J. Med. Chem. 1993,36,157-161
167
o-Chlorobenzenesulfonamidic Derivatives of (Ary1oxy)propanolaminesas 8-BlockinglDiuretic Agents' Violetta Cecchetti; Arnaldo Fravolini,'J Fausto Schiaffella,? Oriana Tabarrini; Giancarlo Bruni? and Giorgio Segret Zstituto di Chimica Farmaceutica e Tecnica Farmaceutica, Uniuersith di Perugia, 06123 Perugia, Italy, and Zstituto di Farmcrcologia, Uniuersiti di Siena, 53100 Siena, Italy Received April 28,1992
A series of compounds lb-f, Sb-f, and 3b-f having an o-chlorobenzenesulfonamidicdiureticmoiety variously linked to the nitrogen sidechain of the 6-blocking (ary1oxy)propanolamie pharmacophore were prepared and tested for their 61-adrenoceptoraffmity. For all the active compounds, .&blocking and diuretic activities were investigated in rata; the structure-activity relationships are discussed. Some of the compounds displayed varying levels of both properties and among these, compounds IC and 2c have been chosen for further development. Introduction In the long-term treatment of essential hypertension, no single drug has yet proved entirely satisfactory. Combined treatment is necessary to evoke an optimal result and a 19-blockerldiureticcombinationis widely used as first line therapy for hypertension management. For the future, an antihypertensive drug having both 6-blocker and moderate diuretic properties2in the same molecule would be of great interest. The advantage of such a compound over a simple combination lies in the fact that the singleentity would be adsorbed,metabolized, and excreted at one rate in a given subject, thus increasing the likelihoodthat the two main biologicalactivitiesremain in balance during the course of drug action. To date, only a few attempts to synthesize hybrid molecules by combining the structures of a diuretic and 6-adrenoceptor antagonist, have been de~cribed.~ The majority of the reported attempts have failed, probably due to the intrinsic difficulty of accommodating the individual sets of structure-activity requirements for the two activities in a single molecule. Our previouslyreported4approach to this symbioticdrug design was achieved by replacing the conventional alkyl substituent at the side chain nitrogen atom of &blockers with a 2-(4-chloro-3-sulfamoylbenzamido)ethylgroup. This type of substitution retained the structural requirements for the interaction with the 8-adrenoceptor, due to the presence of a 2-amidoethyl group known to impart high &blocking potency6 and, at the same time, allowed the diuretic o-chlorobenzenesulfonamidicmoietp to be incorporatedinto the molecule. This replacement was made on oxypropanolamine and (iminoxy)propanolamine derivatives of l,4-benzothiazine, previously reported by us as 6-adrenoceptor antagonists,' on carteolol, selected for isosterism of the carbostyril nucleus with the 1,4-benzothiazine one, and on well-known propranolol. The pharmacological results obtained indicated that the incorporation of o-chlorobenzenesulfonamidicmoiety into the molecule by an amidoethylaminogroup, in some cases, allowed the desired diuretic activity and ale0 maintained the &blocking one. Indeed, some of the resulting compounds (such as la, 2a, 3a) (Figure1) were found to exhibit differently modulated 4-adrenoceptor blocking and diuretic activites.' t
t
Universita di Perugia. Universita di Siena. 0022-2623/9311836-0157$04.00/0
la-t, 2a-1,304
a
x-.
b
X=-@
.=bo c
x
.
o
x-.
1
x . - O
-
m
H
Figure 1. o-Chlorobenzenesulfonamidicderivativea of (aryloxy)-
propanolamines.
As an extensionof this work, we now report the synthesis and pharmacological evaluation of some new (1,Cbenzothiazinyl-, (carbostyril-, and (naphthy1oxy)propanolamine derivatives lb-f, 2b-f, and 3b-I, variously linked to the same diuretic o-chlorobenzenesulfonamidicmoiety. In order to evaluate the role of the bridge linking the two pharmacophore moieties, the former amidoethylamino bridge (NHCH2CH2NHCO)was modifiedby (1)inclusion of the ethylenediaminicgroup in a rigid piperazinic moiety (b,d, 0;(2) isostericsubstitution of the amidic group with a sulfonamidicone (c, d);and (3)eliminationof the amidic group (e, b (Figure 1). Chemistry The (ary1oxy)propanolaminederivatives lb-f, 2b-f, and 3b-f, reported in Figure 1 and Table I were prepared by the following standard chemical procedure: selected epoxide, either purchased or prepared by standard methods, was reacted with an equimolar amount of the appropriate amine derivative 3, I , 7,9, or 10 in refluxed EtOH (Scheme I). The key step in this synthesis was the preparation of amine intermediates 3,6,7,9,and 10 obtained aa in Scheme 11. (Ary1amido)piperazine3 and (arylsulfonamido)pip erazine 7 were syntherizedby acylationof a monoprotected piperazine derivative with 4-chloro-3-sulfamoylbenzoyl Q 1993 American Chemical Society
Cecchetti et
168 Journal of Medicinal Chemietry, 1993, Vol. 36, No.1
Scheme I
chloride and 4-chloro-3-sulfamoylbenzenesulfonylchloride, respectively,and successive hydrolyticdeprotection. In a similar fashion, using N-acetylethylenediamine and 4-chloro-3-sulfamoylbenzenesulfonylchloride, (arylsulfonamid0)ethylamine 6 was obtained. Starting from 4-chloro-3-sulfamoylaniline, N-arylethylenediamine9 was prepared by reaction with 1-chloro-2-nitroethanefollowed by reductive hydrogenation over Raney nickel, while arylpiperazine 10 was synthesized by reaction with bis(2-chloroethyl)amine,according to the general procedure of Brewster et ala8 Pharmacology All target compounds lb-f, 2b-f, and 3b-f were first screened at the receptor level to determine their ability to displace the binding of [SH]dihydroalprenolol (PHIDHA)from turkey erythrocyte membranes (&-adrenoceptors) (Table I). Subsequently, all active compounds lc-3c, le, 38, lf, and Sf, were evaluated for @-blockingpotency in rats by the inhibition of isoprenaline-inducedtachycardiaby both intravenous and oral dosing (Table I). Only the resulting active compoundswere finallyassayed for their diuretic activity in rats after oral administration (Table II).
01.
In all of the tests, in addition to appropriate control drugs,S[(tert-buty~ino)-2-hydroxypropoxyl-3,4-dihydro-3-0xo-W-1,4-bemthiazine(4a)7' and/or N-[(4-chloro-3-sulfamoylbenzamido)ethyllpropanolamine derivatives la-3a,l previously reported by us,were included for comparison. Results and Discussion The object of this study was to evaluate the effects,on both @-blockingand diureticactivities,caused by variations connectingthe (ary1oxy)propanol induced in the bridge (X) @-blockingpharmacophore and the diuretic o-chlorobenzenesulfonamidic moiety. Among the changes made, the inclusion of ethylenediaminic group in a rigid piperazine moiety proved to be very detrimental to binding at fl1-adrenoceptors(Table I), both in amidic (series b) and sulfonamidic (series d) derivatives (compare vs series a and c, respectively). On the other hand, this lose of affinity was not observed in series f where the piperazine group was directly linked to the o-chlorobenzenesulfonamidic moiety (compare vs series e). No correlation was found regarding the aryl ring (A);in series c, the carbostyril derivative2c displayed the highest affinity with a Ki value of 1.4 nM, similar to that of the control drugs, whereas in series e and f only the carbostyril derivatives 28 and 2f showed no affmity. Onlythosecompounds(lc-3~,le,38, lf,and3f)showing affinity in the binding study were tested in vivo by inhibiting isoprenaline-induced tachycardia in rats to
Scheme I1 4
cNwa
I
a
6
oy-maso* -"a:* 8
CI
9
Reagents: (i) 1-formylpiperaeine,benzene, 100 OC; (ii) MeOH, 36% HCI; (iii) AcNHCHzCHzNHz, acetone, EkN; (iv) EtOH, 6 N HCl, d u x ; (VI N-(etho.ycarbOnyl)piperhe,acetone,Et& (vi) 10% NazCOs, reflux; (vii) NO&HZCHZCl, THF; (viii) HdRaney Ni, EtOH, (ix) NH(CH&H&l)THCl, 1-butanol, reflux.
Journal of Medicinal Chemistry, 1993, Vol. 36,No.1 150
6-BlockinglDiuretic Agents Table I. Physical and Biological Properties of (Ary1oxy)propanolaminea
inhibition of inhibition of INA-induced tachycardia in r a d [3H]DHA binding in % mp, purification turkey erythrocytes EDm EDm reaction com& time(h) yield OCb methodc formulad (BIP Ki (nM) ( d k ivy . (dk. DO)^ la 7.4 0.67 0.031 36.24 f 3.466 20 300 0.14 0.039 17.92 f 1.387 3a 820 3.17 0.385 23.13 f 1.157 lb 30 53 145-165 A CzzHzaCNOsSz NA’ NTJ NT 2b 30 71 136-148 A CdbCNOsS NA NT NT 3b 30 65 88-120 A Cd&&lNsOaS NA NT NT IC 30 81 100-130 B CisHz&lWhS3 5.2 0.45 0.016 34.70 f 2.093 2c 30 37 103-115 B CzoHzaClNrO7Sz 1.4 1-80f 0.041 53.78 f 3.185 3c 30 35 87-118 B CziH~C1&0& 160 NA NA Id 15 50 218-223 C CziHzaCNO& NA NT NT 2d 20 39 225-230 B CzzHnCW40782 NA NT NT 3d 12 55 208-212 C Czs.HzsCNOsSz NA NT NT le 30 12 176-186 B CISHBCWMZ 7.8 0.96 0.046 66.37 f 3.498 20 23 22 211-214 B CdzaClN4OsS NA NT NT 30 30 36 70-91 B CziHzrClNs04S 15 NA 40.69 f 2.641 lf 14 63 218-222 D CziHzaCNOsSz 140 NA 66.30 f 22.050 2f 12 62 227-231 D CzzHnCNOaS NA NT NT 3f 8 73 166-170 D Cz3HzsCNW 31 2.91 f 0.083 62.52 3.886 4ah 24 0.05f 0.003 1.63 f 0.026 carte0101 1.9 0.06 f 0.008 4.81 f 0.255 propranolol 2.3 0.18 f 0.023 22.33 f 1.306 0 See Figure 1. Amorphous solids: melting point of these compounds is the temparature at which the white solid became a colorlew g h with decomposition. Solvent used with silica gel column or for recrystallization were as follows: (A) elution with gradient of CHCb to 10% EtOH/CHCb, (B) gradient of CHClsto 5% MeOH/CHCb, (C) isocratic elution with CHCb, (D) recrystallization from EtOH. d All compounde had elemental analyses within +0.4% of theoretical value. e Ki values determined with seven dose levels of each inhibitor in the assay system described in the Experimental Section. f Mean f SE for three separate observations per doaage. g Two minutes after INA (0.12 d k g ) administration. One hour after INA (0.12@kg) admiitration. I NA = no active compound. j NT = not tested. 4a = 8-[(tert-butylamin0)O-hydroxypropoxy]-3,4-dihydro-3-oxo-Wl,4-beneothiazine (see literature7.). ~
* * *
*
Table 11. Diuretic and Saluretic Activity in the Rat (0-5 h) saluretic activity6 dosage urinary (meguiv/kg) compd (mg/kg,po) outputO(mL) Na+ K+ Na+/K+ 8 1.2 f 0.31 0.9 0.8 1.1 la 1.7 f 0.63 1.2 1.1 as 15 30 1.7 1.1 2.0 f 0.46 1.5 2.1 50 4.3 f 0.63 3.1 1.5 1.4 2.8 f 0.31 8 2.6 1.8 2a 2.6 f 0.22 1.3 2.0 1.5 15 1.6 30 3.3 0.66 2.9 1.8 2.1 5.2 i 0.26 2.5 1.2 8 3a 4.8 f 0.36 15 3.1 1.7 1.8 1.2 2.9 f 0.46 30 1.8 1.4 2.5 8 3.5f 0.35 3.2 1.3 IC 2.3 15 3.7 f 0.33 3.0 1.3 1.9 30 5.7 f 0.93 4.5 2.4 2.2 f 0.48 2.0 8 2c 1.8 0.9 3.1 f 0.41 1.9 15 2.3 1.2 2.3 3.4 1.5 4.6 f 0.35 30 2.6 f 0.18 2.1 1.1 1.9 8 le 2.0 15 2.6 1.3 3.3 f 0.27 3.1 f 0.07 2.2 1.2 1.8 30 1.7 8 30 2.8 1.6 2.4 f 0.58 3.2 f 0.31 1.5 15 3.1 2.0 3.5 i 0.77 1.2 30 3.2 2.6 1.3 8 If 2.2 1.7 2.6 f 0.10 15 3.1 f 0.13 1.6 2.9 1.8 30 3.5 f 0.17 1.5 3.0 2.0 8 1.9 f 0.07 2.4 1.3 1.8 31 1.2 15 1.7 1.4 2.2 f 0.12 2.9 i 0.54 1.4 30 3.2 2.0 1.7 8 HCP 4.0 0.29 3.1 1.8 2.0 15 5.9 0.66 3.9 1.9 2.2 30 7.3 f 0.13 5.1 2.3 1.3 saline 1.4 1.1 2.0 f 0.27 0 Mean f SE for five separate observations per dosage. b SE for the saluretic data were calculated and are less than 10% of the mean values. HCT = hydrochlorothiazide.
*
*
evaluate their @-blockingpotency (Table I). In general, when compared with the former derivatives la-3a, the
new (ary1oxy)propanolaminederivatives did not display an improved @-blockingpotency; they were lese potent than the referencecompounds4a,cartmlol,and propaaolol. After intravenous dosing, benzothiazinyl derivative IC, characterized by an sulfonamidoethylaminobridge (X), displayed the best activity (ED60 = 0.45 mg/kg), superior to the other carbostyril analogue 2c (EDw = 1.80 mg/kg) as well as to the all other benzothiazinyl derivatives la-f. The naphthyl derivatives 3 were found to be inactive with the exception of 3f (EDSO= 2.91 mg/kg). In general, these results were confirmed after oral dosing. However,38and lf, usually inactive when administered intravenously, showed an unexpected activity. Although the new derivativesdid not show an improved @-blockingactivity, they had a better diuretic one when compared with the former derivatives la-3a (Table 11). The diureticactivity was especiallysignificantfor the seriea c derivatives which showed an approximately 2-fold urinary output at 30 mg/kg compared to the control. In particular IC,displayingthe best in vivo &blockingactivity after both intravenous and oral dosing, at 30 mg/kg po showed a urinary output equal to 96% of the effect produced by an equimolar hydrochlorothiazidedose of 15 mg/kg and a salureticeffect similarto hydrochlorothiazide at the same dose. In short, the modifications induced in the bridge connecting @-blockingand diuretic pharmacophores confirmed the validity of the proposed symbiotic approach. Specifically, compounds IC and 2c showed the desired pharmacologic profile. It remains to be seen if this dual behavior lowers blood pressure. Nothing can be said, at the present time, about the antihypertensive activity of these compounds because no simple animal model of hypertension is available which responds satisfactory to both @-blockingand diuretic agents. Only after extensivestudies, currently in progrew
160 Journal of Medicinal Chemistry, 1993, Vol. 36, No.1
on compounds IC and 2c, can their presumed efficacy as antihypertensive agents be confirmed.
Experimental Section Melting pointa were determined in capillary tubes (Buchi melting point apparatus) and are uncorrected. Elemental a n a l m were performed on a Carlo Erba Model 1106 elemental analyzer, and the data for C, H, and N are within 10.4 % of the theoretical values. 'H N M R spectra were recorded at 90 MHz (Varian EM 390) or 200 MHz (Bruker AC-200) with M&i as internal standard and MenSO-de as solvent. Chemical shifta are given in ppm (a), and the spectral data are consistent with the assignedstructurea. Massspectra were recorded on Varian MAT 311 A. Reagents and solventa were purchased from common commercial suppliers and were used as received. Column chromatographyseparations were carried out on Merck silica gel 40 (mesh 70-230) and flash chromatography on Merck silica gel 60 (mesh 230-400). Organicsolutionswere dried over anhydrous Naps04 and concentrated with a Buchi rotary evaporator at low pressure. Yields are of purified product and were not optimized. The physical properties of the target compounds lb-f,2b-f, and 3b-f are s u m " d in Table I. 8-(2,3-Epoxypropoxy)-3,4-dihydro-3-oxo-2Hl,4-benzothiazine" and 6-(2,3-epo.ypropoxy)-3,4-carbOatyrilg were prepared by the cited literature methods while 1-(2,3-epoxypropoxy)naphthalene was obtained from Aldrich Chimica. l-(dCbloro-S-rulf~oyl~nzoyl)-dlormylpiperazine (2). A suspension of 4-chloro-3-sulfamoylbenzoylchloride'O (1.8 g, 7.08 "01) in benzene (150 mL) was added portionwise to a stirred solution of 1-formylpiperazine (2.2 g, 19.30 "01) in benzene (50 mL) at room temperature. The mixture was heated at 100 "C for 20 h and then allowed to cool to room temperature. The resulting precipitate was filtered off and washed with saturated aqueous NaHCOa and B O . Recrystallization from EtOH gave 2 (1g, 43%) as a white solid mp 220-222 "C. Anal. (cI?HUC~Sofi)C, H, N. l-(dChloro-3-rulfamoyl~n~yl)pipera~ne (3). The suspension of compound 2 (0.7 g, 2 "01) in MeOH (30 mL) and 36% HCl(2.75 mL) was stirred at room temperature for 18 h. The resulting solutionwas evaporated to dryness to give a white solid (mp 265-267 "C) which was dissolved in H2O (2 mL) and alkalinized (ca.pH 9) by the addition of saturated aqueous NaHCOs The precipitated solid was collected and recrystallized from EtOH to yield 3 (0.34 g, 53 % ) as a white solid mp 234-236 OC; 'H NMR d 2.35-2.65 (4 H, m, CH2NHCH2 piperazine), 2.303.40 (5 H, m, CHgN(CO)CH2 piperazine and NH), 7.45-7.70 (2 H, m, H-5 and H-6), 7.92 (1H, d, J = 2.5 Hz, H-2); MS m/z303.5 (M+). Anal. (CiiHi~ClNsOaS)C, H, N.
N-[ (dChloro-3-rulfnmoylphenyl)rulfonyll-~-acetylethylenediamine (4). A solution of 4-chloro-3-sulfamoylbnzenesulfonyl chloride" (2 g, 6.89 "01) in acetone (30 mL) was added dropwise at room temperature to a stirred solution of N-acetylethylenediamine (0.84 g, 8.22 "01) and triethylamine (0.96 mL, 6.89 "01) in acetone (2 mL). When the addition was complete, the reaction mixture was evaporated to drynessto give a semisolid residue which was dieeolved in warm H20 (30 mL) andth~extractedseveraltimeswithEtOAc.Theorganicphasea were combined, dried,and evaporated to dryneas. The resulting residue was purified by flash chromatography eluting with gradientofCHCbto 10% MeOH/CHCbyielding4 (1.47g,60%) as a white solid mp 155-158 OC. Anal. (cl0HUc~SO&2) C, H, N.
N-[( 4-Chloro-3-rulfamoylphenyl)rulfonyl]ethylene1diamine (6). A suspension of acetyl derivative4 (2.3 g, 6.47 "01) in EtOH (100 mL) and 6 N HCl(23 mL) was refluxed for 32 h. After cooling,the separated white solid was filtered off (mp 251253 OC dec) and then dieeolved in HpO (10 mL). The resulting solutionwas made basic (ca.pH 8)by the addition of a saturated aqueous NasCOs solution and the precipitatedsolid was collectad, dried, and recrystallized from EtOAc/EtOH to yield 6 (1.3 g, 64% 1 as an off-white solid: mp 178-180 O C ; 'H N M R d 2.462.65 and 2.68-2.90 (each 2 H, m, NHCH&H&IHSO2), 5.20 (5 H, br 8, SOaNHl, SOJW and NH3,7.70-7.95 (2 H, m, H-5 and H-6), 8.25 (1H, d, J = 2.5 Hz,H-2). And. (C&I&lN804Sn) C, H, N.
Cecchetti et 01. 1-[ ( 4 - C h l o r o - & r u l l . m o y l p ~ n ~ l ) r ~ o n y l ] - ~ ( e t h o x y ~ bony1)piperarine (6). A solution of 4-chloro-3-sulfamoylbenzBneadfony1chloridei1 (2 g, 6.89 mmol) in acetone (25 mL) WM added dropwise at room temperature to a stirred solution of N-(ethoxycarbony1)piperazine (1.09 g, 6.89 "01) and triethylamine (0.96 mL, 6.89 "01) in acetone (15 mL). After the addition was complete, the reaction mixture was stirred at room temperature for 20 min. The precipitated solid was collected and recrystallized from EtOH to give 6 (1.5 g, 53%) 81) a white solid mp 212-214 "C. Anal. (CisH~&lNaOeSn)C, H, N. 1-[ (4-Chloro-&rulfamoylphenyl)~ulfonyl]piperazine(7). A suspension of 6 (2.4 g, 5.83 "01) in an aqueous solution of 10% NasCOa (30 mL) was refluxed for 20 h. After cooling,the reaction mixturewas adjustedto pH 8by the addition of asolution of 6 N HCl. The precipitated solid was filtered off, washed with H20, dried, and recrystalked from EtOH giving 7 (1.9 g, 96%) as a yellowish-white solid mp 274-276 'C dec; 'H N M R d 2.562.97 (8H, m, piperazine CHs), 7.8Ck7.92 (2 H, m, H-5 and H-6), 8.20 (1H, d, J 2.5 Hz,H-2). Anal. (CidIuClNsO&H&) C, H, N.
N - ( C C ~ ~ r u l t a m o r l p h e n y l ) - 2 n i t r o e t h y l e(8). n A mixture of 4~hloro-3-sulfamoylaniline~~ (11g, 53 "01) and l-chloro-2-nitr0ethane~~ (24g, 219 "01) in dry THF (100 mL) was stirred at room temperature for 8 h. The mixture was then evaporated to dryneas and the residue purified by flash chromatography eluting with 1% MeOH/CH&h to give 8 (4.3 g, 29 % ) as a white solid mp 142-145 "C. Anal. (CsHloClNsOfi) C, H, N. N-(4Chloro-3-r~fpmoylphen~l)et hylenediamine(9). A stirred oolution of 8 (3.7 g, 13.24 "01) in absolute EtOH (400 mL) was hydrogenated over h e y nickel (0.8 g) at room temperature and atmoepheric preasure for 22 h. The mixture was filtered over Celite, and the fdtrate was evaporated to dryness and purified by flash Chromatography eluting with CHnCld a white solid MeOWNH(Cn&)p 73262 to give 9 (2.0 g, 61% mp 257-260 "C; 'H NMR d 2.6CF2.72 and 2.90-3.15 (each 2 H, m, NHpCH&H&IH), 4.18 (4 H, br 8, N H 2 and SOzNHs), 6.036.20 (1H, m, NH),6.60 (1H, dd, J = 3 and 8.4 Hz,H-6), 7.08 (1H, d, J = 3 Hz,H-2),7.14 (1H, d, J = 8.4 Hz,H-5); MS m/z 249.7 (M+). Anal. (C&Ii&lNsO&) C, H, N. l-(dChloro-3-rulfamoylphenyl)pipera~e(10). A mixture of bie(chloroethy1)amine hydrochloride (1.73 g, 9.68 "01) and 4-chloro-3-sulfamoylaniline (2 g, 9.68 "01) in 1-butanol (60 mL) WM refluxed for 48 h and then cooled. Anhydrous Kp COS (1.33 g, 9.6 "01) was then added and the mixture was refluxed for another 48 h. After cooling,the separatedamorphous solid was filtered off and diesolved in Ha0 (10 mL) and the resulting solution was made basic (ca.pH 8)by the addition of a saturated aqueous NaHCOs. The white precipitate was collected, dried, and recryetallized from i-PrOH to give 10 (0.98 g, 37%) as a snow-white solid mp 215-217 "C; 'H N M R d 2.702.95 and 2.98-3.20 (each 4 H, m, piperazine CH2), 6.98 (1H, dd, J = 3 and 9 Hz, H-6),7.28 (1H, d, J 9 Hz,H-5),7.37 (1H, d, J = 3 Hz,H-2); MS m/z 275.7 (M+). Anal. ( c l ~ u c ~ S o & C, ) H, N. ' General P d u r e for the Preparation of (Aryloxy)propanolaminer lb-f,tb-f, Sb-f. Thisprocedure is illustrated for the preparation of l-[4-(4-chloro-3-sulfamoylbenzoyl)-lpiperezinyll -3- [ ( 3 , ~ y ~ 3 - o x ~ W - l , 4 ~ ~ ~ y l ) ~ l propan-2-01 (lb). A mixture of piperazine derivative 3 (0.64 g, 2.11 "01) and 8-(2,3-epoxypropoxy)-3,4-dihydro-3-oxo-2H-l,4-ben~othiazinc'. (0.5 g, 2.11 "01) in absolute EtOH (300mL) was refluxed for 30 h. The solvent was then evaporated to dryneee, and the solid residue was purifies by column chromatography eluting with gradient of CHCb to 10% EtOH/CHCb to give lb (0.6 g, 53%) as a white amorphous solid mp 145-165 "C; 'H NMFt d 2.40-2.60 (6 H, m, CHZN(CH~)CHP), 3.30-3.70 (6 H, m, SCHs and CH2N(CO)CHd,3.90-4.10 (3 H, m, OCH&H(OH)), 4.90 (1 H, br s, OH), 6.60 and 6.70 (each 1H, d, J = 8.1 Hz,H-5 and H-7 benzothidine), 7.10 (1H, t, J = 8.1 Hz,H.6 benzothbine), 7.60 (1H, dd, J = 8.3 and 1.7 Hz,H-6),7.70 (1H, d, J 8.0Hz,H-51, 7.75 (2 H, br I, SOpNHg), 7.96 (1H, d, J = 1.7 Hz, H-2),10.W (1H, 8, NH). Anal. (CaHnsClN4OsSn) C, H, N. Binding Debrminationr. Pellete containing 61type adrenergic receptom were obtained from turkey erythrocyte mem-
/3-BlockinglDiuretic Agents branee as described in the literature.l4 [~HlDihydroalprenolol ([‘HIDHA) obtained from New England Nuclear (NEN), having a specific activity of 99.9 Ci/mmole and a radiochemical purity >98.5%, was used as ligand. &Adrenergicreceptor binding was determined as follow: 100 p L of membranes (431 rcg/mL of protein diluted 1:8 v/v) were incubated for 15 min at 37 OC with 6 nM [‘HIDHA and 100 p L of various concentrations of the teat compound (dissolved in DMSO 6 % ) in 90mM sodium chloride and 12 mM Tris, pH = 7.5 (tofdvolume 1A). The incubationswere stopped by adding 3 mL of cold buffer followed by rapid filtration through glass fiber fiter disks (Whatman GF/B). The samples were subsequently washed with 4.5 mL of the same buffer and placed into scintillation viala; 10 mL of Filter-count (Packard) liquid scintillation cocktail was then added to each vial and counted using a scintillation spectrometer (Packard TRI-CARB 3OOC). Nonspecific binding was d e f i e d as nondisplaceable binding in the presence of 10 pM propranolol and specific binding as the difference between total and nonspecific binding. Blank experiments were carried out to determine the effW of the solvent (5%)on binding. The concentrationof the test compounds that inhibited [*HIDHA or binding by 50% (ICm) were determined by log-probit analysiswith seven concentrationsof the displacers, each performed in duplicate. The ICmvalues obtained were used to calculate apparent inhibition constants (Ki)by the method of Cheng and Prueoff.ls The Ki values for the inhibition of [‘HIDHA binding are reported in Table I and compared with tertbutyl-l,dbenzothiazine derivative 4a, carteolol, and propranolol. Anti-Iropmndne Activity. The 8-adrenoceptorblocking activity was studied in vivo by the inhibition of tachycardia induced by isoprenaline (INA) in rats.18 For this purpose 0.12 rcg/kg of isoprenalinewere injected intravenously (jugular vein) into male Wistar rata, weighing 250-300 g, previously anaesthetized with eodium nembutal(55 mg/kg, ip). The increased in heart rate (HR) was evaluated by electrocardiograph. After several minutes, when the normal heart rate was restored, the test compound was administered intravenously, diseolved in DMSO, or orally, suspended in 1%gum arabic. Responses to isoprenaline were obtained 2 min llfter intravenous or 1h after oral administration. Blank experiments were carried out to determine the effect of the solvent on the test. The comparison was made with two known 8-blockers: carts0101 and propranolol. Three rata were used per group and the results are reported in Table I as EDm values obtained by log-probit analysis. Diuretic Activity. Groups of five male CD rata, weighing 150-170 g, were used. All teat compounds and hydrochlorothiazide (HCT), as control drug,were orally administered dissolved or suspended in 25 mL/kg of saline. Control animals received 25 d / k g of saline only. The rata were starved and deprived of water for 18 h prior to the dosing and, after the administration of the test compounds, were immediately placed singly in metabolic cages. No food or water was supplied during the experimental period. Urine was collected during the 0-5-h interval in volumetric graduate cylinders and was analyzed for sodium and potassium by flame photometry. The results are reported in Table II.
Acknowledgment. We are grateful to Mr. Roberto Bianconi for skillful synthetic assistance. The research was assisted in part by a grant from the Minister0 dell’Universit21 e della Ricerca Scientifica e Tecnologica. Supplementary Material Available: IH NMR data of intermedimtea and target compounds not reportad in the text and antihypertensive activity in S H R model of compound IC (4 pages). Ordering information is given on any current masthead PWe.
References (1) This work was previody p m t e d in part: Cecchetti,V.;Fravolini, A;schiattella.F.; Tabamhi, 0.;Bruni, G.;Segre, 0.An Approach to Diuretic-&Adrenergic Blocking Agents. International Sympo-
Journal of Medicinal Chemistry, 1993, Vol. 36,No.1 161 eium on Medicinal Chemistry,Jerusalem, hael, September 1890, p 41. Gifford, R.W.; Borazanian,R.A. Traditional FhbLme Therapy Overviewof MedicalBenefitsand SideEffecta. Hypertension 1989, 13(1),119-124. (a) Willard, A. K.; Smith, R. L.; Cragoe, E. J. Potential Diuretic&Adrenergic Blocking Agenu: Syntherh of S-[2-[(l,l-Dimethylethyl)amino]-1-hydroIlyethyl]-l,~o~o[2,S-glquinolin~ J.Org.
Chem. 1981,46,3846-3852. (b) Bouley, E.; Teulon, J. M.; C w , M.;Cloarec,A.; Deghenghi,R (p-r(Thienylcarbonyl)aminolp~noxylpropanolamine Derivatives an Diuretic and &Adrenergic Receptor Blocking Agenta. J. Med. Chem. 1966,29,1W103. (c) Kau, S. T.; Howe, B. B.; Li, J. H.-Y.; Smith, L.H.; Kddie, J. R; Barlow, J. J.; Giler, R.E.;Goldberg, M. E.IC1 147,79& A N m l Diuretic Agent with Beta Adrenoceptor Blocking Activity. J. Pharmacol. Exp. Ther. 1987,242,818-826. Cecchetti,V.;Schiaffella,F.; Tabarrini, 0.;Zhou, W.; Fravolini,A.; h i , A.; Bruni, G.; Segre, G.SymbioticApproach to Drug Design:
N-[ ( 4 - C h l o r o - 3 - e u l f a m o y l b e ~ d o ~ e t h y l l p r o ~Derivo~e ativee as &Adrenergic Blocking Agents with Diuretic Activity. Eur. J. Med. Chem. 1991,26,381-386. (a) Large, M.5.;Smith, L. H. &Adrenergic Blocking Agents. 19. J. Med. 1-Phenyl-2-[[(nu~tituted-~mido)allyllaminolethano, Chem. 1980,23,112-117. (b)Large,M.S.;Smith,L.H.@-Adrenergic Blocking Agents. 22. 1-Phenoxy-3-~~~eubtituted-amido)allyllaminol-2-propanob. J.Med. Chem. 1982,26,1288-1292. (c)Large, M.S.;Smith,L. H.&AdrenergicBlockbgAgents. 24. Heterocyclic Subtituted l-(Aryloxy)-3-[[(amido~allyllamino]propsa-2sl.J. Med. Chem. 1982,26,1417-1422. (d) Large, M.8.;Smith, L. H. &Adrenergic Blocking Agents. 23. l-[(Su~tituted-amido)phenoxy1-3- [(subtituted-amido)alkyll amino1propan-2sl. J. Mad. Chem. 1983,26,352-357. (e) Maulean,D.;DolorsPujol,M.;Ro#ll, G. &Adrenergic Antagonints: N-Alkyl and N-Amidoethyl(ary1alkoxy)propanolaminee Related to Propranolol. Eur. J. Med. Chem. 1989,23,421-426. (a) Boiiier, G. R.;Dumont, C.; Labroe, J. Diuretic Activity of 3-Sulfamido-4-chlorobenzoicAcid (SD 141.08)and of S-Suifamidc4-chlorobenzamide (SD 141.12). Theropie 1966,21,331-340. (b) Wild, A.M. Diuretic Jackman, G. B.; Petrow, V.;Stepheneon, 0.; Agents. VI. SomeSulfamoylbenzoicAcids. J.Pharm.Pharmacol. 1962,14,679886. (c) Hoefle, M. L.; Blouin, L. T.; DeWald, H.A; Holmea, A.; Williamn, D. Diuretics. 4-Substituted 3-Sulfamoylbenzoic Acids Hydrazidee. J. Med. Chem. lS(uI,ll, 97073. (a) Cecchetti, V.; Fravolini, A,; Fringueili, R.; S c W e l l a , F.; Mnncellani, G.; Pagella, P. G.; Rugarli, P. L. Syntheeh and &Adrenergic Blockina Activity of 0.yDroDanoIamiiea of 3.4Daydro-~o=o-2H(1,4,benzothiclzine FU&&o Ed. Sci. 1987,42, 61-75. (b) Cecchetti, V.;Fringuelli, R.;Schiaffella, F.; Fravolini, A,; Bruni, G.;Fiaschi, A. I.; Segre, 0. Syntheab and @-Adrenergic Oxime Ethem. Eur. J.Med. Blocking Activityof 1,4-Benzo-e Chem. 1989,24,479-484. (8) Brewnter, K.; Coult, D. B.; Pinder, R. M. 1-PhenylpiperPotential Antagonists of Lwrgic Acids Diethylamide. Chem. Therapeut. 1972,8741. (9) Nakagawa, K.; MuraLami, N.; YoshizaLi, 5.;Tominaga, M.;Mori, H.; Yabuuchi, Y.; Shintani,5.Derivativeaof3 , 4 - D i h ~ h t y r i I an ,%AdrenergicBlocking Agenta. J. Med. Chem. 1974,17,529533. (10) Parke, Davin Co. 4-Halo-3-eulfamoylbemoicAcidn &ten. Brit. 915,259; Chem. Abstr. 1964,61,6962. (11) Boehringer, C. F. & Soehne (3.m.b.H. AnymmetridySubtituted Dbulfonamidee. Brit. 898,243; Chem. Ab&. 1962,67,12386. (12) Jucker,E.; Linden”, A.; Schenker,E.;Flllckiger,E.;T a d e r , M. Synthetic Dmgn. XII. Conntitution and Saluretic Effect of 3-Sulfamoyl-4-chlorobenzoicAcid Derivativer and Wted Compobds. Arzneim.-Forach. 1983, 13,269-280. (13) Zefwov, N. 9.;Koz’miu,A. 5.;Zhdankin,V.V.;Nikulin, A. V.;Zyk, N. V.Lithium Perchloratean a Reagentfor Syntheahof Covalently Bonded Organic Perchlorah via ElectrophilicAdditiom of H a b gena and Nitronium Tetrafluoroborateto Olefm. J. Org. Cham. 1982,47,3679-3684. (14) Mieman, K. P.; Weiland, G. A.; Molinoff,P. B. A Camp" of the Beta-AdrenergicReceptor of the Turkey Erythrocytb with M d i a n Beta1 Betas Receptors. Mol. Pharmacol. 1979,17, 1-7. (15) Cheng, Y. C.; Prueoff, W. H. Relationship between the Inhibition Conatant (KO and the Concentration of Inhibitor which C a m 50% Inhibition (&Q)of an Enzymatic Flaaction. Biochem. Pharmacol. 1973,22,3099-3108. (16) Mylecharane, E.J.; Raper, C. 2-Nitrilophenoxypropanoiaminea: p-AdrenocsptorAntagoninmin the Rat. Arch. In:. Pharmacodyn. 1973,202, 163-170.
