CHIRALITY 4:23&239 (1992)

Synthesis and Pharmacological Investigation of Stereoisomeric Muscarines MARC0 DE AMICI, CLELIA DALLANOCE, CARL0 DE MICHELI, EN20 GRANA, GIUSEPPINA DONDI, HERBERT LADINSKY, GIOVANNBATTISTA SCHIAVI, AND FRANC0 ZONTA Istituto Chimico-Farmaceutico(MDA., CB.),Milan, Dtpartimnto di Scienze Farmaceutiche (CDM.), Trieste, Istituto di Farmacologia (E.G., GD., FZ), Pavia, and Dipartimento di Biochimica e Farmacologia Molecolare (HL.,G.S.), Boehnnger Ingelheim Itah, Milan, ltdy

ABSTRACT The synthesis of the eight stereoisomers of muscarine has been efficiently accomplished by utilizing the two enantiomers of lactic esters as starting material. The synthetic strategy is based on a SnCI4-catalyzedaddition of allyltrimethylsilaneto 0-protected lactic aldehydes followed by an iodocyclization process. All the final derivatives possess an enantiomeric excess higher than 98%. The four pairs of enantiomers bound to M1, MZ,and M3 muscarinic receptor subtypes in membranes from cerebral cortex, heart, and salivary glands, respectively,and recognized heterogeneousstates of the receptors. Of the eight isomers, only natural muscarine (+)-1recognized three affinity states of the M2 receptor. The compound was also the only one to show selectivity in the binding study, demonstrating 37- to 44-fold higher affinity for the M2 than for the MI or M3 receptors. In addition, the compounds were tested in functional assays on isolated guinea pig atria (M2 receptors) and ileum (mixed population of M2 and M3 receptors) and their muscarinic potencies were determined. Among the eight isomers, again only ( + )-1 enantiomer was found to be very active on both tissues. Its potency was more than two orders of magnitude higher than that of its enantiomer ( - )-1 as well as the other six isomers. The eudismic ratios (E.R.) deduced from the two functional tests were 324 and 331. o 1992 Wiley-Liss,Inc. KEY WORDS: total synthesis, chiral muscarines, iodocyclization process, muscarinic receptor subtypes MI, MZ,and M3, affinity states, eudismic ratio

INTRODUCTION Muscarine (+)-1,a natural alkaloid whose structure and stereochemistry is shown in Figure 1, is a cornerstone of medicinal chemistry, being a substance with strong and specific cholinomimetic activity.’” Its effects on smooth muscles of many organs, exocrine glands and heart were termed “muscarinic effects.” Recent pharmacological investigations carried out with highly selective muscarinic cholinergic antagonists have revealed the existence of three distinct subtypes of muscarinic receptors: M1, M2, and M3. 4 On the other hand, up to three affinity states, superhigh (SH),high 0,and low (L) can be distinguished in different tissues with muscarinic agonists. In connection with our studies on the structureactivity relationship of muscarinic ligands6 we became interested in the pharmacologicalselectivity of the diastereomers of muscarine toward the three muscarinic receptor subtypes. For such a purpose, in a previous paper, we reported a preparative route to these compounds via enzyme-catalyzed asymmetric reductions of suitable intermediates. Six out of eight muscarines were prepared in remarkable enantiomeric excess (e.e. 96% or higher) whereas the remaining two were synthesized in 81Yo e.e. Since pharmacological selectivity can be carefully evaluated only on a set of compounds with very close and high e.e. values, a new synthetic route of the eight stereoisomericmuscarines based on the nucleophilicaddition of allyltrimethylsilane D

1992 Wiley-Liss, Inc.

to 0-protected, optically active, lactic aldehyde followed by an iodocyclization process was designed. Pharmacologicalinvestigation of the muscarine diastereomers was carried out both on isolated tissue preparations (functional tests) and on tissue homogenates incubated with a specific radiolabeled ligand (binding tests). CHEMISTRY Large quantities of racemic icdoalcohols ( f )-2, ( f )-3, (f)-4, and ( f ) - 5 (Fig. 2) were prepared by reacting ( f )-methyl lactate with dimethyl maleate followed by appropriate transformations of the intermediate ketoesters. At first glance, we considered the possibility of preparing the required chiral muscarines through an optical resolution of iodoalcohols ( f )-24f )-5after derivatization with a resolving agent. Reaction of the above-mentionedalcoholswith commercially available ( - )-(1s)-camphanicchloride (Scheme 1) yielded a mixture of diastereomeric esters. The diastereomers generated from ( f)-2and ( f )-4 were partially separable by flash chromatography whereas the diastereomers derived from ( f )-3 and (f)-5could not be separated. Due to the difficulties associated with the chromatographic separation, this route was Received for publication October 30, 1991; accepted February 7, 1992. Address reprint requests to Carlo De Micheli, Dipartimento di Scienze Farmaceutiche, Piazzale Europa 1, 34127 Trieste, Italy.

231

STEREOISOMERIC MUSCARWES

abandoned and the total synthesis of 1 and its diastereomers was tackled. Among the numerous approaches to 1 and some of its stereo isomer^,^ one of the most attractive is that described by Chastrette and co-workers.lo In its first application, the authors prepared ( f)-1 in six steps starting from methyl vinyl ketone.'@ The same strategy was later applied to the synthesis of (+ )-1 and (- )-1 by employing D-( + )- and L-( -)-threonine as starting materials.lob As exemplified in Scheme 2, intermediate erythro-(+ )-14a yielded, almost exclusively, the 2,5-&-disubstituted iodoalcohol ( - )-3(cis:trans

HO

xx/

;Me3

1

1-

0

(+)-1 (2S,4R,5S) Fig. 1. Structure and stereochemistry of natural muscarine.

(*)-2

(f)-5

(f)-4

(f)-3

Fig. 2. Racemic iodoalcohols.

6

7

8

9

10

(f)-5

R'COCUPy

R*= (-)-camphanyl Scheme 1.

11

232

DE M C I ET AL.

97:3) which was the immediateprecursor of the final derivative

/

(+)-l.lOb

Since we were interested in the preparation of all the stereoisomeric muscarines, the couples of erythro-threo isomers 14a-16a and 14b-16b were submitted to iodoetherification in order to investigate the stereodirecting effect of the homoallylic hydroxy group. As a method of deriving the required substrates,we opted for the addition of allyltrimethylsilane to a chiral a-alkoxy aldehyde (Scheme 3). l1 Alkylation of commercially available @)-methyl and (S)-ethyl lactate was followed by DIJ3AH reduction to provide the requisite aldehydes 15a and 15b.12SnC14-mediatedaddition of allyltrimethylsilane to 0-benzyl lactic aldehyde 15b afforded a 92.3:7.7 mixture of 16b and 14b (Scheme 3). The corresponding nucleophilic addition to 15a was less selective (16a:14a 85.3:14.7)due to the higher steric hindrance of the 2,6-dichlorobenZyl group. The observed diastereofacial selectivity was interpreted in terms of chelation of SnCl, by the a-alkoxy and carbonyl oxygens of the aldehyde,as shown in Figure 3. l1 Consequently, the prevalent formation of the threo isomers 16a,b derives from the attack of the nucleophile to the less hindered diastereotopic face of the chelated carbonyl moiety. Both the couples 14a/16a and 14b/16b were easily separated by flash chromatography and their ratios were evaluated by capillary GLC analysis. The enantiomeric excess of the compoundswas not evaluated at this stage but in the following step of the reaction sequence. The diastereomers 16qb and 14a,b were separately cyclized at - 15°Cwith iodine in acetonitrile to produce mixtures of diastereomeric 2,5-disubstituted tetrahydrofurans (Scheme 4). As previously reported for 14a,lo cyclizationof the erythro isomers 14a,b was remarkably diastereoselectiveand yielded

OH

C14Sn - - -.-- 0

\

Fig. 3. Nucleophilic addition of allyltrimethylsilane to 0-protected lactic aldehyole chelated with SnCl,.

the cis-2,5-disubstituted tetrahydrofuran derivative 3 as the main product. The 2,6-dichlorobenzyl group of 14a possesses the most appropriate balance of electronicand steric properties to give the highest value of se1ectivity.l3 On the other hand, cyclization of the threo diastereomers 16a,b was by far less selective and gave, in the case of 16b, comparable amounts of 2 and 4. The couples 2-4 and 3-5 were easily separable by column chromatography and their structure was SeCuTed by 'H-NMR and specific rotation. HPK analyses of the Mosher esters of the chiral iodoalcohols showed, for all the compounds, an enantiomeric excess higher than 98%. In summary, the sequence which uses 0-benzyl lactic aldehyde (15b)as starting material is the most convenientroute to the eight stereomeric muscarines since it combines the highest selectivity in the addition of allyltrimethylsilaneto 15b with an almost nonselective iodocyclization process of the main

ODCB 6 steps

COOH

i2/MeCN,

NH2

R

X,X/

OH

D-Threonine(2R,3S)

(-)-3(2S,4R,5S)

(+)-14a(4R,5S)

OR

OH ab

OR

OR

C

L

- + - OH 15a: R = DCB 15b R = Bzl

(+)-16a(85.3%) (+)-16b(92.3%)

OH (+)-14a( 14.7%) (+)-14b(7.7%)

I

233

STEREOISOMERIC MUSCARINES

product 16b. In such a way iodoalcohols 2 and 4 were prepared in reasonable yield and comparable amount. The preparative synthesis of diastereomers 3 and 5 was then &ciently accomplished via a Mitsunobu inversion of conliguration at C-4 (Scheme 5).l4 Treatment of intermediates 2 and 4 with triphenylphosphine (TPP), diethyl azodicarboxylate (DEAD),and benzoic acid at 0°C cleanly produced benzoate 17 and 18, respectively, with complete inversion of configuration and high yield (86 and 74%).Derivatives 17 and 18 were then quantitatively hydrolyzed to 3 and 5 by treatment with potassium carbonate in water-methanol (1:1).Finally the eight stere-

omeric iodoalcohols 2-5 were sequentially reacted with dimethylamine and methyl iodide to produce all the possible muscarine stereoisomers in high enantiomeric excess (>98% e.e.) (Scheme 6). MATERIALS AND METHODS Chemistry ( + )-@)-Methyl lactate, ( - )-@-ethyl lactate, and ( - )-(1s)camphanic chloride were obtained from commercial suppliers and were used as such. (2R)-and (2S)-2-(benzyloxy)propanal were prepared according to literature methods; 15-17 their spe-

+

~I HO

OR 12 - MeCN

v

~

HO +..

I -***,,#/&/

OH

0 (-)-3(%)

Yield %

(+)-5(%)

(+)-14a

80

99.5

0.5

(+)-14b

81

91.8

8.2

OH

(-14%)

Yield %

(+)-4(%)

(+)-16a

75

85.0

15.0

(+)-16b

68

51.1

48.9

Scheme 4.

(-1-2

(+)-2

a

(-)-17 (+)-17

b

(+)-3

(+)-4

(+)-18 (-1-4

a ____)

(-)-18

b

a: Ph3P/DEAD/C6H5COOH; b: K2COdwater-methanol. Scheme 5.

(-)-S

234

DE AMICI ET AL.

(-)-2

-h+a,b

NMe,

0

(+)-2

ab

(-)-3

a,b

(+)-l

1-

(+)-19 (-)-19

a,b

(+)-3

(-)-1

n

(-)-4

a,b

I

(+)-20

(-)-20

-

+

(-)-21

a,b

(-)-7

(-)-21

a: NHMe,/MeOH; b: Mel/Et20. Scheme 6.

Data of 6:yellow viscous oil; Rf 0.30; [WID' O - 23.95 (c 1.378, cific rotations agreed with the value previously reported for (2R)-2-(benzyloxy)propanal. l7 (2S)-2-(2,6-Dichlorobenzyloxy)-CHCI,); lH NMR:1.00 (s,3H, Me); 1.09 (s, 3H, Me); 1.13 (s,3H, propanal was prepared with the same procedure. lH N M R Me); 1.27 (d, 3H, 5-Me;J = 6.2); 1.74 (m, 1H); 1.93 (m, 2H); 2.05 spectra were recorded with a Bruker AC-E 300 (300 MHz) (m, 1H);2.44 (ddd, lH, H-3;J3,4= 4.1;h,, = 10.6;J3,3t= 13.4); 2.59 spectrometer in CDC13 or D20 solution; chemical shifts (6) are (ddd, lH, H-3';j3t,4=6.2;J2,? = 7.8;j& = 13.4);3.24 (dd, lH, H-6; ; (dd, lH, H-6'; J2,e=5.4; J6,e r9.8); expressed in ppm and coupling constants v) in Hertz. Capillary J2,6 =7.8; j6,e ~ 9 . 8 ) 3.31 GLC analyses were conducted on a gas chromatograph 4.054.16 (m, 2H, H-2, and H-5); 5.40 (ddd, lH, H-4; J4.5 -,- =2.0; equipped with a Supelcowax 10 fused silica gel column (15 m, J3,4 = 4.1; Jy,4 = 6.2). Data of 7:yellow viscous oil; R+0.277; TalnZ0+ 12.32 (c 0.25 pm) under the following conditions: 40°C (3 min) to 110°C (5 min), heating rate 10"C/min, to 225"C, heating rate 2.5"C/ 1.112, CHCl,);-]H NMR 1.00 (s,3H; Me); 1.09-6, 3H, Me); 1.13 min. N2 was used as the carrier gas at 0.4 atm. HPLC analyses (s,3H, Me); 1.27 (d, 3H, 5-Me;J=6.2); 1.71 (m, 1H); 1.92 (m, 2H); were performed on a chromatograph equipped with a UV detec- 2.08 (m, 1H); 2.44 (ddd, lH, H-3;J3,4=4.1;J2,3 = 10.6;J3,3~= 13.4); tor (h= 254 nm) and a Whatman Partisil 10 column (250 mm 2.60 (ddd, lH, H-?;J3*,4 = 6.3;J2,3! =7.8J3,3 = 13.4);3.25 (dd, lH, length, 4.6 mm id.); a mixture of n-hexane-ethyl acetate 9:l H-6h,6 =7.8J6,@ =9.8); 3.32 (dd, lH, H-6';]2,& =5.1;J6,@=9.8); was used as the eluent at a flow rate of 1.0 ml/min. Retention 4.054.15 (m, 2H, H-2, and H-5); 5.40 (ddd, lH, H-4;J4,5=2.0; times (Rt)are expressed in minutes. Rotary power determina- J3,4 = 4.1; J3,,4 = 6.3). Data of 10 crystallized from n-hexane:ethyl acetate 20:l as tions were carried out with a Perkin Elmer 241 polarimeter, 1 0.325; [a]D2o + 15.56 (c 0.964, coupled with a Haake N-3B thermostat. Melting points and colorless prisms, mp 8 ~"c;Rf molar purities were obtained from the DSC curves, recorded CHCl,); lH NMR:0.99 (s,3H, Me); 1.08 (s,3H, Me); 1.12 (s, 3H, with a differential scanning calorimeter under the following Me); 1.21 (d, 3H, 5-Me;J = 6.5); 1.72 (m, 1H); 1.95 (m, 2H); 2.10 conditions: sample weight about 2 mg; heating rate 2"C/min. (m, 1H);2.29 (ddd, lH, H-3;J3,4=O.8;J2,3 =6.1;]3,3( =13.4); 2.44 Indium was used as the reference compound. TLC were carried (ddd, lH, H-3'; J3,,4 = 4.2; ]2,3t = 10.5; J3.31 = 13.4); 3.28 (dd, lH, out on commercial silica gel GFZg plates. Liquids were charac- H-6 J2,6 ~ 6 . 6 ;JS,7 =11.0); 3.32 (dd, lH, H-6'; J2,v =4.5; terized by the oven temperature for Kugelrohr distillations. J6,6' = 11.0); 4.24 (m, lH, H-2); 4.34 (dq, lH, H-5; 14,s =3.4; = 6.5); 5.47 (m, lH, H-4). ( + )-(R)-MTPAesters were prepared according to the proceData of 11: colorless viscous oil; Rf 0.30; [ a ]2o~ - 8.97 (c dure described previously. l8Elemental analysis (C,H,N)of new 0.780, CHC1,); ]H NMR 0.99 (s, 3H, Me); 1.08 (s,3H, Me); 1.12 compounds agreed with theoretical values i 0.3%. (s,3H, Me); 1.21 (d, 3H, 5-Me;J= 6.5); 1.71 (m, 1H); 1.95 (m, 2H); General procedure for the preparation of camphanic esters 2.11 (m, 1H);2.30 (ddd, lH, H-3;J3,4=0.8;J2,3 =6.1;J3,y = 13.4); 6-1 3 2.44 (ddd, lH, H3';J3,,4 =4.2;J,,y =10.5;J3.r=13.4); 3.28 (dd, ~4.5; To a solution of iodoalcohols ( f )-24f )-5 (2.0 g, 8 mmol) in lH, H-6 J2,6 =6.6; J6,y = 11.0); 3.32 (dd, 1H, H-6'; pyridine (15 ml) was added, under stirring, (-)-camphanic J6,& = 11.0); 4.23 (m, lH, H-2); 4.34 (dq, lH, H-5; J4,5=3.4; chloride (1.88 g, 8.7 mmol) at 0°C. After stirring overnight at J5,Me = 6.5); 5.47 (m, lH, H-4). We were unable to separate the couples 8-9 and 12-13. room temperature, the mixture was diluted with ether (100 ml) and washed with a saturated aqueous solution of cupric sulfate ( + )-10, ( - )-11 (3x 25 ml). After the usual work-up, the residue was flash Hydrolysis of camphanic esters ( - )-6,( + )-7, To a solution of camphanic ester (1.7 g, 4.0 mmol) in methachromatographed on silica gel (eluent: n-hexane-ethyl acetate nol (15 ml), a 20% aqueous solution of potassium carbonate (20 2:l). Rf values were evaluated with the same mobile phase.

STEREOISOMERIC MUSCARINES

ml) was added. The mixture was stirred at room temperature until disappearance of the starting material. The organic solvent was evaporated under reduced pressure and the aqueous phase extracted with dichloromethane (4 x 25 ml). After the usual workup, the residue was crystallized from n-hexane in and (-)-2,and Kugelrohr distilled at llO°C/ the case of (+)-2 0.5 mm Hg in the case of ( + )-4 and (-)-4. General procedure for the addition of allyltrimethylsilane to aldehydes (-)-15a,(-)-15b,and (+)-15b A solution of stannic chloride (11.2 ml, 0.1 mol) in dry dichloromethane (400 ml) was cooled to - 78"C, under a stream of nitrogen. To the solution was added, dropwise, a solution of the aldehyde (0.096 mol) in dry dichloromethane (50 ml). The solution was stirred for 10 min, then allyltrimethylsilane(16.9 ml) was added in one portion. After stirring at - 78°C for 45 min, the reaction mixture was quenched by a dropwise addition of water (50 ml). The reaction mixture was allowed to warm to room temperature and the organic phase separated. After the usual workup the residue was column chromatographed on silica gel (eluent 15% ethyl acetate-cyclohexane) to produce pure 16a and 14a (or 16b and 14b) in 78-8070 yield. The ratios 16a/14a and 16b/14b were evaluated by capillary GLC under the conditions reported in Materials and Methods.

(+)-14a: Rt 43.29; Rf(15% AcOEt-cyclohexane) 0.56; [N]D 20 + 42.94 (C 1.241, CHC13) [lit.'ob [U]D 20+46.7 (C 4.9, E@H)I. (+)-16a:Rt 41.80; Rf 0.61; [BID + 29.51 (c 1.715, CHCl3). (+)-14b Rt 26.46; Rf (15% AcOEt-cyclohexane) 0.336; [ a ] 20 ~ + 37.40 (C 1.572, CHCl3). (-)-14b [ a ] 20-37.66 ~ (C 1.056, CHCl3). (+ )-16b Rt25.30; Rf 0.378; [ ~ b+ 53.92 (C 0.510, CHC13). (-)-16b [U]D20- 53.87 (C 0.973, CHC13) [lit.17 [ a ] ~-49.0 (C 1.578, CHCl3)].

235

ance of the starting material, the solvent was evaporated off and the residue was column chromatographed (eluent: 10% ethyl acetatsyclohexane) to yield pure (-)-17 (0.368,86%). In analogy, as shown in Scheme 5, we prepared (+)-17, (+)-18,and (-)-18.

(-)-17: colorless prisms from ligroin, mp 68.36"C molar purity 99.32%; Rf 0.435 (10% ethyl acetate-cyclohexane); [U]D 20 - 11.67 (C 0.934, CHCl3). (+)-I7 [ a ] ~ 11.72 (C 0.835, CHC13). (-)-18Yield 74%; colorless oil, Kugelrohr distilled at 175180"/0.5 mm Hg; Rf 0.38 (10% ethyl acetate-qclohexane); [u)D 20 - 33.86 (C 0.863, CHC13). (+ )- 1 8 [U]D 20 +33.97 (C 0.942, CHC13). B. To a solution of (-)-17 (0.368 g) in methanol (20 ml), a 20% aqueous solution of potassium carbonate was added. The mixture was stirred at room temperature until disappearance of the starting material. Methanol was evaporated off and the aqueous phase was extracted with dichloromethane(4 x 25 ml). After the usual work-up, the residue was Kugelrohr distilled at l10°C/0.5 mm Hg to yield 0.245 g (95%) of (-)-3. With the same procedure we prepared (+)-3,(+)-5,and ( - )-5. The enantiomeric excess of iodoalcohols 2-5, as previously reported, was evaluated by HPLC on the corresponding (+ )-(R)-MTPAesters. All the derivatives possessed e.e. values higher than 98%. Rf values were determined in n-hexaneethyl acetate 4:l; Rt values were evaluated under the conditions reported in Materials and Methods.

(+)-2:mp 62.66"C; molar purity 99.78%; Rf 0.21; Rt 28.42; 20 +0.347 (C 1.142, CHCl3). (-)-2[ah20 -0.346 (C 0.997, CHCl3). ( + )-3 Rf 0.14;Rt 31.03; [ub2o + 34.09 (c 0.874, CHC13). @it. General procedure for iodocyclization of ( +)-14a,( + )-14b, [ a ] 20 ~ 30.72 (C 0.874, CHCl3). (-)-3: [BID 20 - 33.72 (C 0.874, CHC13). (-)-14b,(+)-16a,(+)-16b,(-)-16b ( + )-4:Rf 0.14; Rt 30.67; [a]D 20 + 39.86 (c 1.088, CHC13). To a cold ( - 15°C) stirred solution of olefinic benzyl ether ( -)-4 [U]D 20 - 40.74 (C 0.763, CHC13). (0.045mol) in 70 ml anhydrous acetonitrile, under nitrogen, was ( + ) - 5Rf 0.21; Rt 31.27; [ a ]20 ~ + 13.51 (c 1.061, cHc13). added dropwise a solution of iodine (15.3 g, 0.06 mol) in dry (-)-5: [U]D 20 - 13.40 (C 1.292, CHC13). acetonitrile (200 ml). The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was Synthesis of 1, 19,20,21: general procedure treated at room temperature with a saturated Na2S203soluDiastereomeric muscarines 1, 19, 20,21 were prepared tion. Acetonitrile was evaporated under vacuum and the aquefrom the corresponding iodoalcohols in 7&75% by the sequenous layer was extracted with dichloromethane (3x 50 ml). The tial treatment with dimethylamine and methyl iodide, accordorganic extracts were washed twice with an aqueous saturated solution of NaHC03 and dried (Na2SO4). After evaporation of ing to the procedure previously described by US.^ The ammothe solvent, the residue was flash chromatographed on silica nium salts, crystallized from isopropanol, possess analytical gel (eluent:cyclohexaneethyl acetate 7:3) to give pure 2,3,4, data in agreement with the reported values7 The higher e.e. 5.Yields were in the range 6847%. The ratios reported in value of (+ )-21 is reflected in its specific rotation: [a]D 2o +31.09 (C 0.940, EtOH)]. Scheme 4 were obtained from GLC analyses of the reaction +38.06 (C 1.058, EtOH) [lit.7[~]~ mixtures carried out under the conditions reported in Materials Pharmacology and Methods. Binding experimentsl9 ( + )-5, (-)-5 Synthesis of ( + )-3, (--)-3, Male CD-COBS rats, 180-200 g body weight, were killed by A. The following procedure is representative. To a magneti- decapitation. Tissues were removed, cleaned, homogenized in cally stirred and ice-cooled solution of (-)-2 (0.300 g, 1.24 phosphate buffer, pH 7.4, containing KH2PO4 and Na2HP04.2mmol), TPP (1.300 g, 4.96 mmol), and benzoic acid (0.303g, 2.48 H20 (50 mM) (w/v: cerebral cortex, 1:80; whole heart, 1:130; mmol) in dry THF (20 ml) a solution of DEAD (0.785 ml, 4.98 submandibular glands, 1:150) with an Ultra-Turrax at maximmol) in THF (5 ml) was added dropwise. At the disappear- mal speed for 30 sec at O'C, followed by dispersion in a Potter-

DI.[

236

DE AMICI ET AL.

Elvehjem homogenizer (15 strokes) and filtered through two layers of cheesecloth. [ 3H]N-Methylscopolamine([ 3HJNMS), 2.92 TBq (78.9 Ci/ mmol) and [ 3Hlpirenzepine([ 3KJpZ), 3.22 TBq (87.0 Ci/mmol) of radiochemical purity greater than 99% were purchased from NEN, Boston. Racemic quinuclidine benzylate (QNB) was synthesized in the Chemistry Department of Dr. Karl Thomae GmbH, Biberach, Germany. Binding curves for the different compounds were derived indirectly from competition experiments against 0.5 nM[ 3H]PZ labeling the cerebral cortex muscarinic receptor (M1)and 0.3 t~hf[~HJNlvrS for the muscarinic receptors of the heart (M2)and submandibular glands (M3)as described previously.l9 A 1 ml portion of homogenate was incubated for 45 min at 30°C in the presence of the marker ligand and 4 (Table 2) to 13 (Table 3) different concentrations of the muscarine diastereomers. The incubation was terminated by centrifugation (12,000 rpm for 3 min) at room temperature with an Eppendorf microcentrifuge. The resultant pellet was washed twice with 1.5 ml of saline to remove the free radioactivity and the final pellet was allowed to drain. The tips of the tubes containing the pellet were cut off and placed in a scintillation vial containing 200 pl of tissue solubilizer (Lumasolve, Lumac) and left to stand overnight. Radioactivity was then counted after addition of 4 ml of liquid scintillation solution (Lipoluma, Lumac). The muscarine diastereomers were dissolved in distilled water; solutions were prepared freshly for each experiment. Assays were carried out in triplicate and the nonspecific binding was defined as the radioactivity bound or entrapped in the pellet when the incubation medium contained 1 pill QNB (3-quinuclidinylbenzilate racemic mixture). Nonspecific binding averaged less than 3%. The binding curves were analyzed by a SAS-modifiedcomputer program package giving least squares fitting to a sum of up to three superimposed binding sites. The concentration of receptors in each subgroup was expressed as a percentage of total receptors, and hence there were two independent variables for the concentration of sites, but three independent variables for binding constants. In instances in which a third binding site was not found, the computation was repeated with a reduced number of variables. The IC50 values were converted to K, values by correcting for the radioligand occupancy shift according to the Cheng-Prusoff equation: Ki = ICs0/[1+ C/KD],2o where C and KD represent the concentration and the dissociation constant of the radioligand used, respectively. The KD values for NMS used in the equation, were found to be 0.52 nMin heart and 0.54 nMin submandibular glands. For PZ, the KD was found to be 14.2 nM. Hill coefficients (nH) were calculated by linear regression analysis and assessed for statistically significant deviation from unity by Student’s t test. Data from the individual experiments were analyzed independently.

Preparation of guinea pig ileum. The guinea pig ileum was cleared of connective tissue and the lumen was flushed several times with the physiological solution used for the atria. Segments of about 2 cm in length, with one end open, were then placed in a 10 ml organ bath filled with physiological solution, maintained at 37°C. Protocols. The preparations were left to equilibrate for 40-50 min during which time the bath solution was changed every 10 min. Atrial force was recorded with a Statham force transducer connected to a Battaglia Rangoni polygraph. The resting tension was adjusted to 1 g. The responses of the tissues to the drugs were recorded isotonically on a LNI recorder through a Basile transducer. The load was 0.5 g for both the tissues. Cumulative doseresponse curves were obtained by increasing stepwise the concentration of agonist by 0.5 log units (guinea pig ileum) or 0.25 log units (guinea pig atria) until the maximum response was achieved. Racemic muscarine [( f )-11 was used as the reference compound. Measurements. Changes in heart contraction are expressed as percentage reduction of basal values. Responses to drugs of guinea pig ileum are expressed in mm of shortening. Potency values, expressed as - log EC50 (concentrationproducing 50% inhibition in the atrial contraction amplitude or 50% of the maximal contractile response in the ileum), were calculated graphically from each curve. Intrinsic activity (a)was determined by comparing the maximum response to the reference compound [( =k)-l]with that to the other agonists. Compounds (+)-and (-)-1, (+)- and (-)-19, (+)- and(-)-20, (+)- and (-)-21 showed a slope of the doseresponse curve as well as the maximum response similar to that of the standard [( .t)-l].

RESULTS AND DISCUSSION

Muscarine isomers (1, 19, 20, 21) were assessed for agonist activity at muscarinic receptors in guinea pig atria, a tissue homogeneous in M2 receptors,21*22 and guinea pig ileum, a tissue containing a mixed population of mainly M2 and M3 receptors.23-25 The eight isomers were also examined for binding activity at M1, M2, and M3 muscarinic receptors. Affinity values (Ki) and Hill coefficients (nH) of the compounds were derived from displacement of [ 3H]PZ binding to membranes of cerebral cortex (MI receptors) and [ 3HJNMSbinding to membranes from heart and salivary glands (M, and M3 receptors, respectively). In the functional studies, all of the compounds behaved as full agonists (a= 1)in both atria and ileum with the sole exception of epimuscarine (+)-19 which behaved as a partial agonist (cl=O.41) in the atria, but the potencies and E.R.s were very different (Table 1). The natural muscarine (+)-1 showed highest potency on both tissues and demonstrated almost no selectivity between them: - log E&s were 8.13 and 7.69, respectively. In both atria and ileum, the potency of ( + )-1 was more than two orders of magnitude greater than its enantiomer [(-)-11, as reflected by E.R.s of 324 and 331, Functional experiments consistent with the E.R. of 394 found in rabbit ileum.2 PotenMale guinea pigs from a local strain (Bettinardi, Dankin- cies of the other six isomers were generally weaker than that Hartley strain, 50MOO g body weight) were killed by a blow of ( - )-1,with - log EC50 ranging from 4.45 to 5.72; the E.R. to the head and exsanguinated. values among the pairs of enantiomers did not exceed 6.6. As Preparation of guinea pig atria. Spontaneously beating guinea in the functional tests, (+)-1 proved to be the most active pig atria were dissected, isolated, and placed in a 20 ml organ isomer also in the binding studies (Table 2). Furthermore, it bath containing Krebs-Henseleit solution (composition in was the only receptor-selective compound, showing affinity mmol/liter: NaCl, 118; KCl, 5.6 CaCI2, 2.5; MgSO,, 1.19NaH2- ratios of 37 (Ml/M2), and 44 (M3/M2). Its enantiomer (-)-1 PO4, 1.3; NaHC03, 25; glucose, lo), gassed with 5% C02 in O2 showed lower affinity for the three receptor subtypes and prac(pH 7.4), thermoregulated at 29°C. tical no receptor-selectivity(Table 2). The six other compounds,

237

STEREOISOMERIC MUSCARINES

flattened and nHs significantly less than 1.0, a property shown by many other agonists,26but not by antagonists, which have much steeper curves. Such a property indicated that the compound recognized multiple binding sites for each receptor subtype. By contrast, its enantiomer (-)-1 as well as four of the other six compounds[( -)-19, (-)-20,(+)-21,and TABLE 1. In vitro functional studies (- )-211yielded nHs significantly less than unity only at the heart M2 receptor (( + )-19 at both MI and M2 receptors). Guinea pig Guinea pig The results of the application of three component analysis to ileuma E.R. atriaa E.R. Compound the binding of (+)-1and (-)-1 in heart are reported in Table 3 and visualized in Figures 4 and 5. In this tissue, (+ )-1 8.13f0.02 324 7.69f0.05 31 (+)-I recognized three afKnity states of the M2 muscarinic receptor, 5.62 f 0.10 5.17 f 0.09 (-)-I corresponding to superhigh (SH),high 0,and low (L) fractions 4.96 f 0.06 4.52 f 0.04 (+)-19 1.9 of receptor sites (Fig. 4). The ratio KL/KsH was 812 and that of 5.00 f 0.06 4.25f0.11 (-)-19 KL/K" was 39. Comparable afKnity ratios and fractions of the 5.13f 0.08 4.59 f 0.07 (+)-20 1.2 three receptor subpopulations,consistent with the proposition 5.72 f 0.07 3'9 4.66f0.09 ( - )-20 that there is a major component with high affinity and minor (-)-21 5.66 f 0.07 5.27 f 0.06 6.6 components with low and superhigh afKnity, were previously 4.98 f 0.12 48 4.45 f 0.13 (+)-21 found in rat heart for a series of seven different muscarinic 7.44 f0.06 7.23f 0.07 (*)-I receptor agonists.26 On the other hand, in cerebral cortex (MI ) and salivary glands (M3), the binding data for (+ )-1 were "Potencies are expressed as - log EC,. The data represent the means f SEM. found to fit a two-binding site model best, yielding two fracThe number of experiments varied from 6 to 9. All the compounds behave as tions of receptor subpopulations,correspondingto high 0 and full agonists with the exception of (+)19 in the atria (a=0.41). bEudismic ratio. low (L) component sites (Table 3).

similar to ( - )-1, had weak afKnity for each of the receptors and showed virtually no selectivity (at most, 4-fold between subtypes). It was noticed in the binding experiments that the displacement curves for compound (+)-1 in all three tissues were

'"

TABLE 2. Binding affinities and Hill coefficients of the muscarine diastereomers for muscarinic receptor subtypes"

1112

M,

M3

Compound

PY

nH

PY

nH

PY

nH

(+)-I (-)-I (+)-19 (-)-19 ( + )-20 ( - )-20 (-)-21 (+)-21

4.74 3.65 4.25 3.65 3.44 3.83 4.25 3.71

0.76 f 0.01 1.o

6.31 3.98 4.03 3.95 3.84 4.01 4.00 3.99

0.54 f 0.016 0.82 f 0.03

4.67 3.60 3.68 3.46 3.43 3.35 3.67 3.62

0.82 f 0.096 1.o 1.0 1.0 1.0 1.0 1.0 1.o

0.86

1.o 1.o 1.0 1.0 1.0

0.7 0.7 1.o 0.86

0.7 0.86

"Receptor source: MI, cerebral cortex; heart; M3, glands. The data are geometric means (4)or arithmetic meansf SEM (nH)of 3-4 experiments, each performed in triplicate. bSignificantly less than unity, P