Document V3aYNV0Z22qkdBJR0djNp7jxj

TOX1COLOICY A X D *??LIED ?HARMACOLOCjY 21.214-229 (19iz) An Analysis ofthe Convulsant Activity of Substituted Benzenes in the Mouse A. AXGELAND K.3. ROGERS Depcntmcnts of Phrsiolopy and Pbnrtnncology, The Cnhrrsiry, S/~~JJICM,SIO try Eiipld~~d Rccciccd April 20, I971 An Analysis or thc Convulsant Activity of Substituted Bcnzenrc ici '+e Mouse. ANGEL, A,, and ROGERS. K. J. (19721. Toxirol. Appl. Phrr~v.01. 21. 214-219. A series of ti.ronosut.rtitutcd bcnzencs. hydroxj I v . 7 JC:.CI, moncnubstituted phetiob, and hgtlroxynaphth;ilcres were scxc*v4 vor thew ability to product convulsiora in mice. Phenol was the only m-msubstitutcd bcnzenc which posscsscd convulsant activity. C J I C 1C1 1~71h~e ~ most potent hydroxybcnrenc. Substitution of various chemical grk*slps in the u-position m phcnol was foirnd io dccreasc (relative to c;ltecb-J). or abolish the convuls.int activity. Thc diltivc potencies of thc y-bl-p) benzene wcrc dctcrmined from thc CDCO valocs and from dosc.cw I-:...w response cun'cs; close iqrctment kt\sccn ihe 1 mcthndz \\:IS AI. Pyrog~llol(1~.3-Iriliydro~rybcnzcdoicd~nor p r d u n convulsicnt. ).**I the contrary, produced somnolcncc whcn administered IO nonii:ll . I I ~ * : + ~ s ond diminished thc excitatory cllccr of d-ampheumine on locomwv <.:+ vity. ;Monoh\droxyn~phtha!cr.cr or dil.r?droxynipliih~lerrcswi!h +)!h hydroxyl goops on the same ring. did not produce convulsions. (j*lwr dihydroxynaphth;ilcncs t 1.5.. 1.7-, 2.6.. and 2.7) were octiw and h i d potcncics similar to or less than phenol. Certain golyhydroxylic phenols wcrc reported by Briegcr (1579) ant1 ! '"1f!936) to causeconvulsionsin cars. More rcccnrly it has been shown ihat caird~tdI I 2-?111ydroxybenzene) rcadily evokes convuisivc activity in mice wlicrcns pirs:!t!ol ( 1,2,3-trihydroxybentcne) is much less active t -4rigcl and Rogers, 1968; U w m rt d.,1965). Catcchol 3lSo evokcs convulsions in rats. rabbits, cats and monkeys lalngc1. iYfi9).The convulsive spectrum. produced by cntcchol, is 3n unusual one cii~r;ictcrirxJby tremor; aiid violent muscular jerks \\ hich occur boih spontaneouslyanti in rc. P V ~ :to itpylicct tactile or :iuditory stimirli. In the present study P number of plien4c r n y w m l sand subjtitt11cd bcnzenc dernstives have bcc:~tcsted for ability IO p r ~ ~ l i rrnc? ticIonic convulsions in inice, in an nttcmpt lo cluci~lntcthc chcmid s ~ r i ~ c t ir~cr~cp--wil:lfeor tliis convulrant action. Additionnlly. a tcchniqm for the qumilativc analbiis of the convulsant ctkct of polyhydroxybcnzcncs is dcscribed. MJITHODS Assi*s.fmc.nrof Smcrrtrc Actkiry Rclariorrsltips. Male albino mice fSh-lfield stnin) in the weight range 20-25 g wctc uwd. In preliminary studics with uoancrthctucdInice, 0 1972 by A u d n ~ l c?m&fnc 214 I t W3S four bt .111sc or11' IIIC cnnr 311- s ~ h e i i r c IOa strong wi!h ursr lol)g-ln-i:r beforc t t x lllC SUFl'lf pounds u small qu: stcrcd i? dctcrmir. con\ uls; During t the myc Qiim inducec where ( pcndd s11ch a upon 1 bcakcr Mot1c This f volt2 arms being briLi: ninr ?I1 2 and tioti was ou1 ;L s: Oh! sig- Ol? \\ 2 Io ct' r!' d 0 tuted -. . ... . - ... - . . - , ' . . , .. _- L . - _ . . . - .e... . it was found that the end point for myoclonic convulsions \\as d 4 ~ . : t l iIC, cva1~13tc' bc:ause of rhc concomitant bc!raviord eucitc:iicnt which occurred I I I I!:: n n ~ m p~rilor io the convu!sion. H o t w c r , sincc catcchol is able to eliclr myt*clon:iI:r!.\ 111 nninialj anesthctiied sutTicicntly dcepl) 10 'abolish reflex withdr:iv.JI of I !ic hr!:dicg !n :cpoiicc to 3 strong pinch (Angel. 1969).in thc prcscnt intcsiigarinn the nwe 1: i-c' ;1~:4ictizd with urctliane (1g'kg) to acl~icvctlus lcvcl. This ancsthcric was cticl~c:I*~c;.!t!<c. of tlic Iong-lastings1ablcJcpth of nnczthcsici obtained. Thc anest1;ctic \\'ils a d ~ : v t : i ~ ~1c1r5~min biorc thc coiivul.ant compound in all c;iccs. Tlic anesthctited animals N C X p!clcc.d111 the supinc position. and Ihc ambient temperature was maintaincd nt 22 C.31,)kt cnm- p n d j bere dissolvcd in 0.9 1, salinc; in n f w C W ~ S .bowe\cr, it \\as iic;'cwiry t a l ;ldd 3 small qunntily oi sodium hydrovidc to cfrcct wliihiliisllion. h l l soliiiic?nP crc .Iq?niini- siercJ ip, Tlic doses which iirciuccd convulwns i n 501, of the snimalz ~ C I Wn) m dcrsrmincd usiris the rneihad of \Veil( 1952, wilh groups of 6 mice. Tlrc c;:d (.<+tiof ihc convubions \vas clc;irly indicated by myoclonic jerks of thc limbs and i;i!h tt1'ih: rnicc. During rhc cspcrimcnts complctc silcncc was maintaincd in ordcr to avoid prr.ipit;rling i ! ~myoclonic jcrks by auditory stimulus. Qitdi)tilntiVc ~ S , T ~ S J I ~ CoI Ij I ciosc-actirirj* relationsiiips. Thc total motor activity induced by the contulsant chctnical was assccsscd by a method dcscribcd in brkf clw- uhcre (Angel, 1970). I3a,IcJly thc apparatus uwd consistcd of 3 plwtIc bc3kcr %us- p d e d by a stilf wire from a bcnin rigidly clampcd to a short upright iron surport in such 1 way that the heaker I r u q suspcndcd frccly in spec. TIic benn had niiwrlcd cpon it two scmiconductnr strain gauges. The anesthetized mice wr: plxcd in the hedicr ciiher singlq, or in groups of 4; and the chcmicd under tcst wns IrC.iiinistcrc.dip. Motion of any part of the systm rcsultcrd in a forcc transmitred to tlic <trainrlugcs. This force resulted in 311rrlicmtion of clcctrical rcsis~~ncwchich \**iISccn\crt;J into \oltqc change by wing the gauges as two arms ora N'hclttstone hrid!*c. 1h: other iwo i m s of tlrc bridge wcrc coniposcd ofone fixed and onc voriablc rcsi*l:icic.:. tlic h r c r k n g uscd to balance thc bridgc at the start of an cvperimcntal run. Tlic ourrut from thc :tiJge ( & Fig. Ib) \vas amplificd s\iLh ;Idilrcrcntial amplirict ( A I , Fig. Ib). and the q k f i c d voltag was AC coupled into a furthcr siapc of arnplificalion $2. I - i g Ib). vl:Output fromthc sccond amplifier w3s thcn lralf-wave rcctificd with n \.;IC?: diodc. 2 3 the rcctificd signcil was integrated over fixcd intcrvols of timc uith a fi:v icr oycra- '-&d amplifier having a low4eakagc capacitor in its feedback loop. Expr;i~.:Wi!l!iit ''A $how ihai mnveincnt of thc animals in the beaker produced n w1t.y: iiam thc L'S~PUCof A2 (Fig. 1b) which varicd in pcak-pcn k amplitudc from 0.5 to IO Y. !':. i<t.:lii1g a ! ; ~ ~ - W P Y Csignal into thc intcgrator via tlw diodc. it \vas shown that tb: iiilcnrlil . ' m d over fixed jnten.als oftime, \vas dirccily proportional to thc ~ c l t ; ~o;f :q!~*cirp:it ' "-Ial:oveaccrtain value ncedcd io makc thcdmIeconduc1 (Fig.13).\Yl;:ii tii.s"tiim '. * * cQ!t:ige for the diode had Iiccn dctcrminccl tlic output from the \ccoiJ a!:?!ti:*r -:lt:n Eiwn a constant bias equal to this \01!2g!. Thc cipacitor uscd in thc $ d k k ? th: vpcr;ltion;il amplifier to rcriorrn the inrcgrwon was disclrarpcd ai ti:; cnd '.'' .. I*!:pr3ling time period (either 3, 20, or 30 scc) \ia a ticld elkc! trnnciitnr cmi- !x:w.s it. This was iurncd on by applying n puke 10 mwc in duntion pnine from I -,' 7C:o dcrivcd from a digital timing ticvicc. Thc output from tlic intcy,'ctorI ~ I S on a cathode ray oscilloscopc for visual inspection; a permanent rcwrd \vas r -'.1 riilh 3 pen rccorder. The activity of unalrcstheiitcd micc was dcierniiacd by ., . .. .. . . . . . . . . A -... .-_ . . .- - . . -. -. , 216 ANGEL A k D ROGERS replacing the scrniconductor strain gmpe with one consisting cf ;1 cl>ntinuoctslength of silicon rubber tubing filled with mcrcur); and p;?ssed back nntl Iorth bctwcn two prallel supporring bars. The animal.in its normal cage. \vas pl:rccJ nn thc sliilp formcd by the giugc which formed part of n Whcmtone bridgc circuit. I I I tiiisc;lw. movcmcnt of the animal altcrrd !he bore of the rubber tube and hcncc thc rcsistancc of the sling. The rest of thc apparatus was identical to that used for the ar~csthclIzcJmice. *Tsarb 8 '%4 .- 'd f ? v e-c 6 .- loo H t . .-.-. fdik sior tolc an: - - P C f c t RESULTS Structure-Acririrj Rclariotrships ,Vonosubsfirtrrctl bexrtwv. Ucnrenc olonc did not cvnkc nijnclonic convolsicrrlr. Phenol (see Table I ) was the only monosubstituted benzcnc which was aciivc. Thc .. - . f.l- h continiious length of forth hcrnetn 1sr.o i on the sling formcd *!>icsase, movement isrance of the sling. cirzccl mice. COXVULSlO\S FROM SCnSTlTUTED BESZESTS I Y VlCI: 21i Tollowng monosuhstitutcd hentcncs wcrc tcsred. hut Jld nc*r CI I+.C in! oclonic con\ tilsions in sublctha1 doses: m l i n c . fluorohcnzenc. chIorr~!'\ti, :nc. hroruohcrizctr. toluene. niirohenzenc. benzoic acid, bcnzddchrde. bcnz! I .~lidrol.thaophcnol. and anisolc (methoxybcnzcncr. Phenol Ciitcchol Rcmrcinol Quinol P>ropllol Phluroglucinol 12345 OH- - -OH Oti - --Oli --OH - -011 OH - -OH -- - -OH OH OH OH OH 1.01 'C.'13--1.!7) 0.cx l ' t :5.0.41) 002 I').TO-l.IY) O o t l 10 01-1.07) 5.71 (4.07-7.93) --I6 If!.nro.\.\.~cn:eI~cC~a. techol ( I2dihydroxyhenzcne) was found ICI.Cll.ic n w t pc-tcnt convulsant chcrnicd of this series (Table 1). Rcsorcinol and q U i l ~ ~ihdc 1 .?- and 1.4dihydroiybcnzcncs. rcspcctivcly, wcrc similar to plicnol in convulwi x i n i i y whcrcas pyrogallol (1.2.3-trihydroxybcnzcnc). wns convulsant only at w r y hi$h doscs (5.71 rnmolcs~kg)P. hloroglucinol (1,3,5-trihydroxybentenc)was imcwc. -4thv3riou.r !'t l r ~enph u,:he IWO iuharging dsions. c. Thc ,n .- 218 ANGEL AND ROGERS *. --- -. ! I - Sa- phthalene JJ-( Hydrrw)I group sub3t i t kit w n 1.5 1.7 1.G 1.7 I *3 1.4 2.5 CDSO ImM L g (95': 1irnris.t 1.90 (1.55-2.33) 1.90 (1.55-1.33) 1.03 10.85-1.17) 0.77 Vl.34-1.73 I No myoclonic convulsions in rublcthol doas the CSt. VU: lot\ thc con.. rhr. peai in the l;r ( X = 12; incrc:isi I wrcchu,l With I- extensor after whic thc w n v u thtrized n QtrcnrrI of the hyd: CDjO [sc: to Jdcrmi rcl;tuon&i; . _. -c - - ...-. fl c -I . . . .. . . 1 tol~ccniunnccdhy creascd 5) amino'similar poicncy. bstitutcd phenol. x t i v i t y . Salic! IIC I) w r c inxri\c; Iolcrik _c]. -;!pthol did not ,hthalcncs (I$, xtng of simikir t COSVULSIOSS FRO\( SLBSTITLTtD DENZEXtS IN .\tlLl 219 potcncy to phcnoi. Thc dcrivativcs wiih both hydroql groups on tiic sanie rins ( I & , f 1 . 4 . and 3-w)r e inmivc. A summary of the rclntivc ordcr of potcncy of the phenolic con\ tilsants is givctl in i Table 4. !? Quoitrirarire Defenniiiarionof Acririty Elrccr nfawsrlicsio. Comparison oft tic graphs in Fig.2 shows ! h i ihc rime coursc of i thc convulsivc acti\iiy of 3 dosc of c;itcd~ol(0.55 mrnolco Lg, i\ diil<rcltt in thc un311- i csihctized compared to the ancsthcttzcd aninid. In rhc unancstlicwrd ninusc rhc COW I v u l s m acti\ity usually lasts for 3-5 inin iiican 4.68 min z 1.75 S1). ,Y 40) and is rOllowed by I prolongcd pcriod of postictal dcprcssion, whercas in tkc uicsthciizcri itiicc 3(3 1 I fl?. i . iions ! 1 I I 220 c405.- -200 Activity -100 ANGEL AND ROCSRS a. 24Q b I'C o/ i i The activity cun'csfor LIOSCS of catechol of 1.2.3, and 4times the CDSOareshown in Fig. 3iI. 7 tt: A s : rL'\pc)tljZ rellriionbhips for 3 of [he above 4 chemicals arc shown in Fig. 3b. Onl!~U I K WG obtained for I :S~ilrydro~;\naphth3lcnbecause its CD50 a i d LUSU.if; .LIA)-I i.t:ii~ic~lA. s can hc seen from the curves the dosc rcsponse rrh- tiunship for L i i > d:% IW psr.dkl LO those for cotecho1 and quinol, eiihcr uhcn the ! ? t 1.0. 1 1.1.:. : 2.7. and 0.5 i Having dc. decidea to cor ..... . .., , - , . ... * . , . . . . ".".r..--. ..L ..--.-. ....-.'-.... .*...- . * ... -. . * -. * . . . ..-- i x t t \ ity for individual or for groups of animals IS t i k n C l ~ b < . ! y\iinilar rcwlls for thc rel:iti\c porcncits dctcrmined lrom the dose rcspon\c curl cs. tt.1 w a ! l crrcctt (c.g.,thnt produced by 1.5~drli~dro~ynapth~Icwnecr)e, found H licn ct*iqvircd 10 !hose dclcr- mrncd from Ihc CDSO valucs. Thus from the graphs in Fig. 3b the rclaiiw poteiicics arc in Q po; N c n -' ANGEL A N D ROGLRS n .'I :i -.L . -... .... . . . .. .. . .. I '. . . -I ' _ I ," T " . . \ 2 .c .,. . ...._. x IS C ., -. .. .. -. - . - __* ' e - ._..-- . c . . .. .- 0 subsequcntlg applied dose of cafcchol both in thc pc:;k ciTccr cbiaincd and in thc prt*ltw*gation of its tims course. I n f m in this espcnment the Iidf-hrc of thc catccliol cilcct w3s incrcased from 4.5 to 10.5 min. Dcprersiorr nf A etiriry by P v o g n i i d irt L'rrnncstlreri:d .I lice recr on irtrrmsd nctirrty prodtrccti by mrphc:utwic. I n 1hc prmcnt gtudy I I \\:I$ noticed that small doscs of pyrogallol (0.4Y-0.79 rnmolc,hg) ~codcd10 rcJucc tlic pcriod . 224 n -. ANGEL AND ROCERS 8 n I\ LI I2 00 II I300 , II 14.00 I1 15.00 II IbOO I1 17.00 II 1800 tI WOO I moo IW I Fici. 7. Thc XII\II\ in arbiiriry units (ordinates)in 2 groups of unanesthctircd mise (6 animals per group) s s IIII,C t0riiirh hi.d.ird Time). The animals were plaLcJin a "strange" a g e on rhc actiwy n w x r i11 1 2 . W hr: 33 m n ldrr one group was gircn 0.2 ml of saline. the oiher group WY prim pyre y d ~ (I0.9n i l i d e L g ~ l ? e k t did not aitcr the motor response to the stimulant drug. Figure 6 shows the effst i n 3 grciups of ut1.mdirtizc.d mice (6 animals) of I/-aniphemmine alone f Fig. 6b) or uith eirliLr p!r~yiillul (,Fig6.c)or saline (Fig. 63)i n j c h l at the p a t of the amphct- ,niitic eflc;t. E&r yt ; y m g t i f k t I oti iiorittal iocotrruror acririry. O w tlie time period from noon to S.00 pin t l i ~ r c i' s a gradual incrcasa in tlic nwtn.iI acthity of micc. This activity was sirikitiyl, d i y e w r l b) pgropllol. Thus over the 8 hr period then wvasa dccreasc in the total I ~ ~ . m u x; t~i \~i t yr froin 15.362 units in the d i n e injcctbdcontrals to 5684 units in tlio miiili.ih :rcA:cd with pyropllol (0.i9tiiiiiole.Lg).as shown in Fig. 7. . .. , , . ..... . ... .. . - - _--.-.* . .. .-. . .. ' , --.. .. .I- ._. . . ..'. A .I&J* R Convulsant Rchtivc yolcncy ANGEL AND R O G E R S c -e--- c .- -- . .. -l 228 ANGEL AND ROGERS ACKXOIVLEDC 51EST . ' . - .. .-.. . .. . -, .. -*--I. * b .: -.. . . . . .. . - 229