s-1s 3-hc.c,toxyqiiinucliciine Methiorlide. Resolution, iibsolutc Configuration, and Stereospecificity of Interaction w i t h the Acetylcholine Rinding Sites
'rhe general problem of the absolut,e conformation of receptor- arid enzyme-bound quaternary muscarinic agents is briefly reviewed. Special attention is devoted to the siiper-muscarinic agent L(+)-cis-dioxolane (I), i r t which the coriformatioti of the cat,ionic nioiety is not fixed. It is point#ed out that 3-acetogyquinuclidiiie niel hioditle has a ft,ozert c~otrforinationabout the ciiti(itiic. center and thus may provide a . it> :t hiih-trate for AJ. t i l I:. .Jellinek, Acto C r g , 4 . , 10, 277 (19,5i). 18) 13. \V. J . I?llenhroek and .I. .\I. van Rosnuln. A w h . I , i i r i - , i . I ' h c t t ~ r ~ ~ i co(/,,n., 125, 216 (1960). (9) B. Belleau arid J . L. Lavoie, Ccrn. J. Biocheni., 46, 1397 (1968).
I
hut not for analogous dioxolariei differing in geometry or :tbsolute configuration. It does therefore seem that the rAs-dioxolane niay possess the correct absolute COLIfiguration for the induction of large molecular respomes in both the receptor and the enzyme. Some intriguing correlations between the physical and kinetic responseh of AChE and the reiponse of the cholinergic receptors to several classe:, of quaternary salts havc been recently reviewed.Ifl Among the numerous problem3 that remain t o be solved, that of the abhohite co7!formaf7or~ of c.tizpmc- and receptor-bound stimulant\ i- of considcr:ihl(> ititere-t. Thc i i q c of conitrained :innlogs of flexible ligands or -itbitrates i I loyiml :tpproach whicli 1% not, howevw, free of ani1 it ies owing t o t hc p w tence of residual difficultiei in the elucidatioii of the chorrect conformation of the constrained molecule i t i -elution arid in the bourid stat(,. Conformations it1 tliv crystalline state can hc elucidated by X-ray :ttialy\is :ind although tntrinwally informative,' thr rcwiltobt:iinrd by thii method may riot apply to moleculeI I I wlution. .\ cie:ir-cut ex:irnplr. of marl t i o i d disparities betwec,ii tlic ciyqt:tlline in-solution state recently given by us finally allowed the deduction of the absolute coilformution of chymotrypsiii-bound substrates after more thiiii :I decade of controversy regarding the conformation of :t constrained substrate analog.'? As it turned out, thc conformation of this :malog corresponds to the thermodynamically umtable one in the enzyme-bound state, :I cwnclusion oppoiite to that which may be predicted on ( I O J 13. Bellemi in 1'1 ureedings of Buffalo, IS. 1 \ ~ g 1968, 1 I r k , IS. Y., in press. II
lions.
d
Sgmpoulum U I I Drug-Reoeutor Irltri .I Triggle. Ed , Academic Press. Nc I\
(11) R Relleau anti K. Chevalier J A m Che7u SOL.,SO, 6864 (1968, 112) G Hein, R 13 MrQriff, and C. Nieman. ihid , 82, 1830 (1060)
3-XCETOXYQUISUCLIDISE METHIODIDE
September 1969
the basis of theoretical calculations of the probable conformation of noninteracting species under vacuum.13 Returning to the super-muscarinic agent L(+)-c~sdioxolane (I), the problem of the absolute conformation of the freely oscillating cationic moiety in the bound state remains to be solved. Once this is known, the complete chirality of the binding sites may be revealed. The choice of structure for such studies is not an easy one because of the limitations outlined above. However, the r e d t s of Mashkovsky on the muscarinic activity of 3-acetoxyquinuclidine and its methiodide,I4 as well as those of Solter'j on the ability of the latter to act as a substrate for AChE, suggested an approach to our problem because the quaternary moiety of the molecule (11) is virtually frozen in a single conforma-
H A O A C
II
H"0Ac
m
tion. In addition, the bridgehead carbon atom occupies it position equivalent to that of the methyl substituent of fl-methylacetylcholirie (111). However, this raises the question of the configurational equivalence of the two molecules about their respective asymmetric centers. If I1 arid 111 are related as shown, then only the (8) isomer of the former should behave as a substrate for AChE; it should also be significantly more active than the ( R ) isomer as a muscarinic agent. However, pFedictions of this kind are dangerous because of the distinct possibility of stereochemical inversion of specificity of the binding sites owing to special conformational effects in the ligand. Such a phenomenon has been reported by Waser16 for u-muscarone us. L-muscarine :tnd the results have been analyzed and interpreted by us4 in terms of an inversion of conformational stereospecificity similar to that discovered earlier for the case of chymotrypsin.11112I t therefore became necessary to establish the absolute configuration of the optical forms of 11,their behavior toward AChE, and their potencies as muscarinic agents before attempting to use this molecule as a conformational frame for the relevant catioiiic part of the L-cis-dioxolane (I). The purpose of this communication is to report our results on this problem. Experimental Section" Chemical. ( +)-3-Acetoxyquinuclidine methiodide was prepared acc~irdiiigto the literatiirela and recrystallized twice from EtOH; mp 164-165.5' (lit.l* 165-160'). ( 1 3 ) I.. B. Kier, MOL.Phormarol., 8, 487 (1967). (1.1) M. D . Mashkovsky in Proceedings of the 1st International Pharmarology l l r e t i n a , Stockholm, 1962. Vnl. i. IZ. J. Rruningrnnd P. Lindgren. Ed.. Prrgciitilua Prada, L o i d o n , 1~63, p 35~. (15) .I. IT.Rolter. .I. Phnrm. Sci., 64, li5.i (1Htl5). (161 1'. G . \\'mer. /'hnrwucol. !?pi,., 13, 40.5 (1961). ( 1 i) hlelting points and boiling points are uncorrected. JVllere analyses are indicated only by symbols of t h e elements or functions, analytical results ubtained for tLo.ie elernentJ or functions uese witliin 10.4% of the theoreticul values. (18) C . .IGroli, . -1. Kaiser, and R. Renk, Hela. Chim. Acta, 40, 2 1 i O 11957).
849
Resolution of ( f)-3-quinuclidinoI accomplished accordiiig to the method of Sternbach and Kaiser,'g using camphor-10-sidfonic acid. After five recrystallizations from PIIe2CO-i-PrOH the product exhibited constant rot,atioii, [a] *ED - 1.05' (c 5.7, H20), lit.1g [ a I z 5-0.3' ~ (c 3, H20). L)ecomposition of the salt gave (-)-~-quUnuc~idinol, [ a ] 2 s-37.1" ~ (c 3.0, 1 HCl), lit.19 [ C U ] ~ ' I -43.0' I (C 3.0, 1 S HC1). T h e mother liquors yielded crystals, [ a l z 520' ~ (c 3.3, HeO), uiichaiiged after recrvstallizatioiis. The free base had [ c x ] ~ ~ D 18.3" ( e 2.7, 1 S HCI); this corresponds to 7 5 5 optical purity. (+)-3-AcetoxyquinucIidine.-( - )-3-&1iiiiiiclidiiiol (2 g ) was acetylated with AcnO in C;HJT.20 Fractional distillation of the product yielded 1.71 g of a fractioii, bp 12X-130° (28 mm), [ a I z 5 D 28.5' (c 2.94, EtOH), strong baud u t l i 2 5 em-' in the ir (film). Anal. (CSH~NOZ) C, H. ( - )-3-Acetoxyquinuclidine (partially resolved) was prepared as described above for the (+) isomer; [ a . l Z 5 -10.7' ~ (c 2.9, EtOH). The ir spectrum was superimposable upon that of the (+) enantiomer; estimated optical purity, 70%. ( - )-3-Acetoxyquinuclidine methiodide (11) was prepared in the usual manner from (+)-3-acetoxyqiiiiiuclidine and RleI. tallization from EtOH it had nip 203-204', [ a ] z h j o -11" (C 2.03, HZOj. Anal. (C~oHiaIliOn)C, H. (+)-3-AcetoxyquinucIidine me iodide was prepared from ( - )-3-acet~oxyqiiiiniclidine of TO optical purity and AIeI. After 12 recrystallizations from d EtOH, crystals of mp 202203", exhibiting constaiit rotatio were obtained; [ ( Y ] ~ ~11.0 D (C 1.9, HIO). *inal. (CioHJSO~) C, H . Absolute Configuration of (+)-3-Quinuclidinol. ( a ) 3-Quinuclidinyl p-ToIpenesulfinate.-p-Tolueiiesulfinyl chloride21,22 (3.5 g) was dissolved in dry Et20 (15 ml), the solution cooled to -7S0, and while stirring, a solution of 2.2 g of (+)-3-quinuclidinol (7570 optical purity) in a mixture of 10 ml of Et20 and 10 ml of CjHjN was added over 30 min under dry Sf.After 90 min a t -78", the mixture was allowed to attain room temperatiire, diluted with 15 ml of EtzO, and washed Kith 20 ml of 5% XaLXh in HnO, then with HpO. The solution was dried aiid evaporated to give a yellow oil (1.4 g), [ C Y ] ~ ~2.0' D ( e 3.7, Ne2CO), ulllaX (film) 1130 cm-l (>S=O). It was used as such in the next step. (b) ( - )-Methyl p-Tolyl Sulfoxide.-hIehIgI was prepared in Et20 (25 ml) from Me1 (1 g ) and hZg (0.4 g). To this was added slowly over 20 min under Kz, a solution of the preceding quiiiuclidinyl p-toluenesulfinate (1.2 g ) in 25 ml of EtZO. After 1additional hr, HZ0 was added (10 ml) and the Et20 solution was washed with 10% HC1, followed by loc& Na&Oa and HZO. The Et20 was dried and evaporated to yield a yellowish oil whose ir spectrum (film) was ident,ical with that of an authentic specimen of methyl p-tolyl sulfoxide. I t was distilled in vacuo, bp 70-75" (0.15 mm) (bath temperature), to yield colorless material whose nmr spectriim (CDCl,) wad in agreement with the expected structure: [a]% -1.X0 ( e 4.49, EtOH). It follows that, (+):3-quinuclidinol possesses the ( X ) coi~figuratiori.~~ nZ4 Enzymology.-Bovine erythrocyte AChE (Sigma) was used. The velocity of catalyzed hg-drolyrec was niea3iired by the pH Stat method :LS previoii.4y dewribed i i i Iiionbat ions were varried out in a total volrune of 25 nil of enzyme holritioii previously made 0.04 U in lIgC19 arid 0.05 .\I i i i SaC1. The p l I was maintained constant at 7 . A CO?-free 3 2 atmosphere war maintained throughout. The tem was allowed to equilibrate a t 2.5" for .ti miii prior to hubst e additions. The slope of earli init,ial velocity was taken during the second and third minute of incubatioii. The K , arid , , 'T. vdiies wei'e c.ompiited f l o m conveiitional recsipi,oc:al plots (E'igrne 1j. Pharmacology.-'Terminal ileum from guinea pigs (300-400 g ) was suspended in a 1 organ bath of Krebs solutioii a t 3ti'. The usrial mixture of 0 2 aiid 5?; COYwas bubbled through the bath fliiid. Cont 011s of the ileiim were recorded oii a kymograph, rising aii isotoiiic frolital writiiig lever (magnificatioii 8 : 1 at a load (Jf 1 g). Log c:oiic*eiitratioii1's. response to h C h aiitl to the 3-acetoxyquiiiuclidiiie stereoisomers were obtained usiiig ( 1 ~ )1.
ki. d t n . n l r u l ~u r d 3 . liai-ci. .I I,,, C h < m S W ~ ,74. . . ?!?IS (19:21 (20) J . B. Kay, J. B. Rohinson, a n d J . Thomas, J . Chem. Sac., 5112 (191351. (211 17. C. \\-liitmore and F. H. Hamilton, "Organic Syntiteses," till. Vol. I . J o h n R-iley and Sons, Inc.. S e a - Tork, N . I-,, 19-11. p 492. (22) F. Kurzer, O w . Sz/ri.. 84, 93 (195.1). (28) IC. hlislou', 11. A l . Green. P.I.aiIr, .J. .'l' A l ~ l l i l < 'T. ~ , Siiniwns. n i i ~ l .I. L. Ferney. J. A m . Chem. Soc.. 87, I958 (19ti;). (2.1) AI. hI. Green, bl. Axelrod, and I
-
3.0
II
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I
-4000
--2000 2000
2000
4000
6000
I /[SI M / I
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P
September 1969
s51
2- (N,N-DIALKYLAMINO) ETHYL a-(3-PYRIDYL)MASDELATES
@-methylgroup would be nearly eclipsed in the bound state. This conclusion appears improbable on the basis of energy considerations. h skewed conformation such as in V is more appealing; a slightly twisted conformation for I1 is a reasonable approximation of V. If the comparisons are valid, the conclusion emerges that binding on active sites may not necessarily involve the thermodynamically preferred conformation of the ligand, a fact which was recently brought to light in the case of a constrained substrate of chymotrypsin." Results of X-ray studies on crystal^,^,' as well as theoretical ~ a l c u l a t i o n s predict ,~~ opposite conclusions; the obvious reason for this is that no account is taken of the fact that proteins display conformational specificity.l' Owing to internal comperisatiori effect^,^ strained conformations of substrates and inhibitors may be readily stabilized through the translocation of conformational energy within the protein. The reduction of the free energy of activation encountered in enzymecatalyzed reactions has, in fact, been explained by
JencksZ6as resulting from the induction of a strained conformation approaching in structure that of the transition state. Isotope-eff ect studies on the binding of substrates on enzymes have led us to similar con~lusions.~~ * ~ not seem impossible therefore It~ does that IV and V may represent the biologically active conformations a t the binding site level. Finally, it is of interest to note that the configurational handedness of the AChE binding sites is similar to that of the muscarinic receptor binding sites. Acknowledgments.-The authors are grateful to the Defence Research Board of Canada and the Kational Research Council of Canada for the financial support of this work. The 3-quinuclidinol was generously donated by Dr. R. Heggie of the DRB. (26) W. P. Jencks, in "Current Aspects of Biochemical Energetics," N. Kaplan and E. Kennedy, Ed., -4cademic Press, New York, N. Y., 1966, p 273. (27) B. Belleau and J. Moran, Ann. S . Y AcadSct.. 10'7, 822 (1963). (28) B. Belleau, Stud. B t o p k y s . . 4, 95 (1967).
2-(N,N-Dialkylamino)ethyl Esters of ~(3-Pyridy1)mandelicAcids. Synthesis and Pharmacological Evaluation A. SOVELLI, J. R. BBRRIO, Departamento de Quimica Orgcinica AND
Depui luruenlo d e Farmacoloyia, Facultad de Farmacia
H . HCIDOBRO Bioquimica, liniversidad de Uuenos Aires, Uuenos A ires, J r y e n l i n a
Received A p r i l 1, 1969
2-(N,N-Dialkylamino)ethylesters of ~(3-pyridy1)mandelicacids were prepared and screened for pharmacological activity. Compounds VIIb and e compared favorably with benactyzine hydrochloride as inhibitors of spontaneous motility. Some of them (VIIa, b, d, and e) also show anticholinergic, spasmolytic, antihistaminic, and anti-5-HT effects.
Aminoalkyl benzilate esters (I) possess pharmacological effects t,hat have several clinical applicat,ions.* The presence of a pyridyl instead of a phenyl radical should change their pharmacological properties. To prove this assumpt'ion, synthesis of type I1 derivat'ives containing a 3-pyridyl radical was undertaken.
@ @LC/0H 'co2y-7x< , I I
q
1 / I I'
Q C / 0'c0.r-m H J
SCHEME I R
It'
K'
111
I1
T h e general process of synthesis is shown in Scheme 1. IIZa (Table I) was obtained in good yields by condensing ethyl phenylacetate and ethyl nicotinate in NaOEt. ( 1 ) (a) A. Astrom. Acta Pharmncol. Tozicol., 8 , 363 (1952); (b) E. Jaoobsen. Danish M P ~Bull., . 2, 159 (1955); ( c ) E. Jacobnen a n d T. Sliaarup, Actu f'hurniirrol. Torirul., 11, 117 (1955): id) 1,;. Jacobsrn and E. Sonne. ibid., 11, 135 (l(155); (e) zbrd., 12, 310 (1Y56); ( f ) 11. G r e t h e a n d E . Jacobsen, ibid., 18, 125 (1957); ( 9 ) H. Holten and E. Sunne, i b i d . , 11, 148 (1955). ( h ) RI. J. Raymond and C. J. Lucas, Brit. .Wed. J . , 1, 952 (1956): (i) U. Larsen a n d C. H. Holten, Acta Pharmacol. Tozicol., 12, 346 (1956); ( j ) L. Alexander, J . Am. .Wed. Assoc., 162, 966 (1956). ( 3 ( a ) I . h l u r ~ k ~ a.4rto d P . , \ ~ c u ? " / . ,+,I,,,/,, 3 0 , 72y [IYAT,); (1,) 1s. E.l)avies, Brit. M e d . J . . 1, -1 5 6 ) ; ( c ) C . 11. llulten, A c t a I>liarmt~col. 7'oricol.. 15, 113 (1957); (d) JI ardes and SI. Laulan. Presse .We,/,, 65, 180 (1957); (e) A. Coady and E. C. 0. Jewesbury, Brit. Med. J . , 1, 485 ,1956); ( f ) L. Alexander, J . A m . M e d . Assoc.. 166, 1019 (1958).
vI
\'I1
Using the same method, mono- (IIIb) and dimethoxy (IIIc) derivatives were obtained from the ethyl esters uf homoanisic arid homoveratric acids, respectively. Legrand and Lozac'h obtained the p-keto ester by condensing ethyl nicotinate and ethyl phenylacetate i1i low yields o i ~ l y . ~When coiiclensatioii was carried out with ethyl 3-pyridylacetate and ethyl
(lix)
(3) I,. Legrand and N Lozac'li, Bull. Soc Chzm Frnr'ce, 79 (1955).
