Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 4 315
IRREVERSIBLE ENZYME INHIBITORS. 181 HtO-NaBr in fine needles, mp 308' dec. Anal. (C31HzzClN50.2HBr) C, H, N. 7, R = NOz.-Scarlet needles were obtained from EtOH-Hz0HCl, mp > 360". Anal. (C31HzzNeOa.2HCl) C, H, N, C1. Attempted drying of this salt a t elevated temp in vacuo caused loss of HCl and analyses showing a C1- content between 1 and 2 moles of C1 were obtained for samples so dried. For analysis a sample was dried a t room temp in vacuo. 2-Amino-4 [ p - ( p - nitrophenylcarbamoyl)anilino]-6-methylpyrimidine.-4-( p-Nitrophenylcarbamoy1)aniline (2.57 g) was dissolved in hot 2-ethoxyethanol (60 ml) and the soln cooled to 60'. 2-Amino-4-chloro-6-methylpyrimidine( 1.58 g) was added, the mixt was boiled till homogeneous, concd HCl(0.95 ml) was added, and the clear soln was heated on the water bath for 0.5 hr. After a few minutes a product started to crystallize. After thorough cooling the chunky pale yellow prisms of the hydrochloride were collected, suspended in EtOH (15 ml), and stirred with concd NH3 (10 ml) for 0.5 hr. The deep yellow base sepd from DMFHZO-NH~as fine needles, (3.2 g), mp 334-335". Anal. (CISHisNeOs) C, H, N. 2-Amino-4- [ p-( p-aminophenylcarbamoyl)anilino]-6-methylpyrimidine was prepd by Fe19 reduction of the preceding nitro compound in 6593 DMF-H2O. The pure compound sepd from EtOH-H20-NH3 as colorless needles, mp 280-281 O.- Anal. ICBHI~NE,OI C, H. N. %--A sample ofthe above primary amine (0.98 g) and Q-chloroacridine (0.64 g) were dissolved in 65% EtOH (80 ml) by boiling. HCl (12 N , 0.52 ml) was added to the clear soln, yellow crystals of product.2HCl started t o sep shortly afterwards. The reaction mixt was thoroughly chilled after a further 0.5-hr heating on the water bath. The sepd crystals were recrystd from DMFH20-HCl-NaCl. The fine yellow needles of dihydrochloride (1.48 g) had mp 358' dec. Anal. (C31HzsN~0.2HCl)C, H, N, C1. Biological Testing.-The routine screening test consists of ip inoculation of lo5 L1210 cells into 18.5-22.5 g of CSH-DBAz Fl hybrids on day 1 with drug treatment initiated 24 hr later and continued for 5 days. All dosage was in 0.2-ml vol in HzO suspension. Groups of 6 animals/dose level were used with one control group for every 5 tests. The wt change column in Table V records the difference between initial wt and that at day 8 for survivors. The number of animals surviving as long or longer than controls is listed under survivors. Doses have been rounded off to two significant figures. Details of testing of inactive compds have not been given.
-
TABLE V Dose, mg/kg SurWt per day vivors change
Drug
5 , R = CH3,
Average survival, days
T/C, Treated
Control
%
15.9 10.2 156 150 4 -4.2 19.2 10.2 192 dibromide 100 6 -1.8 18.2 1 0 . 2 178 67 6 -0.2 44 6 fO.6 15.1 10.2 148 29 6 $1.1 13.7 10.4 132 20 6 f3.2 12.8 10.4 1 0 . 1 142Q 4 -3.9 14.4 6,R CHa &S 75 10.1 292b 29.5 6 -1.8 bis(p-toluene50 21.7 1 0 . 1 215 sulfonate) 33 6 -2.2 18.3 1 0 . 1 181 22 6 -0.8 15.8 10.1 156 15 6 $0.3 14.0 9 . 8 143 10 6 -0.9 9 . 8 125 6 $3.0 12.2 6.7 6, R = H, a~ 225 6 -3.8 14.5 10.2 142 monochloride 150 6 f0.8 17.8 1 0 . 6 168 100 6 f2.9 17.2 9 . 8 172 67 6 4-2.4 13.4 1 0 . 1 135 44 6 f3.5 12.7 10.4 132 7 , R = NOz, t~ 60 5 -2.4 14.8 1 0 . 4 142 dihydrochloride 40 6 -1.2 16.8 10.4 162 27 6 $0.8 14.0 1 0 . 2 143 18 6 f4.0 13.1 1 0 . 1 132 12 6 f3.8 12.3 1 0 . 1 122 9 . 8 141 6 -5.2 14.4 8,asdihydro350 16.5 10.2 168 6 -2.8 chloride 230 10.4 154 6 $0.2 14.8 155 13.7 10.4 143 100 6 $2.4 1 0 . 1 124 12.3 70 6 $4.2 a Not including one 100-day survivor. * Not including two 100day survivors. a~
Acknowledgments. We are grateful t o Miss Lois Armiger and her assistants for technical assistance in performing the many biological tests. This work was supported by the Auckland Division, Cancer Society of New Zealand (Inc.).
Irreversible Enzyme Inhibitors. 181. Inhibition of Brain Choline Acetyltransferase by Derivatives of 4- Stilbazole 'z2
B. R. BAKER* AND RAYMOND E. GIBSON Department of Chemistry, University of California at Sunta Barbara, Santa Barbara, California 93106 Received November 16, 1970 Forty-three analogs of 4-stilbazole were synthesized and evaluated as inhibitors of choline acetyltransferase from rabbit brain, The most active inhibitor found for the acetyltransferase was 3',4'-dichloro-4-stilbazole (23) which complexed to t,he enzyme 910 times more effectively than the substrate choline and 230 times more effectively than 4-stilbazole. Other highly effective derivatives of choline acetyltransferrtse were the 3'-C1 ( l l ) , 3'-CH3 (12), and 3'-CH30 (13) derivatives of 4-stilbazole which complexed 80- to 130-fold more effectively to the enzyme than choline. Compounds 11-13 and 23 were poor inhibitors of the AChE from rabbit brain; for example, 11 was complexed 130-fold more tightly to choline acetyltransferase than AChE.
The major enzyme in nerve endings involved in nerve impulses is acetylcholinesterase (AChE). The order of events appears to be (a) active transport of choline through the nerve membrane,4 (b) acetylation of (1) This work was generously supported b y Grants CA-08695 and NS09544 from the U . S. Public Health Service.
(2) For the previous paper in this series see B . R . Baker and M . Cory, J. Med. Chem., 14, 119 (1971). (3) L . S . Goodman and A . Gilman, "The Pharmacological Basis of Therrt-
peutics," Macmillan C o . , New York, N. Y . , 1960,
p
422.
choline t o ACh with acetyl-coA mediated by choline a~etyltransferase,~ (c) hydrolysis of ACh t o choline and acetate by AChE during the nerve i m p ~ l s e and ,~ (d) reacetylation of C O A . ~A tremendous amount of (4) L. T. Potter in "The Interaction of Drugs and Subcellular Components in -4nimal Cells,'' P . N. Campbell, Ed., J. and A . Churchill, Ltd., London, 1968, p 293. ( 5 ) (a) Reference 3, p 408; (h) D . Nachmansohn in "Cholinesterases and Anticholinesterases," G . B . Koelle. E d . , Springer-Verlag. Berlin, 1963, p 41. (6) D . Nachmansohn in "Handbuch der Experimentellen Pharmakologie," G . B. Koelle, Ed., Vol. 15, Springer-Verlag, Berlin, 1963, p 40.
316 Journal of Medicinal Chemistry, 1971, Vol. 14, S o . 4
research has been done on inhibition of AChE,' but practically nothing of significant use was done on inhibition of choline a~etyltransferase~ until 1967. In 1967 Cavallito, et al., reported the first* of a series of exciting papersg-12 on inhibition of choline acetyltmnsferase by derivatives of 4-stilbazole (1) ; when the enzyme vas assayed with 5 mill choline, 1 showed
H 1
H 2, RX = HC1
3, RX = CH,I
lo0 times as potent an inhibitor of choline :icetyltransferase than the cholinesterase, whereas 3 \\-as about 400 times as potent against the acetyltransftrase. Furthermore, 2 could pass the appropriate membranes to the brain since it could enhance the behavioral-stimulating effects of nniphetamine in tr:tiiied rats, but 3 showed no activity,13 indicating t hat 3 nith its cationic group could not cross the appropriat e membranes. I n spite of tlie difficulty of membrane passage n.ith quaternary compounds of type 3, Cavallito, et al., performed the remainder of their studies uith quaternary compounds;8-1nthe latter type such as 3 certainly have two advantages over compounds of type 2 in that type 3 are more water soluble and are more potent inhibitors of choline acet? ltransferase in broken cell systems where tlie membrane transport problem has been Since Cavallito, et ul.,@-loassayed only 5 compouiids without a quaternary group, we decided t o make an extended study of 4-stilbazole (1) analogs without :I quaternary group with the expectation that each 10fold increase in potency n-ould approximatel) compensate for a IO-fold loss in solubility. The results are the .ubject of this paper. Enzyme Results.-Choline acetyltransferase and AChE n ere isolated from rabbit brain acetone powder (i) J 1 Colien a n d R \ Oosterhaan in ' Cholinesterases a n d I n t i cholinesterases," G l3 Koelle E d , Springer-Serlag 1963, p 300-362 ( 8 ) J C Smith C J Ca\allito, and F F Foldes Bzochem Pharmncol , 1 6 , 2478 (1967) (U) C J C a i a l l i t o H S k u n J C firnitit a n d P r Foldes, J .lied C h e m , 12, 134 (1'369) (10) C J C a i a l l i t o 11 5. T u n T k a p l a n J C Sinitli, and F F Eoldes i i z d , 13, 221 (1970) (11) f f L \ l l i i t e and C J C a i a l l i t o Bzochzm B i o p h y v 4cti1 206, 2-12 (1870) (12) €1 I, \T hite and C J Caraillito, zbzd 206, 343 (1YiO) (13) hI E Goldberg a n d T R Ciofalo P~zjckophar~nncologzii,1 4 , l i 2 (1969)
by the method of Potter, et al.I4 Choline acctj1tr:insferase M as assayed u-ith 1 m M clioliiic bromide : ~ n d 0.05 i d 1 [lJC]acetyl-CoA b> cuit:iblc motIific:itioii of the nietliod of 1IcCnman arid Hunt :is clc~cribctl111 t l i t , Experimentnl Section. A4CliI; u :is :iss:i> c ~ hl i ii1odificatioii of t l i c nietliod of l'ottcr16 usiiig 1 nrlll ACh 'Cl-. :is dcscribed in t h e 1:xperinitiit:iI SCCI io11 The reported iiihibitioii of clioliiie ncet) 1ti:riisfcuse by 4-itilbazole (1)8 i\:rs confirnicd. 1 lint1 Io0= 470 p J / (Table I) :iud W ~ complexed I ~ t o t!ic cItiz\ tiit' :iboui twice :is \troiigl> :is choline uutler our :iss:ii coliclit 1011s. KO inhibition of liC!iE \\:IS slio\\n b j 1, :I11 liougll wlubility n-as A limiting factor That the b[,iiL(>ii(ariiig \ \ , I \ essential for inhibitioii of choline : i c e t j liraiisf(~1 ,ise :i5 shon 11 by the lack of inhibitioii b\ 3000 p J I 4-\i n ) lpi ridine. A stud? of substituent effects on bindiiig ui ilii. P l i ring of 4-stilbxzole (1) to choline :wet;\ Itratisfern-e \\:is thenniudc. C:iwllito, el al ,b h:id ytudied o r i l ~four a i nlogs of thrk P h moietl of 4-ctilb:izole \\licrt, t l i c 1)) ridiiie K 11 :is not qu:itcriiized , Ilie-e conipoundy Ii:id I lit, Ph moiety of 1 ieplnced h j a-ii:iphtli> 1 (2). t j - i i l c h i > I, 4-biplienj lyl, :ind 4-pyrid11. Tlic moit :ret VI' co~iipound 11 as 2 nit11 1,"= 2,; p J 1 :id[S I]" = 200. The e f f r ~ t sof m g l c m,111 -ubstit u ( ~ 1 ist \\ WP 111'ht investigated. Iiitroductiori of 44'1 (5) or i-3Itx (6) gave :%fold :uid 0-fold increments in biiitliiig t o clioline acet? 1transfer:isc Iiitroductioii of 111c iniorc pol:ri~ CH3O (7), S H ? 18), h c S H (91, 01' SO- (10) 1 ~ t~o 1 decrease in biiiding. Tlius t h t , c#iw of t l i c 4 4 ' 1 :iiid 4-CH3 c:in brl x c o u i i t c d for b> 11) dropliobic hoiidiiig. lntroductioii of :I ?-('I (17) 01 2-('Hd0 (18) lid i o 110 change in bindiiip. Tlie most clrnmatic effects on binding \I ere \c(sii \ \ i t 11 3 substituents. Tlic 3-Cl (11) gave :t GO-fold i~icieiiit~rit in binding :uid the. 3-1Ie (12) and CH,O (13) :i 40-fold increment i l l brinding. Siiicc t I i ( t iii:iwniuni i i i c i c m c > i i i in 1 i dropliobic ~ boilcling tli:rt c\ of bindiiig such :ISelectronic i l f f ect s, point c l o i i o i -:icc.cptor bindiiig, or coiiforni:~tioii~iIcli:iiigc, i n t l i o t * i i i ~n (~' muyt be playing :I role A- 11 e 4iall 4io11 L ~ i i t~l irt w~ 3 3 substituents d l hirid to tlic w n n ~locus 011 i l i r ~ ' I I Z Jn i l > , t h t i., tlir. heiiz;ciw ring is 1101 iuriicd o tlicrc arc' t 11o po\siblr loci for biiidiiig :I nitit < I -ithit l t uerit , sinct. C", c,iniiot biiid b> clonot -:icceptor p o l l i t complexing, i t foIloi\s t l i a i l l i c 3 s u b ~ t i t u t ~ iIiI t~ ~I TP :I pronouiiced electronic cffcct on biii(liiig bt t l i t ' t w i i L t > i i v ring of tlic 4-stilbazolr - \ ' h i e i n A\ltl~ougliI I i ( 1 SHZ group of 14 is stiongl! clectron rel(laGig, i t w pohi ( T = - l.2:3)I7t1i:it i t uould be repul~ecliron1 :I !I\ tlrophobic locu- Tlie po1:ir :mtl electron-\\ ithclrau uig ('S (15) and S O ? (16) group5 :Ire cletrinieiital t o binding t o choline acet vltr'in-fclrase +upporting tlic :irgiiniciit t h a t nonpolar, elect loll-relensirig g r o u p givv llii, bc-t v l i liariceme~iti i i buidiiig Lit t l i f meta positioii. t l i t l v : t r g ~ 12,000
.4cetvlcholinesterasec~
Inhib,
70
[S/Ilo.ae
P M
inhibn
2.1 2000 500 0 620 0 7 >250 0 31 0 0 8 628 0 > 1000 1250 0 250 9 370 10 14 16 3000 130 5000 11 7.8 33 10001 1.0 83 12 12 600 0 >240 loo0 1500 2000 2000 300 120 200 500 0 120 1000 27 16 200 0 0 28 500 12 >200 209 80 350 2.9 30 630 12 >240 2000 670 540 36 1339 200 37 0 509 270 0 39 679 250 0 629 2000 iteric effects on fit. An increase iii size from 4’-CH3 (6) t o 4’-C2Hj (30) led to :t &fold loss in binding. Similarly increase in size from 3’-CH,O (13) t o 3’-CiH;O (34) led t o :in l b fold loss in binding. Such results indicate :I decreased bulk tolerance for the larger groups nitliiii the enz) meinhibitor complex. Hon-ever, it is sometimes po~siblc t o force an energetically unfavorable coiiform:it ioiinl c1i:itigc in t h e eiizyme if by such :I c1i:ingr n e t biiidirig is i~icreased.’~Therefore larger groups :it t h e 3’ xid 4’ poqit ions (28-40) \\‘ere syritliesized for enzymic eva1u:ttioti; unfortunately binding iv:ib riot i n c r e : i d by any of t Iieie large hydrophobic groups. Tlir question xroqe earlier n-hether increased binding by :3’-C1 (11) and 3’-CH30 (13) XIA due to :I point dolior-i1cct.pi or iiit eract ion :IS previously \ecii u-itli :t p-CH30 group on binding of 9-plien>-lguanineto guariiiic de:Lmin:kse.20 If ~ u c h\\-ere the cabe then it is possiblc t h t C1 arid CH30 \\-odd bind iii one conformation ant1 CH, in :I second corifor~natioii:IS slio\\ n by itructures 42A mid 42B; tlierefore 41 TX a b S> r i t hciized for t~valun-
42A
CHI I
42B
H
41A
41B
tion :IS a11 inhibitor of choline :tcetyltraiisferase IT hicli might bind in either conformation 41A or 41B. One of 3 rewlts could be anticipated. :mj one of diicli has a strong bearing on t he enzymic environment of the 1’h moiet?. If there \$ere beparate binding loci for Me arid AIeO (41A or 41B), then 41 bliould be :t better inhibitor than either :3’-metliy1-4-stilb~~zole (12) or 3’-metlioxy-4-stilbazole (13). If tlic :3’-CHJ, CH30, i~iidC1 w r e all bound to the w m e locus ilicri t h e 5’ substituent could be in a noncontact nren of thi’ rrizyme if t h e l’h group interactcdflatl> nit11the enzynic or \\-as in a slot on the enzyme with one face of tlie benzrne ring pointing away from tlie enzyme surface;IO in such n case, 41 11ould be equal t o 12 or 13 R S a n inhibitor. Or the P h moiety might fit into a pocket on the enzyme: if tliere u e r e bull; tolerance on both sidei of tlie 1’11 ring, but binding by a group 011 only one side of the berizeiit ring, t h e n tlie compound (41) would be equal to 12 or 13 :I\ :LII inhibitor. However, if t h e benzene ring n-ould fit into :L pocket \\-here there \ u s bulk tolerance for :I small group on only one side of the benzene ring, then 41 n.ould be :t much poorer iriliibitor t h i n 12 or 13. ( 2 0 ) 13. I?. Ilaker and I T - . F. FT-ood, J . M F ~Chem .
,
11, 644 (1968)
That tlic latter riglit fit in :in enzymic poclwt \\-as t l t r case \Y;IS sho\\-n by ihe 40-fold loss in binding b y 41 compared t o 13. Tlius t h e 1% inoirt\- of 4-stilbazole biiids iii :I flat pocket u-liwc i l ~ is w room for small 3’ or -1’ snbstitur n t s hucli :IS (’I or CH3, but tlicre is iiot room for botli :if:tiid .i’bubstitucliits. \I7iethr~ror i i o t r l i t w is i’ooni for lwge substituc’iits :it the 2‘ ((j’) position tli:it \vould reside outsiclc t 111, poclwt rim:iiiis i o be (let wniiiird i i i future ivork. €To\\-t>vcr.i i is c l e x t l i u t tlir. Iirob:ibilii>. of fiiidiiig :tppreci:rbly better binding at tlit’ 3’.4’ :iiicl ;i’ posit iotis t l i : i i i t l i i ’ ~ ~ ’ , ~ ’ - ~ l i c l i l o r substitwiit ~ ~ ~ ~ l i c ~ of ~yl 23 is negligible. The most poteut compouiid in Table I \\-;is 3’,4’tlichloro-4-stilb:tzol~~(23) n-itli Ir,O = 1.1 pLll; 23 \\-:is complcsed to clioliiie :icetyltrarisi~r:~se 910-fold bi.1 tcr t l i : t i i cliolinr~. .If its ~nnsiinumsolubilii y of 30 pJ1, 23 shou~etlno inhibit ion of XCliE; since :!Oyciiiliibii ioti is rc>adily clctect:iblc, t l i c Ir,o > 120 p-11. ‘I‘liu~ t selectivit!. of iiiliibitioii by 23 bet w e n tlic t \ v o t ~ t i z y m r s is >110. 111 tlieir elcgnni studies on tlic bitiding of 4-srilb:izolci C:ivnllit o. cf a / ., (1) to clioliric :~cct~-ltr:ttisfcr:a~e,”-’” rn:idtl t l i p follon-iiig stroiig point s. (a) Tlir> vinyl grouli n x s iiecessary to traiisniit t~lectroii-cloiior propert ics iroiii t lie bciizerie riiig t (I :I iioiiquaternized pyricliiie ring; t his t rnnsmission could :tlso bel accomplished b). :xi1 a c c t )-lciiic bridge, but : i ~ iE t bridg;c destro>-edt lic a c t ivit;.. (b) .. trans-viti-1 I bridge \\-:is essential Finccl t l i i ’ cis-viii~~l bridge caused loss of activity. (c) 2-Stilbazolc \vas also :\ctive indicating t l i a t tlir pyridine S did i i o t interact i i i :I point donor-accept or complex, but tlic basic ring s\-stem interacted with sonif’ c~lectron-donorlocus oii t h e enzynirl. (dj Quat erniznt ion of 1 lrd t o :I 4O-fold eiili:~nced act ivity. hut :11so iiiere:isetl :let ivit y to\\-:irds ACIiE. The :ictivit\. of 2-sti1b:izolc (43, Table 11) \\-:is confirmed, but i t \\-:I> only :tbout 0.25 :IS active :\? -1-stilb:tzole (1); :3-stilbnxole (44j i\-:is even less df‘ectivc. Siricc tile 1’11 nioiclt J- of 4-stilbnzolc : i p p ~ : ~ rto s be complexcd to tlic enzyme by hydrophobic bondiiig :tnd siricc t lit’ p>-ritliiic ring e:iii lie rot:ttetl i t i its binding locus. it fol1ou.s that ilic pyridiiic. iiioi~tyivill rot a i e t o :illon- tlit> I’ll t o l i : i v ~iii:~ziniiiinli\.tlropliobic iiitcritctioii. Sucli rotation of :I h:isic riiig t (I :1110~~~ii:\simutnI~-\~dropliobic bonding b!. :I li\-clropliobic A l e cliaiii 1i:is been prcviously obs;crvctl \\.it 11 inhibitors of tlih~.cl~~ofol:~t reduc1111
t :Is?.
”‘
(3
.iltliougli C‘:iv:tlIito. ci ~ l . slio\\-td , ~ t1i:it xctiyiiy \v:is c1ccre:ised v . l i ( l i i t lie xTiii),l group of 1 or 2 \\’:is rccliiecd to E t . t lir>csteiii of this dccre:ise ~ ( ~ ~not i l bc d :tscwt:tiii~~d due t CI lack of so1ubilit~-of t h e rrductjoii products; lioivewr, it IV:IS cle:w :it least :i XX-fold loss occurred o i i reduction of 2. ‘l’licrefore, : ~ ’ - n i e t l i y l - 4 - s t i l h ~ i(12) ~~l~~ \\-:I> reducrti t o 45 (Tablt. 11) and evn1u:ited. Sitw 12 Itad I,o = I:! p J f : i i d 45 Iiad I:,O= 2000, 1 1 1 :rppnrrrit ~ loss in biiidiiig u x s 170-fold. Hon-ever, i’ciis s::imc i i i liibitioii by 45 ivoul(1 be obscrvcd if 45 cont:iinss 1 part in 170 of 12, : ~ i iunclct rctablr miouiit ; therefore, the loss i n binding is cqunl t ( I or greater than 170-fold ivlieii tlic viiiJ.1 group of 12 is reduced t o Et (45). 1T711cn t Iic .ubst itucni cfYt>cts 011 4-stilbazole (1) (Tahlc 111) \ v w r cc~nip:ircd\\-it11 tlic substiturnt effects ( 2 1 ) I’,. 1%. 13aker m i l H , 8.5 h a p ~ r o.I. Z’/(c!rrn. S c i . , 66, 308 (l!>titi).
Journal of Medicinal Chemistry, 1971, Vol. 14, Vo. 4 319
IRREVERSIBLE ENZYME INHIBITORS. 181 TABLE I1
INHIBITION" OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE FROM RABBIT BRAINBY
-Choline NO.
Inhib, pM
R
acetyltransferaseb--
% ' inhibn
P M
[S/I]o.sa
11 4-CsH&H=CH 470 2.1 43h 2-CeH&H=CH 1000 35 1800; 0.65 441 3-C6HsCH=CH 1000 0 >4000 c.arefully rinsed with 3 ml of ice-cold H,O, then stirred for 20 niiii with 0.2 J I BC1 :it 0' for 13 miii. The mist n-as centrifuged at 20,000 rpni for 20 mill. The .iipernataiit (18 ml) tored :XI 3". Choliiie awtyltraiw ferase activity gradwilly decreased, but was >ufhcieiitly active up t o storage for 1 month. The AChE activity w:ii stable over aevera1 months. Choline acetyltransferase activity was measured by niodificatioii of the method of LLIcCanian mid Huiit;'j the a.*.say was r n n in the pre.seiice of 10:; DMSO, and inhibitors were added in this solvent. The assay rnixt contained 1 mJ1 choline, 0.1 mJ/ eseriiie, 0.1 .\I KC1: 0.025 J1 Tris bufier, and 0.1 mJ1 acelyl CoA (SniCi,'mmole). The final Reinecke salt in 1lepCO was spotted o n a glass filter paper, dried, and counted in PhAIe containing dimethyl POPOP. nieaiiired bj- modification oi the niethod of a i riin i i i the presence of 2.5C; 1leOEtOE-T and inhibitor-: \yere dissolved in 2!jCT: 31eOEtOII since lor; D>ISO completely inhibited the reaction. The assay contained 1 1nJ1ACh.Cl- (0.9 niCi;mmolej, 0.03 .I1 Tris buffer, and 0.02 .lif MgCl?.
Inhibition of Phenethanolamine N-Methyl Transferase by Ring- Substituted a-Methylphenethylamines (Amphetamines) R l i ~W,FULLER,* JACK~ I I L L ASD S , 1 1 . 4 ~11. ~ I A K S H The Lzlly Research Laboratories, Eli Lally and Company, IndianapolLs, Indaana
Recezved September E?, 1910 Phenethanolamine S-met,hyl transferase ( P N M T ) transfers a Me group from S-adenosylmethionine to phenethylamines with an OH group p to the ?;. Phenethylamines (including a-methylphenethylamines)without such a p substitution combine Lyith and inhibit the enzyme. Amphetamines with various aromatic substituent3 were studied as inhibitors: there was greater than a 1000-fold range in their inhihitor potency. The inhibitor activity of these compourids showed a correlation with the Hammett u arid T (a lipophilic parameter derived from the partition coefficient) associated with the aromatic substituent. The d isomers were more active a.: inhibitors than 2 isomers of amphetamines. d-3,4-Dichloroamphetamiiie was the most active inhibitor in the series and is the most potent inhibitor of P N M T reported to date; its inhibition was reversible and competitive. Inhibitors of P N l I T that are effective in viuo should be of pharmacological importance.
Phenethanolamirie N-methyl transferase (PXlIT) transfers a l l e group from S-adenosylmethionirie to a -\Ie acceptor, nhich apparent15 has to be either a phrnc~thanolamine or a pheriylethylenediamine. '-l The physiological role of P S l I T 1- to convert norepineplirinc into epinephrine, primarily in the adrenal met l ~ i l l a . ~Although the physiological effects of norepinephrine and epinephrine are qualitatively similar in gcneral, there :ire numerous diff ereiices in the resp of v:wioui target organs t o these tv o cateclmlaniiii Thus : L I ~inhibitor of PSlIT uhich altered the ratio of epinephrine : norepinephrine in the adrenal gland ought t o be an interesting pharmacological tool and at leayt potentially useful as a drug. Honever, few studics on PSIIT inhibitors have been published. P N l I T
i, inhibited bs sulfhydryl binding agents.' * J'uller and Hunt reported that Qome phenethylaminez itructurally related to wbstrates hut lacking the 8-OH esbeiitial for \ubstrate activity were inhibitory to P S l I T . Krakoff and Axelrod'O reported the inhibition of PSA\\IT h the monoamine oxidase vera1 amines, among TI one of the nioit poterii itor tranylcypromine \ inhibitors Both the repoi t s by Fuller and Hunt and by I
