J . Am. Chem. SOC.1988, 110, 2975-2976
Highly Effective Mechanism-Based Inactivation of Dopamine &Hydroxylase by a Novel Ketene Thioacetal
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Thomas M. Bargar,*' Robert J. Broersma, Lawrence C. Creemer, and James R. McCarthy2
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Merrell Dow Research Institute Indianapolis Center, P.O. Box 68470 Indianapolis, Indiana 46268-0470
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Jean-Marie Hornsperger,* Paul V. Attwood, and Michel J. Jung3
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Merrell Dow Research Institute, Strasbourg Center, 67084 Strasbourg Cedex, France Received September 21, 1987 Dopamine @-hydroxylase (DBH, E.C. 1.14.17.1), a copperdependent monooxygenase, catalyzes the conversion of dopamine to the sympathetic neurotransmitter n~repinephrine.~Potent, selective inhibitors may find uses in the treatment of diseases resulting from an imbalance in dopamine and norepinephrine level^.^-^ Recently, attention has focused on an understanding of the mechanism of enzyme action and subsequent rational design of inhibitors.' We wish to describe the design and synthesis of 2-( 1,3-dithiolan-2-y1idene)ethanamine (la), a highly potent representative of a new class of sulfur-containing, mechanism-based inhibitors of DBH, which differs from all previous substrates in not having an aromatic ring. Recent studies of oxygenation catalyzed by DBH suggest that the initial event is an electron transfer to an as yet unidentified copper-bound oxygen species, and in this sense DBH functions in a manner analogous to the iron dependent cytochrome P-450 and lipoxygenase enzymes.8 Lipoxygenase can be inhibited with a substrate analogue capable of forming a stable radical cation upon removal of one electron by enzyme bound Fe(II1) species.* In the case of DBH, inhibition can be achieved by providing stabilized radical cations derived from 2-arylallylamine~.~We reasoned that since 2-(2-thienyl)allylamine was so well recOgnized,6 another planar, five-membered sulfur-containing ring capable of stabilizing a radical cation may lead to even better activity. Accordingly, we targeted compound la (Table I) because oneelectron oxidation should lead to a highly stabilized radical cation.l0 The dithiole ring system is well-known for its ability for forming donor-acceptor complexes.' Preparation of compound la was straightforward, and experimental details for this as well as several analogues (Table I) designed to probe structure-activity relationships are found in the (1) Current address: Dow Chemical Company, Agricultural Products Research and Deveopment, 2800 Mitchell Drive, Walnut Creek, CA 94598. (2) Current address: Menell Dow Research Institute, 21 10 E. Galbraith Road, Cincinnati, O H 45215. (3) Current address: Dow Chemical Company, Agricultural Products Research and Development, Letcombe Laboratory, Letcombe Regis, Wantage OXON OX12 9JT, England. (4) (a) Villafranca, J. J. Copper Proteins; Spiro, T. G., Ed.;Wiley: New York, 1981; pp 264-289. (b) Rosenberg, R. C.; Lovenberg, W. Essays Neurochem. Neuropharm. 1980, 4, 163. ( 5 ) Kruse, L. I.; Kaiser, C.; DeWolf, W. E., Jr.; Frazee, J. G.; Erickson, R. W.; Ezekiel, M.; Ohlstein, E. H.; Ruffolo, R. R., Jr.; Berkowitz, B. A. J. Med. Chem. 1986, 29, 887. (6) Bargar, T. M.; Broersma, R. J.; Creemer, L. C.; McCarthy, J. R.; Hornsperger, J.-M.; Palfreyman, M. G.;Wagner, J.; Jung, M. J. J . Med. Chem. 1986, 29, 315. (7) Fitzpatrick, P. F.; Villafranca,J. J. Arch. Biochem. Biophys. 1987,257, 231. (8) Corey, E. J.; Cashman, J. R.; Eckrich, T. M.; Corey, D. R. J. Am. Chem. SOC.1985, 107, 713. ( 9 ) Padgette, S.R.; Wimalasena, K.; Herman, H. H.; Sirimanne, S.R.; May, S. W. Biochemistry 1985, 24, 5826. (10) We also prepared the ring unsaturated derivative 2-(1,3-dithio-2-ylidene)ethanaminetasylate (Ib) (Supplementary Material); however, a control experiment revealed that Ib was not stable under inactivationassay conditions. Compound l a was found to be stable. (1 1) For a leading reference, see: Krief, A. Tetrahedron 1986, 42, 1209.
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IO 15 20 TIME ( m i n ) Figure 1. T i e and concentration dependent inhibition of DBH by la. Purified DBH was incubated with various concentrations of l a under conditions described in the text. At indicated times, aliquots were withdrawn and tested for remaining enzyme activity with use of tyramine as a substrate. The inset shows a Kitz and Wilson plot versus [I]-' from which the kinetic parameters KI and k,, for l a can be calculated.15
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Table I. Kinetic Constants for DBH Inhibitors
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no time-dependent inhibition up to 5 mM
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no time-dependent inhibition up to 5 mM 5b
'Competitive inhibitor; Ki calculated versus tyramine as substrate (see text).
Supplementary Material.12 DBH was isolated and purified from bovine adrenal.13 The enzyme activity was assayed, and the time-dependent inhibition experiments were performed as described previously! The competitive inhibition experiments were performed under standard assay conditions6 except that the (12) All new compounds are fully supported by 'H NMR, mass spectrometry, and elemental analysis, or high-resolution mass spectrometry. (1 3) Aunis, D.; Miras-Portugal, M.T.; Mandel, P. J. Neurochemistry 1975, 24, 425.
0 1988 American Chemical Society
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J. Am. Chem. SOC.1988, 110, 2976-2978
concentration of the substrate tyramine was varied in the range 0.25-5 m M a t each concentration of inhibitor. The rate of turnover of saturated analogue 2 was again performed under standard assay conditions except that 640 pg of DBH and 10.5 p M C u S 0 4 were required to obtain a measurable rate of O2 consumption with use of a Clark oxygen electrode a t 40 m M of 2. Figure 1 shows the psuedo-first-order decay on incubation of DBH with l a at concentrations in the range 0.1-10 p M . Inset is the Kitz and Wilson plot14 from which the kinetic parameters, khaa and KI, can be e ~ t i m a t e d . ' ~In addition, compounds 3 and Sa also produced the expected pseudo-first-order loss of enzymatic activity (seeTable I) and showed linear inactivation plots. In order to produce time-dependent inhibition, 0 2 , Cu*+, and ascorbate were required and tyramine protected from inhibition by la. Extensive dialysis did not result in reversal of the inhibition. These facts suggest that the inhibition is an enzyme-activated phenomenon. Fully saturated derivative 2 was not a time-dependent inhibitor but did exhibit good competitive inhibition (Ki = 11 p M versus tyramine), indicating that the ylidene moiety is essential for time-dependent inactivation but not for active site recognition. Compound 2 was also a poor substrate with a rate of turnover at 40 mM of about 6% of tyramine. Dithiane 3 inactivated DBH a t a much lower rate than dithiolane la. This indicates the importance of mimicking the planarity of an aromatic ring and is reinforced by the fact that the open chain analogue 4 was not an inhibitor. Of the two monosulfur analogues of la, only isomer Sa, with aminomethyl trans to sulfur, is a timedependent inhibitor. The other isomer, 5b, is neither time-dependent nor competitive. This result suggests that the spatial arrangement of the N H 2 relative to S is very important for recognition and indicates that the heterocyclic ring is not simply mimicking the planar aromatic ring of tyramine but implies some positive interaction between one of the S atoms and the active site. One possible mechanism of inhibition by la, analogous to those proposed for other unsaturated inhibitors? proceeds by abstraction of an electron from l a by HOO-Cu(I1) to form a resonance stabilized radical cation whose unpaired electron should combine with the unpaired electron of O=Cu(III) (eq 1). The resulting sulfur-stabilized cation can undergo attack by enzyme nucleophile. Consistent with this mechanism is the observation that prolonged dialysis of inactivated enzyme did not lead to recovery of activity.
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