November, 1963
TROPOLONES AT XDRESERGIC RECEPTORS
(a) From Amino Ketone VII. 1-(4-Methylphenyl)-Z-isopropylaminoethanol Hydrochloride [R, = 4-CH3, Rz = H, R, = H, R4 = CH( CH,),] .-4-lIethyl-~u-isopropylaminoacetophenone (16 g., 0.084 mole) dissolved in 50 ml. of methanol v as reduced a t 0” v i t h 3.2 g. (0.085 mole) of sodium borohydride. After 1 hr. a t 20”, 200 nil. of water mas added and the base was evtracted with ether. Drying over potassium carbonate and evaporation gave an oil which was converted to the h j drochloride. The hydrochloride was recrystallized from methanol-ethyl acetate, m.p. 132-134”. The chlorine analyses of the compounds are listed in Table 11. (b) From Amino Ketone VI1 by Catalytic Hydrogenation. 1-( 3,4-Dimethylphenyl)-2-isopropylaminopropanol Hydrochloride LIX, R1 = 4-CH3, R2 = 3-CH3, RB = CH,, R, = CH(CH3”lJ.3,4-Dimethyl-a-isopropylaminopropiophenone hydrochloride (7.65 g., 0.035 mole) was reduced catalytically nith 1 g of 10% palladium on charcoal in 100 ml. of ethanol. After absorption of 0.035 mole of hydrogen the solution was filtered and evaporated ?n vacuo. The remainder was recrystallized from niethanolethyl acetate, m.p. 214’. Anal. Calcd. for C14H?4ClXO: C, 6j.22; H, !).38; C1, 13.T5. Found: C,65.11; H,9.27; C1, 13.65. This product was identical with a sample prepared by reduction Fvith sodium borohydride.
755
(c) From Bromohydrin VIII. 1-(3,4-DimethylphenyI)-Z-isopropylaminoethanol Hydrochloride [IX, R, = 4-CH3, R2 = 3-CH3, R, = H, Rq = CH(CH),].-1-(3,4-Dimethylphenyl)-2bromoethancl (17.2 g., 0.075 mole) dissolved in 150 nil. of absolute ethanol and 13.2 g . (0.22 mole) of isopropylamine were refluxed for 12 hr. After evaporation, the remainder was dissolved in water and made allidine i\-ith sodium hydroside. Estraction with ether gave an oil Lvhirh was converted to the hydrorhloride. The latter K R S recrystnllizpd from methanol-ethyl acetate, m.p. 1E8-159”. Anal. Calcd. for ClrH&lXO: C, 61.05; H, ‘3.10; C1, 11.54. Found: C, 61.06; H, 9.20; C1, 11.61. Methanol-ethyl acetate \vas used as solvent for recrystallization of all hydrochlorides listed in Table 11.
Acknowledgments.-The authors are indebted to M r . L. Boberg, Miss SI.Ettles, 12rs. C. Burliii, Mr. G. Hallhagen, Ah-. R. Svehii, and Mr. 1;. Tarkkaiieii for skillful1 help in various phases of this work and to the co workers of Dr. Roberts: Dr. R. Ito, Mr. T’. Cairoli, and LIr. R. JJ’. Pritchard, who performed many of the antiarrhythmia experiments.
Occupancy of Adrenergic Receptors and Inhibition of Catechol 0-Methyl Transferase by Tropolones’,2 B. BELLEAU AND J. BURBA Ueparlvient of Chemist, y, Faculty of Pure and Applied Science, Cnicerszty of Ottaua, Ottawa, Ontario Received A p r i l 29, 1963 I n the light of the mechanism of action of catechol-0-methyl transferase (COMT), it was conceived that tropolones should act as specific inhibitors of the enzyme. Confirmation of this postulate was obtained. A variety of tropolones were discovered to be effective inhibitors of COAMT. The earlier claim that these inhibitors act noncompetitively has been withdrawn. Structure-activity relationships were also established. A new assay procedure for CORIT was designed. A structure for the Michaelis complex between COMT and tropolones is proposed. It is shown that the suggested structure for the complex accounts for the behavior of tropolones toward CORIT. The conclusion wa9 reached that catechol and tropolone rings are biochemically isosteric. On the basis of preliminary pharmacological data it %as shown that the catechol-tropolone isosterism also applies t o adrenergic receptors. Depending on the dose of tropolone, in vzoo and zn vitro COMT inhibition as well as preceptor “blockade” could be observed separately. I t would appear that this is the first time that a group of substances that are not amines are slioan to display aKinity for adrenergic receptors.
The mechanisms whereby the catecholamine hormones are inactivated in vivo has been elucidated by A4xelrod, et aL3 It is now recognized that catechol0-methyl transferase (COMT) is the enzyme primarily concerned in the inactivation of circulatiiig catecholamines. The enzyme has been isolated and partially p ~ r i f i e d ,and ~ the nature of its cofactor requirements elucidated. The substrate specificity and cation requirement for enzymic 0-methylation led Senoh, et u Z . , ~ to propose the mechaiiism depicted in I as accounting for the methyl group transfer reaction from adenosylmethioiiine to the meta-phenolic group of substrates. From t’he pharmacological standpoint, the role of COMT acquires special significance because of its (1) T a k e n in p a r t from t h e thesis submitted b y J. Durba in partial fulfillm e n t of t h e requirements for t h e Ph.D. degree, University of O t t a w a , 1962. ( 2 ) For a preliminary account of t h i s work see 13. Relleau a n d J. Burba, Biocliim. Biophys. Acfa, 64, 195 (1961); see text for a correction. (3) T h i s subject has been r e r i e n e d by J. .Ixelrod, in “Adrencrgir Rlectranisins,” A Ciba Foundation Symposiuni, J. R. Vane. Q. E. W. \TolstenIiolme, a n d 11. O’Connor, Editors, J. a n d A. Churchill Ltd.. London, 1960, I). 28. (4) J. Axelrod a n d R. Tomchick, J . Biol. Chem., 233, 702 (l958), ( 5 ) S. Senoh, J. Daly, J. Axelrod, a n d B. Witkop, J . Am. Chem. Soc., 81, 8280 (195Y).
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I involvement in the termination of the physiological action of the circulating catecholamine hormone^.^,' It is in this coiiiiection that accessibility to suitable inhibitors of the enzyme is critical since it is through (6) J ixelrod a n d i\I -J Laroclie Sczence, 130, 800 (1959) ( 7 ) E V & \ a r t s , L Gillespte T C Fleining, a n d A. Sioerdsma, l ’ t o ~ . Soc E r p t l Baol .Wed 98, 74 (1938)
Ii
tioii with the C'OAlT active sites. The prediction is permissible on the hasis of the r:ttional represerit,atioiis I aiid 11, that tropoloiies should act as inhihitors because of their ability to form stable chelates with divalent met,al ioiis,lla property which is shared by catechol riiigs.'2 fIowever, the questiai of their specificity ~f actioii could not be predicted safely, since the possibility existed that they could simply act as metal ion scavciigera as is the case, for instatice, with ethyleiicdianiiiictetrascetic acid (FITl)A).13 If tropoloiies were ts act through an EI>TI-likc iiieclianism, no applications to ,in 2;,ii!ost'udies o n the specific role of CORIT cmld obviously be envisaged. The virtually perfect, isosteristii between catechol aiid t,rop.)lone rings suggests, or1 t,hc other liaiid, that the analogous Ahhaelis complex I1 ought to form initially aiid, provided the coordinatiiig power of tropoloiies toward iriagiiesium does not' exceed that of the enzyme coordinating sites, the r(1sulting inhibition would he rat>lierspecific for COlIl'. If, indeed, the tropolone aiid catechol rings are hiochemically isost'eric as studies with COSIT could conceiv:ihly establish, then tlic possibility would sriw
1 !!lti%l
757
TROPOLONES AT ADRENERGIC RECEPTORS
Sovember, 1963
tested for their inhibitory activity toward the COLITcatalyzed methylation of norepinephrine. The concentration of substrate was adjusted so that maximum reproducibility could be achieved with our method of assay. This necessitated substrate concentrations approaching maximum velocity of methylation. The inhibition constants K , were calculated in the usual fashion.23 The Michaelis constant for norepinephrine was estimated in the conventional manner to be 7 X M, a value in good agreement with the published one.24a The values of K , are assembled in Table I where it can be seen that the presence of ring substituents or their nature has only a marginal effect on the affinity of the tropolone ring for the enzyme. It is interesting to note that 4-methyltropolone (111) is the most active inhibitor of the series, its affinity for COMT being somewhat greater than that of pyrogallol. Both the amino-imino and the amino-thioxo analags XI11 and XV display significant inhibitory activity in agreement with the postulate that a chelation mechanism involving enzyme-bound magnesium is operative. The ability of these nitrogen and sulfur analogs of tropolone to form stable chelates with metal ions has been established.*4b The chelation mechanisni af inhibition is further substantiated by the observation that colchicine which has no free hydroxyl groups is totally inactive as an inhibitor. Furthermore, mesoinositol, which is known to chelate metal ionsz6 but less strongly, also displays some inhibitory activity. Finally, the relative indifference of the enzyme toward substituents, including a norepinephrine side chain as in I X on the tropolone ring, offers strong support in favor of a chelation mechanism of inhibition.
0.10
0.09
0.08
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. 0.06
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0.02
0.01
I
I
I
10
20
30
I
40
1
50 Time (Minutes).
I
I
60
70
Fig. 1.-Typical uninhibited and inhibited ( b y 4-methyltropolone) rate curves for the CORIT-catalyzed methylation of norepinephrine: a, 1-noradrenaline-d-bitartrate 2 X 10-3 S I ; X , 1-Noradrenaline-&bitartrate 2 X 10-3 Jf plus 3 X 10-4 J 1 4 methyltropolone.
data at hand. Even though it appeared possible initially to coiistruct plots which are typical of nonI N H I B I T I O N CoKSTANTS (in mOleS/l.) FOR V A R I O U S SUBSTITUTED competitive inhibition,2 it was observed subsequently T R O P O L O N E S TOTYARD CATECHOL-O-?dETHYL TRAXSFERASE. that the substrate concentrations which had to be used The K , for norepinephrine as substrate was 7 X 10-4 M . were too close to maximum velocity conditions, so that Compound K x 10-5 large experimental errors were inadvertently introduced. 4hfethyltropolone (111) 1.2 On the basis of preliminary pharmacological data (see Stipitat,ic arid (IT) 1.6 following), in vivo COhlT blockade by tropolones has Colchiceine 1.8 the characteristics of competitive rather than non8-Thujaplirin (V) 2.3 competitive inhibition. We have since been informed 7-Thujaplicin ( V I ) 2.5 by Dr. D’IorioZ7that new in vitro data have clearly 4-( 2-Amino-l-h~-droxy)ethyltropolone (IX) 4 5 4-Aicetamidomethyltropolone ( X I ) 3.1 confirmed a competitive type of inhibition in agreement i-HydroxS-4-isopropyltropolone ( X I ) 3.4 with the pharmacological data. l-Methylamino-i-methylimino-l,3,5-cycloheptaThe possibility that tropolones could act by partially triene ( S I I I ) 4.2 depriving COMT of its magnesium ions was examined 1-?*I ethylamino-i-thioxo-l,3,5-cycloheptatriene next. To this end, the effect of magnesium concentra(XIY) 5.6 tion on COMT activity was first established and, in Colchicine m agreement with Axelrod’s observations,4 excess magPyrogallol 1.7 nesium can be seen (Fig. 2 ) to produce a slight inhibimeso-Inosit 01 20.0 tory effect on the rate of methylation. In the presence of 4-niethyltropolone (111) a fivefold relative inIt was of interest to attempt the determination of the crease in magnesium concentration had no effect on the inhibition mechanism and we init’ially reported? that inhibited rate (Fig. 2 ) . It seems clear therefore that tropoloiies act noncompetitively. We now wish to magnesium does not reverse the inhibition, thus sugwithdraw this claim in view of the impossibility to gesting that tropolones form a complex with the enzyme construct reliable Lineweaver-Burk plotsz6 with the active sites normally occupied by the substrate. Xn appreciable degree of specificity may therefoie he (23) li. J. Laidler. “The Cheinical Kinetics of E n z y m e Action,” Oxtord a t the Clarendon Press, 1998, p. 81. expected from this novel class of inhibitors. That the (24) (a) J. R. Crout, Biochem. Pharmacal., 6,47(1961); (b) W.R. 13rasen. active sites responsible for the binding of catechols H. E. Holmquist. and R. E. Renson, J . A m . Chem. Sac., 83, 3125 (1961). TABLEI
(25) A . Lindenbauiu, RI. R. White, and J. Schuhert. Arch. Biochem. ~ i ~ p h ~sa,s .110 , (19.54). (26) H. Lineaeaver and D. Burk, J . A m . Chem. Sue., 6 6 , 658 (1934).
(27) Priiate communication, A . lI’Iorio, C. JIa\rides. and E; JIissAa Can. J . Blochem Ph?/saol., 41, 1 3 1 (1963). We are grateful to 1)r. d’Iorio l o r having inforined us of his result prior to publication.
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0.09
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may be tlic same hen tiopoloiieq are iiivolvcd is e\ ideiiced by the observation that in both tlie substrate uiid inhibitor beries, tlie presence of riiig bubstitucuts produces parallel iiiargiiial variatioiis 011 thc afiiiity for COhIT. It is not improbable therefore that a 1: 1: 1complex is formed betweeu COSIT, magnesium, a i d inhibitor (11) as has beeii postulated for ,2Iichaelis miiplex formation \\ ith substrates I. The ability of tropoloiies to eiigage readily into charge-transfer type3 of complexes2bsuggests, oil the other hand, that additional points of iiiteractioii \\ ith COSIT may be created aiid this may partly account for the or-er-all aifiuity oJ tropoloiies for the enzyme. These in ztztru studies with CO,\IT arid tropoloiies lead to the important coiiclusioii that ctrtrchol and tropolonc rings are biochenzicallg iaoatrrzc: a iiew and potentially useful tool in the study of adrenergic receptor rnechaiiisnis may, therefore, he provided. Some Pharmacological Characteristics of Tropolones.2q--'l'he in vivo t~lockadeof C O N T \vas iiiitially tlcmoiistrated with compouiids ZIT, I-,IX, aiid mlc l i i w i i i ( ~ ?O DCl, acetonitrile as reference) it gave lines characteristic of aromatic protons a t low field and a poorly resolved multiplet characteristic of the side chain protons a t medium field. The areas under these regions of absorption were in the correct ratio of 4 : 3 : in addition, lines characteristic of the presence of small amounts ( - 556) of ethanol (of crystallization?) were observed. Anal. Calcd. for CsHIlTO3: C, 59.66; H, 6.07; S , 7.73. Found: C, 59.06; H , 6.27, N, 7.45. 4-AcetamidomethyltropoIone (XI).-The sequence described by Xozoe, et al., l 8 was applied using 3-carboxy-4-tropoloneacetic acid (X) as starting material. The product ( X I ) thus obtained had m.p. 146-147' (lit.I8 146'); vE:xl 3300, 1630, and 1540 cm. -l Enzymatic Methods.-Catechol-0-methyl transferase (CJRIT) was prepared and purified as describsd by Axelrod and Torn~liick.~ For our specific purposes it was found unnecessary to carry the purification beyond the 6 hr. dialysis against phozphate buffer (PH 7 ) . Incubation Method.-The following technique was used throughout. A mixture of 5 nil. of the COMT preparation, 2 ml. of 0.5 Jf phosphate buffer, pH 7.9, 5 mg. of S-adenosylmethiomine iodide, 400 mmoles of RlgCl, 6H,O, and 3 mmoles of the tropolone to be assayed was brought to a total volume of 11 ml. and the solution vias pre-incubated a t 37" in a Dubnoff metabolic shaker for 10 min. prior to the addition of 20 mmoles of norepinephrine D-bitartrate. Aliquots of 2 ml. were withdrawn a t intervals and mixed with 1 ml. of 0.5 -11borate buffer, p H 10. Controls were run and assayed simultaneously. The quenched aliquots were then processed for the assav of nornietanephrine. The latter was first extracted as follows. Each quenched aliquot mixture was shaken 30 min. with 25 nil. of ethxlene dichlorideisoamvl alcohol (98:2). After standing, 20 ml. of the clear organic phase was withdrawn and extracted for 33 min. with 3 nil. of 0.1 S hydrochloric acid. The acid extract was then assayed for normetanephrine by the nitrosonaphthol method of 17denfriend and Cooper.,, It was established that tropolones, norepinephrine, and vanillin do not give a color by this method. The same techniques were applied in the study of the effect of magnesium ion concentration on the inhibited rates.
+
