698
Journal of Medicinal Chemistry, 1971, Vol. 14, iVo. 8
after each washing: yield 20 mg; mp >260°; uv pH 13 [A,, in (E X 253 (31.3), 363 (7.5); nmr, 2.6 (multiplet, CHr CH,CO), 3.4 (pyrazine CH2CH2), 5.1 (NHCH), 7.35, 7.45, 7.8, 7.9 (phenyl), 8.7 (pyrazine). Anal. (C2,,HJiGOS) C, H, N . mp
Acknowledgments.-The authors express their appreciation to Dr. 'st'. C. Coburn, Jr., and associates for microanalytical and spectral determinations. Some of the analyses \\ere performed by Galbraith Laboratories, Ihoxville, Tenn. Cytotoxicity and antitumor tests n-ere performed by the Chemotherapy
EELSON, ALLEN,A X D VINCENZI
Department of Southern Research Institute under Contract PH43-65-594 with Chemotherapy, National Cancer Institute, Fational Institutes of Health; antitumor testing was carried out under the supervision of Drs. F. IT. Schabel, Jr., and W. R . Laster, Jr., and cytotoxicity testing was under the supervision of Dr. L. J. Wilkoff. Antimalarial screening data \vas supplied to us by the Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C.
Muscarinic Receptors. Derivatives of 7-Oxabicyclo[2.2.1]heptane1 WENDELL. NELSON,*DAVID R. ALLEN,AXD FRAXK F. VINCENZI College of Pharmacy and Department of Pharmacology, Vniverszty of Washington, Seattle, Washington 98105 Received Septeniber 28, 1970 Syntheses of muscarinic analogs endo- arid ero-2-trimethylammonium-7-oxabicyclo[2.2.1]heptaneiodides and endo- and ezo-2-dimethvlaminomethvl-7-oxabicvclo[2.2.1] heptane methiodides are described. Muscarinic aisays are reported
Steric and electronic effects in the drug, as well as conformational differences in drug and in the drugreceptor interaction, have been suggested as major reasons why certain analogs of acetylcholine (ACh) are more active than others at various cholinergic sites.* Preparation and screening of conformationally rigid or semirigid analogs of ACh has met with some success in applications t o muscarinic and AChE sites.* In this study of analogs of various conformers of cholinergic agents, muscarine (1) was chosen as a model, and analogs in the 7-oxabicyclo [2.2.1 ]heptane series n-ere prepared. The C-3 methylene ammonium side chain of muscarine has considerable flexibility in models, and various conformations of it and of muscarone have been suggested to explain the difference in absolute stereochemistry of the most active isomer in each case.3 Calculations (extended Huckel the or^)^-^ are not consistent with earlier drug-receptor propo~als.~ I n this series of compounds the position of the S + l l e 3 is restricted to only certain distances from the ether 0, and the agents incorporate few or no additional atoms in the C skeleton, which potentially allows for accumulation of some evidence concerning the conformation of muscarine in this drug-receptor interaction, although separation of optical isomers would be necessary to
obtain data concerning the muscarine-muscarone controversy. 0
HO. H
+
,
C
V
0 1
NMe3 I-
+ 2
A
I3
H
I +
CH,NMe, ,I4
k 5
Bicyclic analogs 2, 3, 4, and 5 were prepared as rigid desoxymuscarine analogs. Compounds 2 and 3 differ from desoxymuscarine only by connection of the C-2 methyl and C-5 CH2groups through a single C-C bond. Compounds 4 and 5, being homologs of 2 and 3, were (1) (a) An account of this a o r k was presented t o the 161st National prepared because the necessary starting materials were Meeting of t h e American Chemical Society, Lo6 Angeles, Calif.. March intermediates in the preparation of 2 and 3. These 1971, Abstract M E D I 32. (h) This work was supported in part by Grant NS-08121 from the National Institute of Keurological Diseases and Stroke, compounds (4 and 5 ) have the disadvantages of conC . S. Public Health Service, Uethesda, R l d . formational freedom of the quaternary head with (2) For recent reviews see: (a) h l . Friedman, Drugs Aflecting Peripheral Xem. S y ~ t . , 1, 79 ( l Q 6 i ) ; (13) S. Ehrenpreis, J. H. Fleiach, and T. I\-.respect to the 0, but could provide information conLIigyag, Pharmacol. Rev., 21, 131 (1969); (c) P. S.Portoghese, Annu. R e r . cerning possible steric interaction between the y +Me3 Pharmacol., 10, 51 (1970). cation and portions of the bicyclic skeleton when com(3) 1'. G. K a s e r , I ' h ~ r m ~ ~ cRe?.. o l . 13, 465 (1962). (4) 13. Delleau and J. Puranen, J . .?fed. Chem., 6 , 325 (1963). pared to 2 and 3. (5) L. R. Kier, &'oZ. Pharmacal,, 3 , 487 (1967). Initial attempts were made to find a facile route to ( 8 ) L. 13. Kier, in "Fundamental Concepts in Drug-Receptor Interact i o n s , " .J. F. Danielli, J . F. .\loran, and D. J. Triggle, E d . , Academic Press, 7-oxabicyclo [2.2.1Ihept-Bene (8) through Diels-Alder N e w Bork, pi. Y., 1970, pp 15-28. reaction of furan and maleic anhydride to form 7-oxa( 7 ) 9. AI. Liquori, -1.Daimani, and J. L. DeCoen, J . .UoZ. B i d . , 33, 445 bicyclo [2.2.1 ]hept-5-ene-ezo-2,3-dicarboxylic anhy(1968).
Journal of Medicinal Chemistry, 1971, Vol. 14, No. 8 699
MUSCARINIC RECEPTORS
dride (6).8 Although 8 has been prepared by DielsAlder addition of ethylene and furan d i r e ~ t l y ,the ~ conditions of temp and pressures required for this process were not readily available in our laboratory. Anhydride adduct 6 was hydrolyzed and reduced to 71° which was subiected to various conditions to bring about bisdecarboxylktion (Scheme I). Reported procedures SCHEME I
This separation was readily followed by nmr spectroscopy. The difference in 0-Me positions in the nmr spectra of the mixture of unsaturated esters has been described by Ouellette, and provided a monitoring system for determination of the equilibrium for the isomerization of 9 to 10. Separation of the endo and exo isomers permitted complete nmr characterization and the obtainment of data useful in determining the relative stereochemistry at C-2 throughout the series (Table I).
+
TABLE I
COOH
60-Mc NMRDATA
H H 7
6
8 6
using Pb(OAc)a in various solvent^^^-'^ or Pb0214115 failed to provide more than trace amounts of 8, and in one case a small explosion was encountered. Other routes were sought.
&
COOCH3
H
9
10
The mixture of enclo- and exo-2-carbomethoxy-7oxabicyclo [2.2.l]hept-5-enes (9 and 10) has been prepared by Diels-Alder addition of furan to methyl acrylate in generally poor yield after refluxing for severa,l weeks,16 or a t 40" for periods of a month or more. l7 However, st'art'ing materials for this process are readily available and at room temp for 30-60 days yields of up to 50% were obtained by us. Separation of the endo isomer from the mixture of unsaturated carboxylic acids by column chromatography has been reported," although only minimal amounts of the exo isomer are available by this method. Considerable losses were encountered using alumina. However, if reduction of the olefin was performed on the mixture of isomers, facile separation of esters 13 and 14 was accomplished by chromatography on silica gel. Further experimentation showed the unsaturated esters 9 and 10 were readily separable on AgX03-impregnated silica gel. This separation will facilitate further work to compounds closer to muscarine, wit'h oxygen functions in the C-5 and C-6 positions. (8) K . Alder and 0. Diels, Chem. Be?., 62, 557 (1929). (9) W.Nudenberg and L. Butz, J. Amer. Chem. SOC.,66, 307 (1544). (10) K . Alder and K . H. Backendorf, Justus L i e b i g s Ann. Chem., 636, 113 (1938). (11) E. E. van Tamelen and S.P . Pappas, J. Amer. Chem. SOC.,86, 3257 (1963). (12) C . A . Grob, M. Ohta, E. Renk, and A . Weis, Helu. Chim. Acta., 41, 1151 (1958). (13) C. M. Cimarusti and J. Wolinsky, J. Amer. Chem. Soc., BO, 113 (1968). (14) W. von E . Doering, &I. Farber, and A . Sayigh, ibid., 74, 4370 (1952). (15) N. 9. Zefirov, R . A . Ivanova, R . S. Filatova, and Y. K . Yarev, J . Gen. Chem. U S S R , 36, 1901 (1965). (16) R . J. Ouellette, A . Rosenblum, and G . Booth, J . Ore. Chem., 38, 4302 (1968). (17) M. P . Kunstmann, D. S.Tarbell, and R . L. Autrey, J. Amer. Chem. SOC.,84, 4115 (1962).
endo-COOMe (9) J in Hz
6 . 4 3 g,
J6,a
6
ezo-COOMe (10) J in Hz
= 6,
2
J6.1
=
Ja.6
= 6,
6 . 3 7 s, broadened 6 . 2 1 q,
Jj,( 2 5 . 1 6 d, broadened,
Ji ,z-exo
5.00 d, broadened, J4,3-ex0 = 4 3.63 s 3 . 1 7 quintet, J2-exo13-ex0 = J2-cxa,i =
8,
4, = 4 = 10,
J~-exo,a-endo
2.09
octet, J,,, J3-exo,4
=
J3-exo.z-cxo
41
8
2 . 1 6 d or triplets, J w m = 12, J3-exa14 = 4, J~-exo,?-endo =
1 . 5 7 q, J,,,
4
= 12,
J3-endotZ-endo
8
Notable differences were seen in the vinyl region (H, and H6), the H2 and H3-exoand H 3 - e n d o areas. Deshielding effects of the endo-CO.H probably account for the downfield position of H 6 with respect to H5 in 9. Decoupling experiments confirmed the assignment of the downfield signal to H6, as HI is readily identifiable by decoupling of Ha, which then was irradiated to assign H,. The positions and multiplicity of the Hz protons in the endo and exo ester also varied. The large difference in chemical shifts probably results from a deshielding effect of the bridge 0 on the exo proton (in the endo ester) and/or a shielding effect of the 5,6double bond on the endo proton in the exo ester. The former explanation is most consistent with the noted similar difference in chemical shifts in the reduced esters 13 and 14. The differences in multiplicity result from a J 2 - e n d o , l =Y 0, consistent with torsion angle measurement of ca. 90°, giving a quartet for H 2 - e n d o in 10, and J P - ~ x o , ~=Y J ~ - e x o , ~ - e n d o =Y (1/2) J~-exo,3-exo giving a quintet for in 9. The signal of HsSexo is ca. 6 0.6 downfield from H 3 - e n d o probably as a result of the bridge 0 deshielding effect. Alultiplicity differences are a result of J3-endo,4 N 0, as well as torsion angle dependent coupling constants.
700
Iournal of Medicinal Chemistry, 1971, Vol. 14, S o . 8
IO(
2: 8
s e
.F
51
. aE
w
Figure l.-Coiicentration~effect relationship6 of %-substitutedT-oxabicyclo[2.2.1]heptane derivatives. Isometric contraction of guinea pig ileum us. concentration (logarithmic scale j of various compounds as indicated by the numberb. Data are expressed as a percentage of the maximal tissue response, defined as that produced by 10-5 J f ACh. Each point (with one exception, see Experimental Section) represents the mean from at least 3 separate experiments. Bars represent 2 standard errors.
Bridgehead protons HI and Hd show differences in multiplicity based on the torsion angle dependent , J l , 2 and J3,, coupling constants, and the difference in chemical shifts is probably due to the effect of the CO?H on HI in both isomers. Conversion of the isomeric esters 13 arid 14 t o 2 and 3 was accomplished by forniation of the hydrazides 15 and 16, Curtius rearrangement, LAH reduction, K-alkylation using CH,O, and hydrogenation (Pd/C), arid quaternizatiori (Scheme 11). S o change in the
NH2NHz
Curtius
COOCHB 13, endo 14, exo
The homologs 4 and 5 u e r e alao prepared from esters 13 and 14. . h i d e formation with lIe2SH afforded 23 arid 24. Isomerization of eiido to eso iwnier niis noted under the conditions of the reaction, .\le2SH and a trace of HCI at 100" i n [L sealed autoclave. J i k the conditioris for the Diel+L41der addition forniation gave a preponderance of endo ehter 9. thi:, proceis provided :i converiit-rit method t o obtain the ~ \ o isomers, precluding tedious sep:mtion of sni:rll amount. of material from the mixture Separation COUld bc delayed until after amide foiniation, I\ here column chromatography proved t o be vel). buccessful. The nmr hpcctrii of amide, 23 and 24 showed difft-relit h c b of LY-.\Ic group? for each iaonicr jt\\o each) Li- \\ell ai similar diffcrciic,ei 111 t h e IT2 aiid Hd proton. t o 9 and 10. Ailterii:itivcl>,,t h e :iinitles \\ere :ivnilable from t h e free acid. 11 and 12 by forinntioii of the :ic>1 halide followd by rwctioii nith niihyd _\Ie2SH, nithout isomerization. IZeductioii (IJLIH);ifforded t h e COI'I'Csponding tertiary ;\mine*25 :irid 26 xhich \\ (w q u a t t r riized nitli A\I~>I to give 4 : ~ n d5.
& LAH
CON (CHj)?
23, endo 24, exo
0
25, endo 26, exo
Pharmacology. A l l l conipoiinds I\ ere t wted for muscarinic and aritimu.cariiiic activity on guinea pig ileum . E:tc h compound tested produced rnuscari ni c effects as evidenced b>- concn-effect curves approximatel) parallel to that of *iCh,lack of blockade by liexamethonium, and blockade b j atropine. Intrinsic activit) of t h e 4 compound- i i umed to be 1.0, although Yupportive graphical evidence (li'igure 1) is presented only for 4 and 5 . Lo\\ potc.tic) of 2 and 3 prevented completion of thti iespwtivc. csoncii-effect curves. H ~ M (ivcir, the ~ l o p c - of thc-e curve. are approximatelj parallel to thow for tlie o t h w agonisti. arid lack of ar1) qignificant atropine-lil,c actiori (111 thr. part of 2, 3, 4, or 5 v e m s t o ju.til> t h i i :i-.umptioii. Compariwii of t h e potencici nlth ACh (Table 11)
17, endo 18,exo
shape of the iimr signal of the H z proton was observed after hydrazide formation confirming the lack of isomerization at C-2. In samples of the individual hydrazides signals for the respective HS protons were similar to those of unsaturated esters 9 and 10. I n t h e rearrangement products (carbamates 17 and 18) arid I A H reduction products (secondary amines 19 and 20) the nmr spectra were characteristic of the H2-exoas being present in one series and Ha-endo in the other. I n the auaternarv ammonium salts (2 and 3) similar patterns were observed.
suggests that o n l ~ t h e erido :malog, 4, of tlie CHJ compounds ha, +gnificant activit)., bring about l.
