The Metabolism of N-Alkyl-4-bromobenzenesulfonamidesin the Mouse. Correlation with Anticonvulsant Activity
The anticoiivulsaiit poteticy of several N-alk~-l-41~romoberizeriesulfoiixr1iideu i t i iiiice was found to be directly related t o their esteiit of metabolism to 4bromobenzenesulfonamide. The esteiit of in vivo dealkylation of the monoalkylated derivatives varied inversely with the bulk of the alkyl subst.ituent. After oral administration, plasma concentrations of the N-alkylsulfonamides and their dealkylated metabolites were determined at the microgram level b y quantitative thin layer chromatography. Pretreatment of the mice with the metabolic. inhibitor, P-diethylamitioeth~ldipheiiylpropylacetate hydrochloride, decreased the rate of dealkylation slightly t~ril,did [tot sigiiific.:tritly affwt l t t i t i ~ ~ o ~ i v i i I:tc:t,ivity. ~ : ~ ~ i t Pretreat t t i ~ t r t ,of the Iiiic-e with (!CY4, however, coiltic111 aiid I lie :~titi~~~~ttvr~ls:t~it. pult~ric*ies [if 1,lit. ?j-,lkylHiilfunaiiiidt.R. der:it)Iy t l t ~ r r s s e d h [ he estritt o f tieltll Sitiw ? r ' , ? ; - d i t i i c t h y l - I - l ~ r ~ i i i i ~ i ~ ~ e t ~ ~ ~ i i e s was i ~ ~ f oiiit~tabi~lizetl t ~ n ~ t i i t l ~ Io S-i~i~11i~1--l-lir~~iii1~l~siizetiesi1lfoitaiiiidt~ atid 4-bro1nobe1tzeriesi1lfoti:1tnicl~, :I free hydrogen alont oil 1 tic, w1foti:irni~lc: i i i t riigrii is i i ( i t , :L prerequiBit,e for irr riiro dealkylation.
Biological data froiii these laboratories 011 a serie5 of I~eiizeuesulfoiianiides arid related compounds1 liavc shown the 4-bronioheiizenesulfonarnides to be especially act ive anticonvulsant agents as revealed by antagonisni of maximal electroshock seizures and thioseniicarbazide arid strychnine lethality in mice. 4-Bron1obenzeric.sulfonamide (I) and N-ethyl-l-broniobenzenesulfonamide (11), which were testcd most extensively, wercb found to have about equal potency after oral adniiiiistratioii (Table I). The anticonvulsant activity of I was thought to i ~ w l l t froni caarbonic. anhydrasc i t i liibitory activity, whirh is conimonly shown by noitsubstituted sulfonamides.?- The high potelicy of I1 and the other substituted nienibcrs of the scrics \\as unexpected, however, sirice i t is generally avcrptcd that substitution 011 the sulfonamide nitrogcii dcqtimy.; B I, I?
TI, 1: 111, R TT', 13
= = = =
i
H
GH, CHq CHICH-CH?
-
0 S02NHR V, I? VI, I? VII, R \ TIT, R
= 1
=
CH(CHj). (CH?),CHj
(CHz)iCH, OH
carbonic anhydrase inhibitory activity.2 I n i1itt.o carbonic anhydrase inhibitory activity of I as well as lack of such activity by I1 has been established iii these laboratories,6 using the method of ?\Iaren.i The data of r\Iaren5 on 2-acetylamino-1,8.4-thiadiazole-e7-sulfonamide (acetazolamide) and its S5-isopropvl derivative show the latter to be metabolized in vizv t o a carbonic anhydrase inhibitor, thus explaining its activity These data indicated to us that S-alkyl rompounds of the present series (11-YII) rnight be dealkylated to I, which could be responsihie for the observed anticonvulsant activity. However. since Maren5 only presumed the carbonic anhydrase inhibitor to be acetazolaniide (the product was not isolatcd and identified), we could not assume a priori t hat the activity of the N-suhstii uted bromobenzene( 1 ) II II Keasling r: riiiiniann hiid Xeldhatnp, I Ired. C i i r m , 8 , 548 ( l ' l b i i (2) I3 4 Krebs BiochPm I , 43, 5 2 5 (1'148 ( 3 ) Xl' €I. Miller 4 \I Desqert. and R 0 Rohlin Jr 72, 48'1.3 (1930) (4) T hlann a n d I) l i e i l i n \-aiure, 146, 164 (1040) f h a r m a r o l I..rpfl T h e r a p , 117, 383 (1950r ( 0 ) IT. W. Keasltng and I , J Peterson, unpublished results ( T i T H \ l a w n . .I l'harrnarol. I $ r p f l ThPrap , 130, 2 6 (1960)
~ulfonaniidt~si f a s UP 1o nictabolic dealkylat ioii. .\lorcovcr, tlic, siiiiilarity i i i poielicy of 1 aiid 111 i i t niirr was not i i i accord with JIaren's observation that oiily about of l h r Si-mcthyl derivative of awtazolainidc Tvas convc.i*ledto a carbonic anhydraw inhibitor i i i this spccic>. The prcseiit situation 11as wmpli(sated also Ijg the fact Ihat the diniclthyl dri.ivativc. ( I S ) poss*lssccl i~~nsiderablc activity. If 11ir activity nf I X urrv cliic: io its cwuwrbioil t o I , rhis would iwii~r
0
SO,N(CH,)
IX
tradict the huggestioii of Wiseman, et ~ 1 . ~that 8 a frrc, hydrogen 011 the siilfoiianiidr nitrogeii iz noccssai.y f o r in vivo dealkylation of sulfonamides. In the present study, plasma and brain of niicc ~ w r r examined for both the ~-alkyl-4-hromobe1izenesulfoiiamides and possible metabolites by thin layer cbhronia1 ography, followed by ultraviolet measurement. This procedure allowed yuarititation at the microgram levc>l of both drug and metabolitcls and their cwrrelation with pharmacological response.
Results and Discussion 111 preliminary experiments. analysis of niice plasiiia arid brain collected 2 hr. after admiriistratioii of I1 revealed only I. The identity of metabolically produced 1 was established by infrared and ultraviolet spectroqcopy following thin layer chromatography. Sinw most of the pharmacological data for I1 had also beeii obtained 2 hr. after dosing, these preliminary result.; were consistent with the observed similarity in potency of I and 11. They did not prove, however, that IT was devoid of activity. The concentration of I in the brain 2 hr. after oral administration of I1 is essentially identical with that attained 2 hr. after the same dose of I (Table 11). These conceritratioiis are also very similar to tht. corresponding plasma concentrations at 2 hr., indicating that these compounds distribute themselves equally
I
o/
1 8 ) 1- 1{ Wiserrian, I' I , 11, 88 (1962)
I.
iireilirr, a n d R l'inwn
Jr
, Riortirni
f'hrirmn
July 1965
52 1 TABLE I IffJ-ALUES, LTLTR.4VIOLET A I A X I M A , A S D CTLI’R.4VIOLET SELFONAMIDES R’ITH THE GENER.4L S T R U C T U R E
h T I C O S V U L S A N T POTENCIES, OF
.\nticonrulsant ac t i r i t y Ell60 a n d 95% C.I.” a t 30 min.. mdkg. (electroshock)
1t L
(‘otnpd.
Rdatii-e potency 1 = 1
H H H H H
Rib
A\BSORPTIVITIES
Xm,,,
36‘ (33-40) 1. 0 0.32 45 (30-67) 0.80 0.86 I1 56 (42-75) 0.64 0.56 I11 56 (42-75) 0.64 0.74 IT 50 (37-68) 0.72 0.67 Y H 77 (47-106) 0.47 0.76 1-1 H >200 < O . 18 0.68 1-11 H 45 (34-59) 0.80 0.21 TI11 89 (67-118) 0.40 0.78 CHI IX a 95% confidence interval computed by method of Spearman and Karber. Thin layer chromatography on solvent: CHC13-HCOOH (19: 1). Weighed mean of several ED60 determinations.
I
11111
.Ihsorptivity
235 72.0 235.5 54.4 235 65.3 235.5 53.4 236 53.1 235 49.9 235 54.4 235 55.5 235 59.3 silica gel G; developing
TABLEI1 CONCENTRATIONS O F
4-BROMOBENZENESULFONlMIDE
N-ETHYL-4-BROMOBENZEKESULFONAMIDE (11) IN
ORALADMINISTRATION OF 400
-__-~ ,----.ifter Time, min.
of I1
(I) A S D
bhCE
AFTER
MG./KG.~
Plasma concn., T/ml,------
oral administration of 11Total (I 11) of I
+
.ifter oral administration of Ib of I
56.3(6a) 50.7(6e) 53.5
73.8(6a) ___ 67.2(6e) 70.5
130.1(6a) 117.9(6e) 124.0
135.0(6a)
3 8 . 0 (sa) 4 2 . 1 (6e) 1 9 . 3 (6g) __ Alean 33.1
116.1 (6a) 101.7 (6e) 71.4(6g) 96.4
164.1 (6a) 143.8 (6e) 90.7(6g) 129.5
167.5 ( s a )
15 Mean 30
84.6(6bj 8 9 . 4 (6b) 99.2 (6f) __ 94.3
98.9(6b) 100.4 (6b) 99.2 (6f) .~ 108.3
plasiiia. ]io\\ e v ~ l ~ ~ i i i a i ~ i~~ssciitiall? td uiwharigc.d Ailthough50 iiig Lg. of S did decreasr tho rat e of c o i i r v r m i i of I1 to I, relatively laigr ariioiiiiti of I were still fouiid i i i the blood 60 111111. after the adatioii of 11 11 wab not Yiirprisiiig, t1ierefoi.c. that prior adriiitiisltatioii of X did riot produce a sigrtificaiit diffcrence i i i Ihc anticoiivulsaiit activity of IT at 60 inin. ailthough otily about 5 0 t c of' II uas corivertcd 1 0 I at 13 m i I i c'v(w 11it houi prior adiiiimstration of X I tests for aiiticsoiivulsant activity at this tiiiie interval he achvity of TI was potentiated hy tioii of X (Table 111). Swinyaid. orted that S itself exhibits ailticonvulsaiit action, but is iiieffrctive at a dose of 50 riig. tlig. Howevcr, 111 coinbinat ioii with 11, the acition of X may tie potentiated. -1lthough Swinyard. et ul.,14 reported potciitiatioti of tht. aii~ic~mvulsaiitactioii of s2veral d i ~ i g shy S.t tic rerc~rs('inay havcl been the true situat io11 ; t hew \\ orl;erb 13c~ognizedthat prevention 01 ilc~~iiethylat i o i i of t i i t i*ogeii arid inhibit ioii of aliphat 11' yitle-rliaiii oxidat ioii arc i i o t tliv otily iiiecl-iaiiisnih hy nliicli X potciitiatcs ailtiepileptic drugs. To ohtaiii iiiore detinit ire iiiformatioii regarding t hit 1111 riiisic' pot (biicy of 1 lie S-substit uted sulfoiianiides. the effect of pretreatiuent with CC14 was investigated ; thit lat tcr causes liver damagell,lGarid would 1~ CYpcc't ed to irihibit deall CHs > CzHs > CHzCHzCHz > CH(CH3)z > (CHZ)?CH3 > (CHz)3CH3. Thin order is in general agreement with the relative potency of these compounds as anticonvulsant agents (Table I). Statistical analysis of the data revealed that the ED50 values correlate better with the plasma concentrations of I than with the intact drug or total (drug plus metabolite) concentrations [correlation coefficients ( P = 0.05) of -0.60 vs. -0.24 and -0.49, respectively]; ie., even if the Salkyl compounds mere as active as I, the EDso values correlate best with the concentration of I. This further supports the conclusion that metabolically produced I is necessary for activity in this series. Given the consideration that I itself is the most active anticonvulsant agent in this series, inspection of all of the data shows that it is highly unlikely that any of the substituted Compounds have significant intrinsic: anticonvulsant activity. Experimental Materials.--The niethuds of synthesis uf the sulfonalnides have been described in H previous publication from these laboratories.’ Development of the Analytical Procedcres.-Siiice t lie IJOSsibility esisted from the outset that one or more of the N-substituted sulfonamides might be metabolically dealkylated to 4-
l~r~ir~iolierizeriesulfo~i:~~~iide, :in analytical procedure was souglii that' wrould enable simultaneous determination of these iwiiipounds. The similarity of the sulfonamides investigated is sucli that) separation prior to cluttntitation \vas required, c.y.. their ultraviolet maxima do not differ by more than 1 nip. h f t t v t h e investigation of gas ehromatography without .suc~c'ei-, b(:p:tr:it ion \v:i< achieved by thin lnyer clironiat~,gr:tI,h!.. This It'i v:tluei o f the sulfcmirnitie-, which iriiglii i ~ ~ r i i ~ e i v she b l yv i pcc-ted i o occur together iii the s m i e s:iniplP, wci'e 5uf-lic.ieiitIy tlilTereril using silica grl G us tiit. wpport iii:it~rial :iiid c.hloi.iNl'oriii -forink : t d ( l t lie iic~velopiiig~ i l v e i ii~ T:tt)l(~ I Tlie zoiies were l o ~ u t e t lby fliioreswicc ciueiiihing. After (11i:itit i t :ti ive elution wit,li i t kiiowvn voluriir i i f :tl~mlute et h:iiicil, I tir conrent rations were determined by ultraviolet :inall Determination of Sulfonamides in Plasma and Brain Tissue. Six iiiire were usuiilly used for w1.h sulfori:iinidt~determinaticiri :ind were the s m i e iiiicr used i n testing for :tntic~c~iivulsant activity. The blood was hep:irinizetl, p(ioletl, :mil writrifuged, ~ t i i t l 11 froin the i ~ cdls ~ t :t11(1 cwitrifuged :I 8.
iient of pH, the p1:t~rIi:i \vas exlractcYl le of ch1iirr)forni; the CHC1, \\-:IS evapo-
d then tranJerred qunntitatively t,o the with standards. arid developed. Pbsrn:~ from niintreated mice was run iii parallel Fit h the drug-con1 ainiiig saiiipleh. The silica gel, ccirre-ponding t o xiinex, \v:t': i,eruoved from the plate w i t h a miall hptttula arid eluted ~ j t 5h i i i l . I E ;thsolnte eth:~iiol,it being previou4y deterriiiiietl that the zones could he cluuutitativel>. eluted in this miinner. Tlie et,haiiol eluates a e r e exaiiiiiied s~~ec~tro~~licrtoriietrically with u Cary ~ ~ ~ e c t r o p h o t o ~ i (the i e t e rcciiiiplete Fpwtruiii w t e recwrded). ('i)nventrai ion< were cdcul:tt eil froiri t h r ~previously ilet ermiiietl absorptivitic,s (Table I j. The c.ciiic~entr.:~tion;.irf I, ckterniined i n this iri:~iiner,agreed very closely w t hi)-e ) w e d on cs:irbuliicb :tiih~~clr:i~t: inhibitioii.~ 1iifr:ired spe I of t h i n l:iyer chroiiiatogr:iphic eluates a e r e a l h i r u n in :t fevi i':t>e- IO cwnfirm identities. Brain tissues froni gri)up- of 13 1iiic.e were weighed and poulc~l :tiid then :idded t o N I . :>arid then reccinitituted to IO nil. with CIICI.I. 1Iensured voluiiiei of this ,olnt ion were quantitatively I r:in,st'erreti t o a Auorewent, thin layer p1:tte :tlony with standards
