J. Med. Chem. 1985,28,245-248 humidity, and 5% COP Exponentially growing cells (4 X lo5 cells/mL) were incubated with test compounds at concentrations of 1, 10, or 100 pm. Cell counts were taken at 24,48,and 72 h. Cell viability was measured by the trypan blue exclusion technique.ls Stability of all suspensions was monitored throughout the test period by thin-layer chromatography.
(16) Warren, J. R.;Leatherman, D. L.; Metzger, J. F. J.Immunol. 1976, 115, 49.
245
Acknowledgment. This research was supported partly by Grant 903A awarded by the Northeast Louisiana University Research Committee and in part by the Northeast Louisiana School of Pharmacy. Annette Shipp is a Silas M. Burroughs Fellow of the American Foundation for Pharmaceutical Education. Registry No. 2, 51-21-8;3, 93473-97-3;4, 70758-92-8;5, 93473-98-4;6, 93473-99-5;7, 17242-85-2;9, 93474-00-1;11, 93474-01-2; hexamethyldisilazane, 999-97-3.
N - [2-Hydroxy-B-[2- (methy1amino)ethyllphenyllmethanesulfonamide. A Potent Agonist Which Releases Intracellular Calcium by Activation of al-Adrenoceptors Robert M. DeMarinis,* Patricia Lavanchy, J. Paul Hieble, Kam F. Jim, and William D. Matthews Department of Medicinal Chemistry and Pharmacology, Research and Development Division, Smith Kline and French Laboratories, Philadelphia, Pennsylvania 19101. Received May 9, 1984
N-[2-Hydroxy-5-[2-(methylamino)ethyl]phenyl]methanesulfonamide (SK&F 102652) has been prepared and of 25 nM a t al-adrenoceptors as determned characterized pharmacologically. It is a potent agonist with an in the isolated perfused rabbit ear artery. On the presynaptic a2-adrenoceptors of the guinea pig atrium it was considerably weaker, demonstrating an EC50for inhibition of neurotransmission of 1200 nM and thus an overall a1/a2selectivity ratio of 248. In the vascular smooth muscle of the canine saphenous vein an EClm concentration of this agonist in the presence of zero external Ca2+induced 37.9 i 1.4% of the maximal contractile response due to this agent while the endogenous ligand norepinephrine evoked only 14.5 i 0.4% of the maximum. In the presence of low (1 pM)external calcium, this agent produced 78.3 i 5.3% of maximum while norepinephrine gave 45.3 7.4%. This agent produces al-adrenoceptor-mediated contraction primarily by release of intracellular Ca2+and should provide a useful tool for characterizing al-receptor subtypes.
*
In the last several years, it has been amply demonstrated that two distinct types of postsynaptic a-adrenoceptors (al, a2)are present on vascular smooth muscle and that activation of either subtype causes vasoc~nstriction.~-~ Evidence has been produced from both in vitro and in vivo experiments to indicate that there is a pharmacological difference in the Ca2+utilization process of smooth muscle after stimulation of al-or a2-adrenoceptors.4* Studies from a number of laboratories have shown that activation of postsynaptic a2-adrenoceptors causes the influx of extracellular Ca2+across the cell membrane and that it is this translocation of Ca2+that mediates the contractile proOn the other hand, activation of postsynaptic al-adrenoceptors can induce both extracellular Ca2+influx and intracellular Ca2+release to initiate vascular smooth muscle contractions.*ll Activation of the postsynaptic al-adrenoceptor may use either extracellular Ca2+, intracellular Ca2+,or both to ~
~~
Timmermans, P. B. M. W. M.; van Zwieten, P. A. J. Med. Chem. 1982,25, 1389. McGrath, J. C. Biochem. Pharmacol. 1982,31,467. Fowler, P.J.; Grous, M.; Price, W.; Matthews, W. D. J.Pharm. Exp. Ther., in press. (4) Van Meel, J. C. H.; deJonge, A.; Kalkman, H. 0.;Wilffert, B.; Timmermans, P. B. M. W. M.; van Zwieten, P. A. Eur J. Pharmacol. 1981,69, 205. (5) Van Meel, J. C. A.; Timmermans, P. B. M. W. M.; van Zwieten, P. A. J . Cardiouasc. Pharmacol. 1982, 5, 580. (6) Matthews, W. D.; Jim, K. F.; Hieble, J. P.; DeMarinis, R. M. Fed. Proc., Fed. Am. Soc. Expl. Biol. 1984, 43, 2923. (7) Langer, S. Z.; Shepperson, N. B. Br. J. Pharmacol. 1980, 74, 942. (8) Llenas, J.; Massingham, R. J . Pharmacol. 1983,87, 53. (9) Cauvin, C.; Loutzenhiser, R.; Hwang, 0.;van Breeman, C. Eur. J . Pharrnacol. 1982, 84, 233. (10) Langer, S. Z.; Shepperson, N. B. Trends Pharmacol. Sei. 1982, 3, 440. (11) Jim, K.F.;Matthews, W. D. Pharmacologist 1983,25(3), 136.
p N H C H 3 2 . BBr3
NHS02CH3 kH3
N HSOzCH3 OH
3
4
initiate contraction and the pathway taken can be dependent on the chemical structure of the al-agonist employed to produce the contraction. We have shown previously that there are differences in the responses to cy1agonist-mediated vasoconstriction produced by two different chemical classes of a,-agonists.'l These results suggest that agonists such as methoxamine and SK&F 1-89748 (1-1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2naphtha1enamine)l2 primarily use extracellular Ca2+ to produce contractions of smooth muscle. a,-Agonists of a different general structural type, such as phenylephrine or norepinephrine, which have protonic containing substituents on the meta position of the aromatic ring, are able to induce contraction by both translation of extracelular ea2+and by internal release of Ca2+stores.13 The compound described in this report, N-[2-hydroxy-5-[2-(methylamino)ethyl]phenyl]methanesulfonamide (SK&F 102652) is a selective a,-adrenoceptor agonist which has
0022-2623/85/1828-0245$01.50/00 1985 American Chemical Society
246 Journal of Medicinal Chemistry, 1985, Vol. 28, No. 2
Notes Table 1. Effects of Zero External Ca*+ on the Contractions Induced by al-Agonists in the Canine Saphenous Vein" agonist contractions* a-agonists ECm ECim norepinephrine 6.7 f 1.8 9.3 f 0.9 14.5 f 0.4 phenylephrine 4.4 f 1.3 7.3 f 0.7 14.3 f 0.7 4 6.0 f 0.8c 20.2 f 1.8d 37.9 f 1.4' 'Experiments done in the presence of 2 mM EGTA and are the mean of six experiments f SEM. bValues are expressed as percent of control response in 2.5 mM Ca2+physiological saline. cAbsolute concentration 1.5 f 0.7 X lo-' M. dAbsolute concentration 5.0 f 1.5 X lo-' M. 'Absolute concentration 5.0 X M.
I
-9
-8
I
I
I
-7
-6
-5
Log Concentration [MI
Figure 1. Effects of 4 and norepinephrine on the constrictor response of the isolated perfused rabbit ear artery segment. Each point represents the mean of four experiments A SEM.
4 12
4
4
40 120 Concentration (nM)
Figure 2. Effect of increasing concentrations of 4 on the constrictor response of the isolated perfused rabbit ear artery to field
stimulation. the ability to initiate vascular smooth muscle contraction almost entirely by release of Ca2+from internal stores. This agent is the best of a series of compounds which have been evaluated in our laboratory for their ability to release intracellular calcium. It is superior in this respect to either the nonselective endogenous ligand norepinephrine or the classically used al-agonist phenylephrine and it should be of utility as a tool to help elucidate the role of Ca2+in a-receptor-mediated constriction of vascular smooth muscle. Chemistry. The compound of interest was synthesized in seven steps in an overall yield of 12% from previously reported p-hydroxy-m-nitrophenylaceticacid1*as shown in Scheme I. It was characterized as a crystalline hydrobromide salt.15
Results and Discussion The a,-adrenergic potency of 4 as well as that of the endogenous transmitter norepinephrine in the isolated rabbit ear artery segment is shown in Figure 1. In these experiments 4 has an EC50of 25 nM, making it about 8 times more potent than norepinephrine. In this tissue (12) DeMarinis, R. M.; Hieble, J. P. J. Med. Chem. 1983,26,1215.
(13) Jim, K. F.; DeMarinis, R. M.; Matthews, W. D. Eur. J. Pharmacol., in press. (14) Chem. Abstr. 1964, 61, 13359b. (15) This compound has been disclosed as the corresponding hydrochloride in U.S.Patent 3 574741 issued to W. A. Gould and A. A. Larsen. No experimental details are reported. The compound is claimed to be a pressor amine which is in agreement with the a*-agonist activity described by us.
Table 11. Effects of 1 pM External Ca2+on the Contraction Induced by a,-Agonistsin the Canine Saphenous Vein' agonist contractionsb a-agonists ECw. ECm ECim 6.3 f 1.6 6.0 f 2.1
9 f 1.0 45.3 f 7.4 9.6 f 2.4 35.7 f 3.4 4 8.1 f 1.2' 37.6 f 4.8d 78.3 f 5.3' 'Values are the mean of six experiments f SEM. *Values are expressed as percent of control response in 2.5 mM Ca2+. 'Absolute concentration = 1.5 f 0.3 X lo-". dAbsolute concentration = 5.0 f 1.5 X lo-' M. eAbsolute concentration = 5.0 X M. norepinephrine phenylephrine
compound 4 appears to be a full agonist which produces a response equal to the maximum response elicited by norepinephrine. The constrictor response to 4 is blocked competitively by phentolaminewith a dissociation constant of
