446
SHIH-"
CHUA N D HEKKIG . ,\IAUTNEH
\'()I. 11
Sulfur and Selenium Compounds Related to Acetylcholine and Choline. c'11. lsologs of Beiizoylthionocholine and of 2-Diniethylaniinoethyl Thioiiobciizoate','
Iti it coiititiuiiig iiiveatigatioii of compoiiiids isosteric, liiit riot ihoelecttviiic, with choliiic: c>lei.s iiiip1ic:ttetl i i i iierve coiiductioii, benzoylthioiir)choliiie, beiizoylthionothioIcholiiie, beiizoyllliioiioseleiiolcholiiie, aiid their climethylamiiio analogs were synthesized. The activities of these aiid related compoLinds in the giaiit axon of the squid and in the isolated single cell electroplax preparatioii were deterniiiietl.
It has been suggested that acetylcholine plays its crucial role in the conduction of the nerve impulse by intluciiig a conformational change in its receptor polynicr, thus altering membrane permeability to I n an attempt t'o obtain iiiformation about the act,ive sit>esof the depolarizing receptor, a systemat'ic study of the depolarizing and antidepolarizing activities of sulfur and selenium isologs related to choline esters ant1 choline has been initiated. 1Iolecular size is ultercd relatively little when oxygen is rep1:ict.d hy sulfur and still less when sulfur is replaced by selenium. That sulfur and selenium isologs a r t w t s niade likely by the observittioil" that such conipountls almost invariably have identical crystal structures. usually differing from tliose of their oxygen isolops. Sulfur and selenium isologs tend to form ideiitic:iI 1iydr:ttes arid have sirnilnr partition coefficients." idieating theit, such compouiitls exhihit 110 major tlifferenccs in solvation. S-Ray t1ift'r:iction studies coniparing cryst'alline acetylthiolcholine aiid acetylselenolcholine, indicated the packing patterns atid the coilformations of these molecules to bt: strikingly similar.' While it had been shown previously by rneaiis of X-ray diffractioti measurements that' thio- aiid selerio-substitutetl compounds are essentially isosteric,8s9 such siniil:wit,y is even more striking in flexible molecules u c l i 11s thc aforementioned isologs of acetylcholiric~. \\.-heii differences are noted in the biological activities of isosteric compounds, such differen to dificrenccs in the abilities of such isologs to fit receptor sites, but rather tluc t o differences in t,kieir a1)ilitic.s to bind to active sites or to iiiduce configuratiorial changes in receptor biopolyniors. Sirice electroii clistrihutioii is altered ill esters :is their d e - c h a i n :ire replaced by sulfur or seleiiiunl,10it) may hc that diff twnces in the hiological :ictivities of such isologs niust he due t o diffcrciiccs i i i t h i r elect roil (list ribution. It \v:is noted that replacenierit of the side-chaiii osygeii o f :Lcetylcholine with sulfur and selenium" ( I ) 'l'hi- \vork wassupporied, in p a r t , Iii. grants frum t h e National Scienc~? I ' u i i n ~ l a t i o u (GIL4114) a n d t h e S a t i o n a l Cancer I n s t i t u t e ( i f t h e V. d. I'iiblic I-Iealt t i Service (Ci-3937-09). ( 2 ) Parts of this work were presented liefore t h e Section of llediriniil ('lieinistry u t t h e 152nd National Aleetinr of t h e hrnerican Chernicxal Socirty. S+.ii Y u r k , N. Y., S e l i t 1!)6R. 1-1'. i : i j II . Naalimansuirn, IIuri,rU I . e c t i i r 1-11 1). Nachmansohn, Jnrarl J . l i e d . ( 5 ) 1-1. G. h l a n t n e r a n d 11.. 1). Kumle (6) 11. G . Llautner and E. h1. Clayt,on, ibid., 81, 6270 (1!)59). 1,:. Shefter and 11. Q. I I a u t n e r , unpuhlkhed dara. I i . ~ ' ~ P ~ I L O1'11.1 ~IO i. I'l'l$e*iz, I, b'nlr Uiiiirr4Iy. l!lMi. I,:, S I i t ~ f t c r a n dI I . C;. l l a u t n e r . .I. .1m.Cliem. Sor.., 89, 12I!J (1967). (10) 1 1 . f:, I l a i i t n e r . O a k ItidKe Insririitc of S i i c l P a r Stiiilit.>. S \i i i I w ~ i I i t i ~ i n l l i v l i ~ ~ i nKO. t ~ $1, l!lfjG, 1) 4ll!4.
greatly modified t h e activity oi thc c>sterill the guiiirvi pig ileum and frog rectus abdoniiriis preparations'? :iwell ab in the phrenic nerve stimulated rat diaphragni.' T1ie.e results were confirmed axid extended by the us(' of the isolated + , l e - c e l l electroplax preparation.14 shown th:it introduction of sulfur aiid seleriiuiii he side chain of acetylcholine progressively Ionwed depolarizing activity, while replacement of thr oxygen of choline by sulfur or selenium resulted in :t 5triliing iricrcase in depolarizing activity. Cholinethiol was found to he 11iore active in the mercaptan than ill the mercaptide form, xhile methylation of choline, cholinrthiol: :mi choliiieseleiiol increased the (lopolarizing action of these compounds. l 4 In view of the considerable changes in bio1ogic:t 1 activity observed ~ r h e i i tlir side-chain oxygen of cholinergic esterh 11 as rcplitcrd by sulfur and by seleiiiuni, it seemed of interest to irivestigate the replaccinent of the carhonyl oxygeii by sulfur and selenium a < \I(.11. I~enzoylchollrlc I5 clc 1y related to %dinlkylaniiriotthyl beiizoatt+, tlit> latter group of conipounds being of great importance a+ clinically uceful local anesthetic>. It ha3 been claimed that iii para-substituted 2-dialkylaminoethyl benzoates the ability to block the nervv inipulsr c ~ u l dbe related to the bond order of tlic carbonyl g r ~ u p . ' ~Benzoylcholint. itself is on thc borderline of being n depolarizing agent and being :HI i l l hibitor of clepolarizntion ; in the isolated -ingle-cell electroplax preparation it exert': ti mixed effecl . I 7 Hccwitly, L'-tiinietliylaniiiioetliylbenzoate, thr. tertiary aininr arialog of henzoylcholinv, a s R ell a, its thiolcstcr and bcleiiolehter 1~010gsu ere tehted for their wtions i t i hlocking the action potential of squid giant a x o ~ i ~ ~ ~ : i d for their receptor-inhibitory actions in the elect p1:~x prep:ir:tt ion. I q .IS inhibitor< of electrical activi 0qge11 w ~ t hrep1:icctl by wlfur : t r d Ijy sclr~iiuin. undertook the yntliesis o f For thc ttbovc reasoriq thc thiocarbonyl i8+ologsof benzoylcholirie, 2-dimctliyl-
SULFUR AND SELENIUM ANALOGS OF ACETYLCHOLINE.VI1
May 196s
aminoethyl benzoate, and their thiolester and selenolester analogs.
/I
I
CsHjCBCHzCHzB +(CH3)g B = 0, S, Se R = H, CHa
2-Dimethylaminoethyl thionobenzoate was synthesized by the addition of 2-dimethylaminoethanol to thionobenzoyl chloride. A convenient method of preparing the latter compound was reported recently by Hedgley and Fletcher. 2o 2-Dimethylaminoethyl thionothiolbenzoate was prepared by the addition of 2-dimethylaminoethylthiol to thionobenzoyl chloride. Addition of 2-dimethylaminoethylselenol,freshly prepared by the sodium borohydride reduction of the corresponding diselenide, to thionobenzoyl chloride yielded the thionoselenolbenzoate. In all cases, the quaternary ammonium compounds could be prepared from the tertiary amines by the addition of methyl bromide. Experimental Section 2-Dimethylaminoethyl Thionobenzoate Hydrochloride.-A solution of 2-dimethylaminoethanol (0.455 g, 0.005 mole) in CHzClz (10 ml) was added dropwise to a solution of 0.78 g (0.005 mole) of thionobenzoyl chloridez0in 10 ml of the same solvent. The mixture was stirred at room temperature for 10 min. The yellow solution was evaporated to dryness, and the residue taken up in 10 ml of ether. Chilling the mixture, followed by introduction of dry HC1, resulted in precipitation of 1.0 g (8100)of a yellow solid. The product was recrystallized three times from EtOH, yielding yellow crystals melting a t 148-149°;z1 uv spectrum XEtOH mal 289, 247 m p ( emax 13,390, 6140). $nul. Calcd for C11H&lXOS: C, 53.75; H, 6.56; SI 13.04. Found: C, 53.75; H, 6.46; S, 13.02. 2-Dimethylaminoethyl Thionothiolbenzoate Hydrochloride.-A solution of 0.53 g (0.005 mole) of 2-dimethylaminoethylthiol in CH2C12(10 ml) was added to a solution of 0.78 g (0.005 mole) of thionobenzoyl chloride in the same solvent. The orange mixture was worked up in the same fashion as the oxygen analog described above. A 767, yield of orange solid (1.0 g) was obtained. Three recrystallizations from EtOH yielded orange crystals melting at 162-163', uv spectrum 303 mp (E,%, 15,910). Anal. Calcd for C11H&1NSz: C, 50.54; H, 6.16; S, 24.49. Found: C, 50.42; H, 6.16; S, 24.57. 2-Dimethylaminoethyl Thionoselenolbenzoate Hydrochloride. -A solution of 1.0 g of bis(2-dimethylaminoethyl) diselenide" was treated under Nz, with 0.2 g of NaBH4 until the yellow color indicative of the diselenide grouping disappeared. ?JaHC03 (1.0 g) was added, follon-ed by 0.78 g (0.005 mole) of thionobenzoyl chloride; the mixture was then stirred vigorously for 10 min. The product was extracted with several portions of CHzC12, the organic layers were combined, dried (LIgSO1), and evaporated to dryness. The residue was taken up in 10 nll of ether and worked up as the compounds described previously. The red product was obtained in a yield of 547,. On recrystallization from EtOH red needles were obtained; mp 166-167'; uv spectrum 350, 306 mp ( erns, 4960, 13,270). Anal. Calcd for C1lH1&lKSSe: C, 42.79; H I 5.22; S, 10.38; Se, 25.57. Found: C, 42.80; H, 5.21; S, 10.21; Se, 25.42. Thionobenzoylcholine Hydrobromide -A solution of 1.0 g (0.0041 mole) of dimethylaminoethyl thionobenzoate hydrochloride in 10 ml of H 2 0 was hhaken with 20 ml of ice-cold saturated NazSOa solution. The mixture m-as extracted with two 20-ml portions of CHZCl,. The organic extracts were washed (saturated Na2S04)and dried (MgSO1). Evaporation to dryness yielded a residue which was taken lip in RLerCO (20 ml) mixed with MeBr (2.0 nil). A yellow precipitate weighing 0.8 g (677,) was obtained. The product was recrystallized three times from EtOH to yield yellow crystals melting a t 181-182'; uv spectrum 289, 246 mp ( emax 13,623, 6698). __-__ (20) E. J. Hedgley a n d H. G. Fletcher, J. OTV.Chem., SO, 1282 (1965). (21) Melting points were determined with a Gallenkamp melting point apparatus a n d a r e corrected.
447
Anal. Calcd for C1zH18BrKOS: C, 47.36; H, 5.96; S, 10.53; Br, 26.26. Found: C, 47.52; H, 6.16: S, 10.40; Br, 26.07. Thionobenzoylthiolcholine Hydrobromide.-Quaternization with MeBr was carried out as described above. The orange product was obtained in 9470 yield and recrystallized three times from EtOH. Orange crystals melting at 188-189' were obtained; uv spectrum "::A 307 mp ( emBX15,718). Anal. Calcd for C12H18BrKSZ: C, 44.98; H, 5.66; S, 20.00; Br, 24.94. Found: C, 45.06; H, 5.62; S, 19.96; Br, 24.75. Thionobenzoylselenolcholine Hydrobromide.-Quaternization with MeBr, carried out as described above, yielded a purple precipitate (53y0)which was recrystallized from EtOH. Purple plates melting a t 193-194" were obtained; uv spectrum ":'IX 347, 307 mp (emax 5734, 15,340). Measurements were carried out using a Cary Model 13 spectrophotometer. Anal. Calcd for C12H18BrNSSe: C, 39.24; H, 4.94; S, 8.73; Se, 21.52; Br, 21.76. Found: C, 39.49; H, 4.95; S, 8.96; Se, 21.40; Br, 21.72. Determination of Antidepolarizing Activity in the Electroplax Preparation.-The isolated single cell electroplax preparation of the electric eel EIectrophorus electrzcus was set up as described previously.Zz~23Results are summarized in Table I.
TABLE I BLOCKING ACTIVITY IN THE ISOLATED SINGLE-CELL ELECTROPLAX PREP-4RATION R A
I
!I
(CH8)zS+CHzCHlBCCsHs R CHI CH3 CHI CIT3 CH3 CHJ H H
A
B
% blockaden
0 0 0 0 S 60 100 0 Se S 0 60 90 S S 95b S Se 0 0 0 0 S 40 0 Se 1OOb H H S 0 55 S S 60 H H S Se 95b a Per cent blockade of the depolaiizing action induced by 5 X 10-5 N carbamylcholine. * Irreversible action.
Discussion For many years there has been considerable discussion whether acetylcholine is implicated in synaptic transmission of the nerve impulse, in axonal transmission of the nerve impulse, or in both. If, as postulated,3r4acetylcholine is the mediator in axonal transmission and if local anesthetics act by blocking the access of acetylcholine to axonal receptor sites, then one would expect that different analogs related to local anesthetics should exhibit similar relative blocking activities in axonal and in synaptic test preparations. By comparing the blocking activities of the various sulfur and selenium isologs related to benzoylcholine and to 2-dimethylaminoethyl benzoate in the isolated single-cell electroplax (a synaptic preparation) with their blocking activities in the giant axon of the ~ q u i d ' ~ ' ~ ~ (an axonal preparation), it can be seen that replacement of oxygen by sulfur and selenium modifies blocking action in parallel fashion in the two test systems. This observation is compatible with the postulate that similar acetylcholine-binding sites are involved in the receptor biopolymers of these preparations. (22) E. SchotTeiiiels and D. Nschrnansolin, Rzochani Riophu,. Aria 26, 1 (1957). (23) E. SchotTeniels, a b z d , 26, 585 (1957). (24) P. Robenberg and H. G. Mautner, Sczence, 156, 1569 (1967)
