-3cu-methoxycarbonyltropane (Scheme I) in which the y-aminobutyric acid (GABA)skeleton is included.

Results Chemistry-compounds 5a-f were prepared as shown in Scheme I. From the reaction of the ketone 1 4 with ammonium chloride and potassium cyanide in water, the cyanohydrin 2a was the only product isolated. Considering that in the preferred conformation of 1 in solution the pyrrolidine and piperidone rings adopt a flattened N8 envelope and distorted chair conformation puckered at N8 and flattened at C3, respectively, with the N-substituent in the axial position with respect to the piperidine ring,5 the more favorable s a t t a c k of the cyanide anion should lead to the cyano-hydrin 2b. Therefore, the formation of the most stable cyanohydrin 2a [in the case of 2a and 2b the most severe steric interaction is exerted by the -OH group on H6(7)n] could be attributed to a thermodynamic control of the process. By treatment of 2a with anhydrous methanolic HCl, the iminoester hydrochloride 3 was separated; on heating (60 "C) the water solution of 3, the hydroxyester 4 was obtained. From the reaction of 4 with the corresponding acyl chloride, 5a-f were obtained. Structural Study-Descriptwn of Structure of 5d-The main crystallographic data and the structure determinations are given in Table I.R10 Figure 1shows a view of the molecule and the numbering used in the crystallographic study (supplementary material is available from the authors). The bicyclic system, which has a chair-envelope conformation, shows a pseudo-mirror plane through C9, N1, C4,C19,OlO atoms; as C19-CPOlO-Cll and 021-Cl9-CPOlO torsions 794 I Journal of Pharmaceutical Sciences Vol. 82, No. 8, August 1993

Table I-Experlmental Data and Structure Refinement Procedures

Parameter

Value

Crystal data Formula Symmetry Unit cell determination Unit cell dimensions

Packing: V(a3),z Dc, g cm-? M,F(000) p, cm-I

-

Experimental data Technique

C16 H20 0 4 N2 Monoclinic, P2,n Least-squares fit from 25 reflexions (0 <30") 6.136(2),25.188(2),10.160(2)a, 90.0,102.47(2),90.0" 1533.2(6),4 1.3185,304.345,648 0.892

Four circle diffractometer: Nonius Cad-4. Bisecting geometry Graphite oriented monochromator: MoKa w120 scans Detector apertures 1 x 1, up 0 m a . 30".

Number of reflexions: Measured Observed Range of hkl

4468 1782 (341)criterion) -8.8, 0 35,0 14 (sin0ll)mx.0.70

Solution and refinement Solution Refinement Number of variables H atoms w-scheme Final AF peaks Final R and Rw Computer and programs Scattering factors Anomalous dispersion

Direct methods L.S. on Fobs with 1 block 259 Difference fourier synthesis Empirical as to give no trends in vs.and 0.3 eA-3 0.058,0.066 Vax 111750,Multan80,sXray 76,' PESOS,a Parsts Int. Tables for X-Ray Crystallographylo In. Tables for X-Ray Crystallography10

are 56.1(4) and 44.4(4) ', respectively, the two substituent groups are clearly deviated from it. The six-membered ring is puckered at the N1 atom and very flattened at the disubstituted C4 atom, both atoms being 0.884(3) and 0.365(3) 4 respectively, from the plane defined by the remaining four atoms. The 3-pyridincarbonyloxy group is in a pseudoequatorial position because the chair is so distorted at the C4 atom that the ring is nearly intermediate between a chair and a n envelope, as shown by the puckering coordinate@: QT = 0.649(3),0,= 146.3(3), and = 175.9(6).The five-membered ring has an envelope conformation, with the N1 atom 0.645(3) A from the plane defined by the other four atoms. The methyl 0022-3549/93/0800-0794$02.50/0 Q 1993, American Pharmaceutical Association

-4

2b

3

4

I

Scheme I group is in equatorial position. Ring puckering coordinates12 are: Q2 = 0.432(4) and (p2 = 4.3(7). Pyridincarbonyloxyand methoxycarbonyl groups are nearly planar,withmeantorsionatOlO-Cll,Cll-Cl3,andC19-021 of 6.8, 6.3, and 2.8", respectively. These groups are oriented almost perpendicularly; the angle defmed by both groups is 99.2 Bond lengths and angles are as expected, and molecules in the crystal are linked only by van der Waals forces. O.

b

Figure 1-PLUTO view of the molecule (5d) showing the atomic numbering (ref 11).

0

5

Nuclear Magnetic Resonance Spectra-The 'H and "C NMR spectra of 5a-f show a great similarity. Assignments of proton and carbon resonances have been made on the basis of our previous work with tropane derivatives.1.5 Compound 5d has been studied in more detail; its 300 MHz 'H NMR spectrum, homonuclear 2D-COSY-45 (Figure 2), and heteronuclear proton-carbon shift correlation spectral"'* were used to provide the required information. The crossannectivity patterns of homonuclear 2D-COSY spectrum of 5d were analyzed taking into account that the nonredvable wide singlet at 3.3 ppm can be unambiguously assigned to the bridgehead Hl(5) protons. Considering the c o r r e l a t i ~the ~ , following can be established: (1)The doublet centeredat 2.7 ppm due to two protons correlateswith Hl(5)and the signal at 2.4 ppm, and must correspond to H2(4)a owing to the very weak cross-peakwith Hl(5)that is in agreement with a small value for 'JH2(4)rr-H1(5); (2) the doublet of doublets at 2.4 ppm, also due to two protons, with a stronger correlation with H1(5),must correspondto H2(4)P;(3)the signalsat 1.8(two protons) and 2 ppm (two protons) are assigned to H6(7)n and H6(7)x, respectively, owing to their shape and the correlation between the low-field multiplet and Hl(5). In the w e of "C NMR spectra,signal multiplicity obtained from off-resonance decoupled spectra, and the abovementioned proton-carbon shift correlation spectrum of 5d were taken into consideration. The 'H and "C NMR data of 5d and 5e are summarized in Tables I1 and 111,respectively, as representative compoundsof the series studied. Confonnatioml Stzdy-From the 'H and N M R data of 5a-f the following general features for 5a-f (Scheme I) were deduced: (1)The pyrrolidine and piperidine rings in all compounds have a flattened Nsenvelope and distorted chair conformation puckered at N8 and slightly flattened at C3; (2) the conjugated aroyloxy groups in equatorial position lie predominantly in the plane d e h e d by N, C3, and C(0) atoms, with the Journal of Pharmaceutical Sciences I 795 Vol. 82, No. 8, August 1993

i

r

1

A

1.

d h d

n (WJ

e-

s

0

049 Q

9 6

3-

0 4-

s-

8-

F

a-

0

4

Q

Figure 2-Contour plot of the 300 MHz proton COSY spectrum of 5d in CDCI,.

other carbonyl group near to H2(4)P; the methoxycarbonyl groups are attached in axial position, with the carbonyl group near to the ethylene bridgq (3) the N-methyl groups occupy predominantly an equatorial position with resped to the piperidine ring.These conclusionsare supportedby the following: (1)In the ‘H NMX spectra the W%for Hl(5) proton signals of -10 Hz correspond to a tropane system with the piperidine ring in a slightly flattened chair conf0rmation.1~6 In all 3JH2(4)13-Hl(5) = 4 Hz is greater than ‘JH2(4)*H1(5) = 2 Hz (Table 11); consequently, the dihedral angle H2(4~a4-C-H1(5) is slightly greater than H2(4)&C-C-H1(5), according to the Karplus relationship.17 In the “C NMR spectra, the chair conformation adopted by the piperidinering is confirmedby the C2(4)chemical shiRs (TableIII1.16 (2)The 6H and 6C values for the arylcarbonyl moietiea account for a conjugation between these groups; the AXH6(7)~(5tH6(7)n(5)1=0.2 ppm is partially attributed to the field effect exerted by the carbonyl group on H6(7)n18; and the As[H2(4)a(5)-H2(4)p(5)]- 0.4 ppm is partially attributed to the field effect exerted by the carbonyl group on H2(4)p.1g920(3) The shieldingof sC2(4)in 5 with respect to the same carbon atom in 4 (A&C2(4)(5)-C2(4)(4)1= -2 ppm) canbe attributed to the steric compressing effectexertedby the arylcarbonyl group on H2(4)p. 796 I Journal of Pharmaceutical Sciences Vol. 82, No. 8, August 1993

(4) The chemical shift of the N-CH, group is in agreement with an equatorial disposition of this substituent.21 Summarizing, ‘H and 13Cdata of 5a-f in solution show the predominant chair-envelope conformation for the bicyclic system, as in the solid state for 5d. The main differences between crystal and solution conformations are: (1) The Battening of the piperidine ring seems to be greater in the crystal structure. (2) The conformations of the C3 groups are different. These differences are explained as follows: in the “frozen” crystal conformation, the arrangement of the C3 groups and the concomitant flattening at C3 minimizes the steric repulsion between C3 groups, whereas in solution state, the freedom for C3-C = 0 and C3-0 partial rotation seem8 to be predominant, as can be seen from molecular models. A similar conformational behavior is deduced for the hydm chlorides of 5a and 5b from the ‘H and ”C magnetic parameters in CD,OD solutions.The deshielding observed for the proton and carbon signals of the protoned derivativescan be mainly ascribed to the field effect exerted by the positively charged nitrogen atom.3 The ‘JH2(4)/3-H1(5) and ‘JH2(4)a~H1(5)as well as the Wyi of Hl(5) and the A8 between H2(4)a and H2(4)P remain practically invariable after the protonation and support this

Table Il-Pmton Magnetlc Parameters for 5d and 56 (CDCIJ Chemical Shiffs8

H1(5)(brs) H2(4)a(dd) H2(4)p(dd) H6(7)x(m)" H6(7)n(m)" CH,N(s) CH,O(s) H2"d) H4'(dd) H5'(dd) H6'(dd) Harom(m)" CH(s)

6, PPm

5e

5d 3.25

ww = 101 Hz 2.69 2.33 2.03 1.87 2.38 3.76 9.21 7.98 7.61 8.93 -

W%

J, Hz

Coupling

3.23 I : 10 HZ 2.54 2.15 1.96 1.82 2.31 3.64

se

5d

H2(4)aLH2(4)/3 --14.4 -15.1 2.0 2.6 H2(4)41(5) 3.9 4.0 H2(4)w1(5) 1.7 H2'44' 7.9 H4'45' 4.9 H5'46 1.7 H6-H4'

-

-

-

-

a Abbreviations: brs, broad singlet; d, doublet; dd, doublet of doublets; m, multiplet; s, singlet; H1, HPa, and H2p (or H5, H4a, and H4P) form a three-spin AMX system; error: f0.05 ppm. Error: f 0.3 Hz. Tabulated chemical shifts correspond to center of the rnultiplet.

Table lll--'3C Chemlcal Shlfts S (ppm) for 5d and 5e In CDCI, _____

Position

c3. . C6f71 CHiN CH,O C=O(P) C=O(a) C1' C2' C3' C4' C5' C6' CH

Shift, ppm

5d

5e

59.63 38.90 78.17 25.39 38.90 52.41 164.14 172.89 153.36 125.34 136.85 122.96 150.74 -

59.48 38.24 77.85 25.55 38.48 52.12 171.54 173.05 138.05 128.34 128.43 127.09 128.43 128.34 56.51

assumption. Consequently,it seem obvious that the protonated forms, which predominate in a physiological medium, and the free base must exhibit the same predominant chair-envelope conformationof the t r o p e system slightly flattened at C3 and puckered at N8, with the N-methyl group in an equatorial position. Pharmmlogy-GABA is a well-known inhibitory neurotransmitter in the central nervous system.22 GARA receptorsare also present in the peripheral nervous system. The isolated guinea pig ileum myenteric plexus is particularly rich in GABAresponsive cellsand it is a standardbiological preparation for the study of GABA-mediated reeponees.23 GABAB receptor activation results in longitudinal muscle relaxation through an inhibitory preaynaptic action on cholinergic postganglionic neurons, and it is insensitiveto bicucullineor isoguvacine. (S)-(-)-Baclofen, on the other hand, is a selective GABAB reoeptor tqpnist. The title compounds were first investigated as putative GABA, ligands in a binding assay with a rat cerebral preparation and tritiated GABA as the radioligand in the presence of 40 & isoguvacine.24 I Maximum GABAdisplacementwas -20% for

5cand5eand-l5%inthecaseof5b;5a,5d,and5fdidnotcause

M. significant displacement at concentrations up to Compound 5e was further assayed pharmacologicaUyon the electrically stimulated isolated guinea pig myenteric plexus preparation2SAdditionof this compound to the bath resulted in

a decrease in the amplitude of contractions but failed to antagonize baclofen-induced relaxation. Likewise, 5e antagonized exogenous aceiylcholine-induced contractions in this preparation [50% inhibitory concentration (IC,, = 3.6 f 0.7 lo-* M, n = 41. On the other hand, 5e exhibited a dose-dependent inhibitory effect on baclofen-induced relaxation on the isolated guineapigileum (IC50= 3.2 f 0.9 * lO-'M, n = 4). Theseeffeds were statistically significant (p <0.05).

-

Discussion Compound 5e appears to possess anticholinergic properties as assessed in the isolated guinea pig ileum myenteric plexus preparation challenged both electrically and by addition of exogenous acetylcholine, but it also displays baclofen antagonistic properties on guinea pig isolated ileum that can make it a suitable lead compound for the design of novel GABAB antagonist compounds.

Experimental Section Experimental and X-ray structural data for 5d are collected in Table I. All melting points were taken in open capillary tubes in a Electrothermal IA6304 apparatus and are uncorrected. The elemental analyses were made in a Perkin-Elmer elemental analyzer (model 240E). The IR spectra were recorded on a Perkin-Elmer 883 spectrophotometer in the solid state (KBr). The 'H NMR spectra of -4% (w/v)CDC1, solution were recorded at 300 MHz with a Varian UNITY-300 spectrometer. Spectral parameters included sweep widths of 4000 Hz in 24 K memory and acquisition times of 3.0 8 over 64 transients. Resolution enhancement using LB = -0.80, GF = 0.50, and GFS = 0.20 was followed by zero filling into 32 K memory prior to Fourier transformation. The "C NMR spectra were obtained at 20 MHz on a Varian FT-80 A (PFT) spectrometer with -25% (w/v)CDC1, and dimethyl sulfoxide (DMSO) solutions. Two types of spectra were recorded: proton-noise decoupled spectra (to determine the chemical shifts) and offresonance decoupled spectra (to help assign the signals), at a spectral width of 5000 Hz,with an acquisition time of 1.638 8, a delay time of 1.640, and a pulse width of 5 pa. The homonuclear 'H chemical shift correlated 2D diagram of 5d was performed by application of the COSY-45 experiment14 at 300 MHz on aVarian UNITY-300 spectrometer. The COSY spectrum was run with sweep widths of 3004 Hz (memorysize 512 x 512 data points over 256 experiments). Sine bell window functions in both dimensions were used. Each experiment involved eight scans and two dummy scans with an initial delay of 1 s. A heteronuclear proton-carbon correlation experiment was performed for 5d on a Varian UNITY-300 spectrometer with the standard XHCORD pulse sequence1sJ6 with a sweep width of 13889 Hz in the F2 domain ("C) and 2869 Hz in the F1 domain ('HI.The memory size in F2 was 2K and F1 was 1K over 96 experiments, each involving 512 transients. A relaxation delay of 1s was used, and the fmed delay times were calculated from the compromise value 'JqH= 140 Hz. A sine bell window function was applied in both domains. All measurements were done at 303 K with tetramethylsilane (TMS)as the internal reference. The synthesis and purification of 1-4 have been previously described.4 Synthesis of Esters (5a-f): General Procedures-Method A-To a stirred solution of methyl 3fihydroxytropane-3a-carboxylate(4; 0.20 g, 1 mmol) and triethylamine (0.10 g, 1 mmol) in anhydrous methylene chloride (5 mL) was added in a dropwisemanner a solution of the corresponding acyl chloride (1mmol) in anhydrous methylene chloride (5 mL). The reaction mixture was stirred at room temperature for 24 h, and washed with sodium bicarbonate and then with water. The organic layer was dried (anhydrous magnesium sulfate), and the solvent was removed under reduced pressure to give an oil that was purified on a silica gel column prepacked in ethyl acetate. Elution of the column with ethyl acetate/ethanol gave the corresponding ester as a solid, which was further crystallized from the adequate solvent. Method B-A solution of DCC (N,W-dicyclohexylcarbodiimide; 0.23 g, 1.125 mmol) and DMAP (4-dimethylaminopyridine;12 mg, 0.1 Journal of Pharmaceutical Sciences I 797 Vol. 82, No. 8, August 7993

to 3 * lo-' M. The tissue, after washing with Ringer solution, mmol) in anhydrous methylene chloride (5 mL) was added in a was allowed to equilibrate. After equilibration, submaximal doses of dropwise manner to a stirred solution of methyl 3p-hydroxytropanebaclofen M) were added to the bath at 25-min intervals until a 3a-carboxylate (4; 0.20 g, 1 mmol) and the corresponding acid (1 constant response was obtained (thus avoiding any desensitization). mmol) in anhydrous methylene chloride (5 mL). The mixture was This contraction (in mm) was taken as 100%effect. stirred at room temperature for 6 h (48 h for 5d).Then, the reaction Different concentrations of the product studied were added to the mixture was filtered, and the solvent was removed under reduced preparation 15 min before the addition of submaximal concentrations pressure. The residual oil was treated with ethyl ether, the solid that of baclofen. Antagonistic activity is reported as the IC,, value (the precipitated (DCU, N,"-dicyclohexylurea) was filtered, and the concentration that causes 50% inhibition of the maximal relaxation ether was evaporated, yielding the corresponding ester as an oil that obtained with baelofen). The mean ICso values and their standard was purified in the same manner as described in method A. Methyl 3~(4'-Fluorobenzoyloxy)-N-methyl-8-azabicyclo[3.2.11- errors were determined by probit analysis.29 The statistical significance of differences found between vehicle and test compoundsoctane-3ocarboxylate ( 5 a h T h i s compound was obtained (method treated tissues were determined by the t test (awas preset at 0.05). A) in 60%yield, mp 69-71% "C (from ethyl acetate/petroleum ether); IR (KBr); uC0, 1751 and 1712 cm-l. Anal.-Calcd for C17H2,FN0,: C, 63.95; H, 6.27; N, 4.36. Found C, References and Notes 63.76; H, 6.47; N, 4.30. 1. Izquierdo, M. L.; Gglvez, E.; Burgos, C.; Vaquero, J. J.; Florencio, Methyl 3&(4'-Chlorobenzoyloxy)-N-methyl-8-azabicyclo~3.2.11F.; Ojales, A.; Innerarity, A. Eur. J. Med. Chem. 1989,24,123. octane-3twcarboxylate (5b)-This compound was obtained (method 2. Irie a, I.; Lorente, A.; Gfdvez, E.; Florencio, F.; Sanz, J. Eur. J. A) in 5l%yield, mp 86-87 "C (from ethyl acetate/petroleum ether); IR Med: Chem. 1990,25,499. (KBr): uC0, 1745 and 1712 cm-'. 3. Izquierdo, M. L.; Arias, M. S.; Gglvez, E.; Rico, B.; Ardid, I.; Sanz, Anal.-Calcd for C17H,,CIN0,: C, 60.45; H, 5.97; N, 4.15. Found: J.; Fonseca, I.; Orjales, A.; Innerarity, A. J. Pharm. Sci. 1991,80, C, 60.36; H, 6.24; N, 4.18. 554. MethyI 3~(3',4',5'-Trimethoxybenzoyloxy)-N-methyl-8-azabic~ 4. Burgos, C., Thesis, University of AlcalS. de Henares, February clo[3.2.lloctane-3ocarboxylate (5c)-This compound was obtained 1991. (method A) in 70% yield, mp 110-111 "C (from ethyl acetate); IR 5. Arias, M. S.; Gfdvez, E.; Izquierdo, M. L.; Burgos, C. J. Mol. (KBr): d o , 1753 and 1708 cm-'. Struct. 1986,147, 381 and references herein. Anal.-Calcd for: C2,H,,N07: C, 61.06; H, 6.92; N, 3.56. Found: C, 6. Main, P.; Fiske, S. J.; Hull, S. E.; Lessinger, L.; Germain, G.; Declerq, J. P.; Woolfson, M. M. MULTM80. A system of com60.91; H, 7.26; N, 3.87. puter programs for automatic solution of crystal structures from Methyl 3~-(3'-Pyridincarbonyloxy)-N-methyl-8-azabicycloX-ray diffraction alata; Universities of York, England, and Lou[3.2.lloctane-3ocarboxylate (5d)-This compound was obtained vain, Belgium, 1980. (method B) in 88% yield, mp 114-116°C (from ethyl acetate/ 7. Stewart, J. M.; Kundell, F. A.;Baldwin, J. C. TheXRAYSystem. petroleum ether); IR (KBr): uC0, 1743 and 1715 cm-l; 'H NMR (see Computer Science Center; University of Maryland College Park, Table 11); 13C NMR (see Table 111). MD, 1976. Anal.-Calcd for: C16H,,N20,: C, 63.14; H, 6.62; N, 9.20. Found: C, 8. Martinez-Ripoll, M.; Cano, F. H. PESOS. A computerprogram for 63.39; H, 6.65; N, 9.52. the automatic treatment of weighting schemes; Instituto RocaaoMethyl 3~-(Diphenylacetoxy~-N-methyl-8-azabicyclo~3.2.lloclano; Madrid, Spain, 1975. tane-3ocarboxylate (5e)-This compound was obtained as an oil 9. Nardelli, M. Comput. Chem. 1983, 7, 95. (method B) in 67%yield; IR (film): VC0,1752 and 1749 cm-'; 'H NMR 10. Znternational Tables for X-ray Crystallography, Vol. 4; K och. (seeTable 11); 13C NMR (see Table 111). Birmingham, U.K., 1974 (present distributor, D. ReidepDorl drecht, Germany). Methyl 3~-(Xanthen-9'-carbonyloxy)-~-methyl-8-azabicyclo11. Motherwell, W. D. S.; Clergg, W. PLUTO. Program for Plotting [3.2.lloctane-3ocarboxylate (50-This compound was obtained Molecular and Crystal Structures; University of Cambridge: (method B) in 77% yield, mp 111-114 "C (from ethyl acetate); IR Cambridge, U.K., 1978. (KBr): VCO 1752 and 1749 cm'. 12. Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975,97, 1354. Anal.-Calcd for: C2,H,,N0,: C, 70.75; H, 6.18; N, 3.44. Found: C, 13. Derome, A. E. Modern NMR Techniques for Chemistry Research; 70.92; H, 6.18; N, 3.40. Pergamon: Oxford, U.K., 1987. Pharmacological Methods-Tissue Preparation-Female guinea 14. Bax. A.; Freeman. R. J. Maan. Reson. 1981.44, 542. pigs (weighing 300400 g) were killed by a blow to the head and then 15. Perpick-Dumont, M.; Reynoids, W. F.; Enriquez,-R. Magn. Reson. exsanguinated by cutting the jugular vein. Segments of distal ileum Chem. 1988.26, 358. -20 cm long were quickly removed (excision was performed -10 cm 16. Reynolds, W. F.; McLean, S.; Perpick-Dumont, M.; Enriquez, R. above the ileocecal junction) and immediately placed in aerated Magn. Reson. Chem. 1988,26,1068. Ringer solution of the following composition (mM):NaCl(154), KC1 17. Haasnot, C. A. G.; de Leeuw, F. A. A. M.; Altona, C. Tetrahedron (5.66), CaC1, (2.54), NaHCO, (5.95), glucose (2.77), and choline 1980,36, 2783. hydrochloride, (0.002). Strips of guinea pig ileum myenteric plexus 18. Cabezas, N.; Martinez, M.; G W v e z , E.; Arias,M. S.; Florencio, were mounted in an organ bath, superfused with Ringer solution, F.; Garcia-Blanco, S. J. Mol. Struct. 1988, 172, 381. aerated with carbogen, and warmed at 37 "C, according to the method 19. Cabezas, N.; Martinez, M.; GCklvez, E.; Arias, M. S.; Florencio, of Puig et al.25 The plexus was connected to an isotonic transducer F.; Sanz, J. J. Mol. Struct. 1989, 197, 5. under a resting tension of 0.5 g, and contractile activity was 20. Gfdvez, E.; Arias, M. S.;Cabezas, N.; Martinez, M. J.Mol. Struct. 1990,220, 55. registered on an UGO BASILE polygraph. After a I-h equilibration 21. Gglvez, E.; Arias, M. S.; Bellanato, J.; Garcia-Ramos, J. V.; period, the strips were ready for use and were challenged with drugs. Florencio, F.; Smith-Verdier, P.; Garcia-Blanco, S. J. Mol. Struct. Electrical Stimulation-The strips were electrically stimulated by 1985, 127, 185 and references therein. square wave pulses (5 ms) of 70 V at frequencies of 0.15 Hz. This 22. Curtis, D. R.; Johnston, G. A. R. Ergebn. Physiol. 1974,69,97. stimulus induces release of acetylcholine from cholinergic neurons26 23. Bowery, N. G. Trends Pharmacol. Sci. 1982,3,400. and subsequent muscular contraction after interaction of this neu24. Hill. D. R.: Bowerv. N. G. Nature 1981.290. 149. rotransmitter with a population of postsynaptic M,-type cholinergic 25. Puig, M. M.; Gas&, P.; Musacchio, J. M. J. Pharmacol. Exp. receptors.27 GABA modulates this release through an inhibitory Ther. 1978.206. 289. mechanism mediated by GABABreceptors.23 Paton, W. D. M.; Zar, M. A. J. Physiol. 1968, 194, 13. 26. Isolated Ileum-Segments of ileum 2.5 cm long were mounted in a 27. Konno, F.; Takayanagi, I. Eur. J. Phurmacol. 1986, 132, 171. 7-mL organ bath bubbled with a mixture of 5%CO, and 95%0, and 28. Giotti, A.; Luzzi, S.; Spagnesi, S.; Zilletti, L. Br. J. Pharmacol. maintained at 37 "C. The ileum was connected to a n UGO-BASILE 1983, 78, 469. isotonic transducer under a resting tension of 0.5 g, and responses 29. Tallarida, R. J.; Murray, R. B. Manual of Pharmacologic Calcuwere recorded on a polygraph of the same make. Preparations were lations; Springer-Verlag: New York, 1981. allowed to equilibrate for 60 min before drug administration. GABA Antugonistic Activity-Baclofen elicits only relaxation on Acknowledgments the isolated ileum preparation as opposed to the contraction followed by relaxation observed with GABA.28 A concentration-response We thank the Comisi6n Interministerial de Ciencia y TecnoloGa (Grant FAR 88-0440) for the support of this research. curve for this agonistic drug was determined at doses ranging from

798 I Journal of Pharmaceutical Sciences Vol. 82, No. 8, August 7993